Mitochondrial Dysfunction, Oxidative Stress, and Inter-Organ Miscommunications in T2D Progression.

Type 2 diabetes (T2D) is a heterogenous disease, and conventionally, peripheral insulin resistance (IR) was thought to precede islet ß-cell dysfunction, promoting progression from prediabetes to T2D. New evidence suggests that T2D-lean individuals experience early ß-cell dysfunction without significant IR. Regardless of the primary event (i.e., IR vs. ß-cell dysfunction) that contributes to dysglycemia, significant early-onset oxidative damage and mitochondrial dysfunction in multiple metabolic tissues may be a driver of T2D onset and progression. Oxidative stress, defined as the generation of reactive oxygen species (ROS), is mediated by hyperglycemia alone or in combination with lipids. Physiological oxidative stress promotes inter-tissue communication, while pathological oxidative stress promotes inter-tissue mis-communication, and new evidence suggests that this is mediated via extracellular vesicles (EVs), including mitochondria containing EVs. Under metabolic-related stress conditions, EV-mediated cross-talk between ß-cells and skeletal muscle likely trigger mitochondrial anomalies leading to prediabetes and T2D. This article reviews the underlying molecular mechanisms in ROS-related pathogenesis of prediabetes, including mitophagy and mitochondrial dynamics due to oxidative stress. Further, this review will describe the potential of various therapeutic avenues for attenuating oxidative damage, reversing prediabetes and preventing progression to T2D.

Toward Ameliorating Insulin Resistance: Targeting a Novel PAK1 Signaling Pathway Required for Skeletal Muscle Mitochondrial Function.

The p21-activated kinase 1 (PAK1) is required for insulin-stimulated glucose uptake in skeletal muscle cells. However, whether PAK1 regulates skeletal muscle mitochondrial function, which is a central determinant of insulin sensitivity, is unknown. Here, the effect of modulating PAK1 levels (knockdown via siRNA, overexpression via adenoviral transduction, and/or inhibition of activation via IPA3) on mitochondrial function was assessed in normal and/or insulin-resistant rat L6.GLUT4myc and human muscle (LHCN-M2) myotubes. Human type 2 diabetes (T2D) and non-diabetic (ND) skeletal muscle samples were also used for validation of the identified signaling elements. PAK1 depletion in myotubes decreased mitochondrial copy number, respiration, altered mitochondrial structure, downregulated PGC1α (a core regulator of mitochondrial biogenesis and oxidative metabolism) and PGC1α activators, p38 mitogen-activated protein kinase (p38MAPK) and activating transcription factor 2 (ATF2). PAK1 enrichment in insulin-resistant myotubes improved mitochondrial function and rescued PGC1α expression levels. Activated PAK1 was localized to the cytoplasm, and PAK1 enrichment concurrent with p38MAPK inhibition did not increase PGC1α levels. PAK1 inhibition and enrichment also modified nuclear phosphorylated-ATF2 levels. T2D human samples showed a deficit for PGC1α, and PAK1 depletion in LHCN-M2 cells led to reduced mitochondrial respiration. Overall, the results suggest that PAK1 regulates muscle mitochondrial function upstream of the p38MAPK/ATF2/PGC1α-axis pathway.

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic ß cells.

Male mice lacking the androgen receptor (AR) in pancreatic ß cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in ß cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male ß cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male ß cells.

Methylcellulose colony assay and single-cell micro-manipulation reveal progenitor-like cells in adult human pancreatic ducts.

Progenitor cells capable of self-renewal and differentiation in the adult human pancreas are an under-explored resource for regenerative medicine. Using micro-manipulation and three-dimensional colony assays we identify cells within the adult human exocrine pancreas that resemble progenitor cells. Exocrine tissues were dissociated into single cells and plated into a colony assay containing methylcellulose and 5% Matrigel. A subpopulation of ductal cells formed colonies containing differentiated ductal, acinar, and endocrine lineage cells, and expanded up to 300-fold with a ROCK inhibitor. When transplanted into diabetic mice, colonies pre-treated with a NOTCH inhibitor gave rise to insulin-expressing cells. Both colonies and primary human ducts contained cells that simultaneously express progenitor transcription factors SOX9, NKX6.1, and PDX1. In addition, in silico analysis identified progenitor-like cells within ductal clusters in a single-cell RNA sequencing dataset. Therefore, progenitor-like cells capable of self-renewal and tri-lineage differentiation either pre-exist in the adult human exocrine pancreas, or readily adapt in culture.

