Whey Protein Hydrolysate Enhances HSP90 but Does Not Alter HSP60 and HSP25 in Skeletal Muscle of Rats

Whey protein hydrolysate (WPH) intake has shown to increase HSP70 expression. The aim of the present study was to investigate whether WPH intake would also influences HSP90, HSP60 and HSP25 expression, as well as associated parameters. Forty-eight male Wistar rats were divided into sedentary (unstressed) and exercised (stressed) groups, and were fed with three different sources of protein: whey protein (WP), whey protein hydrolysate (WPH) and casein (CAS) as a control, based on the AIN93G diet for 3 weeks. WPH intake increased HSP90 expression in both sedentary and exercised animals compared to WP or CAS, however no alteration was found from exercise or diet to HSP60 or HSP25. Co-chaperone Aha1 and p-HSF1 were also increased in the exercised animals fed with WPH in comparison with WP or CAS, consistent with enhanced HSP90 expression. VEGF and p-AKT were increased in the WPH exercised group. No alteration was found in BCKDH, PI3-Kinase (p85), GFAT, OGT or PGC for diet or exercise. The antioxidant system GPx, catalase and SOD showed different responses to diet and exercise. The data indicate that WPH intake enhanced factors related to cell survival, such as HSP90 and VEGF, but does not alter HSP60 or HSP25 in rat skeletal muscle.     

Moderate intake of BCAA-rich protein improves glucose homeostasis in high-fat-fed mice

Notwithstanding the fact that dietary branched-chain amino acids (BCAAs) have been considered to be a cause of insulin resistance (IR), evidence indicates that BCAA-rich whey proteins (WPs) do not lead to IR in animals consuming high-fat (HF) diets and may instead improve glucose homeostasis. To address the role of BCAA-rich WP as dietary protein in IR and inflammatory response, we fed C57BL/6J mice either high-fat (HF) or low-fat (LF) diets formulated with moderate protein levels (13% w/w) of either WP or hydrolyzed WP (WPH) and compared them with casein (CAS) as a reference. The muscle and plasma free amino acid profiles, inflammatory parameters and glycemic homeostasis were examined. While the LF/CAS diet promoted the rise in triglycerides and inflammatory parameters, the HF/CAS induced typical IR responses and impaired biochemical parameters. No differences in plasma BCAAs were detected, but the HF/WPH diet led to a twofold increase in gastrocnemius muscle free amino acids, including BCAAs. In general, ingestion of WPH was effective at averting or attenuating the damage caused by both the LF and HF diets. No high concentrations of BCAAs in the plasma or signs of IR were found in those mice fed an HF diet along with the hydrolyzed whey proteins. It is concluded that consumption of BCAA-rich whey proteins, especially WPH, does not result in the development of IR.     

Effects of AICAR and exercise on insulin-stimulated glucose uptake, signaling, and GLUT-4 content in rat muscles.

Physical activity is known to increase insulin action in skeletal muscle, and data have indicated that 5'-AMP-activated protein kinase (AMPK) is involved in the molecular mechanisms behind this beneficial effect. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) can be used as a pharmacological tool to repetitively activate AMPK, and the objective of this study was to explore whether the increase in insulin-stimulated glucose uptake after either long-term exercise or chronic AICAR administration was followed by fiber-type-specific changes in insulin signaling and/or changes in GLUT-4 expression. Wistar rats were allocated into three groups: an exercise group trained on treadmill for 5 days, an AICAR group exposed to daily subcutaneous injections of AICAR, and a sedentary control group. AMPK activity, insulin-stimulated glucose transport, insulin signaling, and GLUT-4 expression were determined in muscles characterized by different fiber type compositions. Both exercised and AICAR-injected animals displayed a fiber-type-specific increase in glucose transport with the most marked increase in muscles with a high content of type IIb fibers. This increase was accompanied by a concomitant increase in GLUT-4 expression. Insulin signaling as assessed by phosphatidylinositol 3-kinase and PKB/Akt activity was enhanced only after AICAR administration and in a non-fiber-type-specific manner. In conclusion, chronic AICAR administration and long-term exercise both improve insulin-stimulated glucose transport in skeletal muscle in a fiber-type-specific way, and this is associated with an increase in GLUT-4 content.     

Effects of continuous low-carbohydrate diet after long-term exercise on GLUT-4 protein content in rat skeletal muscle.

Stimulation of AMPK and decreased glycogen levels in skeletal muscle have a deep involvement in enhanced insulin action and GLUT-4 protein content after exercise training. The present study examined the chronic effects of a continuous low-carbohydrate diet after long-term exercise on GLUT-4 protein content, glycogen content, AMPK, and insulin signaling in skeletal muscle. Rats were divided randomly into four groups: normal chow diet sedentary (N-Sed), low carbohydrate diet sedentary (L-Sed), normal chow diet exercise (N-Ex), and low carbohydrate diet exercise (L-Ex) groups. Rats in the exercise groups (N-Ex and L-Ex) were exercised by swimming for 6 hours/day in two 3-hour bouts separated by 45 minutes of rest. The 10-day exercise training resulted in a significant increase in the GLUT-4 protein content (p<0.01). Additionally, the GLUT-4 protein content in L-Ex rats was increased by 29% above that in N-Ex rats (p<0.01). Finally, the glycogen content in skeletal muscle of L-Ex rats was decreased compared with that of N-Ex rats. Taken together, we suggest that the maintenance of glycogen depletion after exercise by continuous low carbohydrate diet results in the increment of the GLUT-4 protein content in skeletal muscle.     

Role of insulin on exercise-induced GLUT-4 protein expression and glycogen supercompensation in rat skeletal muscle.

The purpose of this study was to investigate the role of insulin on skeletal muscle GLUT-4 protein expression and glycogen storage after postexercise carbohydrate supplementation. Male Sprague-Dawley rats were randomly assigned to one of six treatment groups: sedentary control (Con), Con with streptozocin (Stz/C), immediately postexercise (Ex0), Ex0 with Stz (Stz/Ex0), 5-h postexercise (Ex5), and Ex5 with Stz (Stz/Ex5). Rats were exercised by swimming (2 bouts of 3 h) and carbohydrate supplemented immediately after each exercise session by glucose intubation (1 ml of a 50% wt/vol). Stz was administered 72-h before exercise, which resulted in hyperglycemia and elimination of the insulin response to the carbohydrate supplement. GLUT-4 protein of Ex0 rats was 30% above Con in fast-twitch (FT) red and 21% above Con in FT white muscle. In Ex5, GLUT-4 protein was 52% above Con in FT red and 47% above Con in FT white muscle. Muscle glycogen in FT red and white muscle was also increased above Con in Ex5 rats. Neither GLUT-4 protein nor muscle glycogen was increased above Con in Stz/Ex0 or Stz/Ex5 rats. GLUT-4 mRNA in FT red muscle of Ex0 rats was 61% above Con but only 33% above Con in Ex5 rats. GLUT-4 mRNA in FT red muscle of Stz/C and Stz/Ex0 rats was similar but significantly elevated in Ex5/Stz rats. These results suggest that insulin is essential for the increase in GLUT-4 protein expression following postexercise carbohydrate supplementation.     

Post-exercise carbohydrate plus whey protein hydrolysates supplementation increases skeletal muscle glycogen level in rats

Recent studies showed that a combination of carbohydrate and protein was more effective than carbohydrate alone for replenishing muscle glycogen after exercise. However, it remains to be unclear whether the source or degree of hydrolysis of dietary protein influences post-exercise glycogen accumulation. The aim of this study was to compare the effect of dietary protein type on glycogen levels in the post-exercise phase, and to investigate the effects of post-exercise carbohydrate and protein supplementation on phosphorylated enzymes of Akt/PKB and atypical PKCs. Male Sprague-Dawley rats, trained for 3 days, swam with a 2% load of body weight for 4 h to deplete skeletal muscle glycogen. Immediately after the glycogen-depleting exercise, one group was killed, whereas the other groups were given either glucose or glucose plus protein (whey protein, whey protein hydrolysates (WPH), casein hydrolysates or branched-chain amino acid (BCAA) solutions. After 2 h, the rats were killed, and the triceps muscles quickly excised. WPH caused significant increases in skeletal muscle glycogen level (5.01 ± 0.24 mg/g), compared with whey protein (4.23 ± 0.24 mg/g), BCAA (3.92 ± 0.18 mg/g) or casein hydrolysates (2.73 ± 0.22 mg/g). Post-exercise ingestion of glucose plus WPH significantly increased both phosphorylated Akt/PKB (131%) and phosphorylated PKCζ (154%) levels compared with glucose only. There was a significant positive correlation between skeletal muscle glycogen content and phosphorylated Akt/PKB (r = 0.674, P < 0.001) and PKCζ (r = 0.481, P = 0.017). Post-exercise supplementation with carbohydrate and WPH increases skeletal muscle glycogen recovery by activating key enzymes such as Akt/PKB and atypical PKCs.     

