Anders Gudiksen

Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock

Circadian rhythms control metabolism and energy homeostasis, but the role of the skeletal muscle clock has never been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.

Erratum to “Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock” [Mol Metab 3 (2014) 29–41]

[This corrects the article DOI: 10.1016/j.molmet.2013.10.005.].

Lack of Skeletal Muscle IL-6 Affects Pyruvate Dehydrogenase Activity at Rest and during Prolonged Exercise

Pyruvate dehydrogenase (PDH) plays a key role in the regulation of skeletal muscle substrate utilization. IL-6 is produced in skeletal muscle during exercise in a duration dependent manner and has been reported to increase whole body fatty acid oxidation, muscle glucose uptake and decrease PDHa activity in skeletal muscle of fed mice. The aim of the present study was to examine whether muscle IL-6 contributes to exercise-induced PDH regulation in skeletal muscle. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) completed a single bout of treadmill exercise for 10, 60 or 120 min, with rested mice of each genotype serving as basal controls. The respiratory exchange ratio (RER) was overall higher (P<0.05) in IL-6 MKO than control mice during the 120 min of treadmill exercise, while RER decreased during exercise independent of genotype. AMPK and ACC phosphorylation also increased with exercise independent of genotype. PDHa activity was in control mice higher (P<0.05) at 10 and 60 min of exercise than at rest but remained unchanged in IL-6 MKO mice. In addition, PDHa activity was higher (P<0.05) in IL-6 MKO than control mice at rest and 60 min of exercise. Neither PDH phosphorylation nor acetylation could explain the genotype differences in PDHa activity. Together, this provides evidence that skeletal muscle IL-6 contributes to the regulation of PDH at rest and during prolonged exercise and suggests that muscle IL-6 normally dampens carbohydrate utilization during prolonged exercise via effects on PDH.

Impact of β-adrenergic signaling in PGC-1α-mediated adaptations in mouse skeletal muscle

PGC-1α has been suggested to regulate exercise training-induced metabolic adaptations and autophagy in skeletal muscle. The factors regulating PGC-1α, however, have not been fully resolved. The aim was to investigate the impact of β-adrenergic signaling in PGC-1α-mediated metabolic adaptations in skeletal muscle with exercise training. Muscle was obtained from muscle-specific PGC-1α knockout (MKO) and lox/lox mice 1) 3 h after a single exercise bout with or without prior injection of propranolol or 3 h after a single injection of clenbuterol and 2) after 5 wk of wheel running exercise training with or without propranolol treatment or after 5 wk of clenbuterol treatment. A single clenbuterol injection and an acute exercise bout similarly increased the mRNA content of both N-terminal and full-length PGC-1α isoforms, and prior propranolol treatment reduced the exercise-induced increase in mRNA of all isoforms. Furthermore, a single clenbuterol injection elicited a PGC-1α-dependent increase in cytochrome c and vascular endothelial growth factor mRNA, whereas prolonged clenbuterol treatment increased fiber size but reduced capillary density. Exercise training increased the protein content of OXPHOS, LC3I, and Parkin in a PGC-1α-dependent manner without effect of propranolol, while an exercise training-induced increase in Akt2 and p62 protein required PGC-1α and was blunted by prolonged propranolol treatment. This suggests that β-adrenergic signaling is not required for PGC-1α-mediated exercise training-induced adaptations in mitochondrial proteins, but contributes to exercise training-mediated adaptations in insulin signaling and autophagy regulation through PGC-1α. Furthermore, changes observed with acute stimulation of compounds like clenbuterol and propranolol may not lead to corresponding adaptations with prolonged treatment.

PGC‐1α and fasting‐induced PDH regulation in mouse skeletal muscle

The purpose of the present study was to examine whether lack of skeletal muscle peroxisome proliferator‐activated receptor gamma coactivator 1 alpha (PGC‐1α) affects the switch in substrate utilization from a fed to fasted state and the fasting‐induced pyruvate dehydrogenase (PDH) regulation in skeletal muscle. Skeletal muscle‐specific PGC‐1α knockout (MKO) mice and floxed littermate controls were fed or fasted for 24 h. Fasting reduced PDHa activity, increased phosphorylation of all four known sites on PDH‐E1α and increased pyruvate dehydrogenase kinase (PDK4) and sirtuin 3 (SIRT3) protein levels, but did not alter total acetylation of PDH‐E1α. Lack of muscle PGC‐1α did not affect the switch from glucose to fat oxidation in the transition from the fed to fasted state, but was associated with lower and higher respiratory exchange ratio (RER) in the fed and fasted state, respectively. PGC‐1α MKO mice had lower skeletal muscle PDH‐E1α, PDK1, 2, 4, and pyruvate dehydrogenase phosphatase (PDP1) protein content than controls, but this did not prevent the fasting‐induced increase in PDH‐E1α phosphorylation in PGC‐1α MKO mice. However, lack of skeletal muscle PGC‐1α reduced SIRT3 protein content, increased total lysine PDH‐E1α acetylation in the fed state, and prevented a fasting‐induced increase in SIRT3 protein. In conclusion, skeletal muscle PGC‐1α is required for fasting‐induced upregulation of skeletal muscle SIRT3 and maintaining high fat oxidation in the fasted state, but is dispensable for preserving the capability to switch substrate during the transition from the fed to the fasted state and for fasting‐induced PDH regulation in skeletal muscle.

