Browsing by Subject "Pancreatic beta cell"

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    Pancreatic Beta Cell Identity Regulated by the Endoplasmic Reticulum Calcium Sensor Stromal Interaction Molecule 1
    (2021-12) Sohn, Paul; Evans-Molina, Carmella; Elmendorf, Jeffrey; Linnemann, Amelia; Sankar, Uma
    Type 2 diabetes mellitus is a chronic disorder characterized by hyperglycemia, insulin resistance, and insufficient insulin secretion from the pancreatic β cells. To maintain adequate levels of insulin secretion, β cells rely on highly coordinated control of luminal ER Ca2+. Stromal Interaction Molecule 1 (STIM1) is an ER Ca2+ sensor that serves to replenish ER Ca2+ stores in response to depletion by gating plasmalemmal Orai1 channels in a process known as store-operated calcium entry (SOCE). We developed a method for the direct measurement of SOCE in pancreatic β cells and found that deletion of STIM1 in INS-1 cells (STIM1KO) is sufficient to block Ca2+ influx in response to store-depletion. To determine the physiological importance of β cell STIM1, we created mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ) and placed them on a high fat diet (HFD) with 60% of kilocalories derived from fat. After 8 weeks of HFD, female, but not male, STIM1Δβ mice exhibited increased body weight and fat mass as well as significant glucose intolerance and impaired insulin secretion without observable differences in insulin tolerance. Immunohistochemical analysis revealed a reduction of β cell mass and an increase of α cell mass; ELISA of islet lysates revealed a similar significant reduction in insulin content and increased glucagon content. RNA-sequencing performed on STIM1Δβ islets revealed differentially expressed genes for functions related to apoptosis, lipid metabolism, and epithelial cell differentiation, as well as loss of β cell identity. Proteomics analysis of STIM1KO cells phenocopied the metabolic findings, revealing significantly increased glucagon expression. Analysis of islet RNA-sequencing results showed modulation of pathways related to 17-β estradiol (E2) signaling, with notable downregulation of G-protein coupled estrogen receptor 1 (GPER1) expression. Consistently, treatment of female wild-type islets with pharmacological SOCE inhibitors led to reduced expression GPER1, while STIM1KO cells showed lower mobilization of intracellular cAMP levels in response to GPER agonist treatment. Taken together, these findings identify a novel interaction between SOCE and E2 signaling in the female islet and suggest that loss of STIM1 and impairments in SOCE may contribute to diabetes pathophysiology through loss of β cell identity.
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    The Roles of Danio Rerio Nrf2 Paralogs in Response to Oxidative Stress in the Pancreatic Beta Cell
    (2020-06) Doszpoly, Agnes; Linnemann, Amelia; Anderson, Ryan; Wek, Ronald
    Oxidative stress can disrupt cellular homeostasis, leading to cellular dysfunction and apoptosis. The Nrf2 transcription factor regulates the antioxidant response in cells by binding to antioxidant response elements (ARE) in DNA and activating genes of enzymes that combat oxidative stress. During the pathogenesis of diabetes mellitus (DM), β-cells are exposed to increased amounts of reactive oxygen species (ROS) that cause oxidative stress. Zebrafish (ZF) are excellent models for studying the dynamic mechanisms associated with DM pathogenesis, and we recently developed a ZF model of β-cell apoptosis caused by ROS. Two paralogs of Nrf2 have been identified in ZF, Nrf2a and Nrf2b, but their roles in pancreas development and/or β-cell survival are unknown. To investigate their roles, Nrf2a and Nrf2b antisense morpholinos (MO) were injected into Day 0 ZF embryos and analyzed over time. While Nrf2a MO showed no obvious phenotypes compared to WT, Nrf2b MO exhibited reduced pancreas size and islets with disrupted morphology. Ins:NTR Nrf2a MO showed reduced β-cell loss upon exposure to Metronidazole (MTZ) under generation of ROS compared to WT. Sequence analysis of ZF nrf2b in 3-day post-fertilization (dpf) embryos revealed a novel splice variant containing an additional exon that has not been described. Further investigation of Nrf2a and Nrf2b is likely to yield additional insights regarding the function and regulation of the NRF2-signaling pathway and their roles in β-cell protection under oxidative stress.