Improved insulin-stimulated glucose uptake and glycogen synthase activation in rat skeletal muscles after adrenaline infusion: role of glycogen content and PKB phosphorylation

AIM: Effects of in vivo adrenaline infusion on subsequent insulin-stimulated glucose uptake and glycogen synthase activation was investigated in slow-twitch (soleus) and fast-twitch (epitrochlearis) muscles. Furthermore, role of glycogen content and Protein kinase B (PKB) phosphorylation for modulation insulin sensitivity was investigated. METHODS: Male Wistar rats received adrenaline from osmotic mini pumps ( approximately 150 microg kg(-1) h(-1)) for 1 or 12 days before muscles were removed for in vitro studies. RESULTS: Glucose uptake at physiological insulin concentration was elevated in both muscles after 1 and 12 days of adrenaline infusion. Insulin-stimulated glycogen synthase activation was also improved in both muscles. This elevated insulin sensitivity occurred despite the muscles were exposed to hyperglycaemia in vivo. After 1 day of adrenaline infusion, glycogen content was reduced in both muscles; insulin-stimulated PKB ser(473) phosphorylation was increased in both muscles only at the highest insulin concentration. After 12 days of adrenaline infusion, glycogen remained low in epitrochlearis, but returned to normal level in soleus; insulin-stimulated PKB phosphorylation was normal in both muscles. CONCLUSION: Insulin-stimulated glucose uptake and glycogen synthase activation were increased after adrenaline infusion. Increased insulin-stimulated glucose uptake and glycogen synthase activation after adrenaline infusion cannot be explained by a reduction in glycogen content or an increase in PKB phosphorylation. The mechanisms for the improved insulin sensitivity after adrenaline treatment deserve particular attention as they occur in conjunction with hyperglycaemia.     

Caffeine and theophylline block insulin-stimulated glucose uptake and PKB phosphorylation in rat skeletal muscles

Aim: Caffeine and theophylline inhibit phosphatidylinositol 3-kinase (PI3-kinase) activity and insulin-stimulated protein kinase B (PKB) phosphorylation. Insulin-stimulated glucose uptake involves PI3-kinase/PKB, and the aim of the present study was to test the hypothesis that caffeine and theophylline inhibit insulin-stimulated glucose uptake in skeletal muscles. Methods: Rat epitrochlearis muscles and soleus strips were incubated with insulin and different concentrations of caffeine and theophylline for measurement of glucose uptake, force development and PKB phosphorylation. The effect of caffeine was also investigated in muscles stimulated electrically. Results: Caffeine and theophylline completely blocked insulin-stimulated glucose uptake in both soleus and epitrochlearis muscles at 10 mm . Furthermore, insulin-stimulated PKB Ser⁴⁷³ and Thr³⁰⁸ and GSK-3β Ser⁹ phosphorylation were blocked by caffeine and theophylline. Caffeine reduced and theophylline blocked insulin-stimulated glycogen synthase activation. Caffeine stimulates Ca²⁺ release and force development increased rapidly to 10–20% of maximal tetanic contraction. Dantrolene (25 μm ), a well-known inhibitor of Ca²⁺-release, prevented caffeine-induced force development, but caffeine inhibited insulin-stimulated glucose uptake in the presence of dantrolene. Contraction, like insulin, stimulates glucose uptake via translocation of glucose transporter-4 (GLUT4). Caffeine and theophylline reduced contraction-stimulated glucose uptake by about 50%, whereas contraction-stimulated glycogen breakdown was normal. Conclusion: Caffeine and theophylline block insulin-stimulated glucose uptake independently of Ca²⁺ release, and the likely mechanism is via blockade of insulin-stimulated PI3-kinase/PKB activation. Caffeine and theophylline also reduced contraction-stimulated glucose uptake, which occurs independently of PI3-kinase/PKB, and we hypothesize that caffeine and theophylline also inhibit glucose uptake in skeletal muscles via an additional and hitherto unknown molecule involved in GLUT4 translocation.     

