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.

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.

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.

NSC23766, a Known Inhibitor of Tiam1-Rac1 Signaling Module, Prevents the Onset of Type 1 Diabetes in the NOD Mouse Model.

BACKGROUND/AIMS: Type 1 diabetes (T1D) is characterized by absolute insulin deficiency due to destruction of pancreatic ß-cells by cytokines (e.g., interleukin-1ß; IL-1ß) released by invading immune cells. The mechanisms by which these cytokines induce ß-cell dysfunction remain poorly understood. Recent evidence suggests that excessive generation of reactive oxygen species (ROS) by the phagocyte-like NADPH oxidase2 (Nox2), along with significantly low levels of antioxidants in ß-cells, drive them toward oxidative damage. Rac1, a small G-protein, is one of the members of Nox2 holoenzyme. We recently reported that NSC23766, a known inhibitor of Rac1, significantly attenuated cytokine-induced Nox2 activation and ROS generation in pancreatic islet ß-cells in vitro. Herein, we determined the effects of NSC23766 (2.5 mg/kg/day, i.p/daily) on the development of diabetes in the NOD mouse, a model for T1D. METHODS: Two groups of experimental animals (Balb/c and NOD mice) received NSC23766, while the two control groups received equal volume of saline. Body weights and blood glucose were measured every week for 34 weeks. Rac1 activation in pancreatic islets was measured by GLISA activation assay. Rac1 and CHOP expression was determined by Western Blotting. RESULTS: Our findings indicate that administration of NSC23766 significantly prevented the development of spontaneous diabetes in the NOD mice. Furthermore, NSC23766 markedly suppressed Rac1 expression and activity and the endoplasmic reticulum stress (CHOP expression) in NOD islets. CONCLUSIONS: Our findings provide the first evidence implicating the role of Tiam1-Rac1-Nox2 signaling pathway in the onset of spontaneous diabetes in the NOD mouse model.

Metabolic Stress Induces Caspase-3 Mediated Degradation and Inactivation of Farnesyl and Geranylgeranyl Transferase Activities in Pancreatic ß-Cells.

BACKGROUND/AIMS: At least 300 prenylated proteins are identified in the human genome; the majority of which partake in a variety of cellular processes including growth, differentiation, cytoskeletal organization/dynamics and vesicle trafficking. Aberrant prenylation of proteins is implicated in human pathologies including cancer; neurodegenerative diseases, retinitis pigmentosa, and premature ageing syndromes. Original observations from our laboratory have demonstrated that prenylation of proteins [small G-proteins and γ-subunits of trimeric G-proteins] is requisite for physiological insulin secretion. Herein, we assessed the impact of metabolic stress [gluco-, lipotoxicity and ER-stress] on the functional status of protein prenylation pathway in pancreatic ß-cells. METHODS: Farnesyltransferase [FTase] and geranylgeranyltransferase [GGTase] activities were quantified by radioisotopic methods. Caspase-3 activation and FTase/GGTase-α subunit degradation were determined by Western blotting. RESULTS: We observed that metabolic stress activates caspase-3 and induces degradation of the common α-subunit of FTase and GGTase-I in INS-1 832/13 cells, normal rodent islets and human islets leading to functional defects [inactivation] in FTase and GGTase activities. Caspase-3 activation and FTase/GGTase-α degradation were also seen in islets from the Zucker diabetic fatty [ZDF] rat, a model for Type 2 diabetes. Consequential to defects in FTase/GGTase-α signaling, we observed significant accumulation of unprenylated proteins [Rap1] in ß-cells exposed to glucotoxic conditions. These findings were replicated in ß-cells following pharmacological inhibition of generation of prenylpyrophosphate substrates [Simvastatin] or catalytic activity of prenylating enzymes [GGTI-2147]. CONCLUSIONS: Our findings provide the first evidence to suggest that metabolic stress induced dysfunction of the islet ß-cell may, in part, be due to defective protein prenylation signaling pathway.

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.

Tiam1-Rac1 Axis Promotes Activation of p38 MAP Kinase in the Development of Diabetic Retinopathy: Evidence for a Requisite Role for Protein Palmitoylation.

