Impact of skeletal muscle IL-6 on subcutaneous and visceral adipose tissue metabolism immediately after high- and moderate-intensity exercises.

White adipose tissue is a major energy reserve for the body and is essential for providing fatty acids for other tissues when needed. Skeletal muscle interleukin-6 (IL-6) has been shown to be secreted from the working muscle and has been suggested to signal to adipose tissue and enhance lipolysis. The aim of the present study was to investigate the role of skeletal muscle IL-6 in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) lipolysis and glyceroneogenesis with prolonged moderate-intensity exercise and high-intensity exercise in mice. Female inducible muscle-specific IL-6 knockout (IL-6 iMKO) mice and littermate control (Floxed) mice performed a single exercise bout for either 120 min at 16 m/min and 10° slope (moderate intensity) or 30 min at 20 m/min and 10° slope (high intensity), or they remained rested (rest). Visceral and subcutaneous adipose tissues, quadriceps muscles, and blood were quickly obtained. Plasma IL-6 increased in Floxed mice but not in IL-6 iMKO mice with high-intensity exercise. VAT signal transducer and activator of transcription (STAT)3Tyr705 phosphorylation was lower, and VAT hormone-sensitive lipase (HSL)Ser563 phosphorylation was higher in IL-6 iMKO mice than in Floxed mice at rest. Furthermore, HSLSer563 and HSLSer660 phosphorylation increased in VAT and phosphoenolpyruvate carboxykinase protein decreased in SAT with moderate-intensity exercise in both genotypes. On the other hand, both exercise protocols increased pyruvate dehydrogenaseSer232 phosphorylation in VAT only in IL-6 iMKO mice and decreased tumor necrosis factor-α messenger RNA in SAT and VAT only in Floxed mice. In conclusion, the present findings suggest that skeletal muscle IL-6 regulates markers of lipolysis in VAT in the basal state and pyruvate availability for glyceroneogenesis in VAT with exercise. Moreover, skeletal muscle IL-6 may contribute to exercise-induced anti-inflammatory effects in SAT and VAT.

Impact of skeletal muscle IL-6 on regulation of liver and adipose tissue metabolism during fasting.

The liver and adipose tissue are important tissues in whole-body metabolic regulation during fasting. Interleukin 6 (IL-6) is a cytokine shown to be secreted from contracting muscle in humans and suggested to signal to the liver and adipose tissue. Furthermore, skeletal muscle IL-6 has been proposed to play a role during fasting. Therefore the aim of the present study was to investigate the role of skeletal muscle IL-6 in the regulation of substrate production in the liver and adipose tissue during fasting. Male skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and littermate floxed (control) mice fasted for 6 or 18 h (6 h fasting or 18 h fasting) with corresponding fed control groups (6 h fed or 18 h fed) and liver and adipose tissue were quickly obtained. Plasma ß-hydroxybutyrate increased and hepatic glucose, lactate and glycogen decreased with fasting. In addition, fasting increased phosphoenolpyruvate carboxykinase protein and phosphorylation of pyruvate dehydrogenase (PDH) in the liver as well as hormone-sensitive lipase (HSL)Ser660 and HSLSer563 phosphorylation, PDH phosphorylation, adipose triglyceride lipase phosphorylation and perilipin phosphorylation and protein content in adipose tissue without any effect of lack of skeletal muscle IL-6. In conclusion, fasting induced regulation of enzymes in adipose tissue lipolysis and glyceroneogenesis as well as regulation of hepatic gluconeogenic capacity and hepatic substrate utilization in mice. However, skeletal muscle IL-6 was not required for these fasting-induced effects, but had minor effects on markers of lipolysis and glyceroneogenesis in adipose tissue as well as markers of hepatic gluconeogenesis in the fed state.

Training state and skeletal muscle autophagy in response to 36 h of fasting.

The present study aimed at investigating fasting-induced responses in regulators and markers of autophagy in vastus lateralis muscle from untrained and trained human subjects. Untrained and trained subjects (based on maximum oxygen uptake, muscle citrate synthase activity, and oxidative phosphorylation protein level) fasted for 36 h with vastus lateralis muscle biopsies obtained at 2, 12, 24, and 36 h after a standardized meal. Fasting reduced ( P < 0.05) skeletal muscle microtubule-associated protein-1A/1B light chain 3 (LC3)I, LC3II, and adaptor protein sequestosome 1/p62 protein content in untrained subjects only. Moreover, skeletal muscle RAC-alpha serine/threonine-protein kinase (AKT)Thr308, AMP-activated protein kinase (AMPK)Thr172, and Unc-51-like autophagy-activating kinase-1 (ULK1)Ser555 phosphorylation state, as well as Bcl-2-interacting coiled-coil protein-1 (Beclin1) and ULK1Ser757 phosphorylation, was lower ( P < 0.05) in trained than untrained subjects during fasting. In addition, the plasma concentrations of several amino acids were higher ( P < 0.05) in trained than untrained subjects, and the plasma concentration profile of several amino acids was different in untrained and trained subjects during fasting. Taken together, these findings suggest that 36-h fasting has effects on some mediators of autophagy in untrained human skeletal muscle and that training state influences the effect of fasting on autophagy signaling and on mediators of autophagy in skeletal muscle. NEW & NOTEWORTHY This study showed that skeletal muscle autophagy was only modestly affected in humans by 36 h of fasting. Hence, 36-h fasting has effects on some mediators of autophagy in untrained human skeletal muscle, and training state influences the effect of fasting on autophagy signaling and on mediators of autophagy in skeletal muscle.

Training state and fasting-induced PDH regulation in human skeletal muscle.

