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First published online July 17, 2009
Journal of Experimental Biology 212, 2378-2385 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.023408
Skeletal muscle substrate utilization is altered by acute and acclimatory temperature in the American bullfrog (Lithobates catesbeiana)
1 Wellesley College, 106 Central Street, Wellesley, MA 01778, USA
2 Department of Integrative Physiology, University of Colorado, Boulder, CO
80309, USA
* Author for correspondence (e-mail: annmariepetersen{at}gmail.com)
Accepted 7 May 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: lactate, glucose, glycogen, Rana catesbeiana, ectotherm, muscle metabolism
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Hutchinson and Miller, 1978). Studies have demonstrated
clearly that mammalian, amphibian, and reptilian muscles have the capacity to
use glucose and lactate as substrate for ATP generation via oxidative
phosphorylation and for intramuscular glycogen synthesis
(Bendall and Taylor, 1970;
Connett, 1979;
Gleeson, 1985;
Bonen et al., 1990). In fact,
one of the earliest studies on tissue lactate utilization was performed in
amphibian skeletal muscle (Meyerhoff,
1920).
Several substrates are metabolized by vertebrate skeletal muscle, including
carbohydrates (glucose), carboxylic acids (lactate) and fats (fatty acids).
The major metabolic fates for these substrates in vertebrate muscle tissue are
oxidation to CO2, incorporation into glycogen, or carbon exchange
between the glucose and lactate pools. Numerous physiological, seasonal and
ecological factors undoubtedly play a role in substrate and metabolic pathway
selection by skeletal muscle cells of different animals. However, a
comprehensive, comparative analysis of cellular metabolic substrate
utilization is not yet possible due to the limited number of species for which
such data are available. We examined substrate preference in the sartorius
muscle of the American bullfrog, Lithobates catesbeiana [formerly
Rana catesbeiana (Frost et al.,
2006)] and the effect of temperature on the metabolic fate of
these substrates.
The metabolic fate of substrate varies between endotherms and ectotherms.
Mammalian muscle predominantly oxidizes glucose and fatty acids
(Kelley et al., 1988), while
lizard (for a review, see Gleeson,
1991), frog (Bennett and Licht,
1974; Fournier and Guderley,
1992) and fish (Pagnotta and
Milligan, 1991) muscle rely more heavily on glycolytic exchange
between glucose, lactate and glycogen stores for immediate energy supply (for
a review, see Gleeson, 1991).
Resting concentrations of circulating substrates reflect this preference.
Mammals generally are reported to have higher blood glucose than lactate (for
a review, see Brooks et al.,
1996), while the reverse is true in fish and frogs (Hutchinson and
Turney, 1975; Weber et al.,
1986; Petersen and Gleeson,
2007). Tuna have higher turnover of labeled carbon from lactate
than glucose following activity recovery
(Weber et al., 1986). Under
most conditions, ectothermy is characterized by high rates of lactate
production and utilization for ATP generation and slow rates of oxidation of
all substrates. By contrast, muscle from endotherms such as mammals
(Brooks and Gaesser, 1980) and
flying birds (Jenni-Eirmann et al., 2002) possesses relatively fast rates of
oxidative metabolism at rest with preference for glucose (mammals) and fatty
acids (birds). Mouse muscle also mainly utilizes glucose for glycogen
synthesis (Bonen et al., 1990;
Wickler and Gleeson, 1993).
From these data, a pattern emerges: (1) active ectotherms rely more heavily on
glycolytic pathways than endotherms, (2) endotherms are more likely to
completely oxidize substrate and (3) ectotherms also appear to prefer lactate
over glucose specifically for glycogen synthesis and in general as a metabolic
fuel for skeletal muscle.
Divisions in metabolic substrate utilization between thermoregulatory
classes may be correlated to the potent force that temperature exerts on
metabolism. Rates of some physical processes, such as diffusion, are nearly
temperature independent and thus demonstrate a Q10 rate near 1.0
whereas most biochemical pathways exhibit Q10 values upwards of 3.0
(for a review, see Rome et al.,
1992). It is clear that rates of muscle metabolism do increase
with higher temperature (for a review, see
Bennett, 1984). However, it is
not known if substrate preference for metabolism is altered by temperature in
amphibians.
Lithobates catesbeiana is a particularly interesting species of
ectotherm in which to investigate not only skeletal muscle substrate
preference and use but also how acute and chronic temperature exposure affects
this metabolic strategy. This species of frog inhabits a geographic range
stretching from sub-arctic to tropical regions
(Lannoo, 2005) and can be
considered eurythermal, tolerating diurnal changes in pond temperature of up
to 30°C (Carey, 1979;
A.M.P. and T.T.G., unpublished data). Bullfrogs are large, active predators,
and proper allocation of metabolic resources is key to survival in such a wide
array of climates. The purpose of the present study was to investigate the
effects of acute and acclimatory temperature conditions on substrate
preference and substrate metabolic fate in the skeletal muscle of the northern
amphibian L. catesbeiana. Examining substrate preference and the
effects of temperature on these processes may identify patterns of vertebrate
muscle fuel utilization in response to different ecological conditions.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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5°C (Riddle, 1909).
This protocol was approved by the University of Colorado, Boulder,
Institutional Animal Care and Use Committee.
Muscle bundle preparation
On each day of experiment, one bullfrog was sacrificed by decapitation with
a small animal guillotine and double-pithed. The sartorius muscle, with a
small portion of tendon on the proximal and distal ends remaining, was
carefully dissected from each leg. The muscles were immersed in a modified
Ringer solution of 70 mmol l–1 NaCl, 2.5 mmol
l–1 KCl, 1 mmol l–1 MgSO4, 1.8
mmol l–1 CaCl2 and 25 mmol l–1
NaHCO3 in deionized H2O. Each muscle was divided
laterally into three fiber bundles, with care taken to avoid severing fibers.
Bundles were carefully cleaned of extraneous connective tissue and pinned taut
at approximately resting lengths to a Tygon® frame. All bundle dissections
were complete within 60 min of removal from the frog. Framed bundles were then
placed individually in a 12x75 mm glass incubation chamber containing 3
ml of incubation medium with the addition of 1 mmol l–1
D-glucose (J. T. Baker Chemical Company, Phillipsburg, NJ, USA), 5
mmol l–1 lactic acid sodium salt (Sigma-Aldrich, St Louis,
MO, USA), 1 mmol l–1 palmitic acid (Sigma-Aldrich) and 0.4%
bovine serum albumin (Sigma-Aldrich). Concentrations of metabolites in the
bath reflected resting blood levels of these substrates for anuran amphibians
(Hong et al., 1968;
Petersen and Gleeson, 2007).
Incubation medium also included 1 µCi (37,000 Bq) of one of the following
three isotopic labels: D-[U-14C]glucose,
L-[U-14C]lactic acid sodium salt or
[1-14C]palmitic acid (GE Healthcare UK Limited, Buckinghamshire,
UK).
Temperature conditions
Muscles were prepared and incubated under one of three temperature regimen.
Acute exposure bundles were tested at either 5°C, 15°C or 25°C
(normal temperature ranges for most bullfrogs in North America). Following the
2–6-week acclimation period, muscles from frogs acclimated to 5°C
were tested at either 5°C (acclimation exposure) or 15°C
(post-acclimation acute exposure). Muscles from frogs acclimated to 25°C
were tested at either 25°C (acclimation exposure) or 15°C
(post-acclimation acute exposure). Muscle incubation chambers were maintained
at test temperatures by a clear acrylic water jacket flushed with
temperature-controlled (±1°C) water from a circulating water
bath.
Incubation conditions
The incubation medium was continuously bubbled at test temperature with
approximately 2.1% CO2 and 97.9% O2 for at least 45 min
prior to use. This equilibration yielded an incubation pH of 7.9 at 25°C,
which is similar to the extracellular pH of resting ranids at this temperature
(MacKenzie and Jackson, 1978;
Petersen and Gleeson, 2007).
The pH of the medium was allowed to fluctuate with temperature as would occur
in vivo (approximately –0.1 units/+10°C)
(MacKenzie and Jackson, 1978).
The framed muscle was carefully inserted vertically into the chamber and
suspended above a stir bar at the bottom of the chamber. The chamber was
capped with a serum stopper with a gas inlet and outlet that allowed for
constant infusion of the gas mixture into the airspace at the top of the
sealed chamber in order to maintain pH and gas concentration of the medium.
The muscle was incubated for 3 h with continuous stirring of the medium by a
small stir bar at the bottom of the chamber. We conducted preliminary
experiments at room temperature (21±1.5°C) in order to ensure that
the muscle was viable under these conditions for the 3 h time period and found
that oxygen consumption of the muscle is not different between the first hour
of incubation and the third. At the end of the incubation period, the muscle
was removed, blotted dry and then rinsed twice in Ringer solution, blotted
once more and then submerged in liquid nitrogen until completely frozen. The
muscle bundles were then stored for <6 months at –70°C prior to
analysis.
Determination of oxidation to CO2
Immediately following the 3-h incubation, the medium was transferred
via syringe to a 15 ml flask with a hanging reservoir. The reservoir
contained 1 ml of 1:3 ethanolamine:methylcellusolve (Sigma-Aldrich) for
sequestration of CO2. 200 µl of 1 mol l–1 HCl
was added quickly to the chamber, which was then sealed and agitated in order
to ensure mixing. The chamber was left for 2 h, at which time the full 1 ml
content of the sequestration trap was removed and mixed with Scintiverse
scintillation cocktail (Fisher Scientific, Houston, TX, USA) for d.p.m.
enrichment determination using a Wallac Model 1204 scintillation counter
(Perkin-Elmer, Waltham, MA, USA) with internal quench correction.
Quantification of intracellular metabolites
Frozen muscle bundles were weighed and then homogenized in six volumes of
6.0% HClO4. Glycogen from the homogenate supernatant was isolated
by ethanol precipitation, and metabolite levels were determined by
colorimetric assay, as described previously
(Petersen and Gleeson, 2007).
This may underestimate any acid-insoluble proglycogen
(Asp et al., 1999), although
significant amounts of proglycogen in anuran muscle have not yet been
reported. Extracellular space corrections were made using an estimate of 20%
of muscle wet mass based on the findings of Neville and White
(Neville and White, 1979) that
incubation conditions increase extracellular space in frog sartorius
muscle.
[14C] activity
Muscle bundle homogenate was centrifuged at 7200g for 10
min. 150µl aliquots of supernatant were separated into lactate, glucose and
pyruvate fractions by a Dowex 1X8-200 ion-exchange resin column
(Sigma-Aldrich) as described previously
(Donovan and Gleeson, 2006).
Each fraction eluted through the column was then mixed with Scintisafe
scintillation cocktail (Fisher Scientific) and counted as above. Initial
control experiments (N=3) using 14C-labeled glucose and
lactate determined that <0.1% of label was eluted in the inappropriate
fraction (e.g. labeled glucose in the lactate fraction), and therefore our
column separation of lactate, glucose and pyruvate was considered complete.
Aliquots of supernatant were also analyzed for glycogen content and
[14C]glycogen levels as described previously
(Petersen and Gleeson, 2007).
Specific activity of [14C]glucose, [14C]lactate and
[14C]palmitate were calculated by dividing d.p.m.
g–1 tissue by total intracellular levels of each
metabolite.
