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Humans have, throughout history, faced periods of starvation necessitating increased physical effort to gather food. To explore adaptations in muscle function, 13 participants (7 males and 6 females) fasted for seven days. They lost 4.6 ± 0.3 kg lean and 1.4 ± 0.1 kg fat mass. Maximal isometric and isokinetic strength remained unchanged, while peak oxygen uptake decreased by 13%. Muscle glycogen was halved, while expression of electron transport chain proteins was unchanged. Pyruvate dehydrogenase kinase 4 (PDK4) expression increased 13-fold, accompanied by inhibitory pyruvate dehydrogenase phosphorylation, reduced carbohydrate oxidation and decreased exercise endurance capacity. Fasting had no impact on 5’ AMP-activated protein kinase (AMPK) activity, challenging its proposed role in muscle protein degradation. The participants maintained muscle strength and oxidative enzymes in skeletal muscle during fasting but carbohydrate oxidation and high-intensity endurance capacity were reduced. During starvation, muscle strength and endurance, are still critical for survival. Seven days of complete fasting did not reduce maximal strength in leg muscles, but maximal endurance capacity was decreased because carbohydrate oxidation was restrained.

Excess lipid availability causes insulin resistance. We examined the effect of acute exercise on lipid-induced insulin resistance and TBC1 domain family member 1/4 (TBCD1/4)-related signaling in skeletal muscle. In eight healthy young male subjects, 1 h of one-legged knee-extensor exercise was followed by 7 h of saline or intralipid infusion. During the last 2 h, a hyperinsulinemic-euglycemic clamp was performed. Femoral catheterization and analysis of biopsy specimens enabled measurements of leg substrate balance and muscle signaling. Each subject underwent two experimental trials, differing only by saline or intralipid infusion. Glucose infusion rate and leg glucose uptake was decreased by intralipid. Insulin-stimulated glucose uptake was higher in the prior exercised leg in the saline and the lipid trials. In the lipid trial, prior exercise normalized insulin-stimulated glucose uptake to the level observed in the resting control leg in the saline trial. Insulin increased phosphorylation of TBC1D1/4. Whereas prior exercise enhanced TBC1D4 phosphorylation on all investigated sites compared with the rested leg, intralipid impaired TBC1D4 S341 phosphorylation compared with the control trial. Intralipid enhanced pyruvate dehydrogenase (PDH) phosphorylation and lactate release. Prior exercise led to higher PDH phosphorylation and activation of glycogen synthase compared with resting control. In conclusion, lipid-induced insulin resistance in skeletal muscle was associated with impaired TBC1D4 S341 and elevated PDH phosphorylation. The prophylactic effect of exercise on lipid-induced insulin resistance may involve augmented TBC1D4 signaling and glycogen synthase activation.

Impaired Insulin-Stimulated Phosphorylation of Akt and AS160 in Skeletal Muscle of Women With Polycystic Ovary Syndrome Is Reversed by Pioglitazone Treatment Kurt Højlund 1 , Dorte Glintborg 1 , Nicoline R. Andersen 2 , Jesper B. Birk 2 , Jonas T. Treebak 2 , Christian Frøsig 2 , Henning Beck-Nielsen 1 and Jørgen F.P. Wojtaszewski 2 1 Department of Endocrinology, Diabetes Research Centre, Odense University Hospital, Odense, Denmark 2 Department of Exercise and Sport Sciences, Section of Human Physiology Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark Address correspondence and reprint requests to Kurt Højlund, MD, PhD, Department of Endocrinology, Odense University Hospital, Kloevervaenget 6, DK-5000 Odense C, Denmark. E-mail: k.hojlund{at}dadlnet.dk Abstract OBJECTIVE— Insulin resistance in skeletal muscle is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). However, the molecular mechanisms underlying skeletal muscle insulin resistance and the insulin-sensitizing effect of thiazolidinediones in PCOS in vivo are less well characterized. RESEARCH DESIGN AND METHODS— We determined molecular mediators of insulin signaling to glucose transport in skeletal muscle biopsies of 24 PCOS patients and 14 matched control subjects metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry, and we examined the effect of 16 weeks of treatment with pioglitazone in PCOS patients. RESULTS— Impaired insulin-mediated total ( R d ) oxidative and nonoxidative glucose disposal (NOGD) was paralleled by reduced insulin-stimulated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation in muscle of PCOS patients. Akt phosphorylation at Ser473 and Thr308 correlated positively with R d and NOGD in the insulin-stimulated state. Serum free testosterone was inversely related to insulin-stimulated R d and NOGD in PCOS. Importantly, the pioglitazone-mediated improvement in insulin-stimulated glucose metabolism, which did not fully reach normal levels, was accompanied by normalization of insulin-mediated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation. AMPK activity and phosphorylation were similar in the two groups and did not respond to pioglitazone in PCOS patients. CONCLUSIONS— Impaired insulin signaling through Akt and AS160 in part explains insulin resistance at the molecular level in skeletal muscle in PCOS, and the ability of pioglitazone to enhance insulin sensitivity involves improved signaling through Akt and AS160. Moreover, our data provide correlative evidence that hyperandrogenism in PCOS may contribute to insulin resistance. AMPK, AMP-activated protein kinase AS160, Akt substrate of 160 kDa FDR, first-degree relative GAP, GTPase-activating protein IR, insulin receptor IRS-1, insulin receptor substrate-1 NOGD, nonoxidative glucose disposal PCOS, polycystic ovary syndrome PI3K, phosphatidylinositol-3 kinase PPAR, peroxisome proliferator–activated receptor TZD, thiazolidinedione Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 31 October 2007. DOI: 10.2337/db07-0706. Clinical trial reg. no. NCT00145340, clinicaltrials.gov. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted October 26, 2007. Received May 24, 2007. DIABETES

The α2–5′AMP-Activated Protein Kinase Is a Site 2 Glycogen Synthase Kinase in Skeletal Muscle and Is Responsive to Glucose Loading Sebastian B. Jørgensen 1 , Jakob N. Nielsen 1 , Jesper B. Birk 1 , Grith Skytte Olsen 3 , Benoit Viollet 4 , Fabrizio Andreelli 4 , Peter Schjerling 2 , Sophie Vaulont 4 , D. Grahame Hardie 5 , Bo F. Hansen 3 , Erik A. Richter 1 and Jørgen F.P. Wojtaszewski 1 1 Copenhagen Muscle Research Centre, Department of Human Physiology, Institute of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark 2 Department for Molecular Muscle Biology, Rigshospitalet, Copenhagen, Denmark 3 Diabetes Biology, Novo Nordisk, Maaloev, Denmark 4 Department of Genetic, Development, and Molecular Pathology, Institut Cochin, INSERM, CNRS, Rene Descartes University, Paris, France 5 Division of Molecular Physiology, Faculty of Life Sciences, Dundee University, Dundee, Scotland Address correspondence and reprint requests to Jørgen F.P. Wojtaszewski, PhD, Copenhagen Muscle Research Centre, Department of Human Physiology, Institute of Exercise and Sport Sciences, 13 Universitetsparken, University of Copenhagen, DK-2100 Copenhagen, Denmark. E-mail: jwojtaszewski{at}aki.ku.dk Abstract The 5′AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-β-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). In muscle of mice with targeted deletion of the α2-AMPK gene, phosphorylation of GS site 2 was decreased under basal conditions and unchanged by AICAR treatment. In contrast, in α1-AMPK knockout mice, the response to AICAR was normal. Fuel surplus (glucose loading) decreased AMPK activation by AICAR, but the phosphorylation of the downstream targets acetyl-CoA carboxylase-β and GS was normal. Fractionation studies suggest that this suppression of AMPK activation was not a direct consequence of AMPK association with membranes or glycogen, because AMPK was phosphorylated to a greater extent in response to AICAR in the membrane/glycogen fraction than in the cytosolic fraction. Thus, the downstream action of AMPK in response to AICAR was unaffected by glucose loading, whereas the action of the kinase upstream of AMPK, as judged by AMPK phosphorylation, was decreased. The fact that α2-AMPK is a GS kinase that inactivates GS while simultaneously activating glucose transport suggests that a balanced view on the suitability for AMPK as an antidiabetic drug target should be taken. ACC, acetyl-CoA carboxylase AICAR, 5-aminoimidazole-1-β-4-carboxamide ribofuranoside AMPK, 5′AMP-activated protein kinase CK, casein kinase EDL, extensor digitorum longus G6P, glucose-6-phosphate GS, glycogen synthase GSK, glycogen synthase kinase Footnotes S.B.J. and J.N.N. contributed equally to this article. Accepted August 25, 2004. Received May 25, 2004. DIABETES

5´AMP–activated protein kinase (AMPK) is a mediator of a healthy metabolic phenotype in skeletal muscle. Metformin may exacerbate the energy disturbances observed during exercise leading to enhanced AMPK activation, and these disturbances may provoke early muscular fatigue. We studied acute (1 day) and short‐term (4 days) effects of metformin treatment on AMPK and its downstream signaling network, in healthy human skeletal muscle and adipose tissue at rest and during exercise, by applying a randomized blinded crossover study design in 10 lean men. Muscle and fat biopsies were obtained before and after the treatment period at rest and after a single bout of exercise. Metformin treat ment elicited peak plasma and muscle metformin concentrations of 31 μM and 11 μM, respectively. Neither of the treatments affected AMPK activity in skeletal muscle and adipose at rest or during exercise. In contrast, whole‐body stress during exercise was elevated as indicated by increased plasma lactate and adrenaline concentrations as well as increased heart rate and rate of perceived exertion. Also whole‐body insulin sensitivity was enhanced by 4 days metformin treatment, that is reduced fasting plasma insulin and HOMA‐IR. In conclusion, acute and short‐term metformin treatment does not affect energy homeostasis and AMPK activation at rest or during exercise in skeletal muscle and adipose tissue of healthy subjects. However, metformin treatment is accompanied by slightly enhanced perceived exertion and whole‐body stress which may provoke a lesser desire for physical activity in the metformin‐treated patients. Acute or short‐term metformin treatment did not affect the energy status and 5´AMP–activated protein kinase activation at rest or during exercise in skeletal muscle and adipose tissue of healthy subject. However, the metformin treatment was accompanied by enhanced whole‐body stress.

A single bout of exercise improves muscle insulin sensitivity for up to 48 hours via AMPK. Limb ischemia activates AMPK in muscle, and subsequent reperfusion enhances insulin-stimulated vasodilation, potentially eliciting a more pronounced exercise effect with reduced workload. We investigated the combined effect of upper leg intermittent ischemia/reperfusion (IIR) and continuous knee-extension exercise on muscle insulin sensitivity regulation. We found that IIR exercise potentiated AMPK activation and muscle insulin sensitivity. The potentiating effect of IIR exercise on muscle insulin sensitivity was associated with increased insulin-stimulated blood flow in parallel with enhanced phosphorylation of endothelial nitric oxide synthase. Metabolomics analyses demonstrated a suppression of muscle medium-chain acylcarnitines during IIR exercise, which correlated with insulin sensitivity and was consistent with findings in isolated rat muscle treated with decanoyl-l-carnitine. Collectively, combining IIR with low- to moderate-intensity exercise may represent a promising intervention to effectively enhance muscle insulin sensitivity. This approach could offer potential for mitigating muscle insulin resistance in clinical settings and among individuals with lower physical activity levels.

Low Muscle Glycogen and Elevated Plasma Free Fatty Acid Modify but Do Not Prevent Exercise-Induced PDH Activation in Human Skeletal Muscle Kristian Kiilerich 1 , 2 , 3 , Mikkel Gudmundsson 1 , 2 , 3 , Jesper B. Birk 1 , 4 , Carsten Lundby 1 , 5 , Sarah Taudorf 1 , 2 , 6 , Peter Plomgaard 1 , 2 , 6 , Bengt Saltin 1 , 5 , Per A. Pedersen 3 , Jorgen F.P. Wojtaszewski 1 , 4 and Henriette Pilegaard 1 , 2 , 3 1 Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark; 2 Centre of Inflammation and Metabolism, University of Copenhagen, Copenhagen, Denmark; 3 Department of Biology, University of Copenhagen, Copenhagen, Denmark; 4 Section of Human Physiology, Department of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark; 5 Rigshospitalet, Section 7652, Copenhagen, Denmark; 6 Rigshospitalet, Section 7641, Copenhagen, Denmark. Corresponding author: Kristian Kiilerich, kkiilerich{at}bio.ku.dk . Abstract OBJECTIVE To test the hypothesis that free fatty acid (FFA) and muscle glycogen modify exercise-induced regulation of PDH (pyruvate dehydrogenase) in human skeletal muscle through regulation of PDK4 expression. RESEARCH DESIGN AND METHODS On two occasions, healthy male subjects lowered (by exercise) muscle glycogen in one leg (LOW) relative to the contra-lateral leg (CON) the day before the experimental day. On the experimental days, plasma FFA was ensured normal or remained elevated by consuming breakfast rich (low FFA) or poor (high FFA) in carbohydrate, 2 h before performing 20 min of two-legged knee extensor exercise. Vastus lateralis biopsies were obtained before and after exercise. RESULTS PDK4 protein content was ∼2.2- and ∼1.5-fold higher in LOW than CON leg in high FFA and low FFA, respectively, and the PDK4 protein content in the CON leg was approximately twofold higher in high FFA than in low FFA. In all conditions, exercise increased PDHa (PDH in the active form) activity, resulting in similar levels in LOW leg in both trials and CON leg in high FFA, but higher level in CON leg in low FFA. PDHa activity was closely associated with the PDH-E1α phosphorylation level. CONCLUSIONS Muscle glycogen and plasma FFA attenuate exercise-induced PDH regulation in human skeletal muscle in a nonadditive manner. This might be through regulation of PDK4 expression. The activation of PDH by exercise independent of changes in muscle glycogen or plasma FFA suggests that exercise overrules FFA-mediated inhibition of PDH (i.e., carbohydrate oxidation), and this may thus be one mechanism behind the health-promoting effects of exercise. Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received July 15, 2009. Accepted October 2, 2009. © 2010 American Diabetes Association

