The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1723-1730

Feeding a Low Energy Diet and Refeeding a Control Diet Affect Glycolysis Differently in the Slow- and Fast-Twitch Muscles of Adult Male Wistar Rats^1,2,3

David J. Bissonnette⁴ and Khursheed N. Jeejeebhoy^*

School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, QC, Canada, H9X 3V9 and ^* Department of Clinical Science, University of Toronto, Toronto, ON, Canada, M5B 1W8

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Muscle glycogen concentrations in underfed (HYPO) and refed rats (RE) in an earlier study did not correlate with fatigue. We hypothesized that underfeeding slowed glycolysis in the slow-twitch soleus, but not in the fast-twitch extensor digitorum longus (EDL). Thirty adult male Wistar rats were randomly assigned to receive one of two isovolemic and micronutrient-complete liquid diets, a control (CN) energy-complete diet for 10 d or a diet 80% lower in energy (HYPO) for 7 d producing a 20% loss of initial weight. Rats were refed an energy-complete diet for 1 or 4 d (RE1, RE4). Rats were then anesthetized, and the soleus and EDL muscles of the hindlimbs were isolated and electrically stimulated in situ. The pre- and postfatigued muscles were freeze-clamped, lyophilized and stored at 70°C until assayed for specific glycolytic and Krebs cycle metabolites. The HYPO diet caused significantly slower glycolysis in the stimulated soleus but not the EDL compared with the CN diet as supported by the following: 1) a lower fructose-1,6-bisphosphate (F-1,6-P₂)/fructose-6-phosphate (F-6-P) ratio; 2) a greater glucose-6-phosphate (G-6-P)/lactate ratio; 3) a lower lactate/glycogen ratio; and 4) lower lactate concentration. Four days of refeeding normalized the F-1,6-P₂/F-6-P ratio, but did not improve the lactate/glycogen or the G-6-P/lactate ratios. We conclude that undernutrition compromises glycolysis only in slow-twitch muscles and that 4 d of refeeding restores phosphofructokinase activity.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Malnutrition and feeding the malnourished individual significantly influence the endurance and function of skeletal muscle, a system that comprises 40-50% of the adult human weight (Moffet et al. 1993). Muscle fatigue and recovery have been previously observed in malnutrition and refeeding, but the mechanism is not clear, although glycolysis and oxidative phosphorylation in muscle appear to be compromised in the underfed state. Russell et al. (1984) have shown in the gastrocnemius muscle of rats (composed mainly of fast-twitch fibers) that energy-restricted feeding reduced the activity of the rate-limiting enzyme, 6-phosphofructokinase (PFK)⁵ of the glycolytic pathway and reduced the activity of the succinic dehydrogenase enzyme (SDH) of the tricarboxylic acid (TCA) cycle measured in muscle biopsies in vitro. Ardawi et al. (1989) confirmed these results and also showed that in muscle biopsies, the maximal activity of hexokinase, PFK, pyruvate kinase, citrate synthase, -ketoglutarate (-KG) dehydrogenase and 3-hydroxy-acyl-CoA dehydrogenase were reduced with underfeeding. Furthermore, they showed that glycogen phosphorylase in both the soleus and gastrocnemius was greater by ~30% in the low energy-fed rats, that there was reduced in vitro lactate production in soleus-muscle strips and lower in vitro mitochondrial activity in gastrocnemius muscle. These findings suggest a compromised anaerobic glycolysis and oxidative phosphorylation in underfeeding (Pichard et al. 1988). However, Ardawi et al. (1989) measured muscle fatigue only in the fast-twitch gastrocnemius muscle. Consequently, the performance of the slow-twitch muscle could not be coupled with the biochemistry. Subsequently, two P-31 nuclear magnetic resonance studies (Mijan de la Torre et al. 1993, Pichard et al. 1988) showed increased free ADP concentrations in underfed rats and altered rephosphorylation to ATP, further confirming reduced mitochondrial activity. Moreover, ADP concentrations returned to normal with refeeding, an indication that the muscle energetics are dependent on nutrition.