The aim of the present study was to examine the influence of training state on fasting-induced skeletal muscle pyruvate dehydrogenase (PDH) regulation, including PDH phosphorylation. Trained and untrained subjects, matched for skeletal muscle CS activity and OXPHOS protein, fasted for 36 h after receiving a standardized meal. Respiratory exchange ratio (RER) was measured and blood as well as vastus lateralis muscle biopsies were obtained 2, 12, 24, and 36 h after the meal. RER decreased with fasting only in untrained individuals, while PDHa activity decreased from 12 h after the meal in untrained, but only tended to decrease at 36 h in trained. PDH-E1α, PDP1 protein, PDH phosphorylation, and PDH acetylation in skeletal muscle was higher in trained than untrained subjects, but did not change with fasting, while PDK4 protein was higher at 36 h than at 2 h after the meal in both groups. In conclusion, the present results suggest that endurance exercise training modifies the fasting-induced regulation of PDHa activity in skeletal muscle and the substrate switch towards fat oxidation. PDH phosphorylation could not explain the fasting-induced regulation of PDHa activity suggesting other post translational modifications.

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 Cyp's 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 Cyp's, 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.

Impact of fasting followed by short-term exposure to interleukin-6 on cytochrome P450 mRNA in mice.

The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification.

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.

Effects of training status on PDH regulation in human skeletal muscle during exercise.

Pyruvate dehydrogenase (PDH) is the gateway enzyme for carbohydrate-derived pyruvate feeding into the TCA cycle. PDH may play a central role in regulating substrate shifts during exercise, but the influence of training state on PDH regulation during exercise is not fully elucidated. The purpose of this study was to investigate the impact of training state on post-translational regulation of PDHa activity during submaximal and exhaustive exercise. Eight untrained and nine endurance exercise-trained healthy male subjects performed incremental exercise on a cycle ergometer: 40 min at 50% incremental peak power output (IPPO), 10 min at 65% (IPPO), followed by 80% (IPPO) until exhaustion. Trained subjects had higher (P < 0.05) PDH-E1α, PDK1, PDK2, PDK4, and PDP1 protein content as well as PDH phosphorylation and PDH acetylation. Exercising at the same relative intensity led to similar muscle PDH activation in untrained and trained subjects, whereas PDHa activity at exhaustion was higher (P < 0.05) in trained than untrained. Furthermore, exercise induced similar PDH dephosphorylation in untrained and trained subjects, while PDH acetylation was increased (P < 0.05) only in trained subjects. In conclusion, PDHa activity and PDH dephosphorylation were well adjusted to the relative exercise intensity during submaximal exercise. In addition, higher PDHa activity in trained than untrained at exhaustion seemed related to differences in glycogen utilization rather than differences in PDH phosphorylation and acetylation state, although site-specific contributions cannot be ruled out.

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.

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.

Skeletal muscle IL-6 and regulation of liver metabolism during high-fat diet and exercise training.

Interleukin (IL)-6 is released from skeletal muscle (SkM) during exercise and has been shown to affect hepatic metabolism. It is, however, unknown whether SkM IL-6 is involved in the regulation of exercise training-induced counteraction of changes in carbohydrate and lipid metabolism in the liver in response to high-fat diet (HFD) feeding. Male SkM-specific IL-6 KO (MKO) and Floxed mice were subjected to Chow diet, HFD or HFD combined with exercise training (HFD ExTr) for 16 weeks. Hepatic phosphoenolpyruvate carboxykinase (PEPCK) protein content decreased with both HFD and HFD ExTr in Floxed mice, but increased in IL-6 MKO mice on HFD In addition, the intrahepatic glucose concentration was in IL-6 MKO mice higher in HFD than chow. Within HFD ExTr mice, hepatic glucose-6-phosphatase (G6Pase) 36 kDa protein content was higher in IL-6 MKO than Floxed mice. Hepatic pyruvate dehydrogenase kinase (PDK) 4 and PDK2 protein content was in Floxed mice lower in HFD ExTr than Chow. In addition, hepatic ACC1-phosphorylation was higher and ACC1 protein lower in HFD Together this suggests that SkM IL-6 regulates hepatic glucose metabolism, but does not seem to be of major importance for the regulation of oxidative capacity or lipogenesis in liver during HFD or HFD combined with exercise training.

Skeletal muscle interleukin-6 regulates metabolic factors in iWAT during HFD and exercise training.

OBJECTIVE: To investigate the role of skeletal muscle (SkM) interleukin (IL)-6 in the regulation of adipose tissue metabolism. METHODS: Muscle-specific IL-6 knockout (IL-6 MKO) and IL-6(loxP/loxP) (Floxed) mice were subjected to standard rodent diet (Chow), high-fat diet (HFD), or HFD in combination with exercise training (HFD ExTr) for 16 weeks. RESULTS: Total fat mass increased (P < 0.05) in both genotypes with HFD. However, HFD IL-6 MKO mice had lower (P < 0.05) inguinal adipose tissue (iWAT) mass than HFD Floxed mice. Accordingly, iWAT glucose transporter 4 (GLUT4) protein content, 5'AMP activated protein kinase (AMPK)(Thr172) phosphorylation, and fatty acid synthase (FAS) mRNA content were lower (P < 0.05) in IL-6 MKO than Floxed mice on Chow. In addition, iWAT AMPK(Thr172) and hormone-sensitive lipase (HSL)(Ser565) phosphorylation as well as perilipin protein content was higher (P < 0.05) in HFD IL-6 MKO than HFD Floxed mice, and pyruvate dehydrogenase E1α (PDH-E1α) protein content was higher (P < 0.05) in HFD ExTr IL-6 MKO than HFD ExTr Floxed mice. CONCLUSIONS: These findings indicate that SkM IL-6 affects iWAT mass through regulation of glucose uptake capacity as well as lipogenic and lipolytic factors.