Exercise-induced regulation of key factors in substrate choice and gluconeogenesis in mouse liver.

As the demand for hepatic glucose production increases during exercise, regulation of liver substrate choice and gluconeogenic activity becomes essential. The aim of the present study was to investigate the effect of a single exercise bout on gluconeogenic protein content and regulation of enzymes involved in substrate utilization in the liver. Mice were subjected to 1 h of treadmill exercise, and livers were removed immediately, 4 or 10 h after exercise. Glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxylase (PEPCK) mRNA contents in the liver increased immediately after exercise, while the PEPCK protein content increased at 10 h of recovery. Furthermore, 5'AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and pyruvate dehydrogenase (PDH)-E1α Ser(293) phosphorylations decreased immediately after exercise. In addition, PDH kinase 4 (PDK4) mRNA and protein content increased immediately after exercise and at 10 h of recovery, respectively. These findings suggest that acute changes in PEPCK and G6Pase protein contents do not contribute to the regulation of gluconeogenic enzyme activity during 1 h of non-exhaustive exercise. In addition, the observation that PDH-E1α, AMPK, and ACC phosphorylation decreased immediately after exercise may indicate that carbohydrates rather than fatty acids are utilized for oxidation in the liver during non-exhaustive exercise.

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

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

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

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

Role of IL-6 in exercise training- and cold-induced UCP1 expression in subcutaneous white adipose tissue.

Expression of brown adipose tissue (BAT) associated proteins like uncoupling protein 1 (UCP1) in inguinal WAT (iWAT) has been suggested to alter iWAT metabolism. The aim of this study was to investigate the role of interleukin-6 (IL-6) in exercise training and cold exposure-induced iWAT UCP1 expression. The effect of daily intraperitoneal injections of IL-6 (3 ng/g) in C57BL/6 mice for 7 days on iWAT UCP1 expression was examined. In addition, the expression of UCP1 in iWAT was determined in response to 3 days of cold exposure (4°C) and 5 weeks of exercise training in wild type (WT) and whole body IL-6 knockout (KO) mice. Repeated injections of IL-6 in C57BL/6 mice increased UCP1 mRNA but not UCP1 protein content in iWAT. Cold exposure increased iWAT UCP1 mRNA content similarly in IL-6 KO and WT mice, while exercise training increased iWAT UCP1 mRNA in WT mice but not in IL-6 KO mice. Additionally, a cold exposure-induced increase in iWAT UCP1 protein content was blunted in IL-6 KO mice, while UCP1 protein content in iWAT was lower in both untrained and exercise trained IL-6 KO mice than in WT mice. In conclusion, repeated daily increases in plasma IL-6 can increase iWAT UCP1 mRNA content and IL-6 is required for an exercise training-induced increase in iWAT UCP1 mRNA content. In addition IL-6 is required for a full induction of UCP1 protein expression in response to cold exposure and influences the UCP1 protein content iWAT of both untrained and exercise trained animals.

Effects of IL-6 on pyruvate dehydrogenase regulation in mouse skeletal muscle.

Skeletal muscle regulates substrate choice according to demand and availability and pyruvate dehydrogenase (PDH) is central in this regulation. Circulating interleukin (IL)-6 increases during exercise and IL-6 has been suggested to increase whole body fat oxidation. Furthermore, IL-6 has been reported to increase AMP-activated protein kinase (AMPK) phosphorylation and AMPK suggested to regulate PDHa activity. Together, this suggests that IL-6 may be involved in regulating PDH. The aim of this study was to investigate the effect of a single injection of IL-6 on PDH regulation in skeletal muscle in fed and fasted mice. Fed and 16-18 h fasted mice were injected with either 3 ng · g(-1) recombinant mouse IL-6 or PBS as control. Fasting markedly reduced plasma glucose, muscle glycogen, muscle PDHa activity, as well as increased PDK4 mRNA and protein content in skeletal muscle. IL-6 injection did not affect plasma glucose or muscle glycogen, but increased AMPK and ACC phosphorylation and tended to decrease p38 protein content in skeletal muscle in fasted mice. In addition IL-6 injection reduced PDHa activity in fed mice and increased PDHa activity in fasted mice without significant changes in PDH-E1α phosphorylation or PDP1 and PDK4 mRNA and protein content. The present findings suggest that IL-6 contributes to regulating the PDHa activity and hence carbohydrate oxidation, but the metabolic state of the muscle seems to determine the outcome of this regulation. In addition, AMPK and p38 may contribute to the IL-6-mediated PDH regulation in the fasted state.

Direct effects of TNF-α on local fuel metabolism and cytokine levels in the placebo-controlled, bilaterally infused human leg: increased insulin sensitivity, increased net protein breakdown, and increased IL-6 release.

