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.

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.

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.

Hepatic PGC-1α has minor regulatory effect on the liver transcriptome and metabolome during high fat high fructose diet and exercise training.

The prevalence of non-alcoholic fatty liver diseases (NAFLD) has reached epidemic levels during recent years and a major driver of NAFLD are diets high in fat and fructose. A common practice in the treatment of NAFLD are life-style interventions including for example increased physical activity. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) has been shown to be central in mediating the beneficial effects of exercise training by regulating the expression of key metabolic genes. However, the significance of hepatic PGC-1α for high fat high fructose (HFFD) induced changes in gene expression and metabolites associated with NAFLD has not been elucidated. Therefore the aim of the present study was to investigate the effect of hepatic PGC-1α on HFFD and exercise-induced changes in the hepatic transcriptome and metabolome in mice. Using gene-arrays and 1H NMR spectroscopy, the liver transcriptome and metabolome of liver-specific PGC-1α knock-out mice receiving either standard chow, HFFD or HFFD + exercise (HFFD + Ex) were determined. In total 122 genes were identified as differently expressed in mice receiving HFFD for 13 weeks compared to chow, while the loss of hepatic PGC-1α only had very minor effects on the transcriptome. The same was observed for the liver metabolome. The effect of 4 weeks exercise training in combination with 13 weeks of HFFD, had small effects on the transcriptome and metabolome compared to HFFD alone. Together our results highlight a minor regulatory effect of hepatic PGC-1α on the liver transcriptome during high fat high fructose diet and exercise training.

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.

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.

Targeted removal of mitochondrial DNA from mouse and human extrachromosomal circular DNA with CRISPR-Cas9.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is common in eukaryotic cells. Amplification of oncogenes on large eccDNA (ecDNA) can drive biological processes such as tumorigenesis, and identification of eccDNA by sequencing after removal of chromosomal DNA is therefore important for understanding their impact on the expressed phenotype. However, the circular mitochondrial DNA (mtDNA) might challenge the detection of eccDNA because the average somatic cell has hundreds of copies of mtDNA. Here we show that 61.2-99.5% of reads from eccDNA-enriched samples correspond to mtDNA in mouse tissues. We have developed a method to selectively remove mtDNA from total circular DNA by CRISPR/Cas9 guided cleavage of mtDNA with one single-guide RNA (sgRNA) or two sgRNAs followed by exonuclease degradation of the linearized mtDNA. Sequencing revealed that mtDNA reads were 85.9% ± 12.6% removed from eccDNA of 9 investigated mouse tissues. CRISPR/Cas9 cleavage also efficiently removed mtDNA from a human HeLa cell line and colorectal cancer samples. We identified up to 14 times more, and also larger eccDNA in CRISPR/Cas9 treated colorectal cancer samples than in untreated samples. We foresee that the method can be applied to effectively remove mtDNA from any eukaryotic species to obtain higher eccDNA yields.

Beta2 -agonist increases skeletal muscle interleukin 6 production and release in response to resistance exercise in men.

OBJECTIVE: Several tissues produce and release interleukin-6 (IL-6) in response to beta2 -adrenergic stimulation with selective agonists (beta2 -agonists). Moreover, exercise stimulates muscle IL-6 production, but whether beta2 -agonists regulate skeletal muscle production and release of IL-6 in humans in association with exercise remains to be clarified. Thus, we investigated leg IL-6 release in response to beta2 -agonist salbutamol in lean young men at rest and in recovery from resistance exercise. DESIGN: The study employed a randomized controlled crossover design, where 12 men ingested either salbutamol (16 mg) or placebo for 4 days, followed by the last dose (24 mg) administered 1½ h before exercise. Arterial and femoral venous plasma IL-6 as well as femoral artery blood flow was measured before and ½-5 h in recovery from quadriceps muscle resistance exercise. Furthermore, vastus lateralis muscle biopsies were collected ½ and 5 h after exercise for determination of mRNA levels of IL-6 and Tumor Necrosis Factor (TNF)-α. RESULTS: Average leg IL-6 release was 1.7-fold higher (p = 0.01) for salbutamol than placebo, being 138 ± 76 and 79 ± 66 pg min-1 (mean ± SD) for salbutamol and placebo, respectively, but IL-6 release was not significantly different between treatments within specific sampling points at rest and after exercise. Muscle IL-6 mRNA was 1.5- and 1.7-fold higher (p = 0.001) for salbutamol than placebo ½ and 5 h after exercise, respectively, whereas no significant treatment differences were observed for TNF-α mRNA. CONCLUSIONS: Beta2 -adrenergic stimulation with high doses of the selective beta2 -agonist salbutamol, preceeded by 4 consecutive daily doses, induces transcription of IL-6 in skeletal muscle in response to resistance exercise, and increases muscle IL-6 release in lean individuals.

Time-dependent regulation of hepatic cytochrome P450 mRNA in male liver-specific PGC-1α knockout mice.

The circadian rhythm has profound effect on the body, exerting effects on diverse events like sleep-wake patterns, eating behavior and hepatic detoxification. The cytochrome p450 s (Cyps) is the main group of enzymes responsible for detoxification. However, the underlying mechanisms behind circadian regulation of the Cyps are currently not fully clarified. Therefore, the aim of the present study was to investigate the requirement of hepatic peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) for the circadian regulation of the hepatic expression of Cyp1-4 using liver-specific PGC-1α knockout (LKO) mice and littermate controls. The circadian regulator genes Bmal1 and Clock displayed decreased mRNA content at zeitgeber time (ZT) 12, compared to ZT-2 and the mRNA content of Cyp2a4 and Cyp2e1 was higher at ZT-12 than at ZT-2. Moreover, the increase in Cyp2e1 mRNA content was not observed in the PGC-1α LKO mice and hepatic PGC-1α deficiency tended to blunt the rhythmic expression of Clock and Bmal1. However, no circadian regulation was evident at the protein level for the investigated Cyps except for a change in Cyp2e1 protein content in the LKO mice. Of the measured transcription factors, only, the mRNA content of peroxisome proliferator-activated receptor α, showed rhythmic expression. To further analyze the difference between the control and LKO mice, principal component analysis were executed on the mRNA data. This demonstrated a clear separation of the experimental groups with respect to ZT and genotype. Our finding provides novel insight into the role of hepatic PGC-1α for basic and circadian expression of Cyps in mouse liver. This is important for our understanding of the molecular events behind circadian Cyp regulation and hence circadian regulation of hepatic detoxification capacity.

Skeletal muscle adaptations to exercise are not influenced by metformin treatment in humans: secondary analyses of 2 randomized, clinical trials.

Metformin and exercise both improve glycemic control, but in vitro studies have indicated that an interaction between metformin and exercise occurs in skeletal muscle, suggesting a blunting effect of metformin on exercise training adaptations. Two studies (a double-blind, parallel-group, randomized clinical trial conducted in 29 glucose-intolerant individuals and a double-blind, cross-over trial conducted in 15 healthy lean males) were included in this paper. In both studies, the effect of acute exercise ± metformin treatment on different skeletal muscle variables, previously suggested to be involved in a pharmaco-physiological interaction between metformin and exercise, was assessed. Furthermore, in the parallel-group trial, the effect of 12 weeks of exercise training was assessed. Skeletal muscle biopsies were obtained before and after acute exercise and 12 weeks of exercise training, and mitochondrial respiration, oxidative stress and AMPK activation was determined. Metformin did not significantly affect the effects of acute exercise or exercise training on mitochondrial respiration, oxidative stress or AMPK activation, indicating that the response to acute exercise and exercise training adaptations in skeletal muscle is not affected by metformin treatment. Further studies are needed to investigate whether an interaction between metformin and exercise is present in other tissues, e.g., the gut. Trial registration: ClinicalTrials.gov (NCT03316690 and NCT02951260). Novelty: Metformin does not affect exercise-induced alterations in mitochondrial respiratory capacity in human skeletal muscle. Metformin does not affect exercise-induced alterations in systemic levels of oxidative stress nor emission of reactive oxygen species from human skeletal muscle. Metformin does not affect exercise-induced AMPK activation in human skeletal muscle.

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.

High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells.

The influence of glucose and palmitic acid (PA) on mitochondrial respiration and emission of hydrogen peroxide (H2 O2 ) was determined in skeletal muscle-derived microvascular endothelial cells. Measurements were assessed in intact and permeabilized (cells treated with 0.025% saponin) low passage endothelial cells with acute-or prolonged (3 days) incubation with regular (1.7 mM) or elevated (2.2 mM) PA concentrations and regular (5 mM) or elevated (11 mM) glucose concentrations. In intact cells, acute incubation with 1.7 mM PA alone or with 1.7 mM PA + 5 mM glucose (p < .001) led to a lower mitochondrial respiration (p < 0.01) and markedly higher H2 O2 /O2 emission (p < 0.05) than with 5 mM glucose alone. Prolonged incubation of intact cells with 1.7 mM PA +5 mM glucose led to 34% (p < 0.05) lower respiration and 2.5-fold higher H2 O2 /O2 emission (p < 0.01) than incubation with 5 mM glucose alone. Prolonged incubation of intact cells with elevated glucose led to 60% lower (p < 0.05) mitochondrial respiration and 4.6-fold higher H2 O2 /O2 production than incubation with 5 mM glucose in intact cells (p < 0.001). All effects observed in intact cells were present also in permeabilized cells (State 2). In conclusion, our results show that acute and prolonged lipid availability, as well as prolonged hyperglycemia, induces mitochondrial dysfunction as evidenced by lower mitochondrial respiration and enhanced H2 O2/ O2 emission. Elevated plasma substrate availability may lead to microvascular dysfunction in skeletal muscle by impairing endothelial mitochondrial function.

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.

Effect of insulin on natriuretic peptide gene expression in porcine heart.

Gut hormones affect cardiac function and contractility. In this study, we examined whether insulin affects the cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression and release of proANP-derived peptides in pigs. Anaesthetized pigs were included in an experimental study comparing the effect of hyperinsulinemia in 15 pigs submitted to two different protocols versus 11 control pigs receiving saline infusion. Phosphorylation of Akt on Thr308 was determined by western blotting with a pAkt-Thr308 antibody. The mRNA contents of ANP and BNP were determined with real-time PCR; plasma and cardiac tissue proANP was measured with an immunoluminometric assay targeted against the mid-region of the propeptide and a processing-independent assay. Insulin stimulation increased phosphorylation of Akt Thr308 in both left atrium and left ventricle of porcine hearts (p < 0.005). No change was observed in ANP and BNP mRNA contents in the right or left atrium. BNP mRNA contents in the left ventricle, however, decreased 3-fold (p = 0.02) compared to control animals, whereas the BNP mRNA content in the right ventricle as well as ANP mRNA content in the right and left ventricle did not change following hyperinsulinemia. Moreover, the peptide contents did not change in the four cardiac chambers. Finally, proANP concentrations in plasma did not change during the insulin infusion compared to the control animals. These results suggest that insulin does not have direct effect on atrial natriuretic peptide expression but may have a role in the left ventricle.

PGC-1α-mediated regulation of mitochondrial function and physiological implications.