Adrenaline potentiates insulin-stimulated PKB activation in the rat fast-twitch epitrochlearis muscle without affecting IRS-1-associated PI 3-kinase activity

We have previously shown in the rat slow-twitch soleus muscle that adrenaline greatly potentiates insulin-stimulated protein kinase B (PKB) phosphorylation without having an effect alone. However, insulin signalling capacity through the PKB pathway is higher in soleus than in fast-twitch muscles, whereas adrenaline activates phosphorylase more strongly in epitrochlearis. Therefore, the aim of the present study was to investigate the interaction between adrenaline and insulin signalling in the fast-twitch epitrochlearis muscle. Insulin increased insulin receptor substrate-1 (IRS-1)-associated phosphoinositide (PI) 3-kinase activity threefold, and adrenaline did not influence basal or insulin-stimulated PI 3-kinase activity. Insulin but not adrenaline increased PKB activity and phosphorylation of Ser(473) and Thr(308). It is interesting to note that adrenaline potentiated insulin-stimulated PKB activity and PKB Ser(473) and Thr(308) phosphorylation. These effects were mimicked by dibutyryl-cyclic adenosine monophosphate (db-cAMP). Adrenaline and db-cAMP increased glycogen synthase kinase (GSK)-3beta Ser(9) phosphorylation independently of PKB activation and enhanced insulin-stimulated GSK-3beta Ser(9) phosphorylation. Although adrenaline increased GSK-3 phosphorylation (inhibiting activity), phosphorylation of its target sites on glycogen synthase was increased, and adrenaline blocked insulin-stimulated glycogen synthase dephosphorylation of Ser(641) and Ser(645,649,653,657), glycogen synthase activation and glycogen synthesis. Insulin-stimulated glucose transport was not influenced by adrenaline despite the increased PKB activation. In conclusion, as in the slow-twitch soleus muscle, adrenaline potentiates insulin-stimulated PKB activation in the fast-twitch glycolytic epitrochlearis muscle without increasing IRS-1-associated PI 3-kinase activity. Furthermore, adrenaline induces phosphorylation of a pool of GSK-3 that is not involved in the regulation of glycogen metabolism. These results indicate that the combination of adrenaline and insulin may activate novel signalling molecules rather than just summing up their effects on linear pathways.     

Glycogen content regulates insulin‐ but not contraction‐mediated glycogen synthase activation in the rat slow‐twitch soleus muscles

Aim: The aim of this study was to investigate the effect of glycogen content on glycogen synthase (GS) activation and phosphorylation in the slow‐twitch soleus muscles after contraction, during insulin stimulation and when these two stimuli were combined. Methods: Glycogen content was manipulated in vivo with 24 h fasting and fasting followed by 24 h refeeding. Soleus strips were electrically stimulated for 30 min in vitro, and GS activation and phosphorylation were investigated after an additional 30 min incubation with or without insulin. Results: Fasting reduced glycogen content in soleus muscle by 40% and refeeding enhanced by 40%, compared to rats with free access to chow. Insulin‐stimulated GS fractional activity was inversely correlated with glycogen content (R = ?0.95, P < 0.001, n = 24) and rate of glycogen synthesis was also inversely correlated with glycogen content (R = ?0.70, P < 0.001, n = 36). After contraction, GS fractional activity was increased to similar levels in muscles with low, normal and high glycogen content; rate of glycogen synthesis after contraction was also similar. After contraction, insulin additively increased GS activation at all glycogen contents. Group means of GS fractional activity was inversely correlated with GS Ser⁶⁴¹ (R = ?0.93, P < 0.001) and Ser^{645,649,653,657} (R = ?0.85, P < 0.001) phosphorylation, but not with Ser⁷ phosphorylation. Conclusion: Glycogen content regulates insulin‐ but not contraction‐stimulated GS activation and glycogen synthesis in soleus muscles. Furthermore, phosphorylation of GS Ser⁶⁴¹ and Ser^{645,649,653,657} seems to regulate GS activity in soleus.     

Magnetic resonance spectroscopy shows an inverse correlation between intramyocellular lipid content in human calf muscle and local glycogen synthesis rate

Intramyocellular lipid (IMCL) content of skeletal muscle, as measured with ¹H MRS, is inversely correlated with insulin sensitivity as determined by whole body glucose uptake. The latter, however, does not necessarily represent the actual glucose uptake in the corresponding skeletal muscle. In this study, we examined whether IMCL content in human calf muscle correlated with local glucose uptake assessed by measurement of glycogen synthesis rate within the same muscle compartment. We studied 20 subjects belonging to four subgroups of five persons each: young lean, elderly lean, young obese and elderly obese. IMCL content in the soleus and gastrocnemius muscle was determined using ¹H MR spectroscopic imaging and local glycogen synthesis rate in the calf muscle was measured by ¹³C MRS during a euglycaemic hyperinsulinaemic clamp with 20% w/v 30% ¹³C‐1‐labelled glucose infusion. Significantly higher IMCL contents were found in elderly (soleus: p < 0.0001 and gastrocnemius: p < 0.01) and obese subjects (p < 0.01 for both muscles). Local glycogen synthesis rate decreased significantly with obesity (p < 0.01). The principal finding of this study was that the mean IMCL content of the soleus and gastrocnemius muscles was indeed inversely correlated with the local glycogen synthesis rate in the calf muscle (r_s = ?0.50, p < 0.05), with a very similar dependency as the inverse correlation between mean IMCL content and total body glucose uptake (r_s = ?0.54, p < 0.05). We conclude that IMCL content of the soleus and gastrocnemius muscles reflects a measure for local insulin resistance within the same muscle compartment as determined by glycogen synthesis rate. Although the inverse correlation suggests that insulin sensitivity is affected by the local amount of fat present, it remains to be determined if this is a cause or a consequence. Copyright © 2009 John Wiley & Sons, Ltd.     

Additivity of adrenaline and contractions on hormone‐sensitive lipase,but not on glycogen phosphorylase,in rat muscle

Aim: Hormone‐sensitive lipase (HSL) has been proposed to regulate triacylglycerol (TG) breakdown in skeletal muscle. In muscles with different fibre type compositions the influence on HSL of two major stimuli causing TG mobilization was studied. Methods: Incubated soleus and extensor digitorum longus (EDL) muscles from 70 g rats were stimulated by adrenaline (5.5 μm , 6 min) or contractions (200 ms tetani, 1 Hz, 1 min) in maximally effective doses or by both adrenaline and contractions. Results: Hormone‐sensitive lipase activity was increased significantly by adrenaline as well as contractions, and the highest activity (P < 0.05) was seen with combined stimulation [Soleus: 0.40 ± 0.03 (SE) m‐unit mg protein^?1 (basal), 0.65 ± 0.02 (adrenaline), 0.65 ± 0.03 (contractions), 0.78 ± 0.03 (adrenaline and contractions); EDL: 0.18 ± 0.01, 0.30 ± 0.02, 0.26 ± 0.02, 0.32 ± 0.01]. Glycogen phosphorylase activity was always increased more by adrenaline compared with contractions [Soleus: 60 ± 4 (a/a + b)% vs. 46 ± 3 (P < 0.05); EDL: 60 ± 5 vs. 39 ± 6 (P < 0.05)]. After combined stimulation glycogen phosphorylase activity in soleus [59 ± 3 (a/a + b)%] was identical to and in EDL [45 ± 4 (a/a + b)%] smaller (P < 0.05) than the activity after adrenaline only. Conclusions: In slow‐twitch oxidative as well as in fast‐twitch glycolytic muscle HSL is activated by both adrenaline and contractions. These stimuli are partially additive indicating at least partly different mechanisms of action. Contractions may impair the enhancing effect of adrenaline on glycogen phosphorylase activity in muscle.     

Effect of force development on contraction induced glucose transport in fast twitch rat muscle

A previous study has shown that in fast twitch frog sartorius muscle contraction stimulated glucose transport depends only on stimulation frequency and not on workload. In contrast, we have recently shown that in rat slow twitch muscle stimulated to contract at constant frequency, glucose transport varies directly with force development and, in turn, metabolism. The present study was carried out to clarify whether the discrepancy between the earlier studies reflected differences in physiological behaviour between fast and slow twitch muscle. We investigated the effect of force development on glucose transport in incubated fast twitch rat flexor digitorum brevis (rich in type 2a fibres) and epitrochlearis (rich in type 2b fibres) muscle. Muscles were electrically stimulated to perform repeated tetanic contractions at 1 Hz for 10 min. Resting length was adjusted to achieve either no force or maximum force. Glucose transport (2‐deoxyglucose uptake) increased when force was produced compared with when it was not (P < 0.05) in both flexor digitorum brevis (19 ± 7 (basal), 163 ± 14 (no force) and 242 ± 17 (max force) nmol × g^–1 × 5 min^–1) and epitrochlearis (60 ± 4 (basal), 100 ± 7 (no force) and 125 ± 6 (max force) nmol × g^–1 × 5 min^–1). In both muscles glucose transport increased in parallel with metabolic rate, as reflected by muscle lactate concentrations and 5′ AMP‐activated protein kinase activity, during contractions. In conclusion, as previously shown for rat soleus muscle, at a given stimulation frequency glucose transport varies directly with force development in rat flexor digitorum brevis and epitrochlearis muscle. Accordingly, force development enhances glucose transport in all mammalian muscle fibre types. The influence of force development probably reflects effects of enhanced 5′ AMP‐activated protein kinase activity resulting from reduced intra‐cellular energy status and pH.     

Effect of diffusion distance on measurement of rat skeletal muscle glucose transport in vitro

The relationships between muscle size, diffusion distance, and glucose uptake were studied using the Type II b epitrochlearis (13 ± 1 mg intact), Type I soleus (25± 1 mg), and mixed Type II a/II b extensor digitorum longus (25 ± 1 mg) from 60–70 g rats. Using intact muscles, the relative rates of 3-O-methyl-glucose uptake in response to 2 mUml^-1 insulin were soleus = epitrochlearis > extensor digitorum longus, a finding inconsistent with the fibre-type compositions and the relative GLUT-4 protein levels (soleus > extensor digitorum longus > epitrochlearis). To test whether these results were influenced by substrate diffusion limitations in the tubular muscles, soleus and extensor digitorum longus were split longitudinally from tendon to tendon into strips of comparable size (13 ± 1 mg) to the epitrochlearis. Insulin-stimulated rates of 3-O-methyl-glucose uptake were significantly enhanced in the split soleus (+120%) and split extensor digitorum longus (+200%), but not in the epitrochlearis, with the relative rates being soleus > extensor digitorum longus > epitrochlearis. Diffusion distances of the split soleus and extensor digitorum longus, as reflected by [¹⁴C]mannitol space equilibration time, were markedly enhanced (by at least 50%) relative to the intact muscles, and were comparable to that of the epitrochlearis. These results indicate that when muscles of different size and/or shape are used for in vitro measurement of glucose transport, the muscle preparations used must have similar diffusion distances for physiologically meaningful comparisons to be made.     

Effects of gender and prior swim exercise on glucose uptake in isolated skeletal muscles from mice

The purpose of this study was to characterize the effects of prior swim exercise on glucose uptake in isolated skeletal muscles of mice. Male and female mice (C57BL/6) performing 180 min of swimming had significantly decreased glycogen concentration compared to resting controls in soleus, extensor digitorum longus (EDL), and epitrochlearis muscles, regardless of gender. Glucose uptake by isolated muscles was measured using [(3)H]-2-deoxyglucose without insulin or with 180 pmol/l insulin (20, 75, or 200 min post-exercise and sedentary) or 12,000 pmol/l (20 or 200 min post-exercise and sedentary) in the soleus and EDL and without insulin or with 12,000 pmol/l insulin (20 or 200 min post-exercise and sedentary) in the epitrochlearis. Glucose uptake was higher (P < or = 0.01) for female versus male mice at each insulin concentration in the soleus and EDL, but not the epitrochlearis. Although prolonged (180 min) swim exercise did not alter subsequent glucose uptake, a shorter duration exercise protocol (60 min) tested in male mice (20 min post-exercise) led to a 1.5-fold increase in insulin-independent glucose uptake in EDL muscles. However, insulin-stimulated (180 pmol/l) glucose uptake was not altered by 60 min exercise in EDL or soleus. In light of these results, swim exercise is not recommended to evaluate the exercise-induced improvement in insulin-stimulated glucose uptake of muscles of C57BL/6 mice.     

Differential activation of mitogen‐activated protein kinase signalling pathways by isometric contractions in isolated slow‐ and fast‐twitch rat skeletal muscle

Activation of mitogen‐activated protein (MAP) kinases has been implicated in the signal transduction pathways linking exercise to adaptive changes of muscle protein expression. In the present study, we investigated whether contractions of isolated muscles induced phosphorylation of extracellular signal‐regulated kinase 1 and 2 (ERK1/2) and p38 MAPK in a fibre‐type dependent manner. Slow‐twitch (soleus) and fast‐twitch (epitrochlearis, extensor digitorum longus) rat skeletal muscles were exposed to intermittent tetanic stimulation. Compared with the contralateral non‐stimulated muscle, contractions increased ERK1/2 phosphorylation to the same extent in fast‐ and slow‐twitch muscles. Significant increase in phosphorylation of p38 MAPK was observed in the fast‐twitch muscles only. The total amount of ERK1/2 and p38 MAPK proteins was higher in the slow‐twitch soleus muscle. In conclusion, MAP kinase signalling pathways are differentially activated and expressed in slow‐ and fast‐twitch muscles. In addition, this activation is owing to muscle contraction per se and do not demand additional external influence.     

Effects of adrenaline infusion on cAMP and glycogen phosphorylase in fast-twitch and slow-twitch rat muscles

This study investigated the effect of adrenaline infusion on the cAMP content, glycogen phosphorylase activity and the rate of glycogen breakdown in rat extensor digitorum longus (EDL) and soleus muscles. Adrenaline was constantly infused in a dose of 0.15 micrograms kg-1 body wt min-1. The cAMP content increased approximately 2.8-fold in both muscles after 2 min of infusion. Phosphorylase a + b activity was six times higher in fast-twitch muscle (EDL) than in slow-twitch (soleus) and remained unchanged during the infusion. Phosphorylase a activity increased by 8.4-fold in EDL and 2.4-fold in soleus muscles during the infusion period. Glycogen content decreased in EDL muscle by 10% whereas no change was observed in soleus. It is concluded that beta-adrenergic stimulation by adrenaline results in a similar cAMP increase in both muscles. The low rate of glycogen breakdown in EDL and the unchanged content of glycogen in soleus muscle suggest that cAMP mediated transformation of phosphorylase b to a in itself is not adequate for a rapid glycogenolysis in muscle.     

Elevated free fatty acid levels inhibit glucose phosphorylation in slow-twitch rat skeletal muscle

The effect of increased free fatty acid concentrations on glucose metabolism in rat skeletal muscle was investigated at several different steps in glucose metabolism including glucose transport, glucose phosphorylation, glucose oxidation and glycogen synthesis. In isolated soleus (slow-twitch) muscles, insulin-stimulated (100 μml^-1) glucose phosphorylation, but not glucose transport, was inhibited by 26 and 22% in the presence of 1.0 and 2.0 mM oleate, respectively (P< 0.01). Regardless of oleate concentration (0.3 or 2.0 mM), insulin-stimulated glucose 6-phosphate levels were elevated to the same extent over the non-insulin-stimulated levels in soleus muscles {P < 0.01). Insulin-stimulated glucose oxidation was inhibited by 44% in soleus muscles exposed to 2.0 mM oleate (P < 0.05), whereas the rate of glucose incorporation into glycogen was not altered. In insulin-stimulated epitrochlearis (fast-twitch) muscles, elevated concentrations of oleate had no effect on the rates of glucose transport or glucose phosphorylation, or on the level of glucose 6-phosphate. These data suggest that increased free fatty acid availability decreases glucose utilization by selectively inhibiting glucose phosphorylation and oxidation in slow-twitch, but not fast-twitch skeletal muscle.     