It has not been determined, however, to what extent lower biochemical pathway activity affects in situ measured fatigue in both the fast- and slow-twitch muscles in the underfed state. Furthermore, no studies have specifically controlled for micronutrient intake. More recently (Bissonnette et al. 1997), we studied muscle fatigue relative to glycogen concentrations and net glycogenolysis in both the fast- and slow-twitch muscles of energy-restricted rats. We showed that net glycogenolysis in the soleus muscle fell significantly in underfed rats concomitantly with a rise in muscle fatigue, whereas fast-twitch EDL muscles compensated for low energy intake with a supernormal net glycogenolysis that maintained fatigue levels similar to those of controls. The two contrasting muscle types provided internal controls for two distinct metabolic processes, one that is glycolysis dependent (EDL) and another that relies more on oxidative metabolism (soleus). This study further investigates the etiology of this energy-restricted diet-induced fatigue. We hypothesize that underfeeding results in slower glycolysis in the slow-twitch soleus muscle, but not in the fast-twitch EDL. We measured key metabolite concentrations of both the glycolytic and the TCA cycle pathways in the stimulated and unstimulated soleus and EDL muscles of underfed rats and of rats refed for 1 or 4 d. Stimulated and unstimulated muscle data are important in distinguishing between the effects of diet and exercise on muscle metabolite concentrations.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals.  Adult male Wistar rats (Charles River Laboratories, Montreal, Canada) were individually housed, in an environmentally controlled atmosphere at an ambient temperature of 22°C with a 12-h light:dark cycle. The rats adapted to the facility for 2 wk and were given free access to a commercial stock diet (Ralston Purina Rodent Laboratory Chow, Toronto, Canada) before starting the study. After the adaption period, isovolemic liquid diets were fed to the rats and intakes as well as body weights were monitored daily. The initial body weights varied between 370 and 399 g.

Diet. 

Control and energy-restricted diets.  The liquid diets that were used were composed of a mixture of parenteral nutrition products, which included an amino acid mixture (Travasol- electrolyte free; Baxter, Toronto, Canada), a lipid emulsion (Intralipid - 20%; Baxter), and sugar solution (Dextrose 50%; Baxter), to which were added vitamins, electrolytes and trace elements in concentrations that duplicated those published by Hoshino et al. (1991) and Bissonnette et al. (1997) (Table 1). Liquid elemental diets were used to maximize absorption of nutrients, to facilitate the manipulation of macro- and micronutrients and to mimic the nutritional support used in clinical settings. The rats were randomly grouped and then fed either a control (CN) energy-complete diet for 10 d (carbohydrate: 64% of energy; protein: 25% of energy; fat: 11% of energy) or a low energy diet (HYPO) for 7 d (carbohydrate: 73% of energy; protein: 13% of energy; fat: 14% of energy). The CN diet provided an energy density of 3.51 MJ/L (840 kcal/L). The diet intake of a subset of rats was previously monitored (Bissonnette et al. 1997) and CN rats ingested a mean of 325.10 ± 5.44 kJ/d (77.7 ± 1.3 kcal/d). CN rats were fed for 10 d to ensure an exposure to the diet that matched the approximate full duration of the study (7 d energy-restricted feeding and 4 d of refeeding). The HYPO-fed rats consumed a diet with an energy density of 710 kJ/L (170 kcal/L), which induced a 21.8% loss of original weight over 7 d. Previously, we reported that HYPO rats ingested a mean diet intake of 73.22 ± 2.50 kJ/d (17.5 ± 0.6 kcal/d) and described the effect of diet on muscle dry weight and cross-sectional area (Bissonnette, et al. 1997).

Refeeding.  The underfed rats were subsequently refed an energy-complete high carbohydrate diet (carbohydrate: 73% of energy; protein: 13% of energy; fat: 14% of energy), for 1 or 4 d (RE1, RE4). The refeeding diet had an energy density that matched that of the CN diet (3.51 MJ/L). Earlier (Bissonnette et al. 1997), we reported that rats refed for 4 d consumed a mean of 315.06 ± 6.27 kJ/d (75.3 ± 1.5 kcal/d), an amount that was not significantly different from the CN intake. The use of a high carbohydrate liquid diet was intended to duplicate therapeutic diets used clinically for the nutritional support of critically ill patients (Rees et al. 1986). The protein content of the refeeding diet (2.56 g/d) was sufficient to ensure normal growth in this size rat (Altman and Dittmer 1968, NRC 1978, Rogers 1979).

