Isolated Epitrochlearis Muscles Research Articles

Akt substrate of 160kDa is essential for the calorie restriction-induced increase in insulin-stimulated glucose uptake by skeletal muscle of female rats.

We evaluated effects of calorie restriction (CR; consuming 65% of ad libitum (AL) intake) for 8 weeks on female wildtype (WT) and Akt substrate of 160kDa knockout (AS160-KO) rats. Insulin-stimulated glucose uptake (ISGU) was determined in isolated epitrochlearis muscles incubated with 0, 50, 100, or 500 µU/mL insulin. Phosphorylation of key insulin signaling proteins that control ISGU (Akt and AS160) was assessed by immunoblotting (Akt phosphorylation on Threonine-308, pAktThr308 and Serine-473, pAktSer473; AS160 phosphorylation on Serine-588, pAS160Ser588, and Threonine-642, pAS160Thr642). Abundance of proteins that regulate ISGU (GLUT4 glucose transporter protein and hexokinase II) was also determined by immunoblotting. The major results were as follows: (i) WT-CR versus WT-AL rats had greater ISGU with 100 and 500 µU/mL insulin; (ii) CR versus WT-AL rats had greater GLUT4 protein abundance; (iii) WT-CR versus WT-AL rats had greater pAktThr308 with 500 µU/mL insulin; (iv) WT-CR versus WT-AL rats did not differ for pAktSer473, pAS160Ser588, or pAS160Thr642 at any insulin concentration; (v) AS160-KO versus WT rats with each diet had lower ISGU at each insulin concentration, but not lower pAkt on either phosphosite; (vi) AS160-KO versus WT rats had lower muscle GLUT4 abundance regardless of diet; and (vii) AS160-KO-CR versus AS160-KO-AL rats did not differ for ISGU, GLUT4 abundance, pAkt on either phosphosite, or pAS160 on either phosphosite. These novel results demonstrated that AS160 expression, but not greater pAS160 on key phosphosites, was essential for the CR-induced increases in muscle ISGU and GLUT4 abundance of female rats.

Exercise effects on γ3-AMPK activity, phosphorylation of Akt2 and AS160, and insulin-stimulated glucose uptake in insulin-resistant rat skeletal muscle

One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle. Prior research on healthy muscle suggests that enhanced postexercise ISGU depends on elevated γ3-AMPK activity leading to greater phosphorylation of Akt substrate of 160 kDa (pAS160) on an AMPK-phosphomotif (Ser704). Phosphorylation of AS160Ser704, in turn, may favor greater insulin-stimulated pAS160 on an Akt-phosphomotif (Thr642) that regulates ISGU. Accordingly, we tested if exercise-induced increases in γ3-AMPK activity and pAS160 on key regulatory sites accompany improved ISGU at 3 h postexercise (3hPEX) in insulin-resistant muscle. Rats fed a high-fat diet (HFD; 2-wk) that induces insulin resistance either performed acute swim-exercise (2 h) or were sedentary (SED). SED rats fed a low-fat diet (LFD; 2 wk) served as healthy controls. Isolated epitrochlearis muscles from 3hPEX and SED rats were analyzed for ISGU, pAS160, pAkt2 (Akt-isoform that phosphorylates pAS160Thr642), and γ1-AMPK and γ3-AMPK activity. ISGU was lower in HFD-SED muscles versus LFD-SED, but this decrement was eliminated in the HFD-3hPEX group. γ3-AMPK activity, but not γ1-AMPK activity, was elevated in HFD-3hPEX muscles versus both SED controls. Furthermore, insulin-stimulated pAS160Thr642, pAS160Ser704, and pAkt2Ser474 in HFD-3hPEX muscles were elevated above HFD-SED and equal to values in LFD-SED muscles, but insulin-independent pAS160Ser704 was unaltered at 3hPEX. These results demonstrated, for the first time in an insulin-resistant model, that the postexercise increase in ISGU was accompanied by sustained enhancement of γ3-AMPK activation and greater pAkt2Ser474. Our working hypothesis is that these changes along with enhanced insulin-stimulated pAS160 increase ISGU of insulin-resistant muscles to values equaling insulin-sensitive sedentary controls.NEW & NOTEWORTHY Earlier research focusing on signaling events linked to increased insulin sensitivity in muscle has rarely evaluated insulin resistant muscle after exercise. We assessed insulin resistant muscle after an exercise protocol that improved insulin-stimulated glucose uptake. Prior exercise also amplified several signaling steps expected to favor enhanced insulin-stimulated glucose uptake: increased γ3-AMP-activated protein kinase activity, greater insulin-stimulated Akt2 phosphorylation on Ser474, and elevated insulin-stimulated Akt substrate of 160 kDa phosphorylation on Ser588, Thr642, and Ser704.