Resistance training enhances components of the insulin signaling cascade in normal and high-fat-fed rodent skeletal muscle.

Our laboratory recently reported that chronic resistance training (RT) improved insulin-stimulated glucose transport in normal rodent skeletal muscle, owing, in part, to increased GLUT-4 protein concentration (Yaspelkis BB III, Singh MK, Trevino B, Krisan AD, and Collins DE. Acta Physiol Scand 175: 315-323, 2002). However, it remained to be determined whether these improvements resulted from alterations in the insulin signaling cascade as well. In addition, the possibility existed that RT might improve skeletal muscle insulin resistance. Thirty-two male Sprague-Dawley rats were assigned to four groups: control diet (Con)-sedentary (Sed); Con-RT; high-fat diet (HF)-Sed; and HF-RT. Animals consumed their respective diets for 9 wk; then RT animals performed 12 wk of training (3 sets, 10 repetitions at 75% one-repetition maximum, 3x/wk). Animals remained on their dietary treatments over the 12-wk period. After the training period, animals were subjected to hindlimb perfusions. Insulin-stimulated insulin receptor substrate-1-associated phosphatidylinositol-3 kinase activity was enhanced in the red gastrocnemius and quadriceps of Con-RT and HF-RT animals. Atypical PKC-zeta/lambda and Akt activities were reduced in HF-Sed and normalized in HF-RT animals. Resistance training increased GLUT-4 protein concentration in red gastrocnemius and quadriceps of Con-RT and HF-RT animals. No differences were observed in total protein concentrations of insulin receptor substrate-1, Akt, atypical PKC-zeta/lambda, or phosphorylation of Akt. Collectively, these findings suggest that resistance training increases insulin-stimulated carbohydrate metabolism in normal skeletal muscle and reverses high-fat diet-induced skeletal muscle insulin resistance by altering components of both the insulin signaling cascade and glucose transporter effector system.     

Muscle glycogen content affects insulin-stimulated glucose transport and protein kinase B activity

We investigated the possible regulatory role of glycogen in insulin-stimulated glucose transport and insulin signaling in skeletal muscle. Rats were preconditioned to obtain low (LG), normal, or high (HG) muscle glycogen content, and perfused isolated hindlimbs were exposed to 0, 100, or 10,000 microU/ml insulin. In the fast-twitch white gastrocnemius, insulin-stimulated glucose transport was significantly higher in LG compared with HG. This difference was less pronounced in the mixed-fiber red gastrocnemius and was absent in the slow-twitch soleus. In the white gastrocnemius, insulin activation of insulin receptor tyrosine kinase and phosphoinositide 3-kinase was unaffected by glycogen levels, whereas protein kinase B activity was significantly higher in LG compared with HG. In additional incubation experiments on fast-twitch epitrochlearis muscles, insulin-stimulated cell surface GLUT-4 content was significantly higher in LG compared with HG. The data indicate that, in fast-twitch muscle, the effect of insulin on glucose transport and cell surface GLUT-4 content is modulated by glycogen content, which does not involve initial but possibly more downstream signaling events.     

Amylin effect in extrapancreatic tissues participating in glucose homeostasis, in normal, insulin-resistant and type 2 diabetic state

Amylin is co-secreted with insulin, responds to the same stimuli, is anorectic, lowers body weight by reducing fat mass, and is proposed for diabetes treatment. We examined the effect of a 3-day constant infusion of close to physiological doses of amylin in Wistar rats, on glucotransporter expression, glycogen content (G), glycogen synthase a activity (GSa) and glucose transport (GT), in liver, muscle and fat from insulin resistant (IR) and type 2 diabetic (T2D) models, compared to normal (N) animals; plasma glucose and insulin were measured. Plasma insulin in IR was higher than in N or T2D, and amylin normalized the value. In both, IR and T2D, liver G was lower than normal, accompanied by GLUT-2, mRNA and protein, higher and lower, respectively, than in N; amylin normalized G in both groups, without changes in GLUT-2, except for an mRNA increase in T2D. In IR and T2D, muscle GSa was reduced, together with respective over- and under-GLUT-4 expression; amylin induced only a trend toward GSa normalization in both groups. In isolated adipocytes, GT and GLUT-4 in IR and T2D were lower and higher, respectively, than in N; after amylin, not only GT was normalized in both groups but also the response to insulin was much more pronounced, including that in N, without major changes in GLUT-4. This suggests that the beneficial effect of amylin in states running with altered glucose homeostasis could occur by partially acting on the hexose metabolism of the liver and mainly on that of the adipose tissue.     

Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats

We examined the effects of high-fat diet (HFD) and exercise training on insulin-stimulated whole body glucose fluxes and several key steps of glucose metabolism in skeletal muscle. Rats were maintained for 3 wk on either low-fat (LFD) or high-fat diet with or without exercise training (swimming for 3 h per day). After the 3-wk diet/exercise treatments, animals underwent hyperinsulinemic euglycemic clamp experiments for measurements of insulin-stimulated whole body glucose fluxes. In addition, muscle samples were taken at the end of the clamps for measurements of glucose 6-phosphate (G-6-P) and GLUT-4 protein contents, hexokinase, and glycogen synthase (GS) activities. Insulin-stimulated glucose uptake was decreased by HFD and increased by exercise training (P < 0.01 for both). The opposite effects of HFD and exercise training on insulin-stimulated glucose uptake were associated with similar increases in muscle G-6-P levels (P < 0.05 for both). However, the increase in G-6-P level was accompanied by decreased GS activity without changes in GLUT-4 protein content and hexokinase activities in the HFD group. In contrast, the increase in G-6-P level in the exercise-trained group was accompanied by increased GLUT-4 protein content and hexokinase II (cytosolic) and GS activities. These results suggest that HFD and exercise training affect insulin sensitivity by acting predominantly on different steps of intracellular glucose metabolism. High-fat feeding appears to induce insulin resistance by affecting predominantly steps distal to G-6-P (e.g., glycolysis and glycogen synthesis). Exercise training affected multiple steps of glucose metabolism both proximal and distal to G-6-P. However, increased muscle G-6-P levels in the face of increased glucose metabolic fluxes suggest that the effect of exercise training is quantitatively more prominent on the steps proximal to G-6-P (i.e., glucose transport and phosphorylation).     

Cardiac glucose uptake and suppressed expression/translocation of myocardium glucose transport-4 in dogs undergoing ischemia-reperfusion

Impaired glucose metabolism is implicated in cardiac failure during ischemia-reperfusion. This study examined cardiac glucose uptake and expression of glucose transport-4 (GLUT-4) in dogs undergoing ischemia-reperfusion. Cardiac ischemia was induced by cardiopulmonary bypass for 30 min or 120 min in dogs. Plasma insulin and glucose concentrations were measured at pre-bypass (control), and aortic cross-clamp off (ischemia-reperfusion) at 15, 45, and 75 min. At the same time, the left ventricle biopsies were taken for GLUT-4 immunohistochemistry and glycogen content analysis. In dogs receiving 120-min ischemia, coronary arterial and venous glucose concentrations were increased, but the net glucose uptake in ischemia-reperfusion heart were significantly decreased from 25% (control) to zero at 15 and 45 min of reperfusion, and recovered to only 7% after 75 min reperfusion. Myocardium glycogen contents were decreased by 65%. Plasma insulin levels and Insulin Resistant Index were markedly increased in dogs undergoing 120-min ischemia and reperfusion. These changes were relatively mild and reversible in dogs receiving only 30-min ischemia followed by reperfusion. Expression of total GLUT-4 in myocardium was decreased 40% and translocation of GLUT-4 from cytoplasm to surface membrane was decreased 90% in dogs receiving 120-min ischemia followed by 15-min reperfusion. Suppressed translocation of GLUT-4 was also evident in dogs receiving 30-min ischemia, but to a lesser extent. Reduced myocardium glucose uptake, utilization, and glycogen content are clearly associated with ischemia-reperfusion heart injury. This appears to be due, at least in part, to suppressed expression and translocation of myocardium GLUT-4.     

