Dan S. Luciani

Increased islet apoptosis in Pdx1+/– mice

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.

Roles of IP3R and RyR Ca2+ Channels in Endoplasmic Reticulum Stress and β-Cell Death

OBJECTIVE—Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca2+ 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 (IP3Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis. RESEARCH DESIGN AND METHODS—Kinetics of β-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 Ca2+ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)–based probes were used to measure Ca2+ in ER and mitochondria. RESULTS—Neither RyR nor IP3R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca2+ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)–associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP3Rs and RyRs. Conversely, stimulation of ER Ca2+ 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 Ca2+ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca2+ homeostasis and ER stress.

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Obesity is a principal risk factor for type 2 diabetes, and elevated fatty acids reduce β-cell function and survival. An unbiased proteomic screen was used to identify targets of palmitate in β-cell death. The most significantly altered protein in both human islets and MIN6 β-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 Ca2+ flux were also required for CPE proteolysis and β-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 ≈30% using shRNA also increased ER stress and apoptosis. Conversely, overexpression of CPE partially rescued β-cells from palmitate-induced ER stress and apoptosis. Thus, carboxypeptidase E degradation contributes to palmitate-induced β-cell ER stress and apoptosis. CPE is a major link between hyperlipidemia and β-cell death pathways in diabetes.

Ca2+ controls slow NAD(P)H oscillations in glucose‐stimulated mouse pancreatic islets

Exposure of pancreatic islets of Langerhans to physiological concentrations of glucose leads to secretion of insulin in an oscillatory pattern. The oscillations in insulin secretion are associated with oscillations in cytosolic Ca2+ concentration ([Ca2+]c). Evidence suggests that the oscillations in [Ca2+]c and secretion are driven by oscillations in metabolism, but it is unclear whether metabolic oscillations are intrinsic to metabolism or require Ca2+ feedback. To address this question we explored the interaction of Ca2+ concentration and islet metabolism using simultaneous recordings of NAD(P)H autofluorescence and [Ca2+]c, in parallel with measurements of mitochondrial membrane potential (ΔΨm). All three parameters responded to 10 mm glucose with multiphasic dynamics culminating in slow oscillations with a period of ∼5 min. This was observed in ∼90% of islets examined from various mouse strains. NAD(P)H oscillations preceded those of [Ca2+]c, but their upstroke was often accelerated during the increase in [Ca2+]c, and Ca2+ influx was a prerequisite for their generation. Prolonged elevations of [Ca2+]c augmented NAD(P)H autofluorescence of islets in the presence of 3 mm glucose, but often lowered NAD(P)H autofluorescence of islets exposed to 10 mm glucose. Comparable rises in [Ca2+]c depolarized ΔΨm. The NAD(P)H lowering effect of an elevation of [Ca2+]c was reversed during inhibition of mitochondrial electron transport. These findings reveal the existence of slow oscillations in NAD(P)H autofluorescence in intact pancreatic islets, and suggest that they are shaped by Ca2+ concentration in a dynamic balance between activation of NADH‐generating mitochondrial dehydrogenases and a Ca2+‐induced decrease in NADH. We propose that a component of the latter reflects mitochondrial depolarization by Ca2+, which reduces respiratory control and consequently accelerates oxidation of NADH.

Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

Mechanisms of Pancreatic β-Cell Apoptosis in Diabetes and Its Therapies

Diabetes occurs when beta-cells no longer function properly or have been destroyed. Pancreatic beta-cell death by apoptosis contributes significantly in both autoimmune type 1 diabetes and type 2 diabetes. Pancreatic beta-cell death can be induced by multiple stresses in both major types of diabetes. There are also several rare forms of diabetes, including Wolcott-Rallison syndrome, Wolfram syndrome, as well as some forms of maturity onset diabetes of the young that are caused by mutations in genes that may play important roles in beta-cell survival. The use of islet transplantation as a treatment for diabetes is also limited by excessive beta-cell apoptosis. Mechanistic insights into the control of pancreatic beta-cell apoptosis are therefore important for the prevention and treatment of diabetes. Indeed, a substantial quantity of research has been dedicated to this area over the past decade. In this chapter, we review the factors that influence the propensity of beta-cells to undergo apoptosis and the mechanisms of this programmed cell death in the initiation and progression of diabetes.

Glucose and Endoplasmic Reticulum Calcium Channels Regulate HIF-1β via Presenilin in Pancreatic β-Cells

Pancreatic β-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 Ca2+ stores activates calpain-10-dependent apoptosis in β-cells. In the present study, we further characterized intracellular Ca2+ channel expression and function in human islets and the MIN6 β-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 Ca2+ flux through RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of hypoxia-inducible factor (HIF-1β). Moreover, inhibition of RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of presenilin-1. Both HIF-1β and presenilin-1 expression were also induced by low glucose. Overexpression of presenilin-1 increased HIF-1β, suggesting that HIF is downstream of presenilin. Our results provide the first evidence of a presenilin-HIF signaling network in β-cells. We demonstrate that this pathway is controlled by Ca2+ 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 Ca2+ homeostasis and metabolic activity are suppressed in diabetes and islet transplantation.