Exposure of muscles and stimulation protocol.  The muscle exposure technique and muscle stimulation protocols were the same as those previously described (Bissonnette et al. 1997). Briefly, the soleus and EDL were separately stimulated using an electronic stimulator (GRASS - S48, Astro-Med, Longueil, Canada) and studied in situ. Initially, it was determined that 20 V resulted in supramaximal stimulation using square waves that were 75 µs in duration. Using this voltage, the optimal length of the muscle (Lo) needed for a maximal twitch (Pt) was determined. While electrical pulses were delivered at a train duration of 1000 ms and at a frequency of 1 Hz, Pt was determined by adjusting the muscle length. This was done by using a rack and pinion system that could be displaced to obtain the optimal length at which maximal tension was observed. After stimulation, rats were killed by exsanguination from the inferior vena cava. The diet protocol, the surgical procedure for muscle isolation and the muscle sampling and stimulation protocols received approval by the Animal Care Committee of the University of Toronto, which adheres to the guidelines of the Canadian Council on Animal Care.

Bioassays of muscles.  Before stimulation, the muscles of the nonstimulated hindlimbs were freeze-clamped with forceps cooled in liquid nitrogen, lyophilized, stored at 70°C and assayed at a later time. Stimulated muscles were freeze-clamped on the 40th repeated contraction and were treated in the same manner as unstimulated muscles. This sampling approach is advocated for the rate determination of enzyme-catalyzed reactions (Newsholme and Leech 1983). Only that portion of the muscle that was directly clamped by the forceps was assayed for metabolites. Extraneous tissue was not included. The stimulated and unstimulated samples were assayed for glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P), fructose-1,6-bisphosphate (F-1,6-P₂), lactate and -KG concentrations using fluorometric methods described by Bergmeyer (1974).

Statistics.  The data were expressed as means ± SEM. A one-way ANOVA was conducted that allowed a comparison among the four groups (CN, HYPO, RE1 and RE4). Differences with P < 0.05 were considered significant. Unpaired comparisons among the experimental groups were conducted using Duncan's New Multiple Range test for unplanned comparisons. Differences were considered significant when P < 0.01. 

View larger version (20K): [in this window] [in a new window]   Fig 1. Weights of adult Wistar rats fed a control energy- and micronutrient-complete diet (CN from d 1 to 10; n = 9) for 10 d or an 80% energy-restricted but micronutrient-complete diet (HYPO, from d 1 to 7; n = 8) for 7 d and refed an energy and micronutrient-complete diet for 1 d (RE1 on d 8; n = 6) through to 4 d (RE4, on d 11; n = 7). Values are means ± SEM.

	RESULTS

Abstract Introduction Methods Results Discussion References

Body weight.  HYPO-fed rats began the study weighing 371.25 ± 5.16 g, and lost a mean 21.8% of their initial weight over a 7-d feeding period, resulting in a mean weight of 290.25 ± 4.23 g. Regressed over time, weights did improve with refeeding (P < 0.025); however, by d 4 of refeeding, weights were still 15.5% below the mean starting weight. The most dramatic jump in weight was a 7.1% (20.6 g) increase occurring on d 1 of refeeding (Fig. 1).

CN rats weighing 379.44 ± 2.91 g took 4 d to adjust to the liquid diet. During this time, the rats lost 5.3% of their starting weight because of a change in fecal mass and lower volume of diet ingested. After this adaptation period, the rats gained a significant (P < 0.01) 7.5% in body weight over 6 d (d 4-10), representing a mean weight gain of 4.5 g/d (Fig. 1). There was no significant difference between the starting weights of the HYPO and CN rats.

Muscle biochemistry. 

Glycolytic intermediates in the unstimulated and stimulated soleus.  The lactate concentrations were significantly lower in the unstimulated soleus of HYPO and refed rats than in CN. The F-6-P concentrations were unaffected by feeding status, but there were significantly lower unstimulated soleus F-1,6-P₂ concentrations in HYPO and refed rats compared with CN (Table 2). The ratio of F-1,6-P₂/F-6-P in HYPO, RE1 and RE4 rats was significantly lower (P < 0.01) than in CN (Table 3). The -ketoglutarate concentrations were not influenced by the nutritional status.

