Browsing by Author "Bone, Robert"

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    68722 Role of ER calcium in beta cell senescence and diabetes pathophysiology
    (Cambridge University Press, 2021) Weaver, Staci A.; Kono, Tatsuyoshi; Syed, Farooq; Bone, Robert; Evans-Molina, Carmella; Biochemistry and Molecular Biology, School of Medicine
    ABSTRACT IMPACT: The proposed study has the potential to inform new paradigms of type 1 diabetes prevention and therapy with the overall goal of improving β cell health during autoimmunity. OBJECTIVES/GOALS: Type 1 diabetes (T1D) results from immune-mediated destruction of pancreatic βcells. Recent data suggest that activation of senescence and acquisition of a senescence associated secretory phenotype (SASP) by βcells may contribute to T1D pathogenesis. However, the molecular mechanisms responsible for this phenotype are not well understood. METHODS/STUDY POPULATION: We hypothesize that loss of endoplasmic reticulum (ER) Ca2+ induces βcell senescence, SASP as well as mitochondrial dysfunction which drive T1D development. The current study utilizes SERCA2 KO INS-1 βcells (S2KO) exhibiting loss of ER Ca2+ and a SERCA2 haploinsufficient mice on a non-obese diabetic background (NOD-S2+/-) to test the role of ER Ca2+ loss during T1D development. Senescence associated βgalactosidase staining (SA-βgal), expression of senescence markers (RT-qPCR), mitochondrial function (Seahorse, TMRM) and mitochondrial copy number (qPCR) were all measured in S2KO versus WT βcells and are currently being measured in the NOD-S2+/- mouse model at 6, 8, 12, 14, and 16wks of age. RESULTS/ANTICIPATED RESULTS: RT-qPCR assays detecting senescence markers cdkn1a and cdkn2a and mitochondrial specific genes cox1 and nd1 were developed and validated in both INS-1 βcells and mouse islets. Mitochondrial function assay (Seahorse) was optimized for use in INS-1 βcells and is currently under development for use in intact mouse islets. S2KO βcells displayed increased SA- βgal staining as well as increased mitochondrial coupling efficiency (p=0.0146) and baseline mitochondrial copy number (p=0.0053) compared to WT βcells, suggesting a senescence phenotype and altered mitochondrial function. NOD-S2+/- mice exhibited increased expression of the senescence marker cdkn2a in the islet at 12wks (p=0.0117) compared to control mice, whereas cdkn1a remained unchanged across all timepoints tested. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results suggest that loss of SERCA2 and reduced ER Ca2+ alter βcell mitochondrial function and are associated with features of senescence. Future studies will test whether SERCA2 activation and/or senolytic/senomorphic drugs are able to prevent or delay diabetes onset in NOD-S2+/- mice.
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    The Skeletal Phenotype Of The Kk/Ay Murine Model Of Type 2 Diabetes
    (2022-08) Chowdhury, Nusaiba Nahola; Wallace, Joseph; Allen, Matthew; Bone, Robert; Na, Sungsoo
    Type-2-diabetes (T2D) is a progressive metabolic disease characterized by insulin resistance and β-cell dysfunction leading to persistent hyperglycemia. It is a multisystem disease that causes deterioration of multiple organ systems and obesity. Of interest, T2D affects the urinary system and is the leading cause of kidney disease. Both T2D and chronic kidney negatively impacts the skeletal system and increases fracture incidence in patients. Therefore, it is important to establish an animal model that captures the complex multiorgan effects that is common in T2D. In this study, we characterized the metabolic phenotype of the KK/Ay mouse model, a polygenic mutation model of T2D. We concluded that KK/Ay mice closely mimic T2D and are hyperglycemic, hyperinsulinemic and insulin resistant. KK/Ay mice have also had worsened kidney function as supported by elevated levels of blood urea nitrogen, phosphorous, creatinine, and calcium in plasma exhibiting the kidney’s inefficiency in clearing waste from the body. Even though we were able to confirm a metabolic phenotype for T2D and diabetic nephropathy, the skeletal effects of the disease were minimal and major differences in bone physiology were driven by sex differences. This study offered valuable insight into preliminary endpoints for the KK/Ay mouse mode that will decide the direction for future use of this model. We plan to use older mice in future studies to allow a longer time for skeletal effects to more prominently manifest.