Jonas T. Treebak

AMPK-Mediated AS160 Phosphorylation in Skeletal Muscle Is Dependent on AMPK Catalytic and Regulatory Subunits

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 β-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (α1β1γ1 and α2β2γ1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (α2 AMPK knockout [KO], α2 AMPK kinase dead [KD], and γ3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing α2 and γ3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in α2 AMPK KO and KD but not γ3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.

A common Greenlandic TBC1D4 variant confers muscle insulin resistance and type 2 diabetes

The Greenlandic population, a small and historically isolated founder population comprising about 57,000 inhabitants, has experienced a dramatic increase in type 2 diabetes (T2D) prevalence during the past 25 years. Motivated by this, we performed association mapping of T2D-related quantitative traits in up to 2,575 Greenlandic individuals without known diabetes. Using array-based genotyping and exome sequencing, we discovered a nonsense p.Arg684Ter variant (in which arginine is replaced by a termination codon) in the gene TBC1D4 with an allele frequency of 17%. Here we show that homozygous carriers of this variant have markedly higher concentrations of plasma glucose (β = 3.8 mmol l−1, P = 2.5 × 10−35) and serum insulin (β = 165 pmol l−1, P = 1.5 × 10−20) 2 hours after an oral glucose load compared with individuals with other genotypes (both non-carriers and heterozygous carriers). Furthermore, homozygous carriers have marginally lower concentrations of fasting plasma glucose (β = −0.18 mmol l−1, P = 1.1 × 10−6) and fasting serum insulin (β = −8.3 pmol l−1, P = 0.0014), and their T2D risk is markedly increased (odds ratio (OR) = 10.3, P = 1.6 × 10−24). Heterozygous carriers have a moderately higher plasma glucose concentration 2 hours after an oral glucose load than non-carriers (β = 0.43 mmol l−1, P = 5.3 × 10−5). Analyses of skeletal muscle biopsies showed lower messenger RNA and protein levels of the long isoform of TBC1D4, and lower muscle protein levels of the glucose transporter GLUT4, with increasing number of p.Arg684Ter alleles. These findings are concomitant with a severely decreased insulin-stimulated glucose uptake in muscle, leading to postprandial hyperglycaemia, impaired glucose tolerance and T2D. The observed effect sizes are several times larger than any previous findings in large-scale genome-wide association studies of these traits and constitute further proof of the value of conducting genetic association studies outside the traditional setting of large homogeneous populations.

Effects of Endurance Exercise Training on Insulin Signaling in Human Skeletal Muscle Interactions at the Level of Phosphatidylinositol 3-Kinase, Akt, and AS160

The purpose of this study was to investigate the mechanisms explaining improved insulin-stimulated glucose uptake after exercise training in human skeletal muscle. Eight healthy men performed 3 weeks of one-legged knee extensor endurance exercise training. Fifteen hours after the last exercise bout, insulin-stimulated glucose uptake was ∼60% higher (P < 0.01) in the trained compared with the untrained leg during a hyperinsulinemic-euglycemic clamp. Muscle biopsies were obtained before and after training as well as after 10 and 120 min of insulin stimulation in both legs. Protein content of Akt1/2 (55 ± 17%, P < 0.05), AS160 (25 ± 8%, P = 0.08), GLUT4 (52 ± 19%, P < 0.001), hexokinase 2 (HK2) (197 ± 40%, P < 0.001), and insulin-responsive aminopeptidase (65 ± 15%, P < 0.001) increased in muscle in response to training. During hyperinsulinemia, activities of insulin receptor substrate-1 (IRS-1)–associated phosphatidylinositol 3-kinase (PI3-K) (P < 0.005), Akt1 (P < 0.05), Akt2 (P < 0.005), and glycogen synthase (GS) (percent I-form, P < 0.05) increased similarly in both trained and untrained muscle, consistent with increased phosphorylation of Akt Thr308, Akt Ser473, AS160, glycogen synthase kinase (GSK)-3α Ser21, and GSK-3β Ser9 and decreased phosphorylation of GS site 3a+b (all P < 0.005). Interestingly, training improved insulin action on thigh blood flow, and, furthermore, in both basal and insulin-stimulated muscle tissue, activities of Akt1 and GS and phosphorylation of AS160 increased with training (all P < 0.05). In contrast, training reduced IRS-1–associated PI3-K activity (P < 0.05) in both basal and insulin-stimulated muscle tissue. Our findings do not support generally improved insulin signaling after endurance training; rather it seems that improved insulin-stimulated glucose uptake may result from hemodynamic adaptations as well as increased cellular protein content of individual insulin signaling components and molecules involved in glucose transport and metabolism.

Molecular Mechanism by Which AMP-Activated Protein Kinase Activation Promotes Glycogen Accumulation in Muscle

OBJECTIVE 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. RESEARCH DESIGN AND METHODS We recently generated knock-in mice in which wild-type muscle GS was replaced by a mutant (Arg582Ala) that could not be activated by glucose-6-phosphate (G6P), but possessed full catalytic activity and could still be activated normally by dephosphorylation. Muscles from GS knock-in or transgenic mice overexpressing a kinase dead (KD) AMPK were incubated with glucose tracers and the AMPK-activating compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) ex vivo. GS activity and glucose uptake and utilization (glycolysis and glycogen synthesis) were assessed. RESULTS Even though AICAR caused a modest inactivation of GS, it stimulated muscle glycogen synthesis that was accompanied by increases in glucose transport and intracellular [G6P]. These effects of AICAR required the catalytic activity of AMPK. Strikingly, AICAR-induced glycogen synthesis was completely abolished in G6P-insensitive GS knock-in mice, although AICAR-stimulated AMPK activation, glucose transport, and total glucose utilization were normal. CONCLUSIONS We provide genetic evidence that AMPK activation promotes muscle glycogen accumulation by allosteric activation of GS through an increase in glucose uptake and subsequent rise in cellular [G6P].

Impaired Insulin-Stimulated Phosphorylation of Akt and AS160 in Skeletal Muscle of Women With Polycystic Ovary Syndrome Is Reversed by Pioglitazone Treatment

OBJECTIVE— Insulin resistance in skeletal muscle is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). However, the molecular mechanisms underlying skeletal muscle insulin resistance and the insulin-sensitizing effect of thiazolidinediones in PCOS in vivo are less well characterized. RESEARCH DESIGN AND METHODS— We determined molecular mediators of insulin signaling to glucose transport in skeletal muscle biopsies of 24 PCOS patients and 14 matched control subjects metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry, and we examined the effect of 16 weeks of treatment with pioglitazone in PCOS patients. RESULTS— Impaired insulin-mediated total (Rd) oxidative and nonoxidative glucose disposal (NOGD) was paralleled by reduced insulin-stimulated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation in muscle of PCOS patients. Akt phosphorylation at Ser473 and Thr308 correlated positively with Rd and NOGD in the insulin-stimulated state. Serum free testosterone was inversely related to insulin-stimulated Rd and NOGD in PCOS. Importantly, the pioglitazone-mediated improvement in insulin-stimulated glucose metabolism, which did not fully reach normal levels, was accompanied by normalization of insulin-mediated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation. AMPK activity and phosphorylation were similar in the two groups and did not respond to pioglitazone in PCOS patients. CONCLUSIONS— Impaired insulin signaling through Akt and AS160 in part explains insulin resistance at the molecular level in skeletal muscle in PCOS, and the ability of pioglitazone to enhance insulin sensitivity involves improved signaling through Akt and AS160. Moreover, our data provide correlative evidence that hyperandrogenism in PCOS may contribute to insulin resistance.

Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors

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.

AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity

AMP‐activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle‐specific AMPKα1α2 double‐knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle exercise capacity, mitochondrial function, and contraction‐stimulated glucose uptake. Exercise performance was significantly reduced in the mdKO mice, with a reduction in maximal force production and fatigue resistance. An increase in the proportion of myofibers with centralized nuclei was noted, as well as an elevated expression of interleukin 6 (IL‐6) mRNA, possibly consistent with mild skeletal muscle injury. Notably, we found that AMPKα1 and AMPKα2 isoforms are dispensable for contraction‐induced skeletal muscle glucose transport, except for male soleus muscle. However, the lack of skeletal muscle AMPK diminished maximal ADP‐stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial substrate utilization but not baseline mitochondrial muscle content. Together, these results demonstrate that skeletal muscle AMPK has an unexpected role in the regulation of mitochondrial oxidative phosphorylation that contributes to the energy demands of the exercising muscle.—Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J. T., Pehmøller, C., Sanz, N., Sakakibara, I., Saint‐Amand, E., Rimbaud, S., Maire, P., Marette, A., Ventura‐Clapier, R., Ferry, A., Wojtaszewski, J. F. P., Foretz, M., Viollet, B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. FASEB J. 28, 3211–3224 (2014). www.fasebj.org

Sucrose nonfermenting AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle.

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 AMPKα2 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.

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.

Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 proteins in human skeletal muscle.

Phosphorylation signature patterns on TBC1D1 and TBC1D4 proteins in the insulin–glucose pathway were investigated in human skeletal muscle in response to physiological insulin and exercise. In response to postprandial increase in insulin, Akt phosphorylation of T308 and S473 correlated significantly with sites on TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Exercise induced phosphorylation of TBC1D1 (S237, T596) that correlated significantly with activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) with exercise correlated with activity of the α2/β2/γ1 AMPK trimer. TBC1D1 phosphorylation signatures with exercise/muscle contraction were comparable between human and mouse skeletal muscle, and AMPK regulated phosphorylation of these sites in mouse muscle, whereas contraction and exercise elicited different TBC1D4 phosphorylation patterns in mouse compared with human muscle. Our results show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and indicate that Akt and AMPK may be upstream kinases.