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 (≈150 μg 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 ser⁴⁷³ 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.     

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.     

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.     

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.     

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.     

Effect of the nicotine metabolite 5'-hydroxycotinine on glucose transport and glycogen synthase activity in rat skeletal muscle

Cigarette smoke contains many potentially harmful substances, including nicotine and nicotine metabolites, which are likely to contribute to altered glucose homeostasis. We determined the effects of nicotine and nicotine derivatives on glucose transport in skeletal muscle. Split rat soleus muscles were pre-incubated in the presence of nicotine (range 0.01-100 microg/ml) or nicotine metabolites including nicotine 1'-N-oxide, cotinine, trans-3'-hydroxycotinine, 5'-hydroxycotinine, gamma-3-pyridyly-oxo-butyric acid and nicotine iminium ion before measurement of 3-O-methylglucose transport rate and glycogen synthase activity. Nicotine (100 microg/ml) did not alter basal 3-O-methylglucose transport. Insulin-stimulated (0.6 nmol/l) glucose transport was unaltered following acute (50 min) exposure to nicotine (0.01-100 microg/ml). The nicotine metabolite 5'-hydroxycotinine increased basal glucose transport and glycogen synthase activity (up to 50%; P<0.05), with no effect on insulin-stimulated glucose transport and glycogen synthase activity. None of the other nicotine metabolites had any effect on basal or insulin-stimulated glucose transport. Acute exposure of skeletal muscle to the nicotine derivative 5'-hydroxycotinine appears to directly increase basal glucose transport and metabolism. Whether this leads to changes in whole-body glucose homeostasis in cigarette smokers requires further investigation.     

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.     

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.     

Acute and chronic effects of strenuous exercise on glucose metabolism in isolated, incubated soleus muscle of exercise-trained rats

Male and female Wistar rats were exercise-trained for 6 or 11 weeks respectively, to examine the effects of acute exercise or exercise training per se on insulin-stimulated glucose utilization in soleus muscles isolated and incubated in vitro. The maximal activities of hexokinase and 2-oxoglutarate dehydrogenase were significantly elevated (by greater than 50%) in gastrocnemius muscle of exercise-trained male and female rats, indicating an adaptation to the training regime. No significant differences in any of the variables studied were observed between appropriately matched male and female rats. There were no significant differences in the sensitivity or responsiveness of the rates of lactate formation or glycogen synthesis in soleus muscles isolated from exercise-trained and sedentary animals at rest (exercise-trained animals were studied 40 h after the last exercise bout). On the other hand, acute exercise caused significant changes in soleus muscle glucose metabolism. Basal and insulin-stimulated rates of glycogen synthesis were significantly elevated in soleus muscles incubated from both sedentary and exercise-trained rats immediately after an exercise bout. In addition, the responsiveness of glucose utilization to insulin in soleus muscles from exercise-trained rats was significantly increased after acute exercise. The results indicate that significant changes in the control of glucose metabolism by insulin in soleus muscle occur as a result of an acute exercise bout, while no adaptive changes in insulin sensitivity occur in soleus muscle after exercise training.     

Studies of gene expression and activity of hexokinase, phosphofructokinase and glycogen synthase in human skeletal muscle in states of altered insulin-stimulated glucose metabolism