Tumor necrosis factor-α (TNF-α) has widespread metabolic actions. Systemic TNF-α administration, however, generates a complex hormonal and metabolic response. Our study was designed to test whether regional, placebo-controlled TNF-α infusion directly affects insulin resistance and protein breakdown. We studied eight healthy volunteers once with bilateral femoral vein and artery catheters during a 3-h basal period and a 3-h hyperinsulinemic-euglycemic clamp. One artery was perfused with saline and one with TNF-α. During the clamp, TNF-α perfusion increased glucose arteriovenous differences (0.91 ± 0.17 vs. 0.74 ± 0.15 mmol/L, P = 0.012) and leg glucose uptake rates. Net phenylalanine release was increased by TNF-α perfusion with concomitant increases in appearance and disappearance rates. Free fatty acid kinetics was not affected by TNF-α, whereas interleukin-6 (IL-6) release increased. Insulin and protein signaling in muscle biopsies was not affected by TNF-α. TNF-α directly increased net muscle protein loss, which may contribute to cachexia and general protein loss during severe illness. The finding of increased insulin sensitivity, which could relate to IL-6, is of major clinical interest and may concurrently act to provide adequate tissue fuel supply and contribute to the occurrence of systemic hypoglycemia. This distinct metabolic feature places TNF-α among the rare insulin mimetics of human origin.

Direct effects of locally administered lipopolysaccharide on glucose, lipid, and protein metabolism in the placebo-controlled, bilaterally infused human leg.

CONTEXT: Accumulating evidence suggests that chronic exposure to lipopolysaccharide (LPS, endotoxin) may create a constant low-grade inflammation, leading to insulin resistance and diabetes. All previous human studies assessing the metabolic actions of LPS have used systemic administration, making discrimination between direct and indirect effects impossible. OBJECTIVE: We sought to define the direct, placebo-controlled effects of LPS on insulin resistance and protein and lipid metabolism in the infused human leg without systemic interference from cytokines and stress hormones. DESIGN: This was a randomized, placebo-controlled, single-blinded study. PARTICIPANTS AND INTERVENTION: We studied 8 healthy volunteers with bilateral femoral vein and artery catheters during a 3-hour basal and 3-hour hyperinsulinemic-euglycemic clamp period with bilateral muscle biopsies in each period during infusion with saline and LPS. RESULTS: Overall, LPS perfusion significantly decreased leg glucose uptake, and during the clamp LPS decreased glucose arteriovenous differences (0.65 ± 0.07 mmol/L vs 0.73 ± 0.08 mmol/L). Net palmitate release was increased by LPS, and secondary post hoc testing indicated increased palmitate isotopic dilution, although primary ANOVA tests did not reveal significant dilution. Leg blood flows, phenylalanine, lactate kinetics, cytokines, and intramyocellular insulin signaling were not affected by LPS. LPS thus directly inhibits insulin-stimulated glucose uptake and increases palmitate release in the perfused human leg without detectable effects on amino acid metabolism. CONCLUSIONS: These data strongly suggest that the primary metabolic effect of LPS is increased lipolysis and muscle insulin resistance, which, together with secondary insulin resistance, caused by systemic cytokine and stress hormone release may lead to overt glucose intolerance and diabetes.

GLUT4 and glycogen synthase are key players in bed rest-induced insulin resistance.

To elucidate the molecular mechanisms behind physical inactivity-induced insulin resistance in skeletal muscle, 12 young, healthy male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies obtained before and after. In six of the subjects, muscle biopsies were taken from both legs before and after a 3-h hyperinsulinemic euglycemic clamp performed 3 h after a 45-min, one-legged exercise. Blood samples were obtained from one femoral artery and both femoral veins before and during the clamp. Glucose infusion rate and leg glucose extraction during the clamp were lower after than before bed rest. This bed rest-induced insulin resistance occurred together with reduced muscle GLUT4, hexokinase II, protein kinase B/Akt1, and Akt2 protein level, and a tendency for reduced 3-hydroxyacyl-CoA dehydrogenase activity. The ability of insulin to phosphorylate Akt and activate glycogen synthase (GS) was reduced with normal GS site 3 but abnormal GS site 2+2a phosphorylation after bed rest. Exercise enhanced insulin-stimulated leg glucose extraction both before and after bed rest, which was accompanied by higher GS activity in the prior-exercised leg than the rested leg. The present findings demonstrate that physical inactivity-induced insulin resistance in muscle is associated with lower content/activity of key proteins in glucose transport/phosphorylation and storage.

Bed rest reduces metabolic protein content and abolishes exercise-induced mRNA responses in human skeletal muscle.

The aim was to test the hypothesis that 7 days of bed rest reduces mitochondrial number and expression and activity of oxidative proteins in human skeletal muscle but that exercise-induced intracellular signaling as well as mRNA and microRNA (miR) responses are maintained after bed rest. Twelve young, healthy male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies taken before and after bed rest. In addition, muscle biopsies were obtained from six of the subjects prior to, immediately after, and 3 h after 45 min of one-legged knee extensor exercise performed before and after bed rest. Maximal oxygen uptake decreased by 4%, and exercise endurance decreased nonsignificantly, by 11%, by bed rest. Bed rest reduced skeletal muscle mitochondrial DNA/nuclear DNA content 15%, hexokinase II and sirtuin 1 protein content ∼45%, 3-hydroxyacyl-CoA dehydrogenase and citrate synthase activity ∼8%, and miR-1 and miR-133a content ∼10%. However, cytochrome c and vascular endothelial growth factor (VEGF) protein content as well as capillarization did not change significantly with bed rest. Acute exercise increased AMP-activated protein kinase phosphorylation, peroxisome proliferator activated receptor-γ coactivator-1α, and VEGF mRNA content in skeletal muscle before bed rest, but the responses were abolished after bed rest. The present findings indicate that only 7 days of physical inactivity reduces skeletal muscle metabolic capacity as well as abolishes exercise-induced adaptive gene responses, likely reflecting an interference with the ability of skeletal muscle to adapt to exercise.

Exercise-induced pyruvate dehydrogenase activation is not affected by 7 days of bed rest.

To test the hypothesis that physical inactivity impairs the exercise-induced modulation of pyruvate dehydrogenase (PDH), six healthy normally physically active male subjects completed 7 days of bed rest. Before and immediately after the bed rest, the subjects completed an oral glucose tolerance test (OGTT) and a one-legged knee extensor exercise bout [45 min at 60% maximal load (W(max))] with muscle biopsies obtained from vastus lateralis before, immediately after exercise, and at 3 h of recovery. Blood samples were taken from the femoral vein and artery before and after 40 min of exercise. Glucose intake elicited a larger (P ≤ 0.05) insulin response after bed rest than before, indicating glucose intolerance. There were no differences in lactate release/uptake across the exercising muscle before and after bed rest, but glucose uptake after 40 min of exercise was larger (P ≤ 0.05) before bed rest than after. Muscle glycogen content tended to be higher (0.05< P ≤ 0.10) after bed rest than before, but muscle glycogen breakdown in response to exercise was similar before and after bed rest. PDH-E1α protein content did not change in response to bed rest or in response to the exercise intervention. Exercise increased (P ≤ 0.05) the activity of PDH in the active form (PDHa) and induced (P ≤ 0.05) dephosphorylation of PDH-E1α on Ser²9³, Ser²95 and Ser³°°, with no difference before and after bed rest. In conclusion, although 7 days of bed rest induced whole body glucose intolerance, exercise-induced PDH regulation in skeletal muscle was not changed. This suggests that exercise-induced PDH regulation in skeletal muscle is maintained in glucose-intolerant (e.g., insulin resistant) individuals.

PGC-1{alpha} is required for AICAR-induced expression of GLUT4 and mitochondrial proteins in mouse skeletal muscle.

We tested the hypothesis that repeated activation of AMP-activated protein kinase (AMPK) induces mitochondrial and glucose membrane transporter mRNA/protein expression via a peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha)-dependent mechanism. Whole body PGC-1alpha-knockout (KO) and littermate wild-type (WT) mice were given either single or repeated subcutaneous injections of the AMPK activator AICAR or saline. Skeletal muscles were removed either 1 or 4 h after the single AICAR treatment or 24 h after the last injection following repeated AICAR treatment. Repeated AICAR treatment increased GLUT4, cytochrome (cyt) c oxidase I, and (cyt) c protein expression approximately 10-40% relative to saline in white muscles of WT but not of PGC-1alpha-KO mice, whereas fatty acid translocase/CD36 (FAT/CD36) protein expression was unaffected by AICAR treatment in both genotypes. GLUT4, cyt c, and FAT/CD36 mRNA content increased 30-60% 4 h after a single AICAR injection relative to saline in WT, and FAT/CD36 mRNA content decreased in PGC-1alpha-KO mice. One hour after a single AICAR treatment, phosphorylation of AMPK and the downstream target acetyl-coenzyme A carboxylase increased in all muscles investigated independent of genotype, indicating normal AICAR-induced AMPK signaling in the absence of PGC-1alpha. The hexokinase II (HKII) mRNA and protein response was similar in muscles of WT and PGC-1alpha-KO mice after single and repeated AICAR treatments, respectively, confirming that HKII is regulated independently of PGC-1alpha in response to AICAR. In conclusion, here we provide genetic evidence for a role of PGC-1alpha in AMPK-mediated regulation of mitochondrial and glucose membrane transport protein expression in skeletal muscle.