A Novel Role for DOC2B in Ameliorating Palmitate-Induced Glucose Uptake Dysfunction in Skeletal Muscle Cells via a Mechanism Involving ß-AR Agonism and Cofilin.

Diet-related lipotoxic stress is a significant driver of skeletal muscle insulin resistance (IR) and type 2 diabetes (T2D) onset. ß2-adrenergic receptor (ß-AR) agonism promotes insulin sensitivity in vivo under lipotoxic stress conditions. Here, we established an in vitro paradigm of lipotoxic stress using palmitate (Palm) in rat skeletal muscle cells to determine if ß-AR agonism could cooperate with double C-2-like domain beta (DOC2B) enrichment to promote skeletal muscle insulin sensitivity under Palm-stress conditions. Previously, human T2D skeletal muscles were shown to be deficient for DOC2B, and DOC2B enrichment resisted IR in vivo. Our Palm-stress paradigm induced IR and ß-AR resistance, reduced DOC2B protein levels, triggered cytoskeletal cofilin phosphorylation, and reduced GLUT4 translocation to the plasma membrane (PM). By enhancing DOC2B levels in rat skeletal muscle, we showed that the deleterious effects of palmitate exposure upon cofilin, insulin, and ß-AR-stimulated GLUT4 trafficking to the PM and glucose uptake were preventable. In conclusion, we revealed a useful in vitro paradigm of Palm-induced stress to test for factors that can prevent/reverse skeletal muscle dysfunctions related to obesity/pre-T2D. Discerning strategies to enrich DOC2B and promote ß-AR agonism can resist skeletal muscle IR and halt progression to T2D.

Exocytosis Proteins: Typical and Atypical Mechanisms of Action in Skeletal Muscle.

Insulin-stimulated glucose uptake in skeletal muscle is of fundamental importance to prevent postprandial hyperglycemia, and long-term deficits in insulin-stimulated glucose uptake underlie insulin resistance and type 2 diabetes. Skeletal muscle is responsible for ~80% of the peripheral glucose uptake from circulation via the insulin-responsive glucose transporter GLUT4. GLUT4 is mainly sequestered in intracellular GLUT4 storage vesicles in the basal state. In response to insulin, the GLUT4 storage vesicles rapidly translocate to the plasma membrane, where they undergo vesicle docking, priming, and fusion via the high-affinity interactions among the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) exocytosis proteins and their regulators. Numerous studies have elucidated that GLUT4 translocation is defective in insulin resistance and type 2 diabetes. Emerging evidence also links defects in several SNAREs and SNARE regulatory proteins to insulin resistance and type 2 diabetes in rodents and humans. Therefore, we highlight the latest research on the role of SNAREs and their regulatory proteins in insulin-stimulated GLUT4 translocation in skeletal muscle. Subsequently, we discuss the novel emerging role of SNARE proteins as interaction partners in pathways not typically thought to involve SNAREs and how these atypical functions reveal novel therapeutic targets for combating peripheral insulin resistance and diabetes.

Mechanisms by Which Skeletal Muscle Myokines Ameliorate Insulin Resistance.

The skeletal muscle is the largest organ in the body and secretes circulating factors, including myokines, which are involved in various cellular signaling processes. Skeletal muscle is vital for metabolism and physiology and plays a crucial role in insulin-mediated glucose disposal. Myokines have autocrine, paracrine, and endocrine functions, serving as critical regulators of myogenic differentiation, fiber-type switching, and maintaining muscle mass. Myokines have profound effects on energy metabolism and inflammation, contributing to the pathophysiology of type 2 diabetes (T2D) and other metabolic diseases. Myokines have been shown to increase insulin sensitivity, thereby improving glucose disposal and regulating glucose and lipid metabolism. Many myokines have now been identified, and research on myokine signaling mechanisms and functions is rapidly emerging. This review summarizes the current state of the field regarding the role of myokines in tissue cross-talk, including their molecular mechanisms, and their potential as therapeutic targets for T2D.

