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Pathophysiology of Diabetes How to study it and whats the brain

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Pathophysiology of Diabetes
How to study it and what
what’ss the brain
got to do with it?
Christoph
p Buettner, MD, PhD
Euglycemic clamp study
• How do you test insulin action?
• Put the pancreas to rest (somatostatin) and
infuse a set rate of insulin
• The amount of glucose that you need to
infuse to prevent the animal from becoming
hypoglycemic (Glucose Infusion Rate,
Rate
GIR) is a direct measure of insulin action
Physiological effects of insulin
Insulin
li Receptor Knockk Out
Models
Humans lacking insulin receptors show
severe intrauterine growth retardation,
failure to thrive, lipodystrophy, and
hypoglycemia
• "tissue-specific" knockouts are really “Cre
ppromoter-specific."
p
(p
(pattern and extent of
tissue recombination is dependent on the
p
y of the promoter
p
used to drive Cre
specificity
expression).
Interactive Student Presentations
•
•
•
•
•
•
Groups:
Accili et al.
al – Group 1
Bluher et al. – Group 2
B i et al.
Bruning
l 2000 --Group
G
3
Bruning et al. 1998 – Group 4
Michael et al. – Group 5
Early neonatal death in mice
homozygous for a null allele of the
insulin receptor gene
A ili ett al.
Accili
l 1996
General Approach
• Knockout Insulin receptor in whole body
• Watch what happens
Results
• Death between 48-72hrs postnatal of
– hyperglycemia and ketoacidosis
• -/- mice have high plasma insulin
Results
High insulin or IGF1 in brain lead to phosphorylation of
the insulin receptor, IGF-1 receptor, and the IR & IGF-1r
precursors
Results
Discussion
• H
Hyperglycemia
l
i
– No insulin signaling for glucose → glycogen, liver
produces
d
glucose
l
• Ketoacidosis
– Trick starvation – fatty acid breakdown
• Differences between mouse and human
– Human: severe fasting hypoglycemia, no keto
NIRKO
PSB Core I,
I Diabetes
Knockout Mouse Made and Specific
t Brain's
to
B i ' IR
Mice Get Fat; Leptin Resistance
Elevated Blood Glucose +
Triglycerides, Insulin Insensitivity
Fertility Problems from Reduced LH
( till sensitive
(still
iti tto LH)
Conclusions
•“IR in the CNS plays an important functional
role in the regulation of energy homeostasis and
reproductive endocrinology.”
•Increased body
y weight
g
•Insulin and leptin resistance
•Fertility problems from reduced LH
•GnRH mechanism in the hypothalamus?
Adipose Tissue Selective Insulin
R
Receptor
t Knockout
K k t Protects
P t t Against
A i t
Obesity
y and Obesityy – Related
Glucose Intolerance
Bluher, Michael, Peroni, Ueki,
Carter, Kahn, Kahn
Developmental Cell, 2002 3:25-38
Introduction & Background
• Question:
• What is the role
of adipose tissue
in insulin
signaling
i li andd
glucose
homeostasis?
Moller, Nature, 2001
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
MIRKO
LIRKO
Effects of an acute KO of the IR in
liver
li
Introduction
–Increased hepatic glucose production (GP)
is the main cause of fasting hyperglycemia
in patients with diabetes.
–The mechanisms by which insulin controls
GP involve both direct and indirect effects.
–Life-long absence of insulin signaling in
li
liver
leads
l d to
t severe hepatic
h
ti insulin
i
li
resistance.
–Hypothalamic insulin signaling is required
for inhibition of GP.
Rapid Actions of Insulin on GP:
–DIRECT effects, mainly on glycogenolysis
–INDIRECT
INDIRECT effects
ff t via
i li
lipolysis/glucagon
l i / l
–INDIRECT effects via the hypothalamus
yp
Long-term Actions of Insulin on GP:
–DIRECT effects on gene expression
–INDIRECT effects on gene expression
Defects in the long
long-term
term actions of insulin can
in turn have a major impact on the rapid
actions of insulin
Acute down-regulation of the hepatic
insulin receptor by antisense
Buettner et al,J. Clin. Invest. 115:1306-1313 (2005).
Liver histology is unaffected by IRASO treatment
Buettner et al,J. Clin. Invest. 115:1306-1313 (2005).
Euglycemic clamp studies of IR ASO treated mice
Buettner et al,J. Clin. Invest. 115:1306-1313 (2005).
• The acute down regulation of hepatic IR
expression by up to 95% does not result in
hepatic insulin resistance.
• Thus, the rapid control of glucose
production by insulin occurs mainly at
extra-hepatic sites.
• Livers of rats that have been on a high fat
diet ((and in vivo are insulin resistant)) react
normally in vitro to the ability of insulin to
suppress
pp
epinephrin
p p
induced
glycogenolysis.
• Metabolic inflexibility vs insulin resistance
Summary of tissue specific insulin
receptor KO
Insr knockout
Phenotype
Constitutive
Diabetic ketoacidosis
Muscle
Dyslipidemia
Muscle/adipose tissue
Impaired glucose tolerance
Adipocyte
Protection against obesity
Prolonged life span
Liver
Severe insulin resistance
Transient hyperglycemia
Гџ-cell
Impaired glucose tolerance
Brown adipose tissue
Гџ-cell failure
Central nervous system
Obesity, sub-fertility
Endothelium
moderately decreased vascular tone
Insulin sensitivity decreased on a low-salt
diet
Limits of tissue specific KO models
• Promoter specificity
• Developmental effects
• Phenotype not direct consequence of the
tissue KO, but due to change in organ
crosstalk
Organ Crosstalk
• Organs communicate with each other
• Changes in one organ may have profound
effects of the function of another (limits the
conclusions of the tissue specific role of one
protein ie)
Uno, Science 16 June 2006
Uno, Science 16 June 2006
Uno, Science 16 June 2006
Uno, Science 16 June 2006
Yamada, Cell Met, March 2006, Pages 223-229
Yamada, Cell Met, March 2006, Pages 223-229
Yamada, Cell Met, March 2006, Pages 223-229
Yamada, Cell Met, March 2006, Pages 223-229
Brain Control of Metabolism
• Which metabolic functions are regulated by
the brain and how?
• What are the input parameters that the brain
assesses?
•
Organ crosstalk
The Brain as the conductor of the Orchestra
Leptin
Leptin
Food intake
Leptin
Food intake
Insulin
GP
Leptin
Food intake
Insulin
FA oxidation
GP
Leptin
Food intake
Insulin
Leptin
Leptin
GP
Insulin
FFA
GP
Insulin
KATP Blocker
KATP
KATP
Activator
Insulin
ARC
PI3K
PIP3
Hypothalamu
s
PIP2
GLUCOSE
PRODUCTION
Pocai et al 2005, Nature
Vagus Nerve
Stat3 dependent and independent
pathways of leptin
Leptin
Food Intake
Liver Glucose Production
PPARОі
SREBP1c
Luteinizing Hormone
LPL
SCD1
FAS
ACC
Nutrient sensing in the brain
TCA cycle
PPARО±
Citrate CPT1
Citrate
AMPK
ACC
Acetyl-CoA
MCD
Malonyl-CoA FAS Palmitate
?Fatty acid
Buettner, Cell Met. 2007
Food intake
rd
3 ventricle
VMN
Arc
insulin/leptin signaling
g
g
cascade
Nucleus solitarius
Nutrient sensing
g converging
g g
onto malonyl CoA
K(ATP) channel activation
G ucose fluxes
Glucose
u es
VLDL secretion
Vagus
Sympathetic NS
Hormones??
Additional players:
Adiponectin
Resistin
VMN
IL6
IL1 beta
CNTF
GLP1
Ghrelin
Food intake
rd
3 ventricle
Arc
insulin/leptin signaling
g
g
cascade
Nucleus solitarius
Nutrient sensing
g converging
g g
onto malonyl CoA
Neuronal:
Afferent connections
Efferent connections
K(ATP) channel activation
Glucose fluxes
VLDL secretion
Vagus
Sympathetic NS
Pituitary hormones??
Summary
• Hypothalamus integrates hormonal, neuronal and
nutrient clues from the periphery to regulate energy
homeostasis accordingly
• Failure of the central control of metabolism
contributes to the metabolic inflexibilty associated
with insulin resistance and obesity
• Need for multiple approaches to understand
physiology
How to find out if and where a
protein is expressed in the brain?
• Unigene: expression analysis, gives you an
idea in what tissue ggene is expressed
p
• Check Allan Brain Atlas (in situ of all
mouse genes)
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