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Subcellular Organization of Intermediary
Metabolism in the Hepatopancreas of the
Terrestrial Snail, Cepaea nemoralis: A Cytosolic
Department of Zoology, University of Guelph, Guelph, Ontario,
N l G ZWl, Canada
The subcellular distributions of key enzymes of the tricarboxylic acid cycle, electron transport chain, ketone body, amino acid, and carbohydrate metabolism were studied in the
hepatopancreas of the terrestrial snail Cepaea nemoralis. The presence of mitochondria1 carnitine
octanoyl transferase, carnitine palmitoyl transferase, and 3-hydroxyacyl-CoAdehydrogenase indicate an active lipid catabolic pathway in this tissue. Activities of enzymes of ketone body metabolism are similar in magnitude to those of carbohydrate metabolism, suggesting an important
metabolic role for ketone bodies in C. nemoralis. Two enzymes that, in mammals, catalyze the
synthesis of ketone bodies, hydroxymethylglutaryl-CoA synthase and hydroxymethylglutaryl-CoA
lyase, are not detectable in Cepaea hepatopancreas. The activity of 3-oxoacid-CoAtransferase and
a portion of acetoacetyl-CoA thiolase activity are found in the mitochondria. f3-hydroxybutyrate
dehydrogenase is a cytosolic enzyme in this tissue, and preferentially oxidizes the 1,-isomer of phydroxybutyrate. A portion of glutamate dehydrogenase activity is also cytosolic. The subcellular
organization distribution of enzymes of intermediary metabolism in Cepaea hepatopancreas suggests adaptation to periodic anoxia. @ 1996 Wiley-Liss, Inc.
The study of intermediary metabolism in gastropod molluscs has been generally biased toward
carbohydrate-based metabolism in marine species
(see Livingstone and de Zwann, '83 for review).
The metabolism of terrestrial snails is believed
t o be carbohydrate based, and carbohydrate metabolism has been shown t o play a key role in sustaining estivation (Rees and Hand, '90, '93).
However, relatively little is known about the participation of other energy substrates in the intermediary metabolism of gastropods, especially in
non-marine species. The organization and roles
of lipid and ketone body metabolism in these organisms have not been examined in detail. A single study
by Meyer et al.('86) examined the metabolism of ketone bodies, acetoacetate (Am),and p-hydmqbutyrak
(BHB), in the freshwater gastropod Biomphalaria
glabruta. High activities of enzymes of ketone body
metabolism in various tissues and hemolymph levels of Acac and BHB suggest a significant role
for ketone bodies in routine energy metabolism
in B. glabruta. In the hepatopancreas of this
freshwater snail, the enzymes of ketone body
metabolism including 0-hydroxybutyrate dehydrogenase (BHBDH),are localized within the mitochondria (Meyer et al., '86).
In preliminary studies of ketone body metabolism in the terrestrial snail Cepaea nernoralis, we
found that BHBDH, which in virtually all animals
exists bound to the inner membrane of the mitochondrion, is cytosolic in hepatopancreas. We thus
further examined the subcellular distribution of
enzymes of ketone body metabolism in this tissue. To determine the relative importance of ketone
body metabolism in the intermediary metabolism
of this terrestrial gastropod, we also measured the
activities and subcellular distributions of enzymes
of the tricarboxylic acid cycle, the electron transport chain, and carbohydrate, amino acid, and lipid
metabolism in hepatopancreas.
Experimental animals
C. nernoralis were collected on the University
of Guelph campus in the spring of 1994. Snails
were kept in a laboratory terrarium at 20" f 2°C
for up t o 1week prior to experimentation and fed
Received August 22, 1995; revision accepted January 15, 1996.
Address reprint requests to J.S. Ballantyne, Department of Zoology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
ments Inc., Saddlebrook, NJ). Rates of reactions
involving NADH were followed at 340 nm (millimolar extinction coefficient &a40 = 6.22). Reactions
involving 5,5’-dithio-bis-(2-nitrobenzoicacid)
Tissue preparation
(DTNB) were followed at 412 nm (millimolar exSnails were deshelled, the hepatopancreas was tinction coefficient ~ 4 1 213.6). Acetoacetyl-CoA
excised, a n d t h e intestine was removed and thiolase, 3-oxoacid transferase, and hydroxyplaced immediately in 3 mL of ice-cold mito- methylglutaryl-CoA synthase were measured by
chondrial isolation buffer containing 20 mM N- following the accumulation or disappearance of
[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acetoacetyl-CoA at 303 nm (millimolar extinction
acid] (HEPES) and 100 mM sucrose, pH 7.5. All coefficient &303 13.8). Enzyme activities are extissue preparation procedures were carried out on pressed as units per gram of wet tissue wet where
ice, Hepatopancreas was homogenized with three 1 unit equals 1 pmol of substrate converted to
passes of a Potter-Elvejhem homogenizer with a product per minute. For convenience of compariTeflon pestle attached to a drill press, operating son, enzyme activities measured in isolated mitoat c 100 rpm. The homogenate was centrifuged chondria were also expressed as activities per
at 200g for 10 minutes to remove cell debris and tissue wet weight. All enzyme assays were buffnuclei. A 1 mL sample of this supernatant, the ered in 50 mM imidazole, adjusted to appropriate
“whole homogenate,” was taken and treated as pH with KOH and HC1. The following optimal asdescribed below. The remaining homogenate was say conditions were determined with respect to
centrifuged at 10,OOOg for 10 minutes. The result- substrate and cofactor concentrations:
a n t pellet, the “mitochondrial fraction,” was resuspended in 1 mL of mitochondrial isolation TCA cycle:
buffer and treated as described below. This fracMalate dehydrogenase (MDH) (E.C.
tion contained virtually all mitochondria, based
0.15 mM NADH, 5 mM oxaloacetate (omiton activities of cytochome C oxidase, and citrate
ted for control), pH 7.5
synthase. The supernatant from this step was cenCitrate synthase (CS) (E.C. 0.1 mM
trifuged at 40,OOOg for 10 minutes to remove nonDTNB, 0.3 mM acetyl-CoA, 0.5mM oxaloacsoluble materials; it yielded a pellet too small to
etate (omitted for control), pH 8.0
use for enzyme determinations. The resultant su- Electron transport chain
Cytochrome c oxidase (CCO) (E.C. 50
pernatant was considered the “cytoplasmic fraction.” A 1mL sample of this fraction was collected.
pM reduced cytochrome C (omitted for control), pH 8.0
All fraction samples were sonicated with five
bursts of 5 seconds at 60% power output, 25 W, Lipid metabolism
on a Vibra-Cell sonicator (Sonics & Materials Inc.,
3-Hydroxyacyl-CoA dehydrogenase (HOAD)
(E.C. 0.2 mM NADH, 0.1 mM aceDanbury, CT) and subsequently used in enzyme
toacetyl-CoA (omitted for control), pH 8.0
Carnitine palmitoyl transferase (CPT) (E.C.
Enzyme assays 0.1 mM DTNB, 50 pM palmitoylCoA, 100 mM KCl, 5 mM L-carnitine (omitOptimal substrate and cofactor concentrations
were determined for all enzymes assayed with the
ted for control), pH 8.0
Carnitine octanoyl transferase (COT) (E.C.
supernatant of a crude homogenate of C. nemoralis 0.1 mM DTNB, 50 pM octanoylhepatopancreas. For these determinations, hepatoCoA, 100 mM KC1, 5 mM L-carnitine (omitpancreases of deshelled snails were excised, placed
in 2 mL of ice-cold 50 mM imidazole, homogenized
ted for control), pH 8.0
Malic enzyme (ME) (E.C. 1.0 MgC12,
with three passes of a Polytron PTlO tissue ho0.4 mM NADP, 1mM of either D- or L-malate
mogenizer (Knematica Gmbh., Lucerne, Switzerland), and centrifuged at 10,OOOg.
