Redox state and altered pyruvate metabolism contribute to a dose-dependent metformin-induced lactate production of human myotubes.

Metformin-induced glycolysis and lactate production can lead to acidosis as a life-threatening side effect, but slight increases in blood lactate levels in a physiological range were also reported in metformin-treated patients. However, how metformin increases systemic lactate concentrations is only partly understood. Because human skeletal muscle has a high capacity to produce lactate, the aim was to elucidate the dose-dependent regulation of metformin-induced lactate production and the potential contribution of skeletal muscle to blood lactate levels under metformin treatment. This was examined by using metformin treatment (16-776 µM) of primary human myotubes and by 17 days of metformin treatment in humans. As from 78 µM, metformin induced lactate production and secretion and glucose consumption. Investigating the cellular redox state by mitochondrial respirometry, we found metformin to inhibit the respiratory chain complex I (776 µM, P < 0.01) along with decreasing the [NAD+]:[NADH] ratio (776 µM, P < 0.001). RNA sequencing and phospho-immunoblot data indicate inhibition of pyruvate oxidation mediated through phosphorylation of the pyruvate dehydrogenase (PDH) complex (39 µM, P < 0.01). On the other hand, in human skeletal muscle, phosphorylation of PDH was not altered by metformin. Nonetheless, blood lactate levels were increased under metformin treatment (P < 0.05). In conclusion, the findings suggest that metformin-induced inhibition of pyruvate oxidation combined with altered cellular redox state shifts the equilibrium of the lactate dehydrogenase (LDH) reaction leading to a dose-dependent lactate production in primary human myotubes.NEW & NOTEWORTHY Metformin shifts the equilibrium of lactate dehydrogenase (LDH) reaction by low dose-induced phosphorylation of pyruvate dehydrogenase (PDH) resulting in inhibition of pyruvate oxidation and high dose-induced increase in NADH, which explains the dose-dependent lactate production of differentiated human skeletal muscle cells.

Colchicine treatment impairs skeletal muscle mitochondrial function and insulin sensitivity in an age-specific manner.

The aim of the study was to investigate the impact of autophagy inhibition on skeletal muscle mitochondrial function and glucose homeostasis in young and aged mice. The transcriptional co-activator PGC-1α regulates muscle oxidative phenotype which has been shown to be linked with basal autophagic capacity. Therefore, young and aged inducible muscle-specific PGC-1α knockout (iMKO) mice and littermate lox/lox controls were used in three separate experiments performed after either saline or colchicine injections on two consecutive days: (1) Euthanization in the basal state obtaining skeletal muscle for mitochondrial respirometry, (2) whole body glucose tolerance test, and (3) in vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake into skeletal muscle. Muscle PGC-1α was not required for maintaining basal autophagy flux, regardless of age. Colchicine-induced inhibition of autophagy was associated with impairments of skeletal muscle mitochondrial function, including reduced ADP sensitivity and altered mitochondrial redox balance in both young and aged mice. Colchicine treatment reduced the glucose tolerance in aged, but not young mice, and similarly in iMKO and lox/lox mice. Colchicine reduced insulin-stimulated 2-DG uptake in soleus muscle in aged mice, independently of PGC-1α, and without affecting insulin-regulated phosphorylation of proximal or distal mediators of insulin signaling. In conclusion, the results indicate that autophagy regulates the mitochondrial ADP sensitivity and redox balance as well as whole body glucose tolerance and skeletal muscle insulin sensitivity in aged mice, with no additional effects of inducible PGC-1α deletion.

Insulin resistance induced by growth hormone is linked to lipolysis and associated with suppressed pyruvate dehydrogenase activity in skeletal muscle: a 2 × 2 factorial, randomised, crossover study in human individuals.