We have previously shown that overnight fasted women have higher insulin-stimulated whole body and leg glucose uptake despite a higher intramyocellular triacylglycerol concentration than men. Women also express higher muscle mRNA levels of proteins related to lipid metabolism than men. We therefore hypothesized that women would be less prone to lipid-induced insulin resistance. Insulin sensitivity of whole-body and leg glucose disposal was studied in 16 young well-matched healthy men and women infused with intralipid or saline for 7 h. Muscle biopsies were obtained before and during a euglycemic-hyperinsulinemic clamp (1.42 mU · kg⁻¹ · min⁻¹). Intralipid infusion reduced whole-body glucose infusion rate by 26% in women and 38% in men (P < 0.05), and insulin-stimulated leg glucose uptake was reduced significantly less in women (45%) than men (60%) after intralipid infusion. Hepatic glucose production was decreased during the clamp similarly in women and men irrespective of intralipid infusion. Intralipid did not impair insulin or AMPK signaling in muscle and subcutaneous fat, did not cause accumulation of muscle lipid intermediates, and did not impair insulin-stimulated glycogen synthase activity in muscle or increase plasma concentrations of inflammatory cytokines. In vitro glucose transport in giant sarcolemmal vesicles was not decreased by acute exposure to fatty acids. Leg lactate release was increased and respiratory exchange ratio was decreased by intralipid. Intralipid infusion causes less insulin resistance of muscle glucose uptake in women than in men. This insulin resistance is not due to decreased canonical insulin signaling, accumulation of lipid intermediates, inflammation, or direct inhibition of GLUT activity. Rather, a higher leg lactate release and lower glucose oxidation with intralipid infusion may suggest a metabolic feedback regulation of glucose metabolism.

In the Greenlandic Inuit population, 4% are homozygous carriers of a genetic nonsense TBC1D4 p.Arg684Ter variant leading to loss of the muscle-specific isoform of TBC1D4 and an approximately tenfold increased risk of type 2 diabetes 1 . Here we show the metabolic consequences of this variant in four female and four male homozygous carriers and matched controls. An extended glucose tolerance test reveals prolonged hyperglycaemia followed by reactive hypoglycaemia in the carriers. Whole-body glucose disposal is impaired during euglycaemic-hyperinsulinaemic clamp conditions and associates with severe insulin resistance in skeletal muscle only. Notably, a marked reduction in muscle glucose transporter GLUT4 and associated proteins is observed. While metabolic regulation during exercise remains normal, the insulin-sensitizing effect of a single exercise bout is compromised. Thus, loss of the muscle-specific isoform of TBC1D4 causes severe skeletal muscle insulin resistance without baseline hyperinsulinaemia. However, physical activity can ameliorate this condition. These observations offer avenues for personalized interventions and targeted preventive strategies. In Greenlandic Inuit, a TBC1D4 loss-of-function mutation increases type 2 diabetes risk by tenfold. Carriers show severe muscle insulin resistance, impaired glucose disposal and reduced muscle GLUT4, yet exercise mitigates these defects, offering potential for personalized lifestyle interventions.

To investigate pyruvate dehydrogenase (PDH)-E1α sub-unit phosphorylation and whether free fatty acids (FFAs) regulate PDH activity, seven subjects completed two trials: saline (control) and intralipid/heparin (intralipid). Each infusion trial consisted of a 4-h rest followed by a 3-h two-legged knee extensor exercise at moderate intensity. During the 4-h resting period, activity of PDH in the active form (PDHa) did not change in either trial, yet phosphorylation of PDH-E1α site 1 (PDH-P1) and site 2 (PDH-P2) was elevated in the intralipid compared with the control trial. PDHa activity increased during exercise similarly in the two trials. After 3 h of exercise, PDHa activity remained elevated in the intralipid trial but returned to resting levels in the control trial. Accordingly, in both trials PDH-P1 and PDH-P2 decreased during exercise, and the decrease was more marked during intralipid infusion. Phosphorylation had returned to resting levels at 3 h of exercise only in the control trial. Thus, an inverse association between PDH-E1α phosphorylation and PDHa activity exists. Short-term elevation in plasma FFA at rest increases PDH-E1α phosphorylation, but exercise overrules this effect of FFA on PDH-E1α phosphorylation leading to even greater dephosphorylation during exercise with intralipid infusion than with saline. Diabetes 55:3020-3027, 2006