The majority of human energy metabolism occurs in skeletal muscle mitochondria emphasizing the importance of understanding the regulation of myocellular mitochondrial function. The transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) has been characterized as a major factor in the transcriptional control of several mitochondrial components. Thus, PGC-1α is often described as a master regulator of mitochondrial biogenesis as well as a central player in regulating the antioxidant defense. However, accumulating evidence suggests that PGC-1α is also involved in the complex regulation of mitochondrial quality beyond biogenesis, which includes mitochondrial network dynamics and autophagic removal of damaged mitochondria. In addition, mitochondrial reactive oxygen species production has been suggested to regulate skeletal muscle insulin sensitivity, which may also be influenced by PGC-1α. This review aims to highlight the current evidence for PGC-1α-mediated regulation of skeletal muscle mitochondrial function beyond the effects on mitochondrial biogenesis as well as the potential PGC-1α-related impact on insulin-stimulated glucose uptake in skeletal muscle. Novelty PGC-1α regulates mitochondrial biogenesis but also has effects on mitochondrial functions beyond biogenesis. Mitochondrial quality control mechanisms, including fission, fusion, and mitophagy, are regulated by PGC-1α. PGC-1α-mediated regulation of mitochondrial quality may affect age-related mitochondrial dysfunction and insulin sensitivity.

Inducible deletion of skeletal muscle AMPKα reveals that AMPK is required for nucleotide balance but dispensable for muscle glucose uptake and fat oxidation during exercise.

OBJECTIVE: Evidence for AMP-activated protein kinase (AMPK)-mediated regulation of skeletal muscle metabolism during exercise is mainly based on transgenic mouse models with chronic (lifelong) disruption of AMPK function. Findings based on such models are potentially biased by secondary effects related to a chronic lack of AMPK function. To study the direct effect(s) of AMPK on muscle metabolism during exercise, we generated a new mouse model with inducible muscle-specific deletion of AMPKα catalytic subunits in adult mice. METHODS: Tamoxifen-inducible and muscle-specific AMPKα1/α2 double KO mice (AMPKα imdKO) were generated by using the Cre/loxP system, with the Cre under the control of the human skeletal muscle actin (HSA) promoter. RESULTS: During treadmill running at the same relative exercise intensity, AMPKα imdKO mice showed greater depletion of muscle ATP, which was associated with accumulation of the deamination product IMP. Muscle-specific deletion of AMPKα in adult mice promptly reduced maximal running speed and muscle glycogen content and was associated with reduced expression of UGP2, a key component of the glycogen synthesis pathway. Muscle mitochondrial respiration, whole-body substrate utilization, and muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion of AMPKα subunits in adult mice. CONCLUSIONS: Inducible deletion of AMPKα subunits in adult mice reveals that AMPK is required for maintaining muscle ATP levels and nucleotide balance during exercise but is dispensable for regulating muscle glucose uptake, FA oxidation, and substrate utilization during exercise.

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.

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.

Hepatic PGC-1α is not essential for fasting-induced cytochrome p450 regulation in mouse liver.

Fasting has been shown to regulate the expression of the cytochrome p450 (CYP) enzyme system in the liver. However, the exact mechanism behind the fasting-induced regulation of the CYP's remains unknown. In the present study we tested the hypothesis that the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), which is a key-regulator of energy metabolism, is responsible for the fasting-induced regulation of the CYP's. Lox/lox and liver specific PGC-1α (LKO) mice of both sexes, fasted for 18 h and the content of the CYP's as well as the hepatic metabolome was assessed. Fasting increased the mRNA content of Cyp2a4, Cyp2e1, Cyp3a11 and Cyp4a10. The fasting-induced response in Cyp4a10 mRNA content was different between lox/lox and LKO mice, while the absence of PGC-1α had no effect on the fasting-induced response for the other Cyp's. Moreover, the fasting-induced response in mRNA content of Sirtinus 1 and Perilipin 2 was different between lox/lox and LKO mice. Only the CYP1A isoform showed a fasting-induced response at the protein level. Absence of hepatic PGC-1α had no effect on the apparent metabolome, where fasting vs fed was the only discriminate in the following multivariate analysis. In conclusion, hepatic PGC-1α is not essential for the fasting-induced regulation of hepatic CYP's.