718-P: Post-exercise Enhancement of Insulin-Stimulated Glucose Uptake in Insulin-Resistant Rat Skeletal Muscle Concomitant with Greater γ3-AMPK Activity and Insulin-Stimulated AS160 Phosphorylation on Key Regulatory Phosphosites

One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by healthy skeletal muscle, and recently published research linked this outcome to exercise-induced elevation in γ3-AMPK activity which leads to greater phosphorylation of Akt substrate of 160 kDa (pAS160) on an AMPK-phosphomotif (Ser704), which in turn favors greater insulin-stimulated pAS160 on a key Akt-phosphomotif (Thr642) that regulates ISGU. Studying healthy muscle is important, but a more urgent need is to understand the mechanisms for enhanced ISGU in insulin resistant muscle. Our primary goal was to determine if exercise, which increases ISGU, also increases γ3-AMPK activity and pAS160 on key regulatory sites at 3-hours post-exercise (3hPEX) in insulin resistant muscle. Rats fed a high fat diet (HFD; 2 week) known to cause muscle insulin resistance performed acute swim exercise (2 h) or remained sedentary (SED). Other chow-fed (low fat diet, LFD; 2 week) SED rats served as healthy controls. Isolated epitrochlearis muscles from 3hPEX rats (and SED controls) were used for ISGU, pAS160 and γ1- and γ3-AMPK isoform-specific activity. ISGU was lower in HFD SED muscles compared to LFD SED, but this decrement was eliminated in the HFD 3hPEX group. The γ3-AMPK activity, but not γ1-AMPK activity, was elevated in HFD 3hPEX muscles versus both SED controls. In addition, insulin-stimulated pAS160 on Thr642 and Ser704 in HFD 3hPEX muscles was elevated above HFD SED controls and equal to values in LFD SED muscles. These novel results demonstrated, for the first time in an insulin-resistant model, that the post-exercise increase in ISGU was accompanied by enhanced pAS160Ser704 and γ3-AMPK activation. Our working hypothesis is that these key signaling events are necessary for acute exercise to restore the ISGU of insulin resistant muscles to values equal to normal LFD SED controls. Disclosure M.W. Pataky: None. E.B. Arias: None. X. Zheng: None. H. Wang: None. G.D. Cartee: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

Effects of Acute Exercise Combined With Calorie Restriction Initiated Late-in-Life on Insulin Signaling, Lipids, and Glucose Uptake in Skeletal Muscle From Old Rats.

We evaluated effects of calorie restriction (CR: consuming 60-65% of ad libitum [AL] intake) initiated late-in-life with or without acute exercise on insulin-stimulated glucose uptake (ISGU) of skeletal muscle by studying four groups of 26-month-old rats: sedentary-AL, sedentary-CR (8-week duration), 3 hours post-exercise (3hPEX)-AL and 3hPEX-CR. ISGU was determined in isolated epitrochlearis muscles incubated ± insulin. Muscles were assessed for signaling proteins (immunoblotting) and lipids (mass spectrometry). ISGU from sedentary-CR and 3hPEX-AL exceeded sedentary-AL; 3hPEX-CR exceeded all other groups. Akt (Ser473, Thr308) and Akt substrate of 160 kDa (AS160; Ser588, Thr642, Ser704) phosphorylation levels tracked with ISGU. Among the 477 lipids detected, 114 were altered by CR (including reductions in 15 of 25 acylcarnitines), and 27 were altered by exercise (including reductions in 18 of 22 lysophosphatidylcholines) with only six lipids overlapping between CR and exercise. ISGU significantly correlated with 23 lipids, including: acylcarnitine 20:1 (r = .683), lysophosphatidylethanolamine19:0 (r = -.662), acylcarnitine 24:0 (r = .611), and plasmenyl-phosphatidylethanolamine 37:5 (r = -.603). Muscle levels of ceramides (a lipid class previously linked to insulin resistance) were not altered by CR and/or exercise nor significantly correlated with ISGU, implicating other mechanisms (which potentially involve other lipids identified in this study) for greater ISGU and Akt and AS160 phosphorylation with these interventions.

