Browsing by Author "Aslamy, Arianne"

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    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 Medicine
    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.
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    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.
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    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 Medicine
    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 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.
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    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 Medicine
    Loss 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.
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    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 Medicine
    Context: 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.
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    Exocytosis proteins as novel targets for diabetes prevention and/or remediation?
    (American Physiological Society, 2017-05-01) Aslamy, Arianne; Thurmond, Debbie C.; Cellular and Integrative Physiology, School of Medicine
    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 β-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 β-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.