BACKGROUND/AIMS: Evidence in multiple tissues, including retina, suggests generation of reactive oxygen species (ROS) and the ensuing oxidative stress as triggers for mitochondrial defects and cell apoptosis. We recently reported novel roles for Tiam1-Rac1-Nox2 axis in retinal mitochondrial dysfunction and cell death leading to the development of diabetic retinopathy. Herein, we tested the hypothesis that activation of p38 MAP kinase, a stress kinase, represents the downstream signaling event to Rac1-Nox2 activation in diabetes-induced metabolic stress leading to capillary cell apoptosis. METHODS: Activation of p38 MAP kinase was quantified by Western blotting in retinal endothelial cells incubated with high glucose (20 mM) for up to 96 hours, a duration where mitochondrial dysfunction and capillary cell apoptosis can be observed. NSC23766 and 2-bromopalmitate (2-BP) were used to assess the roles of Tiam1-Rac1 and palmitoylation pathways, respectively. RESULTS: Activation of p38 MAP kinase was observed as early as 3 hours after high glucose exposure, and continued until 96 hours. Consistent with this, p38 MAP kinase activation was significantly higher in the retina from diabetic mice compared to age-matched normal mice. NSC23766 markedly attenuated hyperglycemia-induced activation of p38 MAP kinase. Lastly, 2-BP inhibited glucose-induced Rac1, Nox2 and p38 MAP kinase activation in endothelial cells. CONCLUSIONS: Tiam1-Rac1-mediated activation of Nox2 and p38 MAP kinase constitutes early signaling events leading to mitochondrial dysfunction and the development of diabetic retinopathy. Our findings also provide the first evidence to implicate novel roles for protein palmitoylation in this signaling cascade.

TIAM1-RAC1 signalling axis-mediated activation of NADPH oxidase-2 initiates mitochondrial damage in the development of diabetic retinopathy.

AIMS/HYPOTHESIS: In diabetes, increased retinal oxidative stress is seen before the mitochondria are damaged. Phagocyte-like NADPH oxidase-2 (NOX2) is the predominant cytosolic source of reactive oxygen species (ROS). Activation of Ras-related C3 botulinum toxin substrate 1 (RAC1), a NOX2 holoenzyme member, is necessary for NOX2 activation and ROS generation. In this study we assessed the role of T cell lymphoma invasion and metastasis (TIAM1), a guanine nucleotide exchange factor for RAC1, in RAC1 and NOX2 activation and the onset of mitochondrial dysfunction in in vitro and in vivo models of glucotoxicity and diabetes. METHODS: RAC1 and NOX2 activation, ROS generation, mitochondrial damage and cell apoptosis were quantified in bovine retinal endothelial cells exposed to high glucose concentrations, in the retina from normal and streptozotocin-induced diabetic rats and mice, and the retina from human donors with diabetic retinopathy. RESULTS: High glucose activated RAC1 and NOX2 (expression and activity) and increased ROS in endothelial cells before increasing mitochondrial ROS and mitochondrial DNA (mtDNA) damage. N6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine, trihydrochloride (NSC23766), a known inhibitor of TIAM1-RAC1, markedly attenuated RAC1 activation, total and mitochondrial ROS, mtDNA damage and cell apoptosis. An increase in NOX2 expression and membrane association of RAC1 and p47(phox) were also seen in diabetic rat retina. Administration of NSC23766 to diabetic mice attenuated retinal RAC1 activation and ROS generation. RAC1 activation and p47(phox) expression were also increased in the retinal microvasculature from human donors with diabetic retinopathy. CONCLUSIONS/INTERPRETATION: The TIAM1-RAC1-NOX2 signalling axis is activated in the initial stages of diabetes to increase intracellular ROS leading to mitochondrial damage and accelerated capillary cell apoptosis. Strategies targeting TIAM1-RAC1 signalling could have the potential to halt the progression of diabetic retinopathy in the early stages of the disease.

Glucotoxic and diabetic conditions induce caspase 6-mediated degradation of nuclear lamin A in human islets, rodent islets and INS-1 832/13 cells.

Nuclear lamins form the lamina on the interior surface of the nuclear envelope, and regulate nuclear metabolic events, including DNA replication and organization of chromatin. The current study is aimed at understanding the role of executioner caspase 6 on lamin A integrity in islet ß-cells under duress of glucotoxic (20 mM glucose; 24 h) and diabetic conditions. Under glucotoxic conditions, glucose-stimulated insulin secretion and metabolic cell viability were significantly attenuated in INS-1 832/13 cells. Further, exposure of normal human islets, rat islets and INS-1 832/13 cells to glucotoxic conditions leads to caspase 6 activation and lamin A degradation, which is also observed in islets from the Zucker diabetic fatty rat, a model for type 2 diabetes (T2D), and in islets from a human donor with T2D. Z-Val-Glu-Ile-Asp-fluoromethylketone, a specific inhibitor of caspase 6, markedly attenuated high glucose-induced caspase 6 activation and lamin A degradation, confirming that caspase 6 mediates lamin A degradation under high glucose exposure conditions. Moreover, Z-Asp-Glu-Val-Asp-fluoromethylketone, a known caspase 3 inhibitor, significantly inhibited high glucose-induced caspase 6 activation and lamin A degradation, suggesting that activation of caspase 3 might be upstream to caspase 6 activation in the islet ß-cell under glucotoxic conditions. Lastly, we report expression of ZMPSTE24, a zinc metallopeptidase involved in the processing of prelamin A to mature lamin A, in INS-1 832/13 cells and human islets; was unaffected by high glucose. We conclude that caspases 3 and 6 could contribute to alterations in the integrity of nuclear lamins leading to metabolic dysregulation and failure of the islet ß-cell.

Nm23-H1 regulates glucose-stimulated insulin secretion in pancreatic ß-cells via Arf6-Rac1 signaling axis.

BACKGROUND: A growing body of evidence implicates novel roles for nm23-like proteins in the regulation of cellular functions. However, roles of these proteins in islet function and glucose-stimulated insulin secretion (GSIS) remain largely unknown. METHODS: siRNA-nm23-H1 and nucleoside diphosphate kinase and histidine kinase-deficient mutants of nm23-H1 (K12Q and H118F) were used to assess roles of nm23-H1 in GSIS. RESULTS: siRNA-mediated knockdown of the expression of nm23-H1 markedly inhibited GSIS in INS-1 832/13 cells. Nm23-H1 knockdown also resulted in significant inhibition of glucose-mediated activation of Arf6, a small G-protein, which has been implicated in GSIS. Expression of K12Q and H118F mutants of nm23-H1 in INS-1 832/13 cells led to inhibition of glucose-induced translocation and membrane association of Rac1, another small G-protein, which is downstream to Arf6 in the signaling events leading to GSIS. A significant inhibition of GSIS was also seen in these cells expressing K12Q and H118F. CONCLUSIONS: We conclude that the nm23-H1 activation step is upstream of Arf6 activation in signaling events leading to GSIS. NDP kinase and histidine kinase functions of nm23-H1 are necessary for glucose-induced membrane association of Rac1 and ensuing insulin secretion. We present the first evidence for regulation of GSIS by nm23-H1 in pancreatic ß-cells.

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.

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.

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.

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.

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.

A novel histone deacetylase inhibitor prevents IL-1beta induced metabolic dysfunction in pancreatic beta-cells.

The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has recently been shown to inhibit deleterious effects of cytokines on beta-cells, but it is unable to protect beta-cells from death due to its own cytotoxicity. Herein, we investigated novel HDAC inhibitors for their cytoprotective effects against IL-1beta-induced damage to isolated beta-cells. We report that three novel compounds (THS-73-44, THS-72-5 and THS-78-5) significantly inhibited HDAC activity and increased the acetylation of histone H4 in isolated beta-cells. Further, these compounds exerted no toxic effects on metabolic cell viability in these cells. However, among the three compounds tested, only THS-78-5 protected against IL-1beta-mediated loss in beta-cell viability. THS-78-5 was also able to attenuate IL-1beta-induced inducible nitric oxide synthase expression and subsequent NO release. Our data also indicate that the cytoprotective properties of THS-78-5 against IL-1beta-mediated effects may, in part, be due to inhibition of IL-1beta-induced transactivation of nuclear factor kappaB (NF-kappaB) in these cells. Together, we provide evidence for a novel HDAC inhibitor with a significant potential to prevent IL-1beta-mediated effects on isolated beta-cells. Potential implications of these findings in the development of novel therapeutics to prevent deleterious effects of cytokines and the onset of autoimmune diabetes are discussed.