Short-term replacement of starch with isomaltulose enhances both insulin-dependent and -independent glucose uptake in rat skeletal muscle.

Dietary intervention for preventing postprandial increases in glucose level by replacing high-glycemic index (GI) carbohydrates with lower-GI carbohydrate has been proposed as a strategy for treating insulin-resistant metabolic disorders such as type II diabetes. In this study, we examined the effect of short-term replacement of starch with a low-GI disaccharide, isomaltulose, on insulin action in skeletal muscle. Male Wistar rats were fed isomaltulose for 12 h during their dark cycle. In isolated epitrochlearis muscle, insulin-induced glucose uptake was greater in tissue from rats treated with isomaltulose than from those treated with starch. This insulin-sensitizing effect occurred independently of changes visceral fat mass. To determine whether this sensitization was specific to insulin stimulation, we also measured glucose uptake in response to exercise. In isolated epitrochlearis muscles from rats that performed swimming exercise, exercise-induced glucose uptake was higher in isomaltulose-treated than starch-treated animals. This amplification was associated with increased phosphorylation of exercise-induced AMP-activated protein kinase. In conclusion, our results demonstrate that short-term replacement of starch with isomaltulose enhances both insulin-dependent and -independent glucose uptake in isolated skeletal muscle. This transient replacement of carbohydrate with isomaltulose, together with exercise, represents a potentially effective approach for the management of insulin resistance.

Effects of Calorie Restriction and Fiber Type on Glucose Uptake and Abundance of Electron Transport Chain and Oxidative Phosphorylation Proteins in Single Fibers from Old Rats.

Calorie restriction (CR; reducing calorie intake by ~40% below ad libitum) can increase glucose uptake by insulin-stimulated muscle. Because skeletal muscle is comprised of multiple, heterogeneous fiber types, our primary aim was to determine the effects of CR (initiated at 14 weeks old) and fiber type on insulin-stimulated glucose uptake by single fibers of diverse fiber types in 23-26-month-old rats. Isolated epitrochlearis muscles from AL and CR rats were incubated with [3H]-2-deoxyglucose ± insulin. Glucose uptake and fiber type were determined for single fibers dissected from the muscles. We also determined CR-effects on abundance of several key metabolic proteins in single fibers. CR resulted in: (a) significantly (p < .05 to .001) greater glucose uptake by insulin-stimulated type I, IIA, IIB, IIBX, and IIX fibers; (b) significantly (p < .05 to .001) reduced abundance of several mitochondrial electron transport chain (ETC) and oxidative phosphorylation (OxPhos) proteins in type I, IIA, and IIBX but not IIB and IIX fibers; and (c) unaltered hexokinase II abundance in each fiber type. These results demonstrate that CR can enhance glucose uptake in each fiber type of rat skeletal muscle in the absence of upregulation of the abundance of hexokinase II or key mitochondrial ETC and OxPhos proteins.

Calorie restriction leads to greater Akt2 activity and glucose uptake by insulin-stimulated skeletal muscle from old rats.

Skeletal muscle insulin resistance is associated with many common age-related diseases, but moderate calorie restriction (CR) can substantially elevate glucose uptake by insulin-stimulated skeletal muscle from both young and old rats. The current study evaluated the isolated epitrochlearis muscle from ∼24.5-mo-old rats that were either fed ad libitum (AL) or subjected to CR (consuming ∼65% of ad libitum, AL, intake beginning at ∼22.5 mo old). Some muscles were also incubated with MK-2206, a potent and selective Akt inhibitor. The most important results were that in isolated muscles, CR vs. AL resulted in 1) greater insulin-stimulated glucose uptake 2) that was accompanied by significantly increased insulin-mediated activation of Akt2, as indicated by greater phosphorylation on both Thr(309) and Ser(474) along with greater Akt2 activity, 3) concomitant with enhanced phosphorylation of several Akt substrates, including an Akt substrate of 160 kDa on Thr(642) and Ser(588), filamin C on Ser(2213) and proline-rich Akt substrate of 40 kDa on Thr(246), but not TBC1D1 on Thr(596); and 4) each of the CR effects was eliminated by MK-2206. These data provide compelling new evidence linking greater Akt2 activation to the CR-induced elevation of insulin-stimulated glucose uptake by muscle from old animals.