Modulation of glycogen metabolism of rat skeletal muscles by endurance training and testosterone treatment

The effects of training and/or testosterone treatment on glycogen content and the activities of glycogen synthase, glycogen phosphorylase, and fructose-6-phosphate kinase were studied in extensor digitorum longus (EDL) and soleus muscles of intact adult female rats. One group of rats remained sedentary, whereas another group was trained for 7 weeks. Thereafter, both the sedentary and trained rats were subdivided into two control and four testosterone-treated subgroups. Testosterone was administered by a silastic implant. Training was continued for 2 weeks. On the final day of the experiment rats from one trained control and one trained testosterone-treated subgroup ran for 60 min submaximally. Upon testosterone treatment of sedentary rats the glycogen concentration was not changed. However, in the soleus, but not in the EDL, the glycogen content was increased by training (P<0.05) which could, at least partly, be explained by a decrease in activity of active glycogen phosphorylase (P < 0.05). In the EDL of trained rats testosterone treatment increased glycogen content significantly by both an increase in activity of active glycogen synthase and a decrease in activity of active glycogen phosphorylase (P<0.05). In the EDL and soleus of testosterone-treated animals from the exercised subgroup a significant sparing of glycogen was observed, which could be explained by an increase in activity of active glycogen synthase and, in the soleus, could also be explained by a concerted decrease in active glycogen phosphorylase (P<0.05). In the two muscles studied, we also found that testosterone treatment in trained animals shifted the fibre type distribution towards more oxidative fibres in both types of muscle in comparison with the control animals. We conclude that testosterone, at a pharmacological dose, potentiates the training-induced increase in glycogen content of skeletal muscle and induces a glycogen-sparing effect after submaximal exercise.An Established Investigator of the Netherlands Heart Foundation     

Caffeine activates preferentially α1-isoform of 5'AMP-activated protein kinase in rat skeletal muscle

Aim: Caffeine activates 5′AMP‐activated protein kinase (AMPK), a signalling intermediary implicated in the regulation of glucose, lipid and energy metabolism in skeletal muscle. Skeletal muscle expresses two catalytic α subunits of AMPK, α1 and α2, but the isoform specificity of caffeine‐induced AMPK activation is unclear. The aim of this study was to determine which α isoform is preferentially activated by caffeine in vitro and in vivo using rat skeletal muscle. Methods: Rat epitrochlearis muscle was isolated and incubated in vitro in the absence or presence of caffeine. In another experiment, the muscle was dissected after intravenous injection of caffeine. Isoform‐specific AMPK activity, the phosphorylation status of AMPKα Thr¹⁷² and acetyl‐CoA carboxylase (ACC) Ser⁷⁹, the concentrations of ATP, phosphocreatine (PCr) and glycogen, and 3‐O‐methyl‐d ‐glucose (3MG) transport activity were estimated. Results: Incubation of isolated epitrochlearis muscle with 1 mm of caffeine for 15 min increased AMPKα1 activity, but not AMPKα2 activity; concentrations of ATP, PCr and glycogen were not affected. Incubation with 3 mm of caffeine activated AMPKα2 and reduced PCr and glycogen concentrations. Incubation with 1 mm of caffeine increased the phosphorylation of AMPK and ACC and enhanced 3MG transport. Intravenous injection of caffeine (5 mg kg^?1) predominantly activated AMPKα1 and increased 3MG transport without affecting energy status. Conclusion: Our results suggest that of the two α isoforms of AMPK, AMPKα1 is predominantly activated by caffeine via an energy‐independent mechanism and that the activation of AMPKα1 increases glucose transport and ACC phosphorylation in skeletal muscle.     

Effect of creatine supplementation on creatine and glycogen content in rat skeletal muscle