In stimulated soleus, similar observations were made. First, the lactate and F-1,6-P₂ concentrations (Table 4) were significantly lower in HYPO rats (P < 0.01) and, correspondingly, the F-1,6-P₂ /F-6-P ratio (Table 3) was also significantly lower. Second, the G-6-P/lactate ratio (Table 3) was higher only in stimulated soleus of HYPO than in CN rats. In contrast to unstimulated muscle, the F-1,6-P₂/F-6-P ratio, measured in stimulated soleus (Table 4), was restored rapidly by refeeding. The rise of muscle lactate and -ketoglutarate concentrations in the soleus, caused by stimulation and referred to as the delta, was not different among controls, HYPO-fed and refed rats. However, the -ketoglutarate/-glycogen ratio was significantly greater in HYPO and RE1 refed rats than in CN, and finally normalized in RE4 rats (Table 5). This change in the ratio describes a disproportionate rise in the net soleus concentrations of -ketoglutarate relative to the net changes in the concentrations of glycogen, which normalizes with refeeding. The lactate/glycogen ratio (Table 3) in the stimulated soleus was 22% lower (P < 0.01) in HYPO rats; with refeeding, this ratio fell another 97% (P < 0.01) from HYPO values and remained depressed in rats refed on d 4 (RE4).

View this table: [in this window] [in a new window]   Table 5. Change () in key glycolytic and tricarboxylic acid cycle metabolites in the stimulated soleus muscle of control (CN), energy-restricted (HYPO), 1 d (RE) and 4 d (RE4) refed male Wistar rats1,2

Glycolytic intermediates in unstimulated and stimulated EDL.  Lower lactate and -ketoglutarate concentrations were observed in the unstimulated EDL of HYPO and refed rats (P < 0.01) than in CN (Table 6). However, in the unstimulated muscle, the ratio F-1,6-P₂/F-6-P did not differ among the four groups (Table 7).

Unlike the soleus, stimulation of the EDL caused a greater muscle lactate concentration in HYPO and RE1 rats compared with controls (Table 8). Furthermore, the difference () between stimulated and unstimulated lactate and -ketoglutarate (Table 9), in HYPO and in RE1 rats was significant (P < 0.01). F-1,6-P₂ concentrations in the stimulated EDL muscle of HYPO rats were lower than those in CN rats (P < 0.01) (Table 8), but the ratio of F-1,6-P₂/F-6-P (Table 7) in HYPO rats did not differ from CN and refed values. In addition, the G-6-P/lactate ratio (Table 7) was lower in the muscle of HYPO-fed rats (P < 0.01) than in CN, describing a disproportionately more elevated lactate concentration relative to G-6-P concentration after fatigue in underfed rats. The lactate/glycogen ratio (Table 7) was significantly greater (P < 0.01) in the HYPO than in CN rats, a change in ratio that differed from muscle of CN rats and that describes an unusually high concentration of lactate relative to glycogen reserves.

View this table: [in this window] [in a new window]   Table 9. Change () in key glycolytic and tricarboxylic acid cycle metabolites in the electrically stimulated extensor digitorum muscle (EDL) muscle of control (CN), energy-restricted (HYPO), 1 d (RE) and 4 d (RE4) refed male Wistar rats1,2

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Previous studies have demonstrated reduced PFK and succinic dehydrogenase activity in vitro in the gastrocnemius of low energy-fed rats (Ardawi et al. 1989, Russell et al. 1984). In addition, Ardawi et al. (1989) reported increased activity of glycogen phosphorylase in the gastrocnemius and soleus of HYPO rats. Consistent with their findings, we previously showed a reduction in glycogen concentrations in both the soleus and EDL muscles of underfed rats, which was reversed with refeeding (Bissonnette et al. 1997). Despite these observations, it is not clear whether reduced in vitro glucose oxidation translates into an influence of the nutritional states on glycolysis in vivo. With repeated tetanic stimulation, we observed a reduced net glycogenolysis in the soleus and a supernormal net glycogenolysis in the EDL (Bissonnette et al. 1997), two contrasting reactions that suggest that undernutrition slows glycolysis only in slow-twitch-stimulated muscles fibers, and maintains or increases glycolysis in fast-twitch-stimulated fibers. To determine whether glycolysis differs among slow- and fast-twitch muscles of HYPO rats, we measured concentrations of glycolytic intermediate metabolites from in situ unstimulated and stimulated slow-twitch (soleus) (Tables 2, 4) and fast-twitch (EDL) (Tables 6, 8) muscles, changes in metabolite concentrations expressed as net-changes () (Tables 5, 9) and the mass action ratio of F-1,6-P₂/F-6-P.

