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Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2h Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell-surface recruitable GLUT4 pool via kinesin-1 mediated trafficking.

For three-dimensional (3D) bioprinting to fulfill its promise and enable the automated fabrication of complex tissue-mimicking constructs, there is a need for developing bioinks that are not only printable and biocompatible but also have integrated cell-instructive properties. Toward this goal, we here present a scalable technique for generating nanofiber 3D printing inks with unique tissue-guiding capabilities. Our core methodology relies on tailoring the size and dispersibility of cellulose fibrils through a solvent-controlled partial carboxymethylation. This way, we generate partially negatively charged cellulose nanofibers with diameters of ∼250 nm and lengths spanning tens to hundreds of microns. In this range, the fibers structurally match the size and dimensions of natural collagen fibers making them sufficiently large to orient cells. Yet, they are simultaneously sufficiently thin to be optically transparent. By adjusting fiber concentration, 3D printing inks with excellent shear-thinning properties can be established. In addition, as the fibers are readily dispersible, composite inks with both carbohydrates and extracellular matrix (ECM)-derived proteins can easily be generated. We apply such composite inks for 3D printing cell-laden and cross-linkable structures, as well as tissue-guiding gel substrates. Interestingly, we find that the spatial organization of engineered tissues can be defined by the shear-induced alignment of fibers during the printing procedure. Specifically, we show how myotubes derived from human and murine skeletal myoblasts can be programmed into linear and complex nonlinear architectures on soft printed substrates with intermediate fiber contents. Our nanofibrillated cellulose inks can thus serve as a simple and scalable tool for engineering anisotropic human muscle tissues that mimic native structure and function.

Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized along and on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perfused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2h Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. In L6 muscle cells, pharmacological activation of the microtubule motor protein kinesin-1 increased basal and insulin-stimulated GLUT4 translocation, whereas shRNA-mediated knockdown of the kinesin-1 protein encoding gene Kif5B reduced insulin-stimulated GLUT4 translocation. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell-surface recruitable GLUT4 pool via kinesin-1 mediated trafficking.

BackgroundMetabolic dysfunction and cancer cachexia are associated with poor cancer prognosis, yet the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia remain to be defined. A key link between metabolic- and muscle mass-regulation is adenosine monophosphate-activated protein kinase (AMPK). As AMPK could be a potential treatment, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. Here we determined the function of AMPK in cancer-associated metabolic dysfunction, insulin resistance, and cachexia.MethodsIn vastus lateralis muscle biopsies from pre-cachectic and cachectic patients with Non-Small-Cell Lung Carcinoma (NSCLC), AMPK signaling and expression were examined by immunoblotting. To investigate the role of muscle AMPK, male mice overexpressing a dominant-negative AMPKα2 (kinase-dead) specifically in striated muscle (mAMPK-KD) were inoculated with Lewis Lung Carcinoma (LLC) cells. In a subsequent cohort, male LLC-tumor-bearing mice were treated with/without 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13 days. Littermate mice were used as control. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose- and insulin tolerance tests, tissue-specific 2-deoxy- glucose (2-DG) uptake, and immunoblotting.ResultsIn muscle from patients with NSCLC, we found increased expression of AMPK subunits α1, α2, β2, γ1, and γ3; ranging from +27% to +79% compared to healthy control subjects. AMPK subunit expression correlated with indices of cachexia, including cross sectional area and weight loss. Tumor-bearing mAMPK-KD mice presented increased fat loss as well as glucose and insulin intolerance. LLC in mAMPK-KD mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps; −35%, soleus; −49%, EDL; −48%) and the heart (−29%) compared to non-tumor-bearing mice. In skeletal muscle, mAMPK-KD abrogated the tumor-induced increase in phosphorylation of TBC1D4thr642. Additionally, protein expression of TBC1D4 (+26%), pyruvate dehydrogenase (PDH, +94%), and PDH-kinases (PDKs, +45% to +100%), and glycogen synthase (+48%) were increased in skeletal muscle of tumor-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase-II protein expression and normalized phosphorylation of p70S6Kthr389 (mTORC1 substrate) and ACCser212 (AMPK substrate) and rescued the cancer-induced insulin intolerance.ConclusionsUpregulated protein expression of AMPK subunits observed in skeletal muscle of (pre)cachectic patients with non-small-cell lung carcinoma. This seemed protective inferred by AMPK-deficient tumor-bearing mice being highly prone to developing metabolic dysfunction, which included the AMPK-dependent regulation of several proteins involved in glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and cachexia.Abstract Figure

Key points Tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) in mouse does not affect whole-body energy substrate metabolism. AXIN1 imKO does not affect AICAR or insulin-stimulated glucose uptake in adult skeletal muscle AXIN1 imKO does not affect adult skeletal muscle AMPK or mTORC1 signaling during AICAR/insulin/amino acid incubation, contraction and exercise. During exercise, α2/β2/γ3AMPK and AMP/ATP ratio show greater increases in AXIN1 imKO than wild-type in gastrocnemius muscle. Abstract AXIN1 is a scaffold protein known to interact with >20 proteins in signal transduction pathways regulating cellular development and function. Recently, AXIN1 was proposed to assemble a protein complex essential to catabolic-anabolic transition by coordinating AMPK activation and inactivation of mTORC1 and to regulate glucose uptake-stimulation by both AMPK and insulin. To investigate whether AXIN1 is permissive for adult skeletal muscle function, a phenotypic in vivo and ex vivo characterization of tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) mice was conducted. AXIN1 imKO did not influence AMPK/mTORC1 signaling or glucose uptake stimulation, neither at rest nor in response to different exercise/contraction protocols, pharmacological AMPK activation, insulin or amino acids stimulation. The only genotypic difference observed was in exercising gastrocnemius muscle, where AXIN1 imKO displayed elevated α2/β2/γ3 AMPK activity and AMP/ATP ratio compared to wild-type mice. Our work shows that AXIN1 imKO generally does not affect skeletal muscle AMPK/mTORC1 signaling and glucose metabolism, likely due to functional redundancy of its homolog AXIN2. This article is protected by copyright. All rights reserved.

Key points Muscle contractions increase protein synthesis in a mechanistic target of rapamycin (mTOR)-dependent manner, yet it is unclear which/how mTOR complexes regulate muscle protein synthesis. We investigated the requirement of mTOR Complex 2 (mTORC2) in contraction-stimulated muscle protein synthesis. mTORC2 inhibition by muscle-specific Rictor knockout (Rictor mKO) did not prevent contraction-induced muscle protein synthesis. Rapamycin prevented contraction-induced muscle protein synthesis in Rictor mKO but not wild-type mice. Abstract Protein synthesis increases following muscle contractions. Previous studies have shown that inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) suppresses the early but not late muscle protein synthesis response, while inhibition of both mTORC1 and mTORC2 abolishes the two effects. Therefore, we hypothesized that mTORC2 regulates muscle protein synthesis following muscle contractions. To test this, we investigated the effect of mTORC2 inhibition by mouse muscle-specific Rictor knockout (Rictor mKO) on muscle protein synthesis 3 h after contraction. The right gastrocnemius muscles of Rictor mKO and wild-type (WT) mice were isometrically contracted using percutaneous electrical stimulation, while the left gastrocnemius muscles served as controls. Vehicle or the mTORC1 inhibitor rapamycin (1.5 mg/kg) was injected intraperitoneally 1 h before contraction. Treatment of WT mice with rapamycin and Rictor mKO lowered protein synthesis in general, but the response to contractions was intact 3 h after contractions in both conditions. Rapamycin treatment in Rictor mKO mice prevented contraction-stimulated muscle protein synthesis. Notably, signalling traditionally associated with mTORC1 was increased by muscle contractions despite rapamycin treatment. In rapamycin-treated Rictor mKO mice, the same mTORC1 signalling was blocked following contractions. Our results indicate that although neither rapamycin-sensitive mTOR/mTORC1 nor mTORC2 is necessary for contraction-induced muscle protein synthesis, combined inhibition of rapamycin-sensitive mTOR/mTORC1 and mTORC2 synergistically inhibits contraction-induced muscle protein synthesis.

KEY POINTS Muscle-specific genetic ablation of p21-activated kinase (PAK)2, but not whole-body PAK1 knockout, impairs glucose tolerance in mice. Insulin-stimulated glucose uptake partly relies on PAK2 in glycolytic extensor digitorum longus muscle By contrast to previous reports, PAK1 is dispensable for insulin-stimulated glucose uptake in mouse muscle. ABSTRACT The group I p21-activated kinase (PAK) isoforms PAK1 and PAK2 are activated in response to insulin in skeletal muscle and PAK1/2 signalling is impaired in insulin-resistant mouse and human skeletal muscle. Interestingly, PAK1 has been suggested to be required for insulin-stimulated glucose transporter 4 translocation in mouse skeletal muscle. Therefore, the present study aimed to examine the role of PAK1 in insulin-stimulated muscle glucose uptake. The pharmacological inhibitor of group I PAKs, IPA-3 partially reduced (-20%) insulin-stimulated glucose uptake in isolated mouse soleus muscle (P

Key points AMP-activated protein kinase (AMPK)-dependent Raptor Ser792 phosphorylation does not influence mechanistic target of rapamycin complex 1 (mTORC1)-S6K1 activation by intense muscle contraction. α2 -AMPK activity-deficient mice have lower contraction-stimulated protein synthesis. Increasing glycogen activates mTORC1-S6K1. Normalizing muscle glycogen content rescues reduced protein synthesis in AMPK-deficient mice. Abstract The mechansitic target of rapamycin complex 1 (mTORC1)-S6K1 signalling pathway regulates muscle growth-related protein synthesis and is antagonized by AMP-activated protein kinase (AMPK) in multiple cell types. Resistance exercise stimulates skeletal muscle mTORC1-S6K1 and AMPK signalling and post-contraction protein synthesis. Glycogen inhibits AMPK and has been proposed as a pro-anabolic stimulus. The present study aimed to investigate how muscle mTORC1-S6K1 signalling and protein synthesis respond to resistance exercise-mimicking contraction in the absence of AMPK and with glycogen manipulation. Resistance exercise-mimicking unilateral in situ contraction of musculus quadriceps femoris in anaesthetized wild-type and dominant negative α2 AMPK kinase dead transgenic (KD-AMPK) mice, measuring muscle mTORC1 and AMPK signalling immediately (0 h) and 4 h post-contraction, and protein-synthesis at 4 h. Muscle glycogen manipulation by 5 day oral gavage of the glycogen phosphorylase inhibitor CP316819 and sucrose (80 g L-1 ) in the drinking water prior to in situ contraction. The mTORC1-S6K1 and AMPK signalling axes were coactivated immediately post-contraction, despite potent AMPK-dependent Ser792 phosphorylation on the mTORC1 subunit raptor. KD-AMPK muscles displayed normal mTORC1-S6K1 activation at 0 h and 4 h post-exercise, although there was impaired contraction-stimulated protein synthesis 4 h post-contraction. Pharmacological/dietary elevation of muscle glycogen content augmented contraction-stimulated mTORC1-S6K1-S6 signalling and rescued the reduced protein synthesis-response in KD-AMPK to wild-type levels. mTORC-S6K1 signalling is not influenced by α2 -AMPK during or after intense muscle contraction. Elevated glycogen augments mTORC1-S6K1 signalling. α2 -AMPK-deficient KD-AMPK mice display impaired contraction-induced muscle protein synthesis, which can be rescued by normalizing muscle glycogen content.

In mice, exercise is suggested to activate the mechanistic target of rapamycin complex 2 (mTORC2) in skeletal muscle, and mTORC2 is required for normal muscle glucose uptake during exercise. Whether this translates to human skeletal muscle and what signaling pathways facilitate the exercise-induced mTORC2 activation is unknown. We herein tested the hypothesis that exercise increases mTORC2 activity in human skeletal muscle and investigated if β(2)-adrenergic receptor (AR) activation mediates exercise-induced mTORC2 activation. We examined several mTORC2 activity readouts (p-NDRG1 Thr346, p-Akt Ser473, p-mTOR S2481, and p-Akt Thr450) in human skeletal muscle biopsies after uphill walking or cycling exercise. In mouse muscles, we assessed mTORC2 activity readouts following acute activation of muscle β(2)-adrenergic or G(S) signaling and during in vivo and ex vivo muscle contractions. Exercise increased phosphorylation of NDRG1 Thr346 in human soleus, gastrocnemius, and vastus lateralis muscle, without changing p-Akt Ser473, p-Akt Thr450, and p-mTOR Ser2481. In mouse muscle, stimulation of β(2)-adrenergic or G(S) signaling and ex vivo contractions failed to increase p-NDRG1 Thr346, whereas in vivo contractions were sufficient to induce p-NDRG1 Thr346. In conclusion, the mTORC2 activity readout p-NDRG1 Thr346 is a novel exercise-responsive signaling protein in human skeletal muscle. Notably, contraction-induced p-NDRG1 Thr346 appears to require a systemic factor. Unlike exercise, and in contrast to published data obtained in cultured muscles cells, stimulation of β(2)-adrenergic signaling is not sufficient to trigger NDRG1 phosphorylation in mature mouse skeletal muscle. NEW & NOTEWORTHY The mTORC2 readout p-NDRG Thr346 is a novel exercise-responsive protein in human skeletal muscle. β2-AR and G(S) signaling are not sufficient to induce mTORC2 signaling in adult muscle. In vivo, but not ex vivo, contraction induced p-NDRG Thr346, which indicates requirement of a systemic factor for exercise-induced mTORC2 activation.

BackgroundSkeletal muscle wasting is often associated with insulin resistance. A major regulator of muscle mass is the transforming growth factor β (TGF-β) superfamily, including activin A, which causes atrophy. TGF-β superfamily ligands also negatively regulate insulin-sensitive proteins, but whether this pathway contributes to insulin action remains to be determined.MethodsTo elucidate if TGF-β superfamily ligands regulate insulin action we used an adeno-associated virus gene editing approach to overexpress the activin A inhibitor, follistatin (Fst288) in mouse muscle of lean and diet-induced obese mice. We determined basal and insulin-stimulated 2 deoxy-glucose uptake using isotopic tracers in vivo. Furthermore, to evaluate whether circulating Fst and activin A concentrations are associated with obesity, insulin resistance, and weight loss in humans we analysed serum from morbidly obese subjects before, 1 week, and 1 year after Roux-en-Y gastric bypass (RYGB).ResultsFst288 muscle overexpression markedly increased in vivo insulin-stimulated (but not basal) glucose uptake (+75%, pConclusionsWe here present evidence that Fst is a potent regulator of insulin action in muscle and in addition to AKT and p70S6K, we identify TBC1D1, TBC1D4 and PAK1 as Fst targets. A possible role for Fst in regulating glycemic control is suggested because circulating Fst more than doubled post RYGB surgery, a treatment that markedly improved insulin sensitivity. These findings demonstrate the therapeutic potential of inhibiting TGF-β superfamily ligands to improve insulin action and Fst’s relevance to muscle wasting associated insulin resistant conditions in mice and humans.

The cortical cytoskeleton, consisting of the cytoplasmic actin isoforms β and/or γ-actin, has been implicated in insulin-stimulated GLUT4 translocation and glucose uptake in muscle and adipose cell culture. Furthermore, transgenic inhibition of multiple actin-regulating proteins in muscle inhibits insulin-stimulated muscle glucose uptake. The current study tested if γ-actin was required for insulin-stimulated glucose uptake in mouse skeletal muscle. Based on our previously reported age-dependent phenotype in muscle-specific β-actin gene deletion (-/-) mice, we included cohorts of growing 8-14 weeks old and mature 18-32 weeks old muscle-specific γ-actin-/- mice or wild-type littermates. In growing mice, insulin significantly increased the glucose uptake in slow-twitch oxidative soleus and fast-twitch glycolytic EDL muscles from wild-type mice, but not γ-actin-/-. In relative values, the maximal insulin-stimulated glucose uptake was reduced by ~50% in soleus and by ~70% in EDL muscles from growing γ-actin-/- mice compared to growing wild-type mice. In contrast, the insulin-stimulated glucose uptake responses in mature adult γ-actin-/- soleus and EDL muscles were indistinguishable from the responses in wild-type muscles. Mature adult insulin-stimulated phosphorylations on Akt, p70S6K, and ULK1 were not significantly affected by genotype. Hence, insulin-stimulated muscle glucose uptake shows an age-dependent impairment in young growing but not in fully grown γ-actin-/- mice, bearing phenotypic resemblance to β-actin-/- mice. Overall, γ-actin does not appear required for insulin-stimulated muscle glucose uptake in adulthood. Furthermore, our data emphasize the need to consider the rapid growth of young mice as a potential confounder in transgenic mouse phenotyping studies.

Metabolic dysfunction and insulin resistance are emerging as hallmarks of cancer and cachexia, and impair cancer prognosis. Yet, the molecular mechanisms underlying impaired metabolic regulation is not fully understood. To elucidate the mechanisms behind cancer-induced insulin resistance in muscle, we isolated extensor digitorum longus (EDL) and soleus muscles from Lewis Lung Carcinoma tumor-bearing mice. Three weeks after tumor inoculation, muscles were isolated and stimulated with or without a submaximal dose of insulin (1.5 nM). Glucose transport was measured using 2-[3H]Deoxy-Glucose and intramyocellular signaling was investigated using immunoblotting. In soleus muscles from tumor-bearing mice, insulin-stimulated glucose transport was abrogated concomitantly with abolished insulin-induced TBC1D4 and GSK3 phosphorylation. In EDL, glucose transport and TBC1D4 phosphorylation were not impaired in muscles from tumor-bearing mice, while AMPK signaling was elevated. Anabolic insulin signaling via phosphorylation of the mTORC1 targets, p70S6K thr389 and ribosomal-S6 ser235, were decreased by cancer in soleus muscle while increased or unaffected in EDL. In contrast, the mTOR substrate, pULK1 ser757, was reduced in both soleus and EDL by cancer. Hence, cancer causes considerable changes in skeletal muscle insulin signaling that is dependent of muscle-type, which could contribute to metabolic dysregulation in cancer. Thus, skeletal muscle could be a target for managing metabolism in cancer.HighlightsCancer abrogates insulin-stimulated glucose transport selectively in oxidative soleus muscleMultiple TBC1D4 phosphorylation sites are reduced in cancer-associated muscle insulin resistanceCancer leads to increased AMPK signaling in the glycolytic EDL muscleCancer alters anabolic insulin signaling in soleus and EDL muscle

Objective The goal of this study was to determine the glucometabolic effects of acute activation of Gs signaling in skeletal muscle (SKM) in vivo and its contribution to whole-body glucose homeostasis. Methods To address this question, we studied mice that express a Gs-coupled designer G protein-coupled receptor (Gs-DREADD or GsD) selectively in skeletal muscle. We also identified two Gs-coupled GPCRs that are endogenously expressed by SKM at relatively high levels (β2-adrenergic receptor and CRF2 receptor) and studied the acute metabolic effects of activating these receptors in vivo by highly selective agonists (clenbuterol and urocortin 2 (UCN2), respectively). Results Acute stimulation of GsD signaling in SKM impaired glucose tolerance in lean and obese mice by decreasing glucose uptake selectively into SKM. The acute metabolic effects following agonist activation of β2-adrenergic and, potentially, CRF2 receptors appear primarily mediated by altered insulin release. Clenbuterol injection improved glucose tolerance by increasing insulin secretion in lean mice. In SKM, clenbuterol stimulated glycogen breakdown. UCN2 injection resulted in decreased glucose tolerance associated with lower plasma insulin levels. The acute metabolic effects of UCN2 were not mediated by SKM Gs signaling. Conclusions Selective activation of Gs signaling in SKM causes an acute increase in blood glucose levels. However, acute in vivo stimulation of endogenous Gs-coupled receptors enriched in SKM has only a limited impact on whole-body glucose homeostasis, most likely due to the fact that these receptors are also expressed by pancreatic islets where they modulate insulin release., Highlights • A novel mouse model allowed us to study the in vivo metabolic effects of acute activation of Gs signaling in skeletal muscle (SKM). • Acute stimulation of this pathway resulted in impaired glucose tolerance in lean and obese mice due to decreased glucose uptake by SKM. • Acute treatment of mice with selective β2-adrenergic and CRF2 receptor agonists (both receptors couple to Gs and are enriched in SKM) resulted in complex in vivo metabolic outcomes, primarily due to altered insulin release. • Our study provides an excellent example of how different tissue expression patterns of receptors can affect the acute effects of GPCR agonists on whole-body glucose homeostasis • Our findings also highlight the importance of studying both acute and chronic effects of GPCR agonist treatment to properly assess translationally relevant metabolic outcomes.

Skeletal muscle (SKM) insulin resistance plays a central role in the pathogenesis of type 2 diabetes. Because G-protein–coupled receptors (GPCRs) represent excellent drug targets, we hypothesized that activation of specific functional classes of SKM GPCRs might lead to improved glucose homeostasis in type 2 diabetes. At present, little is known about the in vivo metabolic roles of the various distinct GPCR signaling pathways operative in SKM. In this study, we tested the hypothesis that selective activation of SKM Gq signaling can improve SKM glucose uptake and whole-body glucose homeostasis under physiological and pathophysiological conditions. Studies with transgenic mice expressing a Gq-linked designer GPCR selectively in SKM cells demonstrated that receptor-mediated activation of SKM Gq signaling greatly promoted glucose uptake into SKM and significantly improved glucose homeostasis in obese, glucose-intolerant mice. These beneficial metabolic effects required the activity of SKM AMPK. In contrast, obese mutant mice that lacked both Gαq and Gα11 selectively in SKM showed severe deficits in glucose homeostasis. Moreover, GPCR-mediated activation of Gq signaling also stimulated glucose uptake in primary human SKM cells. Taken together, these findings strongly suggest that agents capable of enhancing SKM Gq signaling may prove useful as novel antidiabetic drugs.

Botulinum toxin A (botox) is a toxin used for spasticity treatment and cosmetic purposes. Botox blocks the excitation of skeletal muscle fibers by preventing the release of acetylcholine from motor nerves, a process termed chemical denervation. Surgical denervation is associated with increased expression of the canonical insulin-activated kinase Akt, lower expression of glucose handling proteins GLUT4 and hexokinase II (HKII) and insulin resistant glucose uptake, but it is not known if botox has a similar effect. To test this, we performed a time-course study using supra-maximal insulin-stimulation in mouse soleus ex vivo. No effect was observed in the glucose transport responsiveness at day 1, 7 and 21 after intramuscular botox injection, despite lower expression of GLUT4, HKII and expression and phosphorylation of TBC1D4. Akt protein expression and phosphorylation of the upstream kinase Akt were increased by botox treatment at day 21. In a follow-up study, botox decreased submaximal insulin-stimulated glucose transport. The marked alterations of insulin signaling, GLUT4 and HKII and submaximal insulin-stimulated glucose transport are a potential concern with botox treatment which merit further investigation in human muscle. Furthermore, the botox-induced chemical denervation model may be a less invasive alternative to surgical denervation.

Background: High-intensity muscle contractions (HiMC) are known to increase c-Myc expression which is known to stimulate ribosome biogenesis and protein synthesis in tumor cells. However, whether the increase in c-Myc stimulates ribosome biogenesis and protein synthesis in skeletal muscles remains unknown. Methods: We investigated the effect of adeno-associated virus (AAV)-mediated c-Myc overexpression, with or without fasting or percutaneous electrical stimulation-induced HiMC, on ribosome biogenesis and protein synthesis in adult mouse skeletal muscles. Results: AAV-mediated overexpression of c-Myc in mouse skeletal muscles for 2 weeks increased the DNA polymerase subunit POL1 mRNA, 45S-pre-rRNA, total RNA, and muscle protein synthesis without altering mechanistic target of rapamycin complex 1 (mTORC1) signaling under both ad libitum and fasted conditions. RNA-seq analyses revealed that c-Myc overexpression mainly regulated ribosome biogenesis-related biological processes. The protein synthesis response to c-Myc overexpression mirrored the response with HiMC. No additional effect of combining c-Myc overexpression and HiMC was observed. Conclusion: c-Myc overexpression is sufficient to stimulate skeletal muscle ribosome biogenesis and protein synthesis without activation of mTORC1. Therefore, the HiMC-induced increase in c-Myc may contribute to ribosome biogenesis and increased protein synthesis following HiMC.

Objective Exercise is a cornerstone in the management of skeletal muscle insulin-resistance. A well-established benefit of a single bout of exercise is increased insulin sensitivity for hours post-exercise in the previously exercised musculature. Although rodent studies suggest that the insulin-sensitization phenomenon involves enhanced insulin-stimulated GLUT4 cell surface translocation and might involve intramuscular redistribution of GLUT4, the conservation to humans is unknown. Methods Healthy young males underwent an insulin-sensitizing one-legged kicking exercise bout for 1 h followed by fatigue bouts to exhaustion. Muscle biopsies were obtained 4 h post-exercise before and after a 2-hour hyperinsulinemic-euglycemic clamp. Results A detailed microscopy-based analysis of GLUT4 distribution within seven different myocellular compartments revealed that prior exercise increased GLUT4 localization in insulin-responsive storage vesicles and T-tubuli. Furthermore, insulin-stimulated GLUT4 localization was augmented at the sarcolemma and in the endosomal compartments. Conclusions An intracellular redistribution of GLUT4 post-exercise is proposed as a molecular mechanism contributing to the insulin-sensitizing effect of prior exercise in human skeletal muscle., Graphical abstract Image 1, Highlights • Intramyocellular GLUT4 is redistributed 4 h after exercise in humans. • GLUT4 content is increased in GLUT4 storage vesicles and T-tubuli post-exercise. • Prior exercise + insulin increases sarcolemmal and endosomal GLUT4. • GLUT4 redistribution may thus contribute to post-exercise muscle insulin-sensitization.

The small molecule kinase inhibitor SBI-0206965 was originally described as a specific inhibitor of ULK1/2. More recently, it was reported to effectively inhibit AMPK and several studies now report its use as an AMPK inhibitor. Currently, we investigated the specificity of SBI-0206965 in incubated mouse skeletal muscle, measuring the effect on analog 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-stimulated AMPK-dependent glucose transport and insulin-stimulated AMPK-independent glucose uptake. Pre-treatment with 10 µM SBI-0206965 for 50 min potently suppressed AICAR-stimulated glucose transport in both the extensor digitorum longus (EDL) and soleus muscle. This was despite only a modest lowering of AICAR-stimulated AMPK activation measured as ACC2 Ser212, while ULK1/2 Ser555 phosphorylation was prevented. Insulin-stimulated glucose transport was also potently inhibited by SBI-0206965 in soleus. No major changes were observed on insulin-stimulated cell signaling. No general effect of SBI-0206965 on intracellular membrane morphology was observed by transmission electron microscopy. As insulin is known to neither activate AMPK nor require AMPK to stimulate glucose transport, and insulin inhibits ULK1/2 activity, these data strongly suggest that SBI-0206965 has a non-specific off-target inhibitory effect on muscle glucose transport. Thus, SBI-0206965 is not a specific inhibitor of the AMPK/ULK-signaling axis in skeletal muscle, and data generated with this inhibitor must be interpreted with caution.

AimMuscle contraction stimulates skeletal muscle glucose transport. Since it occurs independently of insulin, it is an important alternative pathway to increase glucose uptake in insulin-resistant states, but the intracellular signalling mechanisms are not fully understood. Muscle contraction activates group I p21-activated kinases (PAKs) in mouse and human skeletal muscle. PAK1 and PAK2 are downstream targets of Rac1, which is a key regulator of contraction-stimulated glucose transport. Thus, PAK1 and PAK2 could be downstream effectors of Rac1 in contraction-stimulated glucose transport. The current study aimed to test the hypothesis that PAK1 and/or PAK2 regulate contraction-induced glucose transport.MethodsGlucose transport was measured in isolated soleus and extensor digitorum longus (EDL) mouse skeletal muscle incubated either in the presence or absence of a pharmacological inhibitor (IPA-3) of group I PAKs or originating from whole-body PAK1 knockout (KO), muscle-specific PAK2 (m)KO or double whole-body PAK1 and muscle-specific PAK2 knockout mice.ResultsIPA-3 attenuated (−22%) the increase in muscle glucose transport in response to electrically-stimulated contraction. PAK1 was dispensable for contraction-stimulated glucose uptake in both soleus and EDL muscle. Lack of PAK2, either alone (−13%) or in combination with PAK1 (−14%), reduced contraction-stimulated glucose transport compared to control littermates in EDL, but not soleus muscle.ConclusionContraction-stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1.

Aim Muscle contraction stimulates skeletal muscle glucose transport. Since it occurs independently of insulin, it is an important alternative pathway to increase glucose transport in insulin‐resistant states, but the intracellular signaling mechanisms are not fully understood. Muscle contraction activates group I p21‐activated kinases (PAKs) in mouse and human skeletal muscle. PAK1 and PAK2 are downstream targets of Rac1, which is a key regulator of contraction‐stimulated glucose transport. Thus, PAK1 and PAK2 could be downstream effectors of Rac1 in contraction‐stimulated glucose transport. The current study aimed to test the hypothesis that PAK1 and/or PAK2 regulate contraction‐induced glucose transport. Methods Glucose transport was measured in isolated soleus and extensor digitorum longus (EDL) mouse skeletal muscle incubated either in the presence or absence of a pharmacological inhibitor (IPA‐3) of group I PAKs or originating from whole‐body PAK1 knockout, muscle‐specific PAK2 knockout or double whole‐body PAK1 and muscle‐specific PAK2 knockout mice. Results IPA‐3 attenuated (−22%) the increase in glucose transport in response to electrically stimulated contractions in soleus and EDL muscle. PAK1 was dispensable for contraction‐stimulated glucose transport in both soleus and EDL muscle. Lack of PAK2, either alone (−13%) or in combination with PAK1 (−14%), partly reduced contraction‐stimulated glucose transport compared to control littermates in EDL, but not soleus muscle. Conclusion Contraction‐stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1., In this study, we show that contraction‐stimulated glucose transport in isolated mouse skeletal muscle is unaffected by whole‐body knockout of PAK1. On the contrary, muscle‐specific knockout of PAK2 either alone or in combination with whole‐body PAK1 knockout partially reduces contraction‐stimulated glucose transport in glycolytic EDL muscle. These data suggest that contraction‐stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1.

Objective Chronic ad libitum low protein-high carbohydrate diet (LPHC) increases health- and life-span in mice. A periodized (p) LPHC regimen would be a more practical long-term human lifestyle intervention, but the metabolic benefits of pLPHC are not known. Also, the interactions between LPHC diet and exercise training have not been investigated. Presently, we aimed to provide proof-of-concept data in mice of the efficacy of pLPHC and to explore the potential interactions with concurrent exercise training. Methods A detailed phenotypic and molecular characterization of mice undergoing different durations of 14 d LPHC (5 E% protein)/14 d control diet cycles for up to 4 months with or without concurrent access to activity wheels allowing voluntary exercise training. Results pLPHC conferred metabolic benefits similar to chronic LPHC, including increased FGF21 and adaptive thermogenesis, obesity-protection despite increased total energy intake and improved insulin sensitivity. The improved insulin sensitivity showed large fluctuations between diet periods and was lost within 14 days of switching back to control diet. Parallel exercise training improved weight maintenance but impaired the FGF21 response to pLPHC whereas repeated pLPHC cycles progressively augmented this response. Both the FGF21 suppression by exercise and potentiation by repeated cycles correlated tightly with Nupr1 mRNA in liver, suggesting dependence on liver integrated stress response. Conclusion These results suggest that pLPHC may be a viable strategy to promote human health but also highlight the transient nature of the benefits and that the interaction with other lifestyle-interventions such as exercise training warrants consideration., Graphical abstract Image 1, Highlights • Periodized (p) low-protein-high-carbohydrate (LPHC) diet confers metabolic benefits. • The metabolic improvements by pLPHC diet are highly transient. • The liver FGF21 and integrated stress response to pLPHC is lowered by exercise and increased by repeated pLPHC cycles.