The effects of high dose creatine feeding (5 g kg(-1) BW day(-1), 5 days) on creatine content, glucose transport, and glycogen accumulation in white gastrocnemius, red gastrocnemius and soleus muscles of the rat was investigated. Isolated rat hindquarters of creatine fed and control rats were perfused with a standard medium containing either insulin alone (0, 100 or 20 000 microU mL(-1)) or in combination with creatine (2 or 10 mmol L(-1)). Furthermore, plasma insulin concentration was measured in normal rats during creatine feeding, as well as in anaesthetized rats during intravenous creatine infusion. Five days of creatine feeding increased (P < 0.05) total creatine content in soleus (+ 20%) but not in red gastrocnemius (+15%, n.s.) and white gastrocnemius (+ 10%, n.s.). In parallel, glycogen content was markedly elevated (P < 0.05) in soleus (+ 40%), less (P < 0.05) in red gastrocnemius (+ 15%), and not in white gastrocnemius (+ 10%, n.s.). Glucose transport rate, muscle GLUT-4 content, glycogen synthase activity in perfused muscles and glycogen synthesis rate were not significantly altered by creatine feeding in either muscle type. Furthermore, high dose creatine feeding raised (P < 0.05) plasma creatine concentration fivefold but did not alter circulating insulin level. It is concluded that short-term high dose creatine feeding enhances creatine disposal and glycogen storage in rat skeletal muscle. However, the creatine and glycogen response to creatine supplementation is markedly greater in oxidative than in glycolytic muscles.     

In-vitro stimulation of the rat epitrochlearis muscle. II. Effects of catecholamines and nutrients on protein degradation and amino acid metabolism

The influence of catecholamines and branched-chain amino acids (BCAA) plus insulin on protein degradation and amino acid metabolism was investigated in isolated and electrically stimulated rat epitrochlearis muscles. 10^-7m adrenaline significantly increased the total amount of muscle tyrosine during 40 min of stimulation with 50 Hz (1 s min^-1) pulse trains. On the other hand, BCAA + insulin at normal and five times normal plasma concentrations had no effect on muscle tyrosine. Muscle 3-methylhistidine was not influenced by any of the treatments. Muscle release and content of aspartate, alanine, glutamate and glutamine showed individual response characteristics to catecholamines and BCAA + insulin. The data indicate that adrenaline can induce an increased total protein degradation in rat fast muscle during acute contractions in vitro and, furthermore, that BCAA + insulin does not retard protein breakdown during acute muscle contraction.     

Effect of diffusion distance on measurement of rat skeletal muscle glucose transport in vitro.

The relationships between muscle size, diffusion distance, and glucose uptake were studied using the Type IIb epitrochlearis (13 +/- 1 mg intact), Type I soleus (25 +/- 1 mg), and mixed Type IIa/IIb extensor digitorum longus (25 +/- 1 mg) from 60-70 g rats. Using intact muscles, the relative rates of 3-O-methyl-glucose uptake in response to 2 mUml-1 insulin were soleus = epitrochlearis greater than extensor digitorum longus, a finding inconsistent with the fibre-type compositions and the relative GLUT-4 protein levels (soleus greater than extensor digitorum longus greater than epitrochlearis). To test whether these results were influenced by substrate diffusion limitations in the tubular muscles, soleus and extensor digitorum longus were split longitudinally from tendon to tendon into strips of comparable size (13 +/- 1 mg) to the epitrochlearis. Insulin-stimulated rates of 3-O-methyl-glucose uptake were significantly enhanced in the split soleus (+120%) and split extensor digitorum longus (+200%), but not in the epitrochlearis, with the relative rates being soleus greater than extensor digitorum longus greater than epitrochlearis. Diffusion distances of the split soleus and extensor digitorum longus, as reflected by [14C]mannitol space equilibration time, were markedly enhanced (by at least 50%) relative to the intact muscles, and were comparable to that of the epitrochlearis. These results indicate that when muscles of different size and/or shape are used for in vitro measurement of glucose transport, the muscle preparations used must have similar diffusion distances for physiologically meaningful comparisons to be made.     

Metabolite accumulation increases adenine nucleotide degradation and decreases glycogenolysis in ischaemic rat skeletal muscle

Adenine nucleotides and glycogen are degraded in skeletal muscle during no-flow ischaemia. Past investigations have ascribed these metabolic changes to the severe energetic stress which arises with the removal of exogenous substrates (principally oxygen). We tested this hypothesis by measuring the high-energy phosphagen and glycogen contents of stimulated rat hindlimb muscles (1 twitch s^?1) prior to and following 40 min of no-flow ischaemia or hypoxic perfusion without glucose (P_ao₂ = 4.6 ± 0.1 torr, plasma glucose = 0.3 ± 0.1 mmol L^?1). Both experimental protocols eliminated exogenous substrate supply; however, the maintenance of flow during hypoxic perfusion ensured the removal of metabolic by-products. A period of forty minutes of skeletal muscle ischaemia was characterized by reductions in the total adenine nucleotide pool, phosphocreatine and glycogen in the slow oxidative soleus, fast oxidative-glycolytic plantaris and the fast glycolytic white gastrocnemius. Compared to ischaemia, the total adenine nucleotide pool was higher (by 7.2–13.3 μmol g^?1 dry wt) and the glycogen content lower (by 10.0–16.6 μmol g^?1 dry wt) in skeletal muscle exposed to hypoxic perfusion without glucose. The ability of hypoxic perfusion to attenuate TAN degradation and augment glycogenolysis can be attributed to metabolic by-product removal. By limiting muscle lactate and PCO ₂ accumulation, hypoxic perfusion without glucose attenuates cellular acidification; this could in turn limit AMP deaminase activation and glycogen phosphorylase inhibition. We conclude that the ischaemia-induced alterations in adenine nucleotide and glycogen metabolism arise in response to the elimination of exogenous substrates and to the accumulation of metabolic by-products.     

Insulin-independent glycogen supercompensation in isolated mouse skeletal muscle: role of phosphorylase inactivation

Glycogen supercompensation (increase in muscle glycogen content above basal) is an established phenomenon induced by unknown mechanisms. It consists of both insulin-dependent and -independent components. Here, we investigate insulin-independent glycogen supercompensation in isolated, intact extensor digitorum longus muscles from mice. Muscles were stimulated electrically, incubated in vitro with 5.5 mM glucose for up to 16 h and then analysed for glycogen, glucose uptake and enzyme activities. Basal glycogen was 84±6 µmol glucosyl units/g dry muscle and was depleted by 80% after 10 min contraction. Glycogen increased after contraction, reaching a peak value of 113±9 µmol glucosyl units/g dry muscle (P<0.05 vs. basal) by 6 h, and returned to basal values by 16 h (84±8). Maximal activities of glycogen synthase, phosphorylase and -glucosidase were not significantly altered by contraction or during the 6-h recovery period. Glycogen synthase fractional activity (0.17/7.2 mM glucose-6-P; inversely related to phosphorylation state of the enzyme) was increased about twofold early after contraction but then decreased and was slightly lower than baseline during the period of supercompensation (4–6 h). Phosphorylase fractional activity (±adenosine monophosphate; directly related to phosphorylation state of the enzyme) decreased to 60% of basal after contraction and decreased further during the initial 4 h of recovery to 40% of basal (P<0.01 vs. basal). After 4 h recovery, glucose uptake was slightly (50%) higher in the stimulated than in the non-stimulated muscle (P<0.01). Thus, insulin-independent glycogen supercompensation involves inactivation of phosphorylase and hence an inhibition of glycogen breakdown.     

Greater filamin C,GSK3α, and GSK3β serine phosphorylation in insulin-stimulated isolated skeletal muscles of calorie restricted 24 month-old rats

Moderate calorie restriction (CR) can improve insulin-stimulated Akt phosphorylation and glucose uptake in muscles from 24 month-old rats, but the specific Akt substrates linking CR-effects on Akt to glucose uptake and other cellular processes are uncertain. We probed CR's influence on site-specific phosphorylation of five Akt substrates (AS160^Ser588, TBC1D1^Thr596, FLNc^Ser2213, GSK3α^Ser21, and GSK3β^Ser9) in predominantly fast-twitch (epitrochlearis) and predominantly slow-twitch (soleus) muscles. We observed no CR-effect on phosphorylation of AS160^Ser588 or TBC1D1^Thr596, but there was a CR-induced increase in insulin-stimulated FLNc^Ser2213, GSK3α^Ser21, and GSK3β^Ser9 phosphorylation for both muscles. These results indicate that CR does not uniformly affect insulin-mediated phosphorylation of Akt substrates in fast- or slow-twitch muscles from 24 month-old rats.