Effect of energy-restricted feeding on the biochemistry of soleus muscle.  The soleus is normally expected to have a low total PFK enzyme concentration, a characteristic of slow-twitch muscles. In the resting state, when PFK activity is markedly down-regulated, any reduction of this enzyme activity by energy-restricted feeding would be expected to have maximal effect on metabolite concentrations. Under these circumstances, energy-restricted feeding would be expected to reduce lactate concentrations as well as the ratio of F-1,6-P₂/F-6-P and raise the G-6-P/lactate ratio relative to unstimulated CN. This is, in fact, what we observed (Table 3).

The relative transformation of substrate concentrations into product in the nonequilibrium reaction, F-6-P  F-1,6-P₂, is propounded as representative of PFK activity (Wakelman and Pette 1982). This holds true especially when metabolite concentration ratios such as F-1,6-P₂/ F-6-P and G-6-P/lactate are measured. The former quotient, when reported to be low, has been used to infer a decreased PFK activity (Wakelman and Pette 1982); similarly, the latter ratio has been used by Hultman et al. (1981) as an indirect measure of PFK activity in humans. Both ratios are a reflection of anaerobic metabolism.

The F-1,6-P₂/F-6-P ratio describes the concentration of product relative to substrate [B]/[A] (Newsholme and Leech 1983) and is an expression of the "Law of Mass Action." The fall in this ratio in the soleus of HYPO rats compared with CN occurred because of a decrease in the formation of F-1,6-P₂, whereas the concentration of F-6-P remained unchanged (Tables 2, 3). There was sufficient F-6-P concentration to favor a downward flux through the glycolytic rate-limiting step in compliance with the Law of Mass Action, thereby decreasing the likelihood of glyconeogenic activity (upward flux).

An increase in glyconeogenic activity is not likely because the lactate/glycogen ratio (Table 3) was significantly reduced (Newsholme and Leech 1983) in the HYPO rats. Glyconeogenic enzyme activity could surpass that of glycolysis when lactate concentrations are elevated and glycogen levels are low (Newsholme and Leech 1983). Furthermore, with refeeding, the lactate/glycogen ratio was 97% (P < 0.01) lower than HYPO values (Table 3), indicating that an even smaller possibility of glyconeogenesis exists and that muscle relies less on glycogen as a fuel.

The downward flux through glycolysis is further supported by the exercise protocol that was followed (Bissonnette et al. 1997). The stimulation protocol, consisting of repeated tetanic contractions provides the basis to assess qualitatively the downward flux direction of glycolysis in stimulated muscles. This is further supported by Newsholme and Leech (1983) who speculated that glycolytic fluxes can increase 1000-fold in human exercised muscle, based on a 25.5-fold increase in the rate of PFK and a 50% fall in fructose-1,6 bisphosphatase.

Therefore, despite abundant F-6-P and a stimulation protocol that should have been sufficient to maintain a normal downward flux through glycolysis and an unchanged F-1,6-P₂/F-6-P ratio, there is a fall in the F-1,6-P₂/F-6-P ratio, suggesting a decrease in PFK activity, which recovers with refeeding (Table 3). Consistent with this finding, we also report a rise in the G-6-P/lactate ratio (Table 3); however, this ratio does not normalize with 4 d of refeeding. Taken in association with the lower lactate/glycogen ratio and the greater -KG/lactate ratio (Table 3), there is evidence for a lower glycolytic flux in HYPO rats that does not promptly recover with refeeding. Moreover, the fall of the F-1,6-P₂/F-6-P ratio was associated with a more fatigable soleus, previously observed in HYPO rats; this fatigability was negatively correlated (r = 0.64) with net glycogenolysis (Bissonnette et al. 1997). Because glycogen utilization is lower in underfed rats, there may be a preferential shift towards an alternate exogenous substrate such as free fatty acids (FFA). We did not measure FFA levels nor did we study FFA metabolism; however, we assume that a shift from glycogen to FFA is likely to have occurred in the soleus for the following reasons: 1) FFA levels normally increase and become more available when insulin levels fall. Consistent with this pattern, we did previously report a fall in insulin and glucose levels in this underfed rat model (Bissonnette et al. 1997). 2) FFA are the preferred fuel of slow-twitch muscles like the soleus. 3) We found a disproportionate build-up of -KG relative to lactate (Table 3) in HYPO and RE1 rats, suggesting that the acetyl CoA that feeds the TCA cycle was coming mainly from the -oxidation of fat and not from pyruvate.

