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.

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.

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.

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.

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.

Depletion of the membrane-fusion regulator Munc18c attenuates caerulein hyperstimulation-induced pancreatitis.

Epithelial pancreatic acinar cells perform crucial functions in food digestion, and acinar cell homeostasis required for secretion of digestive enzymes relies on SNARE-mediated exocytosis. The ubiquitously expressed Sec1/Munc18 protein mammalian uncoordinated-18c (Munc18c) regulates membrane fusion by activating syntaxin-4 (STX-4) to bind cognate SNARE proteins to form a SNARE complex that mediates exocytosis in many cell types. However, in the acinar cell, Munc18c's functions in exocytosis and homeostasis remain inconclusive. Here, we found that pancreatic acini from Munc18c-depleted mice (Munc18c+/-) and human pancreas (lenti-Munc18c-shRNA-treated) exhibit normal apical exocytosis of zymogen granules (ZGs) in response to physiologic stimulation with the intestinal hormone cholecystokinin (CCK-8). However, when stimulated with supraphysiologic CCK-8 levels to mimic pancreatitis, Munc18c-depleted (Munc18c+/-) mouse acini exhibited a reduction in pathological basolateral exocytosis of ZGs resulting from a decrease in fusogenic STX-4 SNARE complexes. This reduced basolateral exocytosis in part explained the less severe pancreatitis observed in Munc18c+/- mice after hyperstimulation with the CCK-8 analog caerulein. Likely as a result of this secretory blockade, Munc18c-depleted acini unexpectedly activated a component of the endoplasmic reticulum (ER) stress response that contributed to autophagy induction, resulting in downstream accumulation of autophagic vacuoles and autolysosomes. We conclude that Munc18c's role in mediating ectopic basolateral membrane fusion of ZGs contributes to the initiation of CCK-induced pancreatic injury, and that blockade of this secretory process could increase autophagy induction.

Novel approaches to restore beta cell function in prediabetes and type 2 diabetes.

The World Health Organization estimates that diabetes prevalence has risen from 108 million in 1980 to 422 million in 2014, with type 2 diabetes accounting for more than 90% of these cases. Furthermore, the prevalence of prediabetes (impaired fasting glucose and/or impaired glucose tolerance) is more than 40% in some countries and is associated with a global rise in obesity. Therefore it is imperative that we develop new approaches to reduce the development of prediabetes and progression to type 2 diabetes. In this review, we explore the gains made over the past decade by focused efforts to improve insulin secretion by the beta cell or insulin sensitivity of target tissues. We also describe multitasking candidates, which could improve both beta cell dysfunction and peripheral insulin sensitivity. Moreover, we highlight provocative findings indicating that additional glucose regulatory tissues, such as the brain, may be key therapeutic targets. Taken together, the promise of these new multi-faceted approaches reinforces the importance of understanding and tackling type 2 diabetes pathogenesis from a multi-tissue perspective.

The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells.

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation.

Inflammatory stress of pancreatic beta cells drives release of extracellular heat-shock protein 90α.

A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1ß, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity.

Exocytosis proteins as novel targets for diabetes prevention and/or remediation?

Diabetes remains one of the leading causes of morbidity and mortality worldwide, affecting an estimated 422 million adults. In the US, it is predicted that one in every three children born as of 2000 will suffer from diabetes in their lifetime. Type 2 diabetes results from combinatorial defects in pancreatic ß-cell glucose-stimulated insulin secretion and in peripheral glucose uptake. Both processes, insulin secretion and glucose uptake, are mediated by exocytosis proteins, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, Sec1/Munc18 (SM), and double C2-domain protein B (DOC2B). Increasing evidence links deficiencies in these exocytosis proteins to diabetes in rodents and humans. Given this, emerging studies aimed at restoring and/or enhancing cellular levels of certain exocytosis proteins point to promising outcomes in maintaining functional ß-cell mass and enhancing insulin sensitivity. In doing so, new evidence also shows that enhancing exocytosis protein levels may promote health span and longevity and may also harbor anti-cancer and anti-Alzheimer's disease capabilities. Herein, we present a comprehensive review of the described capabilities of certain exocytosis proteins and how these might be targeted for improving metabolic dysregulation.

The p21-activated kinase (PAK1) is involved in diet-induced beta cell mass expansion and survival in mice and human islets.

AIMS/HYPOTHESIS: Human islets from type 2 diabetic donors are reportedly 80% deficient in the p21 (Cdc42/Rac)-activated kinase, PAK1. PAK1 is implicated in beta cell function and maintenance of beta cell mass. We questioned the mechanism(s) by which PAK1 deficiency potentially contributes to increased susceptibility to type 2 diabetes. METHODS: Non-diabetic human islets and INS 832/13 beta cells cultured under diabetogenic conditions (i.e. with specific cytokines or under glucolipotoxic [GLT] conditions) were evaluated for changes to PAK1 signalling. Combined effects of PAK1 deficiency with GLT stress were assessed using classic knockout (Pak1 (-/-) ) mice fed a 45% energy from fat/palmitate-based, 'western' diet (WD). INS 832/13 cells overexpressing or depleted of PAK1 were also assessed for apoptosis and signalling changes. RESULTS: Exposure of non-diabetic human islets to diabetic stressors attenuated PAK1 protein levels, concurrent with increased caspase 3 cleavage. WD-fed Pak1 knockout mice exhibited fasting hyperglycaemia and severe glucose intolerance. These mice also failed to mount an insulin secretory response following acute glucose challenge, coinciding with a 43% loss of beta cell mass when compared with WD-fed wild-type mice. Pak1 knockout mice had fewer total beta cells per islet, coincident with decreased beta cell proliferation. In INS 832/13 beta cells, PAK1 deficiency combined with GLT exposure heightened beta cell death relative to either condition alone; PAK1 deficiency resulted in decreased extracellular signal-related kinase (ERK) and B cell lymphoma 2 (Bcl2) phosphorylation levels. Conversely, PAK1 overexpression prevented GLT-induced cell death. CONCLUSIONS/INTERPRETATION: These findings suggest that PAK1 deficiency may underlie an increased diabetic susceptibility. Discovery of ways to remediate glycaemic dysregulation via altering PAK1 or its downstream effectors offers promising opportunities for disease intervention.

VAV2, a guanine nucleotide exchange factor for Rac1, regulates glucose-stimulated insulin secretion in pancreatic beta cells.

AIMS/HYPOTHESIS: Rho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells. METHODS: Co-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA). RESULTS: Western blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016. CONCLUSIONS/INTERPRETATION: Our data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.

YES, a Src family kinase, is a proximal glucose-specific activator of cell division cycle control protein 42 (Cdc42) in pancreatic islet ß cells.

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in ß cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 ß cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed ß cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

Fetal hyperglycemia and a high-fat diet contribute to aberrant glucose tolerance and hematopoiesis in adult rats.

BACKGROUND: Children exposed to gestational diabetes mellitus (GDM) during pregnancy are at increased risk of obesity, diabetes, and hypertension. Our goal was to identify metabolic and hematopoietic alterations after intrauterine exposure to maternal hyperglycemia that may contribute to the pathogenesis of chronic morbidities. METHODS: Streptozotocin treatment induced maternal hyperglycemia during the last third of gestation in rat dams. Offspring of control mothers (OCM) and diabetic mothers (ODM) were evaluated for weight, glucose tolerance, insulin tolerance, and hematopoiesis defects. The effects of aging were examined in normal and high-fat diet (HFD)-fed young (8-wk-old) and aged (11-mo-old) OCM and ODM rats. RESULTS: Young adult ODM males on a normal diet, but not females, displayed improved glucose tolerance due to increased insulin levels. Aged ODM males and females gained more weight than OCM on a HFD and had worse glucose tolerance. Aged ODM males fed a HFD were also neutrophilic. Increases in bone marrow cellularity and myeloid progenitors preceded neutrophilia in ODM males fed a HFD. CONCLUSION: When combined with other risk factors like HFD and aging, changes in glucose metabolism and hematopoiesis may contribute to the increased risk of obesity, type 2 diabetes, and hypertension observed in children of GDM mothers.

Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet ß-cells.

Biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells involves soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) protein-regulated exocytosis. SNARE complex assembly further requires the regulatory proteins Munc18c, Munc18-1 and Doc2b. Munc18-1 and Munc18c are required for first- and second-phase GSIS respectively. These distinct Munc18-1 and Munc18c roles are related to their transient high-affinity binding with their cognate target (t-)SNAREs, Syntaxin 1A and Syntaxin 4 respectively. Doc2b is essential for both phases of GSIS, yet the molecular basis for this remains unresolved. Because Doc2b binds to Munc18-1 and Munc18c via its distinct C2A and C2B domains respectively, we hypothesized that Doc2b may provide a plasma membrane-localized scaffold/platform for transient docking of these Munc18 isoforms during GSIS. Towards this, macromolecular complexes composed of Munc18c, Doc2b and Munc18-1 were detected in ß-cells. In vitro interaction assays indicated that Doc2b is required to bridge the interaction between Munc18c and Munc18-1 in the macromolecular complex; Munc18c and Munc18-1 failed to associate in the absence of Doc2b. Competition-based GST-Doc2b interaction assays revealed that Doc2b could simultaneously bind both Munc18-1 and Munc18c. Hence these data support a working model wherein Doc2b functions as a docking platform/scaffold for transient interactions with the multiple Munc18 isoforms operative in insulin release, promoting SNARE assembly.

Doc2b enrichment enhances glucose homeostasis in mice via potentiation of insulin secretion and peripheral insulin sensitivity.

AIMS/HYPOTHESIS: Insulin secretion from pancreatic beta cells and insulin-stimulated glucose uptake into skeletal muscle are processes regulated by similar isoforms of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) and mammalian homologue of unc-18 (Munc18) protein families. Double C2 domain ß (Doc2b), a SNARE- and Munc18-interacting protein, is implicated as a crucial effector of glycaemic control. However, whether Doc2b is naturally limiting for these processes, and whether Doc2b enrichment might exert a beneficial effect upon glycaemia in vivo, remains undetermined. METHODS: Tetracycline-repressible transgenic (Tg) mice engineered to overexpress Doc2b simultaneously in the pancreas, skeletal muscle and adipose tissues were compared with wild-type (Wt) littermate mice regarding glucose and insulin tolerance, islet function in vivo and ex vivo, and skeletal muscle GLUT4 accumulation in transverse tubule/sarcolemmal surface membranes. SNARE complex formation was further assessed using Doc2b overexpressing L6-GLUT4-myc myoblasts to derive mechanisms relatable to physiological in vivo analyses. RESULTS: Doc2b Tg mice cleared glucose substantially faster than Wt mice, correlated with enhancements in both phases of insulin secretion and peripheral insulin sensitivity. Heightened peripheral insulin sensitivity correlated with elevated insulin-stimulated GLUT4 vesicle accumulation in cell surface membranes of Doc2b Tg mouse skeletal muscle. Mechanistic studies demonstrated Doc2b enrichment to enhance syntaxin-4-SNARE complex formation in skeletal muscle cells. CONCLUSIONS/INTERPRETATION: Doc2b is a limiting factor in SNARE exocytosis events pertinent to glycaemic regulation in vivo. Doc2b enrichment may provide a novel means to simultaneously boost islet and skeletal muscle function in vivo in the treatment and/or prevention of diabetes.

Novel roles for insulin receptor (IR) in adipocytes and skeletal muscle cells via new and unexpected substrates.

The insulin signaling pathway regulates whole-body glucose homeostasis by transducing extracellular signals from the insulin receptor (IR) to downstream intracellular targets, thus coordinating a multitude of biological functions. Dysregulation of IR or its signal transduction is associated with insulin resistance, which may culminate in type 2 diabetes. Following initial stimulation of IR, insulin signaling diverges into different pathways, activating multiple substrates that have roles in various metabolic and cellular processes. The integration of multiple pathways arising from IR activation continues to expand as new IR substrates are identified and characterized. Accordingly, our review will focus on roles for IR substrates as they pertain to three primary areas: metabolism/glucose uptake, mitogenesis/growth, and aging/longevity. While IR functions in a seemingly pleiotropic manner in many cell types, through these three main roles in fat and skeletal muscle cells, IR multi-tasks to regulate whole-body glucose homeostasis to impact healthspan and lifespan.

Positional cloning of a type 2 diabetes quantitative trait locus; tomosyn-2, a negative regulator of insulin secretion.

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lep(ob/ob) and C57BL/6 (B6) Lep(ob/ob) mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16(BT36-38)) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16(BT36-38) mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic ß-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion.

Trefoil factor 2 expressed by the murine pancreatic acinar cells is required for the development of islets and for beta cell function during aging.

Exocrine-to-endocrine crosstalk in the pancreas is crucial to maintain beta cell function. However, the molecular mechanisms underlying this crosstalk are largely undefined. Trefoil factor 2 (Tff2) is a secreted factor known to promote the proliferation of beta cells in vitro, but its physiological role in vivo in the pancreas is unknown. Also, it remains unclear which pancreatic cell type expresses Tff2 protein. We therefore created a mouse model with a conditional knockout of Tff2 in the murine pancreas. We find that the Tff2 protein is preferentially expressed in acinar but not ductal or endocrine cells. Tff2 deficiency in the pancreas reduces beta cell mass on embryonic day 16.5. However, homozygous mutant mice are born without a reduction of beta cells and with acinar Tff3 compensation by day 7. When mice are aged to 1 year, both male and female homozygous and male heterozygous mutants develop impaired glucose tolerance without affected insulin sensitivity. Perifusion analysis reveals that the second phase of glucose-stimulated insulin secretion from islets is reduced in aged homozygous mutant compared to controls. Collectively, these results demonstrate a previously unknown role of Tff2 as an exocrine acinar cell-derived protein required for maintaining functional endocrine beta cells in mice.

Munc18-1 regulates first-phase insulin release by promoting granule docking to multiple syntaxin isoforms.

Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet ß-cells is coincident with type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1(+/-) and ß-cell specific Munc18-1(-/-) knock-out mice, we establish that Munc18-1 is required for the first phase of insulin secretion. Conversely, human islets expressing elevated levels of Munc18-1 elicited significant potentiation of only first-phase insulin release. Insulin secretory changes positively correlated with insulin granule number at the plasma membrane: Munc18-1-deficient cells lacked 35% of the normal component of pre-docked insulin secretory granules, whereas cells with elevated levels of Munc18-1 exhibited a ∼20% increase in pre-docked granule number. Pre-docked syntaxin 1-based SNARE complexes bound by Munc18-1 were detected in ß-cell lysates but, surprisingly, were reduced by elevation of Munc18-1 levels. Paradoxically, elevated Munc18-1 levels coincided with increased binding of syntaxin 4 to VAMP2 at the plasma membrane. Accordingly, syntaxin 4 was a requisite for Munc18-1 potentiation of insulin release. Munc18c, the cognate SM isoform for syntaxin 4, failed to bind SNARE complexes. Given that Munc18-1 does not pair with syntaxin 4, these data suggest a novel indirect role for Munc18-1 in facilitating syntaxin 4-mediated granule pre-docking to support first-phase insulin exocytosis.