Kirsten F. Howlett

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.

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.

Adrenaline and glycogenolysis in skeletal muscle during exercise: a study in adrenalectomised humans

1 The role of adrenaline in regulating muscle glycogenolysis and hormone‐sensitive lipase (HSL) activity during exercise was examined in six adrenaline‐deficient bilaterally adrenalectomised, adrenocortico‐hormonal‐substituted humans (Adr) and in six healthy control individuals (Con). 2 Subjects cycled for 45 min at ∼70 % maximal pulmonary O2 uptake (V̇O2,max) followed by 15 min at ∼86 %V̇O2,max either without (−Adr and Con) or with (+Adr) adrenaline infusion that elevated plasma adrenaline levels (45 min, 4.49 ± 0.69 nmol l−1; 60 min, 12.41 ± 1.80 nmol l−1). Muscle samples were obtained at 0, 45 and 60 min of exercise. 3 In −Adr and Con, muscle glycogen was similar at rest (−Adr, 409 ± 19 mmol (kg dry wt)−1; Con, 453 ± 24 mmol (kg dry wt)−1) and following exercise (−Adr, 237 ± 52 mmol (kg dry wt)−1; Con, 227 ± 50 mmol (kg dry wt)−1). Muscle lactate, glucose‐6‐phosphate and glucose were similar in −Adr and Con, whereas glycogen phosphorylase (a/a+b× 100%) and HSL (% phosphorylated) activities increased during exercise in Con only. Adrenaline infusion increased activities of phosphorylase and HSL as well as blood lactate concentrations compared with those in −Adr, but did not enhance glycogen breakdown (+Adr, glycogen following exercise: 274 ± 55 mmol (kg dry wt)−1) in contracting muscle. 4 The present findings demonstrate that during exercise muscle glycogenolysis can occur in the absence of adrenaline, and that adrenaline does not enhance muscle glycogenolysis in exercising adrenalectomised subjects. Although adrenaline increases the glycogen phosphorylase activity it is not essential for glycogen breakdown in contracting muscle. Finally, a novel finding is that the activity of HSL in human muscle is increased in exercising man and this is due, at least partly, to stimulation by adrenaline.

Effect of adrenaline on glucose kinetics during exercise in adrenalectomised humans

1 The role of adrenaline in regulating hepatic glucose production and muscle glucose uptake during exercise was examined in six adrenaline‐deficient, bilaterally adrenalectomised humans. Six sex‐ and age‐matched healthy individuals served as controls (CON). 2 Adrenalectomised subjects cycled for 45 min at 68 ± 1 % maximum pulmonary O2 uptake (V̇O2,max), followed by 15 min at 84 ± 2 %V̇O2,max without (–ADR) or with (+ADR) adrenaline infusion, which elevated plasma adrenaline levels (45 min, 4.49 ± 0.69 nmol l−1; 60 min, 12.41 ± 1.80 nmol l−1; means ± s.e.m.). Glucose kinetics were measured using [3‐3H]glucose. 3 Euglycaemia was maintained during exercise in CON and –ADR, whilst in +ADR plasma glucose was elevated. The exercise‐induced increase in hepatic glucose production was similar in +ADR and –ADR; however, adrenaline infusion augmented the rise in hepatic glucose production early in exercise. Glucose uptake increased during exercise in +ADR and –ADR, but was lower and metabolic clearance rate was reduced in +ADR. 4 During exercise noradrenaline and glucagon concentrations increased, and insulin and cortisol concentrations decreased, but plasma levels were similar between trials. Adrenaline infusion suppressed growth hormone and elevated plasma free fatty acids, glycerol and lactate. Alanine and β‐hydroxybutyrate levels were similar between trials. 5 The results demonstrate that glucose homeostasis was maintained during exercise in adrenalectomised subjects. Adrenaline does not appear to play a major role in matching hepatic glucose production to the increase in glucose clearance. In contrast, adrenaline infusion results in a mismatch by simultaneously enhancing hepatic glucose production and inhibiting glucose clearance.