Lack of skeletal muscle IL-6 influences hepatic glucose metabolism in mice during prolonged exercise

The liver is essential in maintaining and regulating glucose homeostasis during prolonged exercise. IL-6 has been shown to be secreted from skeletal muscle during exercise and has been suggested to signal to the liver. Therefore, the aim of this study was to investigate the role of skeletal muscle IL-6 on hepatic glucose regulation and substrate choice during prolonged exercise. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice (age, 12-14 wk) and littermate lox/lox (Control) mice were either rested (Rest) or completed a single bout of exercise for 10, 60, or 120 min, and the liver was quickly obtained. Hepatic IL-6 mRNA was higher at 60 min of exercise, and hepatic signal transducer and activator of transcription 3 was higher at 120 min of exercise than at rest in both genotypes. Hepatic glycogen was higher in IL-6 MKO mice than control mice at rest, but decreased similarly during exercise in the two genotypes, and hepatic glucose content was lower in IL-6 MKO than control mice at 120 min of exercise. Hepatic phosphoenolpyruvate carboxykinase mRNA and protein increased in both genotypes at 120 min of exercise, whereas hepatic glucose 6 phosphatase protein remained unchanged. Furthermore, IL-6 MKO mice had higher hepatic pyruvate dehydrogenase (PDH)Ser232 and PDHSer300 phosphorylation than control mice at rest. In conclusion, hepatic gluconeogenic capacity in mice is increased during prolonged exercise independent of muscle IL-6. Furthermore, Skeletal muscle IL-6 influences hepatic substrate regulation at rest and hepatic glucose metabolism during prolonged exercise, seemingly independent of IL-6 signaling in the liver.

Muscle interleukin 6 and fasting-induced PDH regulation in mouse skeletal muscle

Fasting prompts a metabolic shift in substrate utilization from carbohydrate to predominant fat oxidation in skeletal muscle, and pyruvate dehydrogenase (PDH) is seen as a controlling link between the competitive oxidation of carbohydrate and fat during metabolic challenges like fasting. Interleukin (IL)-6 has been proposed to be released from muscle with concomitant effects on both glucose and fat utilization. The aim was to test the hypothesis that muscle IL-6 has a regulatory impact on fasting-induced suppression of skeletal muscle PDH. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) were either fed or fasted for 6 or 18 h. Lack of muscle IL-6 elevated the respiratory exchange ratio in the fed and early fasting state, but not with prolonged fasting. Activity of PDH in the active form (PDHa) was higher in fed and fasted IL-6 MKO than in control mice at 18 h, but not at 6 h, whereas lack of muscle IL-6 did not prevent downregulation of PDHa activity in skeletal muscle or changes in plasma and muscle substrate levels in response to 18 h of fasting. Phosphorylation of three of four sites on PDH-E1α increased with 18 h of fasting, but was lower in IL-6 MKO mice than in control. In addition, both PDK4 mRNA and protein increased with 6 and 18 h of fasting in both genotypes, but PDK4 protein was lower in IL-6 MKO than in control. In conclusion, skeletal muscle IL-6 seems to regulate whole body substrate utilization in the fed, but not fasted, state and influence skeletal muscle PDHa activity in a circadian manner. However, skeletal muscle IL-6 is not required for maintaining metabolic flexibility in response to fasting.