Net incorporation rates into glycogen, CO2, pyruvate, and glucose or lactate were calculated by dividing the measured 14C activity by the specific activity of the intramuscular metabolite, corrected for extracellular space (ECS) 14C by subtracting the estimated d.p.m. in the ECS (20% of the bundle mass x d.p.m. ml–1 incubation medium) from the total d.p.m. measured in the muscle. Extracellular label was assumed to rapidly equilibrate in the tissue, and incorporation rates were assumed to be linear over 3 h of incubation.
StatView 5.01 statistical software was used to conduct data comparisons, with one-way ANOVA and Fisher's PLSD used to determine statistically significant (P<0.05) difference between treatment groups.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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At 5°C, the major fate for lactate is oxidation (35%), followed by storage as glycogen (25%) and conversion to glucose (25%) (Fig. 2B; Table 1), with a surprisingly large portion also found in pyruvate (15%, data not shown). Higher incubation temperatures result in greater rates of oxidation and a proportional shift in metabolic fate of lactate towards glycogen storage. At both 15°C and 25°C, lactate is mostly stored as glycogen, although a significant portion (20%) of lactate is still oxidized at the higher temperatures (Fig. 2B; Table 1).
Muscles acclimated to 5°C metabolized 30x more lactate than glucose, a factor 3-fold higher than when muscles were exposed acutely to 5°C. Suppression of glucose metabolism at cold-acclimation and incubation temperatures is also suggested by the fact that only 10% of net glucose transported into the cell is metabolized at 5°C, while over 50% of transported lactate is used (Fig. 3).
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At 15°C, although there is no measurable difference in uptake and net metabolism in glucose, there is a discernible temperature-dependent shift in the fate of metabolized glucose (Fig. 6A). Muscle from frogs acclimated to 5°C stored the majority (70%) of the glucose as glycogen. Muscle from warm-acclimated (25°C) animals demonstrated a reversal of this pattern: significantly more glucose was converted to lactate (70%, P<0.05) (Fig. 6A).
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In additional experiments, we measured how oxidation of the free fatty acid palmitate is affected by acute and acclimation temperature regimens. We found that acutely exposing the muscles to 15 or 25°C resulted in significantly higher net rates of palmitate oxidation than incubation at 5°C (Fig. 7A, P<0.05). Following acclimation to 5 or 25°C, this pattern persists, as increasing temperature yields significantly higher rates of palmitate oxidation (Fig. 7B, P<0.05). Muscles from frogs acclimated to 5°C and incubated at 15°C were found to oxidize significantly less palmitate than muscles acutely exposed to 15°C (P<0.05, Fig. 7).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Hochochka and Somero, 2002). Since net rates of lactate
metabolism appear to be less temperature sensitive than net glucose metabolism
(Fig. 1;
Table 1), our findings support
the growing body of literature suggesting that lactate is a more utilized
substrate than previously believed (for reviews, see
Brooks et al., 1996;
Gleeson, 1996;
Gladden, 2004). It should be emphasized that our findings reflect net uptake and utilization of substrate label as assessed at a moment in time after 3 h of incubation. Continuous measurement of flux over time would likely reveal a far more complicated pattern of uptake and utilization than is reflected in the present study and might disclose some futile cycling of label or metabolites that these studies cannot detect. The theoretical sources of error in this approach have been reviewed (Gleeson and Dalessio, 1989) and while they may have some impact on quantitative estimates of net flux under some conditions, likely have little import on the findings of substrate use in this study. Our findings do suggest that under these thermal conditions, lactate is the predominantly metabolized substrate by frog muscle and appears to be the substrate whose utilization is less influenced by temperature.
Studies have demonstrated that lactate is used as a preferred fuel by
nerves (Itoh et al., 2003),
some oxidative muscles (Andrade and
McMullen, 2005), and cardiac tissue under some circumstances
(Gertz et al., 1988). We
demonstrate here that lactate may also be the preferred substrate in white
glycolytic muscle of frogs at rest (Fig.