Maturation of Adult β-Cells Revealed Using a Pdx1/Insulin Dual-Reporter Lentivirus

The enigmatic process of beta-cell maturation has significant implications for diabetes pathogenesis, and potential diabetes therapies. This study examined the dynamics and heterogeneity of insulin and pancreatic duodenal homeobox (Pdx)-1 gene expression in adult beta-cells. Insulin and Pdx1 expression were monitored in human and mouse islet cells and MIN6 cells using a Pdx1-monomeric red fluorescent protein/insulin-enhanced green fluorescent protein dual-reporter lentivirus. The majority of fluorescent cells were highly positive for both Pdx1 and insulin. Cells expressing Pdx1 but little or no insulin (Pdx1(+)/Ins(low)) comprised 15-25% of the total population. Time-lapse imaging demonstrated that Pdx1(+)/Ins(low) primary beta-cells and MIN6 cells could convert to Pdx1(+)/Ins(+) cells without cell division. Genes involved in the mature beta-cell phenotype (Glut2, MafA) were expressed at higher levels in Pdx1(+)/Ins(+) cells relative to Pdx1(+)/Ins(low) cells. Conversely, genes implicated in early beta-cell development (MafB, Nkx2.2) were enriched in Pdx1(+)/Ins(low) cells. Sorted Pdx1(+)/Ins(low) MIN6 cells had a higher replication rate and secreted less insulin relative to double-positive cells. Long-term phenotype tracking of Pdx1(+)/Ins(low) cells showed two groups, one that matured into Pdx1(+)/Ins(+) cells and one that remained immature. These results demonstrate that adult beta-cells pass through distinct maturation states, which is consistent with previously observed heterogeneity in insulin and Pdx1 expression in adult beta-cells. At a given time, a proportion of adult beta-cells share similar characteristics to functionally immature embryonic beta-cell progenitors. The maturation of adult beta-cells recapitulates development in that Pdx1 expression precedes the robust expression of insulin and other mature beta-cell genes. These results have implications for harnessing the maturation process for therapeutic purposes.

Suppressed Insulin Signaling and Increased Apoptosis in Cd38-Null Islets

CD38 is a multifunctional enzyme capable of generating metabolites that release Ca2+ from intracellular stores, including nicotinic acid adenine dinucleotide phosphate (NAADP). A number of studies have led to the controversial proposal that CD38 mediates an alternate pathway for glucose-stimulated insulin release and contributes to the pathogenesis of diabetes. It has recently been shown that NAADP mediates Ca2+ mobilization by insulin in human pancreatic β-cells. In the present study, we report altered Ca2+ homeostasis and reduced responsiveness to insulin, but not glucose, in Cd38−/− β-cells. In keeping with the antiapoptotic role of insulin signaling, Cd38−/− islets were significantly more susceptible to apoptosis compared with islets isolated from littermate controls. This finding correlated with disrupted islet architecture and reduced β-cell mass in Cd38−/− mice, both in the context of a normal lab diet and a high-fat diet. Nevertheless, we did not find robust differences in glucose homeostasis in vivo or glucose signaling in vitro in Cd38−/− mice on the C57BL/6 genetic background, in contrast to previous studies by others of Cd38 knockout mice on the ICR background. Thus, our results suggest that CD38 plays a role in novel antiapoptotic signaling pathways but does not directly control glucose signaling in pancreatic β-cells.

Acute effects of insulin on beta-cells from transplantable human islets.

The functional role of autocrine insulin signaling remains unclear despite considerable investigation. In the present study, we tested the effects of high and low doses of exogenous insulin on Ca2+ signaling, insulin synthesis and insulin secretion in dispersed human islet cells using a combination of imaging, radioimmunoassay and patch-clamp electrophysiology. Although 200 nM insulin stimulated Ca2+ signals with larger amplitudes, the percentage of responding cells was lower when compared with 0.2 nM insulin. However, both 0.2 nM insulin and 200 nM insulin led to a transient increase in accessible cellular insulin content under conditions that glucose did not. This pool of insulin likely reflected de novo synthesis as it could be blocked by cyclohexamide or actinomycin D. Blocking endogenous autocrine insulin signaling in quiescent beta-cells with the insulin receptor inhibitor HMNPA led to a reduction in insulin synthesis, suggesting some degree of basal activity of this positive feed-forward loop. Unlike exposure to high glucose, acute treatment with insulin did not stimulate robust insulin exocytosis, as estimated by C-peptide release and capacitance measurements from single beta-cells. Together these data provide further evidence that autocrine insulin signaling can regulate the function of human pancreatic beta-cells. Our findings suggest autocrine insulin signaling directly controls insulin protein levels, but not exocytosis, in beta-cells and demonstrate the functional specificity of insulin signaling and glucose signaling in human islet cells.