Key points• NAD is a substrate for sirtuins (SIRTs), which regulate gene transcription in response to specific metabolic stresses.• Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway.• Using transgenic mouse models, we tested the hypothesis that skeletal muscle Nampt protein abundance would increase in response to metabolic stress in a manner dependent on the cellular nucleotide sensor, AMP-activated protein kinase (AMPK).• Exercise training, as well as repeated pharmacological activation of AMPK by 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), increased Nampt protein abundance. However, only the AICAR-mediated increase in Nampt protein abundance was dependent on AMPK.• Our results suggest that cellular energy charge and nutrient sensing by SIRTs may be mechanistically related, and that Nampt may play a key role for cellular adaptation to metabolic stress. Abstract Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM).Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training-or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related.

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.

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.

Recent evidence suggests that exercise stimulates the degradation of cellular components in skeletal muscle through activation of autophagy, but the time course of the autophagy response during recovery from exercise has not been determined. Furthermore, the regulatory mechanisms behind exercise‐induced autophagy remain unclear, although the muscle oxidative phenotype has been linked with basal autophagy levels. Therefore, the aim of this study was to investigate the role of the key regulator of muscle oxidative capacity, PGC‐1α, in exercise‐induced autophagy at several time points during recovery. Mice with transgenic muscle‐specific overexpression (TG) or knockout (MKO) of PGC‐1α and their respective littermate controls were subjected to a single 1 h bout of treadmill running and euthanized immediately (0 h), 2, 6, and 10 h after exercise. In the PGC‐1α MKO strain, quadriceps protein content of the autophagy marker LC3II was increased from 2 h into recovery in lox/lox control, but not in MKO mice. In the PGC‐1α TG strain, quadriceps protein content of LC3II was increased from 2 h after exercise in TG, but not in WT. Although AMPK and ACC phosphorylation was increased immediately following exercise, the observed exercise‐induced autophagy response was not associated with phosphorylation of the AMPK‐target ULK1. However, lower protein carbonyl content was observed in lox/lox and TG mice after exercise coinciding with the increased LC3 lipidation. In conclusion, the present results suggest a role of skeletal muscle PGC‐1α in coordinating several exercise‐induced adaptive responses including autophagic removal of damaged cellular components.

BackgroundPolyunsaturated n-3 fatty acids (n-3 PUFAs) are reported to protect against high fat diet-induced obesity and inflammation in adipose tissue. Here we aimed to investigate if the amount of sucrose in the background diet influences the ability of n-3 PUFAs to protect against diet-induced obesity, adipose tissue inflammation and glucose intolerance.Methodology/Principal FindingsWe fed C57BL/6J mice a protein- (casein) or sucrose-based high fat diet supplemented with fish oil or corn oil for 9 weeks. Irrespective of the fatty acid source, mice fed diets rich in sucrose became obese whereas mice fed high protein diets remained lean. Inclusion of sucrose in the diet also counteracted the well-known anti-inflammatory effect of fish oil in adipose tissue, but did not impair the ability of fish oil to prevent accumulation of fat in the liver. Calculation of HOMA-IR indicated that mice fed high levels of proteins remained insulin sensitive, whereas insulin sensitivity was reduced in the obese mice fed sucrose irrespectively of the fat source. We show that a high fat diet decreased glucose tolerance in the mice independently of both obesity and dietary levels of n-3 PUFAs and sucrose. Of note, increasing the protein∶sucrose ratio in high fat diets decreased energy efficiency irrespective of fat source. This was accompanied by increased expression of Ppargc1a (peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha) and increased gluconeogenesis in the fed state.Conclusions/SignificanceThe background diet influence the ability of n-3 PUFAs to protect against development of obesity, glucose intolerance and adipose tissue inflammation. High levels of dietary sucrose counteract the anti-inflammatory effect of fish oil in adipose tissue and increases obesity development in mice.

BackgroundThe aim of the present study was to test the hypotheses that 1) a single exercise bout increases UCP1 mRNA in both inguinal (i)WAT and epididymal (e)WAT, 2) UCP1 expression and responsiveness to exercise are different in iWAT and eWAT, 3) PGC-1α determines the basal levels of UCP1 and PRDM16 in WAT and 4) exercise and exercise training regulate UCP1 and PRDM16 expression in WAT in a PGC-1α-dependent manner.MethodsWhole body PGC-1α knockout (KO) and wildtype (WT) littermate mice performed a single treadmill exercise bout at 14 m/min and 10% slope for 1 hour. Mice were sacrificed and iWAT, eWAT and quadriceps muscle were removed immediately after, 2, 6 and 10 hours after running, and from sedentary mice that served as controls. In addition, PGC-1α KO mice and WT littermates were exercise trained for 5 weeks with sedentary mice as untrained controls. Thirty-six-37 hours after the last exercise bout iWAT was removed.ResultsUCP1 mRNA content increased 19-fold in iWAT and 7.5-fold in eWAT peaking at 6 h and 0′ of recovery, respectively, in WT but with no changes in PGC-1α KO mice. UCP1 protein was undetectable in eWAT and very low in iWAT of untrained mice but increased with exercise training to 4.4 (AU) in iWAT from WT mice without significant effects in PGC-1α KO mice.ConclusionThe present observations provide evidence that exercise training increases UCP1 protein in iWAT through PGC-1α, likely as a cumulative effect of transient increases in UCP1 expression after each exercise bout. Moreover, the results suggest that iWAT is more responsive than eWAT in exercise-induced regulation of UCP1. In addition, as PRDM16 mRNA content decreased in recovery from acute exercise, the present findings suggest that acute exercise elicits regulation of several brown adipose tissue genes in mouse WAT.

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The transcriptional coactivator peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC)-1α plays a role in regulation of several metabolic pathways. By use of whole body PGC-1α knockout (KO) mice, we investigated the role of PGC-1α in fasting, acute exercise and exercise training-induced regulation of key proteins in gluconeogenesis and metabolism in the liver. In both wild-type (WT) and PGC-1α KO mice liver, the mRNA content of the gluconeogenic proteins glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) was upregulated during fasting. Pyruvate carboxylase (PC) remained unchanged after fasting in WT mice, but it was upregulated in PGC-1α KO mice. In response to a single exercise bout, G6Pase mRNA was upregulated in both genotypes, whereas no significant changes were detected in PEPCK or PC mRNA. While G6Pase and PC protein remained unchanged, liver PEPCK protein content was higher in trained than untrained mice of both genotypes. The mRNA content of the mitochondrial proteins cytochrome c (Cyt c) and cytochrome oxidase (COX) subunit I was unchanged in response to fasting. The mRNA and protein content of Cyt c and COXI increased in the liver in response to a single exercise bout and prolonged exercise training, respectively, in WT mice, but not in PGC-1α KO mice. Neither fasting nor exercise affected the mRNA expression of antioxidant enzymes in the liver, and knockout of PGC-1α had no effect. In conclusion, these results suggest that PGC-1α plays a pivotal role in regulation of Cyt c and COXI expression in the liver in response to a single exercise bout and prolonged exercise training, which implies that exercise training-induced improvements in oxidative capacity of the liver is regulated by PGC-1α.