Studies in skeletal muscle cell cultures suggest that the cortical actin cytoskeleton is a major requirement for insulin-stimulated glucose transport, implicating the β-actin isoform, which in many cell types is the main actin isoform. However, it is not clear that β-actin plays such a role in mature skeletal muscle. Neither dependency of glucose transport on β-actin nor actin reorganization upon glucose transport have been tested in mature muscle. To investigate the role of β-actin in fully differentiated muscle, we performed a detailed characterization of wild type and muscle-specific β-actin knockout (KO) mice. The effects of the β-actin KO were subtle; however, we confirmed the previously reported decline in running performance of β-actin KO mice compared with wild type during repeated maximal running tests. We also found insulin-stimulated glucose transport into incubated muscles reduced in soleus but not in extensor digitorum longus muscle of young adult mice. Contraction-stimulated glucose transport trended toward the same pattern, but the glucose transport phenotype disappeared in soleus muscles from mature adult mice. No genotype-related differences were found in body composition or glucose tolerance or by indirect calorimetry measurements. To evaluate β-actin mobility in mature muscle, we electroporated green fluorescent protein (GFP)-β-actin into flexor digitorum brevis muscle fibers and measured fluorescence recovery after photobleaching. GFP-β-actin showed limited unstimulated mobility and no changes after insulin stimulation. In conclusion, β-actin is not required for glucose transport regulation in mature mouse muscle under the majority of the tested conditions. Thus, our work reveals fundamental differences in the role of the cortical β-actin cytoskeleton in mature muscle compared with cell culture.

Key points Exercise is a potent physiological stimulus to clear blood glucose from the circulation into skeletal muscle. The mammalian target of rapamycin complex 2 (mTORC2) is an important regulator of muscle glucose uptake in response to insulin stimulation. Here we report for the first time that the activity of mTORC2 in mouse muscle increases during exercise. We further show that glucose uptake during exercise is decreased in mouse muscle that lacks mTORC2 activity. We also provide novel identifications of new mTORC2 substrates during exercise in mouse muscle. Abstract Exercise increases glucose uptake into insulin-resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. The mammalian target of rapamycin complex 2 (mTORC2), a regulator of insulin-controlled glucose uptake, has been reported to interact with ras-related C3 botulinum toxin substrate 1 (Rac1), which plays a role in exercise-induced glucose uptake in muscle. Therefore, we tested the hypothesis that mTORC2 activity is necessary for muscle glucose uptake during treadmill exercise. We used mice that specifically lack mTORC2 signalling in muscle by deletion of the obligatory mTORC2 component Rictor (Ric mKO). Running capacity and running-induced changes in blood glucose, plasma lactate and muscle glycogen levels were similar in wild-type (Ric WT) and Ric mKO mice. At rest, muscle glucose uptake was normal, but during running muscle glucose uptake was reduced by 40% in Ric mKO mice compared to Ric WT mice. Running increased muscle phosphorylated 5′ AMP-activated protein kinase (AMPK) similarly in Ric WT and Ric mKO mice, and glucose transporter type 4 (GLUT4) and hexokinase II (HKII) protein expressions were also normal in Ric mKO muscle. The mTORC2 substrate, phosphorylated protein kinase C α (PKCα), and the mTORC2 activity readout, phosphorylated N-myc downstream regulated 1 (NDRG1) protein increased with running in Ric WT mice, but were not altered by running in Ric mKO muscle. Quantitative phosphoproteomics uncovered several additional potential exercise-dependent mTORC2 substrates, including contractile proteins, kinases, transcriptional regulators, actin cytoskeleton regulators and ion-transport proteins. Our study suggests that mTORC2 is a component of the exercise signalling network that regulates muscle glucose uptake and we provide a resource of new potential members of the mTORC2 signalling network.

The target of rapamycin complex 2 (TORC2) was discovered in 2002 in budding yeast. Its mammalian counterpart, mTORC2, was first described in 2004. Soon thereafter it was demonstrated that mTORC2 directly phosphorylates Akt on Ser473, ending a long search for the elusive 'second' insulin-responsive Akt kinase. In this review we discuss key evidence pertaining to the subcellular localization of mTORC2, highlighting a spatial heterogeneity that relates to mTORC2 activation. We summarize current models for how growth factors (GFs), such as insulin, trigger mTORC2 activation, and we provide a comprehensive discussion focusing on a new exciting frontier, the molecular mechanisms underpinning GF-independent activation of mTORC2.

ObjectiveSkeletal muscle glucose uptake is essential for maintaining whole-body glucose homeostasis and accounts for the majority of glucose disposal in response to insulin. The group I p21-activated kinase (PAK) isoforms PAK1 and PAK2 are activated in response to insulin in skeletal muscle. Interestingly, PAK1/2 signalling is impaired in insulin-resistant mouse and human skeletal muscle and PAK1 has been suggested to be required for insulin-stimulated GLUT4 translocation. However, the relative contribution of PAK1 and PAK2 to insulin-stimulated glucose uptake in mature skeletal muscle is unresolved. The aim of the present investigation was to determine the requirement for PAK1 and PAK2 in whole-body glucose homeostasis and insulin-stimulated glucose uptake in skeletal muscle.MethodsGlucose uptake was measured in isolated skeletal muscle incubated with a pharmacological inhibitor (IPA-3) of group I PAKs and in muscle from whole-body PAK1 knockout (KO), muscle-specific PAK2 (m)KO and double whole-body PAK1 and muscle-specific PAK2 knockout mice.ResultsThe whole-body respiratory exchange ratio was largely unaffected by lack of PAK1 and/or PAK2. Whole-body glucose tolerance was mildly impaired in PAK2 mKO, but not PAK1 KO mice. IPA-3 partially reduced (−20%) insulin-stimulated glucose uptake in mouse soleus muscle. In contrast to a previous study of GLUT4 translocation in PAK1 KO mice, PAK1 KO muscles displayed normal insulin-stimulated glucose uptake in vivo and in isolated muscle. On the contrary, glucose uptake was slightly reduced in response to insulin in glycolytic extensor digitorum longus muscle lacking PAK2, alone (−18%) or in combination with PAK1 KO (−12%).ConclusionsInsulin-stimulated glucose uptake partly relies on PAK2, but not PAK1, in mouse skeletal muscle. Thus, the present study challenges that group I PAKs, and especially PAK1, are major regulators of whole-body glucose homeostasis and insulin-stimulated glucose uptake in skeletal muscle.

Objective: Reactive oxygen species (ROS) have been proposed as signaling molecules mediating exercise training adaptation, but the ROS source has remained unclear. This study aimed to investigate the requirement for NADPH oxidase (NOX)2-dependent redox changes induced by acute and long-term high-intensity interval training (HIIT) in skeletal muscle in a mouse model lacking functional NOX2 complex due to deficient p47phox (Ncf1) subunit expression (ncf1* mutation). Methods: HIIT was investigated after an acute bout of exercise and after a chronic intervention (3x week for 6 weeks) in wildtype (WT) vs. NOX2 activity-deficient (ncf1*) mice. NOX2 activation during HIIT was measured using a genetically-encoded biosensor. Immunoblotting and single-fiber staining were performed to measure classical exercise-training responsive endpoints in skeletal muscle. Results: A single bout of HIIT increased NOX2 activity measured using electroporated p47roGFP oxidation immediately after exercise but not 1h after exercise. After a 6-week of HIIT regime, improvements in maximal running capacity and some muscle training-markers responded less to HIIT in the ncf1* mice compared to WT, including superoxide dismutase (SOD)2, catalase, hexokinase II (HK II), pyruvate dehydrogenase (PDH) and protein markers of mitochondrial oxidative phosphorylation complexes. Strikingly, HIIT-training increased mitochondrial network area and decreased fragmentation in WT mice only. Conclusion: This study provided evidence that HIIT exercise activates NOX2 complex in skeletal muscle and that the presence of functional NOX2 is required for specific skeletal muscle adaptations to HIIT relating to antioxidant defense, glucose metabolism, and mitochondria.

Reactive oxygen species (ROS) act as intracellular compartmentalized second messengers, mediating metabolic stress-adaptation. In skeletal muscle fibers, ROS have been suggested to stimulate glucose transporter 4 (GLUT4)-dependent glucose transport during artificially evoked contraction ex vivo, but whether myocellular ROS production is stimulated by in vivo exercise to control metabolism is unclear. Here, we combined exercise in humans and mice with fluorescent dyes, genetically-encoded biosensors, and NADPH oxidase 2 (NOX2) loss-of-function models to demonstrate that NOX2 is the main source of cytosolic ROS during moderate-intensity exercise in skeletal muscle. Furthermore, two NOX2 loss-of-function mouse models lacking either p47phox or Rac1 presented striking phenotypic similarities, including greatly reduced exercise-stimulated glucose uptake and GLUT4 translocation. These findings indicate that NOX2 is a major myocellular ROS source, regulating glucose transport capacity during moderate-intensity exercise., Reactive oxygen species (ROS) stimulate GLUT4-mediated glucose transport following contraction of isolated muscle, but it is not clear if this occurs in vivo. Here, the authors show in human volunteers that exercise induces ROS increase in muscle and, using loss of-function animal models, they demonstrate that NOX2 is a major ROS source required to stimulate glucose uptake during exercise.

New findings What is the central question of this study? Resolving the mechanism(s) leading to glucose transporter 4 (GLUT4) translocation to the muscle surface membrane has great therapeutic potential. However, the measurement of GLUT4 translocation is technically challenging. Here, we asked whether electroporation of GLUT4-7myc-GFP into skeletal muscle could be used as a tool to study GLUT4 translocation in vivo. What is the main finding and its importance? By acutely inducing GLUT4-7myc-GFP expression in skeletal muscle, we verified that in vivo exercise and AICAR stimulation increased the GLUT4 presence in the sarcolemma measured as myc signal. Importantly, the increased myc signal in the sarcolemma was not accompanied by major visual changes in the distribution of the GFP signal. Abstract Insulin and exercise lead to translocation of the glucose transporter 4 (GLUT4) to the surface membrane of skeletal muscle fibres. This process is pivotal for facilitating glucose uptake into skeletal muscle. To study this, a robust assay is needed to measure the translocation of GLUT4 in adult skeletal muscle directly. Here, we aimed to validate a simple GLUT4 translocation assay using a genetically encoded biosensor in mouse skeletal muscle. We transfected GLUT4-7myc-GFP into mouse muscle to study live GLUT4 movement and to evaluate GLUT4 insertion in the muscle surface membrane after in vivo running exercise and pharmacological activation of AMP-activated protein kinase (AMPK). Transfection led to expression of GLUT4-7myc-GFP that was dynamic in live flexor digitorum brevis fibres and which, upon insulin stimulation, exposed the myc epitope extracellularly. Running exercise, in addition to AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleotide, induced ∼125 and ∼100% increase, respectively, in extracellularly exposure of GLUT4 in the surface membrane of tibialis anterior muscle. Interestingly, the clear increase in surface-exposed GLUT4 content induced by insulin, exercise or AMPK activation was not accompanied by any discernible reorganization of the GLUT4-GFP signal. In conclusion, we provide a detailed description of an easy-to-use translocation assay to study GLUT4 accumulation at the surface membrane induced by exercise and exercise-mimicking stimuli. Notably, our analyses revealed that increased GLUT4 surface membrane accumulation was not accompanied by a discernible change in the GLUT4 localization pattern.

AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α1/α2) is bound to one of two β subunits (β1/β2) and one of three γ subunits (γ1/γ2/γ3). The ability to selectively activate specific isoforms would be a useful research tool and a promising strategy to combat diseases such as cancer and Type 2 diabetes. We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 (unc-51-like autophagy-activating kinase 1) on Ser555, but not proposed AMPK-γ3 substrates such as Ser231 on TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 (TBC1D1) or Ser212 on acetyl-CoA carboxylase subunit 2 (ACC2), nor did it stimulate glucose transport. Surprisingly, however, in human embryonic kidney (HEK) 293 cells expressing human γ1, γ2 or γ3, PT-1 activated all three complexes equally. We were unable to reproduce previous findings suggesting that PT-1 activates AMPK by direct binding between the kinase and auto-inhibitory domains (AIDs) of the α subunit. We show instead that PT-1 activates AMPK indirectly by inhibiting the respiratory chain and increasing cellular AMP:ATP and/or ADP:ATP ratios. Consistent with this mechanism, PT-1 failed to activate AMPK in HEK293 cells expressing an AMP-insensitive R299G mutant of AMPK-γ1. We propose that the failure of PT-1 to activate γ3-containing complexes in muscle is not an intrinsic feature of such complexes, but is because PT-1 does not increase cellular AMP:ATP ratios in the specific subcellular compartment(s) in which γ3 complexes are located.

Exercise is a potent physiological stimulus to clear blood glucose from the circulation into skeletal muscle. The mammalian target of rapamycin complex 2 (mTORC2) is an important regulator of muscle glucose uptake in response to insulin stimulation. Here we report for the first time that the activity of mTORC2 in mouse muscle increases during exercise. We further show that glucose uptake during exercise is decreased in mouse muscle that lacks mTORC2 activity. We also provide novel identifications of new mTORC2 substrates during exercise in mouse muscle.Exercise increases glucose uptake into insulin-resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. The mammalian target of rapamycin complex 2 (mTORC2), a regulator of insulin-controlled glucose uptake, has been reported to interact with ras-related C3 botulinum toxin substrate 1 (Rac1), which plays a role in exercise-induced glucose uptake in muscle. Therefore, we tested the hypothesis that mTORC2 activity is necessary for muscle glucose uptake during treadmill exercise. We used mice that specifically lack mTORC2 signalling in muscle by deletion of the obligatory mTORC2 component Rictor (Ric mKO). Running capacity and running-induced changes in blood glucose, plasma lactate and muscle glycogen levels were similar in wild-type (Ric WT) and Ric mKO mice. At rest, muscle glucose uptake was normal, but during running muscle glucose uptake was reduced by 40% in Ric mKO mice compared to Ric WT mice. Running increased muscle phosphorylated 5' AMP-activated protein kinase (AMPK) similarly in Ric WT and Ric mKO mice, and glucose transporter type 4 (GLUT4) and hexokinase II (HKII) protein expressions were also normal in Ric mKO muscle. The mTORC2 substrate, phosphorylated protein kinase C α (PKCα), and the mTORC2 activity readout, phosphorylated N-myc downstream regulated 1 (NDRG1) protein increased with running in Ric WT mice, but were not altered by running in Ric mKO muscle. Quantitative phosphoproteomics uncovered several additional potential exercise-dependent mTORC2 substrates, including contractile proteins, kinases, transcriptional regulators, actin cytoskeleton regulators and ion-transport proteins. Our study suggests that mTORC2 is a component of the exercise signalling network that regulates muscle glucose uptake and we provide a resource of new potential members of the mTORC2 signalling network.

Objective Reactive oxygen species (ROS) have been proposed as signaling molecules mediating exercise training adaptation, but the ROS source has remained unclear. This study aimed to investigate if increased NADPH oxidase (NOX)2-dependent activity during exercise is required for long-term high-intensity interval training (HIIT) in skeletal muscle using a mouse model lacking functional NOX2 complex due to absent p47phox (Ncf1) subunit expression (ncf1* mutation). Methods HIIT was investigated after an acute bout of exercise and after a chronic intervention (3x/week for 6 weeks) in wild-type (WT) vs. NOX2 activity-deficient (ncf1*) mice. NOX2 activation during HIIT was measured using an electroporated genetically-encoded biosensor. Immunoblotting and single-fiber microscopy was performed to measure classical exercise-training responsive endpoints in skeletal muscle. Results A single bout of HIIT increased NOX2 activity measured as p47-roGFP oxidation immediately after exercise but not 1 h or 4 h after exercise. After a 6-week HIIT regimen, improvements in maximal running capacity and some muscle training-markers responded less to HIIT in the ncf1* mice compared to WT, including superoxide dismutase 2, catalase, hexokinase II, pyruvate dehydrogenase and protein markers of mitochondrial oxidative phosphorylation complexes. Strikingly, HIIT-training increased mitochondrial network area and decreased fragmentation in WT mice only. Conclusion This study suggests that HIIT exercise increases NOX2 activity in skeletal muscle and shows that NOX2 activity is required for specific skeletal muscle adaptations to HIIT relating to antioxidant defense, glucose metabolism, and mitochondria., Graphical abstract Image 1, Highlights • Acute HIIE induces transient NOX2 complex activity in vivo in muscle. • Skeletal muscle adaptations to HIIT were impaired in ncf1-deficient mice. • Functional NOX2 is necessary for HIIT-induced increased expression of antioxidants enzymes. • Ncf1-deficient mice lack HIIT-induced mitochondrial adaptations.

A low‐protein high carbohydrate (LPHC) diet and a high‐protein low carbohydrate (HPLC) diet have been reported to positively and negatively regulate whole‐body glucose tolerance and insulin sensitivity, respectively. Skeletal muscle is quantitatively the most important tissue clearing glucose in the postprandial state, but it is unclear if LPHC and HPLC diets directly influence insulin action in skeletal muscle. To test this, mice were placed on control chow diet, LPHC and HPLC diets for 13.5 weeks at which time the submaximal insulin‐stimulated glucose transport and insulin signaling were evaluated in ex vivo incubated oxidative soleus and glycolytic EDL muscle. At the whole‐body level, the diets had the anticipated effects, with LPHC diet improving glucose tolerance and insulin‐sensitivity whereas HPLC diet had the opposite effect. However, neither insulin‐stimulated Akt/TBC1D4 signaling and glucose transport ex vivo, nor cell signaling in vivo were altered by the diets. These data imply that skeletal muscle insulin sensitivity does not contribute to the whole‐body effects of LPHC and HPLC diets on glucose metabolism.

Reactive oxygen species (ROS) are increasingly recognized as key regulators of cellular metabolism. In skeletal muscle, ROS has been linked to glucose transport in isolated electrically stimulated muscles but the exact source of ROS in the context of in vivo exercise and whether it controls in vivo glucose uptake by glucose transporter 4 (GLUT4) is unknown. Here, we utilized a combination of genetically-encoded biosensors, fluorescent dyes and mouse models lacking the NADPH oxidase 2 (NOX2) regulatory subunits p47phox and Rac1 to demonstrate that NOX2 is the predominant source of ROS during in vivo treadmill exercise in skeletal muscle. Furthermore, p47phox-deficient mice phenocopy key aspects of metabolic dysregulation in Rac1 mKO mice, including decreased in vivo glucose uptake and GLUT4 translocation. These findings provide strong evidence that NOX2 is a major ROS source during in vivo exercise and its activity is crucial to the translocation of GLUT4 to increase glucose uptake.