Browsing by Author "Oh, Eunjin"
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Item Changes in Skeletal Muscle PAK1 Levels Regulate Tissue Crosstalk to Impact Whole Body Glucose Homeostasis(Frontiers, 2022-02-10) Merz, Karla E.; Tunduguru, Ragadeepthi; Ahn, Miwon; Salunkhe, Vishal A.; Veluthakal, Rajakrishnan; Hwang, Jinhee; Bhattacharya, Supriyo; McCown, Erika M.; Garcia, Pablo A.; Zhou, Chunxue; Oh, Eunjin; Yoder, Stephanie M.; Elmendorf, Jeffrey S.; Thurmond, Debbie C.; Anatomy, Cell Biology and Physiology, School of MedicineSkeletal muscle accounts for ~80% of insulin-stimulated glucose uptake. The Group I p21–activated kinase 1 (PAK1) is required for the non-canonical insulin-stimulated GLUT4 vesicle translocation in skeletal muscle cells. We found that the abundances of PAK1 protein and its downstream effector in muscle, ARPC1B, are significantly reduced in the skeletal muscle of humans with type 2 diabetes, compared to the non-diabetic controls, making skeletal muscle PAK1 a candidate regulator of glucose homeostasis. Although whole-body PAK1 knockout mice exhibit glucose intolerance and are insulin resistant, the contribution of skeletal muscle PAK1 in particular was unknown. As such, we developed inducible skeletal muscle-specific PAK1 knockout (skmPAK1-iKO) and overexpression (skmPAK1-iOE) mouse models to evaluate the role of PAK1 in skeletal muscle insulin sensitivity and glucose homeostasis. Using intraperitoneal glucose tolerance and insulin tolerance testing, we found that skeletal muscle PAK1 is required for maintaining whole body glucose homeostasis. Moreover, PAK1 enrichment in GLUT4-myc-L6 myoblasts preserves normal insulin-stimulated GLUT4 translocation under insulin resistance conditions. Unexpectedly, skmPAK1-iKO also showed aberrant plasma insulin levels following a glucose challenge. By applying conditioned media from PAK1-enriched myotubes or myoblasts to β-cells in culture, we established that a muscle-derived circulating factor(s) could enhance β-cell function. Taken together, these data suggest that PAK1 levels in the skeletal muscle can regulate not only skeletal muscle insulin sensitivity, but can also engage in tissue crosstalk with pancreatic β-cells, unveiling a new molecular mechanism by which PAK1 regulates whole-body glucose homeostasis.Item Doc2b enrichment enhances glucose homeostasis in mice via potentiation of insulin secretion and peripheral insulin sensitivity.(Springer, 2014-07) Ramalingam, Latha; Oh, Eunjin; Thurmond, Debbie C.; Biochemistry & Molecular Biology, School of MedicineAIMS/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 β (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.Item DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle(Springer Verlag, 2019-05) Zhang, Jing; Oh, Eunjin; Merz, Karla E.; Aslamy, Arianne; Veluthakal, Rajakrishnan; Salunkhe, Vishal A.; Ahn, Miwon; Tunduguru, Ragadeepthi; Thurmond, Debbie C.; Cellular and Integrative Physiology, School of MedicineAims/hypothesis: Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain β (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. Methods: Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, 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.Item Doc2b Protects β-Cells Against Inflammatory Damage and Enhances Function(American Diabetes Association, 2018-07) Aslamy, Arianne; Oh, Eunjin; Olson, Erika M.; Zhang, Jing; Ahn, Miwon; Moin, Abu Saleh Md; Tunduguru, Ragadeepthi; Salunkhe, Vishal A.; Veluthakal, Rajakrishnan; Thurmond, Debbie C.; Cellular & Integrative Physiology, IU School of MedicineLoss of functional β-cell mass is an early feature of type 1 diabetes. To release insulin, β-cells require soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, as well as SNARE complex regulatory proteins like double C2 domain-containing protein β (Doc2b). We hypothesized that Doc2b deficiency or overabundance may confer susceptibility or protection, respectively, to the functional β-cell mass. Indeed, Doc2b+/- knockout mice show an unusually severe response to multiple-low-dose streptozotocin (MLD-STZ), resulting in more apoptotic β-cells and a smaller β-cell mass. In addition, inducible β-cell-specific Doc2b-overexpressing transgenic (βDoc2b-dTg) mice show improved glucose tolerance and resist MLD-STZ-induced disruption of glucose tolerance, fasting hyperglycemia, β-cell apoptosis, and loss of β-cell mass. Mechanistically, Doc2b enrichment enhances glucose-stimulated insulin secretion (GSIS) and SNARE activation and prevents the appearance of apoptotic markers in response to cytokine stress and thapsigargin. Furthermore, expression of a peptide containing the Doc2b tandem C2A and C2B domains is sufficient to confer the beneficial effects of Doc2b enrichment on GSIS, SNARE activation, and apoptosis. These studies demonstrate that Doc2b enrichment in the β-cell protects against diabetogenic and proapoptotic stress. Furthermore, they identify a Doc2b peptide that confers the beneficial effects of Doc2b and may be a therapeutic candidate for protecting functional β-cell mass.Item Exocytosis Protein DOC2B as a Biomarker of Type 1 Diabetes(Oxford University Press, 2018-05-01) Aslamy, Arianne; Oh, Eunjin; Ahn, Miwon; Moin, Abu Saleh Md; Chang, Mariann; Duncan, Molly; Hacker-Stratton, Jeannette; El-Shahawy, Mohamed; Kandeel, Fouad; DiMeglio, Linda A.; Thurmond, Debbie C.; Cellular and Integrative Physiology, School of MedicineContext: Efforts to preserve β-cell mass in the preclinical stages of type 1 diabetes (T1D) are limited by few blood-derived biomarkers of β-cell destruction. Objective: Platelets are proposed sources of blood-derived biomarkers for a variety of diseases, and they show distinct proteomic changes in T1D. Thus, we investigated changes in the exocytosis protein, double C2 domain protein-β (DOC2B) in platelets and islets from T1D humans, and prediabetic nonobese diabetic (NOD) mice. Design, Patients, and Main Outcome Measure: Protein levels of DOC2B were assessed in platelets and islets from prediabetic NOD mice and humans, with and without T1D. Seventeen new-onset T1D human subjects (10.3 ± 3.8 years) were recruited immediately following diagnosis, and platelet DOC2B levels were compared with 14 matched nondiabetic subjects (11.4 ± 2.9 years). Furthermore, DOC2B levels were assessed in T1D human pancreatic tissue samples, cytokine-stimulated human islets ex vivo, and platelets from T1D subjects before and after islet transplantation. Results: DOC2B protein abundance was substantially reduced in prediabetic NOD mouse platelets, and these changes were mirrored in the pancreatic islets from the same mice. Likewise, human DOC2B levels were reduced over twofold in platelets from new-onset T1D human subjects, and this reduction was mirrored in T1D human islets. Cytokine stimulation of normal islets reduced DOC2B expression ex vivo. Remarkably, platelet DOC2B levels increased after islet transplantation in patients with T1D. Conclusions: Reduction of DOC2B is an early feature of T1D, and DOC2B abundance may serve as a valuable in vivo indicator of β-cell mass and an early biomarker of T1D.Item Fetal hyperglycemia and a high fat diet contribute to aberrant glucose tolerance and hematopoiesis in adulthood(Nature Publishing Group, 2015-02) Blue, Emily K.; Ballman, Kimberly; Boyle, Frances; Oh, Eunjin; Kono, Tatsuyoshi; Quinney, Sara K.; Thurmond, Debbie C.; Evans-Molina, Carmella; Haneline, Laura S.; Department of Pediatrics, IU School of MedicineBackground 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-week-old) and aged (11-month-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.Item Munc18c Depletion Selectively Impairs the Sustained Phase of Insulin Release(American Diabetes Association, 2009-05) Oh, Eunjin; Thurmond, Debbie C.; Biochemistry and Molecular Biology, School of MedicineOBJECTIVE The Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4–mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mechanism(s) using the islet β-cell as a model system. RESEARCH DESIGN AND METHODS Perifusion analyses of isolated Munc18c- (−/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 β-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/fusion of vesicle-associated membrane protein (VAMP)2-containing insulin granules. Electron microscopy was used to gauge changes in granule localization. RESULTS Munc18c (−/+) islets secreted ∼60% less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (−/+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2–Syntaxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion. CONCLUSIONS Collectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion.Item Munc18c: a controversial regulator of peripheral insulin action(Elsevier, 2014-11) Ramalingam, Latha; Yoder, Stephanie M.; Oh, Eunjin; Thurmond, Debbie C.; Department of Biochemistry & Molecular Biology, IU School of MedicineInsulin resistance, a hallmark of impaired glucose tolerance and type 2 diabetes (T2D), arises from dysfunction of insulin action and subsequent glucose uptake by peripheral tissues, predominantly skeletal muscle and fat. Exocytosis of glucose transporter (GLUT4)-containing vesicles facilitated by soluble NSF (N-ethylmaleimide-sensitive factor) attachment receptor (SNARE) protein isoforms, and Munc18c (mammalian homolog of Unc-18c) mediates this glucose uptake. Emerging evidences, including recent human clinical studies, point to pivotal roles for Munc18c in peripheral insulin action in adipose and skeletal muscle. Intriguing new advances are also initiating debates regarding the molecular mechanism(s) controlling Munc18c action. The objective of this review is therefore to present a balanced perspective of new continuities and controversies surrounding the regulation and requirement for Munc18c in the regulation of peripheral insulin action.Item The p21-activated kinase (PAK1) is involved in diet-induced beta cell mass expansion and survival in mice and human islets(Springer, 2016-10) Ahn, Miwon; Yoder, Stephanie M.; Wang, Zhanxiang; Oh, Eunjin; Ramalingam, Latha; Tunduguru, Ragadeepthi; Thurmond, Debbie C.; Department of Pediatrics, IU School of MedicineAIMS/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.Item Potential Site Effects and Transgene Expression Discrepancies in Mouse Lifespan Studies.(Elsevier, 2015-09-01) Thurmond, Debbie C.; Oh, Eunjin; Miller, Richard A.; Department of Pediatrics, IU School of Medicine