Postexercise Improvement in Insulin-Stimulated Glucose Uptake Occurs Concomitant With Greater AS160 Phosphorylation in Muscle From Normal and Insulin-Resistant Rats

Earlier research on rats with normal insulin sensitivity demonstrated that acute exercise increased insulin-stimulated glucose uptake (GU) concomitant with greater phosphorylation of Akt substrate of 160 kDa (pAS160). Because mechanisms for exercise effects on GU in insulin-resistant muscle are unknown, our primary objective was to assess insulin-stimulated GU, proximal insulin signaling (insulin receptor [IR] tyrosine phosphorylation, IR substrate 1–phosphatidylinositol-3-kinase, and Akt phosphorylation and activity), and pAS160 in muscles from acutely exercised (one session) and sedentary rats fed either chow (low-fat diet [LFD]; normal insulin sensitivity) or a high-fat diet (HFD; for 2 weeks, insulin-resistant). At 3 h postexercise (3hPEX), isolated epitrochlearis muscles were used for insulin-stimulated GU and insulin signaling measurements. Although exercise did not enhance proximal signaling in either group, insulin-stimulated GU at 3hPEX exceeded respective sedentary control subjects (Sedentary) in both diet groups. Furthermore, insulin-stimulated GU for LFD-3hPEX was greater than HFD-3hPEX values. For HFD-3hPEX muscles, pAS160 exceeded HFD-Sedentary, but in muscle from LFD-3hPEX rats, pAS160 was greater still than HFD-3hPEX values. These results implicated pAS160 as a potential determinant of the exercise-induced elevation in insulin-stimulated GU for each diet group and also revealed pAS160 as a possible mediator of greater postexercise GU of insulin-stimulated muscles from the insulin-sensitive versus insulin-resistant group.

Sustained postexercise increases in AS160 Thr642and Ser588phosphorylation in skeletal muscle without sustained increases in kinase phosphorylation

Prior exercise by rats can induce a sustained increase in muscle Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) (pAS160(Thr642)). Because phosphorylation of AS160 on both AS160(Thr642) and AS160(Ser588) is important for insulin-stimulated glucose transport (GT), we determined if exercise would also induce a sustained increase in pAS160(Ser588) concomitant with persistently elevated pAS160(Thr642) and GT. Given that the mechanisms for sustained postexercise (PEX) effects on pAS160 were uncertain, we also studied the four kinases known to phosphorylate AS160 (Akt, AMPK, RSK, and SGK1). In addition, because the serine/threonine phosphatase(s) that dephosphorylate muscle AS160 were previously unidentified, we assessed the ability of four serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) to dephosphorylate AS160. We also evaluated exercise effects on posttranslational modifications (Tyr(307) and Leu(309)) that regulate PP2A. In isolated epitrochlearis muscles from rats, GT at 3hPEX with insulin significantly (P < 0.05) exceeded SED controls. Muscles from 0hPEX vs. 0hSED and 3hPEX vs. 3hSED rats had greater pAS160(Thr642) and pAS160(Ser588). AMPK was the only kinase with greater phosphorylation at 0hPEX vs. 0hSED, and none had greater phosphorylation at 3hPEX vs. 3hSED. Each phosphatase was able to dephosphorylate pAS160(Thr642) and pAS160(Ser588) in cell-free assays. Exercise did not alter posttranslational modifications of PP2A. Our results revealed: 1) pAMPK as a potential trigger for increased pAS160(Thr642) and pAS160(Ser588) at 0hPEX; 2) PP1, PP2A, PP2B, and PP2C were each able to dephosphorylate AS160; and 3) sustained PEX-induced elevations of pAS160(Thr642) and pAS160(Ser588) were attributable to mechanisms other than persistent phosphorylation of known AS160 kinases or altered posttranslational modifications of PP2A.

Preventing the calorie restriction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction's effect on glucose uptake in skeletal muscle