1; Table 1). Since
lactate levels rise by up to 20 mmol l–1 following even
moderate activity in amphibians (Petersen
and Gleeson, 2007), the relative abundance of lactate may be the
reason for muscle preference of this substrate. By contrast, blood glucose
levels are highly variable and sometimes so low as to be undetectable
(Farrar and Frye, 1979;
de Roos and Parker, 1982),
hence an unreliable substrate source in these animals.
The predominant trend of glucose use by the muscle was glucose conversion
to lactate at the lowest temperatures, with increasing storage of glucose as
glycogen at higher incubation temperatures
(Fig. 2A;
Table 1). Since conversion of
glucose to lactate (glycolysis) is a major fate at 5°C, glycolysis appears
not to be strongly inhibited by acute exposure to cold temperature. Ohira and
Ohira report that cold-exposed frog muscle has reduced ATP levels
(Ohira and Ohira, 1988), a
condition favorable to glycolysis. Trout acutely (48 h) exposed to low
temperature demonstrated no reduction in concentrations of glycolytic
intermediaries and activators of phosphofructokinase (PFK),
fructose-6-phosphate and glucose-6-bisphospate
(Lehoux and Guderley, 1997),
suggesting minimal inhibition of ectothermic muscle glycolysis by acute
exposure to cold temperature. Maintenance of pathways of lactate formation at
low temperature would seem beneficial to frogs, as lactate could then be
utilized as the substrate throughout the body during variable ambient
temperature conditions (for a review, see
Gladden, 2004).
Under all acute temperature conditions, exchange occurs between lactate,
glycogen and glucose pools, as evidenced by our finding that a small, but
measurable, percentage of lactate carbon was found in the form of free glucose
(Fig. 2;
Table 1). We report here that
significant amounts of carbon from labeled lactate, up to 25% of labeled
lactate in fact, are measured in the form of glucose at the end of the 3-h
incubation (Fig. 2;
Table 1), which would be
consistent with the provocative findings of Fournier and Guderley that muscle
may be a net producer of glucose (Fournier
and Guderley, 1992; Fournier
and Guderley, 1993a; Fournier
and Guderley, 1993b). These findings are in keeping with those of
Fournier et al. (Fournier et al.,
1994), who reported that even when frogs were hepatectomized,
whole-body glucose, lactate and glycogen metabolism were unaltered, suggesting
that the muscle may be able to produce and even export glucose.
Cold-acclimated muscles metabolized 30x more lactate than glucose, a
factor 3x as high as when muscles were exposed acutely to 5°C,
suggesting that one aspect of cold acclimation may be increased capacity to
metabolize lactate, but not glucose (Fig.
3). Disproportionate suppression of glucose vs lactate
metabolism at low temperature (Q10 of 15.0 vs 2.4;
Table 2) suggests that
long-term cold-temperature acclimation inhibits glycolysis much more than
oxidative or gluconeogenic pathways (Fig.
3). Such a case could occur following acclimation to low
temperature if there was long-term, cold-induced, inhibition of muscle PFK or
pyruvate kinase (PK), known regulators of glycolysis. There is evidence for
inhibition of activity of PFK at cold temperature in bird muscle
(Marsh and Dawson, 1982),
although studies in hibernating squirrels
(MacDonald and Storey, 2001)
and at least one species of fish (Lehoux
and Guderley, 1997) suggest that temperature may have only a
limited effect on PFK activity and may be compensated for by long-term
acclimation (Guderley and Gawlicka,
1992).
Muscles from frogs acclimated to 25°C had a significantly higher
conversion rate of glucose to lactate at the higher temperatures and
demonstrated significant sensitivity to temperature change
(Fig. 4A). Consistent with this
finding, Q10 values for glucose-to-lactate conversion are the
highest of any process we measured (Table
2). It is possible that activity of lactate dehydrogenase (LDH)
increases sharply with warm temperature acclimation, as shown in other
ectotherms (Feder, 1983;
Seebacher and James, 2008).
Following 4 months of hibernation, LDH activity is decreased in at least one
species of frog (St Pierre and Boutillier,
2001). A study from the fish literature proposes that at cold
temperature, LDH concentrations and kinetics are altered, in order to favor a
larger pyruvate pool for protection of aerobic potential (Zackhartsev et al.,
2004). Our findings that less glucose is converted to lactate following cold
acclimation could support this observation.
Prior acclimation temperature had no significant effect on net rate of
glucose uptake or metabolism at 15°C
(Fig. 4A). Similarly, striped
bass acclimated to 5°C and 25°C also demonstrated no compensation in
red or white muscle tissues for glucose use but did appear to upregulate
pathways of palmitate oxidation (Jones and
Sidell, 1982).
Metabolic compensation did, however, occur in terms of lactate metabolism
(Fig. 5B). Lactate was
metabolized at 2x the rate by cold-acclimated muscles incubated at
15°C than warm-acclimated muscles at the same temperature
(Fig. 5B). This pattern is in
keeping with the `classic' concept of metabolic compensation in which
low-temperature acclimation results in higher enzyme binding affinity,
activity levels or concentration, resulting in higher disposal rates for
specific substrates (for a review, see
Hochachka and Somero, 2002).
In the present study, increased lactate metabolism occurred in bullfrog muscle
in the absence of changes in glucose utilization. This suggests to us that one
central aspect of frogs' response to cold is an increased utilization of
lactate as a metabolic fuel (Fig.
5; Table 1).
Lactate glyconeogenesis is enhanced in muscles from frogs acclimated to
cold temperature. Since bullfrogs (Stinner
et al., 1994), and other ranids
(Cunjack, 1986), are `cold
active' underwater in winter, it may be ecologically important during cold
seasons for muscle tissues to be able to quickly store substrate that becomes
available following activity. Gleeson suggests that ectotherms in general
utilize glycogen storage as a depot for lactate produced following activity,
allowing muscles to dispose of accumulated lactate while replenishing glycogen
stores for future use (Gleeson,
1991). Fig. 6 is
suggestive of enhancement of glycogenic and glyconeogenic pathways, which
would be a logical strategy if food sources were scarce and energy metabolism
overall is reduced, as would be the case while overwintering.
Prior to overwintering conditions, ranid frogs are known to build up lipid
stores, in the form of fat bodies
(Fitzpatrick, 1976), and
increase glycogen storage in the liver
(Byrne and White, 1975). Our
data do not support a substantial role for fat oxidation in resting muscle
under our conditions (Fig. 7).
It does not appear that palmitate oxidation constitutes a large component of
energy metabolism in these resting muscle cells. Temperature does have a
significant effect on oxidation rates. However, acclimation does not alter
this pattern and no compensatory adjustments appear to exist to increase
palmitate oxidation following acclimation to a colder temperature
(Fig. 7B).
We report here that lactate metabolism is of central importance to amphibian muscle cells, with net rates of metabolism of this substrate far exceeding rates of glucose or palmitate use. Acute exposure to cold temperature results in most glucose being converted to lactate while both substrates are stored mostly as glycogen at warm temperature. Acute exposure to cold temperature also reduces glucose metabolism more markedly than lactate metabolism, signifying a role for lactate as a staple fuel under these conditions. We suggest that ready use of lactate as a substrate in general, and enhancement of glyconeogenic pathways specifically, could play a key role in the tolerance of this species to temperature fluctuation by allowing for storage of available substrate during cold-induced low metabolism.
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) by lactate or glucose (P<0.05,
one-way ANOVA, Fisher's PLSD, N=4–11). Warm acclimation and
incubation results in an increased conversion of glucose to lactate and
increased glycogen synthesis from lactate (P<0.05).
