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Browsing by Author "Yamamoto, Wataru R."
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Item Endoplasmic reticulum stress alters ryanodine receptor function in the murine pancreatic β cell(American Society for Biochemistry and Molecular Biology, 2018-11-12) Yamamoto, Wataru R.; Bone, Robert N.; Sohn, Paul; Syed, Farooq; Reissaus, Christopher A.; Mosley, Amber L.; Wijeratne, Aruna B.; True, Jason D.; Tong, Xin; Kono, Kono; Evans-Molina, Carmella; Biochemistry and Molecular Biology, School of MedicineAlterations in endoplasmic reticulum (ER) calcium (Ca2+) levels diminish insulin secretion and reduce β-cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca2+ loss in β cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP3R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca2+ imaging techniques in INS-1 β cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP3R function. Our results revealed that the RyR is sensitive mainly to ER stress–induced dysfunction, whereas cytokine stress specifically alters IP3R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP3R with xestospongin C prevented ER Ca2+ loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented β-cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca2+ oscillations in tunicamycin-treated INS-1 β cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca2+ transients that depend on both ER Ca2+ leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca2+ dynamics in β cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca2+ may be an early pathogenic event in diabetes.Item Pancreatic and duodenal homeobox protein 1 (Pdx-1) maintains endoplasmic reticulum calcium levels through transcriptional regulation of sarco-endoplasmic reticulum calcium ATPase 2b (SERCA2b) in the islet β cell(American Society for Biochemistry and Molecular Biology, 2014-11-21) Johnson, Justin S.; Kono, Tatsuyoshi; Tong, Xin; Yamamoto, Wataru R.; Zarain-Herzberg, Angel; Merrins, Matthew J.; Satin, Leslie S.; Gilon, Patrick; Evans-Molina, Carmella; Department of Medicine, IU School of MedicineAlthough the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in β cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of β cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca(2+), which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1(+/-) mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca(2+) regulation in Pdx-1-deficient states.