Pancreatic β cells dedicate much of their protein translation capacity to produce insulin to maintain glucose homeostasis. In response to increased secretory demand, β cells can compensate by increasing insulin production capability even in the face of protracted peripheral insulin resistance. The ability to amplify insulin secretion in response to hyperglycemia is a critical facet of β cell function, and the exact mechanisms by which this occurs have been studied for decades. To adapt to the constant and fast changing demands for insulin production, β cells utilize the unfolded protein response of the endoplasmic reticulum. Failure of these compensatory mechanisms contributes to both type 1 and 2 diabetes. Additionally, studies in which β cells are ‘rested’ by reducing endogenous insulin demand have shown promise as a therapeutic strategy that could be applied more broadly. Here we review recent findings in β cells pertaining to the metabolic amplifying pathway, the unfolded protein response, and potential advances in therapeutics based on β cell rest.

No abstract

No abstract

No abstract

Pancreatic islet beta cells require a fine-tuned ER stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical co-treatment experiments we discovered synergy between SW016789 and activators of protein kinase C (PKC) and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

Aims/hypothesisInsulin production and its regulated exocytosis by pancreatic islet beta cells is the major physiological mechanism controlling blood glucose concentrations. Beta cells are susceptible to failure due to genetic and environmental influences, leading to type 1 and 2 diabetes. In this work we aimed to advance understanding of the regulation of beta-cell function by identifying small molecule modulators of secretion.MethodsFor high-throughput screening and routine medium-throughput beta cell function assays we used mouse MIN6 beta cells stably expressing a Gaussia luciferase-linked insulin secretion reporter (InsGLuc-MIN6). We validated hit small molecules using cadaveric human islets in static culture insulin secretion assays. We measured effects on calcium influx and cAMP in MIN6 cells using Fura-2 and a bioluminescence resonance energy transfer reporter assay, respectively. Metabolism was analyzed by targeted metabolomics and Seahorse oxygen consumption assays. We measured relative gene expression and protein amounts by quantitative RT-PCR and SDS-PAGE immunoblotting, respectively.ResultsThrough our high-throughput screen we discovered 2-(3-benzyl-2-iminobenzimidazol-1-yl)-1-thiophen-2-ylethanol (SW016789) which acutely potentiated nutrient-induced calcium influx and insulin secretion. More than 2 hours of exposure to SW016789 transiently induced the unfolded protein response of the endoplasmic reticulum and shut down of insulin secretory function. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or death as determined by multiplexed cytotoxicity assays and a lack of induction of cleaved PARP. This may be due in part to a more rapid induction of Hspa5, and a lesser induction of signalling through the eIF2α kinase PERK and lack of expression of oxidative stress genes like Txnip. We determined that SW016789 acted upstream of the voltage-dependent calcium channel (VDCC) and potentiated nutrient-stimulated, but not KCl-stimulated, calcium influx. The potentiating effects of SW016789 were not due to altered metabolic pathways, mitochondrial function, or actions on G protein-coupled receptors. In chemical co-treatment experiments we discovered synergy between SW016789 and small molecule activators of protein kinase C and VDCCs, suggesting potential involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as co-treatment with dihydropyridine-class VDCC inhibitors protected MIN6 beta cells and human islets from loss of function.Conclusions/interpretationThese data suggest the major mechanism of action of SW016789 in beta cells is to potentiate opening of VDCCs. This activity may partially depend on protein kinase C. Beta cells under pharmacological hypersecretory conditions have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. Further study of the mechanisms underlying these processes will increase understanding of beta cell function in normal and pathophysiological states.Research in contextWhat is already known about this subject? Pharmacologic and environmental chemicals can stimulate aberrant beta cell activity.Hypersecretion of insulin from pancreatic beta cells can cause ER stress and lead to beta cell dysfunction.Defects in beta cell ER stress handling contribute to diabetes pathogenesis.What is the key question? Can the chemical tool SW016789 help uncover novel insulin secretion regulatory pathways and ER proteostasis factors?What are the new findings? SW016789 caused hypersecretion of insulin via enhanced nutrient-stimulated Ca2+ influx followed by transient ER stress and shutdown of beta cell function without apoptosis.Blockade of voltage-dependent Ca2+ channels protected human islets and beta cells from hypersecretion-induced dysfunction.How might this impact on clinical practice in the foreseeable future? These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

PurposeType 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet β-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. ResultsHere, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 β-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. ConclusionGiven the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.

No abstract