Impact of training state on fasting-induced regulation of adipose tissue metabolism in humans

Recruitment of fatty acids from adipose tissue is increased during fasting. However, the molecular mechanisms behind fasting-induced metabolic regulation in human adipose tissue and the potential impact of training state in this are unknown. Therefore the aim of the present study was to investigate 1) fasting-induced regulation of lipolysis and glyceroneogenesis in human adipose tissue as well as 2) the impact of training state on basal oxidative capacity and fasting-induced metabolic regulation in human adipose tissue. Untrained [maximal oxygen uptake (V̇o2max) < 45 ml·min-1·kg-1] and trained subjects (V̇o2max > 55 ml·min-1·kg-1) fasted for 36 h, and abdominal subcutaneous adipose tissue biopsies were obtained 2, 12, 24, and 36 h after a standardized meal. Adipose tissue oxidative phosphorylation complexes, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase (PDH)-E1α protein as well as PDH kinase (PDK) 2, PDK4, and PDH phosphatase 2 mRNA content were higher in trained subjects than in untrained subjects. In addition, trained subjects had higher adipose tissue hormone-sensitive lipase Ser660 phosphorylation and adipose triglyceride lipase protein content as well as higher plasma free fatty acid concentration than untrained subjects during fasting. Moreover, adipose tissue PDH phosphorylation increased with fasting only in trained subjects. Taken together, trained subjects seem to possess higher basal adipose tissue oxidative capacity as well as higher capacity for regulation of lipolysis and for providing substrate for glyceroneogenesis in adipose tissue during fasting than untrained subjects. NEW & NOTEWORTHY This study shows for the first time higher protein content of lipolytic enzymes and higher oxidative phosphorylation protein in adipose tissue from trained subjects than from untrained subjects during fasting. Furthermore, trained subjects had higher capacity for adipose tissue glyceroneogenesis than untrained subjects.

Skeletal Muscle Interleukin-6 Regulates Hepatic Cytochrome P450 Expression: Effects of 16-Week High-Fat Diet and Exercise

High-fat diet (HFD) induces several changes to the pathways regulating energy homeostasis and changes the expression of the hepatic cytochrome p450 (Cyp) enzyme-system. Despite these pervious findings, it is still unclear how the effects of HFD and especially HFD in combination with treadmill running affect hepatic Cyp expression. In this study, we investigated the mRNA and protein expression of selected Cyps in mice subjected to 16 weeks of HFD and treadmill running. To understand the regulatory mechanisms behind the exercise-induced reversion of the HFD-induced changes in Cyp expression, we used a model in which the exercise-induced myokine and known regulator of hepatic Cyps, interleukin-6 (IL-6), were knocked out specifically in skeletal muscle. We found that HFD increased the mRNA expression of Cyp1a1 and Cyp4a10, and decreased the expression of Cyp2a4, Cyp2b10, Cyp2e1, and Cyp3a11. HFD in combination with treadmill running reversed the HFD increase in Cyp4a10 mRNA expression. In addition, we observed increased Cyp1a and Cyp3a protein expression as an effect of exercise, whereas Cyp2b expression was lowered as an effect of HFD. IL-6 effected the response in Cyp3a11 and Cyp1a expression. We observed no changes in the content of the aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, or peroxisome proliferation activator receptor alpha. In conclusion, we show that both HFD and exercise in HFD-fed animals can regulate hepatic Cyp expression and that changes in Cyp3a in response to HFD and exercise are dependent on skeletal muscular IL-6.

Skeletal muscle IL-6 regulates muscle substrate utilization and adipose tissue metabolism during recovery from an acute bout of exercise.

An acute bout of exercise imposes a major challenge on whole-body metabolism and metabolic adjustments are needed in multiple tissues during recovery to reestablish metabolic homeostasis. It is currently unresolved how this regulation is orchestrated between tissues. This study was undertaken to clarify the role of skeletal muscle derived interleukin 6 (IL-6) in the coordination of the metabolic responses during recovery from acute exercise. Skeletal muscle specific IL-6 knockout (IL-6 MKO) and littermate Control mice were rested or ran on a treadmill for 2h. Plasma, skeletal muscle, liver and adipose tissue were obtained after 6 and 10h of recovery. Non-exercised IL-6 MKO mice had higher plasma lactate and lower plasma non-esterified fatty acids than Controls. The activity of pyruvate dehydrogenase in the active form was, in skeletal muscle, higher in IL-6 MKO mice than Controls in non-exercised mice and 6h after exercise. IL-6 MKO mice had lower glucose transporter 4 protein content in inguinal adipose tissue (WAT) than Control in non-exercised mice and 10h after treadmill running. Epididymal WAT hormone sensitive lipase phosphorylation and inguinal WAT mitogen activated kinase P38 phosphorylation were higher in IL-6 MKO than Control mice 6h after exercise. These findings indicate that skeletal muscle IL-6 may play an important role in the regulation of substrate utilization in skeletal muscle, basal and exercise-induced adaptations in adipose tissue glucose uptake and lipolysis during recovery from exercise. Together this indicates that skeletal muscle IL-6 contributes to reestablishing metabolic homeostasis during recovery from exercise by regulating WAT and skeletal muscle metabolism.