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Exercise modulates the insulin-induced translocation of glucose transporters in rat skeletal muscle

Insulin and acute exercise (45 min of treadmill run) increased glucose uptake into perfused rat hindlimbs 5-fold and 3.2-fold, respectively. Following exercise, insulin treatment resulted in a further increase in glucose uptake. The subcellular distribution of the muscle glucose transporters GLUT-1 and GLUT-4 was determined in plasma membranes and intracellular membranes. Neither exercise nor exercise----insulin treatment altered the distribution of GLUT-1 transporters in these membrane fractions. In contrast, exercise, insulin and exercise----insulin treatment caused comparable increases in GLUT-4 transporters in the plasma membrane. The results suggest that exercise might limit insulin-induced GLUT-4 recruitment and that following exercise, insulin may alter the intrinsic activity of plasma membrane glucose transporters.     

Effect of carbohydrate supplementation on postexercise GLUT-4 protein expression in skeletal muscle.

The effect of carbohydrate supplementation on skeletal muscle glucose transporter GLUT-4 protein expression was studied in fast-twitch red and white gastrocnemius muscle of Sprague-Dawley rats before and after glycogen depletion by swimming. Exercise significantly reduced fast-twitch red muscle glycogen by 50%. During a 16-h exercise recovery period, muscle glycogen returned to control levels (25.0 +/- 1.4 micromol/g) in exercise-fasted rats (24.2 +/- 0. 3 micro). However, when carbohydrate supplementation was provided during and immediately postexercise by intubation, muscle glycogen increased 77% above control (44.4 +/- 2.1 micromol/g). Exercise-fasting resulted in an 80% increase in fast-twitch red muscle GLUT-4 mRNA but only a 43% increase in GLUT-4 protein concentration. Conversely, exercise plus carbohydrate supplementation elevated fast-twitch red muscle GLUT-4 protein concentration by 88% above control, whereas GLUT-4 mRNA was increased by only 40%. Neither a 16-h fast nor carbohydrate supplementation had an effect on fast-twitch red muscle GLUT-4 protein concentration or on GLUT-4 mRNA in sedentary rats, although carbohydrate supplementation increased muscle glycogen concentration by 40% (35.0 +/- 0.9 micromol/g). GLUT-4 protein in fast-twitch white muscle followed a pattern similar to fast-twitch red muscle. These results indicate that carbohydrate supplementation, provided with exercise, will enhance GLUT-4 protein expression by increasing translational efficiency. Conversely, postexercise fasting appears to upregulate GLUT-4 mRNA, possibly to amplify GLUT-4 protein expression on an increase in glucose availability. These regulatory mechanisms may help control muscle glucose uptake in accordance with glucose availability and protect against postexercise hypoglycemia.     

Glycogen supercompensation masks the effect of a traininginduced increase in GLUT-4 on muscle glucose transport

Endurance exercise training induces a rapidincrease in the GLUT-4 isoform of the glucose transporter in muscle. Infasted rats, insulin-stimulated muscle glucose transport is increased in proportion to the increase in GLUT-4. There is evidence that highmuscle glycogen may decrease insulin-stimulated glucose transport. Thisstudy was undertaken to determine whether glycogen supercompensation interferes with the increase in glucose transport associated with anexercise-induced increase in GLUT-4. Rats were trained by means ofswimming for 6 h/day for 2 days. Rats fasted overnight after the lastexercise bout had an approximately twofold increase in epitrochlearismuscle GLUT-4 and an associated approximately twofold increase inmaximally insulin-stimulated glucose transport activity. Epitrochlearismuscles of rats fed rodent chow after exercise were glycogensupercompensated (86.4 ± 4.8 µmol/g wet wt) and showed nosignificant increase in maximally insulin-stimulated glucose transportabove the sedentary control value despite an approximately twofoldincrease in GLUT-4. Fasting resulted in higher basal muscle glucosetransport rates in both sedentary and trained rats but did notsignificantly increase maximally insulin-stimulated transport in thesedentary group. We conclude that carbohydrate feeding that results inmuscle glycogen supercompensation prevents the increase in maximallyinsulin-stimulated glucose transport associated with an exercisetraining-induced increase in muscle GLUT-4.

     

Differential regulation of the GLUT-1 and GLUT-4 glucose transport systems by glucose and insulin in L6 muscle cells in culture

The regulation by glucose and insulin of the muscle-specific facilitative glucose transport system GLUT-4 was investigated in L6 muscle cells in culture. Hexose transport activity, mRNA expression, and the subcellular localization of the GLUT-4 protein were analyzed. As observed previously (Walker, P. S., Ramlal, T., Sarabia, V., Koivisto, U.-M., Bilan, P. J., Pessin, J. E., and Klip, A. (1990) J. Biol. Chem. 265, 1516-1523), 24 h of glucose starvation and 24 h of insulin treatment each increase glucose transport activity severalfold. Here we report a differential regulation of the GLUT-4 and GLUT-1 transport systems under these conditions. (a) The level of GLUT-4 mRNA was not affected by glucose starvation and was diminished by prolonged (24 h) administration of insulin; in contrast, the level of GLUT-1 mRNA was elevated under both conditions. (b) Glucose starvation and prolonged insulin administration increased the amount of both GLUT-4 and GLUT-1 proteins in the plasma membrane. (c) In intracellular membranes, glucose starvation elevated, and prolonged insulin administration reduced, the GLUT-4 protein content. In contrast, the GLUT-1 protein content in these membranes decreased with glucose starvation and increased with insulin treatment. Glucose transport was rapidly curbed upon refeeding glucose to glucose-starved cells, with half-maximal reversal after 30 min and maximal reversal after 4 h. This was followed by a marked decrease in the levels of GLUT-1 mRNA without major changes in GLUT-4 mRNA. Neither 2-deoxy-D-glucose nor 3-O-methyl-D-glucose could substitute for D-glucose in these effects. It is proposed that glucose and insulin differentially regulate the two glucose transport systems in L6 muscle cells and that the rapid down-regulation of hexose transport activity by glucose is regulated by post-translational mechanisms.     

Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle

The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr(308) or basal and contraction-stimulated glycogen synthase kinase-3beta (GSK-3beta) Ser(9) phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr(308) phosphorylation and GSK-3alpha Ser(21) phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3alpha Ser(21) phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.     

Mechanisms underlying impaired GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats

Exercise training induces an increase in GLUT-4 in muscle. We previously found that feeding rats a high-carbohydrate diet after exercise, with muscle glycogen supercompensation, results in a decrease in insulin responsiveness so severe that it masks the effect of a training-induced twofold increase in GLUT-4 on insulin-stimulated muscle glucose transport. One purpose of this study was to determine whether insulin signaling is impaired. Maximally insulin-stimulated phosphatidylinositol (PI) 3-kinase activity was not significantly reduced, whereas protein kinase B (PKB) phosphorylation was approximately 50% lower (P < 0.01) in muscles of chow-fed, than in those of fasted, exercise-trained rats. Our second purpose was to determine whether contraction-stimulated glucose transport is also impaired. The stimulation of glucose transport and the increase in cell surface GLUT-4 induced by contractions were both decreased by approximately 65% in glycogen-supercompensated muscles of trained rats. The contraction-stimulated increase in AMP kinase activity, which has been implicated in the activation of glucose transport by contractions, was approximately 80% lower in the muscles of the fed compared with the fasted rats 18 h after exercise. These results show that both the insulin- and contraction-stimulated pathways for muscle glucose transport activation are impaired in glycogen-supercompensated muscles and provide insight regarding possible mechanisms.     

Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter

Glucose and fatty acid metabolism was assessed in isolated working hearts from control C57BL/KsJ-m+/+db mice and transgenic mice overexpressing the human GLUT-4 glucose transporter (db/+-hGLUT-4). Heart rate, coronary flow, cardiac output, and cardiac power did not differ between control hearts and hearts overexpressing GLUT-4. Hearts overexpressing GLUT-4 had significantly higher rates of glucose uptake and glycolysis and higher levels of glycogen after perfusion than control hearts, but rates of glucose and palmitate oxidation were not different. Insulin (1 mU/ml) significantly increased glycogen levels in both groups. Insulin increased glycolysis in control hearts but not in GLUT-4 hearts, whereas glucose oxidation was increased by insulin in both groups. Therefore, GLUT-4 overexpression increases glycolysis, but not glucose oxidation, in the heart. Although control hearts responded to insulin with increased rates of glycolysis, the enhanced entry of glucose in the GLUT-4 hearts was already sufficient to maximally activate glycolysis under basal conditions such that insulin could not further stimulate the glycolytic rate.     

Effects of chronic Akt activation on glucose uptake in the heart

Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake (P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo (P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin.