A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression.

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements.

Acute insulin signaling in pancreatic beta-cells is mediated by multiple Raf-1 dependent pathways.

Insulin enhances the proliferation and survival of pancreatic beta-cells, but its mechanisms remain unclear. We hypothesized that Raf-1, a kinase upstream of both ERK and Bad, might be a critical target of insulin in beta-cells. To test this hypothesis, we treated human and mouse islets as well as MIN6 beta-cells with multiple insulin concentrations and examined putative downstream targets using immunoblotting, immunoprecipitation, quantitative fluorescent imaging, and cell death assays. Low doses of insulin rapidly activated Raf-1 by dephosphorylating serine 259 and phosphorylating serine 338 in human islets, mouse islets, and MIN6 cells. The phosphorylation of ERK by insulin was eliminated by exposure to a Raf inhibitor (GW5074) or transfection with a dominant-negative Raf-1 mutant. Insulin also enhanced the interaction between mitochondrial Raf-1 and Bcl-2 agonist of cell death (Bad), promoting Bad inactivation via its phosphorylation on serine 112. Insulin-stimulated ERK phosphorylation was abrogated by calcium chelation, calcineurin and calmodulin-dependent protein kinase II inhibitors, and Ned-19, a nicotinic acid adenine dinucleotide phosphate receptor (NAADPR) antagonist. Blocking Raf-1 and Ca(2+) signaling resulted in nonadditive beta-cell death. Autocrine insulin signaling partly accounted for the effects of glucose on ERK phosphorylation. Our results demonstrate that Raf-1 is a critical target of insulin in primary beta-cells. Activation of Raf-1 leads to both an ERK-dependent pathway that involves nicotinic acid adenine dinucleotide phosphate-sensitive Ca(2+) stores and Ca(2+)-dependent phosphorylation events, and an ERK-independent pathway that involves Bad inactivation at the mitochondria. Together our findings identify a novel insulin signaling pathway in beta-cells and shed light on insulin's antiapoptotic and mitogenic mechanisms.

Roles of IP3R and RyR Ca2+ channels in endoplasmic reticulum stress and beta-cell death.

OBJECTIVE: Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca(2+) release channels in the ER stress-associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP(3)Rs) and the ryanodine receptors (RyRs) on the induction of beta-cell ER stress and apoptosis. RESEARCH DESIGN AND METHODS: Kinetics of beta-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca(2+) was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure Ca(2+) in ER and mitochondria. RESULTS: Neither RyR nor IP(3)R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca(2+) and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha), C/EBP homologous protein (CHOP)-associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP(3)Rs and RyRs. Conversely, stimulation of ER Ca(2+) release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization. CONCLUSIONS: This study demonstrates that the activity of ER Ca(2+) channels regulates the susceptibility of beta-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in beta-cell apoptosis associated with dysfunctional beta-cell ER Ca(2+) homeostasis and ER stress.

Carboxypeptidase E mediates palmitate-induced beta-cell ER stress and apoptosis.

Obesity is a principal risk factor for type 2 diabetes, and elevated fatty acids reduce beta-cell function and survival. An unbiased proteomic screen was used to identify targets of palmitate in beta-cell death. The most significantly altered protein in both human islets and MIN6 beta-cells treated with palmitate was carboxypeptidase E (CPE). Palmitate reduced CPE protein levels within 2 h, preceding endoplasmic reticulum (ER) stress and cell death, by a mechanism involving CPE translocation to Golgi and lysosomal degradation. Palmitate metabolism and Ca(2+) flux were also required for CPE proteolysis and beta-cell death. Chronic palmitate exposure increased the ratio of proinsulin to insulin. CPE null islets had increased apoptosis in vivo and in vitro. Reducing CPE by approximately 30% using shRNA also increased ER stress and apoptosis. Conversely, overexpression of CPE partially rescued beta-cells from palmitate-induced ER stress and apoptosis. Thus, carboxypeptidase E degradation contributes to palmitate-induced beta-cell ER stress and apoptosis. CPE is a major link between hyperlipidemia and beta-cell death pathways in diabetes.

Glucose and endoplasmic reticulum calcium channels regulate HIF-1beta via presenilin in pancreatic beta-cells.

Pancreatic beta-cell death is a critical event in type 1 diabetes, type 2 diabetes, and clinical islet transplantation. We have previously shown that prolonged block of ryanodine receptor (RyR)-gated release from intracellular Ca(2+) stores activates calpain-10-dependent apoptosis in beta-cells. In the present study, we further characterized intracellular Ca(2+) channel expression and function in human islets and the MIN6 beta-cell line. All three RyR isoforms were identified in human islets and MIN6 cells, and these endoplasmic reticulum channels were observed in close proximity to mitochondria. Blocking RyR channels, but not sarco/endoplasmic reticulum ATPase (SERCA) pumps, reduced the ATP/ADP ratio. Blocking Ca(2+) flux through RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of hypoxia-inducible factor (HIF-1beta). Moreover, inhibition of RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of presenilin-1. Both HIF-1beta and presenilin-1 expression were also induced by low glucose. Overexpression of presenilin-1 increased HIF-1beta, suggesting that HIF is downstream of presenilin. Our results provide the first evidence of a presenilin-HIF signaling network in beta-cells. We demonstrate that this pathway is controlled by Ca(2+) flux through intracellular channels, likely via changes in mitochondrial metabolism and ATP. These findings provide a mechanistic understanding of the signaling pathways activated when intracellular Ca(2+) homeostasis and metabolic activity are suppressed in diabetes and islet transplantation.

Insulin protects islets from apoptosis via Pdx1 and specific changes in the human islet proteome.

Insulin is both a hormone regulating energy metabolism and a growth factor. We and others have shown that physiological doses of insulin initiate complex signals in primary human and mouse beta-cells, but the functional significance of insulin's effects on this cell type remains unclear. In the present study, the role of insulin in beta-cell apoptosis was examined. Exogenous insulin completely prevented apoptosis induced by serum withdrawal when given at picomolar or low nanomolar concentrations but not at higher concentrations, indicating that physiological concentrations of insulin are antiapoptotic and that insulin signaling is self-limiting in islets. Insulin treatment was associated with the nuclear localization of Pdx1 and the prosurvival effects of insulin were largely absent in islets 50% deficient in Pdx1, providing direct evidence that Pdx1 is a signaling target of insulin. Physiological levels of insulin did not increase Akt phosphorylation, and the protective effects of insulin were only partially altered in islets lacking 80% of normal Akt activity, suggesting the presence of additional insulin-regulated antiapoptotic pathways. Proteomic analysis of insulin-treated human islets revealed significant changes in multiple proteins. Bridge-1, a Pdx1-binding partner and regulator of beta-cell survival, was increased significantly at low insulin doses. Together, these data suggest that insulin can act as a master regulator of islet survival by regulating Pdx1.