Effect of energy-restricted feeding on the biochemistry of EDL muscle.  In contrast, there is evidence for an undiminished PFK activity in the stimulated and unstimulated EDL muscle of HYPO rats, as demonstrated by the unchanged F-1,6-P₂/F-6-P quotient and by a significantly smaller G-6-P/lactate ratio (Table 7); the latter describes a disproportionate (P < 0.01) build-up of lactate that is consistent with either a slower TCA cycle flux or supranormal glycolytic flux or both. It is reasonable to expect that a stimulation protocol, consisting of short trains lasting <1 min and applied to a muscle composed mainly of fast-twitch fibers, would result in a more significant flux through glycolysis, thereby producing more lactate. When stimulated, the EDL lactate concentrations rose eightfold in the CN and more than 18-fold in the HYPO rats (Table 8). Such a rise in lactate, in a fast-twitch muscle, occurs because the muscle is composed mainly of glycolytic fibers. Correspondingly, lactate concentrations (Table 8) as well as the  lactate (Table 9) measured in fatigued EDL muscles were more elevated in HYPO and RE1 than in CN. The greater glycolytic flux in the HYPO and RE1 rats corresponds with the supernormal net glycogenolysis previously reported in these two groups (Bissonnette et al. 1997), with which was associated a maintenance of muscle endurance or, in other words, no increase in fatigue (Bissonnette et al. 1997).

The maintained endurance of the fast-twitch EDL muscle is related to greater glycolytic and glycogenolytic activity. This is substantiated by a rise in lactate (P < 0.01) (Table 8) and by a greater lactate/glycogen ratio (P < 0.01) (Table 7). The absence of fatigue in the underfed state is also supported by TCA cycle activity that increased proportionally with glycolysis as denoted by the stable  -KG/-glycogen ratio (Table 7).

On the first day of refeeding, the G-6-P/lactate ratio (Table 7) normalized because the sudden availability of glucose to the system caused greater (P < 0.01) G-6-P concentrations (Table 8) than in HYPO and CN rats. However, hexokinase activity, which would have reflected the extent of glucose availability and of phosphorylation, was not measured in this study.

In conclusion, we previously showed (Bissonnette et al. 1997) that greater soleus fatigability coincided with suboptimal nutrition and a decline in net glycogenolysis, which recovered with 4 d of refeeding. In this study, we also demonstrated that muscle fatigue is strongly linked to depressed PFK activity, which also recovers with 4 d of nutritional support, although there is some evidence that glycolytic flux may not recover as promptly. We did not determine, however, whether the muscle relied more heavily on FFA as an alternate fuel, nor did we measure mitochondrial redox to identify whether there was a shift from glycolysis to oxidative phosphorylation. In contrast to the soleus, our previous work (Bissonnette et al. 1997) found that the predominantly fast-twitch glycolytic EDL muscle adapted to semistarvation by increasing its use of glycogen. In addition, this study has shown that the EDL maintains or increases glycolysis in undernutrition. Earlier work (Bissonnette et al. 1997) showed that this greater reliance on glycogen coincided with an unchanged muscle fatigue compared with controls. As previously shown, fatigue is also related to a reduced rate of oxidative phosphorylation (Mijan de la Torre et al. 1993, Pichard et al. 1988) as well as to altered calcium kinetics (O'Brien et al. 1995). We were not able to substantiate these findings in this study; however, the influence of underfeeding on the glucose/insulin ratio (Bissonnette et al. 1997) and mitochondrial redox and that of altered cell energetics on calcium kinetics may be important metabolic roles that require further elucidation.

	FOOTNOTES

Manuscript received 11 March 1998. Initial reviews completed 13 April 1998. Revision accepted 27 May 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Altman, P. L. & Dittmer, D. S. (1968) Metabolism. In: Federation of American Societies for Experimental Biology (Britton E.C., ed.), pp. 101-112. Bethesda, MD.
• Ardawi, M.S.M., Majzoub, M. F., Masoud, I. M. & Newsholme, E. A. (1989) Enzymatic and metabolic adaptations in the gastrocnemius, plantaris, and soleus muscles of hypocaloric rats.
• Biochem. J. 261: 219-225.
• Bergmeyer, H. U. (1974) Methods of Enzymatic Analysis, Vol.3., 2nd ed. Academic Press, New York, NY.
• Bissonnette D. J., Madapallimatam A., Jeejeebhoy K. N. Effect of hypoenergetic feeding and high carbohydrate refeeding on muscle tetanic tension, relaxation rate, and fatigue in slow and fast twitch muscles in rats. Am. J. Clin. Nutr. 1997; 66:293-303[Abstract/Free Full Text]
• Hoshino E., Pichard C., Greenwood C. E., Kuo G. C., Cameron G., Kurian R., Kearns J. P., Allard J. P., Jeejeebhoy K. N. Body composition and metabolic rate in rats during a continuous infusion of cachectin. Am. J. Physiol. 1991; 260:E27-E36[Abstract/Free Full Text]
• Hultman, E., Sjohölm, H., Sahlin, K. & Edström, L. (1981) Glycolytic and oxidative energy metabolism and contraction characteristics of intact human muscle. In: Ciba Foundation Symposium 82. Human Muscle Fatigue: Physiological Mechanisms (Pitman Medical), pp.19-40. London, UK.
• Mijan de la Torre A., Madapallimattam A., Cross A., Armstrong R. L., Jeejeebhoy K. N. Effect of fasting, hypocaloric feeding and refeeding on the energetics of stimulated rat muscle as assessed by nuclear magnetic resonance spectroscopy. J. Clin. Investig. 1993; 92:114-121
• Moffet, D., Moffett, S. & Schauf, C. (1993) Human Physiology: Foundations & Frontiers, 2nd ed. Mosby-Year Book, Toronto, Canada.
• National Research Council (1978) Nutrient Requirements of Laboratory Rats, 3rd ed., pp. 7-37. National Academy of Sciences, Washington, DC.
• Newsholme, E. A. & Leech, A. R. (1983) Biochemistry for the Medical Sciences. John Wiley and Sons, New York, NY.
• O'Brien, P. J., Shen, H., Bissonette, D. & Jeejeebhoy, K. N. (1995) Effect of hypocaloric feeding and refeeding on myocardial Ca and ATP cycling in the rat. Mol. Cell. Biochem.142: 151-161.
• Pichard C., Vaughan C., Struck R., Armstrong R. L., Jeejeebhoy K. N. Effects of dietary manipulation (fasting, hypocaloric feeding, and subsequent refeeding) on rat muscle energetics assessed by nuclear magnetic resonance. J. Clin. Investig. 1988; 82:895-901
• Rees R.G.P, Keohane P. P., Grimble G. K., Frost P. G., Attrill H., Silk D.B.A. Elemental diet administerd nasogastrically without starter regimens to patients with inflammatory bowel disease. J. Parent. Enteral Nutr. 1986; 10:258-262[Abstract/Free Full Text]
• Rogers, H. E. (1979) Nutrition. In: The Laboratory Rat. Biology and Diseases (Baker, H. G., Lindsey, J. R. & Weisbroth, S. H., eds.), vol 1., pp. 123-152. Academic Press, New York, NY.
• Russell D. McR., Atwood, H. L. ,Whittaker, J. S., Itakura T., Walker P. M., Mickle D.A.G., Jeejeebhoy K. N. The effects of fasting and hypocaloric diets on the functional and metabolic characteristics of rat gastrocnemius muscle. Clin. Sci. (Lond.) 1984; 67:185-194[Medline]
• Wakelam M.J.O., Pette D. The control of glucose 1,6 bisphosphate by developmental state and hormonal stimulation in cultured muscle tissue. Biochem. J. 1982; 204:765-769[Medline]

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences