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Aims/hypothesis Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain β (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. Methods Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, coimmunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B. Results We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM. Conclusions/interpretation These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes. Karla E. Merz and Arianne Aslamy contributed equally to this work.Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00125-019-4824-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Mitochondrial dysfunction is implicated in skeletal muscle insulin resistance. Syntaxin 4 (STX4) levels are reduced in human diabetic skeletal muscle, and global transgenic enrichment of STX4 expression improves insulin sensitivity in mice. Here, we show that transgenic skeletal muscle-specific STX4 enrichment (skmSTX4tg) in mice reverses established insulin resistance and improves mitochondrial function in the context of diabetogenic stress. Specifically, skmSTX4tg reversed insulin resistance caused by high-fat diet (HFD) without altering body weight or food consumption. Electron microscopy of wild-type mouse muscle revealed STX4 localisation at or proximal to the mitochondrial membrane. STX4 enrichment prevented HFD-induced mitochondrial fragmentation and dysfunction through a mechanism involving STX4-Drp1 interaction and elevated AMPK-mediated phosphorylation at Drp1 S637, which favors fusion. Our findings challenge the dogma that STX4 acts solely at the plasma membrane, revealing that STX4 localises at/proximal to and regulates the function of mitochondria in muscle. These results establish skeletal muscle STX4 enrichment as a candidate therapeutic strategy to reverse peripheral insulin resistance.

Skeletal muscle accounts for ~80% of insulin-stimulated glucose uptake. The Group I p21–activated kinase 1 (PAK1) is required for the non-canonical insulin-stimulated GLUT4 vesicle translocation in skeletal muscle cells. We found that the abundances of PAK1 protein and its downstream effector in muscle, ARPC1B, are significantly reduced in the skeletal muscle of humans with type 2 diabetes, compared to the non-diabetic controls, making skeletal muscle PAK1 a candidate regulator of glucose homeostasis. Although whole-body PAK1 knockout mice exhibit glucose intolerance and are insulin resistant, the contribution of skeletal muscle PAK1 in particular was unknown. As such, we developed inducible skeletal muscle-specific PAK1 knockout (skmPAK1-iKO) and overexpression (skmPAK1-iOE) mouse models to evaluate the role of PAK1 in skeletal muscle insulin sensitivity and glucose homeostasis. Using intraperitoneal glucose tolerance and insulin tolerance testing, we found that skeletal muscle PAK1 is required for maintaining whole body glucose homeostasis. Moreover, PAK1 enrichment in GLUT4-myc-L6 myoblasts preserves normal insulin-stimulated GLUT4 translocation under insulin resistance conditions. Unexpectedly, skmPAK1-iKO also showed aberrant plasma insulin levels following a glucose challenge. By applying conditioned media from PAK1-enriched myotubes or myoblasts to β-cells in culture, we established that a muscle-derived circulating factor(s) could enhance β-cell function. Taken together, these data suggest that PAK1 levels in the skeletal muscle can regulate not only skeletal muscle insulin sensitivity, but can also engage in tissue crosstalk with pancreatic β-cells, unveiling a new molecular mechanism by which PAK1 regulates whole-body glucose homeostasis.

Type 2 diabetes afflicts 10% of the US population and is the result of both peripheral insulin resistance (skeletal muscle and adipose tissue) as well as insulin secretion defects (pancreas). Insulin resistance, also commonly termed ‘pre‐diabetes’ is estimated to impact more than 30% of our population, and is caused by the inability of skeletal muscle to clear excess blood glucose from the circulation. Human skeletal muscle accounts for over 80% of glucose clearance in the body, a process important in maintaining glucose homeostasis. Currently the only therapeutics that directly improve muscle glucose uptake are diet and exercise. However, with only 3% of the population leading a “healthy lifestyle”, including exercise, directed therapeutics are required to restore skeletal muscle function in pre‐diabetics. Several studies have identified correlations between insulin resistance and reduced levels of a SNARE protein named Syntaxin 4 (STX4) in human skeletal muscle. STX4 interacts with other SNARE proteins to facilitate the fusion of glucose transporter 4 (GLUT4) vesicles with the plasma membrane, to then channel extracellular glucose to the interior of the muscle cell, ultimately clearing glucose from the circulation.To further define the function of STX4 in muscle, we developed a doxycycline Tet‐on inducible skeletal muscle specific STX4 overexpressing mouse model, under the muscle creatine kinase promoter (skm‐STX4 mice). Chow fed female mice overexpressing STX4 exhibited enhanced insulin sensitivity and improved glucose tolerance. Serum insulin levels from these mice were also reduced compared to littermate controls, concordant with their improved glucose tolerance. The data recapitulated the global overexpression model that we had previously published, suggesting that skeletal muscle is the primary driver in modulating glucose homeostasis. To establish that enriching STX4 can remediate insulin resistance, male skm‐STX4 mice were fed a 45% high fat diet (HF; to mimic a typical western diet), until they became insulin resistant. Upon establishing pre‐diabetes in the mice, STX4 enrichment was induced in the skeletal muscle (HFD+STX4 mice). Remarkably HFD+STX4 mice had the insulin sensitivity of a chow‐fed mouse, resolving the HFD‐linked insulin resistance in full. Moreover, the HFD+STX4 mice had significantly improved voluntary movement and increased respiratory exchange ratio, indicating increased reliance on carbohydrate rather than fat oxidation. The maximal oxygen consumption rate was also increased in primary myotubes from these HFD+ STX4 mice.Taken together, these results suggest that STX4 enrichment carries the potential as a therapeutic to remedy obesity‐linked insulin resistance, potentially via improving mitochondrial function. These results suggest that STX4 could be a viable therapeutic target for treatment of pre‐diabetes.Support or Funding InformationResearch was supported by the NIH (DK067912, DK102233, and DK112917) to D.C.T and by the H.N. & Frances C. Berger Foundation, and Helen & Payson Chu Fellowships to K.E.MThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal muscle accounts for ~80% of insulin‐stimulated glucose uptake. The Group I p21–activated kinase 1 (PAK1) is required for the non‐canonical insulin‐stimulated GLUT4 vesicle translocation in skeletal muscle cells. We have recently found that the abundance of PAK1 protein and its downstream effector in muscle, p41‐ARC, are significantly reduced in the skeletal muscle of human type 2 diabetic (T2D) donors. While whole body PAK1 knockout (KO) mice exhibit glucose intolerance and are insulin resistant, it remains unknown as to the relative contribution by the skeletal muscle to this phenotype. As such, we developed an inducible skeletal muscle‐specific PAK1 KO mouse model, as well as an inducible skeletal muscle‐specific PAK1 overexpressor (OE) mouse model, to discern the requirement for PAK1 in skeletal muscle insulin sensitivity, and the molecular mechanisms involved. Indeed, our skeletal muscle‐specific PAK1‐KO and ‐OE mouse studies demonstrate a requirement and role for PAK1 in skeletal muscle insulin sensitivity. Moreover, overexpression of PAK1 in GLUT4‐myc‐L6 myoblasts conferred protection of insulin‐stimulated GLUT4 translocation under insulin resistant conditions. Unexpectedly however, the skeletal muscle‐specific PAK1‐KO and ‐OE mouse models showed impact upon pancreatic function, suggesting that PAK1 expression levels in the skeletal muscle triggered release of an agent that impacted pancreatic responsiveness to blood glucose levels. Testing this concept, conditioned media (CM) collected from PAK1‐overexpressing myotubes was applied to pancreatic β‐cells in culture. Remarkably, β‐cells treated with CM from PAK1‐overexpressing cells showed enhanced glucose‐stimulated insulin release, akin to that seen in the skeletal muscle‐specific PAK1‐OE mice; this enhancement response was selective to PAK1‐derived CM and was not seen with other CM types tested. Taken together, these data suggest that PAK1 levels in the skeletal muscle can exert feedback to the pancreatic β‐cells, unveiling a new molecular mechanism by which PAK1 regulates whole body insulin sensitivity and glucose homeostasis. Support or Funding Information This work was supported in part by grants to DCT from the NIH (DK102233, DK067912) and a gift from the Joel Katz fund.

Type 2 diabetes results from a combination of peripheral insulin resistance plus insulin secretion defects. Insulin resistance is characterized by a failure of skeletal muscle to clear excess blood glucose from the circulation, and is central to what is commonly called ‘pre‐diabetes'. While type 2 diabetes afflicts ~10% of the US population, pre‐diabetes is estimated to impact more than a third of our population. Over 80% of glucose clearance occurs via the skeletal muscle, there are no directed therapeutics to restore its function in pre‐diabetics. Several studies have identified correlations between insulin resistance and reduced levels of a SNARE protein named Syntaxin 4 (STX4) in human skeletal muscle. STX4 interacts with other SNARE proteins to facilitate the fusion of glucose transporter 4 (GLUT4) vesicles with the plasma membrane, to then channel extracellular glucose to the interior of the muscle cell, ultimately ‘clearing glucose’ from the circulation. Towards understanding how STX4 content is regulated in muscle, we mimicked the pre/diabetic milieu of high glucose and high lipid content in the L6‐GLUT4myc skeletal muscle cells and demonstrated the same STX4 depletion reported in the human studies. Having established this model system, we next assessed the potential for STX4 overexpression to prevent insulin resistance, and found STX4‐overexpressing cells show less Annexin V and Propidium iodide staining compared with sham‐transfected cells, suggesting that STX4 overexpression exerted a protective effect against diabetogenic stimuli‐induced apoptosis. To next test this in vivo, we have developed an inducible skeletal muscle specific STX4 overexpressing mouse model. Remarkably, under standard non‐diabetogenic conditions, female mice overexpressing STX4 show heightened glucose tolerance and insulin sensitivity compared to control mice. Whether STX4‐overexpressing mice are protected from obesity‐induced insulin resistance, or perhaps whether STX4 upregulation in already‐obese mice might reverse insulin resistance and through which mechanism, are questions currently under pursuit. Taken together, these results suggest that STX4 functions in the skeletal muscle to facilitate glucose uptake function and to protect myocytes from diabetogenic stress induced apoptosis. This suggests that STX4 could be a viable therapeutic target for treatment and prevention of pre‐diabetes.Support or Funding InformationThis research was supported by the National Institutes of Health (DK067912, DK102233) to D.C.T. and also by the H.N. & Frances C. Berger Foundation Fellowship to K.MThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.