close

Вход

Забыли?

вход по аккаунту

?

The molecular and metabolic characterization of the CTP:phosphoethanolamine cytidylyltransferase knockout mouse

код для вставкиСкачать
THE MOLECULAR AND METABOLIC CHARACTERIZATION OF THE
CTP:PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE KNOCKOUT
MOUSE
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
MORGAN D. FULLERTON
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
September, 2009
© Morgan D. Fullerton, 2009
1*1
Library and Archives
Canada
Bibliotheque et
Archives Canada
Published Heritage
Branch
Direction du
Patrimoine de I'edition
395 Wellington Street
Ottawa ON K1A 0N4
Canada
395, rue Wellington
Ottawa ON K1A 0N4
Canada
Your file Votre reference
ISBN: 978-0-494-58300-5
Our file Notre reference
ISBN: 978-0-494-58300-5
NOTICE:
AVIS:
The author has granted a nonexclusive license allowing Library and
Archives Canada to reproduce,
publish, archive, preserve, conserve,
communicate to the public by
telecommunication or on the Internet,
loan, distribute and sell theses
worldwide, for commercial or noncommercial purposes, in microform,
paper, electronic and/or any other
formats.
L'auteur a accorde une licence non exclusive
permettant a la Bibliotheque et Archives
Canada de reproduire, publier, archiver,
sauvegarder, conserver, transmettre au public
par telecommunication ou par I'lnternet, preter,
distribuer et vendre des theses partout dans le
monde, a des fins commerciales ou autres, sur
support microforme, papier, electronique et/ou
autres formats.
The author retains copyright
ownership and moral rights in this
thesis. Neither the thesis nor
substantial extracts from it may be
printed or otherwise reproduced
without the author's permission.
L'auteur conserve la propriete du droit d'auteur
et des droits moraux qui protege cette these. Ni
la these ni des extraits substantiels de celle-ci
ne doivent etre imprimes ou autrement
reproduits sans son autorisation.
In compliance with the Canadian
Privacy Act some supporting forms
may have been removed from this
thesis.
Conformement a la loi canadienne sur la
protection de la vie privee, quelques
formulaires secondaires ont ete enleves de
cette these.
While these forms may be included
in the document page count, their
removal does not represent any loss
of content from the thesis.
Bien que ces formulaires aient inclus dans
la pagination, il n'y aura aucun contenu
manquant.
1+1
Canada
ABSTRACT
THE MOLECULAR AND METABOLIC CHARACTERIZATION OF THE
CTP:PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE KNOCKOUT
MOUSE
Morgan D. Fullerton
University of Guelph, 2009
Advisor:
Marica Bakovic
Phosphatidylethanolamine (PE) is a critical inner membrane phospholipid,
important for various cellular functions. CTP:phosphoethanolamine cytidylyltransferase
(Pcyt2), catalyzes the formation of CDP-ethanolamine, which is then combined onto
diacylglycerol (DAG) to form PE via the de novo PE-Kennedy pathway. This thesis aims
to elucidate the role of Pcytl and therefore the de novo pathway in a Pcyt2 knockout
model.
Homozygous disruption of Pcytl results in embryonal lethality prior to 8 days of
development. Pcyt2+/~ mice were phenotypically indistinguishable from wildtype
littermate controls during the first months of development. The Pcytl mRNA and protein
content as well as the in vitro enzyme activity in heterozygous liver, heart, brain and
kidney were -65 % of wildtype levels and therefore up-regulated from the expected 50%,
given the single allele. Despite the alteration in Pcytl expression, there were no changes
in PE tissue content. Phospholipid homeostasis was preserved without compensatory
increases in phosphatidylserine decarboxylation and in spite of a decreased rate of PE
synthesis via that PE-Kennedy pathway, due to a corresponding decrease in the PE
degradation.
Although normal after birth, Pcytl+/~ mice became significantly heavier than
littermate controls at -5-6 months. This phenotype was accompanied by an array of
metabolic disturbances including hepatic and skeletal muscle triglyceride (TG)
accumulation, increased adiposity, hypertriglyceridemia, decreased fatty acid oxidation
and diminished insulin sensitivity. Metabolic radiolabeling experiments revealed that in
Pcyt2+/~ primary hepatocytes, DAG synthesis and degradation as well as TG formation
were increased. Oleate uptake was increased in heterozygous hepatocytes, while no
increase in total content of the main fatty acid transporters were established and in vivo
labeling indicated that hepatic fatty acid synthesis was also increased. Pcyt2+/~ mice had
increased mRNA expression of various lipogenic transcription factors as well as their
downstream targets in both liver and skeletal muscle. These data suggest that a
diminished PE synthesis caused a redirection of DAG toward TG, which was facilitated
by alterations in fatty acid metabolism.
To further investigate the role of Pcyt2 in the progression of the heterozygous
metabolic phenotype, a wildtype or mutant Pcyt2 cDNA construct was transiently
transfected into primary hepatocytes. Expression of Pcyt2 completely restored the
diminished rates of PE synthesis and degradation via the PE-Kennedy pathway, where
the mutant construct (-40% decreased activity) was unable to fully compensate. In
addition, the rates of DAG and TG synthesis from [3H] glycerol were normalized and de
novo fatty acid synthesis was decreased to wildtype levels with Pcyt2 expression. These
data demonstrate that the genetic disruption of Pcyt2 causes a chronic state of decreased
PE production and turnover, altered DAG and TG synthesis and defects in fatty acid
metabolism in Pcyt2+' mice.
PUBLICATIONS
This thesis is based on the following publications:
Fullerton MD, Hakimuddin F, Bakovic M (2007). Developmental and metabolic effects
of disruption of the mouse CTP:phosphoethanolamine cytidylyltransferase gene (Pcytl).
Mol Cell Biol 27:3327-36.
Fullerton MD, Hakimuddin F, Bonen A, Bakovic M (2009). The development of a
metabolic disease phenotype in CTP:phosphoethanolamine cytidylyltransferase deficient
mice. J Biol Chem. Epub July 22.
Fullerton MD, Bakovic M (2009). Phenotypic rescue of CTP:phosphoethanolamine
cytidylyltransferase (Pcytl) deficient primary hepatocytes. Submitted to BBA (BBALIP09-184.)
I would like to acknowledge the work completed by the following collaborators and
companies:
Dr. Fatima Hakimuddin: Performed mitochondrial staining and helped with oleate
uptake.
The group in the Molecular and Cell Biology of Lipids (University of Alberta):
Performed lipoprotein analyses.
Laelie Snook: Completed Western blot analyses on liver and whole muscle homogenates
for fatty acid transport proteins.
Lipid Analytical Laboratories: Performed gas chromatography analyses for lipid fatty
acid composition.
InGenious Targeting Laboratory: For production of the targeting vector and the
generation of the founding hetero2ygous mice.
ACKNOWLEDGEMENTS
I would like to thank my advisor Dr. Marica Bakovic. Thank you for all of the
opportunities you've provided and for your guidance, support and patience over the past
five years. Not many supervisors would trust a new knockout colony to a first year
master's student, but your trust, understanding and expectations were always appreciated.
I have learned more than I believe you thought you were teaching and I wish you all the
best in the future.
Thank you to Dr. David Dyck for serving as a member of my advisory committee.
I've deeply appreciated your input, criticisms and advice on all things academic and
woodworking. Thank you also to Dr. Ray Lu, the last member of my advisory committee,
for helpful advice and assistance with immunohistochemistry.
My research would not have been possible without the help of various
laboratories. Thank you to Dr. Lindsay Robinson's lab (Justine Tishinsky in particular)
for allowing me to save my thumbs and use your multi-channel pipettes, to Dr. Arend
Bonen's lab and specifically Jamie and Laelie for your help. Thank you also to Marcus
Litman, Annette Morrison and the numerous CAF technicians, for your help, guidance
and expertise.
To my friends in the Bakovic lab (past and present), thank you for the laughs and
all of the good times (try to keep the lab clean). Specifically, to Angela Tie, Vera Michel
and Lin Zhu, who make up (for me) the original Bakovic crew, thank you for your help
and your friendship. Thank you to Brianne Thrush and Mark Dekker for countless
scientific discussions and to all friends and colleagues in the department for making five
years go by so quickly.
i
To my family, thank you for understanding. Thank you to my parents, Donnie and
Drew for your unconditional love, support, encouragement and for providing a wonderful
mountain retreat. Thank you to my brother Spencer for your ability to relate to me about
everything but science (that's a good thing). Thank you also to my newer family, Jeff and
Sam and to Debbie and Barry for the safe-haven away from school, for your support and
for your daughter.
Thank you Jessica, my wife and my life. The day has finally come when you
won't have to tell people that your husband is still a student. Thank you for listening to
my short stories long and for understanding that I do that. We are so lucky to be able to
talk science with each other and I am thankful for all of your help, from painful proofreads, to showing me how to do TLC for the first time, to checking on the mice with me.
Our life together has just begun and every day I love you more.
11
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
I
TABLE OF CONTENTS
HI
LIST OF TABLES
VII
LIST OF FIGURES
VIII
LIST OF ACRONYMS
X
CHAPTER ONE
1
INTRODUCTION
1.1 ETHANOLAMINE PHOSPHOLIPIDS
2
1.2 PE BIOSYNTHETIC PATHWAYS
5
1.2.1 PE-Kennedy Pathway
6
1.2.2 PS Decarboxylation
8
1.2.3 Additional Synthetic Pathways
9
1.3 CTP:PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE (EY/PCYT2)
11
1.4 KNOCKOUT MODELS OF PHOSPHOLIPID RELATED GENES
14
1.5 THE LINK BETWEEN NEUTRAL LIPIDS AND PHOSPHOLIPIDS
15
CHAPTER TWO
17
OBJECTIVES OF THE THESIS
2.1 OBJECTIVES OF THE THESIS
18
CHAPTER THREE
20
DEVELOPMENTAL AND METABOLIC EFFECTS OF DISRUPTION OF THE MOUSE
CTP:PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE GENE (PCYT2)
3.1 ABSTRACT:
21
3.2 INTRODUCTION
...22
3.3 EXPERIMENTAL PROCEDURES
25
3.3.1 Targeted disruption of the Pcyt2 gene
25
3.3.2 Animals
25
3.3.3 Genotyping
26
3.3.4 RNA analyses
27
3.3.5 Production of Pcyt2-specific antibody
28
3.3.6 Histology and immunohistochemistry
28
3.3.7 ET immunoblotting
29
111
3.3.8 ET enzymatic activities
30
3.3.9 Phospholipid content
31
3.3.10 Hepatocyte isolation and metabolic labeling
31
3.3.11 Mitochondrial staining
32
3.3.12 In vivo labeling
33
3.3.13 Phospholipid fatty acid composition
33
3.3.14 Statistical analysis
33
3.4 RESULTS
34
3.4.1 Disruption ofPcyt2 results in embryonic lethality
34
3.4.2 Heterozygosity decreases Pcyt2 mRNA expression
38
+
3.4.3 Pcyt2 ' protein expression and enzymatic activity are also diminished.
+
40
3.4.4 Phospholipid content does not change in Pcyt2 ' mice
42
3.4.5 PS decarboxylase and mitochondria are unaltered
43
3.4.6 In vitro and in vivo PE synthesis
45
3.4.7 Hepatic phospholipid fatty acid composition is remodeled
49
3.5 DISCUSSION
52
CHAPTER FOUR
56
THE DEVELOPMENT OF A METABOLIC DISEASE PHENOTYPE IN
CTP:PHOSPHOETHANOLAMINE CYT1DYLYLTRANSFERASE DEFICIENT MICE
4.1 ABSTRACT
57
4.2 INTRODUCTION
58
4.3 EXPERIMENTAL PROCEDURES
62
4.3.1 Animals
62
4.3.2 Plasma lipid analyses and liver histology
62
4.3.3 Rate of VLDL secretion
62
4.3.4 Glucose tolerance test
63
4.3.5 Phospholipid, DAG and TG content and composition
63
4.3.6 Isolation of primary hepatocytes
64
4.3.7 Hepatic fatty acid uptake and transporter expression
65
4.3.8 Hepatic glycerol uptake and glycerolipid metabolism
65
4.3.9 Hepatic fatty acid oxidation
66
4.3.10 In vivo lipid radiolabeling
67
4.3.11 Food intake and energy expenditure
68
4.3.12 RNA isolation and expression analyses
68
4.3.13 Statistical Analyses
68
4.4 RESULTS
70
IV
4.4.1 Pcyt2*' mice have reducedPE synthesis and degradation
70
+/
4.4.2 Pcyt2 ~ mice accumulate liver DAG and TG and develop adult-onset obesity
72
+/
4.4.3 Pcyt2 ~ mice have altered plasma and liver FA composition and hypertriglyceridemia
+
4.4.4 Triglyceride synthesis and lipogenesis are increased in Pcyt2 ' mice
76
80
+
4.4.5 Reduced energy expenditure and reducedfatty acid oxidation in Pcyt2 ' mice
88
+/
4.4.6 Pcyt2 ~ mice develop insulin resistance
90
+
4.4.7 Expression pattern of lipid, glucose and insulin signaling genes is modified in Pcyt2 ' muscle
and liver.
92
4.5 DISCUSSION
98
CHAPTER FIVE
103
PHENOTYP1C RESCUE OF CTP: PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE
(PCYT2) DEFICIENT PRIMARY HEPATOCYTES
5.1 ABSTRACT
104
5.2 INTRODUCTION
105
5.3 EXPERIMENTAL PROCEDURES
107
5.3.7 Primary hepatocyte isolation
107
5.3.2 Transfection of primary hepatocytes
707
5.3.3 Pcyt2-myc Western Blotting
108
5.3.4 Metabolic labeling
109
5.3.5 Pcyt2-H244Y-myc cloning.
770
5.3.6Pcyt2-H244Y-myc characterization in COS-7 cells
777
5.3.7 mRNA expression
777
5.3.8 Statistical Analyses
772
5.4 RESULTS
113
5.4.1 Characterization of the Pcyt2-H244Y-myc mutant in COS-7 cells
775
5.4.2 Expression of Pcyt2-myc but not Pcyt2-H244Y-myc normalizes PE metabolism
775
5.4.3 Radiolabeling of the PE-Kennedy Pathway
777
5.4.4 DAG and TG synthesis is rescued with Pcyt2-myc expression
118
5.4.5 De novo fatty acid synthesis is reduced with Pcyt2-myc rescue
122
5.4.6 The activity of wildtype andPcyt2-H244Y mutant is not additive
124
5.4.7 Pcyt2-myc rescues lipogenic gene expression in Pcyt2 heterozygous hepatocytes
725
5.5 DISCUSSION
127
CHAPTER SIX
132
GENERAL DISCUSSION
6.1 DISCUSSION
133
v
6.1.1 PE Synthesis
133
6.1.2 DAG as a precursor for PE and TG
137
6.1.3 Lipogenesis and FA metabolism in Pcyt2 Heterozygous mice
142
6.1.4 SREBP as a master regulator of lipid homeostasis in Pcyt2 deficiency
144
6.1.5 Genetically engineered mouse models
146
6.1.6 Future work
149
6.2 GENERAL CONCLUSIONS
152
REFERENCES
154
APPENDIX
176
APPENDIX 1
177
APPENDIX II
179
APPENDIX III
180
APPENDIX IV-STANDARD OPERATING PROTOCOL: PCYT2 KNOCKOUT COLONY
182
A/ Generation of Pcyt2 knockout mice
182
B/The first mice at University of Guelph
188
C/Life After Isolation and Breeding
790
D/Colony Maintenance
190
E/ Pcyt2 Knockout Colony Records
194
F/ Procedures
195
VI
LIST OF TABLES
Table 3.1. Genotypes of pups from Pcytl ' intercrosses
36
Table 4.1. Plasma and hepatic TG FA compositions of 36 wk old mice
77
Table 4.2. Hepatic DAG FA compositions
78
Table 4.3. Quantitative summary of hepatic lipids
79
Table 4.4. Real-time PCR results for insulin-regulated genes in Pcyt2+/~ liver from 36 wk
old mice
94
+/
Table 4.5. Real-time PCR results for insulin-regulated genes in Pcyt2 ~ skeletal muscle
from 36 wk old mice
95
Table 5.1. Rates of synthesis for pulse labeling (1-4 h) of primary hepatocytes
121
Table 5.2. Rates of degradation after pulse-chase labeling in primary hepatocytes
122
Appendix III. Table Al. List of primer sequences
180
vn
LIST OF FIGURES
Figure 1.1. PE and phospholipid synthetic pathways
11
Figure 3.1. Targeted disruption of the Pcytl gene
35
Figure 3.2. Embryo histology and developmental Pcyt2 protein expression
37
Figure 3.3. Relative Pcyt2 mRNA expression assessed by semi-quantitative RT-PCR... 39
Figure 3.4. Relative ET protein expression between Pcyt2+ + and Pcyt2+' mice
40
Figure 3.5. ET in vitro enzyme activity
41
Figure 3.6. Comparison of phospholipid content between Pcyt2+/+ and Pcyt2+/~ mice.... 42
Figure 3.7. PisdmKNA expression and mitochondrial staining of hepatocytes
44
Figure 3.8. In vitro contributions of PE biosynthetic pathways
46
Figure 3.9. In vivo contributions of PE biosynthetic pathways
48
Figure 3.10. Comparison of hepatic phospholipid compositions
50
Figure 4.1. CDP-Ethanolamine, DAG and TG synthetic pathways
59
Figure 4.2. Total PE content in isolated hepatocytes
70
Figure 4.3. PE metabolism is altered in heterozygous mice
72
Figure 4.4. Pcyt2+/" mice gain weight chronically
73
Figure 4.5. Pcyt2+/~ mice have fatty liver and hypertriglyceridemia
75
Figure 4.6. Rate of VLDL secretion in young and old mice
80
Figure 4.7. Pcyt2+/~ mice redirect DAG for TG synthesis and have increased hepatic FA
uptake
81
+/
Figure 4.8. Young Pcyt2 ~ mice redirect DAG for TG synthesis
83
Figure 4.9. Western blotting of fatty acid transporters
85
Figure 4.10. In vivo lipogenesis
86
Figure 4.11. Increased de novo lipogenesis in Pcyt2+' livers
87
Figure 4.12. Altered whole-body metabolism in Pcyt2+/~ mice
89
Figure 4.13. Reduced insulin sensitivity and increased muscle lipid accumulation in
Pcyt2+/~ mice
91
Figure 4.14. Altered Pcyt2+/~ hepatic and skeletal muscle mitochondrial and lipogenic
gene expression
93
Figure 5.1. Characterization of the Pcy/2-H244Y-myc mutation
vm
114
Figure 5.2. Pcyt2-myc expression rescues PE metabolism in Pcyt2 "hepatocytes
116
Figure 5.3. DAG and TG metabolism is normalized with expression
119
Figure 5.4. Pcyt2-myc rescue decreases elevated neutral lipids
120
Figure 5.5. Increased lipogenesis in Pcyt2+/~ hepatocytes is corrected by Pcyt2-myc
expression
123
Figure 5.6. The active site mutant Pcyt2-H244Y-myc is not dominant negative
125
Figure 5.7. mRNA expressions of lipogenic genes are affected by Pcyt2 rescue
126
Figure 6.1. Protein sequence alignment for ET and CTa and CTP
134
Figure 6.2. Depiction of acylglycerol recycling
138
Figure 6.3. Glycerolipid synthetic pathways and the consequences of Pcyt2 deficiency.
149
Figure Al. Generation of Pcyt2 Transgenic Mice
177
Figure A2. Pcyt2-myc expression in Pcyt2-TgN livers
179
IX
LIST OF ACRONYMS
Arachidonic acid
AA
Acetyl-CoA carboxylase
ACC
1 -aclyglycerol-3-phosphate acyltransferase
AGPAT
AMP activated-protein kinase
AMPK
Choline/ethanolaminephosphotransferase
CEPT
Chinese hamster ovary
CHO
Carnitine palmitoyl transferase I
CPT-I
CTP:phosphocholine cytidylytransferase
CT
Diacylglycerol
DAG
Diacylglycerol acyltransferase
DGAT
Docosahexaenoic acid
DHA
Early growth response factor 1
EGR-1
Ethanolamine kinase
EK
CDP-ethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase
EPT
Fatty acid
FA
plasma membrane-bound fatty acid binding protein
FABPpm
Fatty acid synthase
FAS
Fatty acid translocase/CD36
FAT/CD36
Fatty acid transport protein
FATP
Glycerol-3-phosphate acyltransferase
GPAT
High-density lipoprotein
HDL
3 -hydroxy-3 -methylglutaryl-CoA reductase
HMG-CoA-R
Low-density lipoprotein
LDL
Lysophosphatidic acid
LPA
Lysosomal phospholipase A2
LPLA2
Mitochondrial-associated membrane
MAM
Monounsaturated fatty acid
MUFA
Neutral lipid storage disease
NLSD
Phosphatidic acid
PA
Phosphatidic acid phosphatase or lipin-1, 2 or 3
PAP
Phosphatidylcholine
PC
CTP: phosphoethanolamine cytidylyltransferase
Pcyt2/ET
Phosphatidylethanolamine
PE
Phosphatidylethanolamine jV-methyltransferase
PEMT
Phospholipase A2
PLA2
Phosphatidylserine
PS
Phosphatidylserine decarboxylase
PSD
Phosphatidylserine synthase
PSS
Polyunsaturated FA
PUFA
Respiratory quotient
RQ
Stearoyl-CoA acid desaturase 1
SCD-1
Saturated fatty acids
SFA
Sterol regulatory element-binding proteins
SREBP
Triglyceride
TG
Very-low density lipoprotein
VLDL
XI
CHAPTER ONE
INTRODUCTION
1.1 Ethanolamine Phospholipids
Phosphatidylethanolamine (PE) is an essential membrane constituent and an
important supplier of biologically active molecules. PE is the most abundant
phospholipid in prokaryotic cell membranes, whereas in eukaryotes PE is secondary to
phosphatidylcholine (PC) (40). In eukaryotes, the majority of PE is distributed on the
inner leaflet of the plasma membrane together with phosphatidylserine (PS), while the
choline-containing phospholipids, PC and sphingomyelin, are mainly located on the outer
leaflet of the bilayer [(35) and recently reviewed in (217)]. PE is a cone-shaped lipid due
to the small size of its head group relative to its large hydrophobic region, which tends to
form non-lamellar structures and thus increases the non-bilayer-forming character of the
membranes (152, 156). This characteristic explains the role of PE in stabilizing integral
and membrane bound proteins (46, 159) and its importance as a molecular chaperone
required for the correct folding and functioning of membrane proteins (22, 213). PE is
specifically involved in membrane fusion, as demonstrated in erythrocyte large
unilamellar vesicles, where vesicles containing PE were shown to be more fusogenic
when compared to PC and sphingomyelin containing vesicles (33). PE also tends to
regulate the dynamic movement of cytoskeletal proteins and subsequent membrane
fusion during cytokinesis in Chinese hamster ovary (CHO) cells (41, 42), becoming
exposed on the cell surface during late telophase/Gl phase of the cellular cycle (95) and
during myoblast/myotubes cell-cell fusion (150, 153). PE deficiency induced by a genetic
defect in Escherichia coli (125) or by surface trapping of PE by a PE-specific antibody in
CHO cells (42), causes an arrest in cell growth. This suggests that PE has an important,
evolutionarily conserved role during cellular division [reviewed in (44)].
2
The loss of membrane phospholipid asymmetry is a signal for important
physiological processes including platelet activation and clearance of apoptotic cells by
phagocytosis. These processes are largely due to increased PS content at the outer layer
of the plasma membrane; however, significant portions of PE also migrate and become
exposed in conjunction with PS (43). Generally, PE cooperates with PS to induce local
membrane perturbations and thus to increase membrane affinity for the binding of
enzyme complexes involved in blood coagulation. Furthermore, a specific import of PE
from both low-density (37, 45) and very-low density lipoproteins (LDL and VLDL
respectively) (76, 94) occurs during platelet activation. This has been shown to further
enhance the procoagulant activity of platelets due to the oxidation products of the
unsaturated fatty acid (FA) component of PE (45, 227).
Cell agonists such as interleukin-1 and phorbol esters can specifically trigger PE
hydrolysis, typically leading to the modulation of selective protein kinase C pathways
(112, 130, 175). Products of PE metabolism such as FAs, diacylglycerols (DAG) and
phosphatidic acid serve as substrates for other lipids and play a critical role as second
messengers in various signaling pathways. The focus of most studies on signaling
cascades has been on inositol-triphosphate and PC-dependent pathways (48); however, it
is clear that PE is also a substrate for different phospholipases (67, 91). In particular,
phospholipase A2 (PLA2) and D isoforms have been associated with PE hydrolysis.
Recently, a lysosomal phospholipase A2 (LPLA2) has been identified (1,2) and shown to
be crucial for macrophage PE hydrolysis in a LPLA2 knockout mouse model (68).
Glycosylated PE is abundant in LDL of diabetics and has been implicated in the
promotion of atherosclerosis in those individuals (147), as it seems to increase LDL-
3
oxidation and could accumulate in atherosclerotic plaques (147). PE is also an
intermediate
donor
of
the
phosphoethanolamine
residue
linking
the
glycosylphosphatidylinositol anchor to the carboxyl-terminus of various proteins (81, 82,
124). Furthermore, in Drosophila, PE substitutes for cholesterol in the feedback
regulation of sterol regulatory element-binding proteins (38), which highlights the central
importance of PE as a membrane sensor for the biosynthesis of FAs and other lipids.
PE species containing a vinyl-ether bond at the sn-\ position represent a unique
type of phospholipid, known as plasmalogens, the synthesis of which requires
alkylacylglycerol produced in peroxisomes. PE-plasmalogens constitute a significant
portion of total ethanolamine phospholipids in mammalian cells (215) and are deficient
in several types of peroxisomal disorders (102). They are particularly abundant in the
membranes of electrically active tissues such as neurons and both cardiac and skeletal
muscle (14, 57, 216). It is estimated that in human heart, one third of total PE and PC is
in plasmalogen form (36, 141). The lymphoid organs, neutrophils, macrophages and
lymphocytes also have high PE-plasmalogen content (36, 110).
The distribution
of
plasmalogens in mammalian tissues has been evaluated at great length; however, their
biological role remains enigmatic. Due to their tendency to form non-lamellar structures
at lower temperatures, relative to their diacyl counterparts, plasmalogens may serve to
better facilitate membrane fusion. This is supported by the abundance of plasmalogens in
tissues that frequently undergo vesicular fusion events such as synaptic membranes (57).
When compared to PE, an increased rate of membrane fusion with vesicles containing
45-50% PE-plasmalogens was also demonstrated in vitro (56).
PE-plasmalogens are the largest endogenous providers of polyunsaturated FA
4
(PUFA) for prostanoid production and cell signaling (5, 53, 166). Plasmalogen-deficient
individuals, most with peroxisomal defects, have decreased n-3 PUFA levels in parts of
the brain (115, 116) and n-6 PUFA (arachidonic acid) levels decreased specifically in the
plasma (117). A PE-plasmalogen selective PLA2 has been identified (218, 219), and
shown to be very active in the brain (49). The release of arachidonic acid from the sn-2
position of PE-plasmalogens is agonist/receptor mediated (218) and it can be stimulated
by ceramide/caspase3 activation of plasmalogen-selective PLA2 (63, 100, 212).
Plasmalogen PLA2 has been localized to PE-plasmalogen rich areas such as neurons and
astrocytes (50), where it may act to cleave docosahexaenoic acid for docosanoid
production (71). Evidence has also shown a role for PE-plasmalogens in cholesterol
transport. High-density lipoprotein (HDL)-mediated cholesterol efflux was reduced in
PE-plasmalogen deficient cells (113) and cholesterol transport from the endocytic
compartments or the cell surface to acyl-CoA-cholesterol-acyltransferase was diminished
in cells deficient
for
dihydroxyacetonephosphate
acyltransferase,
the primary
peroxisomal enzyme for plasmalogen synthesis (129). The specific mechanism by which
PE-plasmalogens affect cholesterol pathways in these cells has yet to be fully elucidated.
1.2 PE Biosynthetic Pathways
There are three pathways by which PE can be synthesized. The PE-Kennedy or
CDP-ethanolamine pathway, is the only route for the de novo synthesis of diacyl PE and
is essential for the production of PE plasmalogens (10, 169, 185, 214). Secondly, the
formation of PE by the decarboxylation of PS in the mitochondria accounts for
significant amounts of mitochondrial PE; however, does not significantly contribute to
5
the production of PE-plasmalogens (10). In various cell models adapted to low amounts
of ethanolamine, PS decarboxylation was able to compensate in the production of PE
formed in vitro (203, 205). Thirdly, PE can be synthesized by base-exchange, through the
action of PS synthase 2 in the mitochondrial associated membrane (165, 171).
1.2.1 PE-Kennedy Pathway
As cells do not synthesize ethanolamine, the PE-Kennedy pathway utilizes
ethanolamine produced metabolically and/or from exogenous lipids supplied by diet (38,
187, 225). A carrier-mediated mechanism for ethanolamine uptake, mainly shared with
choline has been suggested (224); however, no ethanolamine-specific transporters have
ever been cloned or functionally characterized. In this pathway, ethanolamine is first
phosphorylated by ethanolamine kinase (EK) to phosphoethanolamine (6, 109).
Phosphoethanolamine
is
then
converted
to
CDP-ethanolamine
by
CTP:
phosphoethanolamine cytidylyltransferase \Pcyt2 (gene) and ET (protein)] (89). In the
final
stage
of
the
pathway,
CDP-ethanolamine:
1,2-diacylglycerol
ethanolaminephosphotransferase (EPT) (72) catalyzes the formation of PE from CDPethanolamine and diacylglycerol (DAG). ETP alternatively couples CDP-ethanolamine
with alkylacylglycerols, derived in peroxisomes, to synthesize alkylacyl (plasmanyl) PE.
This product contains an ether bond that is further modified in mitochondria to a vinylether plasmalogen product by A'-desaturase (111, 158). Multiple isoforms of kinases and
phosphotransferases have been identified that share substrates from both analogous
branches of the Kennedy pathway (for PC and PE synthesis). Given the overlapping
substrate specificity for these enzymes, it has been assumed that the biosynthesis of PE
6
and PC are similarly regulated. However, there is growing evidence that the two
pathways
and
the
corresponding
cytidylyltransferases
(ET
for
PE
and
CTP:phosphocholine cytidylyltransferase (CTa and CT0 for PC) are differently
regulated (179, 180, 199).
Although the enzymes of the Kennedy pathways mostly show dual-specificity for
ethanolamine and choline, ethanolamine-specific forms have also been discovered.
Choline/ethanolamine kinase exists in three isoforms that are encoded by two separate
genes (7). All are cytosolic proteins, originally regarded to be choline kinases (83);
however, the P isoform possesses both ethanolamine and choline kinase activities (6,
184). Two ethanolamine kinases (EK1 and EK2) have been recently identified and it has
been shown that although EK1 specifically phosphorylates ethanolamine, EK2 has some
dual kinase activity, with a higher affinity for ethanolamine when compared to choline
(109). Phosphotransferases are responsible for the transfer of the CDP-ethanolamine
moiety to DAG. In terms of regulating flux through the pathway, phosphotransferases
operate near-to-equilibrium, utilizing both CDP-ethanolamine and CDP-choline as
substrates (120, 121). Choline/ethanolaminephosphotransferase (CEPT1) is ubiquitously
expressed and shows dual-specificity for ethanolamine and choline (64). Humans also
express choline-specific phosphotransferase, CPT1 (65), that localizes in the Golgi;
where CEPT1 is present in both the endoplasmic reticulum and nuclear membranes (66).
Most recently, a human phosphotransferase that possess strictly ethanolaminephosphotransferase properties has been cloned (72). It also seems that a separate rabbit
myocardial EPT activities exists for PE and plasmalogen synthesis (52).
It is not firmly established how the supply of alkylacylglycerols could regulate
7
the production of plasmalogens; however, DAG availability has been demonstrated to
regulate the PE-Kennedy pathway under certain conditions (86, 181). This led to a
proposal that the PE synthesis is coordinately regulated by the CDP-ethanolamine (via
ET) (170) and DAG supply (199). Ethanolamine supply has also been shown to regulate
PE formation via the de novo pathway (10, 168). In hamster heart, newly imported
ethanolamine is preferentially utilized for PE biosynthesis (122). Other work has shown
that at low ethanolamine concentrations, the ET step is rate-limiting while at higher
concentrations, the EK step becomes rate-limiting (123). Cells grown without
ethanolamine fully adopt the PS decarboxylation pathway to produce ethanolamine and
PE from serine supplied in the media (31, 190, 193, 201, 202). In vivo animal studies
indicate that the PE-Kennedy pathway is the major contributing pathway for the
biosynthesis of PE, corroborating previous work on isolated hepatocytes and human Y79
retinoblastoma cells (179, 221). These experiments were also the first to recognize the
primary role of this pathway for the production of PE-plasmalogens (10).
1.2.2 PS Decarboxylation
Surprisingly, more molecular information is available regarding the biosynthesis
of PE from mitochondrial PS decarboxylation than for de novo biosynthesis via the
Kennedy pathway (194, 201). PS is transported via contact sites (190, 191) from the ER
to the inner mitochondrial membrane, which is the site of PS decarboxylase. PS is then
decarboxylated to PE and the newly formed PE transported to the ER and plasma
membrane (31, 154, 193). The rate of PS transport, rather than the decarboxylation of PS,
is the rate-limiting event in the mitochondrial production of PE (201, 204). In an energy-
8
dependent manner (155), nascent PE is rapidly exported from the mitochondria without
mixing with the inner membrane pool of PE (190), suggesting the presence of a specific
intra-mitochondrial transport system. The exact mechanism for this remains largely
unknown.
1.2.3 Additional Synthetic Pathways
There are several examples where a coordinated control of PE and other
phospholipids biosynthesis is required. For cells to maintain a constant level of PE after
over-expression of the base-exchange enzyme PS synthase I (PPS1), de novo synthesis of
PE was limited (164). Methylation of PE derived from PS, rather than from
ethanolamine, generated a specific pool of PC destined for secretion in lipoproteins
(197). Furthermore, PE derived from ethanolamine and PE derived from PS are
differently metabolized (154) and even newly made PE is utilized differently from preexisting PE (149). These data agree with a so-called channelling model, where the
metabolically related enzymes are functionally organized so that different pools of
substrates are used for different purposes and not readily exchangeable. PE is converted
to PC specifically in the liver through successive methylation reactions. PE Nmethyltransferase is the enzyme responsible for the methylation of PE. Figure 1.1
summarizes the lipid synthetic pathways and how overall lipid metabolism is affected in
Pcytl heterozygous hepatocytes.
9
10
Figure
1.1. PE and phospholipid
ethanolamine
kinase,
ethanolamine
ET;
synthetic
pathways. Abbreviations:
CTP:phosphoethanolamine
phosphotransferase,
PE;
cytidylyltransferase,
phosphatidylethanolamine,
EK;
EPT;
PS;
phosphatidylserine, PSD; phosphatidylserine decarboxylase, PSS1; phosphatidylserine
synthase,
PC;
phosphatidylcholine,
PEMT;
phosphatidylethanolamine
N-
methyltransferase, DAG; diacylglycerol, DGAT; diacylglycerol acyltransferase, PAP;
phosphatidic acid phosphatase or lipin-1, 2 or 3, PA; phosphatidic acid, AGP AT; 1aclyglycerol-3-phosphate acyltransferase, LPA; lysophosphatidic acid, GPAT; glycerol3-phosphate
acyltransferase,
MAM;
mitochondrial
associated
membrane,
ER;
endoplasmic reticulum.
1.3 CTP:Phosphoethanolamine Cytidylyltransferase (ET/Pcyt2)
CDP-ethanolamine formation is an important regulatory step in the PE-Kennedy
pathway (18, 170); however, Pcyt2 has not been studied as extensively compared to its
functional counterpart, the regulatory enzyme of the PC-Kennedy pathway. Around 1956,
Eugene Kennedy (after whom the pathways were named) and colleagues were attempting
to elucidate the mechanism by which ethanolamine and choline phospholipids were
synthesized. It was actually a contaminated vial of ATP, which unbeknownst to the
researchers at the time, contained CTP, that allowed the reaction to occur. This led to the
initial report and characterization of ET and CT activity (89). The ET protein was first
purified in the 1970's from rat liver (169) but more work on subcellular localization and
kinetic properties was performed in more recent years (18, 186, 198, 200). ET is ~50 kDa
cytosolic protein, which exists as a dimer and unlike CT, does not require the presence of
exogenous lipids for optimal activity (198, 200). ET was found to be associated with
membranes of the rough ER (186), which was considered important for the channelling
of CDP-ethanolamine to EPT (an integral ER protein) for the final step of PE
11
biosynthesis (16). However, it is important to note that the localization of ET needs to be
further validated.
Purified
rat
ET
protein
(200)
has
similar
affinity
for
CTP
and
phosphoethanolamine with Km of 53 and 65 uM, respectively. It has a high specificity for
phosphoethanolamine
and less efficiently
utilizes N-methylated
derivatives of
phosphoethanolamine, but it does not react with the CT substrate phosphocholine (198).
An ordered-sequential reaction mechanism was initially proposed in which CTP binds
before phosphoethanolamine, and CDP-ethanolamine is the last product to be released
(169). This mechanism assumes a single binding site for each substrate. The existence of
a second binding site for phosphoethanolamine has also been suggested (198), but its
significance was not firmly established.
In 1996 the yeast Pcyt2 gene was first identified (128) and the human cDNA
isolated by genetic complementation of a conditional ethanolamine auxotroph (133). Rat
(17) and mouse (146) Pcytl were subsequently cloned. Recently, the Pcyt2 gene in the
green algae Chlamydomonas reinhardtii has been shown to be primarily targeted to
mitochondria and has also been cloned (220). C. reinhardtii does not make PC or PS and
offers a unique organism to study PE synthesis via the PE-Kennedy pathway in the
absence of substrate sharing with overlapping phospholipid pathways.
The cloning of yeast and human Pcytl determined that the ET protein is a
member of the cytidylyltransferase super-family, having the characteristic CTP binding
motif, HXGH (133). There also exists a single signature peptide motif typical of the
cytidylyltransferase family, RTXGVSTT, which has been proposed to interact with the
CTP site (142). ET is a unique cytidylyltransferase with two HXGH motifs defining two
12
cytidylyltransferase domains, suggesting that this gene has likely emerged from a unique
internal duplication event. Eukaryotes contain both branches of the Kennedy pathway,
where Pcytl and Pcyt2 most likely evolved from an ancient cytidylyltransferase gene.
The mouse gene and cDNA was cloned and characterized by our lab (146).
Sequence comparisons revealed that the mouse and human coding sequences are highly
conserved; however, their promoters and 3'-UTRs are structurally unrelated, suggesting
species-specific regulation in mouse and human Pcyt2. Recent completion of the
mammalian genome projects allowed the precise mapping of the Pcyt2 gene in numerous
species, including rat, mouse and humans. The mouse Pcyt2 gene is located on
chromosome 11E2, human on chromosome 17q25.3, and rat on chromosome 10q32, all
at similar locations, at the very end of the longer arms, very close to telomeres, and
immediately downstream of the Sirtuin 7 gene.
The Pcyt2 transcript can be alternatively spliced, giving rise to distinct,
catalytically active proteins. The full length transcript (Pcyt2a) experiences a splicing
event that results in the loss of exon 7. The spliced exon corresponds to an 18 amino acid
peptide [180PPHPTPAGDTLSSEVSSQ197] that effectively links the homologous N and C
terminal domains, therefore the shorter variant (Pcyt2/3) lacks the so-called 'linker
peptide'. Sequence comparisons revealed that splicing is highly conserved in insects,
aquatic and avian species as well as an array of mammals.
When cloned into epitope containing vectors and expressed in mammalian cells,
Pcyt2a
and
Pcyt2fl
both
produce
active
enzymes;
however,
the
Km
for
phosphoethanolamine is two-fold higher in the longer a splice variant compared to the
shorter p form. In mice, Pcyt2 mRNA is ubiquitously expressed; however, the expression
13
of the two variants is tissue specific, where in mice the longer splice variant is expressed
at higher levels (178). In addition to moderating catalytic activity, two forms of Pcyt2
may be important for protein-protein interactions and specific cellular localizations (96,
97, 172).
1.4 Knockout Models of Phospholipid Related Genes
Current knockout mice models for phospholipid metabolic genes have been
recently reviewed (195). Various models have addressed PC synthesis via the PCKennedy pathway. Deletion of Pcytla results in embryonic lethality pre-implantation
(209) while Pcytlfr
null mice are viable but experience gonadal dysfunctions (84).
Liver-specific methylation converting PE into PC in PEMT knockout mice was
compensated by the up-regulation of the PC-Kennedy pathway (207), however animals
develop fatty liver and die when fed a choline deficient diet (208). PS synthase 2
knockout mice develop normally due to an increase in PS synthase 1 expression and
decrease in PS degradation but male mice experience testicular abnormalities and
infertility (162). An isoform of ethanolamine kinase, EKI2, has been deleted but no
reduction in the rate of PE synthesis via the PE-Kennedy pathway was observed, likely
due to the compensatory functions ofEKIl and dual choline/ethanolamine kinases. EKI2
null females; however, experience placental thrombosis which is a direct consequence of
EK12 gene disruption (177). The gene encoding PS decarboxylase (Pisd) has also been
disrupted and results in early embryonic lethality and mitochondrial dysfunction (161).
Interestingly, in heterozygous Pisd mice the PE-Kennedy pathway is up-regulated via
increased ET activity, a compensatory mechanism for Pisd deficiency.
14
1.5 The Link Between Neutral Lipids and Phospholipids
The metabolism of phospholipids and triglycerides (TG) are intimately associated.
The regulation of TG and phospholipid metabolism has been shown to play important
roles in the development and pathogenesis of conditions such as type 2 diabetes (T2D),
obesity and cardiovascular disease. The synthesis of both of these lipids begins with the
acylation of glycerol-3-phosphate by the microsomal or mitochondrial isoform of
glycerol-3-phosphate
acyltransferase
(GPAT
and
mtGPAT
respectively)
to
lysophosphatidic acid (LPA). These two isoforms vary in their contribution to total
GPAT activity in a tissue dependent manner and although usually low, mtGPAT accounts
for approximately 40% of GPAT activity in the liver (61). LPA is then modified by up to
five
different
l-aclyglycerol-3-phosphate
acyltransferase
isoforms,
to
become
phosphatidic acid (PA) at the endoplasmic reticulum. Phosphatidic acid phosphatase
(PAP or Lipin-1, 2 or 3), converts PA into DAG also at the level of the ER (148). At this
point in the pathway, DAG can either be esterified to TG via diacylglycerol
acyltransferase-1 or 2 (DGAT1/2), or combined with CDP-ethanolamine/CDP-choline by
CETP to form PE or PC. The synthesis of DAG is therefore essential to both TG and
phospholipid synthesis, an observation first made back in the late 1950's by Kennedy's
group (211). A comprehensive review of the enzymes and regulation of TG synthesis via
the DAG synthetic pathway has recently been published (29).
Obesity is characterized as the accumulation and excess storage of TG in adipose
tissue and remains at the for front of health research due to the numerous pathologies for
which it is a risk factor. Adipose tissue is a key metabolic and endocrine organ, which is
specifically designed and utilized as an energy storage, where energy is stored as TG.
15
When TG storage sites are altered, either within adipose deposits or in ectopic tissues
such as liver, heart or skeletal muscle, there begins a decline in the normal physiology of
these
tissues
resulting
in
insulin
resistance.
Perhaps
the
best
documented
pathophysiological link between phospholipid and TG synthesis is neutral lipid storage
disease (NLSD). An autosomal recessive condition, NLSD manifests as an excess of TG
stored in lipid droplets in most tissues, where there is a defect in the recycling of TGderived acylglycerols to both PC and PE (78, 79). The relationship between the
mechanisms regulating TG and phospholipid metabolism were a focal point of this thesis.
16
CHAPTER TWO
OBJECTIVES OF THE THESIS
17
2.1 Objectives of the Thesis
The purpose to this thesis was to perform a comprehensive characterization of the
Pcyt2 knockout mouse. Of specific interest was the consequence that complete disruption
of this gene would have on phospholipid metabolism and overall development. As well,
the molecular and metabolic phenotype of heterozygous and null animals was to be
assessed, in an effort to better understand the role and regulation of Pcytl.
Previous work had not clearly delineated the definitive role of the PE-Kennedy
pathway and more specifically, Pcytl, in the biosynthesis of PE and phospholipid
homeostasis. The decarboxylation of PS had been shown to fulfill the requirement for PE
in vitro in cells cultured with low concentrations of ethanolamine (203), where as
radiolabeling experiments in which [ 4C]-ethanolamine was introduced into rats in vivo
determined that the PE-Kennedy pathway was responsible for the majority of PE
synthesized in rat heart, kidney and liver (10). The first objective of this thesis was to
elucidate the specific role of Pcytl in murine phospholipid metabolism, throush
characterization of the Pcytl knockout mouse.
Initial characterization of Pcytl deficient mice led to the observation that
heterozygous mice gain weight in a chronic fashion. Many of the knockout mouse models
for genes related to glycerolipid synthesis result in an anti-obesity phenotype. In Pcytl
heterozygotes; however, we observed the opposite, without complete disruption of the
gene. The second objective of this thesis was to determine the mechanisms that facilitate
Pcytl heterozygous mice weight sain and to characterize the alterations in lipid
metabolism.
18
The targeted disruption of a single mouse gene has become an efficient means of
investigating the role of that gene. However, as a loss-of-function experiment, knockout
mice studies possess certain inherent flaws, which should be addressed before
interpreting the results. A single Pcytl allele was silenced in Pcytl heterozygous animals,
rendering a Pcytl deficient state. We proceeded to characterize an observable phenotype
in these mice and identified a logical and relevant metabolic mechanism, which would
explain the observations. In the final objective of this thesis, as a means of investigating
the indisputable role ofPcyt2 in murine lipid metabolism, we sought to rescue the altered
lipid metabolic phenotype in Pcyt2 heterozygous primary hepatocytes via the expression
ofwildtype or a mutated form of Pcytl.
19
CHAPTER THREE
DEVELOPMENTAL AND METABOLIC EFFECTS OF DISRUPTION OF THE
MOUSE CTPrPHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE
GENE (PCYT2)
20
3.1 Abstract:
The CDP-ethanolamine pathway is responsible for the de novo biosynthesis of
ethanolamine phospholipids, where CDP-ethanolamine is coupled with diacylglycerol to
form phosphatidylethanolamine. We have disrupted the mouse gene encoding
CTP:phosphoethanolamine cytidylyltransferase, Pcyt2, the main regulatory enzyme in
this pathway. Intercrossings of Pcyt2+/~ animals resulted in lower litter sizes and
unexpected Mendelian frequencies, with no null mice genotyped. The Pcyt2~'~ embryos
die after implantation, prior to developmental stage E8.5. Examination of mRNA
expression, protein content and enzyme activity in Pcyt2+/~ animals did not reveal the
anticipated 50% decrease due to the gene-dosage effect, but rather a 20-35% decrease.
[14C]ethanolamine radiolabeling of hepatocytes, liver, heart, and brain corroborate Pcyt2
gene expression and activity data and show a decreased rate of phosphatidylethanolamine
biosynthesis in heterozygotes. Total phospholipid content was maintained in Pcyt2+'
tissues; however, this was not due to compensatory increases in the decarboxylation of
phosphatidylserine.
In
heterozygotes
the
fatty
acid
composition
of
hepatic
phosphatidylethanolamine and phosphatidylserine showed decreases in polyunsaturated
fatty acids and increases in saturated fatty acids. These results establish the necessity of
Pcyt2 for mammalian development and demonstrate that a single Pcyt2 allele is up
regulated for heterozygotes to maintain phospholipid homeostasis.
21
3.2 Introduction
Phosphatidylethanolamine (PE) is a dominant inner-leaflet phospholipid in cell
membranes (40), where it plays a role in membrane function by structurally stabilizing
membrane anchored proteins (42, 46, 159), and participates in important cellular
processes such as cell division (42) cell fusion (150, 153), blood coagulation (37, 43, 45)
and apoptosis (43). In Drosophila, where PE is the major membrane phospholipid, the
release of sterol regulatory element-binding proteins (SREBPs) is controlled by PE, as
opposed to sterols in mammalian cells (38). Biosynthesis of PE occurs de novo via the
PE-Kennedy or CDP-ethanolamine pathway (89), which is terminated in the ER, and can
also be produced by the decarboxylation of phosphatidylserine (PS) in mitochondria or
by base-exchange mechanisms (196). Studies using rat liver hepatocytes and hamster
heart have shown that quantitatively, PE is mainly produced via the de novo pathway
(179, 223). In vivo radiolabeling experiments have also shown that the CDPethanolamine pathway is the major route of PE production in rat liver, heart and kidney
(10), which corroborate previous work on isolated hepatocytes and human Y79
retinoblastoma cells (179, 221). In vitro evidence showed that in CHO and baby hamster
kidney cell lines, PS decarboxylation could become the major contributing pathway for
PE production (98, 203).
CTP:phosphoethanolamine cytidylyltransferase (ET) is the main regulatory
enzyme in the PE-Kennedy pathway and catalyzes the formation of CDP-ethanolamine
from phosphoethanolamine and CTP (20, 89, 170). The gene encoding ET, Pcyt2, is a
single gene that is alternatively spliced at exon 7 to produce two transcripts, Pcyt2a and
Pcytlfi (146), which are differentially expressed among tissues. This splicing event is
22
evolutionarily conserved among mammals, birds and amphibians. The analogous ratelimiting enzyme in the PC-Kennedy pathway, CTP:phosphocholine cytidylyltransferase
(Pcytl) has been extensively investigated, while the understanding oiPcyt2 remains in its
infancy.
Various knockout models have been generated to investigate phospholipid
metabolism (195). The liver specific PE-iV-methyltransferase (PEMT), which converts
PE to PC, was disrupted in mice and Pemt1' mice were viable due to the compensation of
the CDP-choline pathway (207). When fed a choline deficient diet; however, Pemt1' mice
experienced steatohepatitis and died within 5 days (208). Various models have also
addressed the isoforms of Pcytl, showing the deletion of Pcytl a results in embryonic
lethality pre-implantation (209) and that Pcytl'bi1' mice are viable but both male and
female mice experience gonadal dysfunction (84). The necessity of the genes responsible
for PS synthesis, PS synthase 1 and 2 (Pssl/2), have been evaluated in a Pss2 knockout
model (15, 162). Pss2~'~ mice develop normally due to an increase in Pssl expression and
decrease in PS degradation (162); however, a percentage of male mice experience
testicular abnormalities and infertility, as Pss2 was determined to be abundant in the
testes (15). Most recently, an isoform of ethanolamine kinase, EKI2, was targeted.
Although there was no reduction in the rate of PE synthesis via the CDP-ethanolamine
pathway, likely due to the presence of both EKI1 and choline kinase, EKI2'1' female mice
experience placental thrombosis (177).
There is evidence supporting the formation of two distinct pools of PE, via the
CDP-ethanolamine and PS decarboxylation pathways, which are utilized for distinct PE
functions (192); however, until recently the necessity for each individual pathway had not
23
been fully appreciated. The rate controlling enzyme in the decarboxylation pathway, PS
decarboxylase (Pisd) has been disrupted in mice and results in early embryonic lethality
around embryonic day (E) 9.0, likely due to malformed and dysfunctional mitochondria
(161). In addition, although Pisct^ mice experienced approximately half of the wildtype
Pisd expression, they were phenotypically indistinguishable from controls. In Pisd
heterozygous animals, ET protein expression as well as the production of PE via the PEKennedy pathway was increased to compensate for the loss of production via the PS
decarboxylation pathway (161). We have created a Pcyt2 knockout model to investigate
the importance of ET and therefore the PE-Kennedy pathway in mammalian
phospholipid biosynthesis. Here we report that the homozygous disruption of this gene
results in embryonic lethality prior to E8.5, while heterozygotes have increased
expression of the remaining Pcyt2 allele required for maintaining necessary levels of PE
for phospholipid homeostasis. At the same time, there is no change in the level of other
phospholipids nor is there compensation from the alternative PE synthetic pathway via
PS decarboxylation.
24
3.3 Experimental Procedures
3.3.1 Targeted disruption of the Pcyt2 gene
As a first step towards generating Pcyt2~'~ mice, we characterized the mouse Pcyt2
gene and promoter (146) and a 12.5kb genomic region used to construct the targeting
vector was first subcloned from a positively identified BAC clone (AY189524). The
targeting vector was created by homologous recombination and was designed such that
the short homology arm (SA) extends ~1.4kb 5' to exon 1. The long homology arm (LA)
starts at the 3' side of exon 3 and is ~8kb long. The neomycin cassette is trapped inside
exon 1, replacing 2.8kb of the gene including the promoter, ATG and exons 2 and 3. The
targeting vector was linearized by digestion with Noil and then transfected by
electroporation of 129 SvEviTL embryonic stem cells (InGenious Targeting, New York).
After selection in G418, surviving clones were screened by PCR using primers ATI
(ATGATTTCTTCATGGTGTAGTCTG)
and
(TGCGAGGCCAGAGGCCACTTGTGTAGC)
Nl
to identify recombinant ES clones
containing the neomycin cassette. Selection screening for homologous recombination
identified two positive ES clones, #286 and #883. Those clones were injected into mouse
blastocysts and implanted into pseudopregnant females. Agouti coloured, chimeric male
offspring were then crossed back to C57B1/6 mice to confirm germline transmission. The
generation of Pcyt2+/~ mice was performed by InGenious Targeting.
3.3.2 Animals
All procedures were approved by the University of Guelph's Animal Care
Committee and were in accordance with guidelines of the Canadian Council on Animal
25
Care. Mice were exposed to a 12-h light/dark cycle beginning with light at 7:00 a.m.
Male and female mice were fed ad libitum a standardized diet (Harlan Teklad S-2335)
and had free access to water. Mice described are of mixed genetic background (C57B1/6
x 129/Sv)
3.3.3 Genotyping
Pcyt2 genotypes were identified from genomic DNA isolated from mouse tails,
embryo yolk sacs and total embryos. Tissues were digested with 200 ug proteinase K at
55°C in a buffer (10 mM Tris-HCl pH 8.0, 0.1 M EDTA pH 8.0, 0.5% SDS, 0.1 M
NaCl). To eliminate RNA, 10 \i% of RNase A was added. Purified genomic DNA was
amplified
using
a
common
upstream
primer
FP
(CCTGGAACTCATGAGATCCTCCTG), in combination with either a downstream
primer RP (ATCGCACCACACCCGCACGA) specific for the wildtype allele or a primer
Nl (TGCGAGGCCAGAGGCCACTTGTGTAGC) specific for the knockout allele.
Confirmatory screenings were performed with separate sets of primers specific to the
neomycin
cassette
(N2F;
GCACCGCTGAGCAATGGAAG,
CGATTGTCTGTTGTGCCCAGTC)
CCTAGAGGAGATTGCCAAGC
of
and
the
ET-RP;
knockout
allele
or
N2R;
(ET-FP;
CTGCCGTGAACAGAGAAGTC)
present only in the wildtype allele. PCR reactions utilized 0.3 units of REDtaq genomic
DNA polymerase (Sigma). Initial denaturation was at 94°C for 5 min, then 32 cycles at
94°C for 1 min, 60°C for 30 s, 72°C for 1 min and a final extension at 72°C for 10 min.
The primer pairs FP/RP, FP/N1, N2F/N2R and ET-FP/ET-RP yielded products of 450,
305, 339 and 167 bp respectively.
26
3.3.4 RNA analyses
Tissues were harvested, immediately frozen in liquid nitrogen and stored at 80°C. 50-100 mg of tissue was homogenized in TRIZOL reagent (Invitrogen) and total
RNA extracted according to the manufacturer's protocol. cDNA was synthesized from 2
jag of total RNA using a poly-dT primer and Superscript II reverse transcriptase
(Invitrogen).
Pcyt2
cDNA
was
(ACCATACTCCGTGACAGCGG)
amplified
and
using
an
upstream
downstream
primer
primer
Fll
R13
(GGTGGGCACAGGGCAAGGGC), corresponding to positions in exons 11 and 13
respectively. The two splice variants of Pcyt2, a and p\ were amplified using F6
(GGAGATGTCCTCTGAGTACCG)
and
R7
(GGCACCAGCCACATAGATGAC)
primers, which flank the spliced region. Pisd was amplified using primers as previously
described (161). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified
using the upstream primer (ACCACAGTCCATGCCATCAC) and the downstream
primer (TCCACCACCCTGTTGCTGTA). PCR for Pcyt2 total, Pcyt2a and p, Pisd and
GAPDH was carried out using the following cycle parameters: 94°C for 30 s, 58°C for 30
s and 72°C for 30 s for 28, 32, 32 and 24 cycles respectively, which were predetermined
as the given phases proportioned to the product concentration. Pcyt2 total, Pcyt2a,
Pcyt2/3, Pisd and GAPDH PCR yielded products of 342 bp, 225 bp, 172 bp, 133 bp and
451 bp respectively, which were resolved on a 1.5% agarose gel, visualized by ethidium
bromide staining and quantified using Image J densitometry software (NIH).
27
3.3.5 Production of Pcyt2-specific antibody
A polyclonal ET antibody that recognizes both a and P splice variants was
generated against the peptide VTKAHHSSQEMSSEYRE, situated at the end of the first
catalytic motif and directly before the spliced peptide (146). Rabbits were immunized
with the hemocyanine-conjugated peptide and immune serum was collected after four
successive immunizations. The antibody (V-5497) was purified on an affinity column
containing immobilized peptide antigen busing 0.2 M Glycine/HCl (pH 2.2) and 250 mM
NaCl. The pure antibody was preserved by adding 0.02 % thimerosal and then stored at 20°C.
3.3.6 Histology and immunohistochemistry
For histological staining, the uterine tissue was dissected from pregnant females at
various stages of pregnancy and fixed in 10% formalin in phosphate buffered saline
(PBS). Tissues were then embedded in paraffin wax, dried with ethanol and sectioned
into 20 urn sections. Slides were then stained with hematoxylin and eosin (H&E). For
immunohistochemistry (IHC), embryos were dissected from pregnant females at various
stages of pregnancy, fixed for 2 h in 4% paraformaldehyde at 4°C and then in 10%
formalin in PBS. Tissues were then embedded in paraffin wax, dried with ethanol and
sectioned into 20 |xm sections and stored at 4°C. Slides were first exposed to xylene to
remove the paraffin and then 100% ethanol, 3% H2O2 in methanol and successively
decreasing concentrations of ethanol for hydration. Slides were then incubated in a citrate
buffer (9.4 mM citric acid and 40 mM sodium citrate, pH 6.0) for 15 min at 94°C for
antigen retrieval and then blocked for 30 min in PBS with 0.2% bovine serum albumin
28
(BSA), 0.5% triton-X-100 and 1% goat serum. The ET antibody was diluted in PBS with
1% BSA (1:50) and slides were incubated at 4°C overnight. After washes with PBS with
0.1% Tween 20, slides were introduced to a goat anti-rabbit IgG secondary conjugated to
horseradish peroxidase (HRP) at a dilution of 1:200 for 30 min. Antigen visualization
was performed for 5 min with a DAB kit (Vector Laboratories) and subsequently
counterstained using Harris' hematoxylin and dehydrated.
3.3.7 ET immunoblotting
Frozen tissue samples were homogenized in a cold lysis buffer (10 mM Tris-HCl
(pH 7.4), 1 mM EDTA and 10 mM NaF) containing protease (1/10) and phosphatase
(1/100) inhibitor cocktails (Sigma). The lysate was centrifuged at 2,000 x g for 20 min at
4°C to remove cell debris. Proteins, 10 ug for liver and 25 |xg for heart and brain, were
separated on 10% SDS PAGE gels and transferred to PVDF membranes. Prior to
blotting, red die (Ponceau S) staining was used to ensure proper protein transfer and
loading. Membranes were blocked with 5% milk in 20 mM Tris-HCl (pH 7.5), 500 mM
NaCl, 0.05% Tween-20 (TBS-T) for 2 h at room temperature followed by brief washing
in TBS-T alone. Membranes were then incubated the ET specific antibody, at a 1:2000
dilution in TBS-T at 4°C overnight. Membranes were then washed three times for 15 min
each in TBS-T. Membranes were incubated with the secondary antibody, an HRP
conjugated goat anti-rabbit IgG, diluted 1:20000 for 1 h at room temperature and then
washed 5 times for 15 min in TBS-T and visualized with enhanced chemiluminescence
(Amersham). For control P-Actin protein, anti-p-Actin antibody (Biovision) was used at a
29
dilution of 1:10000 (1% BSA in TBS-T) and an anti-mouse HRP-conjugated IgG at
1:10000 dilution.
3.3.8 ET enzymatic activities
Tissue homogenates were twice frozen in liquid nitrogen, thawed on ice and then
centrifuged for 2 min at 13,000 * g to pellet cellular debris. The supernatants were then
removed and the enzymatic activity assayed using [14C]phosphoethanolamine (initial
activity was 55 mCi/mmol) (ARC Inc.) as a substrate (182). Briefly, a mixture of 20 mM
Tris/HCl (pH 7.8), 10 mM MgCl2, 5 mM dithiothreitol, 2 mM CTP, 1 mM unlabeled
phosphoethanolamine and 3.6 uM of [14C]phosphoethanolamine (55 uCi/umol) was
incubated with tissue homogenate (50 ug protein) at 37°C for 15 min. Reactions were
terminated by boiling for 2 min. 25 ul was then loaded onto silica G plates (Analtech)
with CDP-ethanolamine and phosphoethanolamine standards and separated in a solvent
system of methanol: 0.5% NaCl: ammonia (50:50:5). Plates were then sprayed with 1%
ninhydrin, heated for 10 min at 80°C and the CDP-ethanolamine band was scraped for
liquid scintillation counting. The ET activity (nmol/min/mg protein) was determined
from the dpm data using a known amount of [14C]phosphoethanolamine as a standard.
This assay is linear for the time performed and up to -150 jug protein as determined
previously (182). Protein content was determined with bicinchoninic acid (BCA) protein
assay (Pierce).
30
3.3.9 Phospholipid content
Tissues were homogenized in PBS and lipids were extracted from homogenates
according to the method of Bligh and Dyer (21). The lower organic phase from the lipid
extraction was dried under a stream of nitrogen gas and lipids were resuspended in a
constant volume of chloroform. Lipids were then separated by thin-layer chromatography
(TLC) on silica gel 60 plates (VWR) in a system of chloroform/methanol/acetic
acid/water, 40:12:2:0.75 (v/v). The fluorescent probe 1,6-diphenylhexatriene was added
to a final fluorophore concentration of 100 uM (80). Plates were dried and phospholipids
were first visualized by excitation at 302 nm and documented. Plates were then sprayed
with 15% sulfuric acid with 0.5% K 2 Cr0 7 and heated for 20 min at 110°C. Plates were
then scanned and phospholipids were quantified using standard curves generated for each
lipid using reflection densitometry. Phosphatidylinositol, PC and PS standards were from
Sigma and PE standard was from Avanti Polar Lipids Inc.
3.3.10 Hepatocyte isolation and metabolic labeling
Hepatocytes were isolated from mice as previously described (177, 209). Briefly,
livers were removed and rinsed briefly in a buffer containing 66.7 mM NaCl, 6.7 mM
KC1, 100 mM HEPES (pH 7.6) and 36 mM glucose. Livers were then minced with a
Mcllwain tissue chopper (Vibratome) and digested in the same buffer plus 5 mM
CaCl2-2H20, collagenase (0.5 mg/ml) and deoxyribonuclease (6 |xg/ml) for 20 min at
37°C three times successively. After each step the supernatant containing the cell
suspension was collected and the combined cell suspension was filtered through 41-urn
Spectra/Mesh nylon (Fisher Scientific). The hepatocytes in the filtrate were collected by
31
centrifugation at 100 x g for 2 min and washed three times with buffer containing 137
mM NaCl, 5 mM KC1, 0.65 mM Mg 2 S0 4 -7H 2 0, 1.2 mM CaCl2-2H20, 10 mM HEPES
(pH 7.4), and BSA (15g/ml). The cells were resuspended in 10 ml of Dulbecco's PBS
containing 5 mM glucose and 3.3 mM pyruvate plus 0.5% BSA. Cells were then plated
on 60 mm collagen-coated plates (BD Biosciences) and incubated at 37°C with 5% C0 2
for 2 h. Media was then removed and fresh media containing 0.2 uCi of
[14C]ethanolamine (initial activity was 55 mCi/mmol) or 2 uCi of L-[3H]serine (initial
activity was 25 Ci/mmol) (ARC Inc.) in the presence of 50 jaM unlabeled substrate was
added for up to 3 h. After incubation, cells were transferred to ice, washed with PBS and
collected for lipid extraction as described above. Phospholipids and water-soluble
intermediates were resolved as described above and the radioactivity associated with each
was determined by liquid scintillation counting. Duplicate plates were used for BCA
protein determination.
3.3.11 Mitochondrial staining
Hepatocytes were isolated and plated as described above. After 20 h the growth
medium was replaced with fresh pre-warmed medium (DMEM plus 17% FBS)
containing 100 nm MitoTracker Red (Molecular Probes). The cells were additionally
incubated for 30 min at 37°C and the media was then replaced with freshly prepared, prewarmed medium containing 4% formaldehyde. Plates were incubated for 15 min at 37°C,
and the fixed cells were washed 2-3 times with PBS before viewing with a fluorescent
microscope.
32
3.3.12 In vivo labeling
Experiments were conducted as previously described (10) with some alterations.
Mice were weighed and injected with the radiolabeled substrate via intraperitoneal
injection. The quantity of radioactive precursor used was 0.5 uCi [l14C]ethanolamine and
5 uCi L-[3H]serine in the presence of 50 |xM unlabeled substrate. Mice were allowed to
recover for 3 h and sacrificed using CO2. Brain, heart, kidney and liver were then
dissected, weighed and immediately homogenized in PBS. Total lipids were extracted as
described above and an aliquot of the total extract as well as the lipid-containing organic
phase were counted for total radioactivity. PE, PC and PS were resolved via TLC as
below and individually counted.
3.3.13 Phospholipid fatty acid composition
Liver
lipid
samples
extracted
as described
above, were
subsequently
transmethylated to investigate fatty acid side chain composition as previously described
(34). Briefly an aliquot of hepatic lipid extract was dried under a stream of nitrogen and
transmethylated with 6% (by volume) H2SO4 in methanol at 80°C for 3 h. After cooling
to room temp, the methylated fatty acids were collected by the addition of petroleum ether
and deionized H2O, and centrifugation at 1000 * g for 15 min at 4°C. Samples were then
stored at-20°C until analysis by GLC (HP 5890 Series II; Hewlett Packard).
3.3.14 Statistical analysis
All significant differences were determined with a Student's t-test using
GraphPad Prism 4.0 software, where a significant value was determined as P<0.05.
33
3.4 Results
3.4.1 Disruption ofPcytl results in embryonic lethality
Chimeric mice were generated as described in the Materials and Methods, chosen
for agouti colouration and subsequently bred to C57B1/6 to determine if germline
transmission was achieved. Eight Pcyt2+/~ mice were generated as founders and crossed
to generate Pcyt2~/~ mice. Two individual mouse lines were generated from positive ES
clones #286 and #883; however, no differences were established between the two.
Although Pcyt2+/~ mice were fertile, initial intercrosses yielded low litter sizes.
34
&
.^
3?
4?
/
FP
xp
WT-F WT-R^?'fr
/
1*1 |***|
••!«»
SIRT7
2
Nl N2F N2R
v i Fg.
3
/
4
5
Wild-type allele
* * '
Vector Sequence
„AT1
f
SIRT7
Li
Neo
#286
Targeting vector
/
f\ f\
fi
R
^yp^ny
y
yr^yr^yiny u y^r^
7
8 9
LA
8Kb
H
4
SA
1.4 Kb
B
Neo
5 6
n
B
n n
10 11 12
H H
13 14 H
Knockout allele
#883
453 bp (FP/RP)
305 bp (FP/N1)
1.5 Kb
Figure 3.1. Targeted disruption of the Pcyt2 gene. (A) The targeting vector was created
by replacing a 2.8 kb fragment, including exons 1 to 3 and the transcriptional start site
with a neomycin cassette. Short and long homologous arms of 1.4 and 8 kb respectively
flank the neo cassette. Genotyping primer locations are indicated. (B) PCR confirmation
of two recombinant ES cells, #286 and #883, using primers ATI and Nl (1.5 kb). (C)
Genotyping of mouse genomic DNA using a common forward primer FP and two
specific reverse primers, RP and Nl for the wildtype and knockout alleles respectively.
35
PCR genotyping (Fig. 3.1 A and C) revealed no homozygous mutants from a total of 119
mice (Table 3.1), which led to the hypothesis that Pcytl1' mice are lethal in utero.
Table 3.1. Genotypes of pups from Pcyt2+/~ intercrosses
Genotype
Age"
3 wks
E13.5
E10.5
E8.5 C
Total -
119
21
41
56
+/+
+/-
-/-
49
4
10
15
70
12
22
30
0
0
0
3
• Resorbed *
na
5
9
8
a Heterozygous mice were intercrossed and embryos and pups were collected at the
specified developmental stage and were genotyped according to materials and methods,
b The uterus was examined for evidence of resorbed embryos,
c Genomic DNA from E8.5 embryos was isolated using the whole embryo.
To further investigate this, embryos were collected from timed matings and genotyped.
Embryos from El3.5 were dissected and the yolk sac genotyped. At this stage, no Pcyt2~f~
embryos were identified; however, there were identifiable resorption sites on the uterus.
E10.5 embryos were then dissected and genotyped with a similar distribution of
genotypes and similar pattern of resorption (Fig. 3.2A and B). Finally, embryos were
isolated at E8.0-E9.0. At this stage of development, the entire embryo was used for
genotyping and from a total of 56 embryos, only three Pcytl1' were identified.
36
B
;
D
X
D
Tmm
1mm
/
jr."
/'
?•-*
? ^ \ , - > • •'
fa-.,.
IU: •./;.-,•:
•
"
"
"
*
;
'
, ) ' * *
- ' •*
Figure 3.2. Embryo histology and developmental Pcyt2 protein expression. (A) H&E
staining of a wildtype El0.5 embryo and (B) an implantation site where the embryo has
been completely resorbed. The ubiquitous expression of Pcytl is shown by IHC in
wildtype E8.5 embryos (5x), where (C) Pcytl
stained, (D) counterstained with
hematoxylin and (E) a two-fold magnification of the expression in the neural tube and
somites of E8.5 embryos are demonstrated. For a later developmental stage, E12 (1.5x),
(F) Pcytl stained and (G) counterstained embryos are shown, each with a magnified view
of expression in the developing ventricle. Figures are representative of at least 4 embryos
37
at each developmental stage (D- decidua; M- mesencephalon; Am- amnion; N- neural
tube; S- somites). Pcyt2 staining is indicated in brown (C and F) and counterstaining
indicated in blue (D, E and G).
Upon dissection, it was noticeable that the embryos that had been genotyped as Pcyt2
null were necrotic. Unlike the later stages of embryonic development, no distinguishable
resorption sites were observed on the uterus and certain implantation sites were void of
recognizable embryos, indicating that these embryos were being resorbed at earlier stages
of development and had mostly completed the resorption process. These results force the
conclusion that Pcytl1' embryos implant and are lethal prior to E8.5. The pattern of
expression of ET in wildtype embryos at this stage was established using IHC. As shown
in Fig 3.2C-G, the protein was ubiquitously detected throughout the embryo at gestational
stages E8.5 and E12, demonstrating that ET is essential in the early stages of
embryogenesis. IHC utilizing only the HRP-conjugated secondary antibody was used as a
negative control to test for endogenous peroxidase activity and for non-specific staining
(not shown). It has also been demonstrated previously that ET is expressed in rat liver
between days E17 and E22 (birth), showing a gradual increase in activity which then
remained constant during adult stages (3). The rat liver ET protein had a similar profile to
that of ET activity (3). Thus the embryonic lethality of Pcyt2 homozygotes led us to
further investigate the consequences of the single Pcytl allele in the heterozygous state.
3.4.2 Heterozygosity decreases Pcyt2 mRNA expression
We next investigated the transcriptional consequences of a single Pcyt2 allele by
measuring Pcyt2 mRNA in various tissues. As shown in Fig. 3.3, mRNA levels were
38
reduced 20-35% in liver, heart, brain, kidney and adipose tissue of both male and female
Pcyt2+/~ mice.
A
p Pcyt2+'+ I Pcyt2
+/-
B
Liver Heart Brain Kidney Fat
Liver Heart Brain Kidney Fat
Figure 3.3. Relative Pcyt2 mRNA expression assessed by semi-quantitative RT-PCR.
Primers are common to both Pcyt2 isoforms and the amplified product is shown relative
to GAPDH expression for (A) females and (B) males. Data shown represent mean±SEM
for four mice of each gender and genotype quantified in triplicate, where * indicates
significance (P<0.05).
It is firmly established that ET is encoded by a single gene; however, it can exist as two
distinct transcripts due to the alternative splicing of exon 7 (146), for which there is a
tissue specific pattern of expression. We investigated if the disruption of a single Pcyt2
allele had effects on the mRNA expression of the two splice variants in the various
tissues and found that there were no differences between genotype and gender in
heterozygous mice. The levels of Pcyt2 mRNA expression were not decreased to the
expected level in the heterozygous state, as predicted by the gene dosage effect.
Therefore to further investigate we next assessed the characteristics of ET protein
expression and activity.
39
3.4.3 Pcyt2+ 'protein expression and enzymatic activity are also diminished
To establish ET protein content in tissues of Pcyt2+/+ and Pcyt2+' mice,
immunoblotting was conducted using a polyclonal ET antibody (Fig 3.4).
(3-Actin
Pcyt2
+/+ +/-49 kDa
Liver!
Heart
Brain!
*T7"T"
sp!|p$F " ^ w 8 * ^ .
"f-/in,
"37 kDa
-49 kDa ^ —*• — j*-37 kDa
-37 kDa
-49 kDa
Pcvt2*'~
Liver Heart Brain
Female
Liver Heart Brain
Male
Figure 3.4. Relative ET protein expression between Pcyt2+/+ and Pcyt2+/~ mice. (A)
The left panel shows total Pcyt2 (-49 kDa) and the right panel shows P-Actin (-37 kDa)
as an internal control. Blots are representative of four mice of each gender and genotype.
(B) Densitometry plots for Pcyt2 expression were normalized to P-Actin expression and
compared to wildtype levels (set at 1). Data shown represent mean±SEM for four mice of
each gender and genotype quantified in duplicate.
Although the single Pcyt2 gene gives rise to two splice variants, the molecular weight
differs by less than 2 kDa and therefore since the antibody recognizes a common peptide
on both splice variants, they are indistinguishable when resolved by SDS-PAGE mini
gels. Thus the band corresponding to ~49 kDa, we have designated to be total ET. We
established that total ET protein content in Pcyt2+/~ liver, heart and brain were reduced
40
similarly for both genders, and to the approximate level corresponding to mRNA levels,
using P-Actin as a control. We next examined the enzyme activity of ET in the liver,
heart, brain and kidney (Fig. 3.5).
Liver Heart Brain Kidney Liver Heart Brain Kidney
Figure 3.5. ET in vitro enzyme activity. Activity was assessed in tissue homogenates
from Pcyt2+/+ and Pcyt2+/~ mice using [14C]phosphoethanolamine as a substrate for (A)
females and (B) males. Data are expressed as nmol/min/mg protein and represent
mean±SEM for at least four mice from each gender and genotype quantified in triplicate,
where * indicates significance (P<0.05).
Importantly, there were significant decreases (20-35%) in Pcyt2+/~ animals relative to
littermate controls in all tissues and for both genders. These data demonstrate that
although ET activity is decreased in heterozygous animals, the actual values do not
decrease to 50% of the wildtype levels. This suggests that there is an up regulation of the
remaining functional allele occurs in heterozygous animals, which is consistently
observed at various levels of gene expression.
41
•>+/-
3.4.4 Phospholipid content does not change in Pcyt2 ' mice
We next investigated the tissue content of the major phospholipids, PE, PC and
PS in liver, heart, brain and kidney of Pcyt2+/~ and control mice from both genders (Fig.
3.6).
•
100
Pcyt2+/*
• Pcyt2+A
£. Lnsz
<u
754
toft
504
bfi
254
PE
PC
PS
PE
P
Brain
PE
PC
PS
Kidney
PE
PS
PC
PC
PS
Figure 3.6. Comparison of phospholipid content between Pcyt2+/+ and Pcyt2+/~ mice.
Phospholipids were isolated from tissues (A) liver, (B) heart (C) brain and (D) kidney as
described in the materials. Data represent mean±SEM results from at least seven mice
from each gender and genotype and are expressed as ug lipid/mg tissue.
Tissues from a total of seven mice of both genders were analyzed; however, since no
difference was observed between gender, results were combined for analyses. In the
42
heterozygous state, the level of PE did not differ between genotypes and the amounts of
PC and PS were also consistent. Unaltered levels of PE regardless of decreased
expression and activity of ET in heterozygous animals prompted the possibility that there
could be a compensatory increase in PE production via PS decarboxylation or that the
single Pcytl allele may be sufficient for PE production via the CDP-ethanolamine
pathway. To address these questions we first investigated the limiting enzyme in the
decarboxylation of PS in the mitochondria.
3.4.5 PS decarboxylase and mitochondria are unaltered
Pcyt2 heterozygous mice maintain PE homeostasis in spite of diminished Pcyt2
expression, therefore we questioned if the decarboxylation of PS in the mitochondria
would be up regulated as a means of maintaining PE levels. The mRNA expression of
Pisd was not altered in the liver, heart or brain of Pcyt2+/' mice (Fig. 3.7A).
43
A
mPcytl^
Liver
Heart
MPcytl
Brain
Figure 3.7. Pisd mRNA expression and mitochondrial staining of hepatocytes. (A)
The amplified product is normalized to GAPDH mRNA and expressed relative to
wildtype (set at 1). Data shown represent mean±SEM for four mice of each gender and
genotype quantified in triplicate. Fluorescence micrographs of 20 h cultured primary
hepatocytes from (B) Pcyt2+,+ and (C) Pcyt2+/~ mouse stained with the mitochondrial
probe MitoTracker Red.
The results were first normalized to GAPDH and expressed relative to wildtype levels, so
that each tissue is compared relative to a value of one. Due to the possibility of an altered
state of PS decarboxylation in the mitochondria, we addressed the relative state of the
mitochondria by introducing MitoTracker Red to isolated hepatocytes (Fig 3.7B and C).
It was hypothesized that the mitochondria of Pcyt2+/~ mice would be unaffected in
relation to their wildtype controls and this is shown to be the case, as both Pcyt2+/+ and
Pcyt2+/~ binuclear hepatocytes show similar distribution of mitochondria around the
nucleus. Although there were no increases in Pisd mRNA or changes in mitochondrial
44
distribution, we additionally investigated the contribution of the PS decarboxylation
pathway to the synthesis of PE in isolated hepatocytes.
3.4.6 In vitro and in vivo PE synthesis
To investigate the contributions of the PE-Kennedy and PS decarboxylation
pathways in vitro, [14C]ethanolamine and [3H]serine were introduced to hepatocytes
isolated from the livers of Pcyt2+/+ and Pcy/2+/"animals. Hepatocytes were incubated for a
total of 3 h, with samples taken at 1, 2 and 3 h time points. [14C]ethanolamine labeling of
hepatocytes revealed a decreased synthesis of PE via the CDP-ethanolamine pathway in
Pcyt2+/~ hepatocytes (Fig 3.8A).
45
Pcyt2+/+
Pcyt2+/-
B
[14C]-PE
[14C]-PC
Time (h)
[»C]-
PC]-
1 qnnPhosphoethanol ami nc
CDP-ethanol am! ne
Time (h)
E
1A-,
/•—•v
.3 14O
o 12,„
s.fcO ~
£ 10o
eEX «-
F
[3H]-PE
/
V
/
/
Ai
a
•<B
«
T
9-
"3 8B
a. 7
.—1
Time (h)
-""'
C^
•
I
V'
,
11-
ex 10-
%-•
6J1—.
PH]-PC
^
I
i
i
i
1
2
TIUM:(h)
3
Figure 3.8. In vitro contributions of PE biosynthetic pathways. Primary hepatocytes
were incubated with [14C]ethanolamine or [3H]serine as described in the materials and
methods. After ethanolamine incubation, the synthesis of [14C]-PE and [14C]-PC are
shown in (A) and (B) respectively. Levels of [14C]-phosphoethanolamine and [14C]-CDPethanolamine are shown in (C) and (D) respectively. After [ H]-serine incubation, the
46
synthesis of [3H]-PE and [3H]-PC are shown in (E) and (F) respectively. All data are
mean±SEM expressed as pmol/mg protein and are representative of at least two livers
and performed in triplicate, where * indicates significance (PO.01).
The diminished rate of PE synthesis is due to lower levels of ET activity as demonstrated
by corresponding differences in the water-soluble intermediates, showing an increase in
[l4C]-phosphoethanolamine (Fig 3.8C) and a decreased level of [14C]-CDP-ethanolamine
(Fig 3.8D). The results were consistent with the hypothesis that there is a decreased rate
of PE synthesis by the CDP-ethanolamine pathway that was limited by lowered activity
of ET in heterozygous animals.
PE conversion to PC by the methylation pathway was measured by the amount of
PC produced after [14C]ethanolamine labeling in hepatocytes. Data show that the
methylation of PE derived via the CDP-ethanolamine pathway was not altered between
genotypes (Fig 3.8B). Finally, hepatocytes were radiolabeled with [3H]serine to look at
the PE produced from PS. These experiments showed that there were no differences in
the decarboxylation of PS to form PE between genotypes (Fig 3.8E), as determined by
the amount of [3H]-PE produced; there were also no differences between genotypes in the
amount of [3H]-PC formed from [3H]serine (Fig 3.8F).
To investigate the contributions of the CDP-ethanolamine and PS decarboxylation
pathways in vivo, [14C]ethanolamine and [3H]serine were injected into heterozygous and
wildtype animals and incorporation into PE, PC and PS were measured. Upon
intraperitoneal injections, [14C]-radiolabeled products were quickly found in all tissues
examined, although
in different
quantities. After
47
3 h, the incorporation
of
[ C]ethanolamine into PE in the liver, kidney, heart and brain (Fig. 3.9A) was reduced
(20-35%) in Pcyt2•>+/-.' mice compared to wildtype littermates.
BPcyt2+/*
[14C]-PE
00
Q
[14C]-PE
lo-i
lOf
8H
S 8H
6i
•5?
6-
o
4-
Ll
Kidney
&2
Heart
Liver
B [14C]-PC
Liver
mPcyt2+A
m
Brain
3
Liver
H]-PC
Liver
Figure 3.9. In vivo contributions of PE biosynthetic pathways. (A) The incorporation
of [14C]ethanolamine into PE and (B) the incorporation of [3H]serine into PE in liver. (C)
The amount of PC derived from the incorporation of [14C]-PE (CDP-ethanolamine
pathway) and (D) PC derived from [3H]-PE (PS decarboxylation pathway) via the PEMT
pathway in the liver. Data represent mean±SEM of four mice quantified in duplicate and
are expressed as dpm (xlOn)/g of tissue, where * indicates significance (P<0.05).
The amount of PC formed from [r 14,CJethanolamine via PE methylation was also
determined (Fig. 3.9C); however, no differences between genotypes were observed.
Finally, after [3H]serine injections, there were no differences in the amount of PE formed
48
in liver after 3 h (Fig. 3.9B), again suggesting that the production of PE by
decarboxylation was not affected by Pcyt2 deletion. The in vivo labeling experiments also
indicated that in the liver, serine radiolabeling of PC was not altered in Pcyt2+/~ mice (Fig
3.9D).
3.4.7 Hepatic phospholipid fatty acid composition is remodeled
We next investigated whether there was a remodeling of fatty acid side chains in
the major phospholipid classes in liver. PE from male and female heterozygous mice
revealed slightly different profiles (Fig. 3.10A).
49
UPcya*'* mPeyii*
25 f
25
20-
£g"<
15-
2 101
10
5 i
5 •
•r^ipn^
cc-c
... ,, .,. ., flfl ,y, Eft ,|l
0
JjU-4aJiite
-. * h *. \- -i *fr I
cv.c
TOovcv
*&&&<!>
Male
Female
Figure 3.10. Comparison of hepatic phospholipid compositions. Analyses of
individual fatty acid side chains of phospholipids for Pcyt2+/+ and Pcyt2+/~ livers
expressed as a percent of total. Results are for three mice of each gender and genotype.
Data for female and male analyses for PE, PS and PC are shown in A, B and C
respectively, where * indicates significance (P<0.05).
50
Pcyt2 ' liver PE generally contained more saturated fatty acids (SFA), palmitic and
stearic acids and less unsaturated fatty acids. Females show a general decrease in
polyunsaturated fatty acids (PUFA), while males have significantly lower levels of co-3
fatty acids only. Although PE was expected to be the most affected by the disruption of
Pcyt2, the compositions of PC and PS were also examined. The fatty acid composition of
liver PS also demonstrated a significant increase in SFA (palmitic and stearic acid) and
decreased PUFA content in Pcyt2+/' mice, specifically arachidonic acid (AA) and
docosahexaenoic acid (DHA) (Fig. 3.1 OB). Although PE and PS experienced greater
fluctuation in fatty acid composition, PC composition remained relatively undisturbed in
heterozygotes of both genders (Fig. 3. IOC).
51
3.5 Discussion
In the current investigation, we describe the phenotype resulting from the
complete and partial disruption of the Pcyt2 gene in mice. Pcyt2 is a single gene, which
can be alternatively spliced to code for two distinct isoforms. The importance of ET
within the PE-Kennedy pathway, as well as the importance of the entire pathway to
mammalian PE biosynthesis had not been fully elucidated. However, by targeting the
Pcyt2 gene, we show that its complete disruption in mice results in early embryonic
lethality prior to stage E8.5, which makes the Pcyt2 gene and de novo pathway for PE
biosynthesis indispensable for murine development.
Moreover, heterozygous embryos show no overt phenotypic differences during
embryonal development and pups are indistinguishable from the wildtype littermate
controls. Pcyt2 mRNA and protein expression was decreased in heterozygous mice
relative to their littermate controls, although not to the expected levels that are associated
with a true gene dosage effect, as seen in other heterozygous knockout models (161,
209). Most importantly, no variations were detected in the amount of total PE and other
phospholipids in heterozygous mice, and we demonstrate that a compensatory up
regulation of the Pcyt2 single allele is established to maintain normal phospholipid
homeostasis.
The effect of Pcyt2 deletion on the PE-Kennedy pathway was established at
multiple levels in Pcyt2+/~ and control mice. Radiolabeling of primary hepatocytes
demonstrated that the de novo pathway was diminished in Pcyt2+/~ cells, as indicated by a
decreased
rate
of
PE
production,
increased
levels
of
the
ET
substrate
phosphoethanolamine and decreased levels of the ET product CDP-ethanolamine (Fig
52
3.8). In support of the in vitro experiments, the whole animal incorporation of
[14C]ethanolamine into PE in the liver, kidney, heart and brain closely resembled the
expression pattern and the in vitro activity of ET, with an overall reduction. Our results
show a reduced production rate of PE in Pcyt2+/~ hepatocytes and mice as well as
consistent phospholipid levels between genotypes in all of the tissues examined.
Therefore we addressed the possibility that an alternative PE synthetic pathway may have
been increased in Pcyt2+/' animals to compensate for the diminished rate of PE synthesis.
It has been previously demonstrated that phospholipid content could still be maintained
regardless of the disruption of certain phospholipid synthetic pathways. In mice, liver PC
production is maintained by the PC-Kennedy pathway when the PE methylation pathway
is targeted by the deletion of the Pemt gene (207), in Pss2~'~ mice, phospholipid levels
were preserved by the presence of Pssl (162), and in mice heterozygous for Pisd, PE
levels were compensated by up regulation of ET and associated increase in PE production
via the PE-Kennedy pathway (161). To investigate if the reverse phenomenon occurs in
Pcyt2+/~ mice, we investigated the expression of Pisd as well as the radiolabeling of the
decarboxylation pathway by [3H]serine, both in hepatocytes and in whole animals. Our
results indicate that there is no significant change in the decarboxylation of PS to
compensate for PE production in Pcyt2+/' animals. An alternative fate for PE in the liver
is the specific role for the conversion to PC by PEMT. It has been shown that PC
produced by PE methylation is destined for bile and lipoprotein secretion (197) and that
PE produced by decarboxylation is preferable for methylation (197). Our results
demonstrate that PS decarboxylation to PE and PC production are not altered in the
heterozygous liver.
53
Pcyt2 was up regulated from the anticipated levels due to gene dosage at all of the
stages of gene expression analyzed. It is tempting to speculate therefore that since the
levels of ET expression effectively mimicked that of the transcriptional output, the
increase in ET regulation is initiated at the transcriptional level. In spite of the up
regulation of the remaining Pcyt2 allele, data demonstrate a slower production of PE in
Pcyt2+/' mice compared to controls (Fig 3.8). The important question that remains;
however, is how this occurs without a change in total PE content. To account for the
diminished rate of synthesis, it is most likely that the PE turnover is also slowed, to
effectively maintain the PE levels in heterozygous animals. We establish that complete
Pcytl disruption results in embryonic lethality and that these embryos ultimately begin
resorption at an early developmental stage, likely due to the absence of sustainable
amounts of PE. However in Pcyt2+/~ embryos and mature mice, the critical level of
membrane phospholipids is maintained by increasing ET protein expression from the
single allele.
Changes in PE turnover could also be seen from the changes in phospholipid fatty
acid profiles of Pcyt2+/~ mice. We analyzed the fatty acid composition of PE, PS and PC
in liver and although there were no changes in total phospholipid content, SFA of PE and
PS, but not PC, increased in both male and female Pcyt2+/' mice, while their PUFA
content decreased. Although the ramifications and significance of these specific
alterations in PE and PS composition have yet to be established, numerous other studies
and models have demonstrated that fatty acid side chains could affect membrane fluidity
and prostanoid production (139), and that PUFA deficiency is strongly associated with
fetal development (173) and numerous chronic disorders such as mood disorders (143),
54
neurodegenerative conditions (51, 163) and cardiovascular disease (26). We postulate
that a decreased PE phospholipid turnover in Pcyt2 heterozygote mice may ultimately
lead to altered PUFA/SFA ratio, which would be the first physiological demonstration of
how PE production via Kennedy pathway is coupled to the 'Lands' pathway (99) for the
fine-tuning of fatty acid composition of individual phospholipids.
Regardless of past investigations where the necessity of the PE-Kennedy pathway
in mammalian cells has been questioned, lethality of Pcyt2 null embryos demonstrates
that both Pcyt2 and therefore the PE-Kennedy pathway are indispensable for murine
development. The presence of a single Pcyt2 allele results in a haplo-insufficient state,
which consequently results in an up-regulation of that allele in heterozygous animals to
maintain normal levels of PE for cellular functions. This up-regulation of Pcyt2 in
heterozygous animals is critical since no compensatory increases in the PE production via
the mitochondrial PS decarboxylation pathway were established.
55
CHAPTER FOUR
THE DEVELOPMENT OF A METABOLIC DISEASE PHENOTYPE IN
CTPcPHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE DEFICIENT
MICE
56
4.1 Abstract
Phosphatidylethanolamine (PE) is an important inner-membrane phospholipid mostly
synthesized de novo by the CDP-ethanolamine pathway and by decarboxylation of
phosphatidylserine. CTP:phosphoethanolamine cytidylyltransferase (Pcytl) catalyses the
formation of CDP-ethanolamine, which is often the rate-regulatory step in de novo
pathway. In the current investigation, we show that a reduction in the rate of CDPethanolamine formation in Pcyt2+/~ mice limits the rate of PE synthesis. This metabolic
disturbance increases the availability of diacylglycerol and results in the increased
formation of triglycerides, which is facilitated by an increase in hepatic fatty acid uptake
and de novo synthesis as well as reduced fatty acid utilization as an energy substrate.
Pcyt2+/~ mice gain weight with age, where older heterozygous mice have increased
plasma triglycerides, accumulate diacylglycerol and triglycerides in liver and muscle.
Accordingly, gene expression analyses demonstrated the up-regulation of the main
lipogenic genes, down-regulation of fatty acid P-oxidation genes and perturbations in
genes involved in insulin signaling pathways. These data demonstrate that in order to
preserve membrane PE phospholipids, Pcytl deficiency generates compensatory changes
in triglyceride and energy substrate metabolism resulting in a progressive development of
liver steatosis, hypertriglyceridemia, obesity and insulin resistance, the main features of
the metabolic syndrome.
57
4.2 Introduction
Phosphatidylethanolamine (PE) is the major inner-leaflet phospholipid in cellular
membranes and it plays important roles in membrane integrity, cell division, cytokinesis,
autophagy and blood coagulation (13). Phospholipases degrade PE to the secondary
substrates diacylglycerols (DAG), phosphatidic acid and free fatty acids (FA) which are
involved in energy metabolism and various cell-signaling cascades. Specifically, Nacylated PE is a main source of endogenous cannabinoids including N-arachidonylethanolamine anandamide (118) and PE is a donor of the phosphoethanolamine moiety of
glycosylphosphatidylinositol anchors, ensuring the linking of the carboxyl-terminus of
various proteins to the plasma membrane (124). PE also serves as a natural ligand for
steroidogenic factor-1 (161) and liver receptor homologue-1 (140), transcription factors
involved in the regulation of gene function. In Drosophila, PE completely substitutes for
cholesterol in the feedback regulation of sterol regulatory element binding proteins
(SREBPs) (38), which highlights its importance in the nuclear control of lipid
metabolism.
The CDP-ethanolamine (PE-Kennedy) pathway (Figure 4.1) is responsible for
the de novo synthesis of PE and is essential for the production of PE plasmalogens (10,
19), where as the decarboxylation of phosphatidylserine (PS) produces a significant
portion of mitochondrial PE (25) and in some cell cultures can become a dominant
pathway for PE synthesis (205).
58
CDP-Ethanolamine
Pathway
DAG/TG Synthesis
Pathway
^
Eth
acyl-CoA
Glycerol
G3P GPD1
C02
Figure 4.1. CDP-Ethanolamine, DAG and TG synthetic pathways. Abbreviations as
follows: Eth, ethanolamine; EK, ethanolamine kinase; P-Eth, phosphoethanolamine;
ET/Pcyt2,
CTP:phosphoethanolamine
Ethanolamine;
EPT,
cytidylyltransferase;
CDP-ethanolamine
CDP-Eth,
CDP-
1,2-diacylglycerol
ethanolaminephosphotransferase; PE, phosphatidylethanolamine; GPD1, glycerol-3phosphate dehydrogenase 1; G3P, glycerol-3-phosphate; GPAT, glycerol 3-phosphate
acyltransferase; LPA, lysophosphatidic acid; AGPAT,
l-acylglycerol-3-phosphate
acyltransferase; PA, phosphatidic acid; PAP/Lipin-1, phosphatidic acid phosphatase;
DAG, diacylglycerol; DGAT1/2, diacylglycerol acyltransferase 1/2; TG, triglyceride; PS,
phosphatidylserine; PSD, PS decarboxylase, arrows indicate direction up/down-regulated
with Pcyt2 deficiency.
59
PE may also be formed from pre-existing phopsholipids by base-exchange
mechanisms; however, this is a quantitatively minor pathway (171). In the PE-Kennedy
pathway,
ethanolamine
phosphoethanolamine,
is
first
which
phosphorylated
is
converted
by
to
ethanolamine
kinase
CDP-ethanolamine
to
by
CTP:phosphoethanolamine cytidylyltransferase (ET). In the final step of the pathway,
CDP-ethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (ETP) catalyzes
the formation of PE from CDP-ethanolamine and DAG. ETP alternatively couples CDPethanolamine with alkylacylglycerols, derived in peroxisomes, to produce alkylacyl
(plasmanyl) PE, which is further modified in the mitochondria to the final vinyl-ether
(plasmalogen) PE. Multiple isoforms of kinases and phosphotransferases have been
identified that share substrates for both the choline and ethanolamine branches of the
Kennedy pathway, yet ET is produced by a single gene (Pcytl) and specific for the
ethanolamine branch of the pathway (13).
The Pcyt2 gene has been cloned and characterized for yeast, human, rat and
mouse (17, 128, 133, 146). It gives rise to two distinct, evolutionarily conserved splice
variants, with distinct tissue expression and catalytic properties (178). Murine and human
Pcyt2 promoters have been isolated and initially characterized (87, 146). The human
promoter can be regulated by early growth response factor 1 (EGR1) and nuclear factor
kappa B and a lower de novo synthesis and PE content in human breast cancer cells was
attributed to decreased Pcyt2 gene regulation by EGR1 (226). The regulation and
function of the Pcyt2 gene has been recently reviewed (13).
Although it is not firmly established how the supply of alkylacylglycerols regulate
the production of plasmalogens, DAG availability can control the flux through the PE-
60
Kennedy pathway under certain conditions (86, 181). The possibility that substrate
supply controls the Kennedy pathway has also been shown for ethanolamine (10, 75).
Thus it is very likely that the PE synthesis via the Kennedy pathway is coordinately
regulated by the availability of both substrates, CDP-ethanolamine (via Pcytl) and DAG,
which is very relevant for the current investigation.
We have previously described the deletion of Pcytl in mice, where null embryos
are lethal prior to embryonic day 8.5 and heterozygous (Pcyt2+/') mice maintain normal
PE levels despite reduced formation of CDP-ethanolamine and reduced flux through the
de novo pathway (54). This demonstrated that Pcyt2 is essential during murine
development
and PE content
cannot be
compensated
by the
mitochondrial
decarboxylation of PS. In the current investigation, we establish that reduced formation
of CDP-ethanolamine in Pcyt2+/~ mice limits the rate of PE synthesis. This increases the
availability of DAG and causes a shift in triglyceride (TG) and energy substrate
metabolism leading to development of adult-stage obesity, hypertriglyceridemia and a
resistance to the effects of insulin.
61
4.3 Experimental Procedures
4.3.1 Animals
Pcyt2+/~ mice were generated as described previously (54). All procedures
conducted were approved by the University of Guelph's Animal Care Committee and
were in accordance with guidelines of the Canadian Council on Animal Care. The mice
were exposed to a 12-h light/dark cycle beginning with light at 7:00 a.m. Male and
female mice were fed ad libitum a standardized diet (Harlan Teklad S-2335) and had free
access to water. Aside from weight gain analyses, male mice were used for all
experiments.
4.3.2 Plasma lipid analyses and liver histology
Plasma collected from mice fasted 12-16 h was assayed for total cholesterol,
glucose, free fatty acids (FA) and TG using standard kits (Wako Chemicals). Insulin was
determined using an ELISA kit (Linco). Livers were dissected and fixed in 10% formalin
in phosphate buffered saline (PBS) and embedded in paraffin. 10 urn sections were
stained with hematoxylin and eosin.
4.3.3 Rate of VLDL secretion
Mice (2 and 8 month old) were injected with poloxamer 407 (P407), to inhibit
lipoprotein lipase, via intraperitoneal (IP) injection. Blood was sampled via the
saphenous vein at baseline, 1, 2 and 4 h, plasma isolated and total TG was determined as
above.
62
4.3.4 Glucose tolerance test
Mice were fasted 4 h and after a baseline saphenous vein blood sample, 2 mg/kg
glucose (in 0.9% saline) was administered via IP injection. Blood samples for glucose
and insulin determination were taken for up to 2 h, where blood glucose was determined
by a monitoring system (Ascensia Elite XL).
4.3.5 Phospholipid, DAG and TG content and composition
Tissues were homogenized in PBS or plasma was aliquoted and lipids were
extracted by the method of Bligh and Dyer (21). In brief, lipids were extracted by adding
chloroform: methanol (1:2) to tissue homogenates and vortexing for 30 s. Chloroform
and water were subsequently added, each with a brief vortex resulting in a final ratio of
chloroform:methanol:water (1:1:0.9). The lipid-containing chloroform phase was dried
under a stream of nitrogen gas and lipids were resuspended in a constant volume of
chloroform or isopropanol, depending on the experiment. For the separation of
phospholipid species, lipids were spotted onto silica gel 60 plates and resolved in a
solvent system of chloroform/methanol/acetic acid/water (40:12:2:0.75, v/v/v/v). Neutral
lipids were separated in a solvent system of heptane/isopropyl ether/acetic acid (60:40:3,
v/v/v). Authentic standards were spotted in parallel to the samples and the plates sprayed
with aniline naphthalene sulfonic acid and exposed to UV light for visualization. The
amount of PE was also determined in primary hepatocytes as previously described (226).
Briefly, cells were radiolabeled with 0.5 |aCi [14C]ethanolamine with 50 uM unlabeled
ethanolamine per well for 24 h. Lipids were extracted and resolved as described above
and radioactivity of PE was determined by liquid scintillation counting. The composition
63
of PE, PC and PS were established after separated by thin layer chromatography as
described above/After isolation, individual species were subsequently transmethylated
with 6% (by volume) H2SO4 in methanol at 80°C for 3 h. Samples were then cooled to
room temperature and the methylated FA were collected by the addition of petroleum
ether and deionized water, followed by centrifugation at 1,000 x g for 15 min at 4°C.
Samples were analyzed by gas-liquid chromatography (HP 5890 Series II; Hewlett
Packard) as previously (34). DAG and TG composition and content were determined by
gas-liquid chromatography as above. Total TG content was additionally measured by an
enzymatic kit (Wako Chemicals) and total DAG and TG by thin layer chromatography,
as previously described (80), where separated total DAG and TG were quantified using
standard curves and reflection densitometry. Hepatic cholesterol was assessed using total
cholesterol kit (Wako Chemicals), where lipids were resuspended in isopropanol prior to
use.
4.3.6 Isolation of primary hepatocytes
Primary hepatocytes were isolated as previously described (92, 93) with slight
modification. Livers were first perfused with an EGTA solution and second with a
collagenase solution, through the inferior vena cava after clamping of the superior vena
cava and cutting of the portal vein. Cell viability was assessed using trypan blue
exclusion and cell number counted using a hemocytometer (viability was always greater
than 95%). Cells were plated on 6-well collagen coated plates (10x5 cells/60 mm dish).
Cells were allowed to attach for 2 h and then media (Williams' Medium E; Gibco) and
cells in suspension were removed and replaced with complete media (Williams' Medium
64
E with 10% fetal bovine serum (Sigma) and 1% antibiotic-antimyotic solution (Gibco)).
Cells were incubated overnight at 37°C and used for labeling experiments the following
day.
4.3.7 Hepatic fatty acid uptake and transporter expression
Hepatocytes (~0.5 x 106 cells) were grown overnight and labeled with BSAcomplexed [3H]oleate (Perkin Eylmer) (0.5 uCi/well, initial activity was 22.7 Ci/mmol)
in the presence of 40 uM unlabeled (BSA-complexed Na-oleate). Cells were labeled for
1, 3, 5 and 8 min, after which the uptake was stopped, radiolabeled cells were washed
three times, lysed in a homogenization buffer and cell associated radioactivity determined
by liquid scintillation counting. For FA transporter expression, proteins were isolated and
investigated as previously described (24), Ponceau S was utilized as a loading control.
4.3.8 Hepatic glycerol uptake and glycerolipid metabolism
For pulse experiments, cells were labeled with 1 juiCi [3H]glycerol (initial activity
was 20 Ci/mmol) (ARC Inc.) in the presence of 50 uM unlabeled glycerol or 0.5 uCi
[3H]oleate for 1, 2 and 4 h. [3H]glycerol was added directly to the cells, where as labeled
[3H]oleate bound to BSA was first pre-incubated with BSA-complexed Na-oleate (molar
ratio was 4:1). For pulse-chase experiments, hepatocytes were pulsed with 0.1 (xCi
[14C]ethanolamine (initial activity 55 mCi/mmol) (ARC Inc.) in the presence of 50 |JM
unlabeled ethanolamine or 2.5 uCi [3H]glycerol in the presence of 50 uM unlabeled
glycerol for 2 h in complete media. Radiolabeled media was then removed, cells were
washed with PBS and chased with a media containing an excess (250 uM) of unlabeled
65
ethanolamine or glycerol for 1,2 and 4 h. For analyses, cells were washed twice with icecold PBS and collected in PBS. Lipids were extracted as described for plasma and tissues
above. Total phospholipids, DAG and TG were scraped and radioactivity determined by
liquid scintillation counting. Phospholipids were separated as outlined above. Glycerol
uptake was assayed as described (104), with modifications. Isolated hepatocytes were
incubated in PBS, supplemented with 6 mM glucose and 1 |j.Ci [3H]glycerol for 1, 2 and
3 min to ensure linearity in glycerol uptake. After each incubation, cells were washed
twice with ice cold PBS containing increasing concentrations of unlabeled glycerol (10,
50 and 100 mM) and lysed. The glycerol uptake in nmol/mg/min was determined from
the cell-associated radioactivity as described above for the [3H]-glycerol pulse and pulsechase experiments.
4.3.9 Hepatic fatty acid oxidation
Primary hepatocytes were cultured as described above with slight modifications.
Cells were plated onto 35 mm dishes (-1.2 x 106 cells/dish), pre-coated with collagen.
After 24 h, cells were incubated with an assay mixture consisting of 250 uM 'cold'
oleate complexed to 2.5% fatty acid-free BSA in complete William's medium E, 1 mM
carnitine and 1 uCi/well [14C]oleate (initial activity 55 mCi/mmol) (ARC Inc.) for 1.5 h
(104). After the addition of the radiolabeled medium (3 ml), the 35 mm dishes containing
the cells were placed without lids into clean 100 mm culture dishes with two rubber
stoppers fitted on the lid. A small cup was placed in a holder inside the larger dish, the
system was made air-tight with parafilm and the plates were incubated at 37°C. After the
incubation period, 200 ul of 1 M benzethonium hydroxide was added by syringe into the
66
small cup through the rubber stopper. 3 ml of 3 M H2SO4 was added through another
rubber stopper directly to the cells to liberate both trapped [14C]C02 and the tricarboxylic
acid cycle intermediates contained within the cells. The labeled CO2 trapped by the
benzethonium hydroxide was collected for 2 h and then added to scintillation fluid and
radioactivity determined by liquid scintillation counting. Duplicate plates were utilized
for protein determination using the BCA protein kit (Pierce).
4.3.10 In vivo lipid radiolabeling
Mice were fasted for 4 h prior to an IP injection of 5 uCi of [3H]acetate (initial
activity was 20 Ci/mmol) (ARC Inc.) diluted in 0.9% saline in the presence of 250 uM
unlabeled Na-acetate. Mice were sacrificed after 1 h and the liver was extracted. 100-200
mg portions of liver were immediately homogenized in two volumes of PBS. Total lipids
were extracted by the method of Bligh and Dyer as described above then saponified in
ethanolic 0.5 M NaOH for 3 h at 70°C. Non-saponifiable lipids were extracted three
times with petroleum ether and radioactivity determined by liquid scintillation counting.
Remaining saponifiable lipids were acidified with 6 N HC1, extracted three times with
petroleum ether and evaporated to dryness. Samples were resuspended in a constant
volume of chloroform and aliquots were taken for radioactivity determination by liquid
scintillation counting. The incorporation of [ HJacetate into total phospholipids,
sphingomyelin, DAG, TG, free FA, free cholesterol and cholesterol esters was
determined by thin layer chromatography, resolving total lipids as described above.
67
4.3.11 Food intake and energy expenditure
Mice from each genotype were housed individually for 5 days, after which the
consumption of their diet was weighed at 8:00 am for 10 consecutive days. For energy
expenditure, mice were weighed prior to the removal of all food. After 24 h and ad
libitum access to water, mice were weighed and an estimation of energy expenditure
made from the change in body weight. Indirect calorimetry (Oxymax; Columbus
Instruments) was performed over a 24 h period, from which the respiratory quotient (RQ:
VC0 2 /V0 2 ) was determined.
4.3.12 RNA isolation and expression analyses
Tissue harvesting, RNA extraction and first strand cDNA synthesis was
performed as described previously (54), where semi-quantitative as well as real-time PCR
was utilized for mRNA analyses. For semi-quantitative PCR, each gene was analyzed in
the linear phase of PCR, using the optimal reaction cycle conditions. Liver and skeletal
muscle mRNA was expressed relative to P-Actin. Primers and cycle conditions are
available upon request. For real-time PCR, cDNA was added to each well of the Mouse
Insulin Signaling Pathway Array (RT2 Profiler PCR array; SuperArray), and data
analyzed according to the manufacture's instructions.
4.3.13 Statistical Analyses
All data are expressed as mean ± SEM. Statistical significance (for Pcyt2+/~
relative to wild-type littermate controls) were determined using two-tailed, unpaired
Students t-test or linear regression. Growth curve and metabolic chamber data were
68
analyzed by two-way ANOVA. GraphPad Prism 4 software was used for all statistical
calculations. For all radiolabeling experiments, specific activity was adjusted to account
for the amount of unlabeled substrate. Rates of synthesis and degradation were assessed
by linear regression of the natural log (In) of the amount of product (nmol/mg).
69
4.4 Results
4.4.1 Pcyt2•>+/- ' mice have reduced PE synthesis and degradation.
In our previous study (54) we performed specific radiolabeling of the PEKennedy pathway with [14C]ethanolamine and established that the formation of CDPethanolamine was significantly reduced and was limiting PE synthesis in 8-10 wk old
Pcyt2+/~ mice. We also established that the synthesis of phosphatidylcholine (PC) and PS
as well as the total tissue phospholipid content (PE, PS and PC) in the liver, kidney, heart
and brain remained unmodified in heterozygous animals (54). We show here, albeit in
older animals (40 wks), that in isolated hepatocytes the steady-state levels of PE as
measured by [14C]ethanolamine incorporation, also remained unchanged between
genotypes (Figure 4.2).
«=]
o~ _ , _
+->
^
BBVB]
•BVB]
^^^^^^^^H
O
v_
Q.
^
.
HBVBj
o> 2£
^^H
^^^B
•*••»•.
^^^^^^^H
LU
BBVH
Q_
—
1O
E
c
0-
Pcyt2+/+
^^^H
••VH
BftVH
^^H
^^M
!'"
HH
Figure 4.2. Total PE content in isolated hepatocytes. To ensure that previous results
were consistent in isolated hepatocytes, cells were radiolabeled with [14C]ethanolamine
for 24 h. PE was isolated and radioactivity determined (n=3 performed in triplicate). Data
are expressed as nmol PE formed/ mg of protein.
70
We further demonstrate that the rate of PE synthesis from [3H]glycerol was
similarly reduced in the Pcyt2+/~ hepatocytes compared to littermate controls (0.08±0.02
vs. 0.17±0.03 nmol/mg/h) (Figure 4.3A), as it was after radiolabeling with
-14,
['XJethanolamine
(54).
Pcyt2+/+
Pcyt2+/025
o
Q.
O)
0.20
E
0.15
o
E
c
0.10
[3H]Glycerol Pulse
•*-*•
ill
a. 0.0b
X
m
0.00
B
[ H]Glycerol Pulse-chase
000
2
4
Time (h)
r H CjEfrianolamine
o
Q.
O)
I
"o
E
c
I
O
71
Pulse-chase
Figure 4.3. PE metabolism is altered in heterozygous mice. (A) Rate of PE synthesis
from [3H]glycerol pulse experiments in hepatocytes. (B) and (C) The rate of PE
degradation in hepatocytes using [l4C]ethanolamine and [3H]glycerol respectively;
studies utilized at least 4 livers per group from 32-36 wk old mice and were performed in
quadruplicate. Rates of synthesis and degradation are described in text and calculated as
described in the methods.
To measure PE turnover, pulse-chase experiments were performed using
[14C]ethanolamine (the PE-Kennedy pathway only) and [3H]glycerol (the PE-Kennedy
and PS decarboxylation pathways) and they similarly revealed a slower rate of PE
degradation in Pcyt2+/~ hepatocytes compared to controls (Figure 4.3B and C)
([3H]glycerol: 0.095±0.012 vs. 0.26±0.05 and [14C]ethanolamine: 0.056±0.021 vs.
0.14±0.02 nmol/mg/h). This demonstrates that reduced PE catabolism is a critical
adaptation because of a slower PE synthesis via the Kennedy pathway that allows
Pcyt2+/~ animals to maintain constant membrane PE content, since mitochondrial PS
decarboxylation is not affected (54). Furthermore, mice deficient of PS synthase 2 also
had a decreased rate of PS degradation as an adaptive mechanism for maintaining
constant membrane PS (162).
4.4.2 Pcyt2+/~ mice accumulate liver DAG and TG and develop adult-onset obesity.
The weights of both male and female mice were examined for 40-50 wks and it
was established that Pcyt2+/~ mice gained weight gradually with age and became
significantly heavier compared to littermate controls after approximately 24 wks (Figure
4.4A).
72
A
504
--*-- Male +/•
40
3
~
30
<D
^
P<0.05
20
-•--• Female +/-•— Female +/+
10
0
— i
0
1
5
i
i
i
1
1
1
1
r~
10 15 20 25 30 35 40 45 50
Time (wks)
B
3 Pcyt2+t+ ^m
Epi.
Subcut.
Renal
Pcyt2+l-
Liver
•>+/-
Figure 4.4. Pcyt2 ' mice gain weight chronically. (A) Increased body weight (from 450 wks) for Pcyt2>+/-' and control littermates of both genders (n= at least 16 per group);
inset shows Pcytl i+/-' and littermate control comparison at ~40 wk of age. (B) Adipose and
liver weights as a percent of body weight (n= 8 per group) (epi; epididymal, subcut;
subcutaneous).
73
In older animals (-32 wks), adipose tissue and liver weight significantly increased
in Pcyt2+/~ mice relative to controls (Figure 4.4B); where at 8 wks there were no
alterations in the weights of adipose deposit or liver between genotype. As a percent of
body weight, epididymal, subcutaneous and renal adipose mass were increased 24.5, 13.4
and 23.0% respectively, where Pcyt2+/~ livers were 27.9% (P=0.02) heavier than controls.
Total hepatic TG was elevated in Pcyt2+/~ mice approximately 2-fold (Figure 3.5A),
while liver DAG content was also elevated (1.8±0.1 vs. 1.3±0.1 mg/g liver or 38%) in the
older Pcyt2+/~ mice (Figure 4.5B).
74
B
Pcyt2+/+
• Pcyt2+I-
Pcyt2+/-
Pcyt2+/+
2.5-
P=0.02
1.041
&
0.5
O.QJL
D
~200-
100
VLDL
CD
HDL
CO
j<
;j
5
Pcyt2+I+
Pcyt2+I-
o
si
O
R>
E
u>
1
II
\l
_JL
1200
E,
"o
a>
[
'?!'
600
_l
"o>
*i
a.
--**
O
H TO
E
<
to/>
Q.
LDL
1800
t
«-—=?=—
2400
jo
Q.
60
JL
=• 50
o
o
^; 40H
£30
O
E
J5
0.
10
I
3000
Elution Time (s)
•>+/-
Figure 4.5. Pcyt2 ' mice have fatty liver and hypertriglyceridemia. (A) Liver TG
content (n= 8 per group). (B) Liver DAG content (n=6 per group). (C) Histological
analyses of Pcyt2+~ and control livers, fixed in paraffin, sectioned at 10 uM and stained
with hematoxylin and eosin (xlOO); representative of at least 4 mice for each genotype.
(D) Total plasma TG (n=8 per group) and (E) lipoprotein profile of plasma TG (n= 6 per
group). (F) Total plasma cholesterol (n=8 per group). (G) Total plasma PC (n=6 per
group). Results are from 32-36 wk old mice (VLDL, very low-density lipoprotein; LDL,
low-density lipoprotein; HDL, high-density lipoprotein).
Histological analyses of Pcyt2,+/-' liver revealed large vacuole-like formations
corresponding to lipid droplets, in which TG had accumulated (Figure 4.5C). In addition
to no changes in adiposity in 8 wk old mice, DAG and TG levels were consistent between
75
genotypes, demonstrating a progressive state of lipid accumulation and weight gain in
heterozygous animals.
4.4.3 Pcyt2+~ mice
have
altered plasma
and
liver
FA
composition
and
hypertriglyceridemia.
At 32 wks of age, Pcyt2+/~ mice had altered plasma lipid profiles compared to lean
littermate controls. Total plasma TG was significantly elevated (55.6%; P=0.001) in
Pcyt2+/~ animals (Figure 4.5D), where increases in TG were predominantly associated
with very low-density lipoprotein (VLDL) fractions (Figure 4.5E). In addition, Pcyt2+/~
plasma TG FA composition was altered, where significant increases in the amount of
monounsaturated FAs (MUFA; P=0.043) and decreases in total polyunsaturated FAs
(PUFA; P=0.023) were observed (Table 4.1).
76
Table 4.1. Plasma and hepatic TG FA compositions of 36 wk old mice
C14:0
C15:0
C16:0
C18:0
C18:l
C18:2n6
C18:3n6
C18:3n3
C18:4n3
C20:l
C20:2n6
C20:3n6
C20:4n6
SFA
MUFA
PUFA
Omega-3
Omega-6
Plasma
+/+
4.02±1.65
3.98±1.9
28.37±5.81
4.82±2.83
8.97±4.47
10.98±1.27
0.26±0.27
1.13±0.83
0.73±0.55
3.24±1.85
0.51±0.22
2.4±0.86
2.02±2.05
45.46±5.83
28.56±3.45
23.48±4.94
5.63±1.41
17.85±4.21
(n=4 per group)
+/5.15±1.42
5.67±1.73
26.04±5.31
5.55±0.83
18.69±8.99
6.91±3.9
0.73±0.88
0.53±0.33
0.55±0.43
4.33±4.07
1.19±0.99
1.1±0.52
0.92±0.73
43.18±11.82
39.81±12.8
18.27±2.54
4.23±1.56
14.03±2.2
P-value
0.165
0.085
0.418
0.503
*0.036
0.061
0.184
0.089
0.515
0.504
0.095
*0.003
0.185
0.635
*0.043
*0.023
0.081
*0.045
Liver (n=5 per group)
+/+
+/1.29±0.47
1.32±0.47
0.44±0.36
0.36±0.25
22.66±3.32
25.71±2.75
2.27±0.81
2.16±0.68
37.1±5.43
43.76±3.94
20.45±6.26
13.19±3.05
0.52±0.29
0.27±0.14
0.69±0.46
0.45±0.38
0.42±0.4
0.28±0.41
1.32±0.56
1.43±0.54
0.22±0.11
0.33±0.23
0.63±0.45
0.52±0.25
1.49±0.99
1.24±0.63
26.85±3.2
29.91±2.56
44.11±4.85
51.41 ±4.43
26.2±8.07
18.68±5.41
1.94±1.17
1.36±0.56
22.59±8.79
17.32±0.43
P-value
0.905
0.642
0.141
0.812
*0.038
*0.039
0.129
0.349
0.549
0.723
0.326
0.620
0.616
0.124
*0.021
0.091
0.309
0.235
SFA-Saturated FA; MUFA- monounsaturated FA and PUFA- polyunsaturated FA. *
represents a significant difference (P<0.05) compared to littermate control (data
represent percent of FA total).
Based on individual FA species, a dramatic 2-fold elevation in plasma TG oleic acid
(C18:l; P=0.036) and significant decrease in C20:3n6 (P=0.003) and total omega-6 FA
(P=0.045) were present in Pcyt2+/~ mice. The FA profile of Pcyt2+/~ hepatic TG mimicked
plasma TG, with elevated C18:l (P=0.038), decreased C18:2n6 (P=0.039) and increased
total MUFA (P=0.021) content (Table 4.1). Increased oleic acid content in Pcyt2+/"TG is
indicative of an elevated stearoyl-CoA acid desaturase 1 (SCD1) activity which produces
oleic acid as well as an elevated DAG acyltransferase (DGAT) activity, for which oleic
acid is a preferred substrate in the final step of TG synthesis (136) and their roles are
discussed below. Reduced PUFA content was previously established for Pcyt2+/~ hepatic
PE (54), where significant reductions were observed in arachidonic acid (AA) and
77
docosahexaenoic acid (DHA). Liver PS (21) and DAG (Table 4.2) also had lower AA,
while PC FA composition did not significantly differ from control animals (54).
Table 4.2. Hepatic DAG FA compositions
Liver (n=5 per group)
C14:0
C14:l
C15:0
C16:0
C16:l
C18:0
C18:l
C18:2n6
C18:3n6
C18:3n3
C18:4n3
C20:0
C20:l
C20:2n6
C20:3n6
C20:4n6
C20:3n3
C20:4n3
C20:5n3
C22:0
C22:l
C22:2n6
C22:4n6
C22:5n6
C22:5n3
C22:6n3
C24:0
C24:l
Saturated
Monounsaturated
Polyunsaturated
Omega-3
Omega-6
Omega-3/Omega-6
+/+
2.98±1.20
0.80±0.71
1.49±0.38
26.27±3.50
3.06±1.25
5.99±1.48
26.40±5.77
9.60±2.76
0.41±0.33
0.87±0.77
2.63±1.26
0.38±0.47
1.96±2.14
0.43±0.24
0.56±0.34
2.16±1.20
0.55±0.59
0.70±0.35
1.07±0.54
0.88±1.12
3.13±2.28
0.46±0.67
0.58±0.51
1.52±1.11
0.82±1.13
1.87±0.81
0.80±0.32
1.61±1.32
38.79±5.93
36.97±5.94
24.24±5.51
8.51±3.53
15.73±4.02
0.56±0.23
+/3.16±1.17
0.70±0.57
1.23±0.32
25.95±6.36
2.94±0.08
6.97±2.11
25.21±2.37
8.45±2.63
0.57±0.43
0.59±0.30
3.41±0.81
0.72±0.50
3.54±2.08
0.77±0.50
0.35±0.31
1.53±0.52
0.79±0.51
0.31±0.18
0.77±0.64
0.52±0.59
3.22±2.80
1.06±0.43
1.10±1.08
1.29±0.56
0.92±0.47
1.99±0.61
1.11±0.88
0.84±0.33
39.66±8.43
36.45±4.51
23.89±4.56
8.77±2.23
15.11±2.86
0.58±0.13
P-value
0.792
0.779
0.234
0.918
0.840
0.376
0.655
0.476
0.496
0.433
0.236
0.264
0.223
0.179
0.281
0.276
0.465
*0.043
0.398
0.504
0.952
0.103
0.319
0.663
0.857
0.778
0.452
0.218
0.841
0.870
0.906
0.882
0.767
0.814
SFA-Saturated FA; MUFA- monounsaturated FA and PUFA- polyunsaturated FA. *
represents a significant difference (P<0.05) compared to littermate control (data
represent percent of FA total).
78
Interestingly, total liver DAG content was elevated in older Pcyt2 ' mice (Figure 4.5B),
where DAG FA composition had generally higher saturated FA and lower MUFA
compared to TG. Total plasma PC and cholesterol contents were unchanged between
genotypes (Figure 4.5F and G) and the distribution of cholesterol and PC among various
lipoprotein fractions was also not affected. Furthermore the FA composition of plasma
PC was not altered. All together, these data show that Pcyt2 deficient mice develop
obesity, fatty liver and hypertriglyceridemia and have elevated MUFA in TG and reduced
PUFA content in PE, DAG and TG. A quantification of total phospholipid, PE, PS, PC,
DAG and TG amounts are summarized in Table 4.3.
Table 4.3. Quantitative summary of hepatic lipids
Pcyt2+I+
Pcyt2+I-
PL;
Total PL
15.94 ±3.24
PE
2.90 ±1.01
15.02 ±2.61
2.34 ±0.57
phospholipid,
PE;
PS
0.65 ±0.17
0.62 ± 0.09
PC
9.06 ±1.58
8.24 ±1.32
phosphatidylethanolamine,
DAG
1.24 ±0.18
1.87 ±0.39*
TG
13.91 ±5.33
20.88 ± 4.64*
PS; phosphatidylserine,
PC;
phosphatidylcholine, DAG; diacylglycerol and TG; triglyceride. Significant differences
are represented by*; P<0.01 and #; P<0.04 compared to littermate control (data represent
mg lipid per g tissue).
As Pcyt2+/~ mice experience elevated plasma triglycerides, we investigated the rate of
VLDL secretion. P407 was used to inhibit lipoprotein lipase, as it has been shown to have
less of an adverse affect on cholesterol metabolism compared to Triton WR-1339 (126).
The rates of VLDL secretion were unchanged between genotype of younger (8 wk old)
mice. In older (32-36), there was a trend towards an increased rate in Pcyt2+/~ livers;
however, this was not statistically significant (Figure 4.6).
79
Time (h)
Figure 4.6. Rate of VLDL secretion in young and old mice. After P407 injection to
inhibit lipoprotein lipase, plasma TG was assessed and secretion expressed as (timol/kg
(n=at least 6 mice in each group).
This suggests that in Pcyt2+/~ mice PE and TG homeostasis are tightly linked,
independently of other glycerolipids, most likely via a common intermediate (DAG)
(Figure 4.1), which we investigate further in the next sections.
4.4.4 Triglyceride synthesis and lipogenesis are increased in Pcyt2+' mice.
We next sought to delineate the possible mechanisms facilitating the metabolic
phenotype of Pcyt2 heterozygous animals. The rates of synthesis (pulse) and degradation
(pulse-chase) of total phospholipids (mainly PC), DAG and TG from [3H]glycerol were
evaluated in primary hepatocytes from 32-36 wks old mice (Figure 4.7).
80
Pcyt?h
--Pcyt?'B
3
[ H]-PLs
H]-DAG
1
2
3
Time (h)
teiG_
0.7
2
Time (h)
3
[3H]-DAG (Pulse-chase)
"S 0.6-
J--
Q.
_-- •
JL
P=0.04
en 0.5-
E
^ 0.4E
c 0.3§0.2.
Q
±0.1-
4
6
Time (min)
•>+/-
Figure 4.7. Pcyt2 ' mice redirect DAG for TG synthesis and have increased hepatic
FA uptake. (A-C) Pulse experiments with [3H]glycerol demonstrating incorporation into
total phospholipids (PL), DAG and TG. (D and E) Degradation of DAG and TG assessed
by pulse-chase experiments using [3H]glycerol, where all rates are calculated as described
in the methods. (F) Hepatic FA uptake ([3H]oleate) (for labeling experiments, at least 4
livers per group performed in quadruplicate).
81
As a measure of DAG incorporation into PE from all possible pathways, the rate of PE
synthesis from [ HJglycerol was reduced in Pcytl ' hepatocytes (Figure 4.3). This was in
agreement with previously established rates of PE synthesis from [14C]ethanolamine, a
measure of PE synthesized only via the PE-Kennedy pathway (54).
Pulse experiments for total phospholipid synthesis from [3H]glycerol (mainly PC)
was unaltered between genotypes (Figure 4.7A). While the synthesis of DAG (0.11=1=0.04
vs. 0.04±0.01 nmol/mg/h) and TG (0.18±0.05 vs. 0.09±0.02 nmol/mg/h) from
[3H]glycerol were higher in Pcyt2+/~ hepatocytes compared to control hepatocytes
respectively (Figure 4.7B and C). Moreover, [ H]glycerol pulse labeling of hepatocytes
from younger (8-12 wks) mice (Figure 4.8), revealed a similar pattern, with no change in
total phospholipid and increased DAG and TG synthesis,
demonstrating that these
alterations are a direct consequence of Pcyt2 gene disruption and not the obese
phenotype, which develops later at ~30 wks of age.
82
1
2
3
Tlme(h)
o
i_
Q.
£
"5
E
c
O
ht
mI
Figure 4.8. Young Pcyt2,+/-' mice redirect DAG for TG synthesis. (A-C) Pulse
experiments with [3H]glycerol demonstrating incorporation into total PL, DAG and TG.
Hepatocytes cultured from 8-10 wk old mice, with 2 livers per group performed in
quadruplicate).
83
In terms of glycerolipid turnover, pulse-chase experiments with [3H]glycerol
revealed a faster DAG utilization in Pcyt2+/~ hepatocytes (0.25±0.06 vs. 0.11±0.04
nmol/mg/h) and no changes in TG degradation relative to controls (Figure 4.7D and E).
Together, these data suggest that the reduced rate of PE synthesis in Pcyt2+/~ hepatocytes
is consistent with the increased rate of DAG production and utilization, which facilitates
the redirection of DAG away from PE and towards TG synthesis. Consequently, an
accelerated formation of TG rather than its degradation (lipolysis) is responsible for the
observed TG accumulation in Pcyt2+/hepatocytes (Figure 4.5A) and for the development
of liver steatosis (Figure 4.5C).
As shown in Figure 4.1, additional FAs (acyl-CoA) are required for an elevated
TG synthesis from DAG, to complete the final step of DAG esterification catalyzed by
DGAT. To establish if external FAs are used for DAG and TG synthesis we performed
radiolabeling experiments with [3H]oleate. However, we established that the synthetic
rates of DAG and TG from [3H]oleate were unchanged, yet the overall amounts
incorporated into DAG and TG were elevated in Pcyt2+/~ hepatocytes compared to
controls. This prompted the investigation of FA uptake (Figure 4.7F), which showed that
[3H]oleate uptake was 2.3-fold higher in Pcyt2+/~ hepatocytes compared to littermate
controls (23.8±3 versus 10.5±2 pmol/mg/min). Interestingly, the protein content of
plasma membrane-fatty acid binding protein, fatty acid translocase and fatty acid
transport proteins 1 and 4 were unchanged in liver and skeletal muscle (Figure 4.9)
showing that their content is not a reflection of the elevated FA transport efficiency.
84
B
\Pcyt2+l-
CZZJ Pcyt2+/+
g>
FABPpm FAT/CD36
FATP1
FABPpm
FATP4
FAT/CD36
FATP4
Figure 4.9. Western blotting of fatty acid transporters. Protein expression of (A)
hepatic (n=4) and (B) skeletal muscle (n=5); FABPpm- plasma membrane-bound fatty
acid binding protein; FAT/CD36- fatty acid translocase/CD36; FATP1/4- fatty acid
transport protein 1 and 4. Pcyt2+/~ expression is relative to littermate control. For all
experiments, mice used were 32-36 wks old. (AU= arbitrary units, where control was set
tol).
We next examined the contribution of de novo FA synthesis in 8 and 36 wk old
-a
animals by measuring the in vivo incorporation of [ HJacetate into various lipid fractions.
An altered distribution of radiolabel was observed in Pcyt2+/~ livers of 36-wk old animals,
where [3H] acetate incorporation increased into the saponifiable fraction (glycerolipids)
and decreased into non-saponifiable fractions (mainly cholesterol) (Figure 4.10).
85
Pcyt2+/+
8 wks
i Pcyt2+l-
36wks
Figure 4.10. In vivo lipogenesis. Distribution of [ HJacetate into hepatic saponifiable
(Sap) and non-saponifiable (Non-Sap) lipid fractions in 8 and 36 wk old mice (n=6 per
group).
The incorporation of [3H] acetate into free FAs, sphingomyelin as well as total
phospholipids were similar between genotypes; however, Pcyt2+/~ livers had increased
labeling of DAG and TG and decreased labeling of free cholesterol and cholesterol esters,
which was observed at both ages (Figure 4.11).
86
EE3 Pcyt2+/+
mi Pcyt2H-
30
PL
FA
SM
DAG
TG
Choi
CE
PL
FA
8 wks
SM
DAG
TG
Choi
CE
36 wks
Figure 4.11. Increased de novo lipogenesis in Pcyt2 ' livers. (A) The in vivo
incorporation of [3H]acetate into hepatic total PL, sphingomyelin (SM), FA, DAG, TG,
cholesterol and cholesterol esters (CE) for 8 and 36 wk old mice (n=6 per group).
This demonstrates that in Pcyt2+/~ mice, the genetic defect in the PE-Kennedy pathway is
linked to early increases in lipogenesis and adjustments in overall lipid metabolism prior
to the manifestation of symptoms of fatty liver and obesity.
Since acetate (acetyl-CoA) is required for de novo synthesis of FA and used in the
synthesis of cholesterol (mevalonate pathway) the above data further show that de novo
FA synthesis (and their incorporation into DAG and TG) in the Pcyt2+/' mice were
elevated at least in part at the expense of cholesterol biosynthesis/esterification. These
data suggest that in addition to reduced PE metabolism, cholesterol metabolism is also
reduced in Pcyt2+/~ mice, which could be required for the proper maintenance of
membrane homeostasis in heterozygous animals.
87
4.4.5 Reduced energy expenditure and reducedfatty acid oxidation in Pcytl ' mice.
To test if hyperphagia was contributing to weight gain in Pcyt2+/~ mice, we
monitored food intake for 10 days. At 32 wks of age, an equal amount of food was
consumed between Pcyt2+/~ and control littermates (3.8±0.8 g/day vs. 3.9±0.8 g/day).
Food intake was also equal in younger animals (8 wks), indicating no specific role of
food consumption in Pcyt2+/~ obesity. As an initial investigation into the energy
expenditure of the mice, we initiated a 24 h fast in both younger and older animals, after
which animals were weighed and weight loss recorded. Pcyt2+/~ animals displayed
resistance to weight loss compared to control animals at all ages examined (30% at 8 and
20 wks and 35% at 36 wks of age; Figure 4.12A), which is indicative of a general defect
in energy substrate metabolism starting prior to the development of obesity.
88
C O Pcyt2+/+
I Pcy12+/-
-5
T
5-7
20
36
Age (Weeks)
JC *»w"
Pcyt2+I-
is
o
~r
Q.
::
U:
'^S! >xv':| ' '/
O)
£ 30^©
|||J:||*:5
P=0.01
-f1
^1^V>^^
O
a
-o
S 20Q.
<N
o
o
O
•t
10-
"Z_J
"o
i—i—P—i—i—i—r
~i—I—i—i—!—i—i—i—r
8:30am
c
0J
"i
7:30am
7:30pm
Figure 4.12. Altered whole-body metabolism in Pcy/2•»+/-" mice. (Aj Mice (8, 20 and 36
wks of age) were subjected to a 24 h fast, and weight loss expressed as a percentage of
body weight (n=10 per group). (B) Respiratory quotient (VCO2/VO2) determined by
indirect calorimetry over a 24 h period; 36 wk old mice were fasted during the light cycle
(7:30 am - 7:30 pm) and fed during the dark cycle (7:30 pm - 7:30 am) (n=5 per group). *
represents statistical significance (P<0.05) as determined by one-way ANOVA. (C)
[14C]oleate oxidation in primary hepatocytes, where data are expressed as nmol CO2
produced/mg prot/h (n= at least 3 livers analyzed in triplicate).
Indirect calorimetry measurements from older mice displayed an upward shift in the RQ
-.+/•
in Pcyt2 ' animals during fasting and during early hours of feeding (Figure 4.12B),
•»+/-
indicating that Pcytl ' mice have a decreased capacity for FA utilization as an energy
89
substrate during fasting and an impaired switching to carbohydrate substrate during the
fasted-to-fed transition. Indeed, [14C]oleate oxidation in hepatocytes from older mice was
blunted 20% in Pcyt2+/~ animals compared to littermate controls (41.27±1.8 vs. 52.83±2.7
nmol/mg/h respectively) (Figure 4.12C). This indicates that at the level of the liver,
Pcyt2+/~ mice have a decreased capacity for FA utilization as an energy substrate. Muscle
and liver peroxisome proliferator activated receptor a, peroxisome proliferator activated
receptor co-activator la, acyl-CoA oxidase and the mitochondrial proton carrier
uncoupling protein-1 expressions were all reduced in Pcyt2+/~ mice (as shown later in
Figure 4.14 and table 4.4). These changes in gene expression corroborate impairments in
FA oxidation seen in Pcyt2+/'mice as demonstrated by 24 h fasting and [14C]oleate
oxidation.
4.4.6 Pcyt2+' mice develop insulin resistance.
Since obesity and hepatic steatosis have been frequently associated with insulin
resistance (114, 144), we also investigated measures of whole body glucose and insulin
sensitivity in both young and old Pcyt2+' mice. Although elevated, fasting levels of
Pcyt2+/~ plasma insulin, glucose and free FAs were not significantly different from
controls in older animals (Figure 4.13A-C).
90
B
— 7
| 6-
I5
CZ3 Pcyt2+/+
IH! Pcyt2+/-
8 4
o 3H
55 2
(B
o. o
D
25
20
0w
O
O
O 10
E
«
1'rJ
11 5
0-
L
-*-Pcyt2+/+ (8 wks)
-•-- Pcyt2+I- (8 wks)
-+-Pcyt2+I+ (36 wks)
- * - Pcyt2+/- (36 wks)
/'!
'/
'/
M
JS 5
30
60
90
120
30
60
90
120
Time (min)
Time (min)
Figure 4.13. Reduced insulin sensitivity and increased muscle lipid accumulation in
Pcyt2+/~ mice. (A-C) Fasting plasma insulin, glucose and non-esterified FA levels
respectively (n= at least 10 per group of 32-36 wk old mice for). (D) IP glucose tolerance
test (IPGTT); where glucose levels were from baseline to 120 min (n=6 per group). (E)
IPGTT insulin levels from baseline to 120 min (n=6 per group). (F) Area under the curve
for IPGTT glucose (36 wk old mice). (G) Area under the curve for IPGTT insulin (36 wk
old mice). For G and H, * represents statistical significance (P<0.05). (H) Total PL,
DAG and TG content of skeletal muscle of 36 wk old mice (n=8).
91
However, when challenged with an intraperitoneal glucose load, both glucose (Figure
4.13D) and insulin (Figure 4.13E) were elevated in 32-36 wk old Pcyt2+/' mice relative to
controls post-injection, where the areas under the curve were significantly increased
compared to wild-type (P=0.049 (-30%) and P=0.01 (-50%) (Figure 4.13F and G). As
in the liver, skeletal muscle DAG and TG content were also significantly increased in 36
wk old Pcyt2+/~ mice (Figure 4.13H). Importantly, 8 wk old Pcyt2+' mice remain insulin
sensitive (Figure 4.13D and E), although they had increased TG synthesis, altered FA
composition, increased lipogenesis and reduced FA oxidation as shown in previous
sections.
4.4.7 Expression pattern of lipid, glucose and insulin signaling genes is modified in
Pcyt2+' muscle and liver.
The gene expression patterns of the 36-wk old Pcyt2+/~ muscle and liver insulin,
glucose and lipid related genes are shown in Figure 4.14 and Tables 4.4 and 4.5.
92
Em Pcyt2 +/+
2.5-1
• • Pcyi2 +/-
Figure 4.14. Altered Pcyt2 ' hepatic and skeletal muscle mitochondrial and
lipogenic gene expression. (A and B) Hepatic and skeletal muscle mRNA expression,
relative to P-Actin as an endogenous control and relative to littermate controls, set to 1
(n=8 per group, 36 wk old mice). Analyses were performed in triplicate. Ppar;
peroxisome proliferator activated receptor, Srebp; sterol regulatory element binding
protein, Chrebp; carbohydrate response element binding protein, Fas; fatty acid synthase,
Scdl;
stearoyl-CoA
mitochondrial
desaturase-1, Dgat; diacylglycerol
glycerol
3-phosphate
methylglutaryl-CoA reductase, Acoxl;
acyltransferase,
acyltransferase,
Hmgcr;
acyl-CoA oxidase-1, Pgc-la;
mtGpat;
3-hydroxy-3peroxisome
proliferator activated receptor co-activator la. * represents statistical significance
(P<0.05).
93
-* ~. ~. t^ t^- t*- en m v>
t^- t~~- t^- r*- t^- t^- oo
~- in «n 10 *n »n m
^_:
IT) »T)
•
•
•
•
• O O
*
• — *— ^ - r n r ^ —«
CN
«—' '—'
I
I
I
I
I
—
^O v-> v-i
•
• ' •
' .
I
§
v=
•«
o
60
O
hom
c
3
43
3
3
X)
3
I
a>
c
8
o
U
§
<u'
oo 3
so K
° c
43
o
43
"o 2
E
5 <52
2'i
o
r- •
—
-§.S
'£
o
I H
.3
43
< '
C
in
i_
O
i
o 2
60 4 |
3
U
g s '5 2 p . eg V] 6 f l
2§> 2 - _;
5 o. o P>re
S
c
_
^
s
> 3 o.
4
00 l - E « 2 4 3
60 * - b
E
c
c
O
4
5
•S
.5 .S c
fr.S
s
' 5 43
3
3-
•~
V3
43
3
O
a.
p-
•8
o
a
c
i-<
•«-»
3
^
£ W
[A
O
o
K
a. £
XJ
111
u
42^
60
3
c« C
?1
fcw
<u"
O
4^
3
•S
60
V
D.
^^
O
3
O
3
<L>
3
tt
U +^
so a
o
i-
I 7 42 ox , m > UH w :
'
S pa
c H
S <"
3
&
5 ffi
60
3
,
1
2g
g
«
m
'S ^
i 3
i 43
X
O
^
cj
d
•2
T
O
43
O
4=
3 'K
^>
^o
Il 53
^ o--So. -5a
(J 0> o
Q.
W
W5
o
«> 2
P.
!
W
Ig
•S e
-U ,
4>
•5
8
60
O 43
L> 3
3
O
ex o
£ O
C
O
pa
I.
i
60
.S I -3 *
4—i
III
w
(N
s •*
ex
I/)
>
«
-o
mo
o
a
—
C
n
s,
o
n
BJl
l
c
60
O
ece
pie
" 'E
epair
o
!
o
I
&0 g
u .5
S
1 H
°
^5
o o
I 43
O
I U <
»n
»n
Ov
(Nr-^omONr-omc^ONoOTi-O'^Ocooocsr--o<N^©r^^©r-oo- m
— »n oo ON r-- o — 0\ m r*-t — oo — oo
o ^ o o r ^
— — o — — — o—• O
o — o o —
o o o o o o o o o
I I I I I I I I I I I I I
^ o o
r ^ f S
O N —
O N t
O O
o \ t o \ h n o
i ^ O \ ' n o o < N
' I ^ r ^ O O O O
^ O O —
—
O O O O
O
<N vo ^- r^ 0\ O — —
0 0 0\
o o
O
— •*t CM — cs oo t~- r^ o tr>
^ - O
—
O — O ON V~I O OO —
— — O — — O
CS
' O O
O O O O O O
O
O O
o
—
4^
—
W
i0-
O
C/3
" ^ CL r^
3 43 4^
_,
(^
ro
a £ < a i <; s
2
£ a oa U
CN
—
U
O
—
u. m i4
43
„
60 —
>>
43
*£
Q.
—
x
O
43
o
P.
<N
U
O.
£-a-SI3fs
o
+
O
u
G
o
•a
(D
'5.
43
C
T3
3
ca
43
H
<U at
P i PH
3
U
S E
(U
u
a,
u
•—
43 g
° "B
S3 iJ
£5
u 2
94
<
*
fc,
« ^ ^ » m
°° "*«••> NO r--
£
<
M
M
-
-
-
-
m
00
v i N ^O
OO ON O —
m
—
\o
1^
CN
O
-
- - ri N ri N
— 00
MD U-l
r*
( N Ov r f
(N m
Tf
oo co *r\ »ri >n ^o v) h oo -_ r-;
cs oi fS —- ^ J ^ J —' ^-* ^-1 fs] r o
£
a.
>-,
"5
a.
C
3
Xi
3
%
U
O
••c
«
C
•
•
co
—
.t;
w
O
o c
X.
ft.
d
S.g
I * •E
O
a)
.£
ft^
1) - f t 1 o
u IS
• - . • s o .
tw 3
o
o- E
c o
•53 E
i—i
•g..s c
2 ;_o
x:
Q £ £ ft, H ft, ft.
— V">
0 4 ON
— "3- 0 0
»
(N v j
©
O
O
—
•s3K •2>3
to
c £.-§
ft-._
JZ 0 0 - *
00 C
1
a Ho£
• ^
*
C/3 W
•<
— u
3 •£
2 S
Q £ £
Xi
*^
<u E
x: o
B (N 00 12
>- 2 O X
12
cd
i
c
•5
c
15
v.
(L>
H
U
O
o *
m
(^
t
oo
O
—
r^
N
o
—
- o
<;
tn
ft, U
a
•S S
&.
a
s <s
g> g
o
c
•5 P P
j
x:
o oi
11 s -J I
o
^ 2 x;
.
P._cO o.
§ .a
T3
C
O
>x P
o 00
•S .E '
>%x>
X
I
•8B
g.E
i
oo .5 •
o -o
<= .5 >, a o o
<L>
oo 3
_ c .5
^
•S c
o
o o 3 "P °
C
&
§ g>
o S
= E
a. u
°
a z I£ U
o — o o — — — o -* — — o o
o o o o o o o o — o o o o
'
s ss s
I . I I
I
I
I
I
I
I
o
oo
f
»—
—
—
m
m
—
<N
oo
<^
m
rO
0 0 0 0
Tt r ^ — vo c o <^
<^ M >£ in vo in
o \ »
O t * -J
oo ON oo m o \ m
<N O O <N O
—
0
0
Io
°| | | | |°l ° I I I I °
2ZZZZZ z z z z z
I
z z z z z
<2 -
——
"°
-g 5 <£ J*
- o M *
co C —
j ^ q ^2 -P
^
f
i
S
O
O
O T S °O us—X£I
X! ft. ft. <
OJ
- - > , «
— <N
Si2
II
-g-o
X> ft"
U NO
+
0
Z Z 2 Z
,£i —
i
BC >
o o - o o a o - O ^ v i n n o o o o
0
°l°i r i
o
X>
—
o. j * .S
0 0 =
•*
ft,
.PQ
13
m
0
00
-* - - « m O N - i o - O f f i - 0
n - ^ - o o o r - r - o r ^ - N o r - N O c n T r N O
O O C N C O C O © — o
— r*iTj-r-r«ioo
o o oo •*
o
'•% as S ^
JS
2
(U
S §
c
J3
o •3 s
o00
o
o MJc •So2
c
'3
E
o
x:
N
<N
U .£ ro
•S
I
<
>
c '5 u '53 EC
I oo S
«
c «
I. "o
—
3
"
i-
S«
'£
" c
• M
o
a
oo
c
•3
c
oo
s.>co
U
S
-
00
O
2&
§ (N
§ 2
•5
Pi
PQ
3
XI
T3
X
ne,
e u
c
3
JO
3
00
_o
o
p
o
-
O
<u
G
c3
^ " ft. - 0
O . O <H
j a c« o u ft. U O Z 5 U o
c2
J3
'B.
(U
<u
•o
1 &
§-1
a
c
2 E
O eS
ft< ft.
•O .2
>> "5
••=•
S
xi 2
u
U 2
o £ «
95
**<
Although there was some variation in the degree of expression between the liver and
muscle, components of the insulin signaling cascade {Irs2, Pik3rl, Ppplc, Akt2, AktS,
Ptpnl) were generally up-regulated, while mRNA for insulin receptor associated proteins
and adaptors (Dok, Dok2, Sosl, Sorbinl) and downstream targets (Prkcz, Prkcc, eIF2b,
Adrald, Tg, Nos2) were mostly down-regulated in both liver and muscle of the Pcyt2+/~
mice.
Based on the expression analyses of the key liver genes, the production of free
glucose by gluconeogenesis and glycogenolysis were likely inhibited (G6pc; -3.73-fold),
while the production of glucose-6-phosphate and glycerol-3 -phosphate by glycolysis
were likely promoted {Gck; 2.14-fold and Gpdl; 1.52-fold), suggesting the presence of
rising levels of glucose in Pcyt2+/~ liver (Table 4.4). Elevated Gpdl in Pcyt2+/~ livers may
further assure an elevated glycerol-3-phosphate supply for the biosynthesis of
glycerolipids (i.e. increased DAG and TG production). Interestingly, mitochondrial
glycerol-3-phosphate acyltransferase and Lipin-1 (phosphatidic acid phosphatase), the
subsequent enzymes involved in the formation of lyso-phosphatidic acid and DAG were
not altered in Pcyt2+/~ mice (Figure 4.14). Elevated expression of the Gpdl gene that
regulates supply of glycerol-3-phosphate for glycerolipid synthesis was associated with
the elevated expression of genes involved in de novo FA synthesis (lipogenesis) and
reduced expression of genes for FA oxidation, described in the previous sections. The
insulin and glucose responsive Srebpl, a key regulator of lipogenesis, was 2-3-fold upregulated in both Pcyt2+/~ liver and muscle (Table 4.4 and 4.5), which may have in turn
been responsible for the increased expression of Fas and Scdl in both tissues (Figure
4.14) and is in keeping with [3H]acetate radiolabeling data. Also, Pcyt2+/~ livers had
reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (the rate-limiting
96
enzyme in cholesterol biosynthesis) (Figure 4.14A) and low-density lipoprotein receptor
were observed in Pcyt2+/~ livers (Table 4.4). Pcyt2+/~ liver and muscle also had enhanced
expression of both Dgatl and Dgat2. Therefore the gene expression pattern also show
that Pcyt2+/~ mice experience shifts in lipid metabolism and in the balance between
energy
storage
and
energy
oxidation,
which
progresses
hypertriglyceridemia, liver steatosis, obesity and insulin resistance.
97
into
adult-onset
4.5 Discussion
In the past, there have been select lines of evidence that have highlighted the
relationship between phospholipids (mainly PC), DAG and TG metabolism (27, 79, 188).
DAG and FA released from phospholipids can be utilized for TG synthesis (77) and
alterations in cellular PC content is sufficient to cause changes in TG metabolism (27). In
rat hepatocytes, an elevated TG synthesis occurred as a result of excess DAG when CDPcholine was limiting PC synthesis and also where DGAT activity was maximal (160). It
has
also
been
established
that
mutations
inhibiting
CTP:phosphocholine
cytidylyltransferase (CT) of the PC-Kennedy pathway in CHO cells results in a
redirection of DAG from phospholipids to TG (27). Interestingly, in Drosophila,
inhibition of PC synthesis increased TG content in lipid droplets, mainly by altering the
size and the morphology of the droplets (59). In a mammalian context, a liver-specific
CTa knockout model was shown to have decreased VLDL secretion and therefore
accumulated TG in the liver due to reduced PC synthesis (85).
The specific interaction between PE and TG metabolism has been largely
unexplored. Here we described an animal model with genetically reduced PE synthesis,
which similarly to the inhibition of PC synthesis via the Kennedy pathway in CHO cells,
leads to elevated DAG and TG (27). Surprisingly, this also leads to the development of a
metabolic disease phenotype. Our results demonstrate a causal effect of Pcytl disruption
on the redirection of DAG toward TG formation. A decreased rate of CDP-ethanolamine
formation in Pcyt2+/~ mice reduces the rate of PE synthesis. As a direct consequence,
DAG is made available for TG formation, which is further facilitated by an increased
availability of FA (Figures 4.1).
98
It is common for the up-regulation of certain genes in DAG and TG synthesis to
result in symptoms of obesity (105, 127, 132); however, most genetic knock-out models
result in the amelioration of obesity, hepatic steatosis and insulin resistance (4, 61, 157).
Previously, it was not known that obesity and insulin resistance could manifest due to
irregularities in membrane phospholipid homeostasis. Pcyt2+/~ mice are indistinguishable
from littermate controls during the first stages of postnatal development, and remain so
until approximately 24 wks of age. We establish that young (8-20 wk) mice experience a
metabolic phenotype similar to the older (obese) animals, which is characterized by
chronic state of decreased FA utilization as an energy substrate and increased hepatic
lipogenesis, resulting in an increase in TG formation (Figure 4.10 and 4.11). This
represents an inherent shift in glycerolipid metabolism together with an increased
partitioning of FA toward storage (away from oxidation), causing a shift to a more
positive energy balance and being most likely responsible for the chronic disease
progression. There appears to be a threshold (-28 wks), after which the heterozygous
animals become significantly heavier than control animals. Hypertriglyceridemia is
observed without a significant increase in VLDL secretion from the liver (127), which
may contribute to hepatic TG accumulation and insulin resistance.
Various lipid intermediates such as DAG, ceramide and free FAs negatively
regulate the insulin signaling cascade and are frequently elevated in obesity and insulin
resistance (183). We believe that increased DAG availability due to reduced production
of CDP-ethanolamine may play a major role in the development of obesity and insulin
resistance in Pcytl deficient mice, although the role of ceramides in the progression of
this phenotype cannot be ruled out in the current investigation. Though young Pcyt2+/~
animals possess a metabolic phenotype similar to the heavier, older animals (reduced PE
99
synthesis, increased lipogenesis/TG formation and decreased FA oxidation), they are
neither obese, nor have they lost their sensitivity to insulin. It would seem logical that the
alterations in lipid metabolism are adaptive measures to prevent the accumulation of toxic
amounts of DAG, unused in PE synthesis. Since additional FAs are needed for DAG
esterification, our metabolic labeling and gene expression studies showed that they were
made available by several processes including a reduction in FA oxidation (an increased
utilization of glucose as a source of energy and acetyl-CoA production), elevated de novo
FA
synthesis
and
by
reduced
acetyl-CoA
participation
in
cholesterol
synthesis/esterification. The mechanisms that interfere with insulin's ability to regulate
glucose metabolism in Pcyt2 deficient mice required further analyses and investigations
are currently under way. It is also interesting that in spite of a protective mechanism to
redirect DAG toward TG storage, older Pcyt2+/~ mice maintained elevated DAG levels in
both liver and muscle, suggesting that the rate at which DAG can be utilized for TG
esterification is slower than the rate of DAG formation (observed by glycerol labeling).
This also suggests the existence of distinct pools of DAG, which based on the
composition of the fatty acid side chains (Table 4.2) may be destined for different cellular
processes such as de novo synthesis or lipolysis (30).
Studies have shown an inverse relationship between intramyocelluar TG storage
and insulin sensitivity in both rodent and human models (145), though, trained athletes
have the highest levels of both intramuscular TG and insulin sensitivity (183). There is
increasing evidence that supports a role for increased levels of active lipid intermediates
rather than TG itself, which may play more critical roles in the impairment of insulin
signaling (167). Over-expression of Dgatl in mouse skeletal muscle (resulting in a 3-fold
increase in acyltransferase activity) increased TG formation, yet improved insulin
100
sensitivity by decreasing levels of DAG (106). Although we believe that young Pcyt2
animals potentially use similar means to eliminate excess DAG, our model suggests the
possibility that a threshold may exist, where at a certain point the elimination of DAG via
endogenous TG synthesis (DGAT pathway) may no longer be capable of acting as a
protective mechanism. Particularly, this could be the case developmentally. We have
shown that older Pcyt2+/~ mice maintain the same lipogenic mechanisms as young mice;
however, they develop fatty liver, obesity and insulin resistance. The important
consequence of chronically elevated lipogenesis in Pcyt2+/~ mice is an increased FA
uptake and DAG accumulation which may eventually become lipotoxic in older animals.
This accumulation would most likely act by modulating the activities of DAG-dependent
novel protein kinase C isoforms (58, 222), the well established players in insulin
resistance (151). We show that key metabolic genes as well as the insulin signaling genes
become altered in the liver and skeletal muscle of old Pcyt2+/~ mice. It remains to be
determined when exactly during development the expressions of these specific genes
become critically impaired as well as which key metabolic and regulatory pathways are
the main triggers of the Pcyt2+/~ phenotype.
The disruption of Pcyt2 is not only responsible for alteration in DAG and TG
synthesis but also affects other aspects of cellular lipid metabolism, such as reduced
cholesterol synthesis and FA oxidation. Pcyt2 heterozygous animals possess an inherent
defect in energy metabolism, which could be an over-compensation in response to the
altered DAG levels; however, could also be a result of impaired lipolysis and reduced PE
availability as a source of FA, which is an interesting area for further investigation.
Considerable efforts have been made to understand phospholipid catabolism and multiple
lipases/phospholipases have been established as significant contributors to the above
101
pathologies. However, it is equally important to understand the anabolic aspects of
phospholipid metabolism, including de novo PE biosynthesis. The Pcyt2 deficient model
is unique and offers a new description for the development of obesity related disorders. It
will increase our understanding of the PE-Kennedy pathway at the whole-body level and
further affirm the importance of the Pcyt2 gene in lipid metabolism.
102
CHAPTER FIVE
PHENOTYPIC RESCUE OF CTP: PHOSPHOETHANOLAMINE
CYTIDYLYLTRANSFERASE (PCYT2) DEFICIENT PRIMARY HEPATOCYTES
103
5.1 Abstract
The
CTP:phosphoethanolamine
cytidylyltransferase
(ET)
gene
{Pcytl)
regulates production of CDP-ethanolamine, which combines with diacylglycerol to form
the
membrane
lipid,
phosphatidylethanolamine
(PE).
[14C]-ethanolamine
and
[3H]glycerol pulse and pulse-chase experiments established that PE synthesis and
turnover are reduced in Pcyt2 deficient hepatocytes relative to controls. [3H]glycerol
radiolabeling also revealed an increased formation of both diacylglycerol and triglyceride
and only elevated turnover of diacylglycerol, consistent with increased triglyceride
accumulation. [ H]acetate radiolabeling showed that de novo fatty acid synthesis was also
increased in Pcytl deficient hepatocytes. Expression of the mouse Pcy/2-myc/His cDNA
into deficient hepatocytes increased ET protein by 2-fold and normalized PE synthesis
and turnover. Reconstitution oiPcyt2 simultaneously reduced diacylglycerol, triglyceride
and de novo fatty acid synthesis to levels comparable to wildtype hepatocytes. Although
increased wildtype Pcyt2-myc normalized lipid formation and degradation, a Pcyt2
mutant H244Y, with 60% catalytic activity was unable to normalize any of the
parameters investigated. These data suggest that the increased lipogenic phenotype in
Pcyt2 deficient hepatocytes is a direct consequence of Pcyt2 gene disruption and
therefore reduced PE formation via the Kennedy pathway. The PE-Kennedy pathway was
re-established only when fully active Pcyt2 was reintroduced and reversed the formation
of DAG and TG to levels more comparable to the wildtype hepatocytes.
104
5.2 Introduction
Phosphatidylethanolamine (PE) is a primary lipid on the inner-leaflet of cellular
membranes and has been shown to regulate various cellular processes including
cytokenesis, coagulation, autophagy and cell signaling (13). There are two main synthetic
pathways for the production of PE. Decarboxylation of phosphatidylserine (PS) mostly
contributes to mitochondrial PE (25), and can become dominant in the absence of
ethanolamine in vitro (205). PE is synthesized de novo by the CDP-ethanolamine or PEKennedy pathway (89), where by ethanolamine is taken into the cell and phosphorylated
by ethanolamine kinase. CTP:phosphoethanolamine cytidylyltransferase (protein; ET and
gene; Pcytl) then catalyzes the formation of CDP-ethanolamine through the addition of
CTP
to
phosphoethanolamine
and
finally
1,2-diacylglycerol
ethanolaminephosphotransferase adds CDP-ethanolamine to diacylglycerol (DAG) in the
endoplasmic reticulum to form PE.
We have characterized a Pcytl heterozygous mouse model as homozygous
disruption was embryonic lethal prior to embryonic day 8 (54). The most direct
consequence of heterozygous Pcyt2 disruption is decreased PE synthesis via the PEKennedy pathway, which coupled with a decrease in the rate of degradation, preserves
cellular PE levels. However, this leads to a redirection of DAG, the common
intermediate, away from the PE-Kennedy pathway and towards the triglyceride (TG)
synthesis. This inherent metabolic change is present at early adulthood, yet strongly
manifests at 5- 6 months of age and the Pcytl heterozygotes experience an accumulation
of TG in tissues and plasma, which leads to the development of obesity and insulin
resistance. In addition, fatty acid metabolism is affected in young Pcytl+/~ mice, such that
there is an increase in de novo synthesis, and increase in FA uptake and a reduction in
105
fatty acid p-oxidation. The fatty acid composition of plasma and hepatic lipids are also
modified in heterozygous mice. Hepatic PE has an increase in saturated fatty acids
(mainly stearic and palmitic acid), where hepatic TG has significantly increased oleic
acid. Both PE and TG have decreased polyunsaturated fatty acids, where the composition
of PE is mimicked by phosphatidylserine but not phosphatidylcholine (PC) (54).
In the current investigation, we demonstrate that the expression of an active
wildtype but not a mutated form of Pcyt2 in primary hepatocytes isolated from Pcyt2+'
mice completely re-established the PE-Kennedy pathway and consequently normalizes
DAG and TG metabolism.
106
5.3 Experimental Procedures
5.3.1 Primary hepatocyte isolation.
Primary hepatocytes were isolated from 32-36 wk old Pcyt2 heterozygous and
wildtype littermate controls as previously described (92, 93) with slight modifications.
Livers were perfused with an EGTA buffer (140 mM NaCl, 6.7 mM KC1, 10 mM HEPES
and 50 uM EGTA, pH 7.4) solution and then with a solution (67 mM NaCl, 6.7 mM KC1,
5 mM CaCl2-2H20 and 100 mM HEPES, pH 7.6) containing 0.5% collagenase, through
the inferior vena cava after clamping of the superior vena cava and cutting of the portal
vein. Hepatocyte viability was assessed using trypan blue exclusion and cell number
counted using a hemocytometer (viability was always greater than 90%). Hepatocytes
were plated on 6-well plates (BD Biosciences) (lxl0 5 cells/60 mm dish), allowed to
attach for 2-4 h and then Williams' Medium E (Gibco) and floating cells were removed
and replaced with a complete media (Williams' Medium E with 10% fetal bovine serum
and 1% antibiotic-antimyotic solution (Gibco)).
5.3.2 Transfection of primary hepatocytes.
Isolated hepatocytes were incubated overnight in complete medium at 37°C and
transfected the following day. After 16-20 h, overnight culture medium was replaced with
a fresh medium containing 50 uM ethanolamine. Hepatocytes were transfected using
JetPEI™ transfection reagent (Polyplus transfection), where 6 ul of JetPEI in 100 ul of
100 mM NaCl was combined with 3 |ig of plasmid DNA (Pcyf2-myc/His or Pcyt2H244Y-myc/His) in 100 ul of 100 mM NaCl and the mixture then incubated at room
temperature for 30 min. To each well, 200 ul of transfection mixture was added directly
into 2 ml of culture medium. Cells were then incubated for 48 h in medium containing 50
107
juM ethanolamine, at which point labeling experiments commenced. As a measure of
transfection efficiency, 64 ng of pRL-CMV Renilla luciferase vector (Promega) was cotransfected in each well and an aliquot of total cell suspension analyzed for luciferase
activity (average efficiency was 1247 ± 96.35 relative light units/mg prot). The myc/His
tagged Pcyt2a cDNA was previously described (178), and cloning of Pcyt2-H244Y-myc
mutant is described below.
5.3.3 Pcyt2-myc Western Blotting.
Transfected hepatocytes were collected into cold lysis buffer (10 mM Tris-HCl (pH 7.4),
1 mM EDTA and 10 mM NaF), containing protease (1/10) and phosphatase (1/100)
inhibitor cocktails (Sigma) and lysed further with two freeze-thaw cycles. Lysates were
centrifuged at 13 000 rpm for 10 min at 4°C to remove cell debris and supernatant
transferred to a new tube. Protein concentration was determined using the BCA method
(Pierce). Cell lysates (25 ng) were resolved on 10% SDS- PAGE and semi-dry
transferred to a PVDF membrane. Proper protein transfer and equal loading were verified
using Ponceau S staining, after which membranes were blocked with 5% milk in 20 mM
Tris-HCl (pH 7.5), 500 mM NaCl, 0.05% Tween-20 (TBS-T) for 1 h at room temperature
followed by overnight incubation at 4°C with either anti-ET antibody (1:2000 in 5%
milk-TBS-T) or anti-Myc antibody (Invitrogen) (1:5000 in 5% milk-TBS-T). After three
wash steps (5-10 min), membranes were incubated with a goat anti-rabbit or goat antimouse secondary antibody for anti-Pcyt2 and anti-Myc respectively (1:20 000) for 1 h at
room temperature and then visualized with enhanced chemiluminescence (Amersham).
108
5.3.4 Metabolic labeling.
Radiolabeling experiments were conducted by adding 0.1
uCi/well of
[14C]ethanolamine (initial activity 55 mCi/mmol), 2.5 uGi/well of [3H]glycerol (initial
activity was 20 Ci/mmol) or 2.5 uCi/well of [3H]acetate (initial activity was 20 Ci/mmol)
(ARC Inc.) for 1, 2, 4 or 24 h. Plates pulsed from 1-4 h were used to determine synthetic
rates of PE, DAG and TG, where 24 h labeling was performed to assess their steady-state
amounts, as described previously (226). For pulse-chase experiments, cells were pulsed
with the [3H]glycerol for 2 h, after which the media was removed and replaced with
media containing an excess of unlabeled substrate (250 uM glycerol) and collected after
1, 2 and 4 h. Cells were washed twice with ice-cold PBS and collected in 300 ul of PBS,
where 50 ul was used for measurements of protein concentration and luciferase
luminescence. Total lipids were extracted by the method of Bligh and Dyer (21), where
the water-soluble ethanolamine intermediates of the PE-Kennedy pathway remained in
the aqueous phase and PE was in the organic (chloroform) phase. The radiolabeled
ethanolamine, phosphoethanolamine, and CDP-ethanolamine in the aqueous phase were
separated in by TLC in a solvent system of methanol/0.5% NaCl/ammonia (50:50:5,
v/v/v) and analyzed as described previously (54). Total ethanolamine incorporation into
PE
was
determined
after
the
separation
of
PE
in
a
TLC
system
of
chloroform/methanol/acetic acid/water (25:15:4:2, v/v/v/v). Spots corresponding to PE
standard were scraped and subjected to liquid scintillation analysis. For the determination
of neutral lipid fractions after radiolabeling with [ H]glycerol, lipids were separated in a
solvent system of heptane/isopropyl ether/acetic acid (60:40:3, v/v/v), and radioactivity in
total phospholipids, DAG and TG determined by liquid scintillation counting.
109
For [3H]acetate radiolabeling (4 h), the chloroform phase was saponified with 200
ul of ethanolic 0.5 M NaOH for 3 h at 70°C and 200 ul of water was added after cooling.
The non-saponifiable fraction (mainly cholesterol) was extracted three times with 500 |xl
of petroleum ether and radioactivity determined by liquid scintillation counting. The
saponifiable fraction (phospholipids, DAG and TG) was acidified with 300 ul of 6 N
HC1, extracted three times with 500 ul of petroleum ether, evaporated to dryness,
resuspended in a constant volume of chloroform (200 ul) and radioactivity determined by
liquid scintillation counting.
5.3.5 Pcyt2-H244Y-myc cloning.
Pcjtf2-H244Y-myc cloning. A mutation in the second putative active site
conferring a Tyr at position 244 in place of a His (H244YIGH) was introduced into the
mouse Pcyt2a-myc/His clone, previously generated (178). Overlapping PCR was utilized
to alter the His to Try (cac—>tat). The first reaction used a forward primer also containing
an 5' EcoRV site (primer A: 5'-gatatcgccgccaggatttgcggg-3') and a mutant reverse
primer (primer B: 5'- ccacgtgcccgatatagaacaggtcaaagg-3'), which yielded a 752 bp
fragment containing the mutation (shown in bold). The second reaction used a forward
primer complementary to mutant primer B (primer C: 5'- cctttgacctgttctatatcgggcacgtgg3') and a more downstream reverse primer containing an 3' Xhol site (primer D: 5'ctegaggtcaatctcccctccagg-3'), which yielded a 498 bp fragment. The 752 and 498 bp
amplified PCR products were then combined and used as template for a third PCR
reaction with the most external primers A and D to yield a single 1250 bp product
corresponding to the full length Pcytla cDNA. The amplified fragment was cloned into a
PCR vector, pGEM-T Easy vector (Promega), isolated by digestion with EcoRV and
110
Xhol and then subcloned into the pcDNA4/myc-His mammalian vector (Invitrogen) to
yield the clone Pcytl-H244Y-myc. Sequencing of Pcy/2-H244Y-myc ensured that the
correct mutation was present and that no other errors were introduced by PCR.
5.3.6 Pcyt2-H244Y-myc characterization in COS-7 cells.
COS-7 cells grown on 6-well plates were transfected with 2.5 ug of plasmid DNA
pcDNA4/myc-His (empty control), Pcyt2-myc (wildtype) or Pcy/2-H244Y-myc (mutant)
using 10 \xl of Lipofectamine (Invitrogen). 48 h post-transfection, the cells were
incubated for 1 h with [14C]ethanolamine (0.2 uCi/well) in the presence of 50 uM
unlabeled ethanolamine. Radiolabeled cells were washed twice in ice-cold PBS and
collected by trypsinization. PE and water-soluble intermediates were extracted and
radioactivity determined as described above for hepatocytes. ET enzyme activity after
/>c>t2-H244Y-myc transfection was determined by measuring the conversion of
[l4C]phosphoethanolamine to [14C]CDP-ethanolamine in vitro as previously described
(54).
5.3.7 mRNA expression
Pcyt2 heterozygous hepatocytes were isolated and 48 h-transfected as described
above. RNA was extracted using Trizol, as per the manufactures instructions. cDNA
synthesis as well as semi-quantitative PCR were conducted as previously described (54),
where all PCR reactions were analyzed in the linear phase and using optimal cycle
conditions. All genes are expressed relative to P-Actin and normalized to non-transfected
heterozygous controls (primers and cycle conditions available upon request).
Ill
5.3.8 Statistical Analyses.
All data are reported as mean ± SEM, where significance was determined by
Student's t-test or by one-way ANOVA. For all radiolabeling experiments, specific
activity was adjusted to account for the amount of unlabeled substrate. Synthetic rates
were calculated by linear regression and the rates of degradation were assessed by linear
regression of the natural log (In) of the amount of product (nmol/mg). All P-values
reported describe the differences in parameters between heterozygous and wildtype
hepatocytes.
112
5.4 Results
5.4.1 Characterization of the Pcyt2-H244Y-myc mutant in COS-7 cells
ET is a unique cytidylyltransferase due to the presence of two putative catalytic
CTP- binding motifs (HYGH and HIGH in the N and C-terminal regions respectively).
We sought to evaluate the activity of a catalytic mutation of the second active site, as no
studies have described this previously and to use this mutant in our rescue experiment
with Pcyt2 heterozygous hepatocytes. After successful cloning and sequence verification
of the Pcyt2-H244Y-myc construct, we initially transiently transfected the mutant and
wildtype Pcyt2 plasmids into COS-7 cells to determine the consequence of the mutation
on ET activity. The expression of the wildtype and mutant Pcyt2 were verified by the
detection of the Myc epitope with both plasmids (Figure 5.1 A).
113
Pcyt2-H244Y-myc
WT
2a.
-r-
E
o
E
H i
Pcyt2-myc
HET + Pcyf2-H244Y-myc
P=0.05
•Hi
c
LU
a.
o
D £
«• 1.25-
TO
E 1.00"o
E 0.75c
P=0.01
*l
3
•B 0.50-
tS °- 2l_ E
£
O
0.25-I
o
ao
UJ o
c
~
°
1
0
p=o.oor
I
Figure 5.1. Characterization of the .Pcjrt2-H244Y-myc mutation. COS-7 cells
transfected for 48 h with Pcyt2-myc or Pcyt2-H244Y-myc
and radiolabeled with
[l4C]ethanolamine for 1 h. A) Anti-myc immunoblot demonstrating expression oiPcytlmyc and Pcyt2-H244Y-myc tagged proteins. B) Amount of end product [14C]PE, C)
substrate [14C]phosphoethanolamine and D) product [14CJCDP-ethanolamine after
lh
incubation (data are expressed as nmol/mg prot). E) After 48 h expression, total cell
lysate ET activity was assessed and expressed as nmol/mg prot/min. All experiments
were performed in triplicate and repeated at least 3 times. P-values are by Student's t-test.
114
We show that the in vitro enzyme activity of Pcyf2-H244Y-myc was 60% of the wildtype
protein (Figure 5.IB). This was further corroborated by the in vivo study with
[14C]ethanolamine (Figure 5.1C-E). Pcyt2-H244Y-myc
expressing cells had a 40%
increased level of [14C]phosphoethanolamine (ET substrate), a 25% decreased amount of
[14C]CDP-ethanolamine (ET product) and a 15% lower amount of [14C]PE formed after 2
h of labeling. This demonstrated that the mutation H244Y within the second catalytic
motif reduced the activity of ET and therefore decreased production of PE via the
Kennedy pathway.
5.4.2 Expression ofPcyt2-myc but not Pcyt2-H244Y-myc normalizes PE metabolism
Reduced PE synthesis in Pcytl heterozygous mice resulted in no changes in PC,
but caused liver TG accumulation and elevated DAG utilization for TG synthesis,
metabolically coupling the PE-Kennedy pathway with TG synthesis (55). We aimed to
demonstrate that the accumulation of DAG and TG is the direct consequence of Pcyt2
disruption by re-introducing Pcyt2-myc or •Pcyf2-H244Y-myc into heterozygous primary
hepatocytes to elevate ET activity and therefore repair the PE-Kennedy pathway.
Successful transfections and normalized expression of both Pcyt2 constructs were
maintained by simultaneously monitoring luciferase activity from co-transfected CMVRenilla luciferase vector and ET levels by Western blotting (Figure 5.2A).
115
I
IWT
HET
Anti-Myc
1 2
EE3 HET + Pcy/2-myc
B
3
4
5
HET + Pcyf2-H244Y-myc
rC]PE
0.75
Synthesis
!~1
50kDa!
0.50-
J,
l l
Anti-ET
50kDa I
0.00
r^S
r4->
rX,
111 '-'-• H I
i l l 111 I
Time (h)
0.25
I
0.20 H
o
0.15H
C]Phos phoethanolam ine
f14
D
C]CPP-Ethanolamine
0.10
—
O
0.054
0.00
Time (h)
Time (h)
f*H]PE Synthesis
0.45
[•*H]PE Degradation
!
IU
Figure 5.2. Pcyt2-myc expression rescues PE metabolism in Pcyt2,+/-' hepatocytes
Primary hepatocytes were transfected for 48 h and pulse labeled with [rI4,CJethanolamine.
A) Immunoblot demonstrating expression in primary hepatocytes using anti-myc (above)
and anti-Pcyt2 (below) antibodies. Lane 1; non-transfected Pcyt2+/~, lanes 2 and 4; Pcyt2H244Y-myc and lanes 3 and 5; Pcyt2-myc. B) The incorporation of [14C] into PE, C)
phosphoethanolamine and D) CDP-ethanolamine. E) Hepatocytes were also pulse labeled
with [ Hjglycerol to measure the incorporation of [ H] into PE (rate of synthesis) or F)
pulse-chased to measure the rate of PE degradation. All data are expressed as nmol/mg
prot. Wildtype livers (n=2) and heterozygous livers (n=4 transfected/4 non-transfected),
where each time point was performed in triplicate. * represents at least P<0.05 by oneway ANOVA.
116
Non-transfected cells served as a negative control for the Pcyt2-myc tag (with the antiMyc antibody) transfections and for the amount of introduced ET protein into
heterozygous hepatocytes (probed with the anti-ET antibody). Similar levels of
expression between the wildtype and mutant constructs and approximately 2-fold over
non-transfected hepatocytes were consistently observed (Figure 5.2A). The established
expression levels of ET were then used for the metabolic radiolabeling experiments,
described in the next sections.
5.4.3 Radiolabeling of the PE-Kennedy Pathway
Pulse-labeling experiments with [14C]ethanolamine revealed that after expression
of Pcyt2-myc PE synthesis via the PE-Kennedy pathway was normalized. This was
evidenced by the increased rate of [14C]PE production (22%), by a 36% decrease in
[14C]phosphoethanolamine and a 21% increase in [14C]CDP-ethanolamine production
(Figure 5.2B-D). A 2-fold expression of the Pcyt2-H244Y-myc was also able to increase
the rate of PE synthesis in untransfected heterozygous hepatocytes (15%), which
demonstrates a dosage effect as the rate was not completely normalized to wildtype
levels. Therefore we concluded that the wildtype Pcyt2-myc and Pcyt2-H244Y-my c were
able to restore the PE synthesis in a dose-dependent manner in deficient hepatocytes. To
test if expression would also impact on PE turnover, we performed pulse and pulse-chase
experiments with [3H]glycerol. [3H]PE synthesis directly mimicked that of [14C]PE
synthesis (Figure 5.2E). Furthermore, data show that the rate of [ H]PE degradation in
deficient cells expressing Pcyt2-myc was increased and was completely normalized
compared to untransfected heterozygous hepatocytes (P<0.05) (Figure 5.2F). This
117
demonstrated that not only PE synthesis, but also PE degradation could be rescued with
Pcyt2-myc in Pcyt2 heterozygotes.
5.4.4 DAG and TG synthesis is rescued with Pcyt2-myc expression
We proposed that the limited supply of CDP-ethanolamine decreases the
formation of PE, which causes an increased utilization of DAG for TG synthesis in Pcyt2
heterozygous animals (54). Here we tested this hypothesis more directly and demonstrate
that upon Pcyt2-myc expression, there was an attenuation in the formation of both DAG
and TG to levels of wildtype cells, as assessed by [3H]glycerol pulse labeling (Figure
5.3A and B).
118
I
IWT
HET
J
1.25-t
[ H]DAG Synthesis
CZ3 HET + Pcyt2-myc
B
8-
•
HET+ Pcyf2-H244Y-myc
3
J HJTG_Syiithesis
~ e-
I 7-
s
f
I
I 0,5«
0.2SI
3 5-
I 4-
2
Time (h)
4
3
0,8-r
6
H]DAG Degradation
D
2,5
^HjTG Degradation
S 2.0
0,6!
E 0,4-1
3
£
!
1.0
5- 0.5
Q 0.24
H.
0.0
2
Tlme{h)
4
Figure 5.3. DAG and TG metabolism is normalized with expression. Primary
hepatocytes were transfected for 48 h and labeled with [3H]glycerol. A) Pulse labeling to
measure the incorporation of [3H]glycerol into DAG and C) TAG. Pulse-chase labeling
with [3H]glycerol to measure the degradation of [3H] from D) DAG and E) TG. All data
are expressed as nmol/mg prot. Wildtype livers (n=2) and heterozygous livers (n=4
transfected/4 non-transfected), where each time point was performed in triplicate. *
represents at least P<0.05 by one-way ANOVA.
As was the case for the formation of PE, expression of the Pcyt2-H244Y-myc mutant was
only partially able to normalize the formation of DAG (30%; P<0.05) and had no effect
on TG. The degradation of DAG that was high in untransfected Pcyt2+/~ hepatocytes was
reduced almost 2-fold with Pcyt2-myc expression (Figure 5.3C). TG degradation was
unaltered in any of the cells compared to wildtype (Figure 5.3D) as expected since TG
degradation was not affected by Pcyt2 deletion (55).
119
Since Pcyt2-myc expression resulted in alterations in both DAG and TG, we
further examined their steady-state levels after 24 h of radiolabeling with [3H]glycerol.
As expected, Pcyt2-myc expression decreased the DAG levels compared to untransfected
heterozygous controls (0.87 ± 0.09 vs. 1.17. ± 0.12 nmol/mg prot; P<0.05), and was
similar to wildtype levels (0.81 ± 0.08 nmol/mg prot) (Figure 5.4A).
WT
Ifei HET + Pcyt2-myc
KIHET
B
I
(nmoi
i
t>
~r
6T
4-
CD 2m
0-
1
•
T
I11
V
1
Figure 5.4. Pcyt2-myc rescue decreases elevated neutral lipids. 48 h transient
transfection and 24 h [3H]glycerol labeling of primary hepatocytes. A) Total cellular
DAG and B) total cellular TG. Data are expressed as nmol/mg prot, where wildtype (n=2)
and heterozygous (n=4 transfected/ 4 non-transfected), performed in triplicate. *
represents at least P<0.05 by one-way ANOVA.
TG content was lowered in Pcyt2-myc transfected cells compared to untransfected
heterozygous cells (5.59 ± 0.29 vs. 6.47 ± 0.46 nmol/mg prot; P<0.13), to an amount that
was closer to wildtype cells (4.82 ± 0.35 nmol/mg prot) (Figure 4b). There was no
difference in the steady-state amount of PE measured in any of the treatments, consistent
with our previously established mechanism, where PE homeostasis is maintained by
reduced PE degradation. For a complete summary, tables 5.1 and 5.2 describe the rates of
120
formation and degradation assessed by linear regression analyses for all of the
radiolabeling experiments.
Table 5.1. Rates of synthesis for pulse labeling (1-4 h) of primary hepatocytes
Product
(Radiolabel)
WT
HET
Pcyt2-myca
Pcyt2-H244Y-myc
[14C] PE (Eth) b
0.23 ± 0.04 c
0.14 ±0.04
0.21 ±0.04*
0.19 ±0.05*
[ 3 H]PE(Gly)
0.23 ±0.06
0.16 ±0.04
0.23 ± 0.07*
0.18 ±0.03*
[ 3 H]DAG(Gly)
0.11 ±0.05
0.29 ±0.10
0.12 ±0.04**
0.17 ±0.05*
[ 3 H]TG(Gly)
0.18 ±0.05
0.23 ± 0.04
0.16 ±0.07**
0.22 ± 0.06
[3H] DAG (Ace)
0.09 ±0.02
0.22 ±0.04
0.12 ±0.04*
na
[3H]TG(Ace)
0.33 ± 0.06
0.45 ± 0.05
0.32 ±0.05*
na
a
Pcyt2-myc and Pcy/2-H244Y-myc transfections were in heterozygous hepatocytes.
b
Abbreviations: Eth; ethanolamine, Gly; glycerol, Ace; acetate.
c
Rates are expressed as nmol/mg prot/h, and calculated from linear regression of the In
transformed concentration data. Wildtype livers (n=2) and heterozygous livers (n=4
transfected/4 non-transfected), where each time point was performed in triplicate. * and
** represent significance compared to untransfected HET, P<0.05 and PO.001
respectively.
121
Table 5.2. Rates of degradation after pulse-chase labeling in primary hepatocytes.
Product
(Radiolabel)
WT
HET
Pcyt2-myca
[ 3 H]PE(Gly) b
0.29±0.08 c
0.14 ±0.06
0.26 ±0.07*
[ 3 H]DAG(Gly)
0.12 ±0.03
0.24 ±0.05
0.13 ±0.07*
[ 3 H]TG(Gly)
0.081 ±0.02
0.079 ±0.04
0.074 ±0.04
a
Pcyt2-myc and Pcyt2-H244Y-myc transfections were in heterozygous hepatocytes.
b
Abbreviations: Gly; glycerol.
c
Rates are expressed as nmol/mg prot/h, and calculated from linear regression of the In
transformed concentration data. Wildtype livers (n=2) and heterozygous livers (n=4
transfected/4 non-transfected), where each time point was performed in triplicate. *
represent significance compared to untransfected HET (P<0.05).
5.4.5 De novo fatty acid synthesis is reduced with Pcyt2-myc rescue
We used [3H] acetate to measure de novo fatty acid synthesis and incorporation
into DAG and TG in isolated hepatocytes. With in vivo animal studies we observed
increased incorporation into DAG and TG but not total phospholipids (55). Here, there
were also no differences in the incorporation of acetate into total phospholipids in
transfected and untransfected hepatocytes; however, there was reduced FA incorporation
into DAG and TG in transfected heterozygous cells compared to increased levels in
untransfected heterozygous control cells (Figure 5.5A and B). We assessed the
incorporation of [3H]acetate into saponifiable (total glycerolipids) and non-saponifiable
(mainly cholesterol) fractions and demonstrate that the expression of Pcyt2 in
heterozygous hepatocytes corrected the levels in both fractions (Figure 5.5C). These data
corroborate [ H] glycerol labeling experiments and also demonstrate an increased rate of
122
formation of both DAG and TG in Pcyt2 heterozygous hepatocytes, which is reduced
upon Pcyt2 expression.
WT
HET E B HET+ Pcyf2-myc
[3H] Acetate into DAG
1
2
4
Time (h)
r3
B
H]Acetate into TG
1.00
2 0.75
a.
E
"o
E
e
0.50
0.25
0.00
si
!
i 1
• i
sg
T
"T"
i
o a.
H o.
i
a?
phQAoetate into total lipids
__i 1 i
o
4.5-,
4.03.53,02.52.01.51.00.50.0-
i
Non-Saponifiable
1
-
Saponif iable
•>+/-
Figure 5.5. Increased lipogenesis in P c j ^ " hepatocytes is corrected by Pcyt2-myc
expression. The incorporation of [3H]acetate into A) DAG and B) TG as an indication of
de novo lipogenesis in primary hepatocytes after 48 h transfection. C) Total [ H]acetate
incorporation
into glycerolipid
(saponifiable)
123
and cholesterol
(non-saponifiable)
fractions. All data are expressed as nmol/mg prot, where wildtype (n=2) and
heterozygous (n=4 transfected/ 4 non-transfected), performed in triplicate. * represents at
least P<0.05 by one-way ANOVA.
5.4.6 The activity of wildtype and Pcyt2-H244Y mutant is not additive
Characterization of the Pcyt2-H244Y-myc construct revealed that mutation within
the second putative catalytic motif conferred a reduced enzymatic activity cells (Figure
5.1). To investigate this mutation further, we co-transfected equal amounts of the Pcyt2myc and the Pcy/2-H244Y-myc mutant into heterozygous hepatocytes and compared the
incorporation of [3H]glycerol into PE after 2 h, relative to untransfected hepatocytes. The
results in Figure 5.6 demonstrate that co-transfection of the mutant had no effect on the
ability of Pcyt2-myc to rescue the synthesis of PE in heterozygous hepatocytes and there
was no additive increase in PE synthesis. Therefore results suggest that co-expression of
wildtype and mutant Pcyt2 clones resulted in normal PE formation, as seen in separate
experiments with strictly wildtype Pcyt2.
124
CZ1 WT
M HET
S ^ HET + Pcytf-H244Y-myc + Pcytf-myc
JL
*—*
,1
2
a.
1
"o
0.50-
JL
X
E
5T
CO
I
1
0.25-
LU
I
0.00-
1
1
2
4
IM
Time(h)
Figure 5.6. The active site mutant Pcyt2-H244Y-myc is not dominant negative.
Primary hepatocytes were co-transfected with both wildtype Pcyt2-myc and the mutant
Pcyt2-H244Y-myc for 48 h and pulse labeled with [3H]glycerol. The incorporation of
labeled glycerol into PE was measured and Pcyt2-H244Y-myc did not interfere with the
ability of Pcyt2-myc to rescue PE synthesis. Data are expressed as nmol/mg prot, where
wildtype (n=2) and heterozygous (n=4 transfected/ 4 non-transfected), performed in
triplicate. * represents at least P<0.05 by one-way ANOVA.
5.4.7 Pcyt2-myc rescues lipogenic gene expression in Pcyt2 heterozygous hepatocytes
We had previously reported that Pcyt2+/~ mice experienced an increased mRNA
expression of various important lipogenic genes (55). The expression of Pcyt2-myc in
heterozygous hepatocytes lowers the expression of sterol regulatory element binding
protein- 1c (Srebp-lc), fatty acid synthase (Fas) and diacylglycerol acyltransferase 1 and
2 (Dgatl/2), with no alterations in peroxisome proliferator activated receptor co-activator
la (Figure 7).
125
HET + Pcyf2-myc
HET
HET + Pcyt2-rryc
HET
1.5
11111
I I II I I I I
SREBP-1c
FAS
DGAT1
DGAT2
PGC-1a
Figure 5.7. mRNA expressions of lipogenic genes are affected by Pcyt2 rescue. A)
Representative summary of the mRNA expression for lipogenic genes up-regulated in
Pcyt2+/~ mice, in non-transfected control heterozygous and Pcyt2-myc transfected
primary hepatocytes. B) mRNA expression expressed as arbitrary units of density, where
mRNA expression was normalized to 0-Actin and then expressed relative to nontransfected control heterozygous hepatocytes (n=2 livers, where each treatment was
conducted in quadruplicate and the analyses conducted at least twice). * represents
P<0.05 as determined by student's t-test.
Therefore, it was concluded that the disruption of Pcyt2 is responsible for the increased
mRNA expressions of these genes in heterozygous livers.
126
5.5 Discussion
In the current investigation we demonstrate that a redirection of DAG away from
PE synthesis and toward TG formation is directly responsible for the fatty liver
phenotype observed in Pcyt2+/~ mice (55). Our previous work had shown the metabolic
evidence for this conclusion; however, the role of Pcyt2 deletion in the accumulation of
DAG and TG in Pcyt2+/~ mice might still have been considered indirect. Here we
endogenously added Pcyt2 by expressing a Pcy/2-tagged construct into heterozygous
hepatocytes to demonstrate that Pcyt2 genetic deficiency is directly responsible for the
elevated DAG, TG and ultimately development of liver steatosis in Pcyt2+/~ mice.
Inherently, genetic knockout models are loss-of-function experiments, and
although powerful insight into complex biological systems may be revealed using these
models, conclusions may be tenuous. Numerous investigations have employed the use of
genetic reconstitution, to reverse an observable phenotype through transient or stable
expression in primary cell culture, adenoviral expression or transgenic rescue (88, 138,
210). A mutant Chinese hamster ovary cell line (MT-58) possesses impaired PC synthesis
due to a mutation in CTP:phosphocholine cytidylyltransferase (CT) (47), the analogous
cytidylyltransferase to ET of the PE-Kennedy pathway. PC synthesis was rescued by
expression of the wildtype CT construct, where expression of a gene encoding an
alternate PC synthetic pathway (phosphatidylethanolamine 7V-methyltransferase) failed to
compensate (74, 206).
The relationship between phospholipid and TG synthesis has been noted in the
literature (27, 59, 79); however, mice deficient for genes involved in PC synthesis
(CTP:phosphocholine cytidylyltransferase and PE-JV methyltransferase) did not result in
obesity or direct perturbations in TG synthesis (188). It is also interesting that
127
diacylglycerol acyltransferase (DGAT) knockout mice have normal phospholipids (PE
and PC) (157), however DGAT over-expression in human lung fibroblasts caused a
reduction in phospholipid synthesis (PE and PC) (12). Disruption of mitochondrial
glycerol-3-phosphate acyltransferase (GPAT), the first committed step in glycerolipid
synthesis, resulted in an alteration in PE and PC fatty acid composition, but not content
(61). Finally, deletion of stearoyl-CoA desaturase 1, the rate-limiting enzyme in
monounsaturated fatty acid synthesis and critical component in TG synthesis, resulted in
increases in PC and PE content by 40 and 23% respectively (39).
In Pcyt2 deficient hepatocytes, ET is limiting the flux through the PE-Kennedy
pathway, resulting in a build up of the phosphoethanolamine (substrate) and a decrease in
CDP-ethanolamine (product) (54). Expression of Pcyt2-myc increases the activity of ET
and normalizes the pathway intermediates and corrects PE homeostasis. Pcyt2-myc
expression also rescues the concomitant changes in DAG and TG. This demonstrates that
with expression, the increased availability of CDP-ethanolamine increases the DAG
incorporation into PE synthesis and was sufficient normalize TG synthesis in
heterozygous hepatocytes.
As well as an increased rate of incorporation into TG in Pcyt2+/~ hepatocytes, an
increase in the incorporation of [3H]glycerol into DAG suggested an increased synthesis
of DAG. Our previous investigations did not show any increase in mitochondrial
glycerol-3-phosphate acyltransferase or phosphatidic acid phosphatase (lipin-1) mRNA
expression. It is therefore unknown how a decrease in PE synthesis would result in an
increase in the synthesis of DAG in Pcyt2+/~ animals. We clearly establish that DAG
destined for PE formation was redirected from PE to TG in heterozygous hepatocytes.
The hepatic and skeletal muscle mRNA expression profile of heterozygous mice display
128
an altered expression of genes involved in glucose metabolism (glucokinase, glycerol-3phosphate
dehydrogenase
and
glucose-6-phosphatase)
(Fullerton
and
Bakovic
unpublished results). Perhaps this leads not only to increased FA synthesis but also
increased production of glycerol-3-phosphate, which is the initial substrate in the DAG
and glycerolipid synthetic pathways (29).
However, there is also an inherent defect in energy substrate metabolism in
Pcyt2+/~ mice stemming from reduced FA oxidation and increased FA formation from
glucose (de novo lipogenesis); therefore it is not surprising that DAG synthesis was also
up-regulated since required to accommodate the newly made FA and to store them in the
form of both DAG and TG. Total TG content was not significantly reduced after 48 h of
Pcyt2-myc expression even though the rate of TG synthesis was decreased compared to
untransfected heterozygous hepatocytes, likely because of transient transfection
expression. As there was no change in lipolysis in transfected hepatocytes and given a
slower rate of synthesis, perhaps total TG content would be reduced to wildtype levels in
stable-transfected Pcyt2-myc expressing hepatocytes and/or a longer transient incubation
period.
In addition to the elevated DAG, the increased formation and accumulation of TG
in Pcyt2+/~ mice is facilitated by an increase in de novo fatty acid synthesis. Importantly,
just as expression of Pcyt2-myc decreased the DAG content, the increases in FA
synthesis associated with the heterozygous phenotype was also normalized in hepatocytes
expressing Pcy/2-myc. mRNA expression analyses revealed lower expressions of the
main lipogenic genes increased in Pcyt2+' livers. Although the mechanism by which
Pcyt2 may influence the expression of Srebp-lc is not known, in heterozygous
hepatocytes, our results indicate a reciprocal relationship whereby it may be possible that
129
Srebp-lc expression would be increased in response to the metabolic consequences of
Pcyt2 disruption, hence increasing transcription of Fas. In addition, we show that the
functional expression of Pcyt2 has a direct effect on the expression of Dgatl and Dgat2,
which may facilitate the corrections in DAG utilization and TG formation.
Pcyt2 encodes a distinctive cytidylyltransferase, where unlike other family
members, it possesses two putative CTP catalytic sites, beginning at residues 35 (HYGH)
and 244 (HIGH). The first site is situated in the N-terminal region, as in most
cytidylyltransferases. The focus here was on the significance of the second binding site
and was addressed by introducing a Tyr at amino acid position 244 in substitution of a
His. The Tyr containing mutant had 40% lower ET enzyme activity and approximately
20% reduced flux through the PE-Kennedy pathway relative to the wildtype enzyme
suggesting the importance of the His residue as well as the second active site (HIGH) in
the catalytic function of ET.
Heterozygous disruption of the Pcyt2 gene causes an array of consequences,
including obesity and insulin resistance. It is well known that the accumulation of DAG
has a negative effect on insulin signaling (222); therefore in Pcyt2+/~ mice, the reduction
of DAG with Pcy/2-myc expression may improve insulin signaling in vivo. To establish if
reconstitution of Pcyt2 in the deficient mice would normalize lipid metabolism and
insulin sensitivity, we attempted to generate Pcyt2 transgenic mice; however, we were
unsuccessful in two attempts. As we could not rescue Pcyt2~/~ in vivo, expression in
isolated hepatocytes of heterozygous mice was the best alternative .
The phenotype of Pcyt2+/~ animals stems from inborn reduction in PE synthesis
and homeostatic responses in PE, FA and DAG, which results in chronic ectopic
accumulation of TG in liver and skeletal muscle. As well this results in increased
130
adiposity, leading towards diminished sensitivity to insulin (55). The results of the
current investigation prove for the first time the existence of a direct functional coupling
between de novo PE synthesis and TG accumulation. We demonstrate that DAG and TG
related lipid disorders could originate from impairments in membrane phospholipid
homeostasis regulated by the PE-Kennedy pathway, where functional restoration by
Pcyt2 also rescues the lipid metabolic phenotype.
131
CHAPTER SIX
GENERAL DISCUSSION
132
6.1 Discussion
PE is an integral component of cellular membranes and plays many structural and
non-structural roles. PE production is facilitated by de novo synthesis via the PEKennedy pathway, the decarboxylation of PS in the mitochondria and to a lesser extent,
calcium dependent base-exchange mechanisms. Although the synthetic pathways are well
documented, knowledge into the regulation and overall contribution, as well as individual
components within each pathway has only recently been gained. The discoveries of
phospholipid synthetic pathways led to their investigation in yeast and in vitro cell
culture models and laid the foundation upon which our current knowledge rests. The use
of genetically engineered mice have allowed for whole animal, in vivo analyses of the
genes involved in phospholipid metabolic pathways.
6.1.1 PE Synthesis
Eugene Kennedy and colleagues first described the analogous synthetic pathways
of PE and PC (89). The abundance and importance of PC within mammalian cells drove
the examination of PC biosynthesis, such that the kinetics and regulation of the pathway
were documented in great detail, well before the PE-Kennedy pathway. The first step of
each branch of the pathway is the phosphorylation of ethanolamine or choline by various
specific and dual-role kinases. The final conversion to PE or PC is also completed by an
enzyme conferring single or dual-specificity. Although this may suggest a similar or
over-lapping control, the respective cytidylyltransferases are quite unique both in
structure and regulation. CT is the rate-determining enzyme in the PC-Kennedy pathway
and is reversibly translocated from the cytosol to membranes for activation (196). On the
contrary, ET has only been shown to be rate-limiting under certain conditions (170),
133
whereby DAG availability may also govern flux through the pathway (181). ET has
mainly been shown to be located in the cytosol, although recent work in our lab suggests
co-localization with nuclear and microsomal fractions (Tie 2009, unpublished results).
Perhaps the most striking contrast between these analogous proteins is their gene and
protein structure (Figure 6.1).
ET_
CTaCT(5-
MIRNGHGAASAAGLKGP
IDAQSSAKVNSRKRRKEAPGPNGATEEDGIPSKVQRCAVGLRQPAPFSDEIEVDFSKPYVRVTMEEACRGT
§PVLTTDAESETGIPKSLSNEPPSETMEEXEHTCPQPRLTLTAPAPFADESSCQCQAPHEKLTVAQARLGT
ET- GDQRIVRVWCDGCYDMVgY ggSNQLRQARAM--GDYLIVGVHTDEEIAKHKGPPVFTQEERYKMVQAIKWV
CTa- PCERPVRVYADGIFDLFSS eSARALMQAKNLFPNTYLIVGVCSDELTHNFKGFTVMNENERYDAVQHCRYV
CT|3- PVDRPVRVYADGIFDL: !S^ARALMQAKTLFPNSYLLVGVCSDDLTHKFKGFTVMNEAERYEALRHCRYV
ETCTaCTP-
DEWPAAE'YVTTLETiDKHNCDFCVHGNDITLTVDGRDTYEEVKQAGRYRECKRTQGVSTTDLVGRMLLVT
DEWRNAPHTLTPEFLAEHRIDFVAHDDIPYSSAGSDDVYKHIKDAGMFAPTQRTEGISTSDIITRIVRDY
DEVIRDAPWTIiTPEFLEKHK'IDFVAHDDIPYSSAGSDDVYKHIKEAGMFVPTQRTEGISTSDIITRIVRDY
E T - KAHHSSQEMSSEYREYADSFGI PPHPTPAGDTLSSEVSSC gCPGGQSPWTGVSQFLQTSQKIIQFASGKEPQ
.-"••..
'• . ; . - ' . ".
J2ERVDKVKKKVKDVEEKSKEFVQKVEEKSIDLIQKWEEKSRE
T B ' • ":•
::: . '
" '.•:"
••'•'
"QNQVDKMKEKVKNVEERSKEFVNRVEEKSHDLIQKWEEKSRE
CTaC
ETCTaCTP-
PGETVIYVAGAFDLFgjlgJjVDFLQEVHKLAKRPYVIAGLHFDQEVNRYKGKNYPIMNLHERTLSVLACRYV
FIGSFLiMFGPEGALKHMLKiGKGRMLQAlSPK^PSSSWHERSPSPS
FJGNFfcELFGPDGAWKQMFQERSSRMIiQALjSPKQSPVSSPTRSRSFSRSPSPTFSWLPNKTSPPSSPKAAS
E T - SEWIGAPYSVTAELLNHFKVDLVCHGKTEIVPDRDGSDPYQEPKRRGIFYQIDSGSDLTTDLIVQRIIKN
C T a - |vTCDliSEDEED
C T P - |siSSMSEGDEDEK
ETCTaCTP-
RLEYEARNQKKEAKELAFLEATKQQEAPPGGEID
Figure 6.1. Protein sequence alignment for ET and CTa and CTp. Active site motifs
(HXGH) as well as the 'linker' peptide corresponding to exon 7 (not present in ET13) are
shaded in black.
There are two separate genes encoding CT, Pcytla and Pcytlfi, where the P isoform
possesses two splice variants and the CT protein possesses one putative CTP binding
domain. In contrast, Pcytl is a single gene that is alternatively spliced at exon 7 to
produce two functional variants, both variants containing a duplicate active site motif
134
(HXGH). ET is the only cytidylyltransferase that contains two putative active site motifs
(refer to figure 6.1), and although Ala mutagenesis has yet to specifically determine the
importance of each domain, it was determined in this study that a relatively passive
mutation of the His to a Tyr in the second active site confers a diminished ET activity.
In addition to in vitro structural and functional assessments of the ET protein, the
question remained as to the importance of both Pcyt2 as well as the PE-Kennedy pathway
for PE biosynthesis. The first insight into pathway requirements in vivo stemmed from
the disruption of the mouse Pisd gene by Steenbergen and colleagues (161). Pisd' mice
were lethal in utero, most likely due to a lack of mitochondrial PE leading to
abnormalities, where as Pisdt'' had a compensatory increase in the PE-Kennedy pathway
and were phenotypically indistinguishable from littermate controls. This result
demonstrated the necessity for the mitochondrial production of PE via decarboxylation as
well as a key role of Pcytl and the de novo pathway. What remained to be addressed was
the role that Pcytl played in PE and overall phospholipid metabolism.
We sought to address this question head on, by disrupting Pcyt2 in mice. The
necessity for Pcyt2 was demonstrated by the early embryonal death of Pcyt2~/~ embryos
prior to day 8 of embryonic development. Identification of partially resorbed embryos at
that stage implies that additional pathways were able to compensate for the production of
PE, such that implantation could occur. However since these embryos do not survive, a
threshold, albeit very early in development, must exist after which there are insufficient
levels of PE necessary for homeostasis. The adaptation of cultured baby hamster kidney
cells to low levels of ethanolamine and reliance on PS decarboxylation for PE seen by
Voelker (203) may indeed hold true; however, these results may not translate to in vivo
135
mammalian systems. Pcyt2+/~ mice, as were the Pisct "mice, are phenorypically normal
compared to littermate controls during the first months of post-natal development.
We considered that Pisd may have been increased in Pcyt2+', as many genetically
modified mice with alterations in phospholipid related genes experience compensation
from alternative pathways. In the Pemt'1' mouse, levels of PC were maintained by
increased expression of CTa in the liver (207). In a tissue specific manner, PSS1 and CT|3
are reciprocally up-regulated in Pss2 null mice (162) and Pcytla heterozygous embryos
respectively (209). The results indicate that in Pcyt2+' mice, the production of PE was
not facilitated by a transcriptional up-regulation of Pisd. In vitro and in vivo radiolabeling
with [3H] serine did not indicate any discrepancy between genotypes, also supporting the
conclusion that PS decarboxylation is not significantly altered in Pcyt2 heterozygotes.
This conclusion, however, does not take into consideration i) that [3H] serine can be
converted to phosphoethanolamine via sphingomyelin metabolism, ii) the in vitro activity
of PSD (not measured) or iii) the incorporation of serine into DAG or acyl-CoAs, all of
which could result in [ H]PE synthesis (189, 197). A recent investigation identified that
less than ~2% of labeled serine is converted to phosphoethanolamine in rat hepatoma
cells (19) and investigation into the over-expression of EKI, strictly monitored the
incorporation of the [ H] label from serine into total PE (109). Although there is support
in the literature for our measurements of PS decarboxylation, these phenomenon have the
potential to alter the interpretation of the [3H] serine labeling and therefore future studies
are warranted.
PE levels were maintained by decreasing the rate of PE degradation, a
phenomenon also documented in Pss2 null animals. It is of interest to note that Pssl'''
/Pss2+/~ or Pssl^'/PssZ'- mice were viable in spite of a -90% reduction in PSS activity,
136
resulting in a 40 and 30% decrease in PS and PE levels respectively (9). On top of
highlighting the necessity for PSS activity, it also demonstrates that fluctuations in PE
and PS levels are tolerated in certain systems. One may speculate that in Pcyt2+/~ mice, a
decreased rate of PE formation stemming from a single Pcyt2 allele results in a lower
total amount. In this instance, a threshold is reached whereby to maintain phospholipid
and most likely membrane homeostasis, a decreased rate of PE degradation is adopted.
Assessment of the total cellular PE content did not involve quantification of total
phosphorus, as is routinely performed (134, 162). We sought to measure the amount of
PE via densitometry, using visualized TLC plates and standard concentration curves (80).
The total hepatic PE content was verified by GC analyses and also revealed no changes
between genotype. The current studies also do not assess phospholipase activity, a likely
source of altered degradation; therefore, potential exists for further investigation.
6.1.2 DAG as a precursor for PE and TG
Central to the production of all glycerolipids is the incorporation of acyl-CoA and
in the case of phospholipids, a polar head group, onto a glycerol backbone. DAG serves
as a branch point for the de novo synthesis of the main group of phospholipids (PC, PE
and indirectly PS) and TG. Complex regulation governs the recycling and re-acylation of
acylglycerols after hydrolysis, as partially summarized in figure 6.2 (78); however, the
main focus of this thesis was an investigation into the de novo synthetic pathways.
137
endoplasmic reticulum
Figure 6.2. Depiction of acylglycerol recycling. Adapted from (78) where abbreviations
not in text: MAG; monoacylglycerol, PG; phosphatidylglycerol, PI; phosphatidylinositol.
The disruption of Pcyt2 resulted in embryonic lethality in null animals, which
restricted the analyses to a comparison of heterozygous and wildtype mice. In addition to
altered rates of PE synthesis and degradation, we unexpectedly observed a chronic and
subtle weight increase in the Pcyt2+/~ mice. At approximately 6 months of age, Pcyt2+'
mice on average weighed significantly more than littermate controls. We hypothesized
that a decreased flux through the PE-Kennedy pathway due to a decreased amount of
CDP-ethanolamine, caused a bottleneck in the pathway and a pooling of DAG fated for
PE synthesis (as described visually in Figure 1.1). Radiolabeling with [3H]glycerol in
isolated primary hepatocytes determined that the rate of glycerol incorporation into DAG
and subsequently TG, was increased in the Pcyt2+/~ cells, reflecting an increased DAG
138
synthesis as well as utilization (Figure 4.7). In vitro labeling with [ HJoleate and
[ HJacetate as well as an in vivo measure of lipogenesis with [ H]acetate, corroborate this
as a plausible mechanism. The strongest evidence in support of our hypothesis and that
links phospholipid (PE) metabolism to DAG incorporation into TG was the functional
expression of Pcyt2 back into Pcyt2+/~ hepatocytes. Transient transfections rescued the
rate of PE synthesis as well as degradation by increasing ET activity and availability of
CDP-ethanolamine. This compensated CDP-ethanolamine was then combined with DAG
at a rate which supplied sufficient amounts of PE to cellular membranes and such that an
altered rate of degradation (via unexplored mechanisms, most likely phospholipase
activity modulation) was not required. The normalization of PE metabolism restored the
rate of both DAG synthesis and incorporation into TG (TG synthesis) to wildtype levels.
Pcyt2+/~ mice experience an array of metabolic disturbances, some of which stem
from the genetic disruption, such as TG accumulation and an inherent decrease in FA
oxidation (at least in the liver), and some of which are a consequence of the former, such
as insulin insensitivity and chronic weight gain. Genetic reconstitution of Pcyt2 in
primary hepatocytes allowed for the determination of rates of formation and degradation
as well as total lipid amounts; however, lacking from the experimental design was how
this would chronically affect the mice. Would the expression of Pcyt2 in heterozygous
mice result in a leaner, more metabolically stable phenotype? To address this question
we attempted to generate Pcyt2 transgenic mice using a verified Pcyt2-myc transgene.
After the plasmid was linearized and purified, it was sent to Sick Kids Transgenic
Facility for micro-injection into embryos. We obtained two cohorts of pups (from two
separate attempts), both of which had several pups that were positive for the transgene at
the DNA level, indicating that successful incorporation had occurred. In both instances
139
however, the presence of DNA and in one instance, mRNA expression of the transgene
did not translate into expression at the protein level (See appendix I). However, if the
level of functional expression in a transgenic animal reached wildtype levels or higher,
we would expect that Pcyt2'/'/MYC+ mice would be viable and exhibit a phenotype similar
if not indistinguishable from wildtype controls across all stages of development.
Previous work has demonstrated a link between phospholipid and TG synthesis,
or simply the overall relationship of glycerolipids (27, 79, 174). In the current studies,
loss of Pcyt2 results in a disturbed metabolic phenotype. A similar end point was reached
by hepatic over-expression of mtGPAT by adenoviral transduction. Increased mtGPAT
resulted in a 7- and 12-fold increase in hepatic DAG and TG respectively, where
phospholipid synthesis was unchanged (105). As in Pcyt2+/~ mice, hepatic mtGPAT overexpression caused an increase in SCD-1, with subsequent increases in oleic acid (18:1) in
TG as well as a decrease in FA oxidation. A 2-fold increase in VLDL secretion was
observed in mtGPAT over-expressing livers where no significant increase was observed
in Pcyt2+/~ mice. Although results were quite parallel, the phenotype of Pcyt2+/' mice is
not as severe as seen with over-expression of mtGPAT. However, the mechanism is
common to both models.
We have shown evidence of increased TG synthesis in Pcyt2+/~ mice due to a
redirection of DAG. Reconciliation of this increased synthesis may be difficult for
various reasons. Firstly, DAG synthesis is increased in Pcyt2+/~ mice. A reduction of
CDP-ethanolamine causing a decreased synthesis of PE would be expected to underutilize DAG. However, one would expect a simple redirection of normally available
DAG toward another fate (TG synthesis), not increased DAG formation. Secondly,
although Pcyt2 disruption is ubiquitous, we specifically investigated this phenotype in the
140
liver. The major hepatic phospholipid is PC, with PE constituting approximately 25-30%
of total hepatic phospholipids (161). If there is a 30% reduction in the rate of PE
synthesis, which in itself makes up only 30% of total hepatic phospholipid, the altered
DAG would only be equivalent to - 9 % of total phospholipids, which is an even smaller
portion of the total glycerolipid pool. It is tempting to speculate that the insult to hepatic
glycerolipid metabolism, although minor, becomes chronically problematic, as evidenced
by a lack of observable phenotype in Pcyt2+/~ mice until ~28 weeks. We do not report
increased mRNA expression of mtGPAT, although we did not analyze the microsomal
isoforms, nor did we characterize the protein expression. Although specific GPAT
activity was not measured, our [ Hjglycerol labeling experiments suggest an increased
activity of either of the GPAT isoforms.
In addition to an increased rate of DAG synthesis and incorporation into TG, we
observed accumulation of DAG in liver as well as skeletal muscle. As there is most likely
a point at which the level of PE is essential for cellular homeostasis, the incorporation of
DAG (redirected from phospholipids and newly formed) into TG may also reach a
critical threshold. DAG esterification into TG is a positive adaptation, where by a
reactive lipid intermediate is sequestered into a safe location (TG) as seen in muscle
specific DGAT over-expression via adenovirus (3-fold) (106). It is possible that over
time, the endogenous up-regulation of DGAT isoforms in liver and skeletal muscle in
Pcyt2 heterozygotes, confirmed only indirectly with mRNA and [3H]glycerol labeling, is
unable to convert DAG to TG, leading to accumulation. It may also be possible that
certain pools of DAG, distinguished by their FA composition, would be destined for
selective fates (30). Further work is required to elucidate the intricacies of the proposed
mechanism; however, lipid accumulation in skeletal muscle illustrates that this phenotype
141
is not particular to the liver, and possibly specific to tissues with lipogenic capacities.
Taken together, these studies provide the first evidence to highlight the relationship
between alterations in PE synthesis, TG synthesis and ultimately accumulation.
6.1.3 Lipogenesis and FA metabolism in Pcyt2 Heterozygous mice
The objective of this work was to elucidate the role of Pcyt2 and PE in murine
development. Unexpectedly, heterozygous disruption of Pcyt2 resulted in an array of
metabolic consequences. It was observed that Pcyt2+/~ livers had increased FA uptake and
decreased FA oxidation in isolated hepatocytes and an increased rate of de novo
lipogenesis. Of the lipogenic disturbances described, some explanations are intuitive,
where others require speculation and warrant further investigation before conclusions can
be drawn.
For PE synthesis, the modification of DAG would normally be the covalent
attachment of a polar head group. However the formation of TG requires the
esterification of an additional acyl-CoA. We anticipated that for our "divergent DAG"
hypothesis to have merit there would need to be an increased supply of FA for the final
esterification catalyzed by DGAT. The uptake of [3H]oleate into isolated primary
hepatocytes was assessed and a 2-fold increase in the rate of uptake in Pcyt2+/' cells was
observed. As a means of investigating this further, total protein content of key FA
transporters were assessed and it was shown that there was no change between genotypes.
A limiting factor was the measurement of only total cellular content and not the subcellular distribution. As seen in recent studies, it is not always the total amount of
transport proteins (mainly FAT/CD36), rather their cellular distribution and presence at
the plasma membrane that is important (70).
142
An increase in the rate of FA uptake may indeed facilitate the final esterification
of redirected DAG. We also demonstrate that FA ([14H]oleate) oxidation is diminished in
Pcyt2+/~ hepatocytes and that de novo lipogenesis ([3H]acetate incorporation) is increased.
Acetyl-CoA carboxylase (ACQ catalyzes the formation of malonyl-CoA, which acts as a
crucial modulator of lipid metabolism in lipogenic tissues. Particularly in the liver, the
cytosolic ACC1 has been shown to supply malonyl-CoA to FAS, for the initiation of de
novo lipogenesis (3). The malonyl-CoA produced by the mitochondrial-associated ACC2
has been demonstrated to be the main pool of malonyl-CoA acting to allosterically inhibit
the uptake of long-chain FA into the mitochondria via carnitine palmitoyl transferase I
(CPT-1) (119). Recently, the liver-specific knockout of ACC1 in mice resulted in an upregulation of ACC2 (~2-fold) as compensation, which suggests that although site-specific
pools of malonyl-CoA may exist, ACC2 is able to contribute to de novo lipogenesis (62).
Our results suggest that Pcyt2+'
livers experience an increase in de novo lipogenesis,
which may be indicative of ACC1 transcriptional up-regulation or possibly impaired
phosphorylation via AMP activated-protein kinase (AMPK), which inactivates ACC1 via
phosphorylation of serine residue 79 (60). Pcyt2+' livers also experience impaired FA
oxidation, which in addition to an up-regulation of ACC2, may stem from decreased
ACC2 phosphorylation by AMPK resulting in increased inhibition of CPT-1 and
perturbations in long-chain FA import into the mitochondria (CPT-1), from changes in
mitochondrial FAT/CD36 and/or from defects at any point in the tricarboxylic acid cycle.
Although the exact mechanism by which this phenotype is facilitated remain elusive, we
show that the altered lipogenesis is a direct result of Pcyt2 disruption, as transient
transfection of Pcy12-myc into heterozygous hepatocytes results in a normalization of the
increased [ HJacetate incorporation into glycerolipids.
143
As it was hard to reconcile the chronic redirection and accumulation of DAG due
to such seemingly minor perturbations to PE metabolism, so to are the alterations to
lipogenesis and FA metabolism. Increased DAG availability leading to increased TG
formation would require an excess supply of FA; however, it is difficult to reason why
the cell would compensate by i) increasing FA uptake ii) decreasing FA oxidation and iii)
initiating de novo lipogenesis. Moreover, in addition to an increased incorporation of
labeled acetate into glycerolipids, this seemed to occur at the expense of acetate
incorporation into the cholesterol fractions, as seen by in vivo and in vitro labeling.
Although labeling revealed a decrease in lipogenesis directed toward cholesterol, total
hepatic cholesterol was not significantly different between Pcyt2+/' and littermate
controls (though slightly decreased). mRNA expression of the rate-limiting enzyme in
cholesterol biosynthesis (HMG-CoA reductase) was also slightly, but significantly downregulated. However, the extrapolation of a decreased mRNA expression leading to a
diminished overall activity would be purely speculative. Overall, disruption of Pcyt2
results in multiple points of compensation, such that the system may alter cholesterol
metabolism so that FA homeostasis (uptake, oxidation, synthesis and esterification) can
accommodate neutral lipid synthesis, which has been altered due to perturbations in
phospholipid (PE) homeostasis. It is clear that future investigation into the exact
mechanisms by which Pcyt2 deficiency affects each facet of lipid metabolism is
important.
6.1.4 SREBP as a master regulator of lipid homeostasis in Pcyt2 deficiency
SREBPs are a family of nuclear transcription factors that can modulate the
transcription of various lipogenic gene targets (73). The inactive SREBP molecules are
144
bound to various adaptor proteins, where activation of SREBPs occurs via a sterol
sensing domain, which upon low sterol conditions the complex travels from the ER to the
Golgi network (135). This initiates the cleavage of the immature peptide to the active,
nuclear form, which can then bind its consensus sequence on target promoters. Two
isoforms of SREBP1 (la and lc) are produced from alternative splicing, which mainly
influence FA metabolism. SREBP2 is transcribed from a separate gene and regulates
cholesterol biosynthesis (73).
We have indirect evidence to suggest that SREBP-lc is elevated in both liver and
skeletal muscle of older Pcyt2+/~ mice (32 wk). We are limited in our interpretation as
although the mRNA expression was elevated, this does not infer that there were also
increases in SREBPlc protein content or activation. A measure of nuclear SREBPlc
would have provided better evidence, although we also determined there to be increased
mRNA expression of downstream targets (FAS and SCD-1). There is also a nutritional
and hormonal regulation to SREBP1 activation, which is induced by insulin (90, 101).
Although it is unclear if SREBP1 influences the metabolic adaptations seen in young
Pcyt2+/~ mice, as the phenotype progresses, the mice become slightly hyperinsulinemic
(although not statistically significant). This chronic state of increased insulin, may affect
the expression of SREBP1, which would then act to reinforce the phenotype. In addition,
Pcyt2+/~ mice have decreased PUFA in PE as well as TG. Increased PUFA have been
demonstrated to inhibit activation of SREBP1, therefore it may be possible that a
reciprocal relationship could exist, although this is purely conjecture.
The argument that SREBP may govern or in part contribute to the phenotype
observed in Pcyt2+/~ mice partially stems from an investigation that demonstrated an
intimate relationship between PE and SREBP in Drosophila (38). In mammalian systems
145
cholesterol acts as the main regulator of SREBP activation through interactions with
cleavage proteins. However, work from the lab of Brown and Goldstein has demonstrated
that PE acts to regulate the activation of SREBP in Drosophila. Unlike mammalian cells,
PE is the main membrane constituent in Drosophila and no sterols are produced (28).
Dissimilar as the systems may be, the regulation of SREBP via PE and sterols in
Drosophila and mammalian cells respectively is remarkable similar. It was speculated
that perhaps the structural, non-lamellar properties of PE may play a role in influencing
membrane fluidity that could mimic membrane regulation by cholesterol (38). We do not
suggest that there is a direct relationship between PE and SREBP in mammalian cells;
however, it would be very interesting to investigate how Pcyt2 disruption may or may not
affect the activation of SREBP 1 in this model.
6.1.5 Genetically engineered mouse models
The insight gained from the use of genetically engineered mice has been
invaluable. The ability to abolish a particular gene from a mouse has allowed for the
characterization of countless pathways and biological processes. The knockout mouse
project is a trans-NIH initiative to generate embryonic stem cells or mice harbouring
targeted deletion of every gene in the mouse genome (8). The novelty of such an
approach; however, has become blunted in the past year, as there are inherent flaws with
targeting a mouse gene for ubiquitous disruption. For conclusions to have merit, the
limitations and biases of genetic models must first be understood and justified within
each investigation.
Pcyt2 knockout mice were generated by standard methods and maintained on a
mixed genetic background (C57B1/6 x 129/Sv). The mice were not back-crossed to
146
achieve a congenic strain; however, this was justified by the numerous genetically
engineered models for lipid related genes that were reported on mixed genetic
backgrounds (61, 69, 157, 162, 207). Although it may be possible that on a different or
pure genetic background, a modified phenotype might have been observed, our studies
compare heterozygous animals to wildtype littermate controls; therefore, there would
have been no effect of genetic background on any of the parameters.
A decade ago, whole-body targeted disruption of a single gene was considered a
complex endeavor, which yielded powerful results. The realization has been made that
the universal deletion of a single gene over the span of development is not necessarily the
best course of action. We have characterized a mouse model, in which the mice possess a
single Pcyt2 allele throughout their lifespan; therefore, we are left to observe a Pcyt2
deficient state and describe a chronic situation. Conditional and inducible knockdown or
knock-in mice, as well as tissue-specific and inducible over-expressing mice are now
being generated. This makes possible the acute characterization of biological pathways
and enzymes. Regardless of the type of genetic alteration, consideration must be made
that the levels of over- or under-expression are not always physiological. This thesis has
aimed to characterize Pcytl heterozygous mice, where at Pcytl expression is decreased
30-40%. Therefore this model is within physiological considerations (i.e. not up- or
down-regulated n-fold) and results in complex metabolic adaptations. Figure 6.3
illustrates the potential pathway interactions of a Pcyt2 heterozygous hepatocyte.
147
148
Figure 6.3. Glycerolipid synthetic pathways and the consequences of Pcyt2
deficiency. Abbreviations: EK; ethanolamine kinase, ET; CTP:phosphoethanolamine
cytidylyltransferase,
EPT;
phosphatidylethanolamine,
PS;
ethanolamine
phosphotransferase,
phosphatidylserine,
PSD;
PE;
phosphatidylserine
decarboxylase, PSS1; phosphatidylserine synthase, PC; phosphatidylcholine, PEMT;
phosphatidylethanolamine
Af-methyltransferase,
DAG;
diacylglycerol,
DGAT;
diacylglycerol acyltransferase, TG; triglyceride, PAP; phosphatidic acid phosphatase or
lipin-1,
2 or
acyltransferase,
3, PA; phosphatidic
LPA;
acid,
lysophosphatidic
AGPAT;
acid,
l-aclyglycerol-3-phosphate
GPAT;
glycerol-3-phosphate
acyltransferase, FAT/CD36; fatty acid translocase/CD36, FABPpm; fatty acid binding
protein plasma membrane, FA; fatty acid, MAM; mitochondrial associated membrane,
ER; endoplasmic reticulum.
6.1.6 Future work
The investigations summarized in this thesis have created a basic understanding
for the role and regulation of Pcyt2 in murine development and lipid homeostasis;
however, a number of unanswered questions remain. At what stage are the Pcyt2 null
embryos lethal? We identified partially resorbed knockout embryos at E8.5, suggesting
that the threshold for PE compensation via alternative pathways had been crossed and
apoptosis/necrosis had been initiated. Investigations into the specific developmental
abnormalities and morphological consequences of full Pcyt2 disruption may be
beneficial.
Although not specifically addressed in this thesis, CDP-ethanolamine is combined
with alkylacylglycerol from the peroxisome as one of the last steps in PE-plasmalogen
formation. Therefore in tissues, how does partial Pcyt2 disruption affect PE-plasmalogen
formation and content? We determined that there was no difference in the total amount of
149
PE in brain, heart, liver and kidney and this determination was inclusive of PEplasmalogen. However, it may be possible that the ratio of diacyl- to alkylacyl-PE is
skewed in heterozygous mice. As plasmalogens are mainly found in electrically active
tissues (cardiac and neuronal) (131), investigation into content in these tissues may be
important. Other than weight gain, our initial characterization demonstrated no overt
phenotypic changes in Pcyt2+/~ mice; however, a complete neuronal or cardiac
characterization may demonstrate subtle modifications.
We have demonstrated that Pcyt2+/~ mice experience a shift in lipogenic gene
expression at 32 wk. However, two important questions remain. Do young Pcyt2+' mice
experience altered gene expression and at what point during development? If the
alterations in transcription result in corresponding alterations in protein content? As
mentioned above, SREBPlc is up-regulated in Pcyt2+/~ mice; however, it is unknown if
the increased transcription is present at all stages of development. Is SREBPlc
coordinately up-regulated as a means of compensating for the redirection of DAG or is
the increase simply as a result of the phenotype later in development? Perhaps a more
crucial question would be does the increased transcription in fact lead to increased
translation and/or activity? Studies can sometimes reveal changes in mRNA expression
that do not correlate with protein content (108). We have performed labeling experiments
that address the alterations in metabolism in Pcyt2+/~ mice and implicate the involvement
of certain enzyme activity, such as increased DAG and TG formation via GPAT and
DGAT activities respectively. However we have only initiated investigations into the
individual molecular players involved by examining gene expression. Proteomic
comparison between genotypes at various developmental stages would be informative as
to the up- and down-regulated proteins.
150
As well as TG accumulation in the liver, Pcyt2 heterozygous mice have elevated
plasma TG content. Although it would be useful to investigate TG secretion via
metabolic labeling of VLDL particles using [35S]methionine for the determination of
apolipoprotein B, results using P407 injections and subsequent plasma TG analyses
revealed no significant difference in the rate of VLDL secretion from the liver. Therefore
alternative mechanisms must be responsible for the plasma increase. Work is currently in
progress to determine the rate of TG clearance as well as intestinal absorption in
heterozygous mice relative to littermate controls, as well as plasma and tissue LPL
activity assays. If perturbed, these phenomenon provide an avenue by which plasma TG
may be increased in Pcyt2+' animals.
The specific mechanisms that lead to Pcyt2+/~ insulin resistance also have yet to
be elucidated; as hepatic steatosis, obesity and DAG accumulation remain strictly
associative. An IP glucose tolerance test was used as an initial investigation, and directly
challenged the pancreas to produce and secrete insulin. We observed that older
heterozygous mice were still able to mount an insulin response; however, had become
desensitized to insulin. What remains to be seen are the exact sites of insulin resistance,
be that skeletal muscle or the liver. This should be determined using a hyperinsulinemiceuglycemic clamp, which through the infusion of labeled tracers will reveal the rate of
glucose turnover and subsequently the differentiation between hepatic and peripheral
insulin resistance (11). In addition to the site of insulin resistance, the mechanism by
which the insulin-stimulated glucose uptake is blunted should also be determined. We
have performed real-time PCR for the basal mRNA expression of a number of insulin and
glucose related genes; however, even though there were some discrepancies between
genotype, these measurements were made in the basal state. Basal and insulin stimulated
151
protein expression/phosphorylation of important players should be assessed in both
isolated primary hepatocytes, or a perfused liver set up (23) as well as isolated skeletal
muscle strips (176). Also, in the current studies, mRNA expression and lipid amounts
were determined in a quadriceps skeletal muscle preparation, which consisted of a variety
of muscle fibre types. The "whole" muscle preparation may provide a more
comprehensive interpretation given the contribution of the various fibre types; however,
in future studies, delineation should be made between muscle fibres. It could be
hypothesized that the accumulation of DAG in a chronic fashion becomes critical in the
later stages of increased adiposity where DAG interferes with novel protein kinase C
isoforms (58, 183, 222). Also, the amount of ceramide in.Pcyt2+/~ tissues (not evaluated
in this thesis) should be determined, as reports suggest that ceramide accumulation
interferes with the insulin signaling cascade by inhibiting Akt/PKB, among others (167).
The accumulation of reactive intermediates (DAG and ceramides) may play a detrimental
role in insulin signaling in older heterozygous mice; however, as adiposity increases it
may also result in a state of chronic inflammation (32, 107). This could be characterized
by macrophage infiltration into adipose tissue and the release of inflammatory cytokines
such as tumor necrosis factor which may also impair insulin signaling (137). These
aspects of the Pcyt2 deficient model require further investigation.
6.2 General conclusions
The disruption ofPcyt2 in mice has demonstrated that Pcyt2 and therefore the PEKennedy pathway is necessary for murine and possibly mammalian development. In
heterozygous animals, transcription, translation and enzyme activity are decreased 3040% resulting in a similar decrease in the rate of PE synthesis in all tissues examined. PE
152
content is maintained in heterozygotes, not by a compensatory increase in PS
decarboxylation, but a decrease in PE degradation. As a direct consequence of the
decreased flux through the PE-Kennedy pathway, the unused DAG is redirected toward
TG esterification in the liver. In support of our observations, a recent investigation
specifically deleted Pcyt2 from the liver, which resulted in a 10-fold increase in hepatic
TG accumulation due to the redirection of DAG. This was accompanied by an increase in
FA, DAG, cholesterol and cholesterol esters in the liver, as well as alterations in
molecular species in PE (103). In our model, the redirection of DAG toward TG is
facilitated by a chronic alteration in FA metabolism, with increased FA uptake and de
novo lipogenesis as well as decreased FA oxidation in the liver. Heterozygous mice are
significantly heavier than controls at a point that mimics adult-onset obesity, at which
point they are hypertriglyceridemic and insulin resistant. This work demonstrates direct
and indirect evidence for the integral relationship between PE and DAG/TG metabolism.
153
REFERENCES
1.
Abe, A., and J. A. Shayman. 1998. Purification and characterization of 1-0acylceramide synthase, a novel phospholipase A2 with transacylase activity. J
Biol Chem 273:8467-74.
2.
Abe, A., J. A. Shayman, and N. S. Radin. 1996. A novel enzyme that catalyzes
the esterification of N-acetylsphingosine. Metabolism of C2-ceramides. J Biol
Chem 271:14383-9.
3.
Abu-Elheiga, L., A. Jayakumar, A. Baldini, S. S. Chirala, and S. J. Wakil.
1995. Human acetyl-CoA carboxylase: characterization, molecular cloning, and
evidence for two isoforms. Proc Natl Acad Sci U S A 92:4011-5.
4.
Abu-Elheiga, L., W. Oh, P. Kordari, and S. J. Wakil. 2003. Acetyl-CoA
carboxylase 2 mutant mice are protected against obesity and diabetes induced by
high-fat/high-carbohydrate diets. Proc Natl Acad Sci U S A 100:10207-12.
5.
Akoh, C. C , and R. S. Chapkin. 1990. Composition of mouse peritoneal
macrophage phospholipid molecular species. Lipids 25:613-7.
6.
Aoyama, C , K. Nakashima, and K. Ishidate. 1998. Molecular cloning of
mouse choline kinase and choline/ethanolamine
kinase: their
sequence
comparison to the respective rat homologs. Biochim Biophys Acta 1393:179-85.
7.
Aoyama, C , N. Yamazaki, H. Terada, and K. Ishidate. 2000. Structure and
characterization of the genes for murine choline/ethanolamine kinase isozymes
alpha and beta. J Lipid Res 41:452-64.
8.
Araki, M., K. Araki, and K. Yamamura. 2009. International Gene Trap Project:
towards gene-driven saturation mutagenesis in mice. Curr Pharm Biotechnol
10:221-9.
9.
Arikketh, D., R. Nelson, and J. E. Vance. 2008. Defining the importance of
phosphatidylserine synthase-1 (PSS1): unexpected viability of PSS1-deficient
mice. J Biol Chem 283:12888-97.
10.
Arthur, G., and L. Page. 1991. Synthesis of phosphatidylethanolamine and
ethanolamine plasmalogen
by the CDP-ethanolamine
and
pathways in rat heart, kidney and liver. Biochem J 273(Pt 1): 121-5.
154
decarboxylase
11.
Ayala, J. E., D. P. Bracy, O. P. McGuinness, and D. H. Wasserman. 2006.
Considerations in the design of hyperinsulinemic-euglycemic clamps in the
conscious mouse. Diabetes 55:390-7.
12.
Bagnato, C , and R. A. Igal. 2003. Overexpression of diacylglycerol
acyltransferase-1 reduces phospholipid synthesis, proliferation, and invasiveness
in simian virus 40-transformed human lung fibroblasts. J Biol Chem 278:5220311.
13.
Bakovic, M., M. D. Fullerton, and V. Michel. 2007. Metabolic and molecular
aspects
of
ethanolamine
phospholipid
biosynthesis:
the
role
of
CTP:phosphoethanolamine cytidylyltransferase (Pcyt2). Biochem Cell Biol
85:283-300.
14.
Balakrishnan, S., H. Goodwin, and J. N. Cumings. 1961. The distribution of
phosphorus-containing lipid compounds in the human brain. J Neurochem 8:27684.
15.
Bergo, M. O., B. J. Gavino, R. Steenbergen, B. Sturbois, A. F. Parlow, D. A.
Sanan, W. C. Skarnes, J. E. Vance, and S. G. Young. 2002. Defining the
importance of phosphatidylserine synthase 2 in mice. J Biol Chem 277:47701-8.
16.
Bladergroen, B. A., M. J. Geelen, A. C. Reddy, P. E. Declercq, and L. M. Van
Golde.
1998.
Channelling
of
intermediates
in
the
biosynthesis
of
phosphatidylcholine and phosphatidylethanolamine in mammalian cells. Biochem
J334(Pt3):511-7.
17.
Bladergroen, B. A., M. Houweling, M. J. Geelen, and L. M. van Golde. 1999.
Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA
from rat liver. Biochem J 343 Pt 1:107-14.
18.
Bladergroen, B. A., and L. M. van Golde. 1997. CTP:phosphoethanolamine
cytidylyltransferase. Biochim Biophys Acta 1348:91-9.
19.
Bleijerveld, O. B., J. F. Brouwers, A. B. Vaandrager, J. B. Helms, and M.
Houweling. 2007. The CDP-ethanolamine pathway and phosphatidylserine
decarboxylation generate different phosphatidylethanolamine molecular species. J
Biol Chem 282:28362-72.
20.
Bleijerveld, O. B., W. Klein, A. B. Vaandrager, J. B. Helms, and M.
Houweling. 2004. Control of the CDPethanolamine pathway in mammalian cells:
155
effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the
amount of intracellular diacylglycerol. Biochem J 379:711-9.
21.
Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and
purification. Can J Biochem Physiol 37:911-7.
22.
Bogdanov, M., M. Umeda, and W. Dowhan. 1999. Phospholipid-assisted
refolding of an integral membrane protein. Minimum structural features for
phosphatidylethanolamine to act as a molecular chaperone. J Biol Chem
274:12339-45.
23.
Boini, K. M., D. Graf, A. M. Hennige, S. Koka, D. S. Kempe, K. Wang, T. F.
Ackermann, M. Foller, V. Vallon, K. Pfeifer, E. Schleicher, S. Ullrich, H. U.
Haring, D. Haussinger, and F. Lang. 2009. Enhanced insulin sensitivity of
gene-targeted mice lacking functional KCNQ1. Am J Physiol Regul Integr Comp
Physiol 296:R1695-701.
24.
Bonen, A., X. X. Han, D. D. Habets, M. Febbraio, J. F. Glatz, and J. J.
Luiken. 2007. A null mutation in skeletal muscle FAT/CD36 reveals its essential
role in insulin- and AICAR-stimulated fatty acid metabolism. Am J Physiol
Endocrinol Metab 292:E 1740-9.
25.
Borkenhagen, L. F., E. P. Kennedy, and L. Fielding. 1961. Enzymatic
Formation and Decarboxylation of Phosphatidylserine. J. Biol. Chem. 236:PC2830.
26.
Breslow, J. L. 2006. n-3 fatty acids and cardiovascular disease. Am J Clin Nutr
83:1477S-1482S.
27.
Caviglia, J. M., I. N. De Gomez Dumm, R. A. Coleman, and R. A. Igal. 2004.
Phosphatidylcholine
deficiency
upregulates
enzymes
of
triacylglycerol
metabolism in CHO cells. J Lipid Res 45:1500-9.
28.
Clark, A. J., and K. Block. 1959. The absence of sterol synthesis in insects. J
Biol Chem 234:2578-82.
29.
Coleman, R. A., and D. P. Lee. 2004. Enzymes of triacylglycerol synthesis and
their regulation. Prog Lipid Res 43:134-76.
30.
D'Santos, C. S., J. H. Clarke, R. F. Irvine, and N. Divecha. 1999. Nuclei
contain two differentially regulated pools of diacylglycerol. Curr Biol 9:437-40.
156
31.
Daum, G., and J. E. Vance. 1997. Import of lipids into mitochondria. Prog Lipid
Res 36:103-30.
32.
de Luca, C , and J. M. Olefsky. 2008. Inflammation and insulin resistance.
FEBS Lett 582:97-105.
33.
Deeba, F., H. N. Tahseen, K. S. Sharad, N. Ahmad, S. Akhtar, M.
Saleemuddin, and O. Mohammad. 2005. Phospholipid diversity: correlation
with membrane-membrane fusion events. Biochim Biophys Acta 1669:170-81.
34.
Denomme, J., K. D. Stark, and B. J. Holub. 2005. Directly quantitated dietary
(n-3) fatty acid intakes of pregnant Canadian women are lower than current
dietary recommendations. J Nutr 135:206-11.
35.
Devaux, P. F. 1991. Static and dynamic lipid asymmetry in cell membranes.
Biochemistry 30:1163-73.
36.
Diagne, A., J. Fauvel, M. Record, H. Chap, and L. Douste-Blazy. 1984.
Studies on ether phospholipids. II. Comparative composition of various tissues
from human, rat and guinea pig. Biochim Biophys Acta 793:221-31.
37.
Dobner, P., E. Koller, and B. Engelmann. 1999. Platelet high affinity low
density lipoprotein binding and import of lipoprotein derived phospholipids.
FEBS Lett 444:270-4.
38.
Dobrosotskaya, I. Y., A. C. Seegmiller, M. S. Brown, J. L. Goldstein, and R.
B. Rawson. 2002. Regulation of SREBP processing and membrane lipid
production by phospholipids in Drosophila. Science 296:879-83.
39.
Dobrzyn, A., P. Dobrzyn, M. Miyazaki, H. Sampath, K. Chu, and J. M.
Ntambi. 2005. Stearoyl-CoA desaturase 1 deficiency increases CTP: choline
cytidylyltransferase
translocation
into
the
membrane
and
enhances
phosphatidylcholine synthesis in liver. J Biol Chem 280:23356-62.
40.
Dowhan, W. 1997. Molecular basis for membrane phospholipid diversity: why
are there so many lipids? Annu Rev Biochem 66:199-232.
41.
Emoto, K., H. Inadome, Y. Kanaho, S. Narumiya, and M. Umeda. 2005.
Local change in phospholipid composition at the cleavage furrow is essential for
completion of cytokinesis. J Biol Chem 280:37901-7.
157
42.
Emoto, K., T. Kobayashi, A. Yamaji, H. Aizawa, I. Yahara, K. Inoue, and M.
Umeda. 1996. Redistribution of phosphatidylethanolamine at the cleavage furrow
of dividing cells during cytokinesis. Proc Natl Acad Sci U S A 93:12867-72.
43.
Emoto, K., N. Toyama-Sorimachi, H. Karasuyama, K. Inoue, and M. Umeda.
1997. Exposure of phosphatidylethanolamine on the surface of apoptotic cells.
Exp Cell Res 232:430-4.
44.
Emoto, K., and M. Umeda. 2001. Membrane lipid control of cytokinesis. Cell
Struct Funct 26:659-65.
45.
Engelmann, B., B. Schaipp, P. Dobner, M. Stoeckelhuber, C. Kogl, W. Siess,
and
A.
Hermetter.
1998. Platelet
agonists
enhance
the
import
of
phosphatidylethanolamine into human platelets. J Biol Chem 273:27800-8.
46.
Escriba, P. V., A. Ozaita, C. Ribas, A. Miralles, E. Fodor, T. Farkas, and J.
A. Garcia-Sevilla. 1997. Role of lipid polymorphism in G protein-membrane
interactions: nonlamellar-prone phospholipids and peripheral protein binding to
membranes. Proc Natl Acad Sci U S A 94:11375-80.
47.
Esko, J. D., M. M. Wermuth, and C. R. Raetz. 1981. Thermolabile CDPcholine synthetase in an animal cell mutant defective in lecithin formation. J Biol
Chem 256:7388-93.
48.
Exton, J. H. 1990. Signaling through phosphatidylcholine breakdown. J Biol
Chem 265:1-4.
49.
Farooqui, A. A., and L. A. Horrocks. 2004. Brain phospholipases A2: a
perspective on the history. Prostaglandins Leukot Essent Fatty Acids 71:161-9.
50.
Farooqui, A. A., and L. A. Horrocks. 2001. Plasmalogens: workhorse lipids of
membranes in normal and injured neurons and glia. Neuroscientist 7:232-45.
51.
Farooqui, A. A., W. Y. Ong, and L. A. Horrocks. 2003. Plasmalogens,
docosahexaenoic acid and neurological disorders. Adv Exp Med Biol 544:335-54.
52.
Ford, D. A. 2003. Separate myocardial ethanolamine phosphotransferase
activities responsible for plasmenylethanolamine and phosphatidylethanolamine
synthesis. J Lipid Res 44:554-9.
53.
Ford, D. A., and R. W. Gross. 1989. Plasmenylethanolamine is the major
storage depot for arachidonic acid in rabbit vascular smooth muscle and is rapidly
158
hydrolyzed after angiotensin II stimulation. Proc Natl Acad Sci U S A 86:347983.
54.
Fullerton, M. D., F. Hakimuddin, and M. Bakovic. 2007. Developmental and
metabolic effects of disruption of the mouse
CTP:phosphoethanolamine
cytidylyltransferase gene (Pcyt2). Mol Cell Biol 27:3327-36.
55.
Fullerton, M. D., F. Hakimuddin, A. Bonen, and M. Bakovic. 2009. The
development of a metabolic disease phenotype in CTP:phosphoethanolamine
cytidylyltransferase deficient mice. J Biol Chem. Epub July 22.
56.
Glaser, P. E., and R. W. Gross. 1994. Plasmenylethanolamine facilitates rapid
membrane fusion: a stopped-flow kinetic investigation correlating the propensity
of a major plasma membrane constituent to adopt an HII phase with its ability to
promote membrane fusion. Biochemistry 33:5805-12.
57.
Gremo, F., G. E. De Medio, G. Trovarelli, S. Dessi, and S. Porru. 1985.
Mature and immature synaptosomal membranes have a different
lipid
composition. Neurochem Res 10:133-44.
58.
Griffin, M. E., M. J. Marcucci, G. W. Cline, K. Bell, N. Barucci, D. Lee, L. J.
Goodyear, E. W. Kraegen, M. F. White, and G. I. Shulman. 1999. Free fatty
acid-induced insulin resistance is associated with activation of protein kinase C
theta and alterations in the insulin signaling cascade. Diabetes 48:1270-4.
59.
Guo, Y., T. C. Walther, M. Rao, N. Stuurman, G. Goshima, K. Terayama, J.
S. Wong, R. D. Vale, P. Walter, and R. V. Farese. 2008. Functional genomic
screen reveals genes involved in lipid-droplet formation and utilization. Nature
453:657-61.
60.
Ha, J., S. Daniel, S. S. Broyles, and K. H. Kim. 1994. Critical phosphorylation
sites for acetyl-CoA carboxylase activity. J Biol Chem 269:22162-8.
61.
Hammond, L. E., P. A. Gallagher, S. Wang, S. Hiller, K. D. Kluckman, E. L.
Posey-Marcos, N. Maeda, and R. A. Coleman. 2002. Mitochondrial glycerol-3phosphate acyltransferase-deficient
mice have reduced weight and liver
triacylglycerol content and altered glycerolipid fatty acid composition. Mol Cell
Biol 22:8204-14.
62.
Harada, N., Z. Oda, Y. Hara, K. Fujinami, M. Okawa, K. Ohbuchi, M.
Yonemoto, Y. Ikeda, K. Ohwaki, K. Aragane, Y. Tamai, and J. Kusunoki.
159
2007. Hepatic de novo lipogenesis is present in liver-specific ACC1-deficient
mice. Mol Cell Biol 27:1881-8.
63.
Hazen, S. L., D. A. Ford, and R. W. Gross. 1991. Activation of a membraneassociated phospholipase A2 during rabbit myocardial ischemia which is highly
selective for plasmalogen substrate. J Biol Chem 266:5629-33.
64.
Henneberry, A. L., and C. R. McMaster. 1999. Cloning and expression of a
human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine
and phosphatidylethanolamine. Biochem J 339 (Pt 2):291-8.
65.
Henneberry, A. L., G. Wistow, and C. R. McMaster. 2000. Cloning, genomic
organization, and characterization of a human cholinephosphotransferase. J Biol
Chem 275:29808-15.
66.
Henneberry, A. L., M. M. Wright, and C. R. McMaster. 2002. The major sites
of cellular phospholipid synthesis and molecular determinants of Fatty Acid and
lipid head group specificity. Mol Biol Cell 13:3148-61.
67.
Hii, C. S., Y. S. Edwards, and A. W. Murray. 1991. Phorbol ester-stimulated
hydrolysis
of
phosphatidylcholine
and
phosphatidylethanolamine
by
phospholipase D in HeLa cells. Evidence that the basal turnover of
phosphoglycerides does not involve phospholipase D. J Biol Chem 266:20238-43.
68.
Hiraoka, M., A. Abe, Y. Lu, K. Yang, X. Han, R. W. Gross, and J. A.
Shayman. 2006. Lysosomal phospholipase A2 and phospholipidosis. Mol Cell
Biol 26:6139-48.
69.
Hofmann, S. M., L. Zhou, D. Perez-Tilve, T. Greer, E. Grant, L. Wancata, A.
Thomas, P. T. Pfluger, J. E. Basford, D. Gilham, J. Herz, M. H. Tschop, and
D. Y. Hui. 2007. Adipocyte LDL receptor-related protein-1 expression modulates
postprandial lipid transport and glucose homeostasis in mice. J Clin Invest
117:3271-82.
70.
Holloway, G. P., J. J. Luiken, J. F. Glatz, L. L. Spriet, and A. Bonen. 2008.
Contribution of FAT/CD36 to the regulation of skeletal muscle fatty acid
oxidation: an overview. Acta Physiol (Oxf) 194:293-309.
71.
Hong, S., K. Gronert, P. R. Devchand, R. L. Moussignac, and C. N. Serhan.
2003. Novel docosatrienes and 17S-resolvins generated from docosahexaenoic
160
acid in murine brain, human blood, and glial cells. Autacoids in antiinflammation. J Biol Chem 278:14677-87.
72.
Horibata, Y., and Y. Hirabayashi. 2006. Identification and characterization of a
human ethanolaminephosphotransferasel (hEPTl). J Lipid Res.
73.
Horton, J. D., J. L. Goldstein, and M. S. Brown. 2002. SREBPs: activators of
the complete program of cholesterol and fatty acid synthesis in the liver. J Clin
Invest 109:1125-31.
74.
Houweling,
M.,
Z.
Cui,
and
D.
E. Vance.
1995. Expression
of
phosphatidylethanolamine N-methyltransferase-2 cannot compensate for an
impaired CDP-choline pathway in mutant Chinese hamster ovary cells. J Biol
Chem 270:16277-82.
75.
Houweling, M., L. B. Tijburg, W. J. Vaartjes, and L. M. van Golde. 1992.
Phosphatidylethanolamine metabolism in rat liver after partial hepatectomy.
Control of biosynthesis of phosphatidylethanolamine by the availability of
ethanolamine. Biochem J 283 ( Pt 1):55-61.
76.
Ibrahim, S., A. Djimet-Baboun, V. Pruneta-Deloche, C. Calzada, M.
Lagarde, and G. Ponsin. 2006. Transfer of very low density lipoproteinassociated phospholipids to activated human platelets. J Lipid Res 47:341-8.
77.
Igal, R. A., J. M. Caviglia, I. N. de Gomez Dumm, and R. A. Coleman. 2001.
Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is
used for triacylglycerol synthesis. J Lipid Res 42:88-95.
78.
Igal, R. A., and R. A. Coleman. 1996. Acylglycerol recycling from
triacylglycerol to phospholipid, not lipase activity, is defective in neutral lipid
storage disease fibroblasts. J Biol Chem 271:16644-51.
79.
Igal, R. A., and R. A. Coleman. 1998. Neutral lipid storage disease: a genetic
disorder with abnormalities in the regulation of phospholipid metabolism. J Lipid
Res 39:31-43.
80.
Igal, R. A., Pallanza de Stringa, M.C., and Tacconi de Gomez Dumm, I.N. .
1994. Simple method for quantitation of neutral and polar lipids by TLC and
fluorescence emission. Int. J. Biochromatogr. 1:137-142.
161
81.
Imhof, I., E. Canivenc-Gansel, U. Meyer, and A. Conzelmann. 2000.
Phosphatidylethanolamine is the donor of the phosphorylethanolamine linked to
the alpha 1,4-linked mannose of yeast GPI structures. Glycobiology 10:1271-5.
82.
Imhof, I., I. Flury, C. Vionnet, C. Roubaty, D. Egger, and A. Conzelmann.
2004. Glycosylphosphatidylinositol (GPI) proteins of Saccharomyces cerevisiae
contain ethanolamine phosphate groups on the alpha 1,4-linked mannose of the
GPI anchor. J Biol Chem 279:19614-27.
83.
Ishidate, K. 1997. Choline/ethanolamine kinase from mammalian tissues.
Biochim Biophys Acta 1348:70-8.
84.
Jackowski, S., J. E. Rehg, Y. M. Zhang, J. Wang, K. Miller, P. Jackson, and
M. A. Karim. 2004. Disruption of CCTbeta2 expression leads to gonadal
dysfunction. Mol Cell Biol 24:4720-33.
85.
Jacobs, R. L., C. Devlin, I. Tabas, and D. E. Vance. 2004. Targeted deletion of
hepatic CTP:phosphocholine cytidylyltransferase alpha in mice decreases plasma
high density and very low density lipoproteins. J Biol Chem 279:47402-10.
86.
Jamil, H., A. K. Utal, and D. E. Vance. 1992. Evidence that cyclic AMPinduced inhibition of phosphatidylcholine biosynthesis is caused by a decrease in
cellular diacylglycerol levels in cultured rat hepatocytes. J Biol Chem 267:175260.
87.
Johnson, C. M., Z. Yuan, and M. Bakovic. 2005. Characterization of
transcription factors and cis-acting elements that regulate human CTP:
phosphoethanolamine cytidylyltransferase
(Pcyt2). Biochim Biophys Acta
1735:230-5.
88.
Karpova, T., R. R. Maran, J. Presley, S. P. Scherrer, L. Tejada, and L. L.
Heckert. 2005. Transgenic rescue of SF-1-null mice. Ann N Y Acad Sci
1061:55-64.
89.
Kennedy, E. P., and S. B. Weiss. 1956. The function of cytidine coenzymes in
the biosynthesis of phospholipides. J Biol Chem 222:193-214.
90.
Kim, J. B., P. Sarraf, M. Wright, K. M. Yao, E. Mueller, G. Solanes, B. B.
Lowell, and B. M. Spiegelman. 1998. Nutritional and insulin regulation of fatty
acid synthetase and leptin gene expression through ADD1/SREBP1. J Clin Invest
101:1-9.
162
91.
Kiss, Z., and W. B. Anderson. 1989. Phorbol ester stimulates the hydrolysis of
phosphatidylethanolamine in leukemic HL-60, NIH 3T3, and baby hamster
kidney cells. J Biol Chem 264:1483-7.
92.
Klaunig, J. E., P. J. Goldblatt, D. E. Hinton, M. M. Lipsky, J. Chacko, and B.
F. Trump. 1981. Mouse liver cell culture. I. Hepatocyte isolation. In Vitro
17:913-25.
93.
Klaunig, J. E., P. J. Goldblatt, D. E. Hinton, M. M. Lipsky, and B. F. Trump.
1981. Mouse liver cell culture. II. Primary culture. In Vitro 17:926-34.
94.
Klein, S., M. Spannagl, and B. Engelmann. 2001. Phosphatidylethanolamine
participates in the stimulation of the contact system of coagulation by very-lowdensity lipoproteins. Arterioscler Thromb Vase Biol 21:1695-700.
95.
Kobayashi, T., and R. E. Pagano. 1989. Lipid transport during mitosis.
Alternative pathways for delivery of newly synthesized lipids to the cell surface. J
Biol Chem 264:5966-73.
96.
Kogo, H., and T. Fujimoto. 2000. Caveolin-1 isoforms are encoded by distinct
mRNAs. Identification Of mouse caveolin-1 mRNA variants caused by
alternative transcription initiation and splicing. FEBS Lett 465:119-23.
97.
Kogo, H., K. Ishiguro, S. Kuwaki, and T. Fujimoto. 2002. Identification of a
splice variant of mouse caveolin-2 mRNA encoding an isoform lacking the Cterminal domain. Arch Biochem Biophys 401:108-14.
98.
Kuge, O., M. Nishijima, and Y. Akamatsu. 1986. Phosphatidylserine
biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and
characterization of phosphatidylserine auxotrophs. J Biol Chem 261:5790-4.
99.
Lands, W. E. M. a. C , C.G.. 1976. p.3-85 In Martonosi, A. (ed.), The Enzymes
of Biological Membranes,. 2.
100.
Latorre, E., M. P. Collado, I. Fernandez, M. D. Aragones, and R. E. Catalan.
2003. Signaling events mediating activation of brain ethanolamine plasmalogen
hydrolysis by ceramide. Eur J Biochem 270:36-46.
101.
Le Lay, S., I. Lefrere, C. Trautwein, I. Dugail, and S. Krief. 2002. Insulin and
sterol-regulatory element-binding protein-lc (SREBP-1C) regulation of gene
expression in 3T3-L1 adipocytes. Identification of CCAAT/enhancer-binding
protein beta as an SREBP-1C target. J Biol Chem 277:35625-34.
163
102.
Lee, T. C. 1998. Biosynthesis and possible biological functions of plasmalogens.
Biochim Biophys Acta 1394:129-45.
103.
Leonardi, R., M. W. Frank, P. D. Jackson, C. O. Rock, and S. Jackowski.
2009. Elimination of the CDP-Ethanolamine pathway disrupts hepatic lipid
homeostasis. J Biol Chem. Epub Aug 7.
104.
Lewin, T. M., S. Wang, C. A. Nagle, C. G. Van Horn, and R. A. Coleman.
2005. Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic
fate of exogenous fatty acids in hepatocytes. Am J Physiol Endocrinol Metab
288:E835-44.
105.
Linden, D., L. William-Olsson, A. Ahnmark, K. Ekroos, C. Hallberg, H. P.
Sjogren, B. Becker, L. Svensson, J. C. Clapham, J. Oscarsson, and S.
Schreyer. 2006. Liver-directed overexpression of mitochondrial glycerol-3phosphate acyltransferase results in hepatic steatosis, increased triacylglycerol
secretion and reduced fatty acid oxidation. Faseb J 20:434-43.
106.
Liu, L., Y. Zhang, N. Chen, X. Shi, B. Tsang, and Y. H. Yu. 2007.
Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal
muscle and protects against fat-induced insulin resistance. J Clin Invest 117:167989.
107.
Lumeng, C. N., S. M. Deyoung, J. L. Bodzin, and A. R. Saltiel. 2007.
Increased inflammatory properties of adipose tissue macrophages recruited during
diet-induced obesity. Diabetes 56:16-23.
108.
Luquet, S., J. Lopez-Soriano, D. Hoist, A. Fredenrich, J. Melki, M.
Rassoulzadegan, and P. A. Grimaldi. 2003. Peroxisome proliferator-activated
receptor delta controls muscle development and oxidative capability. Faseb J
17:2299-301.
109.
Lykidis, A., J. Wang, M. A. Karim, and S. Jackowski. 2001. Overexpression of
a mammalian ethanolamine-specific kinase accelerates the CDP-ethanolamine
pathway. J Biol Chem 276:2174-9.
110.
MacDonald, J. I., and H. Sprecher. 1989. Distribution of arachidonic acid in
choline- and ethanolamine-containing phosphoglycerides in subfractionated
human neutrophils. J Biol Chem 264:17718-26.
164
111.
Mancini, A., F. Del Rosso, R. Roberti, P. Orvietani, L. Coletti, and L.
Binaglia. 1999. Purification of ethanolaminephosphotransferase from bovine liver
microsomes. Biochim Biophys Acta 1437:80-92.
112.
Mandal, A., Y. Wang, P. Ernsberger, and M. Kester. 1997. Interleukin-1induced ether-linked diglycerides inhibit calcium-insensitive protein kinase C
isotypes. Implications for growth senescence. J Biol Chem 272:20306-11.
113.
Mandel, H., R. Sharf, M. Berant, R. J. Wanders, P. Vreken, and M. Aviram.
1998. Plasmalogen phospholipids are involved in HDL-mediated cholesterol
efflux: insights from investigations with plasmalogen-deficient cells. Biochem
Biophys Res Commun 250:369-73.
114.
Marchesini, G., S. Natale, R. Manini, and F. Agostini. 2005. Review article:
the treatment of fatty liver disease associated with the metabolic syndrome.
Aliment Pharmacol Ther 22 Suppl 2:37-9.
115.
Martinez, M. 1992. Tissue levels of polyunsaturated fatty acids during early
human development. J Pediatr 120:S129-38.
116.
Martinez, M., and I. Mougan. 1999. Fatty acid composition of brain
glycerophospholipids in peroxisomal disorders. Lipids 34:733-40.
117.
Martinez, M., I. Mougan, M. Roig, and A. Ballabriga. 1994. Blood
polyunsaturated fatty acids in patients with peroxisomal disorders. A multicenter
study. Lipids 29:273-80.
118.
McFarland, M. J., and E. L. Barker. 2004. Anandamide transport. Pharmacol
Ther 104:117-35.
119.
McGarry, J. D., G. F. Leatherman, and D. W. Foster. 1978. Carnitine
palmitoyltransferase I. The site of inhibition of hepatic fatty acid oxidation by
malonyl-CoA. J Biol Chem 253:4128-36.
120.
McMaster, C. R., and R. M. Bell. 1997. CDP-choline:l,2-diacylglycerol
cholinephosphotransferase. Biochim Biophys Acta 1348:100-10.
121.
McMaster, C. R., and R. M. Bell. 1997. CDP-ethanolamine:l,2-diacylglycerol
ethanolaminephosphotransferase. Biochim Biophys Acta 1348:117-23.
122.
McMaster, C. R., and P. C. Choy. 1992. Newly imported ethanolamine is
preferentially utilized for phosphatidylethanolamine biosynthesis in the hamster
heart. Biochim Biophys Acta 1124:13-6.
165
123.
McMaster, C. R., P. G. Tardi, and P. C. Choy. 1992. Modulation of
phosphatidylethanolamine
biosynthesis
by
exogenous
ethanolamine
and
analogues in the hamster heart. Mol Cell Biochem 116:69-73.
124.
Menon, A. K., and V. L. Stevens. 1992. Phosphatidylethanolamine is the donor
of the ethanolamine residue linking a glycosylphosphatidylinositol anchor to
protein. J Biol Chem 267:15277-80.
125.
Mileykovskaya, E., Q. Sun, W. Margolin, and W. Dowhan. 1998. Localization
and function of early cell division proteins in filamentous Escherichia coli cells
lacking phosphatidylethanolamine. J Bacteriol 180:4252-7.
126.
Millar, J. S., D. A. Cromley, M. G. McCoy, D. J. Rader, and J. T. Billheimer.
2005. Determining hepatic triglyceride production in mice: comparison of
poloxamer 407 with Triton WR-1339. J Lipid Res 46:2023-8.
127.
Millar, J. S., S. J. Stone, U. J. Tietge, B. Tow, J. T. Billheimer, J. S. Wong, R.
L. Hamilton, R. V. Farese, Jr., and D. J. Rader. 2006. Short-term
overexpression of DGAT1 or DGAT2 increases hepatic triglyceride but not
VLDL triglyceride or apoB production. J Lipid Res 47:2297-305.
128.
Min-Seok, R., Y. Kawamata, H. Nakamura, A. Ohta, and M. Takagi. 1996.
Isolation and characterization of ECT1 gene encoding CTP: phosphoethanolamine
cytidylyltransferase of Saccharomyces cerevisiae. J Biochem (Tokyo) 120:10407.
129.
Munn, N. J., E. Arnio, D. Liu, R. A. Zoeller, and L. Liscum. 2003. Deficiency
in ethanolamine plasmalogen leads to altered cholesterol transport. J Lipid Res
44:182-92.
130.
Musial, A., A. Mandal, E. Coroneos, and M. Kester. 1995. Interleukin-1 and
endothelin stimulate distinct species of diglycerides that differentially regulate
protein kinase C in mesangial cells. J Biol Chem 270:21632-8.
131.
Nagan, N., and R. A. Zoeller. 2001. Plasmalogens: biosynthesis and functions.
Prog Lipid Res 40:199-229.
132.
Nagle, C. A., J. An, M. Shiota, T. P. Torres, G. W. Cline, Z. X. Liu, S. Wang,
R. L. Catlin, G. I. Shulman, C. B. Newgard, and R. A. Coleman. 2007.
Hepatic overexpression of glycerol-sn-3-phosphate acyltransferase 1 in rats
causes insulin resistance. J Biol Chem 282:14807-15.
166
133.
Nakashima, A., K. Hosaka, and J. Nikawa. 1997. Cloning of a human cDNA
for CTP-phosphoethanolamine cytidylyltransferase by complementation in vivo
of a yeast mutant. J Biol Chem 272:9567-72.
134.
Noga, A. A., Y. Zhao, and D. E. Vance. 2002. An unexpected requirement for
phosphatidylethanolamine N-methyltransferase in the secretion of very low
density lipoproteins. J Biol Chem 277:42358-65.
135.
Nohturfft, A., D. Yabe, J. L. Goldstein, M. S. Brown, and P. J. Espenshade.
2000. Regulated step in cholesterol feedback localized to budding of SCAP from
ER membranes. Cell 102:315-23.
136.
Ntambi, J. M., and M. Miyazaki. 2004. Regulation of stearoyl-CoA desaturases
and role in metabolism. Prog Lipid Res 43:91-104.
137.
Odegaard, J. I., and A. Chawla. 2008. Mechanisms of macrophage activation in
obesity-induced insulin resistance. Nat Clin Pract Endocrinol Metab 4:619-26.
138.
Okamoto, H., J. Nakae, T. Kitamura, B. C. Park, I. Dragatsis, and D. Accili.
2004. Transgenic rescue of insulin receptor-deficient mice. J Clin Invest 114:21423.
139.
Onuki, Y., M. Morishita, Y. Chiba, S. Tokiwa, and K. Takayama. 2006.
Docosahexaenoic acid and eicosapentaenoic acid induce changes in the physical
properties of a lipid bilayer model membrane. Chem Pharm Bull (Tokyo) 54:6871.
140.
Ortlund, E. A., Y. Lee, I. H. Solomon, J. M. Hager, R. Sail, Y. Choi, Z. Guan,
A. Tripathy, C. R. Raetz, D. P. McDonnell, D. D. Moore, and M. R. Redinbo.
2005. Modulation of human nuclear receptor LRH-1 activity by phospholipids
and SHP. Nat Struct Mol Biol 12:357-63.
141.
Panganamala, R. V., L. A. Horrocks, J. C. Geer, and D. G. Cornwell. 1971.
Positions of double bonds in the monounsaturated alk-1-enyl groups from the
plasmalogens of human heart and brain. Chem Phys Lipids 6:97-102.
142.
Park, Y. S., P. Gee, S. Sanker, E. J. Schurter, E. R. Zuiderweg, and C. Kent.
1997. Identification of functional conserved residues of CTP:glycerol-3phosphate cytidylyltransferase. Role of histidines in the conserved HXGH in
catalysis. J Biol Chem 272:15161-6.
167
143.
Parker, G., N. A. Gibson, H. Brotchie, G. Heruc, A. M. Rees, and D. HadziPavlovic. 2006. Omega-3 fatty acids and mood disorders. Am J Psychiatry
163:969-78.
144.
Petersen, K. F., and G. I. Shulman. 2006. Etiology of insulin resistance. Am J
Med 119: SI 0-6.
145.
Phillips, D. I., S. Caddy, V. Ilic, B. A. Fielding, K. N. Frayn, A. C. Borthwick,
and R. Taylor. 1996. Intramuscular triglyceride and muscle insulin sensitivity:
evidence for a relationship in nondiabetic subjects. Metabolism 45:947-50.
146.
Poloumienko, A., A. Cote, A. T. Quee, L. Zhu, and M. Bakovic. 2004.
Genomic organization and differential splicing of the mouse and human Pcyt2
genes. Gene 325:145-55.
147.
Ravandi,
A.,
A.
Kuksis,
phosphatidylethanolamine
and
promotes
N.
A.
macrophage
Shaikh.
uptake
1999.
of
low
Glycated
density
lipoprotein and accumulation of cholesteryl esters and triacylglycerols. J Biol
Chem 274:16494-500.
148.
Reue, K., and D. N. Brindley. 2008. Thematic Review Series: Glycerolipids.
Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism.
J Lipid Res 49:2493-503.
149.
Samborski, R. W., N. D. Ridgway, and D. E. Vance. 1990. Evidence that only
newly made phosphatidylethanolamine is methylated to phosphatidylcholine and
that phosphatidylethanolamine is not significantly deacylated-reacylated in rat
hepatocytes. J Biol Chem 265:18322-9.
150.
Santini, M. T., P. L. Indovina, A. Cantafora, and I. Blotta. 1990. The cesiuminduced delay in myoblast membrane fusion is accompanied by changes in
isolated membrane lipids. Biochim Biophys Acta 1023:298-304.
151.
Schmitz-Peiffer, C. 2002. Protein kinase C and lipid-induced insulin resistance in
skeletal muscle. Ann N Y Acad Sci 967:146-57.
152.
Seddon, J. M., G. Cevc, and D. Marsh. 1983. Calorimetric studies of the gelfluid (L beta-L alpha) and lamellar-inverted hexagonal (L alpha-HII) phase
transitions in dialkyl- and diacylphosphatidylethanolamines.
22:1280-9.
168
Biochemistry
153.
Sessions, A., and A. F. Horwitz. 1981. Myoblast aminophospholipid asymmetry
differs from that of fibroblasts. FEBS Lett 134:75-8.
154.
Shiao, Y. J., G. Lupo, and J. E. Vance. 1995. Evidence that phosphatidylserine
is imported into mitochondria via a mitochondria-associated membrane and that
the majority of mitochondrial phosphatidylethanolamine is derived from
decarboxylation of phosphatidylserine. J Biol Chem 270:11190-8.
155.
Simbeni, R., F. Paltauf, and G. Daum. 1990. Intramitochondrial transfer of
phospholipids in the yeast, Saccharomyces cerevisiae. J Biol Chem 265:281-5.
156.
Singer, S. J., and G. L. Nicolson. 1972. The fluid mosaic model of the structure
of cell membranes. Science 175:720-31.
157.
Smith, S. J., S. Cases, D. R. Jensen, H. C. Chen, E. Sande, B. Tow, D. A.
Sanan, J. Raber, R. H. Eckel, and R. V. Farese, Jr. 2000. Obesity resistance
and multiple mechanisms of triglyceride synthesis in mice lacking Dgat. Nat
Genet 25:87-90.
158.
Snyder, F., Lee, T.-C, and Wykle, R.L.. 1985. Ether-linked glyceroplipids and
their bioactive species: enzymes and metabolic regulation, vol. 2. Plenum Press,
New York.
159.
Soulages, J. L., Z. Salamon, M. A. Wells, and G. Tollin. 1995. Low
concentrations of diacylglycerol promote the binding of apolipophorin III to a
phospholipid bilayer: a surface plasmon resonance spectroscopy study. Proc Natl
Acad Sci U S A 92:5650-4.
160.
Stals, H. K., W. Top, and P. E. Declercq. 1994. Regulation of triacylglycerol
synthesis in permeabilized rat hepatocytes. Role of fatty acid concentration and
diacylglycerol acyltransferase. FEBS Lett 343:99-102.
161.
Steenbergen, R., T. S. Nanowski, A. Beigneux, A. Kulinski, S. G. Young, and
J. E. Vance. 2005. Disruption of the phosphatidylserine decarboxylase gene in
mice causes embryonic lethality and mitochondrial defects. J Biol Chem
280:40032-40.
162.
Steenbergen, R., T. S. Nanowski, R. Nelson, S. G. Young, and J. E. Vance.
2006. Phospholipid homeostasis in phosphatidylserine synthase-2-deficient mice.
Biochim Biophys Acta 1761:313-23.
169
163.
Stillwell, W., S. R. Shaikh, M. Zerouga, R. Siddiqui, and S. R. Wassail. 2005.
Docosahexaenoic acid affects cell signaling by altering lipid rafts. Reprod Nutr
Dev 45:559-79.
164.
Stone, S. J., Z. Cui, and J. E. Vance. 1998. Cloning and expression of mouse
liver phosphatidylserine synthase-1 cDNA. Overexpression in rat hepatoma cells
inhibits
the
CDP-ethanolamine
pathway
for
phosphatidylethanolamine
biosynthesis. J Biol Chem 273:7293-302.
165.
Stone, S. J., and J. E. Vance. 2000. Phosphatidylserine synthase-1 and -2 are
localized to mitochondria-associated membranes. J Biol Chem 275:34534-40.
166.
Sugiura, T., T. Fukuda, Y. Masuzawa, and K. Waku. 1990. Ether
lysophospholipid-induced production of platelet-activating factor in human
polymorphonuclear leukocytes. Biochim Biophys Acta 1047:223-32.
167.
Summers, S. A. 2006. Ceramides in insulin resistance and lipotoxicity. Prog
Lipid Res 45:42-72.
168.
Sundler, R. 1973. Biosynthesis of rat liver phosphatidylethanolamines from
intraportally injected ethanolamine. Biochim Biophys Acta 306:218-26.
169.
Sundler, R. 1975. Ethanolaminephosphate cytidylyltransferase. Purification and
characterization of the enzyme from rat liver. J Biol Chem 250:8585-90.
170.
Sundler, R., and B. Akesson. 1975. Regulation of phospholipid biosynthesis in
isolated rat hepatocytes. Effect of different substrates. J Biol Chem 250:3359-67.
171.
Sundler, R., B. Akesson, and A. Nilsson. 1974. Quantitative role of base
exchange in phosphatidylethanolamine synthesis in isolated rat hepatocytes.
FEBS Lett 43:303-7.
172.
Tabuchi, M., N. Tanaka, J. Nishida-Kitayama, H. Ohno, and F. Kishi. 2002.
Alternative splicing regulates the subcellular localization of divalent metal
transporter 1 isoforms. Mol Biol Cell 13:4371-87.
173.
Tarn, O., and S. M. Innis. 2006. Dietary polyunsaturated fatty acids in gestation
alter fetal cortical phospholipids, fatty acids and phosphatidylserine synthesis.
Dev Neurosci 28:222-9.
174.
Testerink, N., M. M. van der Sanden, M. Houweling, J. B. Helms, and A. B.
Vaandrager. 2009. Depletion of phosphatidylcholine affects ER morphology and
protein traffic at the Golgi complex. J Lipid Res.
170
175.
Thompson, M. G., S. C. Mackie, A. Thorn, and R. M. Palmer. 1997.
Regulation of phospholipase D in L6 skeletal muscle myoblasts. Role of protein
kinase c and relationship to protein synthesis. J Biol Chem 272:10910-6.
176.
Thrush, A. B., G. J. Heigenhauser, K. L. Mullen, D. C. Wright, and D. J.
Dyck. 2008. Palmitate acutely induces insulin resistance in isolated muscle from
obese but not lean humans. Am J Physiol Regul Integr Comp Physiol 294:R120512.
177.
Tian, Y., P. Jackson, C. Gunter, J. Wang, C. O. Rock, and S. Jackowski.
2006. Placental thrombosis and spontaneous fetal death in mice deficient in
ethanolamine kinase 2. J Biol Chem.
178.
Tie,
A.,
and
M.
Bakovic.
2007.
Alternative
splicing
of
CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ
in catalytic properties. J Lipid Res 48:2172-81.
179.
Tijburg, L. B., M. J. Geelen, and L. M. Van Golde. 1989. Biosynthesis of
phosphatidylethanolamine via the CDP-ethanolamine route is an important
pathway in isolated rat hepatocytes. Biochem Biophys Res Commun 160:127580.
180.
Tijburg, L. B., M. J. Geelen, and L. M. van Golde. 1989. Regulation of the
biosynthesis
of
triacylglycerol,
phosphatidylcholine
and
phosphatidylethanolamine in the liver. Biochim Biophys Acta 1004:1-19.
181.
Tijburg, L. B., M. Houweling, M. J. Geelen, and L. M. Van Golde. 1989.
Inhibition of phosphatidylethanolamine synthesis by glucagon in isolated rat
hepatocytes. Biochem J 257:645-50.
182.
Tijburg, L. B., P. S. Vermeulen, and L. M. van Golde. 1992. Ethanolaminephosphate cytidylyltransferase. Methods Enzymol 209:258-63.
183.
Timmers, S., P. Schrauwen, and J. de Vogel. 2008. Muscular diacylglycerol
metabolism and insulin resistance. Physiol Behav 94:242-51.
184.
Uchida, T. 1994. Regulation of choline kinase R: analyses of alternatively spliced
choline kinases and the promoter region. J Biochem (Tokyo) 116:508-18.
185.
van den Bosch, H. 1974. Phosphoglyceride metabolism. Annu Rev Biochem.
43:243-277.
171
186.
van Hellemond, J. J., J. W. Slot, M. J. Geelen, L. M. van Golde, and P. S.
Vermeulen. 1994. Ultrastructural localization of CTP:phosphoethanolamine
cytidylyltransferase in rat liver. J Biol Chem 269:15415-8.
187.
Van Veldhoven, P. P., S. Gijsbers, G. P. Mannaerts, J. R. Vermeesch, and V.
Brys. 2000. Human sphingosine-1 -phosphate lyase: cDNA cloning, functional
expression studies and mapping to chromosome 10q22(l). Biochim Biophys Acta
1487:128-34.
188.
Vance, D. E. 2008. Role of phosphatidylcholine biosynthesis in the regulation of
lipoprotein homeostasis. Curr Opin Lipidol 19:229-34.
189.
Vance, J. E. 1988. Compartmentalization of phospholipids for lipoprotein
assembly on the basis of molecular species and biosynthetic origin. Biochim
Biophys Acta 963:70-81.
190.
Vance,
J.
E.
1991.
Newly
made
phosphatidylserine
and
phosphatidylethanolamine are preferentially translocated between rat liver
mitochondria and endoplasmic reticulum. J Biol Chem 266:89-97.
191.
Vance, J. E. 1990. Phospholipid synthesis in a membrane fraction associated with
mitochondria. J Biol Chem 265:7248-56.
192.
Vance, J. E., E. J. Aasman, and R. Szarka. 1991. Brefeldin A does not inhibit
the movement of phosphatidylethanolamine from its sites for synthesis to the cell
surface. J Biol Chem 266:8241-7.
193.
Vance, J. E., and Y. J. Shiao. 1996. Intracellular trafficking of phospholipids:
import of phosphatidylserine into mitochondria. Anticancer Res 16:1333-9.
194.
Vance, J. E., and R. Steenbergen. 2005. Metabolism and functions of
phosphatidylserine. Prog Lipid Res 44:207-34.
195.
Vance, J. E., and D. E. Vance. 2005. Metabolic insights into phospholipid
function using gene-targeted mice. J Biol Chem 280:10877-80.
196.
Vance, J. E., and D. E. Vance. 2004. Phospholipid biosynthesis in mammalian
cells. Biochem Cell Biol 82:113-28.
197.
Vance, J. E., and D. E. Vance. 1986. Specific pools of phospholipids are used
for lipoprotein secretion by cultured rat hepatocytes. J Biol Chem 261:4486-91.
172
198.
Vermeulen, P. S., M. J. Geelen, and L. M. van Golde. 1994. Substrate
specificity of CTP: phosphoethanolamine cytidylyltransferase purified from rat
liver. Biochim Biophys Acta 1211:343-9.
199.
Vermeulen, P. S., Geelen, H.J.M., Tijburg, L. B. and Van Golde, L.M.. 1997
Adv. In Lipobiology 2:287-322.
200.
Vermeulen, P. S., L. B. Tijburg, M. J. Geelen, and L. M. van Golde. 1993.
Immunological characterization, lipid dependence, and subcellular localization of
CTP:phosphoethanolamine
cytidylyltransferase
purified
from
Comparison with CTP:phosphocholine cytidylyltransferase.
rat
liver.
J Biol Chem
268:7458-64.
201.
Voelker, D. R. 2000. Interorganelle transport of aminoglycerophospholipids.
Biochim Biophys Acta 1486:97-107.
202.
Voelker, D. R. 1997. Phosphatidylserine decarboxylase. Biochim Biophys Acta
1348:236-44.
203.
Voelker, D. R. 1984. Phosphatidylserine functions as the major precursor of
phosphatidylethanolamine in cultured BHK-21 cells. Proc Natl Acad Sci U S A
81:2669-73.
204.
Voelker, D. R. 1989. Reconstitution of phosphatidylserine import into rat liver
mitochondria. J Biol Chem 264:8019-25.
205.
Voelker, D. R., and J. L. Frazier. 1986. Isolation and characterization of a
Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for
growth and exhibiting defective phosphatidylserine synthase activity. J Biol Chem
261:1002-8.
206.
Waite,
K.
A.,
and
D.
E.
Vance.
2000.
Why
expression
of
phosphatidylethanolamine N-methyltransferase does not rescue Chinese hamster
ovary cells that have an impaired CDP-choline pathway. J Biol Chem 275:21197202.
207.
Walkey, C. J., L. R. Donohue, R. Bronson, L. B. Agellon, and D. E. Vance.
1997. Disruption of the murine gene encoding phosphatidylethanolamine Nmethyltransferase. Proc Natl Acad Sci U S A 94:12880-5.
208.
Walkey, C. J., L. Yu, L. B. Agellon, and D. E. Vance. 1998. Biochemical and
evolutionary significance of phospholipid methylation. J Biol Chem 273:27043-6.
173
209.
Wang, L., S. Magdaleno, I. Tabas, and S. Jackowski. 2005. Early embryonic
lethality
in
mice
with
targeted
deletion
of
the
CTP:phosphocholine
cytidylyltransferase alpha gene (Pcytla). Mol Cell Biol 25:3357-63.
210.
Watt, M. J., B. J. van Denderen, L. A. Castelli, C. R. Bruce, A. J. Hoy, E. W.
Kraegen, L. Macaulay, and B. E. Kemp. 2008. Adipose triglyceride lipase
regulation of skeletal muscle lipid metabolism and insulin responsiveness. Mol
Endocrinol 22:1200-12.
211.
Weiss, S. B., E. P. Kennedy, and J. Y. Kiyasu. 1960. The enzymatic synthesis
of triglycerides. J Biol Chem 235:40-4.
212.
Wolf, R. A., and R. W. Gross. 1985. Identification of neutral active
phospholipase
C
which
hydrolyzes
choline
glycerophospholipids
and
plasmalogen selective phospholipase A2 in canine myocardium. J Biol Chem
260:7295-303.
213.
Xie,
J.,
M.
Bogdanov,
P.
Heacock,
and
W.
Dowhan.
2006.
Phosphatidylethanolamine and monoglucosyldiacylglycerol are interchangeable
in supporting topogenesis and function of the polytopic membrane protein lactose
permease. J Biol Chem 281:19172-8.
214.
Xu, F. Y., K. O, and P. C. Choy. 1997. Biosynthesis of plasmenylethanolamine
(l-0-alk-l'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine)
in the guinea pig
heart. J Lipid Res 38:670-9.
215.
Xu, Z. L., D. M. Byers, F. B. Palmer, M. W. Spence, and H. W. Cook. 1991.
Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-,
alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells. J Biol
Chem 266:2143-50.
216.
Yamada, K., K. Imura, M. Taniguchi, and T. Sakagami. 1976. Studies on the
composition of phospholipids in rat small intestinal smooth muscle. J Biochem
(Tokyo) 79:809-17.
217.
Yamaji-Hasegawa, A., and M. Tsujimoto. 2006. Asymmetric distribution of
phospholipids in biomembranes. Biol Pharm Bull 29:1547-53.
218.
Yang, H. C , A. A. Farooqui, and L. A. Horrocks. 1996. Characterization of
plasmalogen-selective phospholipase A2 from bovine brain. Adv Exp Med Biol
416:309-13.
174
219.
Yang, H. C , A. A. Farooqui, and L. A. Horrocks. 1996. Plasmalogen-selective
phospholipase A2 and its role in signal transduction. J Lipid Mediat Cell Signal
14:9-13.
220.
Yang, W., C. B. Mason, S. V. Pollock, T. Lavezzi, J. V. Moroney, and T. S.
Moore. 2004. Membrane lipid biosynthesis in Chlamydomonas reinhardtii:
expression
and
characterization
of
CTP:phosphoethanolamine
cytidylyltransferase. Biochem J 382:51-7.
221.
Yorek, M. A., R. T. Rosario, D. T. Dudley, and A. A. Spector. 1985. The
utilization of ethanolamine and serine for ethanolamine phosphoglyceride
synthesis by human Y79 retinoblastoma cells. J Biol Chem 260:2930-6.
222.
Yu, C , Y. Chen, G. W. Cline, D. Zhang, H. Zong, Y. Wang, R. Bergeron, J.
K. Kim, S. W. Cushman, G. J. Cooney, B. Atcheson, M. F. White, E. W.
Kraegen, and G. I. Shulman. 2002. Mechanism by which fatty acids inhibit
insulin
activation
of
insulin
receptor
substrate-1
(IRS-l)-associated
phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 277:50230-6.
223.
Zelinski, T. A., and P. C. Choy. 1982. Phosphatidylethanolamine biosynthesis in
isolated hamster heart. Can J Biochem 60:817-23.
224.
Zha, X., F. T. Jay, and P. C. Choy. 1992. Effects of amino acids and
ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby
hamster kidney-21 cells. Biochem Cell Biol 70:1319-24.
225.
Zhou, J., and J. D. Saba. 1998. Identification of the first mammalian
sphingosine phosphate lyase gene and its functional expression in yeast. Biochem
Biophys Res Commun 242:502-7.
226.
Zhu, L., C. Johnson, and M. Bakovic. 2008. Stimulation of the human CTP:
phosphoethanolamine cytidylyltransferase gene by early growth response protein
1. J Lipid Res.
227.
Zieseniss, S., S. Zahler, I. Muller, A. Hermetter, and B. Engelmann. 2001.
Modified phosphatidylethanolamine as the active component of oxidized low
density lipoprotein promoting platelet prothrombinase activity. J Biol Chem
276:19828-35.
175
APPENDIX
176
Appendix I
F l l R12
f
CMV
B
=^
1
Ml
^-
o*=>
2 3 4 5 6 7 8 9 10 11 12 13
14
V5
M,c
A
BGH
PolyA
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19
M 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 P
M N
N
5
6
8
10 11 12 13
M 34 27 23 20 19
--3EE1I
D
M
13 19 20 23 27 34 P
Figure Al. Generation of Pcyt2 Transgenic Mice. The full length cDNA for mouse
Pcyt2 (a variant) was digested from the pcDNA4 vector (Invitrogen) using Mfel and Psil.
This digestion ensured the presence of the CMV promoter and BGH poly A tail, as
shown by a schematic representation (A). DNA was purified and injected into
blastocysts, which were then implanted into pseudopregnant females. Pups that were born
were weaned at 3 weeks of age and genotyped for the cDNA transgene using a forward
primer specific to ex on 12 and a reverse primer specific to the Myc epitope tag sequence,
unique to the vector and transgene (B). Genotyping was performed on genomic DNA,
177
isolated from the tail. Upon confirmation of the transgene a the genomic level, mRNA
was isolated from the ear punches from the positive mice. cDNA was then derived using
a 3' AP primer for the reverse transcription reaction. Using cDNA from the genomic
positive mice, a genomic negative mouse and a C57B1/6 wildtype mouse, PCR was
performed using two primers specific for Pcyt2 (exon 12 and exon 13) as a positive
control for the RT reaction (C). The next PCR utilized the same genomic primers for
exon 12 and the Myc tag, which will detect any expression of the transgene at the mRNA
level. One mRNA positive was obtained from 12 genomic positive mice (D).
178
Appendix II
13
19
20
23
27
34
Figure A2. Pcyt2-myc expression in Pcyt2-Tg^i livers. Western blot with anti-Myc
antibody, as described in Chapter 5. P= positive control, pure protein.
179
Appendix III. Table Al. List of primer sequences
Pcyt2 primers
Alternate phospholipid pathways
DAG, TG and fatty acid synthesis
Fatty acid oxidation
Cholesterol Homeostasis
Pcyt2-FP
Pcyt2WT-KP
Pcyt2K0-KP
KOcon-FP
KOcon-RP
ETcon FP
ETcon RP
F6
R7
F l l - Pcyt2
R13- Pcyt2
CTaFP
CTaRP
Pisd-FP
Pisd-RP
DGAT1-FP
DGAT1-RP
DGAT2-FP
DGAT2-RP
SREBPlc-FP
SREBPlc-RP
mtGPAT-FP
mtGPAT-RP
PPARg-FP
PPARg-RP
FAS-FP
FAS-RP
ACC1-FP
ACC1-RP
ACC2-FP
ACC2-RP
SCD1-FP
SCD1-RP
Lipin-1 FP
Lipin-1 RP
ChREBP-FP
ChREBP-RP
PPARaFP
PPARaRP
PGC1FP
PGC1 RP
ACOxFP
ACOxRP
SREBP2 FP
SREBP2 RP
HMGCR-FP
HMGCR-RP
180
cctggaactcatgagatcctcctg
atcgcaccacacccgcacga
tgcgaggccagaggccacttgtgtagc
gcaccgctgagcaatggaag
cgattgtctgttgtgcccagtc
cctagaggagattgccaagc
ctgccgtgaacagagaagtc
ggagatgtcctctgagtaccg
ggcaccagccacatagatgac
accatactccgtgacagcgg
ggtgggcacagggcaagggc
ggtggaggagaagagcatcg
ggaagtcttgccagagaagg
gtttgctgtcacgtgcctgtg
cagtgcaagccacatacggg
atccagacaacctgacctaccg
gaccgccagctttaagagacgc
ggctggtaacttccggatgcc
gatcagctccatggcgcaggg
tcacaggtccagcaggtccc
ggtactgtggccaagatggtcc
gacggaggctcgatgaaacccc
agcattctgataacgcctctcgcc
cagaagtgccttgctgtgggg
cttggctttggtcagcggg
cttcgagatgtgctcccagctgc
cttagtgataaggtccacggaggc
ccaggccatgtgggctttggg
ccctttccctcctcctccctc
ctagcctgggcactctgtccc
gaacatctcgtaggcccagcg
cgcatctctatggatatcgcccc
ctcagctactcttgtgactcccg
ccctcgatttcaacgtaccc
gcagcctgtggcaattcacc
ctggggacctaaacaggagc
gaagccaccctatagctccc
gctcacagaatttgccaagg
gtcatccagttctaaggcattg
ggtgttcggtgagattgtagagtg
cttgcttttcccagatgaggg
acattaacagcctggacagcct
tgaatcttggggagtttatct
ggtggacagtgatgtggacttg
gaccaacagcttcacaaagacgc
ggagatcatgtgctgcttcggc
cccaaggaaaccttagcctgctc
id transporters
Pcytl H244Y mutation
FAT/CD36-FP
FAT/CD36-RP
FATP1-FP
FATP1-RP
FATP4-FP
FATP4-RP
FABPpm-FP
FABPpm-RP
Primer A
Primer B
Primer C
Primer D
181
ccccgaggaccacactgtgtc
tggttgtctggattctggagggg
atacttctgggaccaccgggc
gaccttgccgtccatgttggc
gtactatggattccgcatgcgg
gacacgtaccaaacggataggg
gagccaggagaactttgagcc
gttcagtcacggactttatgcc
gatatcgccgccaggatttgcggg
ccacgtgcccgatatagaacaggtcaaagg
cctttgacctgttctatacgggcacgtgg
ctcgaggtcaatctcccctccagg
Appendix IV- Standard Operating Protocol: PCYT2 knockout colony
A/ Generation of Pcyt2 knockout mice. A ~12.5kb region used to construct the targeting
vector was first sub cloned from a positively identified BAC clone using a homologous
recombination based method. The region was designed such that the short homology arm
(SA) extends ~1.4kb 5' to ex on 1. The long homology arm (LA) starts at the 3' side of
exon 3 and is ~8kb long. The Neo cassette replaces ~3kb of the gene including exons 1-3
and the ATG in exon 1.
The targeting vector is confirmed by restriction analysis after each modification step and
by sequencing using primers designed to read from the selection cassette into the SA
(Nl) and the LA (N7). P6 and T7 primers anneal to the vector sequence and read into the
5' and 3' ends of the BAC sub clone. The sequence of the backbone vector is attached at
the end of the document.
N
p H
1
Vector
P
T7
Vector seq
5 6 7
SA
8
9 10111213
LA
8kb
1.4k
b
Primer sequences
Primer Nl 5'-TGCGAGGCCAGAGGCCAGTTGTGTAGC-3'
Primer N7 5'-ATGTGTCAGTTTCATAGCCTGAAG-3'
Primer P6 5'-ATTTAGGTGACACTATAGAACTC-3'
Primer T7 5'-ATTATGCTGAGTGATATCCCTCT-3'
182
BAC Sub Clone Sequencing Data and Alignment with Genomic Sequence
P6 sequencing data from t a r g e t i n g construct
Query: 9205 c g t t t a t a a t a t g t g g t t c t a c a t t t c a a t c a g a c a c a t g a t t t c t t c a t g g t g t a g t c t
llllllllllllllllllllllllllll
Sbjct: 108
III M i l l
9264
111 M 11111111 l l l l l l l
cgtttataatatgtggttctacatttca-tca-acaca-gatttcttcatggngtagtct
Query: 9265 gtaggccacacctccccagtcaaccggcctcttcccgcccccatccctgtcacgcagtct
164
9324
IIIIMIMIIIIIIIIMIIIIIMIIIIIIIIIIIIIIIIMMMIIIIIIMIIII
Sbjct: 165 gtaggccacacctccccagtcaaccggcctcttcccgcccccatccctgtcacgcagtct 224
Query: 9325 gggaaagggaaaagggtctggtccatctgcccctatccatacaagtgagagacggggccc 9384
IIIIIIIIIIIIIIIIMIIMIIIIIIIIMMIIIIIIIIIIIIIIIIIMIIIIIII
Sbjct: 225
Query: 93 85
gggaaagggaaaagggtctggtccatctgcccctatccatacaagtgagagacggggccc
agagttgctagcaagttatccttggaggtgaggccatcctgtggctagcactaccccttc
284
9444
IIIIIMMMIIIIIMIIIIIIIIMIIMIIIIIINIMMIIIMIIIIIIIIII
Sbjct: 285
agagttgctagcaagttatccttggaggtgaggccatcctgtggctagcactaccccttc
Query: 9445 ctatgtgtgcagctcccaccaacagtgggtgagacagaagaggctgtcgagcgagggctg
imilllllllllllllllllMIIIIIIIIIIMMI
Sbjct: 345
344
9504
IIMIIIMIIIIIIMIII
ctatgtgtgcagctcccaccaacagtgggtgagacagaanaggctgtcgagcgagggctg
Query: 9505 ctgctgccctgaagactttcccctgcagcttggtgtcccctgcagcttggtgtcactgac
404
9564
MIIIIIIIIIIIIIIIIIIIIMIIIIIIIIIIIIIIIIIIIIIIMIIIIMIIIIII
Sbjct: 405
ctgctgccctgaagactttcccctgcagcttggtgtcccctgcagcttggtgtcactgac
Query: 9565 gctgcttccagccaaagcccagattaagacataggtgggatggcaggtggcctcagacag
iiiiMiiiiiiiiimiiiiiiiiiiiiiiiiiiiimii
Sbjct: 465
in
ii i n
464
9624
MI
gctgcttccagccaaagcccagattaagacataggtgggatggnaggnggnctcnnacan
524
Query: 9625 ctttcctgtgcctgggaggg 9644
IIIIIII
ilium
II
S b j c t : 525 n t t t c c t g n g c c t g g g a n g g 544
Query: genomic sequence from ENSEMBL
S b j c t : P6 sequencing d a t a from t a r g e t i n g c o n s t r u c t
T7
sequencing data from t a r g e t i n g construct
Query: 20920 tgccttcttctggcctctataggcactgggagaatgcatgtatgcacagaaacgcttgca
Query: 20860 t g c g c a g g t g g c t c a c a g g g c a g t t c a c a t c t g t c t g g a a c t c t a g t t c c a g g g g t c a g a
II II II II II IIIIIIIIIIIIIIIIIIIIIIIIIIIMIIIII II II II II II MI II II II II II II IIIIIIMIIIIIIIIIIIIMIIIIMIIIIIIII II II II MI II II II
Sbjct:
Sbjct: 690
630
20979
20919
tgcgcaggtggctcacagggcagttcacatctgtctggaactctagttccaggggtcaga
tgccttcttctggcctctataggcactgggagaatgcatgtatgcacagaaacgcttgca 631
571
Query: 20980 ggcaaaacatccacacacataaaatgtaaagacattataaagaggaagaaagaaaatgtc
21039
IMIIIIIIIIIIIMIIIIIIIIIIIIIIIIIIIIIMIIMIIIIIIIIIIIIIIIII
Sbjct: 570
ggcaaaacatccacacacataaaatgtaaagacattataaagaggaagaaagaaaatgtc
Query: 21040 ctacaggcctgccctcagcctgatcttacggaggcattttcccaactgaggatcacgcct
511
21099
IIIIIIIIIIIIIIIIIIIIMIIIIIIIIMIIIIIIIIIIIIIMIIIIIIIIIIMI
Sbjct: 510
ctacaggcctgccctcagcctgatcttacggaggcattttcccaactgaggatcacgcct
Query: 21100 ctcagaggactctagttcacatcaagttaacataacatcagccaccacaggggccttgct
451
21159
Sbjct: 450
ctcagaggactctagttcacatcaagttaacataacatcagccaccacaggggccttgct
Query: 21160 tattctttggtcctgtctaggctcaccaacagggtctcactctataacctatattggcct
391
21219
llllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
IIIIIIIIIIIIIIIIIIIIIIIMIIIIIIIIIIIIIIIIIIIIIIIMIIIIIIIIII
Sbjct: 390
tattctttggtcctgtctaggctcaccaacagggtctcactctataacctatattggcct
Query: 21220 cgaacttggagaaaataactcatcttgccaatgcaacaaggttggagttgttttggtgtg
331
21279
IIIIIIIIIIIIIIIIIIIIMIIIIIIIMIIIMIIIIIIIIIIMIIIIIIIMIII
Sbjct: 330
cgaacttggagaaaataactcatcttgccaatgcaacaaggttggagttgttttggtgtg
Query: 21280 gtgctcacggtggaagactctgatgctgggcttgccctttctgaggagaagtgttgcgct
271
21339
IIIMIMIIIMIIIIIIIIIIIMIMIIIIIIIIIIIIIIIIIIIIIIMIIIIIII
Sbjct: 270
gtgctcacggtggaagactctgatgctgggcttgccctttctgaggagaagtgttgcgct
183
211
Query: 21340 caggctccaaccttcagaaaagcctgggcaagcaactctggaccctaggggcaagtaact
21399
IIIIIIIIIMIIIIMMIIIIIIIIIIIIIIIIIIIIIIIIIMIIIIIIIIIIIMI
Sbjct: 210
caggctccaaccttcagaaaagcctgggcaagcaactctggaccctaggggcaagtaact
Query: 21400 gccctgcccacgctacaccagtgggtatggcctaggaaccgtccccacctgtacttagca
151
21459
IMIIIIIIIIIIIMIIIIIIIIIIIIIIIIIIMIIMIIIIIIIII IIIIIIMII
Sbjct: 150
gccctgcccacgctacaccagtgggtatggcctaggaaccgtccccaccagtacttagca 91
Query: 21460 aacagtgactcgaatactggacacaggagggtaacatgtaagagacttgtgg
MIIIIIIIMMIIIIIIIIIIIIIII
21511
IIMIIMIIIIIIIIIIIIII
Sbjct: 90
aacagtgactcgaatactggacacagga--gtaacatgtaagagacttgtgg 41
Query: genomic sequence from ENSEMBL
Sbjct: T7 sequencing data from targeting construct
Please note that alignments are generated from the raw sequencing
(ENSEMBL).
BAC Sub Clone Restriction Analysis
1kb
P
H
X
<•»
P: PstI
H: Hindlll
X: Xbal
S.Okbp
3-Okbp
The restriction patterns agree with that predcited by the ENSEMBL database.
184
data
using
BLAST
Backbone Vector Sequence
3'end of BAC clone joins here
ATCGATGATATCAGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGG
TTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTT
CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTC
ACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA
GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAG
GCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA
CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA
CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAG
CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGA
ACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT
AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATG
TAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTAT
TTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCG
GCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA
AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAA
ACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAA
ATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC
AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTG
ACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAAT
GATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAA
GGGCCGAGCGCAGAAGTGGTCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG
GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGG
CATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGG
CGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT
GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT
ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAG
AATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCAC
ATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGA
TCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC
TTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGG
GAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCA
TTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA
TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCA
TGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATG
ACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGAT
GCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCT
TAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGACATATTGTCGTTAGA
ACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGTGACACTATAC
CTGCAGGCGCGCCATTTAAATGCGGCCGC
5' end of BAC clone joins here
185
PCR screening strategy of recombinant clones
Ten micrograms of the targeting vector was linearized by NotI and then transfected by
electroporation of 129 SvEv iTL embryonic stem cells. After selection in G418, surviving
clones were expanded for PCR analysis to identify recombinant ES clones. Primers Al,
A2 and A3 were designed upstream (5') to the short homology arm (SA) outside the
region used to generate the targeting construct. PCR reactions using Al, A2 or A3 with
the Nl primer at the 5' end of the Neo cassette amplify 1.5, 1.55 and 1.7kb fragments
respectively. The control PCR reaction was done using Nl and primer 1 which is at the 5'
end of the SA inside the region used to create the targeting construct.
Targeting Construct
A1,2,3
{
AT1
AT2
N1
<—
—•
I—
n
nnr
n
Neo
Vector seq
Vector seq
SA
1.4kb
4
5 6 7
8
9 10 111213 14
LA
8kb
Oligos for PCR screening
Al: 5'- ccacattgttgatgttgttattgg -3'
A2: 5'- gtatgttatgtttgtggtgttctg-3'
A3: 5'- tagacttggtgtgtatattgagtg-3'
ATI: 5'- atgatttcttcatggtgtagtctg-3'
AT2: 5'- ccgtgccttaagcattcttaggag-3'
N1: 5' -tgcgaggccagaggccacttgtgtagc-3'
PCR Parameters
94°C 20sec, 62°C 60secs, 72°C 120secs (35 cycles)
Pooled PCR (3x96-well plates)
The positive clones of the pooled samples were identified using Al/Nl primers with the
expected PCR product of 1.5kb (indicated by the arrow). The positive control using
AT1/AT2 gives a band of 1.42kb.
186
Al/Nl
a 2T
ill! I rnrn^ ?M& • —* ^
;:
'::WV^lw^;'-;:;:::\::::-::\.:^^J:;;::';\./
r
""" -">£:i:i:i::: !:.:'tort N
• «';•::
51' n » si JT **s>W
y
^ ** ^
^
u
^ '
Individual PCRs
Individual clones were screened with A2/N1 primers. ES clones #286 & 883 were
identified as recombinant clones.
ES clones #286 and 883 were grown and injected into C57B1/6 blastocyts and injected
into pseudo-pregnant females. Positive pups were identified by agouti colouring and
crossed to C57 wildtype mice to establish germline transmission.
187
B/ The first mice at University of Guelph. Eight mice were shipped from InGenious
Laboratories at Stony Brook, N.Y. All mice were heterozygous for the Pcyt2 gene
(hetero). Five males (#24, 41, 43, 44 and 45) and three females (#34. 48 and 49) were
sent. Upon arrival to the University, the mice were housed in the isolation unit of the
Central Animal Facility (CAF). Due to incompatible housing conditions between the
facilities at Stony Brook and Guelph, the mice were placed in isolation to ensure they
were free of disease.
The mice were past the age at which tail snips could be taken, therefore 200ul of
blood was taken from the saphenous vein and genomic DNA extracted with a Qiagen kit.
PCR was performed as detailed above, however not with success. DNA was amplified
with the Genomiphi kit from Amersham (GE Healthcare), however still, no PCR results
were obtained. A new PCR genotyping scheme was designed, resulting in smaller PCR
products. DNA isolation protocol was also designed for tail snips and ear notches.
DNA Isolation Protocol.
1- Remove 0.5 cm or tail (weanling age) or the full ear notch from notching
2- Incubate in extraction buffer at 55°C o/n
3- Add 250 ul of 5 M NaCl (to precipitate out proteins) and shake gently for 20 min
4- Add 700 ul of chloroform (in fume hood) (this cleans the DNA and ensures good
separation from other debris)
5- Spin at 3000 rpm at 4°C for 10 min
6- Remove aqueous (top) phase (-700 ul) with a 1ml pipette tip (cut the tip off so
that the opening to the tip is larger, a small tip point can shear genomic DNA),
place into new 1.5 ml tube.
7- Add 700 ul of 100% ethanol (to precipitate DNA) and incubate at -20°C for 30
min (cold helps DNA precipitation)
8- Spin at 12500 rpm at 4°C for 10 min and then pour off supernatant (a pellet
should remain, which is the DNA)
9- Add 700 ul-1 ml of 75% ethanol (to wash remaining salts) and spin at 12500 rpm
at 4°C for 5 min
188
10- Pour off supernatant and ethanol dissolve by placing tube upside down (ethanol in
the DNA sample can interfere with downstream applications, so it's important to
remove ethanol... However!! It's also very bad to let the DNA pellet dry out
completely, so be careful. 5-10 min is usually enough, but check each sample)
11-Resuspend DNA pellet in 50 ul of TE pH 8.0 and heat to 55°C to help dissolve
DNA for 10 minutes. Do not vortex DNA. It is possible to gently pipette up and
down to dissolve pellet. Store DNA at 4°C for the short term applications and at 80°C for long term. Repeated freeze/thawing can shear genomic DNA.
Extraction Buffer;
10 mM Tris-HCl pH 8.0, 0.1 M EDTA pH 8.0, 0.5% SDS, 0.1 M NaCl, 10 ug of RNase
A and 200 ug proteinase K. 500 ul of Buffer is added to each sample, then prot K and
RNAse A is added (make master mix!).
PCR Primers and conditions;
FP- CCTGGAACTCATGAGATCCTCCTG
RP- ATCGCACCACACCCGCACGA
Nl- TGCGAGGCCAGAGGCCACTTGTGTAGC
N2F- GCACCGCTGAGCAATGGAAG
N2R- CGATTGTCTGTTGTGCCCAGTC
ET-FP- CCTAGAGGAGATTGCCAAGC
ET-RP- CTGCCGTGAACAGAGAAGTC
94°C for 5 min, then 32 cycles
94°C for 1 min, 60°C for 30 s, 72°C for 1 min and a final extension at 72°C for 10 min.
FP/RP = 450, FP/N1 = 305, N2F/N2R = 339 and ET-FP/ET-RP = 167.
SEE FIGURE 3.1
The PCR genotyping is based on the strategy that a WT mouse will only amplify the 453
bp PCR fragment, which indicates the WT allele, a KO mouse will therefore only amplify
the 305 bp, which indicates the KO allele and a hetero will have both. You must run two
separate PCR reactions for each allele (a combined PCR can be run, but there is primer
and reagent competition, so separate works best), however once the PCR is complete, the
two reactions can be added to the same well in the agarose gel, so that you can see which
alleles are present.
The mice in isolation were the bred together to obtain true KO mice (null animals). The
pups were cross-fostered to another female (foster-mother). This was to ensure that the
189
pups didn't have Helicobacter (the main reason that they were in isolation). If the pups
were clean, then the mothers didn't pass it to them via the birthing process. Two negative
test results were needed to then transfer the mice to the CAF regular housing unit.
C/Life After Isolation and Breeding. The mice were transferred from the isolation unit to
CAF in January of 2005. CAF requires a magnetic key pad to gain access (24 h) and is in
Morgan Fullerton's name (until he leaves, upon which it will be transferred to another's
name). Record sheets of all breedings, procedures and movements of mice is kept outside
of the room. All activities pertaining to the mice must be recorded their (mainly for CAF
record, but also for Pcyt2 mouse records). Breeding is conducted at the CAF.
Breeding:
1- Place male and female together in either a new cage (male first for the day, so that
he can assume dominance and then add female later that day). If a male is housed
individually in his own cage, the female(s) can be added at any time. A male
should not be added to a female's cage, because she will assume dominance (or
can) and breeding will not occur.
2- If plugs are being checked -a vaginal plug is a white, mucus plug in the vaginal
canal, which is the only assumption of conception. If a timed mating is being set
up, or when you need to know the gestational day of the pregnancy, assume that
the morning (7-8:30am) that a plug is identified is gestational day 0.5 (assuming
that the mating happened the previous night). Mice have a 21 day gestation, so
embryonic day X can be determined from there.
3- Usually, plugs are not checked, and so a mouse is deemed pregnant upon visual
inspection. The male can be removed from the cage after pregnancy is confirmed,
however leaving the male in is not detrimental to the pups, and after giving birth,
the mother is immediately in heat again, and the male will breed her that night,
with very high rates for a second pregnancy.
D/Colony Maintenance. After breeding, the mice remain with the mother for 3-4 weeks,
at which point they are weaned and sexed.
1- At 3-4 weeks, each mouse is removed and identified as male or female (sexed)
2- They are placed in a new cage, without the mother (females and males separate)
190
3- Once separated for sex, each mouse is then ear notched (in the past DNA was
obtained from tail tips, however under the new AUP, DNA is to be obtained from
the ear notch. For this reason, a new ear notching scheme has been designated so
that a full ear notch will be obtained from each mouse, below is the old and new
scheme)
4- Mice are placed on top of the cage lid and restrained appropriately
5- Take the ear punch, position it on the ear and quickly and strongly, punch the ear.
This is somewhat painful for the mice and works best for everyone involved if it
works first time, so be confident and firm.
6- The ear notch is collected for DNA genotyping and the ear punch is sterilized in
between each mouse.
The mice are housed at CAF, under the immediate care of the CAF technicians (who do
most of the weaning, sexing and notching). This costs the investigator money per cage,
per day. For this reason, at ~2 months of age (8 weeks), mice are transported from CAF
to the animal wing of the ANNU third floor (rm 379c). Usually:
1- the necessary cages are taken from ANNU to CAF and placed in the loading dock
(must be in a sealed garbage bag)
2- the CAF staff will disinfect the cages and transfer the mice that you deem for
transfer to the new cages
3- Once in ANNU cages, the water bottles and cage card holders are left in the CAF
mouse room and the mice are loaded onto a cart and wheeled to the elevator and
down to the loading dock
4- The mice are placed into the CAF van and transported to ANNU 379c.
Panos Mavronicolas (pmavroni@uoguelph.ca x54309) is the contact at CAF for the van.
191
Main ear notching schematic:
O
02
O
Secondary ear notching schematic:
^3333
Ten is " 1 " on the other ear.
** Important**- the number of pups in each litter dictates the highest notching number,
for example: If a litter has 11 pups, then the pups are separated into male and female
(does not matter which sex goes first), and then are notched 1-11. The next litter is notch
the same way. This places the utmost importance on the proper housing of the mice.
Some colonies are notched 1-500 or higher, but here, we notch each litter and then ensure
that during transportation and weekly cage maintenance and housekeeping, that the mice
are kept in the same cage as the cage card designates. If there is a breakout or a dropping
of multiple cages, then there is no way to tell the number 1 from one litter from the
number 1 from another.
Mice are kept in rm 379c of the HHNS animal wing. Key #48 opens the main
doors to the animal wing, while key #24 opens all doors within the wing, including the
surgery room, supply rooms and the actual mouse room.
Mice are to be monitored daily, with exceptions made on the weekend. Daily
monitoring is for food and water levels as well as overall health. Cages should be
changed every 1.5-2 weeks.
1- Count the number of small and large cages needed for the change (large cages are
the square ones, not the long ones)
2- Lids need only be replaced after 3-4 months
192
3- Bring the new cages to the room on one of the clean carts, however do not bring
the cart into the mouse room. Transfer the empty cages into the room and store on
the floor
4- Return the cart
5- Bed-a-cob is the bedding used for the mice and is found in the storage room, north
of the elevator
6- Begin by placing a layer of bedding into each of the new cages (it's best to set up
the cages on the metal surface and fill them 6 (for large) or 12 (for small) at a
time.
7- Place 2 crumpled paper towels for a large cage and 1 per small cage, along with 4
or 2 nestlett, for large and small cage respectively (nestlett is a white square the
mice can then make nests out of (also found where the bed-a-cob is kept)
8- After bedding all cages, remove the water bottles from the old mouse cages and
place them in the sink.
9- For small cages, it's best to line up 6 new cages against the wall and put the 6 old
cages (with mice inside) in front
10-Lift the lid and transfer the mice from old to new cage, along with the mouse hut
(red little house., if extremely dirty, then new houses are kept under the table, in
the cabinet, however most times, huts can be used many weeks in a row)
11-Top up the food in the cage lid by filling it while the lid is over the old cage...
otherwise the mice have food dust all over them and it can get in their eyes
12-Place lid on new cage and MAKE SURE to transfer the cage card to the new
cage. This comes back to the necessity for accurate cage changes... The card from
the old cage must be placed on the corresponding new cage for the identification
system to hold true.
13-Place the mice back on the shelf (after having swept/wiped the shelf clean, if
necessary)
14-The room is swept, and new water bottles filled and put in place. Large cages use
the large water bottles
15-Lastly, the old water bottles are emptied and placed in the dirty side of the cage
cleaning room, as well, the old cages are emptied of bedding. Empty the bedding
into the garbage that is in the room, but bring it into the hall. Wear a mask while
193
emptying the cages and take the paint scraper from the cage cleaning room to
ensure that the corner and sides of the cages are free of bedding.
16-Take old dirty cages around to the dirty side of cage wash (never through the
clean side) and place in front of loading platform.
That is the cage cleaning process in a nutshell. It is inevitable the mice will become too
old for experiments or simply will do unused. When this happens, we euthanize the mice
with CO2. The animal surgery room is 389.
1- Take the mouse cage to the room and turn on the CO2 tank.
2- Turn the air on and let the tank equilibrate for a minute.
3- Place the mouse inside
4- Suffocation usually takes 30 s
5- Watch that the mouse takes its last breath
6- Carcasses go in -20 walk-in freezer
E/ Pcyt2 Knockout Colony Records. From the founding animals shipped from the US,
there has been an excel file that documents the identification, breeding records etc.
-
Each litter is designated a letter, called the mating ID. That letter (capital)
designates the mating pair that produced the litter and the date of birth. A number
follows the letter, indicating the number of litters that particular mating pair
generates (ex. A2= the second litter from the mating pair designated by "A")
-
With in the letter and litter number designations, the pups are numbered
sequentially from 1 —> n, where n is the total number of pups in the litter
Because there were originally two individual positive clones from which the mice
were generated, a record was kept of which mice were from which clone (#883 or
#286). Since there is no phenotypic difference between the mice, the lines were
mixed and the clone is no longer important.
The AUP letter 'R' or 'G' represents on which AUP the mice are euthanized. Any
mouse euthanized but not used for experimental purposes are under G. The rest of
the mice should be designated R. This makes the yearly audit of how many
animals are used on each protocol easier when the time comes.
194
Weights were filled in earlier in the project, however formal growth curves were
recorded separately and hence, the weights have not been recorded consistently.
Comments, pertaining to how the mice were used may be helpful, but not
necessary for records
Sample of the inventory spreadsheet:
1 ID | Sex
| Genotype | Mating ID | DOB | POD | Age | Weight
| Clone | AUP | Comments
**One person should be responsible for the record and colony maintenance. That is not to
say that cage changing responsibilities should fall on one person, but for the maintenance
of the spreadsheet and dictation of mice used and bred, one person should be responsible.
It is too hard to organize with two people.
F/ Procedures.
Terminal Heart Bleed: requires that you observe the animal take its last breath. After
which, place the mouse in the supine position (on its back) and feel for the solar plexus
(the bottom of the ribs in the middle). Have a lcc needle prepared and insert the needle at
a 10-20° angle. Expel the syringe slightly and look for blood, if none, then withdraw the
needle slightly and re-enter after slightly shifting the needle horizontally. Once you see
blood, pull the syringe until there is no more blood. Make sure that you have the
appropriate tubes for collecting blood (ie. for serum or plasma).
IP Injection: restrain the mouse by placing on the cage lid and grabing behind the neck.
Your grip should ensure that the mouse can't turn its head to bite. With your pinky finger,
restrain the tail and place the mouse so that its underside is face up. Inject the needle on
the right side of the midline, at about the level of the lower limb. Insert the needle at a
20° angle and ensure that it's in far enough that it's not a subcutaneous injection. After
the needle is in, expel the plunger first to ensure that you haven't hit any organs (mainly
the bladder), then plunge the syringe slowly. After, slowly take out the needle and place
mouse back in cage.
Saphenous Vein Blood Collection: restrain the mouse by placing inside a cylindrical
tube (50 ml falcon tube). The right hind leg is left out and you restrain the leg by
195
pinching the skin behind the quad, this ensures that the mouse can't bend its leg. Take the
clippers and shave the thigh, exposing the saphenous vein. Some put a dap of Vaseline or
petroleum jelly on the leg, so that the blood will not spread all over, but stay pooled (this
is a good idea). Plunge the needle into the vein so that the blood vessel is pierced and
then watch the blood pool. Collect the blood via capillary action the collection vials by
placing the animal upside down (use gravity). Flow can be encouraged by gently flicking
the toes and can be stopped by flexing the foot. At one time, only 200 ul of blood can be
taken without euthanizing the animal. Apply pressure with gauze after to stop bleeding
and place mouse in separate cage over night to ensure healing of the wound.
Hepatocyte Isolation: Prepare in advance (store at 4°C)
HEPES BUFFER I (1000ml)
8.3g NaCl
0.5g KCL
2.4g HEPES
pH to 7.4 with 5N NaOH
HEPES BUFFER II (1000ml)
3.9g NaCl
0.5g KCL
0.7g CaCl2 2H 2 0
24g HEPES
ph to 7.6 with 5N NaOH
-
Materials Needed
Collagen (to coat plates) or use Sarstedt Cell+ 6-well plates
WILLIAMS MEDIUM E with L-Glutamine (Gibco; cat# 31500-028; lOxlL)
make according to manufacturer, pH to 7.2
ANTIBIOTIC-ANTIMYCOTIC mixture (Gibco; cat# 15240-0391; 20mL); stored
at -20°C
COLLAGENASE Type IV (Sigma; cat # C5138; for hepatocyte isolation; lg);
stored at -20°C
EGTA
Cell Strainers 70-100uM pore size ( BD Falcon)
Peristaltic Pump using Tubing size 6mm O.D.
Thread, 28 gauge needle
Two surgery clamps, 2 scissors, 2 fine forceps and 1 angle super fine forceps
Day before Perfusion
1) Soak instruments and tubing with 70% EtOH overnight.
196
2) If using 6 well plates, you can coat 6 well dishes or 35mm dishes with 20-25|^g
of fibronectin using a sterile rubber policeman (can just soak in 70% ethanol).
Make sure the material is spread to cover the whole well and allowed to coat
overnight at room temperature.
3) Prepare media:
-450ml Williams' Media E
-50ml FBS
-5ml antibiotic-antmycotic mixture
*add 5ml of L-glutamine if the media is more than one
month old
Day of perfusion
1) Set circulating water bath to 40°C
2) Warm complete media to 37°C
3) Prepare the following solutions:
Collagenase solution - 50mg in 100ml of HEPES BUFFER II
** Oxygenate the buffer 5-10 min before adding collagenase (shake).
b)
EGTA solution - 19mg in 100ml of HEPES I
**Oxygenate the solution 5-10 min after the EGTA has dissolved (shake).
a)
**STERILE FILTER BOTH SOLUTIONS BEFORE USE**
4) Flush tubing with sterile water. Be sure to attach the butterfly needle to the end
of the tubing.
5) Wet pieces of gauze with EGTA and soak suture as well.
Perfusion
1) Use the protocol set out by animal care for "Anesthesia of mice", place mouse
under funnel (attached to isoflorene). 02 should be at 2, and isoflorene at 2.5.
Watch for mouse to loose balance and quickly transfer mouse from under funnel
to the surgical board, switch the gas so that isoflorene is now coming through the
tube into the mouth piece (place mouse head directly inside). Turn isoflorene up
to 3 if needed. Watch for slowed breathing. Mouse has reached surgical plane
anesthesia, when there is no to pinch reflex (pinch the toe).
2) After the mouse goes down, pin it to the dissecting board and spray wash with
70% EtOH.
3) Cut open the abdomen and pin the flap down. With a piece of wet gauze or
kimwipe, push the gut to the right to expose the inferior vena cava. By pushing
197
the liver anteriorly, the portal vein will be exposed. TAKE CARE NOT TO NICK
THE LIVER!!
4) Slip the wet suture under the lower vena cava and make a loose knot. Insert the
butterfly needle into the vena cava (try to place the needle midway and not too far
up or down). While weighing it down in place with a pair of forceps, pull the
loose knot tight onto the needle. QUICKLY cut open the chest cavity, clamp the
upper vena cava and aorta and snip the portal vein. While you snip the portal
vein, turn on the peristaltic pump, which is set at a flow rate of 8ml/min. Perfuse
the liver with 50ml of ETGA. The liver should turn pale right away.
5) Switch to COLLAGENASE and perfuse at a flow rate of 6ml/min. The liver
should enlarge and become 11/2 times the original size. Perfuse the liver with
50ml of collagenase.
6) Carefully cut the liver out and put into a sterile Petri dish containing 10 ml
Williams' Media E. Take to tissue culture hood. Gently tease the liver cells out of
the sac with two fine forceps. Transfer the cells, WITHOUT LETTING AIR
BUBBLES INTO THE PIPET, to the sterile bottle with the cell strainer (to filter
cells). Rinse the Petri dish with media a few times and transfer to the same bottle
(should be no more than 5ml). Centrifuge at 1000 rpm for 5-7 minutes.
7) Discard supernatant and resuspend the cells in 30ml of media (CAREFULLY
PIPET UP AND DOWN WITHOUT LETTING AIR BUBBLES INTO THE
PIPET; ie: pipette up 10ml and only let out 9ml of resuspended cells, go up and
down a few times.) Centrifuge at lOOOrpm for 5-7 minutes.
8) Discard supernatant and resuspend the cells (as above) in 20ml of complete media
(Williams' media with 10% FBS and 1% antibiotic/antimycotic mixture).
9) Do TRYPAN BLUE EXCLUSION METHOD: Put 100|iL of filtered cell
suspension into 800 uL of media and 100|iL of Trypan Blue. Do a cell count in a
hemocytometer (4x4 grid). Count both live and dead cells and calculate the
percent of viability. 75% and up are usable. Multiply the average b 10,000 and
the dilution factor (10).
10) Generally plate between 1-1.5 106 cells per well (6 well dishes) if you want them
at least 85% confluent the next day. Otherwise plate at whatever density you
want. The 6-well dishes hold 2ml of media. Plate in complete media (see # 8).
11) Incubate for a minimum of 2 hours before removing dead cells. Rinse with lmL
and replace with 2mL of fresh complete media. Incubate overnight before using
for experiments.
198
Immunohistochemistry Protocol - Paraffin Embedded Tissue (Adapted from A
Bendall)
Preparation
Time:
Two Day Protocol (Day 1: 2hr 45min; Day 2: 2hr 15min)
Slides:
Remove treated slides from 4°C and bring to room temperature before
initiating IHC
Reagents:
Methanol/3% H 2 0 2
-Add 5 ml of 30% H 2 0 2 to 45 ml Methanol
Citrate Buffer
-Mix 57 ml of 0.1M Citric Acid with 249 ml 0.1M
Sodium Citrate and add 300 ml dH 2 0; mix well
- pH to 6.0 with NaOH if required
PBT
-To desired volume of IX PBS (500ml) add 0.2%
BSA (l.Og) and 0.5% Triton-X-100 (2.5 ml)
Blocking Solution
-To 10 ml PBT add 135 ul Goat Serum (2° origin)
PBS/0.1% Tween 20 -To 50 ml IX PBS add 50 ul Tween 20 and mix
PBS/1% BSA
-Dissolve 0.5g of BSA in 50 ul IX PBS
1° and 2° Antibody
DAB (Vector Kit)
-Dilute required volume of 1° or 2° in PBS/1% BSA
-To 5 ml dH 2 0 add 2 drops of Buffer stock solution
-Add 4 drops of DAB stock solution and mix well
-Add 2 drops Hydrogen Peroxide solution and mix
-DAB solution will stain brown but can add 2 drops
of Nickel solution for grey/black staining
0.1 % NH4OH
-Dilute 1.43 ml of 3.5% Ammonium Hydroxide in
48.57mlofdH 2 0
1 % HCl/70% EtOH
-Dilute 0.5 ml of HC1 in 49.5 ml of 70% EtOH
Deparaffinization and Hydration
Submerge slides in the following series of solutions:
Xylene
Xylene
100% EtOH
10 minutes
10 minutes
2 minutes
199
100% EtOH
Methanol/3% H 2 0 2
100% EtOH
95% EtOH
70% EtOH
50% EtOH
30% EtOH
Tap H 2 0
dH 2 0
2 minutes
20 minutes
2 minutes
2 minutes
2 minutes
2 minutes
2 minutes
5 minutes
5 minutes
Antigen Retrieval
Utilizing the hotplate, preheat the Citrate Buffer, pH 6.0 to 95°C before treatment:
Citrate Buffer, pH 6.0 at 95°C
Cool Down
PBT
PBT
15 minutes
15 minutes
5 minutes
5 minutes
Blocking
Place the slides on soaked Whatman paper overtop damp paper towel and perform the
blocking step within a 37°C incubator (use a PAP pen to keep the solution on the tissue
section):
Blocking Solution
PBS/0.1% Tween 20
PBS/0.1% Tween 20
30 minutes
5 minutes
5 minutes
Primary Antibody
Perform 1° Antibody binding within the cold room (4°C) within a moist chamber made
out of damp paper towel:
1° Antibody
PBS/0.1% Tween 20
PBS/0.1% Tween 20
PBS/0.1% Tween 20
Overnight
5 minutes
5 minutes
5 minutes
Secondary Antibody
Utilizing a 2° designed to target the 1°, perform the binding at room temperature within a
moist chamber:
2° Antibody
PBS/0.1% Tween 20
PBS/0.1% Tween 20
60 minutes
5 minutes
5 minutes
200
PBS/0.1% Tween 20
5 minutes
Antigen Visualization
Flood the slides individually with DAB solution, and stop the reaction when the target
area is significantly stained:
DAB Solution
dH 2 0
4 minutes (or until adequately stained)
5 minutes
Counterstaining and Dehydration
(Adapted from A. Vince)
Harris' Hematoxylin
Rinse with cold tap H 2 0
l%HCl/70%EtOH
Rinse with cold tap H 2 0
0.1% NH4OH
Rinse with cold tap H 2 0
dH20
30% EtOH
50% EtOH
70% EtOH
95% EtOH
100% EtOH
Xylene
Xylene
1-2 minutes
4-5 dips
4-5 dips
5 minutes
2 minutes
2 minutes
2 minutes
2 minutes
2 minutes
5 minutes
5 minutes
Mounting
Mount slides with one or two drops of Permount and cover with a coverslip
Gently invert slides and press on the reverse side of the slide to remove any air bubbles
Clean excess glue with Chloroform and a Q-Tip if required
Store slides in the dark to prevent bleaching of the DAB stain
201
Документ
Категория
Без категории
Просмотров
0
Размер файла
2 676 Кб
Теги
sdewsdweddes
1/--страниц
Пожаловаться на содержимое документа