(individually) (omitted for control), pH 7.5
ATP-citrate lyase (ATPCL) (E.C. 20 mM
Maximal enzyme activities were determined usMgC12, 0.1 mM NADH, 1 mM DTT, 0.4 mM
ing a Hewlett Packard HP8452 diode array specCoA, 10 mM ATP, 25 units MDH, 20 mM citrophotometer (Hewlett Packard, Mississauga,
trate (omitted for control), pH 8.0
Ontario, Canada), equipped with a thermostated
Isocitrate dehydrogenase (ICDH) (E.C.
cell changer maintained at 20°C with a Haake D8
5 mM MgC12, 0.4 mM nicotinamide adenine
circulating water bath (Haaek Buchler Instrulettuce ad libitum. Humidity within the terrarium
was kept between 50% and 100%with moist sphagnum moss.
diphosphate (NADP), 0.5 mM threo-DsLsisocitrate (omitted for control), pH 8.0
Ketone body metabolism
Acetoacetyl-CoA thiolase (AAT) (E.C.
10 mM MgC12, 0.16 acetoacetyl-CoA, 0.2 mM
CoA (omitted for control), pH 8.0
3-Oxoacid-CoA-transferase: 0.4 mM succinylCoA, 5 mM MgC12, 5 mM iodoacetamide, 10
mM acetoacetate (omitted for control), pH 8.0
BHBDH (E.C. 2 mM DTT, 2 mM NAD,
40 mM DL-P-hydroxybutyrate (omitted for
control), pH 8.0
Hydroxymethylglutaryl-CoA synthase (HMS)
(E.C. (modified from Quant et al., '89):
10 mM MgC12,2 mM DTT, 5 mM acetyl phosphate, 10 pM acetoacetyl-CoA, 100 pM acetylCoA and 10 units phosphotransacetylase
(PTA) (both omitted for control), pH 8.0
Hydroxymethylglutaryl-CoA lyase (HML) (E.C. (modified from Ballantyne and Berges,
'91): 10 mM MgC12, 0.2 mM NADH, 1 unit
BHBDH, 0.2 mM HMG-CoA (omitted for control), pH 8.0
Amino acid metabolism
Glutamate dehydrogenase (GDH) (E.C.
250 mM ammonium acetate, 0.1 ,mM EDTA,
0.05 mM NADH, 1 mM ADP, 7 mM a-ketoglutarate (aKG) (omitted for control), pH 8.0
Glutamate-pyruvate transaminase (GPT) (E.C. 200 mM L-alanine, 0.2 mM NADH,
0.025 mM pyridoxal-5'-phosphate, 1unit lactate dehydrogenase (LDH), 10.5 mM aKG
(omitted for control), pH 7.5
Glutamate-oxaloacetate transaminase (GOT)
(E.C. 0.2 mM NADH, 0.25 mM pyridoxal-5'-phosphate, 30 mM aspartate, 1
unit MDH, 3.5 mM aKG (omitted for control), pH 7.5
Carbohydrate metabolism
LDH (E.C. 0.4 mM NADH, 1 mM
pyruvate (omitted for control), pH 7.5
Pyruvate kinase (PK) (E.C. 10 mM
MgC12,0.4 mM NADH, 1.3 mM ADP, 2 units
LDH, 10 mM phosphoenolpyruvate (omitted
for control), pH 7.5
Hexokinase (HK) (E.C. 10 mM MgC12,
0.16 mM NADP, 1mM ATP, 2 units glucose6-phosphate dehydrogenase, 10 mM D-glucose (omitted for control), pH 7.5
Glucose-6-phosphate dehydrogenase (G6PDH)
(E.C. 7 mM MgC12,0.4 mM NADP,
1 mM glucose-6-phosphate (omitted for control), pH 7.5
a-Glycerophosphate dehydrogenase (aGPDH)
(E.C. 0.2 mM NADH, 0.4 mM dihydroxyacetone phosphate (omitted for control),
pH 8.0.
The maximal activities of BHBDH with the Dor L-stereoisomers were determined. BHBDH activity with each isomer is the mean of six measurements, determined on homogenates prepared
as described above.
The levels of enzymes of various metabolic pathways and their proportional distribution between
mitochondria and cytosol provide a qualitative
view of the metabolic organization of C. nemoralis
The activities of all enzymes measured in the
hepatopancreas are presented in Table 1.The percentage of this activity recovered in the summed
mitochondrial and cytosolic fractions, and the percentage of this amount appearing in either the
mitochondrial or cytosolic fractions individually,
are presented in Table 2.
TABLE 1 . Maximal activities of enzymes in
C. nemoralis heDatoDancreas'
Whole homogenate activitv
TCA cycle
Oxidative metabolism
Ketone body metabolism
Lipid metabolism
Amino acid metabolism
Carbohydrate metabolism
0.629 & 0.089
8.848 f 3.787
40.718 f 7.535
2.676 2 0.432
3.909 2 0.810
0.795 2 0.202
0.825 2 0.096
0.032 2 0.020
0.149 2 0.043
0.296 0.051
0.067 2 0.025
0.198 2 0.051
3.263 2 0.933
11.653 +: 0.830
0.420 2 0.051
0.298 f 0.024
4.264 f 1.325
2.072 f 0.761
1.394 2 0.216
'Activities (pmollminig wet tissue weight) are means of six separate
determinations, f SE. ND, not detected.
TABLE 2. Recovery of whole homogenate enzyme activities in summed mitochondrial and cytosolic fractions and distribution
of enz.yme activities between mitochondrial and cytosolic fractions'
% of recovered
recovered in
summed fractions
activity in
9k of recovered
activity in
104.9 9.1
87.8 9.2
76.2 f 11.9
90.6 f 2.1
22.9 0.9
17.7 1.0
9.4 f 2.1
77.1 -1- 0.9
82.3 2 1.0
98.9 r 6.6
98.2 2 6.6
1.8 2 0.3
133.7 f 17.6
108.7 2 11.9
150.6 f 37.0
33.3 f 6.5
92.8 f 1.4
66.7 -1- 6.5
7.2 2 1.4
57.0 2 18.0
107.8 2 29.0
200.4 2 65.1
83.4 2 8.0
74.3 2 11.5
16.6 2 8.0
25.7 11.5
129.2 2 30.7
100.1 f 3.8
96.3 7.8
59.4 2 8.4
28.6 3.1
23.1 1.3
40.6 f 8.4
71.4 f 3.1
76.9 2 1.3
117.0 f 10.9
84.9 f 14.6
103.5 f 15.4
92.4 27.5
88.4 f 7.2
8.8 4.2
3.4 f 0.9
10.5 0.3
2.1 f 0.6
90.2 f 4.8
96.6 z 0.9
89.5 0.3
97.9 f 0.6
% Activity
TCA cycle
Oxidative metabolism
Ketone body metabolism
Lipid metabolism
Amino acid metabolism
Carbohydrate metabolism
'Values represent average percent recovery of six separate determinations. ND, not determined.
TCA cycle
Enzymes that can provide substrate and reducing
equivalents (NADPH) to fuel cytoplasmic lipid
CS and MDH activities are detectable in the
were examined. Malic enzyme, which
mitochondrial fraction of the hepatopancreas. Alcatalyzes
formation of pyruvate from malate
though CS appears t o be exclusively mitochondrial
with the concomitant generation of NADPH, is un(91% in mitochondrial fraction), most of the MDH
detectable in all cellular fractions. NADP+-depenactivity is cytosolic (82%).
dent ICDH also provides NADPH for cytosolic lipid
The relatively high ICDH activity is priElectron transport chain
marily cytosolic, with a small proportion of total
CCO activity is exclusively mitochondrial, with activity detected in the mitochondrial fraction.
98% of its activity in this fraction.
ATPCL catalyzes what is believed to be an important step in lipid synthesis in some biological sysLipid metabolism
tems (Newsholme and Leech, '83), the production
The enzymes involved in the transport and
of acetyl-CoA from citrate produced in and exported
P-oxidation of fatty acids are substantially mi- from the mitochondria. ATPCL is present in approxitochondrial. CPT and COT, which catalyze the mately equal proportions in the mitochondrial and the
carnitine-dependent transport of long and me- cyt~solicfractions. The summed activities in these two
dium chain fatty acids, respectively, into the mi- fractions is about twice that measured in the whole
tochondrion, are not detectable in the cytosolic homogenate. The basis for this is not known.
fraction. HOAD catalyzes the P-oxidation of fatty
Ketone body metabolism
acids. HOAD is primarily a mitochondrial activActivities of BHBDH, AAT, and 30AT are of the
ity, with 16.6% of recovered activity appearing in
the cytosolic fraction.
same order of magnitude as those of key enzymes
(PK and HK) of carbohydrate metabolism. BHBDH,
normally a mitochondrial membrane-bound enzyme,
is exclusively cytosolic in C. nemoralis hepatopancreas, with no detectable mitochondrial activity.
30AT, which catalyzes the reversible oxidation of
acetoacetate, is mitochondrial, with less than 10%
appearing in the cytosolic fraction. AAT produced
acetoacetyl-CoA from acetyl-CoA. AAT activity is
largely cytosolic, with about 30% of total activity
appearing in the mitochondrial fraction. HMS and
HML catalyze the synthesis of acetoacetate from
acetoacetyl-CoA in mammals, through the transient production of hydroxymethylglutaryl-CoA.
Neither of these enzymes is detectable in any cellular fraction. The validity of the assays was verified using rat and mouse liver (data not shown).
Amino acid metabolism
The amino acid transaminases, GPT and GOT,
have high proportions of cytoplasmic activity, with
lesser activities appearing in the mitochondrial
fraction. Activity of GOT is greater t h a n GPT,
GDH, which catalyzes the oxidative deamination
of glutamate to a-ketoglutarate, is approximately
equally distributed between mitochondrial and
cytosolic fractions.
Carbohydrate metabolism
HK, which catalyzes the first reaction in the
intracellular metabolism of glucose, is present in
the cytosol, with minor contamination of the mitochondrial fraction. PK, which catalyzes what is
considered to be one of the rate-limiting steps in
glycolysis, the production of pyruvate from phosphoenolpyruvate, is detectable exclusively in the
cytosol. Virtually no LDH activity is seen in the
mitochondrial fraction. All LDH activity measured
in the whole homogenate is recovered in the cytosol. aGPDH is involved in the cytoplasmic arm of
the a-glycerophosphate shuttle, which participates
in balancing mitochondrial and cytoplasmic redox.
The activity of this enzyme is also virtually exclusively cytosolic. G6PDH catalyzes the first committed reaction of the pentose phosphate pathway
and generates NADPH, which can be used to drive
lipid synthesis. GGPDH is entirely cytoplasmic in
C. nemoralis hepatopancreas.
Stereospecificity of BHBDH for D- or L-BHB
Maximal BHBDH activity with L-BHB was optimized at a n L-BHB concentration of 20 mM. The
mean activity with the L-isomer was 1.758 2 0.235
pmoVmidg wet tissue weight. Optimal BHBDH
activity with D-BHB was achieved with 40 mM
D-BHB. Mean activity with the D-isomer was
0.515 f 0.039 ymol/min/g wet tissue weight.
As the major storage site for lipid (Voogt, '831,
the hepatopancreas plays a n important role in the
energy metabolism of terrestrial snails (Heeg, '77;
Krupanidhi et al., '78). Although the tissue itself
is capable of lipid catabolism, as we have demonstrated, it is unclear if lipid stored in the hepatopancreas can be transported to other tissues. The
most metabolically active transport form of lipid
in many organisms are non-esterified fatty acids
(NEFAs). The very low solubility of NEFAs i n
aqueous solutions requires that they be transported bound t o a carrier. There is no evidence of
a fatty acid binding protein in gastropod molluscs,
and the transport of fatty acids in hemolymph may
not contribute substantially to mollusc energy
metabolism (Allen, '77). Lipid carbon stored in
hepatopancreas may therefore be transported in
another, more soluble, form. In some animals this
is achieved through the partial oxidation of lipid
in the hepatopancreas to more freely soluble metabolic intermediates, like ketone bodies, which can
be readily exported t o peripheral tissues (Zammit
and Newsholme, '79). This may be a strategy for
distribution of stored energy reserves in Cepaea.
Based on enzyme activities, the metabolism of
ketone bodies in the hepatopancreas is substantial. In Cepaea hepatopancreas, activities of enzymes of ketone body metabolism are of the same
order of magnitude as those of carbohydrate metabolism. In most other animals these activities
are only about 10% of activities of key enzymes of
carbohydrate metabolism (Newsholme and Leech,
'83j. Similarly, the concentration of BHB in fed B.
glabrata hemolymph (0.6 mM) (Meyer et al., '86)
is comparable t o that of glucose (0.62 mM) (see
Livingstone and de Zwaan, 83). This suggests that
ketone bodies are possibly as important as carbohydrates in the intermediary metabolism of freshwater and terrestrial gastropods. The data of
Meyer et al. ('86) support a role for ketone bodies
in routine energy metabolism in B. glabrata, as
opposed to their more specialized role i n starvation
in most other animals (Newsholme and Leech, '83).
The organization of the pathways of ketone body
metabolism in C. nemoralis differs from that of
mammals in several respects. In most mammals,
ketogenesis occurs primarily through reactions catalyzed by HMS and HML, whereas oxidation occurs
through 30AT (Newsholme and Leech, '83). HMS
and HML are undetectable in both the mitochon-
BHB t--
Acac c--
glucose 4-
Fig. 1. Proposed organization of aerobic metabolism, such
as could occur following a bout of anoxia, in C. nemoralis
hepatopancreas. (For clarity, some enzymes and intermediates have been omitted.) Intermediates in bold letters indicate anaerobic endproducts and importediexported substrates.
Note particularly that anaerobic endproducts lactate and alanine are reoxidized by LDH and a GPTIGDH couple, respec-
tively. NADH generated in these processes is reoxidized by
cytosolic BHBDH and gluconeogenesis, and redox balance is
maintained. Pyruvate incorporation into glucose andor glycogen is maximized by: 1) exchange with Acac; 2) maintenance of high mitochondrial redox, promoting malate export;
and 3) low mitochondrial concentrations of oxaloacetate, promoting synthesis of Acac for export from fatty acids.
acetoacetyl-CoA thiolase
P-hydroxybutyrate dehydrogenase
carnitine palmitoyl transferase
glutamate-oxaloacetate transaminase
glutamate dehydrogenase
glutamate-pyruvate transaminase
lactate dehydrogenase
malate dehydrogenase
drial and cytoplasmic fractions of Cepaea hepato- tively prevents the loss of acetoacetyl-CoA via the
pancreas, indicating that this pathway of ketone combined actions of acetyl-CoAhydrolase and AAT,
body synthesis is not present. The lack of HMS and by virtually eliminating the accumulation of CoA
HML in Cepaea hepatopancreas contrasts with the through an acetyl-CoA regenerating system. The
results of Meyer et al. ('86), who measured HMS assay used by Meyer et al. ('86) did not account for
and HML activities in the hepatopancreas of the acetoacetyl-CoA lost through the latter pathway and
freshwater snail B. glabratus. We assayed HMS us- therefore may have substantially overestimated the
ing the method of Quant et al. ('891, which effec- activity of HMS in B. glabrata.
Ketone body synthesis and degradation do not
depend on the presence of HMS and HML. Acac
can also be synthesized through the reversal of
the reaction catalyzed by 30AT (Fig. 1). Correspondingly, the levels of 30AT are threefold higher
in Cepaea hepatopancreas than in rat liver (0.21
pmol/min/g) (Zammit and Newsholme, ,791, which
may indicate a major role for this enzyme in ketogenesis. A ketogenic role for 30AT has been suggested in fish (Philips and Hird, '771, where activity
of this enzyme is also quite high, for example, 12
pmol/min/g in bass (Zammit and Newsholme, '79).
In starved elasmobranch fish, an increase in blood
ketone body levels is paralleled by a threefold increase in the activity of liver 30AT (Zammit and
Newsholme, '79). Thus, ketogenesis in Cepaea
hepatopancreas must be catalyzed by AAT, 30AT,
and BHBDH (Fig. l),without the involvement of
Another important finding of the present study
is the subcellular distribution of BHBDH, which
is exclusively cytosolic in C. nemoralis hepatopancreas (Fig. 1).In most animals it is a mitochondrial enzyme, found associated with the inner
mitochondria1 membrane (Wang et al., '88). A cytosolic BHBDH is, however, found also in ruminant liver and kidney (see Zammit, '90, for
review). Zammit ('90) suggests that cytosolic
BHBDH may promote gluconeogenesis from pyruvate under ketogenic conditions. Gluconeogenesis
is the main source of glucose and glycogen in ruminants, as virtually no glucose is produced by
ruminant gut (Heitmann et al., '87).
Gluconeogenesis in Cepaea hepatopancreas, in
the fed state, is probably less important than in
ruminants. Gastropods possess a variety of carbohydrases, and transport of sugars from the gut
has been demonstrated (Livingstone and de
Zwaan, '83), suggesting that, unlike ruminants,
gut is an important source of glucose in gastropods in the fed state. Gluconeogenesis is, however,
very important during recovery from anoxia, when
glycogen reserves are depleted (Livingstone and
DeZwaan, '83). Cytosolic BHBDH may play a role
in promoting gluconeogenesis in these situations.
Most terrestrial snails have a well-developed
anaerobic metabolism (Livingstone and DeZwaan,
,831,adapted to a life history that includes prolonged anoxia during hibernation (Oudejans and
van der Horst, '74) and intermittent bouts of
anaerobic metabolism during estivation (Barnhart
and McMahon, '88). During periods of anoxia, terrestrial gastropods deplete their endogenous
glycogen reserves and accumulate alanine and
large amounts of lactate in the hepatopancreas
(Churchill and Storey, '89). Upon resumption of
aerobic metabolism, both anaerobic endproducts
are reoxidized to pyruvate, which may then be
used t o regenerate glucose and glycogen (Fig. 1).
Ketogenesis in Cepaea hepatopancreas from
stored lipid (Fig. 11,occurring in parallel with the
reincorporation of pyruvate into carbohydrates,
would stimulate this process in two ways. First,
it would provide intramitochondrial Acac for export, which would stimulate pyruvate uptake
through the mitochondria monocarboxylate carrier
(Zammit, '90). Second, cytosolic reduction of Acac
t o BHB would aid in maintenance of cytosolic redox through reoxidation of NADH generated by
reformation of pyruvate from lactate, via LDH,
and from alanine, through the combined actions
of GPT and cytosolic GDH (Fig. 1).Cytoplasmic
redox might otherwise become too highly reduced
following anaerobic bouts, as hepatopancreas must
reoxidize endogenous pools of alanine and lactate,
as well as pools of these substrates transported from
other tissues via the hemolymph (Livingstone and
DeZwann, '83; Churchill and Storey, '89) (Fig. 1).
A role for lipid in anaerobic metabolism has been
variously suggested (Oudejans and van der Horst,
'74; Zs-Nagy, '79; DeZwaan, '83), and there is some
evidence that fatty acids may function as terminal electron acceptors. In hepatopancreas of the
estivating giant African snails Achatina, free fatty
acid content cycles over 24 days of estivation, showing periods of increase and decrease (Umezerike and
Iheanacho, '83). Interestingly, 0, consumption and
COBemission show similar cyclic fluxes during estivation, as snails alternate between aerobic and
anaerobic metabolism (Barnhart and McMahon,
'88). These data support a possible biphasic role
for lipids in estivation, with anaerobic synthesis
providing an electron sink and aerobic partial oxidation, leading to the synthesis of ketone bodies.
The cytoplasmic BHBDH of C. nemoralis hepatopancreas is also similar to that of ruminant liver in
that it preferentially oxidizes the L-stereoisomer of
BHB (Williamson and Kuenzel, '71). Oxidation of
L-BHB may not be the result of non-specificity of
the cytosolic BHBDH. A necessary step t o clarify
the relative physiological importance of the L- and
D-isomers is the measurement of hemolymph levels of both stereoisomers of BHB.
Other aspects of the subcellular organization of
C. nemoralis hepatopancreas indicating adaptation t o episodic anoxic conditions include the significant proportions of GDH, GPT, and GOT
activities that occur in the cytosol (Hochachka,
Fig. 2. Proposed pathways for the generation of anaerobic endproducts in C. nenoralis hepatopancreas. (For clarity,
some enzymes and intermediates have been omitted.) Intermediates in bold indicate accumulated anaerobic endproducts.
Note particularly the cyclic fluxes of glutamate and aKG, and
redox coupling. NADH produced through anaerobic glycolysis is reoxidized via LDH and via the cytosolic GPT/GDH
'SO). The predominantly anaerobic rat tapeworm,
Hymenolepis diminuta (Cestoda) has an exclusively cytosolic GDH (Mustafa et al., '78), with
lactate and alanine among the accumulated
anaerobic endproducts (Mustafa et al., '78). These
authors have suggested that the shift of GDH from
mitochondria to cytosol may facilitate the maintenance of cytosolic redox in H . diminuta. Similarly, the subcellular distributions of GDH and
GPT in Cepaea hepatopancreas suggest that the
balance of cytosolic redox in this tissue could be
achieved through the reduction of pyruvate to alanine, via a coupled cytosolic GPT and GDH (Fig.
2). This arrangement would not deplete glutamate
or aKG pools, since it provides cyclic fluxes of
these metabolites. This pathway results in the net
consumption of NH3 (Walsh and Henry, '911, which
may be of further benefit in providing a proton
sink, reducing the rate of acidification of the cytosol. This proposed strategy differs from that of
anaerobic marine molluscs, which use a much
larger intracellular aspartate pool (12 pmoVg wet
weight in Busycon contrarium ventricle; Ellington,
'81) to regenerate glutamate from aKG (Fig. 2).
With aspartate pools an order of magnitude lower
in tissues of terrestrial snails (0.85 pmoVg wet
weight in 0. lactea hepatopancreas; Churchill and
Storey, '89), glutamate pools could be thus sustained for only the early stages of anoxia.
In summary, the organization of ketone body
metabolism in the hepatopancreas of the terrestrial snail C. nemoralis differs from that of freshwater snails and most mammals and is similar
in several respects t o that of ruminants. Based
on our observations, we suggest that there are
links between ketone body metabolism and gluconeogenesis and amino acid metabolism in this
tissue. The subcellular distribution of enzymes of
intermediary metabolism in this tissue are consistent with adaptations to periodic anoxia, which
typify terrestrial snail life history. Further study
of the tissue distribution of these enzymes and
hemolymph concentrations of substrates are required t o define the roles of ketone bodies in the
intermediary metabolism of these organisms.
This research was funded through a Natural
Sciences and Engineering Research Council of
Canada (NSERC) operating grant t o J.S.B.
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