Investigating the Role of ER Stress on Mouse Pancreatic Beta-Cell Function

Abstract

Type 2 diabetes mellitus (T2DM) is a chronic disease characterized by impaired glucose-stimulated insulin secretion in the setting of increased insulin resistance, a condition in which the body fails to respond to insulin properly. The endoplasmic reticulum (ER) serves as an essential quality control organelle and a Ca2+ reservoir. ER stress has been proposed to cause T2DM, but the specific effects of ER stress on beta-cell function are incompletely understood. To determine the interrelationship between ER stress and beta-cell function, the work in this dissertation primarily used the well-known ER stress inducer tunicamycin (TM) to trigger ER stress in insulin-secreting INS-1(832/13) cells or isolated mouse pancreatic islets. Beta-cell function, as exemplified by the tight regulation of ER or cytosolic [Ca2+], or membrane potential oscillations and insulin secretion, and beta-cell apoptosis were quantified at various time points, in parallel with well-established ER stress response markers. We observed that the induction of ER stress resulted in decreased ER [Ca2+], increased cytosolic free Ca2+ oscillations, membrane potential oscillations and increased insulin secretion, even under sub-threshold glucose conditions (e.g. 5 mM glucose) that are typically associated with only minimal insulin release. As ER Ca2+ depletion is generally known to activate store-operated Ca2+ entry (SOCE) in many cell types, we then used YM58483, a selective SOCE blocker. We found that the cytosolic Ca2+ and membrane potential oscillations and increased insulin secretion we observed resulted from increased SOCE, as these were all acutely blocked by the application of YM58483.

Further dissection of TM-triggered ER [Ca2+] loss was carried out by studying the effect of TM on the ER Ca2+ channels, inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), and how their activity was tied to subsequent alterations in beta-cell Ca2+ homeostasis. Mouse islets were treated with dantrolene (Dan, a RyR1 isoform inhibitor) or xestospongin C (XeC, an IP3Rs inhibitor) along with TM overnight. We found that TM-induced ER Ca2+ depletion, cytosolic Ca2+ and mitochondrial Ca2+ oscillations were inhibited by co-treatment with Dan, whereas XeC was without effect on the oscillations. While RyR1 transcript was increased after TM exposure, transcripts corresponding to IP3R1 and IP3R2 were decreased by TM. Taken together, TM appeared to deplete ER [Ca2+] by increasing RyR1 level and triggering the subsequent activation of SOCE, resulting in increased cytosolic Ca2+ oscillations occurring despite the presence of sub-threshold glucose concentration.

Finally, we characterized the role of an inward rectifier K+ channel, Kir2.1, in the beta-cell. Our lab previously published data supporting the hypothesis that Kir2.1 channels compensated for the loss of functional KATP channels in islets from KATP (SUR1) deficient mice in the production of cytosolic Ca2+ oscillations. We tested here whether Kir2.1 channels might also be involved in ER stress-induced beta-cell dysfunction, by using the selective Kir2.1 channels inhibitor ML133. We found that overnight exposure of beta cells to ML133 suppressed TM-triggered cytosolic Ca2+ oscillations in 5 mM glucose solution, likely due to ML133’s inhibitory effect on the activation of the ER stress response marker spliced XBP1.

In summary, this dissertation maps the complex process of ER stress altering beta-cell function. In the process, we identified several novel mechanisms, including SOCE, RyR1 and Kir2.1 channels, that may augment insulin secretion in T2DM patients, and their clinical potentials as drug targets.