Browsing by Author "Thurmond, Debbie C."
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Item The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells(American Society for Biochemistry and Molecular Biology, 2017-11-17) Tunduguru, Ragadeepthi; Zhang, Jing; Aslamy, Arianne; Salunkhe, Vishal A.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Thurmond, Debbie C.; Biochemistry and Molecular Biology, School of MedicineDefects 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.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 Death-Associated Protein Kinase Regulates Vascular Smooth Muscle Cell Signaling and Migration(2011-03-16) Blue, Emily Keller; Gallagher, Patricia J.; Elmendorf, Jeffrey S.; Herring, B. Paul; Rhodes, Simon J.; Thurmond, Debbie C.Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.Item DOC2B enhancement of beta cell function and survival(2018-03-08) Aslamy, Arianne; Thurmond, Debbie C.; Elmendorf, Jeffrey S.; Evans-Molina, Carmella; Baucum, Anthony J.Diabetes mellitus is a complex metabolic disease that currently affects an estimated 422 million people worldwide, with incidence rates rising annually. Type 1 diabetes (T1D) accounts for 5-10% of these cases. Its complications remain a major cause of global deaths. T1D is characterized by autoimmune destruction of β-cell mass. Efforts to preserve and protect β-cell mass in the preclinical stages of T1D are limited by few blood-borne biomarkers of β-cell destruction. In healthy β-cells, insulin secretion requires soluble n-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes and associated accessory regulatory proteins to promote the docking and fusion of insulin vesicles at the plasma membrane. Two target membrane (t)-SNARE proteins, Syntaxin 1/4 and SNAP25/23, and one vesicle-associated (v)-SNARE protein, VAMP2, constitute the SNARE core complex. SNARE complex assembly is also facilitated by the regulatory protein, Double C2-domain protein β (DOC2B). I hypothesized that DOC2B deficiency may underlie β-cell susceptibility to T1D damage; conversely , overexpression of DOC2B may protect β-cell mass. Indeed, with regard to DOC2B abundance, my studies show reduced levels of DOC2B in platelets and islets of prediabetic rodents and new-onset T1D humans. Remarkably, clinical islet transplantation in T1D humans restores platelet DOC2B levels, indicating a correlation With regard to protection/functional effects, DOC2B deficiency enhances susceptibility to T1D in mice, while overexpression of DOC2B selectively in β-cells protects mice from chemically induced T1D; this correlates with preservation of functional β-cell mass. Mechanistically, overexpression of DOC2B and the DOC2B peptide, C2AB, protects clonal β-cell against cytokine or thapsigargin-induced apoptosis and reduces ER stress; this is dependent on C2AB’s calcium binding capacity. C2AB is sufficient to enhance glucose stimulated insulin secretion (GSIS) and SNARE activation in clonal β-cells to the same extent as full-length DOC2B. In summary, these studies identify DOC2B as a potential biomarker and novel therapeutic target for prevention/management of T1D.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 Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet β-cells(Portland Press, 2014-12) Ramalingam, Latha; Lu, Jingping; Hudmon, Andy; Thurmond, Debbie C.; Department of Biochemistry & Molecular Biology, IU School of MedicineBiphasic glucose-stimulated insulin secretion (GSIS) from pancreatic β-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 β-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.Item Dual regulation of voltage- and ligand-gated calcium channels by collapsin response mediator protein 2(2013-10-07) Brittain, Joel Matthew; Khanna, Rajesh; Cummins, Theodore R.; Oxford, G. S.; Quilliam, Lawrence; Thurmond, Debbie C.Synaptic transmission is coordinated by a litany of protein-protein interactions that rely on the proper localization and function of pre- and post-synaptic Ca2+ channels. The axonal guidance/specification collapsin response mediator protein-2 (CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+ channel (CaV2.2). CRMP-2 bound directly to CaV2.2 in two regions; the channel domain I-II intracellular loop and the distal C-terminus. Both proteins co-localized within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to EGFP caused a significant increase in Ca2+ channel current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2–overexpressing neurons. Both activity- and CRMP-2-phosphoryation altered the interaction between CaV2.2 and CRMP-2. I identified a CRMP-2-derived peptide (called CBD3) that bound CaV2.2 and effectively disrupted the interaction between CaV2.2 and CRMP-2. CBD3 peptide fused to the HIV TAT protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, and reversed neuropathic hypersensitivity produced by an antiretroviral drug. Unchecked Ca2+ influx via N-methyl-D-aspartate receptors (NMDARs) has been linked to activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2 was suggested to affect NMDAR trafficking and possibly involved in neuronal survival following excitotoxicity. Based upon these studies, I hypothesized that a peptide from CRMP2 could preserve neurons in the face of excitotoxic challenges. Lentiviral–mediated CRMP2 knockdown or treatment with TAT-CBD3 blocked neuronal death following glutamate exposure likely via blunting toxicity from NMDAR-mediated delayed calcium deregulation. TAT-CBD3 induced internalization of the NMDAR subunit NR2B in dendritic spines without altering somal surface expression. TAT-CBD3 reduced NMDA-mediated Ca2+-influx and currents in cultured neurons. The presented work validates CRMP-2 as a novel modulator of pre- and post-synaptic Ca2+ channels and provides evidence that the TAT-CBD3 peptide could be useful as a potential therapeutic for both chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults.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.