Loss of functional β-cell mass is an early feature of type 1 diabetes. To release insulin, β-cells require soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, as well as SNARE complex regulatory proteins like double C2 domain-containing protein β (Doc2b). We hypothesized that Doc2b deficiency or overabundance may confer susceptibility or protection, respectively, to the functional β-cell mass. Indeed, Doc2b knockout mice show an unusually severe response to multiple-low-dose streptozotocin (MLD-STZ), resulting in more apoptotic β-cells and a smaller β-cell mass. In addition, inducible β-cell-specific Doc2b-overexpressing transgenic (βDoc2b-dTg) mice show improved glucose tolerance and resist MLD-STZ-induced disruption of glucose tolerance, fasting hyperglycemia, β-cell apoptosis, and loss of β-cell mass. Mechanistically, Doc2b enrichment enhances glucose-stimulated insulin secretion (GSIS) and SNARE activation and prevents the appearance of apoptotic markers in response to cytokine stress and thapsigargin. Furthermore, expression of a peptide containing the Doc2b tandem C2A and C2B domains is sufficient to confer the beneficial effects of Doc2b enrichment on GSIS, SNARE activation, and apoptosis. These studies demonstrate that Doc2b enrichment in the β-cell protects against diabetogenic and proapoptotic stress. Furthermore, they identify a Doc2b peptide that confers the beneficial effects of Doc2b and may be a therapeutic candidate for protecting functional β-cell mass.

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.

Impaired skeletal muscle (skm) insulin signaling results in insulin resistance and the development of type 2 diabetes (T2D). Insulin binding to its receptors on skm cells leads to an intracellular signaling cascade resulting in glucose transporter type 4 (GLUT4) translocation to the plasma membrane (PM) and glucose uptake. The p21–activated kinase 1 (PAK1) is a required element for skm insulin sensitivity. It has been shown that whole body PAK1−/− knock‐out (KO) mice exhibit glucose intolerance and insulin resistance. In clonal skm cells (L6‐GLUT4myc) PAK1 inhibition was shown to impair insulin‐induced filamentous actin (F‐actin) remodeling and GLUT4 translocation; coupled with a loss of normal insulin‐stimulated cofilin dephosphorylation (i.e. activation). Recently, we have shown that PAK1 is essential in the process of actin polymerization via p41‐ARC (Actin‐related protein 2/3 complex subunit 1B). However, the effect of skm specific PAK1 modulations on in vivo glucose homeostasis, and downstream effectors of PAK1 are yet to be fully determined.We hypothesize that PAK1 is essential for insulin signaling in skm, and that upon diabetogenic stress, i) reduced PAK1 levels in skm will adversely affect whole body insulin sensitivity, whereas ii) PAK1 enrichment can preserve or restore insulin sensitivity. To study the effects of PAK1 modulation on insulin sensitivity in vivo, state‐of‐the‐art inducible skm‐specific PAK1 KO and overexpressing mouse models are used. L6‐GLUT4myc clonal cells are used to identify key downstream effectors of PAK1 using cutting‐edge imaging, mass spectrometry, and metabolomic analyzes.Importantly, both PAK1 and p41‐ARC protein levels were found to be significantly reduced in the skm of human T2D donors compared to healthy human donors (n=3–4). We have generated a doxycycline‐inducible skm‐specific, i) PAK1 depletion mouse model (skm‐PAK1‐KO) and ii) PAK1 overexpressing transgenic mouse model (skm‐PAK1‐Tg). PAK1 deletion in skm resulted into impaired in vivo insulin sensitivity in skm‐PAK1‐KO mice compared to control mice (n=10, per group), studied by intraperitoneal insulin tolerance test (IPITT). Moreover, this was coupled with reduced insulin‐induced GLUT4 translocation in skm of skm‐PAK1‐KO mice compared to the control mice (n=2). Conversely, overexpression of PAK1 in L6‐GLUT4myc cells significantly improved GLUT4 translocation under insulin resistance condition (5 nM of insulin for 12 h) compared to control cells (n=4). Altogether, these findings emphasize the i) importance of PAK1 in skm and thereby whole body glucose homeostasis and ii) potential protective effect of PAK1 enrichment on skm insulin sensitivity under diabetogenic conditions. Moreover, this study in future is aimed at determining key downstream effectors of PAK1, which might serve as insulin sensitizers.Support or Funding InformationThis study was supported by grants from the National Institutes of Health (DK067912 and DK102233 to D.C.T.), the American Heart Association (15PRE21970002 to RT, 17POST33661194 to JZ), as well a gift from the Ruth and Robert Lanman Endowment (to D.C.T.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.