Calorie restriction (CR; ~60% of ad libitum, AL, consumption) improves insulin-stimulated glucose uptake in skeletal muscle. The precise cellular mechanism for this healthful outcome is unknown, but it is accompanied by enhanced insulin-stimulated activation of Akt. Previous research using Akt2-null mice demonstrated that Akt2 is essential for the full CR-effect on insulin-stimulated glucose uptake by muscle. However, because Akt2-null mice were completely deficient in Akt2 in every cell throughout life, it would be valuable to assess the efficacy of transient, muscle-specific Akt inhibition for attenuation of CR-effects on glucose uptake. Accordingly, we used a selective Akt inhibitor (MK-2206) to eliminate the CR-induced elevation in insulin-stimulated Akt2 phosphorylation and determined the effects on Akt substrates and glucose uptake. We incubated isolated epitrochlearis muscles from 9-month-old AL and CR (~60–65% of AL intake for 6months) rats with or without MK-2206 and measured insulin-stimulated (1.2nM) glucose uptake and phosphorylation of the insulin receptor (Tyr1162/1163), pan-Akt (Thr308 and Ser473), Akt2 (Thr308 and Ser473), AS160/TBC1D4 (Thr642), and Filamin C (Ser2213). Incubation of isolated skeletal muscles with a dose of a selective Akt inhibitor that eliminated the CR-induced increases in Akt2 phosphorylation prevented CR's effects on insulin-stimulated glucose uptake, pAS160Thr642 and pFilamin CSer2213 without altering pIRTyr1162/1163. These data provide compelling new evidence linking the CR-induced increase in insulin-stimulated Akt2 phosphorylation to CR's effects on insulin-mediated phosphorylation of Akt substrates and glucose uptake in skeletal muscle.

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

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. 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 Thr(172) and acetyl-CoA carboxylase (ACC) Ser(79) , the concentrations of ATP, phosphocreatine (PCr) and glycogen, and 3-O-methyl-d-glucose (3MG) transport activity were estimated. 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. 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.

Leucine supplementation in rats induced a delay in muscle IR/PI3K signaling pathway associated with overall impaired glucose tolerance

Although activation of the mammalian target of rapamycin complex/p70 S6 kinase (S6K1) pathway by leucine is efficient to stimulate muscle protein synthesis, it can also exert inhibition on the early steps of insulin signaling leading to insulin resistance. We investigated the impact of 5-week leucine supplementation on insulin signaling and sensitivity in 4-month old rats fed a 15% protein diet supplemented (LEU) or not (C) with 4.5% leucine. An oral glucose tolerance test was performed in each rat at the end of the supplementation and glucose transport was measured in vitro using isolated epitrochlearis muscles incubated with 2-deoxy- d-[ 3H]-glucose under increasing insulin concentrations. Insulin signaling was assessed on gastrocnemius at the postabsorptive state or 30 and 60 min after gavage with a nutrient bolus. Tyrosine phosphorylation of IRβ, IRS1 and PI3 kinase activity were reduced in LEU group 30 min after feeding (−36%, −36% and −38% respectively, P<.05) whereas S6K1, S6rp and 4EBP1 phosphorylations were similar. Overall glucose tolerance was reduced in leucine-supplemented rats and was associated with accumulation of perirenal adipose tissue (+27%, P<.05). Conversely, in vitro insulin-response of muscle glucose transport tended to be improved in leucine-supplemented rats. In conclusion, dietary leucine supplementation in adult rats induced a delay in the postprandial stimulation in the early steps of muscle insulin signaling without muscle resistance on insulin-induced glucose uptake. However, it resulted in overall glucose intolerance linked to increased local adiposity. Further investigations are necessary to clearly define the beneficial and/or deleterious effects of chronic dietary leucine supplementation in healthy subjects.

Kinetics of GLUT4 Trafficking in Rat and Human Skeletal Muscle

OBJECTIVEIn skeletal muscle, insulin stimulates glucose transport activity three- to fourfold, and a large part of this stimulation is associated with a net translocation of GLUT4 from an intracellular compartment to the cell surface. We examined the extent to which insulin or the AMP-activated protein kinase activator AICAR can lead to a stimulation of the exocytosis limb of the GLUT4 translocation pathway and thereby account for the net increase in glucose transport activity.RESEARCH DESIGN AND METHODSUsing a biotinylated photoaffinity label, we tagged endogenous GLUT4 and studied the kinetics of exocytosis of the tagged protein in rat and human skeletal muscle in response to insulin or AICAR. Isolated epitrochlearis muscles were obtained from male Wistar rats. Vastus lateralis skeletal muscle strips were prepared from open muscle biopsies obtained from six healthy men (age 39 ± 11 years and BMI 25.8 ± 0.8 kg/m2).RESULTSIn rat epitrochlearis muscle, insulin exposure leads to a sixfold stimulation of the GLUT4 exocytosis rate (with basal and insulin-stimulated rate constants of 0.010 and 0.067 min−1, respectively). In human vastus lateralis muscle, insulin stimulates GLUT4 translocation by a similar sixfold increase in the exocytosis rate constant (with basal and insulin-stimulated rate constants of 0.011 and 0.075 min−1, respectively). In contrast, AICAR treatment does not markedly increase exocytosis in either rat or human muscle.CONCLUSIONSInsulin stimulation of the GLUT4 exocytosis rate constant is sufficient to account for most of the observed increase in glucose transport activity in rat and human muscle.