When whole body insulin-stimulated glucose disposal rate is measured in man applying the euglycaemic, hyperinsulinaemic clamp technique it has been shown that approximately 75% of glucose is taken up by skeletal muscle. After the initial transport step, glucose is rapidly phosphorylated to glucose-6-phosphate and routed into the major pathways of either glucose storage as glycogen or the glycolytic/tricarboxylic acid pathway. Glucose uptake in skeletal muscle involves-the activity of specific glucose transporters and hexokinases, whereas, phosphofructokinase and glycogen synthase hold critical roles in glucose oxidation/glycolysis and glucose storage, respectively. Glucose transporters and glycogen synthase activities are directly and acutely stimulated by insulin whereas the activities of hexokinases and phosphofructokinase may primarily be allosterically regulated. The aim of the review is to discuss our present knowledge of the activities and gene expression of hexokinase II (HKII), phosphofructokinase (PFK) and glycogen synthase (GS) in human skeletal muscle in states of altered insulin-stimulated glucose metabolism. My own experimental studies have comprised patients with disorders characterized by insulin resistance like non-insulin-dependent diabetes mellitus (NIDDM) and insulin-dependent diabetes mellitus (IDDM) before and after therapeutic interventions, patients with microvascular angina and patients with severe insulin resistant diabetes mellitus and congenital muscle fiber type disproportion myopathy as well as athletes who are in a state of improved insulin sensitivity. By applying the glucose insulin clamp method in combination with nuclear magnetic resonance 31P spectroscopy to normoglycaemic or hyperglycaemic insulin resistant subjects impairment of insulin-stimulated glucose transport and/or phosphorylation in skeletal muscle has been shown. In states characterized by insulin resistance but normoglycaemia, the activity of HKII measured in needle revealed any genetic variability that contributes to explain the decreased muscle levels of GS mRNA or the decreased activity and activation of muscle GS in NIDDM patients and their glucose tolerant but insulin resistant relatives. Thus, the causes of impaired insulin-stimulated glycogen synthesis of skeletal muscle in normoglycaemic insulin resistant subjects are likely to be found in the insulin signalling network proximal to the GS protein. In insulin resistant diabetic patients the impact of these yet unknown abnormalities may be accentuated by the prevailing hyperglycaemia and hyperlipidaemia. Endurance training in young healthy subjects results in improved insulin-stimulated glucose disposal rates, predominantly due to an increased glycogen synthesis rate in muscle, which is paralleled by an increased total GS activity, increased GS mRNA levels and enhanced insulin-stimulated activation of GS. These changes are probably due to local contraction-dependent mechanisms. Likewise, one-legged exercise training has been reported to increase the basal concentration of muscle GS mRNA in NIDDM patients to a level similar to that seen in control subjects although insulin-stimulated glucose disposal rates remain reduced in NIDDM patients. In the insulin resistant states examined so far, basal and insulin-stimulated glucose oxidation rate at the whole body level and PFK activity in muscle are normal. In parallel, no changes have been found in skeletal muscle levels of PFK mRNA and immunoreactive protein in NIDDM or IDDM patients. In endurance trained subjects insulin-stimulated whole body glucose oxidation rate is often increased. However, depending on the intensity and frequency, physical exercise may induce an increased, a decreased or an unaltered level of muscle PFK activity. In athletes the muscle PFK mRNA is similar to what is found in sedentary subjects whereas the immunoreactive PFK protein concentration is decreased.     

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.     

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.     

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.     

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.     

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.     

The effects of testosterone on insulin sensitivity in male rats.

In order to examine the effects of testosterone (T) on insulin sensitivity, male rats were castrated or sham-operated, and exposed to low or high doses of T to substitute normal or to produce high serum T concentrations. Insulin sensitivity was followed by euglycaemic, hyperinsulinaemic glucose clamp measurements. An index of insulin-stimulated glucose transport was obtained in the white gastrocnemius (WG), extensor digitorum longus (EDL), red gastrocnemius (RG) and soleus (SOL) muscles after a bolus dose of [2-3H]deoxyglucose (2-DOG) when steady state was obtained in the clamp measurements. Glycogen synthesis was followed similarly with [U-14C]glucose as a labelled precursor after isolation of glycogen in the muscles mentioned, and in the liver. Castration and high T were followed by a marked insulin resistance in the clamp measurements. This was paralleled by a diminished insulin stimulation of glucose incorporation into glycogen down to about 50% of control values, apparently equally pronounced in all muscles but not found in liver glycogen synthesis. 2-DOG uptake was diminished by castration in the WG and RG muscles but was unaffected by high doses of T. Substitution of castrated rats with a low dose of T, restoring their serum T concentrations to the normal range, completely abolished these perturbations of insulin sensitivity. It is concluded that T is an important regulator of muscular insulin sensitivity, which seems to be highest in a 'window' of normal serum T concentrations.     

Sustained phosphorylation and activation of protein kinase B correlates with brain-derived neurotrophic factor and insulin stimulated survival of cerebellar granule cells.

Brain-derived neurotrophic factor (BDNF) and insulin promote the survival of 6-7 day old post-natal rat cerebellar granule cells. Previous studies using the PI3 kinase inhibitor, wortmannin and the over-expression of protein kinase B (PKB) have indicated that both PI3 kinase and PKB activation are central for insulin-stimulated survival of these neurones. Here we report that BDNF, insulin and epidermal growth factor (EGF) all cause the phosphorylation and stimulation of endogenous PKB activity, though with differing profiles. The addition of BDNF, or insulin resulted in a rapid and sustained phosphorylation and stimulation of PKB activity, whilst EGF stimulation, which does not promote survival, caused a more transient phosphorylation and stimulation of PKB activity. We also investigated the involvement of the PKB substrate, glycogen synthase kinase 3 (GSK 3). All three growth factors caused the inactivation of GSK-3beta, suggesting that the inactivation of GSK-3beta does not correlate with survival.     

The effects of testosterone on insulin sensitivity in male rats

In order to examine the effects of testosterone (T) on insulin sensitivity, male rats were castrated or sham-operated, and exposed to low or high doses of T to substitute normal or to produce high serum T concentrations. Insulin sensitivity was followed by euglycaemic, hyperinsulinaemic glucose clamp measurements. An index of insulin-stimulated glucose transport was obtained in the white gastrocnemius (WG), extensor digitorum longus (EDL), red gastrocnemius (RG) and soleus (SOL) muscles after a bolus dose of [2-³H]deoxyglucose (2-DOG) when steady state was obtained in the clamp measurements. Glycogen synthesis was followed similarly with [U-14C]glucose as a labelled precursor after isolation of glycogen in the muscles mentioned, and in the liver. Castration and high T were followed by a marked insulin resistance in the clamp measurements. This was paralleled by a diminished insulin stimulation of glucose incorporation into glycogen down to about 50% of control values, apparently equally pronounced in all muscles but not found in liver glycogen synthesis. 2-DOG uptake was diminished by castration in the WG and RG muscles but was unaffected by high doses of T. Substitution of castrated rats with a low dose of T, restoring their serum T concentrations to the normal range, completely abolished these perturbations of insulin sensitivity. It is concluded that T is an important regulator of muscular insulin sensitivity, which seems to be highest in a ‘window’ of normal serum T concentrations.     

Effect of endurance and sprint exercise on the sensitivity of glucose metabolism to insulin in the epitrochlearis muscle of sedentary and trained rats

Summary The effects of two types of acute exercise (1 h treadmill running at 20 m· min^–1, or 6 × 10-s periods at 43 m · min^–1, 0° inclination), as well as two training regimes (endurance and sprint) on the sensitivity of epitrochlearis muscle [fast twitch (FT) fibres] to insulin were measured in vitro in rats. The hormone concentration in the incubation medium producing the half maximal stimulation of lactate (la) production and glycogen synthesis was determined and used as an index of the muscle insulin sensitivity. A single period of moderate endurance as well as the sprint-type exercise increased the sensitivity of la production to insulin although the rate of la production enhanced markedly only after sprint exercise at 10 and 100 U· ml^–1 of insulin. These effects persisted for up to 2 h after the termination of exercise. Both types of exercise significantly decreased the muscle glycogen content, causing a moderate enhancement in the insulin-stimulated rates of glycogen synthesis in vitro for up to 2 h after exercise. However, a significant increase in the sensitivity of this process to insulin was found only in the muscle removed 0.25 h after the sprint effort. Training of the sprint and endurance types increased insulin-stimulated rates of glycolysis 24 h after the last period of exercise. The sensitivity of this process to insulin was also increased at this instant. Both types of training increased the basal and maximal rates of glycogen snythesis, as well as the sensitivity of this process to insulin at the 24^th following the last training session. It was concluded that in the epitrochlearis muscle, containing mainly FT fibres, both moderate and intensive exercise (acute and repeated) were effective in increasing sensitivity of glucose utilization to insulin. Thus, the response in this muscle type to increased physical activity differs from that reported previously in the soleus muscle, representing the slow-twitch, oxidative fibres in which sprint exercise did not produce any changes in the muscle insulin sensitivity.     

Effect of hyperglycaemia on muscle glycogen mobilization during muscle contractions in the rat

Summary In the rat, muscle glycogen is mobilized during the first stage of exercise, despite normoglycaemia. The aim of the present study was to examine if this process could be prevented or reduced by hyperglycaemia. Three experiments were carried out: in the first, rats were forced to run on a treadmill; in the second the gastrocnemius muscle group was made to contract by stimulation of the sciatic nerve and in the third adrenaline was administered subcutaneously. Each group was divided into two subgroups: control and enriched with glucose (hyperglycaemic). It was shown that hyperglycaemia has no effect on running-induced glycogen mobilization in hind-limb muscles of different fibre composition but prevented it totally in diaphragm muscle. Hyperglycaemia also did not affect the glycogen mobilization induced by stimulation of the sciatic nerve. However, it delayed and reduced markedly the glycogenolytic effect of adrenaline. It is concluded that increased glycogenolysis in muscles at the beginning of exercise may be a consequence of a delay in the activation of glucose transporting mechanisms in muscle cells.