The signaling mechanisms that mediate the important effects of contraction to increase glucose transport in skeletal muscle are not well understood, but are known to occur through an insulin-independent mechanism. Muscle-specific knockout of LKB1, an upstream kinase for AMPK and AMPK-related protein kinases, significantly inhibited contraction-stimulated glucose transport. This finding, in conjunction with previous studies of ablated AMPKalpha2 activity showing no effect on contraction-stimulated glucose transport, suggests that one or more AMPK-related protein kinases are important for this process. Muscle contraction increased sucrose nonfermenting AMPK-related kinase (SNARK) activity, an effect blunted in the muscle-specific LKB1 knockout mice. Expression of a mutant SNARK in mouse tibialis anterior muscle impaired contraction-stimulated, but not insulin-stimulated, glucose transport. Whole-body SNARK heterozygotic knockout mice also had impaired contraction-stimulated glucose transport in skeletal muscle, and knockdown of SNARK in C2C12 muscle cells impaired sorbitol-stimulated glucose transport. SNARK is activated by muscle contraction and is a unique mediator of contraction-stimulated glucose transport in skeletal muscle.

The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells.

Dietary supplementation of nicotinamide adenine dinucleotide (NAD+) precursors has been suggested as a treatment for non-alcoholic fatty liver disease and obesity. In the liver, NAD+ is primarily generated by nicotinamide phosphoribosyltransferase (NAMPT), and hepatic levels of NAMPT and NAD+ have been reported to be dependent on age and body composition. The aim of the present study was to identify time course-dependent changes in hepatic NAD content and NAD+ salvage capacity in mice challenged with a high-fat diet (HFD). We fed 7-week-old C57BL/6JBomTac male mice either regular chow or a 60% HFD for 6, 12, 24, and 48 weeks, and we evaluated time course-dependent changes in whole body metabolism, liver steatosis, and abundance of hepatic NAD-associated metabolites and enzymes. Mice fed a 60% HFD rapidly accumulated fat and hepatic triglycerides with associated changes in respiratory exchange ratio (RER) and a disruption of the circadian feeding pattern. The HFD did not alter hepatic NAD+ levels, but caused a decrease in NADP+ and NADPH levels. Decreased NADP+ content was not accompanied by alterations in NAD kinase (NADK) abundance in HFD-fed mice, but NADK levels increased with age regardless of diet. NAMPT protein abundance did not change with age or diet. HFD consumption caused a severe decrease in protein lysine malonylation after six weeks, which persisted throughout the experiment. This decrease was not associated with changes in SIRT5 abundance. In conclusion, hepatic NAD+ salvage capacity is resistant to long-term HFD feeding, and hepatic lipid accumulation does not compromise the hepatic NAD+ pool in HFD-challenged C57BL/6JBomTac male mice.

Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear.

Circadian rhythms can be perturbed by shift work, travel across time zones, many occupational tasks, or genetic mutations. Perturbed circadian rhythms are associated with the increasing problem of obesity, metabolic dysfunction, and insulin resistance. We hypothesized that insulin sensitivity in skeletal muscle follows a circadian pattern and that this pattern is important for overall metabolic function. This hypothesis was verified using mice as a model system. We observed circadian rhythmicity in whole body insulin tolerance, as well as in signaling pathways regulating insulin- and exercise-induced glucose uptake in skeletal muscle, including AKT, 5'-adenosine monophosphate-activated protein kinase (AMPK) and TBC1 domain family member 4 (TBC1D4) phosphorylation. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissues in vivo also differed between day- and nighttime. However, the rhythmicity of glucose uptake differed from the rhythm of whole-body insulin tolerance. These results indicate that neither skeletal muscle nor adipose tissue play a major role for the circadian rhythmicity in whole-body insulin tolerance. To study the circadian pattern of insulin sensitivity directly in skeletal muscle, we determined glucose uptake under basal and submaximal insulin-stimulated conditions ex vivo every sixth hour. Both insulin sensitivity and signaling of isolated skeletal muscle peaked during the dark period. We next examined the effect of exercise training on the circadian rhythmicity of insulin sensitivity. As expected, voluntary exercise training enhanced glucose uptake in skeletal muscle. Nevertheless, exercise training did not affect the circadian rhythmicity of skeletal muscle insulin sensitivity. Taken together, our results provide evidence that skeletal muscle insulin sensitivity exhibits circadian rhythmicity.

Loss-of-function (LoF) mutations in KCNQ1, encoding the voltage-gated K+ channel Kv7.1, lead to long QT syndrome 1 (LQT1). LQT1 patients also present with post-prandial hyperinsulinemia and hypoglycaemia. In contrast, KCNQ1 polymorphisms are associated with diabetes, and LQTS patients have a higher prevalence of diabetes. We developed a mouse model with a LoF Kcnq1 mutation using CRISPR-Cas9 and hypothesized that this mouse model would display QT prolongation, increased glucose-stimulated insulin secretion and allow for interrogation of Kv7.1 function in islets. Mice were characterized by electrocardiography and oral glucose tolerance tests. Ex vivo, islet glucose-induced insulin release was measured, and beta-cell area quantified by immunohistochemistry. Homozygous mice had QT prolongation. Ex vivo, glucose-stimulated insulin release was increased in islets from homozygous mice at 12-14 weeks, while beta-cell area was reduced. Non-fasting blood glucose levels were decreased at this age. In follow-up studies 8-10 weeks later, beta-cell area was similar in all groups, while glucose-stimulated insulin secretion was now reduced in islets from hetero- and homozygous mice. Non-fasting blood glucose levels had normalized. These data suggest that Kv7.1 dysfunction is involved in a transition from hyper- to hyposecretion of insulin, potentially explaining the association with both hypoglycemia and hyperglycemia in LQT1 patients.

Nicotinamide phosphoribosyltransferase (NAMPT) converts nicotinamide to NAD+. As low hepatic NAD+ levels have been linked to the development of nonalcoholic fatty liver disease, we hypothesized that ablation of hepatic Nampt would affect susceptibility to liver injury in response to diet-induced metabolic stress. Following 3 weeks on a low-methionine and choline-free 60% high-fat diet, hepatocyte-specific Nampt knockout (HNKO) mice accumulated less triglyceride than WT littermates but had increased histological scores for liver inflammation, necrosis, and fibrosis. Surprisingly, liver injury was also observed in HNKO mice on the purified control diet. This HNKO phenotype was associated with decreased abundance of mitochondrial proteins, especially proteins involved in oxidoreductase activity. High-resolution respirometry revealed lower respiratory capacity in purified control diet-fed HNKO liver. In addition, fibrotic area in HNKO liver sections correlated negatively with hepatic NAD+, and liver injury was prevented by supplementation with NAD+ precursors nicotinamide riboside and nicotinic acid. MS-based proteomic analysis revealed that nicotinamide riboside supplementation rescued hepatic levels of oxidoreductase and OXPHOS proteins. Finally, single-nucleus RNA-Seq showed that transcriptional changes in the HNKO liver mainly occurred in hepatocytes, and changes in the hepatocyte transcriptome were associated with liver necrosis. In conclusion, HNKO livers have reduced respiratory capacity, decreased abundance of mitochondrial proteins, and are susceptible to fibrosis because of low NAD+ levels. Our data suggest a critical threshold level of hepatic NAD+ that determines the predisposition to liver injury and supports that NAD+ precursor supplementation can prevent liver injury and nonalcoholic fatty liver disease progression.

The circadian clock is a cell-autonomous transcription-translation feedback mechanism that anticipates and adapts physiology and behavior to different phases of the day. A variety of factors including hormones, temperature, food-intake, and exercise can act on tissue-specific peripheral clocks to alter the expression of genes that influence metabolism, all in a time-of-day dependent manner. The aim of this study was to elucidate the effects of exercise timing on adipose tissue metabolism. We performed RNA sequencing on inguinal adipose tissue of mice immediately following maximal exercise or sham treatment at the early rest or early active phase. Only during the early active phase did exercise elicit an immediate increase in serum nonesterified fatty acids. Furthermore, early active phase exercise increased expression of markers of thermogenesis and mitochondrial proliferation in inguinal adipose tissue. In vitro, synchronized 3T3-L1 adipocytes showed a timing-dependent difference in Adrb2 expression, as well as a greater lipolytic activity. Thus, the response of adipose tissue to exercise is time-of-day sensitive and may be partly driven by the circadian clock. To determine the influence of feeding state on the time-of-day response to exercise, we replicated the experiment in 10-h-fasted early rest phase mice to mimic the early active phase metabolic status. A 10-h fast led to a similar lipolytic response as observed after active phase exercise but did not replicate the transcriptomic response, suggesting that the observed changes in gene expression are not driven by feeding status. In conclusion, acute exercise elicits timing-specific effects on adipose tissue to maintain metabolic homeostasis.

Metabolic disorders such as obesity represent a major health challenge. Obesity alone has reached epidemic proportions, with at least 2.8 million people worldwide dying annually from diseases caused by overweight or obesity. The brain-metabolic axis is central to maintain homeostasis under metabolic stress via an intricate signaling network of hormones. Protein interacting with C kinase 1 (PICK1) is important for the biogenesis of various secretory vesicles, and we have previously shown that PICK1-deficient mice have impaired secretion of insulin and growth hormone.

We investigated the phosphorylation signatures of two Rab-GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers exercised in the fasted or fed state and muscle biopsies were taken before and immediately after exercise. We identified TBC1D1/4 phospho-sites that (1) did not respond to exercise or postprandial increase in insulin (TBC1D4: S666), (2) responded to insulin only (TBC1D4: S318), (3) responded to exercise only (TBC1D1: S237, S660, S700; TBC1D4: S588, S751), and (4) responded to both insulin and exercise (TBC1D1: T596; TBC1D4: S341, T642, S704). In the insulin-stimulated leg, Akt phosphorylation of both T308 and S473 correlated significantly with multiple sites on both TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Interestingly, in the exercised leg in the fasted state TBC1D1 phosphorylation (S237, T596) correlated significantly with the activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) correlated with the activity of the α2/β2/γ1 AMPK trimer. Our data show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK regulated phosphorylation of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring findings between species is problematic.

Metformin-induced activation of the 5'-AMP-activated protein kinase (AMPK) has been associated with enhanced glucose uptake in skeletal muscle, but so far no direct causality has been examined. We hypothesized that an effect of in vivo metformin treatment on glucose uptake in mouse skeletal muscles is dependent on AMPK signaling. Oral doses of metformin or saline treatment were given to muscle-specific kinase dead (KD) AMPKα2 mice and wild-type (WT) littermates either once or chronically for 2 wk. Soleus and extensor digitorum longus muscles were used for measurements of glucose transport and Western blot analyses. Chronic treatment with metformin enhanced insulin-stimulated glucose uptake in soleus muscles of WT (∼45%, P < 0.01) but not of AMPK KD mice. Insulin signaling at the level of Akt protein expression or Thr(308) and Ser(473) phosphorylation was not changed by metformin treatment. Insulin signaling at the level of Akt and TBC1D4 protein expression as well as Akt Thr(308)/Ser(473) and TBC1D4 Thr(642)/Ser(711) phosphorylation were not changed by metformin treatment. Also, protein expressions of Rab4, GLUT4, and hexokinase II were unaltered after treatment. The acute metformin treatment did not affect glucose uptake in muscle of either of the genotypes. In conclusion, we provide novel evidence for a role of AMPK in potentiating the effect of insulin on glucose uptake in soleus muscle in response to chronic metformin treatment.

The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin β1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle-an observation found to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle.

During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically, chronic activation of AMPK causes an increase in glycogen accumulation in skeletal and cardiac muscles, which in some cases is associated with cardiac dysfunction. The aim of this study was to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation.

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.

The glucose tolerance test (GTT) is widely used in preclinical research to investigate glucose metabolism, but there is no standardised way to administer glucose. The aim of this study was to directly compare the effect of the route of glucose administration on glucose and insulin kinetics during a GTT in mice.

BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disease ranging from simple hepatic steatosis (NAFL) to non-alcoholic steatohepatitis (NASH) which may progress to cirrhosis and liver cancer. NAFLD is rapidly becoming a global health challenge, and there is a need for improved diagnostic- and prognostic tools and for effective pharmacotherapies to treat NASH. The molecular mechanisms of NAFLD development and progression remain incompletely understood, though ample evidence supports a role of microRNAs (miRNAs) - small non-coding RNAs regulating gene expression - in the progression of metabolic liver disease.

The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning.