Mark Hargreaves

Exercise, GLUT4, and Skeletal Muscle Glucose Uptake

Glucose is an important fuel for contracting muscle, and normal glucose metabolism is vital for health. Glucose enters the muscle cell via facilitated diffusion through the GLUT4 glucose transporter which translocates from intracellular storage depots to the plasma membrane and T-tubules upon muscle contraction. Here we discuss the current understanding of how exercise-induced muscle glucose uptake is regulated. We briefly discuss the role of glucose supply and metabolism and concentrate on GLUT4 translocation and the molecular signaling that sets this in motion during muscle contractions. Contraction-induced molecular signaling is complex and involves a variety of signaling molecules including AMPK, Ca(2+), and NOS in the proximal part of the signaling cascade as well as GTPases, Rab, and SNARE proteins and cytoskeletal components in the distal part. While acute regulation of muscle glucose uptake relies on GLUT4 translocation, glucose uptake also depends on muscle GLUT4 expression which is increased following exercise. AMPK and CaMKII are key signaling kinases that appear to regulate GLUT4 expression via the HDAC4/5-MEF2 axis and MEF2-GEF interactions resulting in nuclear export of HDAC4/5 in turn leading to histone hyperacetylation on the GLUT4 promoter and increased GLUT4 transcription. Exercise training is the most potent stimulus to increase skeletal muscle GLUT4 expression, an effect that may partly contribute to improved insulin action and glucose disposal and enhanced muscle glycogen storage following exercise training in health and disease.

AMP-Activated Protein Kinase Regulates GLUT4 Transcription by Phosphorylating Histone Deacetylase 5

OBJECTIVE—Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms. RESEARCH DESIGN AND METHODS AND RESULTS—Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription. CONCLUSIONS—These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.

Exercise increases serum Hsp72 in humans

Abstract Recent evidence suggests that heat shock proteins (Hsps) may have an important systemic role as a signal to activate the immune system. Since acute exercise is known to induce Hsp72 (the inducible form of the 70-kDa family of Hsp) in a variety of tissues including contracting skeletal muscle, we hypothesized that such exercise would result in the release of Hsp72 from stressed cells into the blood. Six humans (5 males, 1 female) ran on a treadmill for 60 minutes at a workload corresponding to 70% of their peak oxygen consumption. Blood was sampled from a forearm vein at rest (R), 30 minutes during exercise, immediately postexercise (60 minutes), and 2, 8, and 24 hours after exercise. These samples were analyzed for serum Hsp72 protein. In addition, plasma creatine kinase (CK) was measured at these time points as a crude marker of muscle damage. With the exception of the sample collected at 30 minutes, muscle biopsies (n = 5 males) were also obtained from the vastus lateralis at the time of blood sampling and analyzed for Hsp72 gene and protein expression. Serum Hsp72 protein increased from rest, both during and after exercise (0.13 0.10 vs 0.87 ± 0.24 and 1.02 ± 0.41 ng/mL at rest, 30 and 60 minutes, respectively, P < 0.05, mean SE). In addition, plasma CK was elevated (P < 0.05) 8 hours postexercise. Skeletal muscle Hsp72 mRNA expression increased 6.5-fold (P < 0.05) from rest 2 hours postexercise, and although there was a tendency for Hsp72 protein expression to be elevated 2 and 8 hours following exercise compared with rest, results were not statistically significant. The increase in serum Hsp72 preceded any increase in Hsp72 gene or protein expression in contracting muscle, suggesting that Hsp72 was released from other tissues or organs. This study is the first to demonstrate that acute exercise can increase Hsp72 in the peripheral circulation, suggesting that during stress these proteins may indeed have a systemic role.

Effect of carbohydrate feedings on muscle glycogen utilization and exercise performance.

Ten men were studied during 4 h of cycling to determine the effect of solid carbohydrate (CHO) feedings on muscle glycogen utilization and exercise performance. In the experimental trial (E) the subjects ingested 43 g of sucrose in solid form along with 400 ml of water at 0, 1, 2 and 3 h of exercise. During the control trial (C) they received 400 ml of an artificially sweetened drink without solid CHO. No differences in VO2, heart rate, or total energy expenditure were observed between trials; however, respiratory exchange ratios were significantly (P less than 0.05) higher during E. Blood glucose was significantly (P less than 0.05) elevated 20 min post-feeding in E; however, by 50 min no differences were observed between trials until 230 min (E = 4.5 +/- 0.2 mmol X l-1 vs C = 3.9 +/- 0.2, means +/- SE; P less than 0.05). Muscle glycogen utilization was significantly (P less than 0.05) lower during E (100.7 +/- 10.2 mmol X kg-1 w.w.) than C (126.2 +/- 5.5). During a sprint (100% VO2max) ride to exhaustion at the end of each trial, subjects performed 45% longer when fed CHO (E = 126.8 +/- 24.7 s vs C = 87.2 +/- 17.5; P less than 0.05). It was concluded that repeated solid CHO feedings maintain blood glucose levels, reduce muscle glycogen depletion during prolonged exercise, and enhance sprint performance at the end of such activity.

