Exercise increases phosphorylation of the putative mTORC2 activity readout NDRG1 in human skeletal muscle.

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 ß2-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 ß2-adrenergic or GS 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 ß2-adrenergic or GS 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 ß2-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. ß2-AR and GS 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.

AXIN1 knockout does not alter AMPK/mTORC1 regulation and glucose metabolism in mouse skeletal muscle.

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 signalling during AICAR/insulin/amino acid incubation, contraction and exercise. During exercise, α2/ß2/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 signalling or glucose uptake stimulation at rest or 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/ß2/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 signalling and glucose metabolism, probably due to functional redundancy of its homologue AXIN2.

c-Myc overexpression increases ribosome biogenesis and protein synthesis independent of mTORC1 activation in mouse skeletal muscle.

High-intensity muscle contractions (HiMCs) are known to increase c-Myc expression that is known to stimulate ribosome biogenesis and protein synthesis in most cells. However, although c-Myc mRNA transcription and c-Myc mRNA translation have been shown to be upregulated following resistance exercise concomitantly with increased ribosome biogenesis, this connection has not been tested directly. 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. AAV-mediated overexpression of c-Myc in mouse skeletal muscles for 2 wk 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-sequencing (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. Our results suggest that 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.NEW & NOTEWORTHY Resistance exercise is known to increase c-Myc expression, which is known to stimulate ribosome biogenesis and protein synthesis in a variety of cells. However, whether the increase in c-Myc stimulates ribosome biogenesis and protein synthesis in skeletal muscles remains unknown. We found that c-Myc overexpression is sufficient to stimulate skeletal muscle ribosome biogenesis and protein synthesis without activation of mTORC1.

Rapamycin and mTORC2 inhibition synergistically reduce contraction-stimulated muscle protein synthesis.

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.

Contraction-regulated mTORC1 and protein synthesis: Influence of AMPK and glycogen.

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.

Insulin-stimulated glucose uptake partly relies on p21-activated kinase (PAK)2, but not PAK1, in mouse skeletal muscle.

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 < 0.001). However, because there was no phenotype with genetic ablation of PAK1 alone, consequently, the relative requirement for PAK1 and PAK2 in whole-body glucose homeostasis and insulin-stimulated muscle glucose uptake was investigated. Whole-body respiratory exchange ratio was largely unaffected in whole-body PAK1 knockout (KO), muscle-specific PAK2 KO and in mice with combined whole-body PAK1 KO and muscle-specific PAK2 KO. By contrast, glucose tolerance was mildly impaired in mice lacking PAK2 specifically in muscle, but not PAK1 KO mice. Moreover, while PAK1 KO muscles displayed normal insulin-stimulated glucose uptake in vivo and in isolated muscle, insulin-stimulated glucose uptake was slightly reduced in isolated glycolytic extensor digitorum longus muscle lacking PAK2 alone (-18%) or in combination with PAK1 KO (-12%) (P < 0.05). In conclusion, glucose tolerance and insulin-stimulated glucose uptake partly rely on PAK2 in glycolytic mouse muscle, whereas PAK1 is dispensable for whole-body glucose homeostasis and insulin-stimulated muscle glucose uptake.

The ULK1/2 and AMPK Inhibitor SBI-0206965 Blocks AICAR and Insulin-Stimulated Glucose Transport.

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.

ß-Actin shows limited mobility and is required only for supraphysiological insulin-stimulated glucose transport in young adult soleus muscle.

Studies in skeletal muscle cell cultures suggest that the cortical actin cytoskeleton is a major requirement for insulin-stimulated glucose transport, implicating the ß-actin isoform, which in many cell types is the main actin isoform. However, it is not clear that ß-actin plays such a role in mature skeletal muscle. Neither dependency of glucose transport on ß-actin nor actin reorganization upon glucose transport have been tested in mature muscle. To investigate the role of ß-actin in fully differentiated muscle, we performed a detailed characterization of wild type and muscle-specific ß-actin knockout (KO) mice. The effects of the ß-actin KO were subtle; however, we confirmed the previously reported decline in running performance of ß-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 ß-actin mobility in mature muscle, we electroporated green fluorescent protein (GFP)-ß-actin into flexor digitorum brevis muscle fibers and measured fluorescence recovery after photobleaching. GFP-ß-actin showed limited unstimulated mobility and no changes after insulin stimulation. In conclusion, ß-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 ß-actin cytoskeleton in mature muscle compared with cell culture.

Mammalian target of rapamycin complex 2 regulates muscle glucose uptake during exercise in mice.

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.

PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain.

AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α1/α2) is bound to one of two ß subunits (ß1/ß2) 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.

Transparent and Cell-Guiding Cellulose Nanofiber 3D Printing Bioinks.

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.

Adenosine monophosphate-activated protein kinase is elevated in human cachectic muscle and prevents cancer-induced metabolic dysfunction in mice.

BACKGROUND: Metabolic dysfunction and cachexia are associated with poor cancer prognosis. With no pharmacological treatments, it is crucial to define the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia. Adenosine monophosphate-activated protein kinase (AMPK) connects metabolic and muscle mass regulation. As AMPK could be a potential treatment target, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. We therefore established AMPK's roles in cancer-associated metabolic dysfunction, insulin resistance and cachexia. METHODS: In vastus lateralis muscle biopsies from n = 26 patients with non-small cell lung cancer (NSCLC), AMPK signalling and protein content were examined by immunoblotting. To determine the role of muscle AMPK, male mice overexpressing a dominant-negative AMPKα2 (kinase-dead [KiDe]) specifically in striated muscle were inoculated with Lewis lung carcinoma (LLC) cells (wild type [WT]: n = 27, WT + LLC: n = 34, mAMPK-KiDe: n = 23, mAMPK-KiDe + LLC: n = 38). Moreover, male LLC-tumour-bearing mice were treated with (n = 10)/without (n = 9) 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13 days. Littermate mice were used as controls. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose and insulin tolerance tests, tissue-specific 2-[3H]deoxy-d-glucose (2-DG) uptake and immunoblotting. RESULTS: Patients with NSCLC presented increased muscle protein content of AMPK subunits α1, α2, ß2, γ1 and γ3 ranging from +27% to +79% compared with control subjects. In patients with NSCLC, AMPK subunit protein content correlated with weight loss (α1, α2, ß2 and γ1), fat-free mass (α1, ß2 and γ1) and fat mass (α1 and γ1). Tumour-bearing mAMPK-KiDe mice presented increased fat loss and glucose and insulin intolerance. LLC in mAMPK-KiDe mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps: -35%, soleus: -49%, extensor digitorum longus: -48%) and the heart (-29%) than that in non-tumour-bearing mice. In skeletal muscle, mAMPK-KiDe abrogated the tumour-induced increase in insulin-stimulated TBC1D4thr642 phosphorylation. The protein content of TBC1D4 (+26%), pyruvate dehydrogenase (PDH; +94%), PDH kinases (+45% to +100%) and glycogen synthase (+48%) was increased in skeletal muscle of tumour-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase II protein content and normalized phosphorylation of p70S6Kthr389 (mTORC1 substrate) and ACCser212 (AMPK substrate) and rescued cancer-induced insulin intolerance. CONCLUSIONS: Protein contents of AMPK subunits were upregulated in skeletal muscle of patients with NSCLC. AMPK activation seemed protectively inferred by AMPK-deficient mice developing metabolic dysfunction in response to cancer, including AMPK-dependent regulation of multiple proteins crucial for glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and possibly cachexia.

NOX2 deficiency exacerbates diet-induced obesity and impairs molecular training adaptations in skeletal muscle.

The production of reactive oxygen species (ROS) by NADPH oxidase (NOX) 2 has been linked to both insulin resistance and exercise training adaptations in skeletal muscle. This study explores the previously unexamined role of NOX2 in the interplay between diet-induced insulin resistance and exercise training (ET). Using a mouse model that harbors a point mutation in the essential NOX2 regulatory subunit, p47phox (Ncf1*), we investigated the impact of this mutation on various metabolic adaptations. Wild-type (WT) and Ncf1* mice were assigned to three groups: chow diet, 60% energy fat diet (HFD), and HFD with access to running wheels (HFD + E). After a 16-week intervention, a comprehensive phenotypic assessment was performed, including body composition, glucose tolerance, energy intake, muscle insulin signaling, redox-related proteins, and mitochondrial adaptations. The results revealed that NOX2 deficiency exacerbated the impact of HFD on body weight, body composition, and glucose intolerance. Moreover, in Ncf1* mice, ET did not improve glucose tolerance or increase muscle cross-sectional area. ET normalized body fat independently of genotype. The lack of NOX2 activity during ET reduced several metabolic adaptations in skeletal muscle, including insulin signaling and expression of Hexokinase II and oxidative phosphorylation complexes. In conclusion, these findings suggest that NOX2 mediates key beneficial effects of exercise training in the context of diet-induced obesity.

AMPKγ3 Controls Muscle Glucose Uptake in Recovery From Exercise to Recapture Energy Stores.

Exercise increases muscle glucose uptake independently of insulin signaling and represents a cornerstone for the prevention of metabolic disorders. Pharmacological activation of the exercise-responsive AMPK in skeletal muscle has been proven successful as a therapeutic approach to treat metabolic disorders by improving glucose homeostasis through the regulation of muscle glucose uptake. However, conflicting observations cloud the proposed role of AMPK as a necessary regulator of muscle glucose uptake during exercise. We show that glucose uptake increases in human skeletal muscle in the absence of AMPK activation during exercise and that exercise-stimulated AMPKγ3 activity strongly correlates to muscle glucose uptake in the postexercise period. In AMPKγ3-deficient mice, muscle glucose uptake is normally regulated during exercise and contractions but impaired in the recovery period from these stimuli. Impaired glucose uptake in recovery from exercise and contractions is associated with a lower glucose extraction, which can be explained by a diminished permeability to glucose and abundance of GLUT4 at the muscle plasma membrane. As a result, AMPKγ3 deficiency impairs muscle glycogen resynthesis following exercise. These results identify a physiological function of the AMPKγ3 complex in human and rodent skeletal muscle that regulates glucose uptake in recovery from exercise to recapture muscle energy stores. ARTICLE HIGHLIGHTS: Exercise-induced activation of AMPK in skeletal muscle has been proposed to regulate muscle glucose uptake in recovery from exercise. This study investigated whether the muscle-specific AMPKγ3-associated heterotrimeric complex was involved in regulating muscle glucose metabolism in recovery from exercise. The findings support that exercise-induced activation of the AMPKγ3 complex in human and mouse skeletal muscle enhances glucose uptake in recovery from exercise via increased translocation of GLUT4 to the plasma membrane. This work uncovers the physiological role of the AMPKγ3 complex in regulating muscle glucose uptake that favors replenishment of the muscle cellular energy stores.

Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle.

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 2-hr 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.

Gene deletion of γ-actin impairs insulin-stimulated skeletal muscle glucose uptake in growing mice but not in mature adult mice.

The cortical cytoskeleton, consisting of the cytoplasmic actin isoforms ß 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 ß-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 ß-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.

In vivo metabolic effects after acute activation of skeletal muscle Gs signaling.

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 (ß2-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 ß2-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.

Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells.

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with ß2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective ß2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of ß-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle ß2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic ß2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating ß2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.

The p21-activated kinase 2 (PAK2), but not PAK1, regulates contraction-stimulated skeletal muscle glucose transport.

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.