AIMS/HYPOTHESIS: Growth hormone (GH) causes insulin resistance that is linked to lipolysis, but the underlying mechanisms are unclear. We investigated if GH-induced insulin resistance in skeletal muscle involves accumulation of diacylglycerol (DAG) and ceramide as well as impaired insulin signalling, or substrate competition between fatty acids and glucose. METHODS: Nine GH-deficient male participants were randomised and examined in a 2 × 2 factorial design with and without administration of GH and acipimox (an anti-lipolytic compound). As-treated analyses were performed, wherefore data from three visits from two patients were excluded due to incorrect GH administration. The primary outcome was insulin sensitivity, expressed as the AUC of the glucose infusion rate (GIRAUC), and furthermore, the levels of DAGs and ceramides, insulin signalling and the activity of the active form of pyruvate dehydrogenase (PDHa) were assessed in skeletal muscle biopsies obtained in the basal state and during a hyperinsulinaemic-euglycaemic clamp (HEC). RESULTS: Co-administration of acipimox completely suppressed the GH-induced elevation in serum levels of NEFA (GH versus GH+acipimox, p < 0.0001) and abrogated GH-induced insulin resistance (mean GIRAUC [95% CI] [mg min-1 kg-1] during the HEC: control, 595 [493, 718]; GH, 468 [382, 573]; GH+acipimox, 654 [539, 794]; acipimox, 754 [618, 921]; GH vs GH+acipimox: p = 0.004). GH did not significantly change either the accumulation of DAGs and ceramides or insulin signalling in skeletal muscle, but GH antagonised the insulin-stimulated increase in PDHa activity (mean ± SEM [% from the basal state to the HEC]: control, 47 ± 19; GH, -15 ± 21; GH+acipimox, 3 ± 21; acipimox, 57 ± 22; main effect: p = 0.02). CONCLUSIONS/INTERPRETATION: GH-induced insulin resistance in skeletal muscle is: (1) causally linked to lipolysis; (2) not associated with either accumulation of DAGs and ceramides or impaired insulin signalling; (3) likely to involve substrate competition between glucose and lipid intermediates. TRIAL REGISTRATION: ClinicalTrials.gov NCT02782208 FUNDING: The work was supported by the Grant for Growth Innovation (GGI), which was funded by Merck KGaA, Darmstadt, Germany. Graphical abstract.

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.

PGC-1α regulates mitochondrial properties beyond biogenesis with aging and exercise training.

Impaired mitochondrial function has been implicated in the pathogenesis of age-associated metabolic diseases through regulation of cellular redox balance. Exercise training is known to promote mitochondrial biogenesis in part through induction of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Recently, mitochondrial ADP sensitivity has been linked to reactive oxygen species (ROS) emission with potential impact on age-associated physiological outcomes, but the underlying molecular mechanisms remain unclear. Therefore, the present study investigated the effects of aging and exercise training on mitochondrial properties beyond biogenesis, including respiratory capacity, ADP sensitivity, ROS emission, and mitochondrial network structure, in myofibers from inducible muscle-specific PGC-1α-knockout mice and control mice. Aged mice displayed lower running endurance and mitochondrial respiratory capacity than young mice. This was associated with intermyofibrillar mitochondrial network fragmentation, diminished submaximal ADP-stimulated respiration, increased mitochondrial ROS emission, and oxidative stress. Exercise training reversed the decline in maximal respiratory capacity independent of PGC-1α, whereas exercise training rescued the age-related mitochondrial network fragmentation and the impaired submaximal ADP-stimulated respiration in a PGC-1α-dependent manner. Furthermore, lack of PGC-1α was associated with altered phosphorylation and carbonylation of the inner mitochondrial membrane ADP/ATP exchanger adenine nucleotide translocase 1. In conclusion, the present study provides evidence that PGC-1α regulates submaximal ADP-stimulated respiration, ROS emission, and mitochondrial network structure in mouse skeletal muscle during aging and exercise training.

Prior exercise training improves cold tolerance independent of indices associated with non-shivering thermogenesis.

KEY POINTS: Mammals defend against cold-induced reductions in body temperature through both shivering and non-shivering thermogenesis. The activation of non-shivering thermogenesis is primarily driven by uncoupling protein-1 in brown adipose tissue and to a lesser degree by the browning of white adipose tissue. Endurance exercise has also been shown to increase markers of white adipose tissue browning. This study aimed to determine whether prior exercise training would alter the response to a cold challenge and if this would be associated with differences in indices of non-shivering thermogenesis. It is shown that exercise training protects against cold-induced weight loss by increasing food intake. Exercise-trained mice were better able to maintain their core temperature, independent of differences in markers of non-shivering thermogenesis. ABSTRACT: Shivering is one of the first defences against cold, and as skeletal muscle fatigues there is an increased reliance on non-shivering thermogenesis. Brown and beige adipose tissues are the primary thermogenic tissues regulating this process. Exercise has also been shown to increase the thermogenic capacity of subcutaneous white adipose tissue. Whether exercise has an effect on the adaptations to cold stress within adipose tissue and skeletal muscle remains to be shown. Male C57BL/6 mice were either subjected to voluntary wheel running or remained sedentary for 12 days. Exercise led to decreased body weight and increased glucose tolerance. Mice were then divided into groups kept at 25°C room temperature or a cold challenge of 4°C for 48 h. Exercised mice were protected against cold-induced reductions in weight and in parallel with increased food intake. Providing exercised mice with the same amount of food as sedentary mice eliminated the protection against cold-induced weight loss. Cold exposure led to greater reductions in rectal temperature in sedentary compared to exercised mice. This protective effect was not explained by differences in the browning of white adipose tissue or brown adipose tissue mass. Similarly, the ability of the ß3 -adrenergic agonist CL 316,243 to increase energy expenditure was attenuated in previously exercised mice, suggesting that the activation of uncoupling protein-1 in brown and/or beige adipocytes is not the source of protective effects. We speculate that the protection against cold-induced reductions in rectal temperature could potentially be linked to exercise-induced alterations in skeletal muscle.

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 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 ß-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 ß-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 ß-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.

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.

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 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.

AMPKα is essential for acute exercise-induced gene responses but not for exercise training-induced adaptations in mouse skeletal muscle.

Exercise training increases skeletal muscle expression of metabolic proteins improving the oxidative capacity. Adaptations in skeletal muscle by pharmacologically induced activation of 5'-AMP-activated protein kinase (AMPK) are dependent on the AMPKα2 subunit. We hypothesized that exercise training-induced increases in exercise capacity and expression of metabolic proteins, as well as acute exercise-induced gene regulation, would be compromised in muscle-specific AMPKα1 and -α2 double-knockout (mdKO) mice. An acute bout of exercise increased skeletal muscle mRNA content of cytochrome c oxidase subunit I, glucose transporter 4, and VEGF in an AMPK-dependent manner, whereas cluster of differentiation 36 and fatty acid transport protein 1 mRNA content increased similarly in AMPKα wild-type (WT) and mdKO mice. During 4 wk of voluntary running wheel exercise training, the AMPKα mdKO mice ran less than WT. Maximal running speed was lower in AMPKα mdKO than in WT mice but increased similarly in both genotypes with exercise training. Exercise training increased quadriceps protein content of ubiquinol-cytochrome c reductase core protein 1 (UQCRC1), cytochrome c, hexokinase II, plasma membrane fatty acid-binding protein, and citrate synthase activity more in AMPKα WT than in mdKO muscle. However, analysis of a subgroup of mice matched for running distance revealed that only UQCRC1 protein content increased more in WT than in mdKO mice with exercise training. Thus, AMPKα1 and -α2 subunits are important for acute exercise-induced mRNA responses of some genes and may be involved in regulating basal metabolic protein expression but seem to be less important in exercise training-induced adaptations in metabolic proteins.

Muscle PGC-1α modulates hepatic mitophagy regulation during aging.

Aging has been suggested to be associated with changes in oxidative capacity, autophagy, and mitophagy in the liver, but a simultaneous evaluation of these key cellular processes is lacking. Moreover, skeletal muscle transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α has been reported to mediate inter-organ signaling through myokines with regulatory effects in the liver, but the potential role of muscle PGC-1α on hepatic changes with age remains to be resolved. The aim of the present study was therefore to investigate 1) the effect of aging on mitochondrial autophagy and mitophagy capacity in mouse liver and 2) whether muscle PGC-1α is required for maintaining autophagy and mitophagy capacity in the liver during aging. The liver was obtained from young (Young) and aged (Aged) inducible muscle-specific PGC-1α knockout (iMKO) and floxed littermate control mice (Lox). Aging increased liver p62, Parkin and BCL2/adenovirus E1B 19 kDa protein-interacting protein (BNIP)3 protein with no effect of muscle specific PGC-1α knockout, while liver Microtubule-associated protein 1A/1B-light chain 3(LC3) II/I was unchanged with age, but tended to be lower in iMKO mice than in controls. Markers of liver mitochondrial oxidative capacity and oxidative stress were unchanged with age and iMKO. However, Parkin protein levels in isolated liver mitochondria were 2-fold higher in Aged iMKO mice than in Aged controls. In conclusion, aging had no effect on oxidative capacity and lipid peroxidation in the liver. However, aging was associated with increased levels of autophagy and mitophagy markers. Moreover, muscle PGC-1α appears to regulate hepatic mitochondrial translocation of Parkin in aged mice, suggesting that the metabolic capacity of skeletal muscle can modulate mitophagy regulation in the liver during aging.

Impact of Aging and Lifelong Exercise Training on Mitochondrial Function and Network Connectivity in Human Skeletal Muscle.

Aging is associated with metabolic decline in skeletal muscle, which can be delayed by physical activity. Moreover, both lifelong and short-term exercise training have been shown to prevent age-associated fragmentation of the mitochondrial network in mouse skeletal muscle. However, whether lifelong endurance exercise training exerts the same effects in human skeletal muscle is still not clear. Therefore, the aim of the present study was to examine the effect of volume-dependent lifelong endurance exercise training on mitochondrial function and network connectivity in older human skeletal muscle. Skeletal muscle complex I+II-linked mitochondrial respiration per tissue mass was higher, but intrinsic complex I+II-linked mitochondrial respiration was lower in highly trained older subjects than in young untrained, older untrained, and older moderately trained subjects. Mitochondrial volume and connectivity were higher in highly trained older subjects than in untrained and moderately trained older subjects. Furthermore, the protein content of the ADP/ATP exchangers ANT1 + 2 and VDAC was higher and of the mitophagic marker parkin lower in skeletal muscle from the highly trained older subjects than from untrained and moderately trained older subjects. In contrast, H2O2 emission in skeletal muscle was not affected by either age or exercise training, but SOD2 protein content was higher in highly trained older subjects than in untrained and moderately trained older subjects. This suggests that healthy aging does not induce oxidative stress or mitochondrial network fragmentation in human skeletal muscle, but high-volume exercise training increases mitochondrial volume and network connectivity, thereby increasing oxidative capacity in older human skeletal muscle.

TBC1D4-S711 Controls Skeletal Muscle Insulin Sensitization After Exercise and Contraction.

The ability of insulin to stimulate glucose uptake in skeletal muscle is important for whole-body glycemic control. Insulin-stimulated skeletal muscle glucose uptake is improved in the period after a single bout of exercise, and accumulating evidence suggests that phosphorylation of TBC1D4 by the protein kinase AMPK is the primary mechanism responsible for this phenomenon. To investigate this, we generated a TBC1D4 knock-in mouse model with a serine-to-alanine point mutation at residue 711 that is phosphorylated in response to both insulin and AMPK activation. Female TBC1D4-S711A mice exhibited normal growth and eating behavior as well as intact whole-body glycemic control on chow and high-fat diets. Moreover, muscle contraction increased glucose uptake, glycogen utilization, and AMPK activity similarly in wild-type and TBC1D4-S711A mice. In contrast, improvements in whole-body and muscle insulin sensitivity after exercise and contractions were only evident in wild-type mice and occurred concomitantly with enhanced phosphorylation of TBC1D4-S711. These results provide genetic evidence to support that TBC1D4-S711 serves as a major point of convergence for AMPK- and insulin-induced signaling that mediates the insulin-sensitizing effect of exercise and contractions on skeletal muscle glucose uptake.

Ameliorating Effects of Lifelong Physical Activity on Healthy Aging and Mitochondrial Function in Human White Adipose Tissue.

Growing old is patently among the most prominent risk factors for lifestyle-related diseases and deterioration in physical performance. Aging in particular affects mitochondrial homeostasis, and maintaining a well-functioning mitochondrial pool is imperative in order to avoid age-associated metabolic decline. White adipose tissue (WAT) is a key organ in energy balance, and impaired mitochondrial function in adipocytes has been associated with increased low-grade inflammation, altered metabolism, excessive reactive oxygen species (ROS) production, and an accelerated aging phenotype. Exercise training improves mitochondrial health but whether lifelong exercise training can sufficiently maintain WAT mitochondrial function is currently unknown. Therefore, to dissect the role and dose-dependence of lifelong exercise training on aging WAT metabolic parameters and mitochondrial function, young and older untrained, as well as moderately and highly exercise trained older male subjects were recruited and abdominal subcutaneous (s)WAT biopsies and venous blood samples were obtained to measure mitochondrial function and key metabolic factors in WAT and plasma. Mitochondrial intrinsic respiratory capacity was lower in sWAT from older than from young subjects. In spite of this, maximal mitochondrial respiration per wet weight, markers of oxidative capacity, and mitophagic capacity were higher in sWAT from the lifelong highly exercise trained group than all other groups. Furthermore, ROS emission was generally lower in sWAT from lifelong highly exercise trained subjects than older untrained subjects. Taken together, aging reduces intrinsic mitochondrial respiration in human sWAT, but lifelong high-volume exercise training increases oxidative capacity by increasing mitochondrial volume likely contributing to healthy aging.

GDF15 is an exercise-induced hepatokine regulated by glucagon and insulin in humans.

Objective: Growth differentiation factor (GDF)-15 is implicated in regulation of metabolism and circulating GDF15 increases in response to exercise. The source and regulation of the exercise-induced increase in GDF15 is, however not known. Method: Plasma GDF15 was measured by ELISA under the following conditions: 1) Arterial-to-hepatic venous differences sampled before, during, and after exercise in healthy male subjects (n=10); 2) exogenous glucagon infusion compared to saline infusion in resting healthy subjects (n=10); 3) an acute exercise bout with and without a pancreatic clamp (n=6); 4) healthy subjects for 36 hours (n=17), and 5) patients with anorexia nervosa (n=25) were compared to healthy age-matched subjects (n=25). Tissue GDF15 mRNA content was determined in mice in response to exhaustive exercise (n=16). Results: The splanchnic bed released GDF15 to the circulation during exercise and increasing the glucagon-to-insulin ratio in resting humans led to a 2.7-fold (P<0.05) increase in circulating GDF15. Conversely, inhibiting the exercise-induced increase in the glucagon-to-insulin ratio blunted the exercise-induced increase in circulating GDF15. Fasting for 36 hours did not affect circulating GDF15, whereas resting patients with anorexia nervosa displayed elevated plasma concentrations (1.4-fold, P<0.05) compared to controls. In mice, exercise increased GDF15 mRNA contents in liver, muscle, and adipose tissue. Conclusion: In humans, GDF15 is a "hepatokine" which increases during exercise and is at least in part regulated by the glucagon-to-insulin ratio. Moreover, chronic energy deprivation is associated with elevated plasma GDF15, which supports that GDF15 is implicated in metabolic signalling in humans.

Redundancy in regulation of lipid accumulation in skeletal muscle during prolonged fasting in obese men.

Fasting in human subjects shifts skeletal muscle metabolism toward lipid utilization and accumulation, including intramyocellular lipid (IMCL) deposition. Growth hormone (GH) secretion amplifies during fasting and promotes lipolysis and lipid oxidation, but it is unknown to which degree lipid deposition and metabolism in skeletal muscle during fasting depends on GH action. To test this, we studied nine obese but otherwise healthy men thrice: (a) in the postabsorptive state ("CTRL"), (b) during 72-hr fasting ("FAST"), and (c) during 72-hr fasting and treatment with a GH antagonist (GHA) ("FAST + GHA"). IMCL was assessed by magnetic resonance spectroscopy (MRS) and blood samples were drawn for plasma metabolomics assessment while muscle biopsies were obtained for measurements of regulators of substrate metabolism. Prolonged fasting was associated with elevated GH levels and a pronounced GHA-independent increase in circulating medium- and long-chain fatty acids, glycerol, and ketone bodies indicating increased supply of lipid intermediates to skeletal muscle. Additionally, fasting was associated with a release of short-, medium-, and long-chain acylcarnitines to the circulation from an increased ß-oxidation. This was consistent with a ≈55%-60% decrease in pyruvate dehydrogenase (PDHa) activity. Opposite, IMCL content increased ≈75% with prolonged fasting without an effect of GHA. We suggest that prolonged fasting increases lipid uptake in skeletal muscle and saturates lipid oxidation, both favoring IMCL deposition. This occurs without a detectable effect of GHA on skeletal muscle lipid metabolism.

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.