Exercise-induced histone modifications in human skeletal muscle

Skeletal muscle adaptations to exercise confer many of the health benefits of physical activity and occur partly through alterations in skeletal muscle gene expression. The exact mechanisms mediating altered skeletal muscle gene expression in response to exercise are unknown. However, in recent years, chromatin remodelling through epigenetic histone modifications has emerged as a key regulatory mechanism controlling gene expression in general. The purpose of this study was to examine the effect of exercise on global histone modifications that mediate chromatin remodelling and transcriptional activation in human skeletal muscle in response to exercise. In addition, we sought to examine the signalling mechanisms regulating these processes. Following 60 min of cycling, global histone 3 acetylation at lysine 9 and 14, a modification associated with transcriptional initiation, was unchanged from basal levels, but was increased at lysine 36, a site associated with transcriptional elongation. We examined the regulation of the class IIa histone deacetylases (HDACs), which are enzymes that suppress histone acetylation and have been implicated in the adaptations to exercise. While we found no evidence of proteasomal degradation of the class IIa HDACs, we found that HDAC4 and 5 were exported from the nucleus during exercise, thereby removing their transcriptional repressive function. We also observed activation of the AMP‐activated protein kinase (AMPK) and the calcium–calmodulin‐dependent protein kinase II (CaMKII) in response to exercise, which are two kinases that induce phosphorylation‐dependent class IIa HDAC nuclear export. These data delineate a signalling pathway that might mediate skeletal muscle adaptations in response to exercise.

Exercise Increases Ca2+–Calmodulin‐Dependent Protein Kinase II Activity in Human Skeletal Muscle

There is evidence in rodents that Ca2+‐calmodulin‐dependent protein kinase II (CaMKII) activity is higher in contracting skeletal muscle, and this kinase may regulate skeletal muscle function and metabolism during exercise. To investigate the effect of exercise on CaMKII in human skeletal muscle, healthy men (n= 8) performed cycle ergometer exercise for 40 min at 76 ± 1 % peak pulmonary O2 uptake (V̇O2peak), with skeletal muscle samples taken at rest and after 5 and 40 min of exercise. CaMKII expression and activities were examined by immunoblotting and in vitro kinase assays, respectively. There were no differences in maximal (+ Ca2+, CaM) CaMKII activity during exercise compared with rest. Autonomous (‐ Ca2+, CaM) CaMKII activity was 9 ± 1 % of maximal at rest, remained unchanged at 5 min, and increased to 17 ± 1 % (P < 0.01) at 40 min. CaMKII autophosphorylation at Thr287 was 50‐70 % higher during exercise, with no differences in CaMKII expression. The effect of maximal aerobic exercise on CaMKII was also examined (n= 9), with 0.7‐ to 1.5‐fold increases in autonomous CaMKII activity, but no change in maximal CaMKII activity. CaMKIV was not detected in human skeletal muscle. In summary, exercise increases the activity of CaMKII in skeletal muscle, suggesting that it may have a role in regulating skeletal muscle function and metabolism during exercise in humans.

Effect of carbohydrate feeding frequencies and dosage on muscle glycogen use during exercise

Nine men were studied during three 4-h cycling bouts to determine the effect of frequency and dosage of solid carbohydrate (CHO) feedings (86 g) on muscle glycogen utilization and exercise performance. In the frequency trial (F), the subjects ingested 10.75 g of CHO along with 200 ml of water at 30-min intervals; in the dosage trial (D), the subjects ingested 21.5 g of CHO with 400 ml of water at 60-min intervals. During the control trial (C), the subjects ingested 400 ml of an artificially sweetened placebo at 60-min intervals. Respiratory exchange ratios were significantly elevated in both trials D and F (P less than 0.05). Blood glucose was significantly elevated in trial D 20 min post-feeding but had returned to control levels by 50 min. In trial F, blood glucose was maintained at a constant level throughout the entire 4 h. In trial C, blood glucose declined steadily during the entire 4 h. Despite the differences in blood glucose levels between the three trials, there were no significant differences in the rate of muscle glycogen utilization in any of the trials (D = 82.9 +/- 6.6 [SE] mmol X kg-1 vs C = 80.9 +/- 6.9 mmol X kg-1 vs F = 74.4 +/- 12.2 mmol X kg-1). In a sprint ride (100% VO2max) to exhaustion at the end of each trial, the subjects performed significantly longer in trial F compared to C (120.97 +/- 9.6 vs 81.0 +/- 7.1 s).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenaline increases skeletal muscle glycogenolysis, pyruvate dehydrogenase activation and carbohydrate oxidation during moderate exercise in humans

1 To evaluate the role of adrenaline in regulating carbohydrate metabolism during moderate exercise, 10 moderately trained men completed two 20 min exercise bouts at 58 ± 2 % peak pulmonary oxygen uptake (V̇O2,peak). On one occasion saline was infused (CON), and on the other adrenaline was infused intravenously for 5 min prior to and throughout exercise (ADR). Glucose kinetics were measured by a primed, continuous infusion of 6,6‐[2H]glucose and muscle samples were obtained prior to and at 1 and 20 min of exercise. 2 The infusion of adrenaline elevated (P < 0.01) plasma adrenaline concentrations at rest (pre‐infusion, 0.28 ± 0.09; post‐infusion, 1.70 ± 0.45 nmol l−1; means ±s.e.m.) and this effect was maintained throughout exercise. Total carbohydrate oxidation increased by 18 % and this effect was due to greater skeletal muscle glycogenolysis (P < 0.05) and pyruvate dehydrogenase (PDH) activation (P < 0.05, treatment effect). Glucose rate of appearance was not different between trials, but the infusion of adrenaline decreased (P < 0.05, treatment effect) skeletal muscle glucose uptake in ADR. 3 During exercise muscle glucose 6‐phosphate (G‐6‐P) (P = 0.055, treatment effect) and lactate (P < 0.05) were elevated in ADR compared with CON and no changes were observed for pyruvate, creatine, phosphocreatine, ATP and the calculated free concentrations of ADP and AMP. 4 The data demonstrate that elevated plasma adrenaline levels during moderate exercise in untrained men increase skeletal muscle glycogen breakdown and PDH activation, which results in greater carbohydrate oxidation. The greater muscle glycogenolysis appears to be due to increased glycogen phosphorylase transformation whilst the increased PDH activity cannot be readily explained. Finally, the decreased glucose uptake observed during exercise in ADR is likely to be due to the increased intracellular G‐6‐P and a subsequent decrease in glucose phosphorylation.

Pre-exercise carbohydrate and fat ingestion: effects on metabolism and performance

A key goal of pre-exercise nutritional strategies is to maximize carbohydrate stores, thereby minimizing the ergolytic effects of carbohydrate depletion. Increased dietary carbohydrate intake in the days before competition increases muscle glycogen levels and enhances exercise performance in endurance events lasting 90 min or more. Ingestion of carbohydrate 3–4 h before exercise increases liver and muscle glycogen and enhances subsequent endurance exercise performance. The effects of carbohydrate ingestion on blood glucose and free fatty acid concentrations and carbohydrate oxidation during exercise persist for at least 6 h. Although an increase in plasma insulin following carbohydrate ingestion in the hour before exercise inhibits lipolysis and liver glucose output, and can lead to transient hypoglycaemia during subsequent exercise in susceptible individuals, there is no convincing evidence that this is always associated with impaired exercise performance. However, individual experience should inform individual practice. Interventions to increase fat availability before exercise have been shown to reduce carbohydrate utilization during exercise, but do not appear to have ergogenic benefits.

Carbohydrate Nutrition and Fatigue

SummaryCarbohydrates are important substrates for contracting muscle during prolonged, strenuous exercise, and fatigue is often associated with muscle glycogen depletion and/or hypoglycaemia. Thus, the goals of carbohydrate nutritional strategies before, during and after exercise are to optimise the availability of muscle and liver glycogen and glood glucose, with a view to maintaining carbohydrate availability and oxidation during exercise. During heavy training, the carbohydrate requirements of athletes may be as high as 8 to 10 g/kg bodyweight or 60 to 70% of total energy intake. Ingestion of a diet high in carbohydrate should be encouraged in order to maintain carbohydrate reserves and the ability to train intensely. Ingestion of a high carbohydrate meal 3 to 4 hours prior to exercise ensures adequate carbohydrate availability and enhances exercise performance. Although hyperinsulinaemia associated with carbohydrate ingestion in the hour prior to exercise may result in some metabolic alterations during exercise, it may not necessarily impair exercise performance and may, in some cases, enhance performance. Carbohydrate ingestion during prolonged, strenuous exercise, where performance is often limited by carbohydrate availability, delays fatigue. This is due to maintenance of blood glucose levels and a high rate of carbohydrate oxidation, rather than a slowing of muscle glycogen utilisation, although liver glycogen reserves may be spared. During recovery from exercise, muscle glycogen resynthesis is critically dependent upon the ingestion of carbohydrate. Factors influencing the rate of muscle glycogen resynthesis include the timing, amount and type of carbohydrate ingested and muscle damage. Adequate carbohydrate availability before, during and after exercise will maintain carbohydrate oxidation during exercise and is associated with enhanced exercise performance.