DOC2b Enhances ß-Cell Function via a Novel Tyrosine Phosphorylation-Dependent Mechanism.

Double C2 domain Β (DOC2b) protein is required for glucose-stimulated insulin secretion (GSIS) in ß-cells, the underlying mechanism of which remains unresolved. Our biochemical analysis using primary human islets and human and rodent clonal ß-cells revealed that DOC2b is tyrosine phosphorylated within 2 min of glucose stimulation, and Src family kinase member YES is required for this process. Biochemical and functional analysis using DOC2bY301 mutants revealed the requirement of Y301 phosphorylation for the interaction of DOC2b with YES kinase and increased content of VAMP2, a protein on insulin secretory granules, at the plasma membrane (PM), concomitant with DOC2b-mediated enhancement of GSIS in ß-cells. Coimmunoprecipitation studies demonstrated an increased association of DOC2b with ERM family proteins in ß-cells following glucose stimulation or pervanadate treatment. Y301 phosphorylation-competent DOC2b was required to increase ERM protein activation, and ERM protein knockdown impaired DOC2b-mediated boosting of GSIS, suggesting that tyrosine-phosphorylated DOC2b regulates GSIS via ERM-mediated granule localization to the PM. Taken together, these results demonstrate the glucose-induced posttranslational modification of DOC2b in ß-cells, pinpointing the kinase, site of action, and downstream signaling events and revealing a regulatory role of YES kinase at various steps in GSIS. This work will enhance the development of novel therapeutic strategies to restore glucose homeostasis in diabetes.

Changes in Skeletal Muscle PAK1 Levels Regulate Tissue Crosstalk to Impact Whole Body Glucose Homeostasis.

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 ß-cells in culture, we established that a muscle-derived circulating factor(s) could enhance ß-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 ß-cells, unveiling a new molecular mechanism by which PAK1 regulates whole-body glucose homeostasis.

Enrichment of the exocytosis protein STX4 in skeletal muscle remediates peripheral insulin resistance and alters mitochondrial dynamics via Drp1.

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.

Syntaxin 4 Enrichment in ß-Cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice.

Syntaxin 4 (STX4), a plasma membrane-localized SNARE protein, regulates human islet ß-cell insulin secretion and preservation of ß-cell mass. We found that human type 1 diabetes (T1D) and NOD mouse islets show reduced ß-cell STX4 expression, consistent with decreased STX4 expression, as a potential driver of T1D phenotypes. To test this hypothesis, we generated inducible ß-cell-specific STX4-expressing NOD mice (NOD-ißSTX4). Of NOD-ißSTX4 mice, 73% had sustained normoglycemia vs. <20% of control NOD (NOD-Ctrl) mice by 25 weeks of age. At 12 weeks of age, before diabetes conversion, NOD-ißSTX4 mice demonstrated superior whole-body glucose tolerance and ß-cell glucose responsiveness than NOD-Ctrl mice. Higher ß-cell mass and reduced ß-cell apoptosis were also detected in NOD-ißSTX4 pancreata compared with pancreata of NOD-Ctrl mice. Single-cell RNA sequencing revealed that islets from NOD-ißSTX4 had markedly reduced interferon-γ signaling and tumor necrosis factor-α signaling via nuclear factor-κB in islet ß-cells, including reduced expression of the chemokine CCL5; CD4+ regulatory T cells were also enriched in NOD-ißSTX4 islets. These results provide a deeper mechanistic understanding of STX4 function in ß-cell protection and warrant further investigation of STX4 enrichment as a strategy to reverse or prevent T1D in humans or protect ß-cell grafts.

Emerging Roles of Small GTPases in Islet ß-Cell Function.

Several small guanosine triphosphatases (GTPases) from the Ras protein superfamily regulate glucose-stimulated insulin secretion in the pancreatic islet ß-cell. The Rho family GTPases Cdc42 and Rac1 are primarily involved in relaying key signals in several cellular functions, including vesicle trafficking, plasma membrane homeostasis, and cytoskeletal dynamics. They orchestrate specific changes at each spatiotemporal region within the ß-cell by coordinating with signal transducers, guanine nucleotide exchange factors (GEFs), GTPase-activating factors (GAPs), and their effectors. The Arf family of small GTPases is involved in vesicular trafficking (exocytosis and endocytosis) and actin cytoskeletal dynamics. Rab-GTPases regulate pre-exocytotic and late endocytic membrane trafficking events in ß-cells. Several additional functions for small GTPases include regulating transcription factor activity and mitochondrial dynamics. Importantly, defects in several of these GTPases have been found associated with type 2 diabetes (T2D) etiology. The purpose of this review is to systematically denote the identities and molecular mechanistic steps in the glucose-stimulated insulin secretion pathway that leads to the normal release of insulin. We will also note newly identified defects in these GTPases and their corresponding regulatory factors (e.g., GDP dissociation inhibitors (GDIs), GEFs, and GAPs) in the pancreatic ß-cells, which contribute to the dysregulation of metabolism and the development of T2D.

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee ß-cell Function and Protection.

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic ß-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to ß-cells improves whole-body glucose homeostasis, enhances ß-cell function, and surprisingly, protection of ß-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of ß-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.

Syntaxin 4 Mediates NF-κB Signaling and Chemokine Ligand Expression via Specific Interaction With IκBß.

Enrichment of human islets with syntaxin 4 (STX4) improves functional ß-cell mass through a nuclear factor-κB (NF-κB)-dependent mechanism. However, the detailed mechanisms underlying the protective effect of STX4 are unknown. For determination of the signaling events linking STX4 enrichment and downregulation of NF-κB activity, STX4 was overexpressed in human islets, EndoC-ßH1 and INS-1 832/13 cells in culture, and the cells were challenged with the proinflammatory cytokines interleukin-1ß, tumor necrosis factor-α, and interferon-γ individually and in combination. STX4 expression suppressed cytokine-induced proteasomal degradation of IκBß but not IκBα. Inhibition of IKKß prevented IκBß degradation, suggesting that IKKß phosphorylates IκBß. Moreover, the IKKß inhibitor, as well as a proteosomal degradation inhibitor, prevented the loss of STX4 caused by cytokines. This suggests that STX4 may be phosphorylated by IKKß in response to cytokines, targeting STX4 for proteosomal degradation. Expression of a stabilized form of STX4 further protected IκBß from proteasomal degradation, and like wild-type STX4, stabilized STX4 coimmunoprecipitated with IκBß and the p50-NF-κB. This work proposes a novel pathway wherein STX4 regulates cytokine-induced NF-κB signaling in ß-cells via associating with and preventing IκBß degradation, suppressing chemokine expression, and protecting islet ß-cells from cytokine-mediated dysfunction and demise.

A requirement for PAK1 to support mitochondrial function and maintain cellular redox balance via electron transport chain proteins to prevent ß-cell apoptosis.

OBJECTIVE: p21 (Cdc42/Rac1) activated Kinase 1 (PAK1) is a candidate susceptibility factor for type 2 diabetes (T2D). PAK1 is depleted in the islets from T2D donors, compared to control individuals. In addition, whole-body PAK1 knock out (PAK1-KO) in mice worsens the T2D-like effects of high-fat diet. The current study tested the effects of modulating PAK1 levels only in ß-cells. MATERIALS/METHODS: ß-cell-specific inducible PAK1 KO (ßPAK1-iKO) mice were generated and used with human ß-cells and T2D islets to evaluate ß-cell function. RESULTS: ßPAK1-iKO mice exhibited glucose intolerance and elevated ß-cell apoptosis, but without peripheral insulin resistance. ß-cells from ßPAK-iKO mice also contained fewer mitochondria per cell. At the cellular level, human PAK1-deficient ß-cells showed blunted glucose-stimulated insulin secretion and reduced mitochondrial function. Mitochondria from human PAK1-deficient ß-cells were deficient in the electron transport chain (ETC) subunits CI, CIII, and CIV; NDUFA12, a CI complex protein, was identified as a novel PAK1 binding partner, and was significantly reduced with PAK1 knockdown. PAK1 knockdown disrupted the NAD+/NADH and NADP+/NADPH ratios, and elevated ROS. An imbalance of the redox state due to mitochondrial dysfunction leads to ER stress in ß-cells. PAK1 replenishment in the ß-cells of T2D human islets ameliorated levels of ER stress markers. CONCLUSIONS: These findings support a protective function for PAK1 in ß-cells. The results support a new model whereby the PAK1 in the ß-cell plays a required role upstream of mitochondrial function, via maintaining ETC protein levels and averting stress-induced ß-cell apoptosis to retain healthy functional ß-cell mass.

Role of Skeletal Muscle in Insulin Resistance and Glucose Uptake.

The skeletal muscle is the largest organ in the body, by mass. It is also the regulator of glucose homeostasis, responsible for 80% of postprandial glucose uptake from the circulation. Skeletal muscle is essential for metabolism, both for its role in glucose uptake and its importance in exercise and metabolic disease. In this article, we give an overview of the importance of skeletal muscle in metabolism, describing its role in glucose uptake and the diseases that are associated with skeletal muscle metabolic dysregulation. We focus on the role of skeletal muscle in peripheral insulin resistance and the potential for skeletal muscle-targeted therapeutics to combat insulin resistance and diabetes, as well as other metabolic diseases like aging and obesity. In particular, we outline the possibilities and pitfalls of the quest for exercise mimetics, which are intended to target the molecular mechanisms underlying the beneficial effects of exercise on metabolic disease. We also provide a description of the molecular mechanisms that regulate skeletal muscle glucose uptake, including a focus on the SNARE proteins, which are essential regulators of glucose transport into the skeletal muscle. © 2020 American Physiological Society. Compr Physiol 10:785-809, 2020.

Recent Insights into Beta-cell Exocytosis in Type 2 Diabetes.

As one of the leading causes of morbidity and mortality worldwide, diabetes affects an estimated 422 million adults, and it is expected to continue expanding such that by 2050, 30% of the U.S. population will become diabetic within their lifetime. Out of the estimated 422 million people currently afflicted with diabetes worldwide, about 5% have type 1 diabetes (T1D), while the remaining ~95% of diabetics have type 2 diabetes (T2D). Type 1 diabetes results from the autoimmune-mediated destruction of functional ß-cell mass, whereas T2D results from combinatorial defects in functional ß-cell mass plus peripheral glucose uptake. Both types of diabetes are now believed to be preceded by ß-cell dysfunction. T2D is increasingly associated with numerous reports of deficiencies in the exocytosis proteins that regulate insulin release from ß-cells, specifically the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE protein's functionality is further regulated by a variety of accessory factors such as Sec1/Munc18 (SM), double C2-domain proteins (DOC2), and additional interacting proteins at the cell surface that influence the fidelity of insulin release. As new evidence emerges about the detailed mechanisms of exocytosis, new questions and controversies have come to light. This emerging information is also contributing to dialogue in the islet biology field focused on how to correct the defects in insulin exocytosis. Herein we present a balanced review of the role of exocytosis proteins in T2D, with thoughts on novel strategies to protect functional ß-cell mass.

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle.

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 ß (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, co-immunoprecipitation 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.

Syntaxin 4 Expression in Pancreatic ß-Cells Promotes Islet Function and Protects Functional ß-Cell Mass.

Syntaxin 4 (Stx4) enrichment in human and mouse islet grafts improves the success of transplants in reversing streptozotocin (STZ)-induced diabetes in mice, although the underlying molecular mechanisms remain elusive. Toward a further understanding of this, human islets and inducible transgenic mice that selectively overexpress Stx4 in islet ß-cells (ßTG-Stx4) were challenged with proinflammatory stressors in vitro and in vivo. Remarkably, ßTG-Stx4 mice resisted the loss of ß-cell mass and the glucose intolerance that multiple low doses of STZ induce. Under standard conditions, glucose tolerance was enhanced and mice maintained normal fasting glycemia and insulinemia. Conversely, Stx4 heterozygous knockout mice succumbed rapidly to STZ-induced glucose intolerance compared with their wild-type littermates. Human islet ß-cells overexpressing Stx4 exhibited enhanced insulin secretory capability; resilience against proinflammatory cytokine-induced apoptosis; and reduced expression of the CXCL9, CXCL10, and CXCL11 genes coordinate with decreased activation/nuclear localization of nuclear factor-κB. Finding ways to boost Stx4 expression presents a novel potential therapeutic avenue for promoting islet function and preserving ß-cell mass.