Glucose production during strenuous exercise in humans : role of epinephrine

The increase in hepatic glucose production (HGP) that occurs during intense exercise is accompanied by a simultaneous increase in epinephrine, which suggests that epinephrine may be important in regulating HGP. To further investigate this, six trained men were studied twice. The first trial [control (Con)] consisted of 20 min of cycling at 40 ± 1% peak oxygen uptake (V˙o 2 peak) followed by 20 min at 80 ± 2%V˙o 2 peak. During the second trial [epinephrine (Epi)], subjects exercised for 40 min at 41 ± 2%V˙o 2 peak. Epinephrine was infused during the latter 20 min of exercise and resulted in plasma levels similar to those measured during intense exercise in Con. Glucose kinetics were measured using a primed, continuous infusion of [3-3H]glucose. HGP was similar at rest (Con, 11.0 ± 0.5 and Epi, 11.1 ± 0.5 μmol ⋅ kg-1 ⋅ min-1). In Con, HGP increased ( P < 0.05) during exercise to 41.0 ± 5.2 μmol ⋅ kg-1 ⋅ min-1at 40 min. In Epi, HGP was similar to Con during the first 20 min of exercise. Epinephrine infusion increased ( P < 0.05) HGP to 24.0 ± 2.5 μmol ⋅ kg-1 ⋅ min-1at 40 min, although this was less ( P< 0.05) than the value in Con. The results suggest that epinephrine can increase HGP during exercise in trained men; however, epinephrine during intense exercise cannot fully account for the rise in HGP. Other glucoregulatory factors must contribute to the increase in HGP during intense exercise.The increase in hepatic glucose production (HGP) that occurs during intense exercise is accompanied by a simultaneous increase in epinephrine, which suggests that epinephrine may be important in regulating HGP. To further investigate this, six trained men were studied twice. The first trial [control (Con)] consisted of 20 min of cycling at 40 +/- 1% peak oxygen uptake (VO2 peak) followed by 20 min at 80 +/- 2% VO2 peak. During the second trial [epinephrine (Epi)], subjects exercised for 40 min at 41 +/- 2% VO2 peak. Epinephrine was infused during the latter 20 min of exercise and resulted in plasma levels similar to those measured during intense exercise in Con. Glucose kinetics were measured using a primed, continuous infusion of [3-3H]glucose. HGP was similar at rest (Con, 11.0 +/- 0.5 and Epi, 11.1 +/- 0.5 micromol. kg-1. min-1). In Con, HGP increased (P < 0.05) during exercise to 41.0 +/- 5.2 micromol. kg-1. min-1 at 40 min. In Epi, HGP was similar to Con during the first 20 min of exercise. Epinephrine infusion increased (P < 0.05) HGP to 24.0 +/- 2.5 micromol. kg-1. min-1 at 40 min, although this was less (P < 0.05) than the value in Con. The results suggest that epinephrine can increase HGP during exercise in trained men; however, epinephrine during intense exercise cannot fully account for the rise in HGP. Other glucoregulatory factors must contribute to the increase in HGP during intense exercise.

Resistance exercise and insulin regulate AS160 and interaction with 14-3-3 in human skeletal muscle

A single bout of aerobic exercise can enhance insulin action, but whether a similar effect occurs after resistance exercise is unknown. Hyperinsulinemic-euglycemic clamps were performed on eight male subjects at rest and after a single bout and three repeated bouts of resistance exercise over 7 days. Skeletal muscle biopsies were taken before and after the clamp and immediately after a single exercise bout. Whole-body insulin action measured by glucose infusion rate decreased (P < 0.05) after a single exercise bout, whereas in response to repeated bouts of resistance exercise, the glucose infusion rate was similar to the rest trial. In skeletal muscle, Akt substrate of 160 kDa (AS160) phosphorylation, an Akt substrate implicated in the regulation of GLUT4 translocation, and its interaction with 14-3-3 was decreased (P < 0.05) only after a single exercise bout. Insulin increased (P < 0.05) phosphorylation of AS160 and its interaction with 14-3-3, but the insulin response was not influenced by resistance exercise. Phosphorylation of insulin receptor substrate-1 and Akt were similar to changes in AS160 phosphorylation after exercise and/or insulin. In conclusion, a single bout of resistance exercise impairs whole-body insulin action. Regulation of AS160 and interaction with 14-3-3 in skeletal muscle are influenced by resistance exercise and insulin but do not fully explain the effect of resistance exercise on whole-body insulin action.

Metabolic remodelling in obesity and type 2 diabetes: pathological or protective mechanisms in response to nutrient excess?

Altered metabolism in tissues such as the liver, skeletal muscle and adipose tissue is observed in metabolic diseases characterized by nutrient excess and energy imbalance, such as obesity and type 2 diabetes. These alterations in metabolism can include resistance to the hormone insulin, lipid accumulation, mitochondrial dysfunction and transcriptional remodelling of major metabolic pathways. The underlying assumption has been that these same alterations in metabolism are fundamental to the pathogenesis of metabolic diseases. An alternative view is that these alterations in metabolism occur to protect cell and tissue viability in the face of constant positive energy balance. This speculative review presents evidence that many of the metabolic adaptations that occur in metabolic diseases characterized by nutrient excess can be viewed as protective in nature, rather than pathogenic per se for disease progression. Finally, we also briefly discuss the usefulness and potential pitfalls of therapeutic approaches that attempt to correct these same metabolic defects when energy balance is not altered, and the potential links between metabolic survival responses and other chronic diseases such as cancer.

Exercise-induced muscle glucose uptake in mice with graded, muscle-specific GLUT-4 deletion

To investigate the importance of the glucose transporter GLUT‐4 for muscle glucose uptake during exercise, transgenic mice with skeletal muscle GLUT‐4 expression approximately 30–60% of normal (CON) and approximately 5–10% of normal (KO) were generated using the Cre/Lox system and compared with wild‐type (WT) mice during approximately 40 min of treadmill running (KO: 37.7 ± 1.3 min; WT: 40 min; CON: 40 min, P = 0.18). In WT and CON animals, exercise resulted in an overall increase in muscle glucose uptake. More specifically, glucose uptake was increased in red gastrocnemius of WT mice and in the soleus and red gastrocnemius of CON mice. In contrast, the exercise‐induced increase in muscle glucose uptake in all muscles was completely abolished in KO mice. Muscle glucose uptake increased during exercise in both red and white quadriceps of WT mice, while the small increases in CON mice were not statistically significant. In KO mice, there was no change at all in quadriceps muscle glucose uptake. No differences in muscle glycogen use during exercise were observed between any of the groups. However, there was a significant increase in plasma glucose levels after exercise in KO mice. The results of this study demonstrated that a reduction in skeletal muscle GLUT‐4 expression to approximately 10% of normal levels completely abolished the exercise‐induced increase in muscle glucose uptake.

Regulation of glucose kinetics during intense exercise in humans: Effects of α- and β-adrenergic blockade

This study examined the effect of combined [alpha]- and [beta]-adrenergic blockade on glucose kinetics during intense exercise. Six endurance-trained men exercised for 20 minutes at approximately 78% of their peak oxygen consumption (V2) following ingestion of a placebo (CON) or combined [alpha]- (prazosin hydrochloride) and [beta]- (timolol maleate) adrenoceptor antagonists (BLK). Plasma glucose increased during exercise in CON (0 minutes: 5.5 ± 0.1; 20 minutes: 6.5 ± 0.3 mmol · L-1, P < .05). In BLK, the exercise-induced increase in plasma glucose was abolished (0 minutes: 5.7 ± 0.3; 20 minutes: 5.7 ± 0.1 mmol · L-1). Glucose kinetics were measured using a primed, continuous infusion of [6,6-2H] glucose. Glucose production was not different between trials; on average these values were 25.3 ± 3.9 and 30.9 ± 4.4 [mu]mol · kg-1 · min-1 in CON and BLK, respectively. Glucose uptake during exercise was greater (P < .05) in BLK (30.6 ± 4.6 [mu]mol · kg-1 · min-1) compared with CON (18.4 ± 2.5 [mu]mol · kg-1 · min-1). In BLK, plasma insulin and catecholamines were higher (P < .05), while plasma glucagon was unchanged from CON. Free fatty acids (FFA) and glycerol were lower (P < .05) in BLK. These findings demonstrate that adrenergic blockade during intense exercise results in a blunted plasma glucose response that is due to enhanced glucose uptake, with no significant change in glucose production.

The effect of insulin and exercise on c-Cbl protein abundance and phosphorylation in insulin-resistant skeletal muscle in lean and obese Zucker rats.

Aims/hypothesisRecruitment of the protein c-Cbl to the insulin receptor (IR) and its tyrosine phosphorylation via a pathway that is independent from phosphatidylinositol 3′-kinase is necessary for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. The activation of this pathway by insulin or exercise has yet to be reported in skeletal muscle.MethodsLean and obese Zucker rats were randomly assigned to one of three treatment groups: (i) control, (ii) insulin-stimulated or (iii) acute, exhaustive exercise. Hind limb skeletal muscle was removed and the phosphorylation state of IR, Akt and c-Cbl measured.ResultsInsulin receptor phosphorylation was increased 12-fold after insulin stimulation (p<0.0001) in lean rats and threefold in obese rats. Acute exercise had no effect on IR tyrosine phosphorylation. Similar results were found for serine phosphorylation of Akt. Exercise did not alter c-Cbl tyrosine phosphorylation in skeletal muscle of lean or obese rats. However, in contrast to previous studies in adipocytes, c-Cbl tyrosine phosphorylation was reduced after insulin treatment (p<0.001).Conclusions/interpretationWe also found that c-Cbl associating protein expression is relatively low in skeletal muscle of Zucker rats compared to 3T3-L1 adipocytes and this could account for the reduced c-Cbl tyrosine phosphorylation after insulin treatment. Interestingly, basal levels of c-Cbl tyrosine phosphorylation were higher in skeletal muscle from insulin-resistant Zucker rats (p<0.05), but the physiological relevance is not clear. We conclude that the regulation of c-Cbl phosphorylation in skeletal muscle differs from that previously reported in adipocytes.