Branched-Chain Amino Acid-Containing Dipeptides, Identified from Whey Protein Hydrolysates, Stimulate Glucose Uptake Rate in L6 Myotubes and Isolated Skeletal Muscles

In earlier studies we showed that dietary whey protein increased skeletal muscle and liver glycogen content in exercise-trained rats. However, little is known about whether ingredients of whey protein stimulate skeletal muscle glycogen accumulation. The aim of this study was to identify bioactive peptides in whey protein hydrolysates (WPH) which stimulated glucose uptake and glycogen synthesis rate in skeletal muscles. Branched-chain amino acid (BCAA)-containing dipeptides in WPH were identified using LC/MS/MS. L6 myotubes and isolated epitrochlearis muscles were used for the glucose uptake assays. The myotubes and muscles were incubated with or without 1 mM dipeptides, LY294002 a phosphoinositide 3-kinase (PI3-kinase) inhibitor, or GF102903X an atypical protein kinase C (aPKC) inhibitor, followed by measurement of 2-deoxyglucose uptake. Isolated muscles were incubated for 3 h with or without 1 mM Ile-Leu to determine glycogen synthesis rate. The BCAA-containing dipeptides, Ile-Val, Leu-Val, Val-Leu, Ile-Ile, Leu-Ile, Ile-Leu, and Leu-Leu were detected in the WPH by LC/MS/MS. These dipeptides caused significant stimulation in glucose uptake rate in the L6 myotubes. Ile-Leu, the main component in WPH, also stimulated glucose uptake in isolated skeletal muscles. Stimulation of glucose uptake by Ile-Leu was completely inhibited by treatment with either LY294002, or GF109203X in both L6 cells and isolated muscles. Ile-Leu increased glycogen contents in isolated muscles. These results suggest that BCAA-containing bioactive dipeptides in WPH stimulate glucose uptake in skeletal muscles via the PI3-kinase and aPKC pathways, resulting in increased skeletal muscle glycogen contents.

Acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA without increasing proteolysis in skeletal muscle

Acute alcohol intoxication decreases muscle protein synthesis, but there is a paucity of data on the ability of alcohol to regulate muscle protein degradation. Furthermore, various types of atrophic stimuli appear to regulate ubiquitin-proteasome-dependent proteolysis by increasing the muscle-specific E3 ligases atrogin-1 and MuRF1 (i.e., "atrogenes"). Therefore, the present study was designed to test the hypothesis that acute alcohol intoxication increases atrogene expression leading to an elevated rate of muscle protein breakdown. In male rats, the intraperitoneal injection of alcohol dose- and time-dependently increased atrogin-1 and MuRF1 mRNA in gastrocnemius, the latter of which was most pronounced. A comparable change was absent in the soleus and heart. The ability of in vivo-administered ethanol to increase atrogene expression was independent of the route of alcohol administration (intraperitoneal vs. oral), as well as of nutritional status (fed vs. fasted) and gender (male vs. female). The increase in atrogin-1 and MuRF1 was independent of alcohol metabolism, and the overproduction of endogenous glucocorticoids and could not be prevented by maintaining the circulating concentration of insulin-like growth factor-I. Despite marked changes in atrogene expression, acute alcohol in vivo did not alter the release of either 3-methylhistidine (MH) or tyrosine from the isolated perfused hindlimb, suggesting that the rate of muscle proteolysis remains unchanged. Moreover, alcohol did not increase the directly determined rate of protein degradation in isolated epitrochlearis muscles or cultured myocytes. Finally, no increase in atrogene expression or 3-MH release was detected in muscle from rats fed an alcohol-containing diet. Our results indicate that although acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA preferentially in fast-twitch skeletal muscle, this change was not associated with increased rates of muscle proteolysis. Therefore, the loss of muscle mass/protein in response to chronic alcohol abuse appears to result primarily from a decrement in muscle protein synthesis, not an increase in degradation.

Dissociation Between Acute Alcohol (EtOH) Intoxication‐Induced Increases in Atrogin‐1 and MuRF1 mRNA and Rates of Proteolysis in Skeletal Muscle

EtOH abuse causes skeletal muscle wasting resulting, in part, from a reduction in protein synthesis. However, whether alcohol enhances muscle proteolysis remains controversial. The current study determined whether acute EtOH stimulated muscle protein breakdown by measuring the mRNA content for the muscle‐specific ubiquitin E3 ligases atrogin‐1 and MuRF1 (aka atrogenes) and assessing the in vitro rate of proteolysis. IP injection of EtOH in rats increased atrogene mRNA in a time‐dependent manner, peaking at 8 h (4‐fold) and returning to baseline by 24 h. The EtOH‐induced rise in atrogenes was dose‐dependent, with increases observed in rats with blood alcohol levels &gt;150 mg/dL. In contrast, there was no EtOH‐induced change in atrogene mRNA in heart or soleus under these conditions. Elevated atrogene expression appears mediated indirectly because neither perfusion of an EtOH‐containing buffer into the isolated hindlimb of control rats nor incubation of C2C12 myocytes with EtOH increased atrogene mRNA. Finally, IP injection of a maximally effective dose of EtOH showed no change in proteolysis determined in the isolated epitrochlearis muscle. These data demonstrate EtOH selectively increases atrogene mRNA in fast‐twitch skeletal muscle, most likely via production of a secondary mediator, but this change was not associated with a concomitant stimulation of muscle protein breakdown. (AA11290 and AA12814).

Decreased contraction-stimulated glucose transport in isolated epitrochlearis muscles of pregnant rats

Late pregnancy is characterized by insulin resistance for glucose transport in skeletal muscle. The main purpose of this study was to investigate the effect of late pregnancy on contraction-stimulated glucose transport in isolated rat skeletal muscle after in vitro electrical stimulation. Isolated epitrochlearis muscles of 19-day pregnant and aged-matched nonpregnant control rats were studied. One muscle from each rat was stimulated to contract, and the contralateral muscle served as a resting control. Tension developed during contractile activity, 3-O-methylglucose (3-MG) transport rate, and glycogen concentration were determined. Epitrochlearis muscles from other rats were used to measure insulin-stimulated 3-MG transport. There was no detectable difference between the nonpregnant and pregnant groups for contractile performance (peak tension, total tension, or fatigue). Pregnancy was not associated with significant changes in muscle glycogen concentration (resting or after contractile activity) or the contraction-stimulated decrement in glycogen concentration. For muscles from pregnant vs. nonpregnant groups, there was a 22% reduction (P < or = 0.05) in contraction-stimulated glucose transport, a 28% decrease (P < or = 0.05) in insulin-stimulated glucose transport, and unchanged basal glucose transport. In conclusion, isolated epitrochlearis muscles from pregnant vs. nonpregnant rats had a relative decrement in contraction-stimulated glucose transport that was similar to the relative decline in insulin-stimulated glucose transport. The decrement in contraction-stimulated glucose transport was not attributable to pregnancy-related changes in tension development or glycogen levels. The similar relative decline in insulin- and contraction-stimulated glucose transport raises the possibility that pregnancy impairs a distal process that is common to mechanisms whereby each stimulus activates glucose transport.

5'-aminoimidazole-4-carboxyamide-ribonucleoside-activated glucose transport is not prevented by nitric oxide synthase inhibition in rat isolated skeletal muscle.

1. The nucleoside intermediate 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) activates skeletal muscle AMP-activated protein kinase (AMPK) and increases glucose uptake. The AMPK phosphorylates neuronal nitric oxide synthase (nNOS)mu in skeletal muscle fibres. There is evidence that both AMPK and nNOSmu may be involved in the regulation of contraction-stimulated glucose uptake. 2. We examined whether both AICAR- and contraction-stimulated glucose uptake were mediated by NOS in rat skeletal muscle. 3. Rat isolated epitrochlearis muscles were subjected in vitro to electrically stimulated contractions for 10 min and/or incubated in the presence or absence of AICAR (2 mmol/L) or the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 micromol/L). 4. Muscle contraction significantly (P < 0.05) altered the metabolic profile of the muscle. In contrast, AICAR and L-NMMA had no effect on the metabolic profile of the muscle, except that AICAR increased muscle 5'-aminoimidazole-4-carboxyamide-ribonucleotide (ZMP) and AICAR content. Nitric oxide synthase inhibition caused a small but significant (P < 0.05) reduction in basal 3-O-methylglucose transport, which was observed in all treatments. 5'-Aminoimidazole-4-carboxyamide-ribonucleoside significantly increased (P < 0.05) glucose transport above basal, with NOS inhibition decreasing this slightly (increased by 209% above basal compared with 184% above basal with NOS inhibition). Contraction significantly increased glucose transport above basal, with NOS inhibition substantially reducing this (107% increase vs 31% increase). 5'-Aminoimidazole-4-carboxyamide-ribonucleoside plus contraction in combination were not additive on glucose transport. 5. These results suggest that NO plays a role in basal glucose uptake and may regulate contraction-stimulated glucose uptake. However, NOS/nitric oxide do not appear to be signalling intermediates in AICAR-stimulated skeletal muscle glucose uptake.

Insulin-nonspecific reduction in skeletal muscle glucose transport in high-fat-fed rats

High-fat feeding diminishes insulin-stimulated glucose transport in skeletal muscle. However, conflicting results are reported regarding whether phosphatidylinositol (PI)-3 kinase-independent glucose transport is also impaired in insulin-resistant high-fat-fed rodents. The aim of the present study was to study whether non-insulin-dependent mechanisms for stimulation of glucose transport are defective in skeletal muscle from high-fat-fed rats. Rats were fed normal chow diet or high-fat diet for 4 weeks and isolated epitrochlearis muscles were used for measuring glucose transport. Insulin-stimulated glucose transport was significantly lower in rats fed the high-fat diet compared with chow-fed rats ( P < .05). Hypoxia-stimulated glucose transport was also reduced in high-fat-fed rats ( P < .05). Nevertheless, hypoxia-stimulated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation (Thr 172) level was not affected by high-fat feeding. Glucose transport by sodium nitroprusside stimulation was reduced in high-fat-fed rats ( P < .05). Protein content of glucose transporter (GLUT)-4 and AMPK-α, and glycogen content were comparable between both groups. Our findings provide evidence that high-fat feeding can affect not only insulin but also non-insulin-stimulated glucose transport. A putative defect in common steps in glucose transport may play a role to account for impaired insulin-stimulated glucose transport in rats fed a high-fat diet.

The Effect of Pregnancy on Tension Development by Electrically Stimulated Rat Epitrochlearis Muscle

0223 Gorski et al. (Eur J Appl Physiol 55:390, 1986) measured tension development of the gastrocnemius-plantaris-soleus in perfused hindlimbs of pregnant and nonpregnant rats during electrical stimulation via the sciatic nerve. Peak tension was similar between groups, but muscles from pregnant rats fatigued more rapidly than controls. PURPOSE: The major aim was to determine if pregnancy alters tension development of isolated rat skeletal muscle that is stimulated to contract directly rather than via the nerve. Muscle glycogen concentration was also measured to determine if pregnancy altered the contraction-induced glycogen depletion. METHODS: Sprague-Dawley rats (19-day pregnant, PR; and nonpregnant controls, N-PR) were studied. Both epitrochlearis muscles were dissected out, mounted in a bath containing oxygenated Krebs-Henseleit buffer supplemented with 8mM glucose, and attached to an isometric force transducer. One muscle was stimulated to contract (100 pulses/s, 0.5ms pulse duration, 1 train/min, 10 s train duration for 10 min) while the contralateral muscle served as a resting control. Subsequently, muscles were freeze-clamped, weighed, and homogenized. Glycogen concentration was assessed using the amyloglucosidase method. RESULTS: No significant differences between PR and N-PR were observed for peak tension (PR = 31.5 ± 0.2g and N-PR = 31.6 ± 0.3g). Tension declined for both groups over the course of the ten tetani, but there was no evidence for fatigue rate to differ between the PR and N-PR rats. Interpretation is unchanged when data are expressed relative to epitrochlearis mass which was unaffected by pregnancy. Glycogen concentration of resting muscles was approximately 20% higher for PR vs. N-PR (p<0.05), but post-contraction glycogen values were not significantly different. CONCLUSIONS: Pregnancy did not alter peak tension or fatigue of electrically stimulated, isolated epitrochlearis muscles. The Influence of pregnancy on muscle fatigue may depend on methodology (e.g., indirect stimulation via the nerve vs. direct stimulation, hindlimb vs. epitrochlearis muscles, etc.) rather than being an inevitable consequence of pregnancy. Supported by The Virginia Horne Henry Fund for Women's Physical Education.