The role of mitochondrial apoptotic pathway in islet amyloid-induced ß-cell death.

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to ß-cell death in type 2 diabetes. We previously showed that extracellular hIAPP aggregates promote Fas-mediated ß-cell apoptosis. Here, we tested if hIAPP aggregates can trigger the mitochondrial apoptotic pathway (MAP). hIAPP aggregation in Ad-hIAPP transduced INS-1 and human islet ß-cells promoted cytochrome c release, caspase-9 activation and apoptosis, which were reduced by Bax inhibitor. Amyloid formation in hIAPP-expressing mouse islets during culture increased caspase-9 activation in ß-cells. Ad-hIAPP transduced islets from CytcKA/KA and BaxBak ßDKO mice (models of blocked MAP), had lower caspase-9-positive and apoptotic ß-cells than transduced wild-type islets, despite comparable amyloid formation. Blocking Fas (markedly) and Bax or caspase-9 (modestly) reduced ß-cell death induced by extracellular hIAPP aggregates. These findings suggest a role for MAP in amyloid-induced ß-cell death and a potential strategy to reduce intracellular amyloid ß-cell toxicity by blocking cytochrome c apoptotic function.

Cellular metabolism constrains innate immune responses in early human ontogeny.

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.

Bcl-2 Regulates Reactive Oxygen Species Signaling and a Redox-Sensitive Mitochondrial Proton Leak in Mouse Pancreatic ß-Cells.

In pancreatic ß-cells, controlling the levels of reactive oxygen species (ROS) is critical to counter oxidative stress, dysfunction and death under nutrient excess. Moreover, the fine-tuning of ROS and redox balance is important in the regulation of normal ß-cell physiology. We recently demonstrated that Bcl-2 and Bcl-xL, in addition to promoting survival, suppress ß-cell glucose metabolism and insulin secretion. Here, we tested the hypothesis that the nonapoptotic roles of endogenous Bcl-2 extend to the regulation of ß-cell ROS and redox balance. We exposed mouse islet cells and MIN6 cells to the Bcl-2/Bcl-xL antagonist Compound 6 and the Bcl-2-specific antagonist ABT-199 and evaluated ROS levels, Ca(2+) responses, respiratory control, superoxide dismutase activity and cell death. Both acute glucose stimulation and the inhibition of endogenous Bcl-2 progressively increased peroxides and stimulated superoxide dismutase activity in mouse islets. Importantly, conditional ß-cell knockout of Bcl-2 amplified glucose-induced formation of peroxides. Bcl-2 antagonism also induced a mitochondrial proton leak that was prevented by the antioxidant N-acetyl-L-cysteine and, therefore, secondary to redox changes. We further established that the proton leak was independent of uncoupling protein 2 but partly mediated by the mitochondrial permeability transition pore. Acutely, inhibitor-induced peroxides promoted Ca(2+) influx, whereas under prolonged Bcl inhibition, the elevated ROS was required for induction of ß-cell apoptosis. In conclusion, our data reveal that endogenous Bcl-2 modulates moment-to-moment ROS signaling and suppresses a redox-regulated mitochondrial proton leak in ß-cells. These noncanonical roles of Bcl-2 may be important for ß-cell function and survival under conditions of high metabolic demand.

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.

Cardiomyocyte ATP production, metabolic flexibility, and survival require calcium flux through cardiac ryanodine receptors in vivo.

Ca(2+) fluxes between adjacent organelles are thought to control many cellular processes, including metabolism and cell survival. In vitro evidence has been presented that constitutive Ca(2+) flux from intracellular stores into mitochondria is required for basal cellular metabolism, but these observations have not been made in vivo. We report that controlled in vivo depletion of cardiac RYR2, using a conditional gene knock-out strategy (cRyr2KO mice), is sufficient to reduce mitochondrial Ca(2+) and oxidative metabolism, and to establish a pseudohypoxic state with increased autophagy. Dramatic metabolic reprogramming was evident at the transcriptional level via Sirt1/Foxo1/Pgc1α, Atf3, and Klf15 gene networks. Ryr2 loss also induced a non-apoptotic form of programmed cell death associated with increased calpain-10 but not caspase-3 activation or endoplasmic reticulum stress. Remarkably, cRyr2KO mice rapidly exhibited many of the structural, metabolic, and molecular characteristics of heart failure at a time when RYR2 protein was reduced 50%, a similar degree to that which has been reported in heart failure. RYR2-mediated Ca(2+) fluxes are therefore proximal controllers of mitochondrial Ca(2+), ATP levels, and a cascade of transcription factors controlling metabolism and survival.

Bcl-2 and Bcl-xL suppress glucose signaling in pancreatic ß-cells.

B-cell lymphoma 2 (Bcl-2) family proteins are established regulators of cell survival, but their involvement in the normal function of primary cells has only recently begun to receive attention. In this study, we demonstrate that chemical and genetic loss-of-function of antiapoptotic Bcl-2 and Bcl-x(L) significantly augments glucose-dependent metabolic and Ca(2+) signals in primary pancreatic ß-cells. Antagonism of Bcl-2/Bcl-x(L) by two distinct small-molecule compounds rapidly hyperpolarized ß-cell mitochondria, increased cytosolic Ca(2+), and stimulated insulin release via the ATP-dependent pathway in ß-cell under substimulatory glucose conditions. Experiments with single and double Bax-Bak knockout ß-cells established that this occurred independently of these proapoptotic binding partners. Pancreatic ß-cells from Bcl-2(-/-) mice responded to glucose with significantly increased NAD(P)H levels and cytosolic Ca(2+) signals, as well as significantly augmented insulin secretion. Inducible deletion of Bcl-x(L) in adult mouse ß-cells also increased glucose-stimulated NAD(P)H and Ca(2+) responses and resulted in an improvement of in vivo glucose tolerance in the conditional Bcl-x(L) knockout animals. Our work suggests that prosurvival Bcl proteins normally dampen the ß-cell response to glucose and thus reveals these core apoptosis proteins as integrators of cell death and physiology in pancreatic ß-cells.

Glucose-induced endothelial heparanase secretion requires cortical and stress actin reorganization.

AIMS: Heparanase, which specifically cleaves carbohydrate chains of heparan sulfate, has been implicated in the pathology of diabetes-associated complications. Using high glucose (HG) to replicate hyperglycaemia observed following diabetes, the present study was designed to determine the mechanism by which HG initiates endothelial heparanase secretion. METHOD AND RESULTS: To examine the effect of HG on endothelial heparanase, bovine coronary artery endothelial cells were incubated with 25 mM glucose. Strategies using different agonists and antagonists were used to determine the mechanism behind HG-induced heparanase secretion. In endothelial cells, heparanase colocalized with lysosomes predominately around the nucleus, and HG caused its dispersion towards the plasma membrane for subsequent secretion. ATP release, purinergic receptor activation, cortical actin disassembly, and stress actin formation were essential for this HG-induced heparanase secretion. With HG, phosphorylation of filamin likely contributed to the cortical actin disassembly, whereas Ca(2+)/calmodulin-dependent protein kinase II and p38 mitogen-activated protein kinase /heat shock protein 25 phosphorylation mediated stress actin formation. The endothelial secreted heparanase in response to HG demonstrated endoglucuronidase activity, cleaved heparan sulfate, and released attached proteins like lipoprotein lipase and basic fibroblast growth factor. CONCLUSION: Our results suggest that HG is a potent stimulator of endothelial heparanase secretion. These data may assist in devising new therapeutic strategies to prevent or delay the cardiovascular complications associated with diabetes.

AMP-activated protein kinase confers protection against TNF-{alpha}-induced cardiac cell death.

AIMS: Although a substantial role for 5' adenosine monophosphate-activated protein kinase (AMPK) has been established in regulating cardiac metabolism, a less studied action of AMPK is its ability to prevent cardiac cell death. Using established AMPK activators like dexamethasone (DEX) or metformin (MET), the objective of the present study was to determine whether AMPK activation prevents tumour necrosis factor-alpha (TNF-alpha) induced apoptosis in adult rat ventricular cardiomyocytes. METHODS AND RESULTS: Cardiomyocytes were incubated with DEX, MET, or TNF-alpha for varying durations (0-12 h). TNF-alpha-induced cell damage was evaluated by measuring caspase-3 activity and Hoechst staining. Protein and gene estimation techniques were employed to determine the mechanisms mediating the effects of AMPK activators on TNF-alpha-induced cardiomyocyte apoptosis. Incubation of myocytes with TNF-alpha for 8 h has increased caspase-3 activation and apoptotic cell death, an effect that was abrogated by DEX and MET. The beneficial effect of DEX and MET was associated with stimulation of AMPK, which led to a rapid and sustained increase in Bad phosphorylation. This event reduced the interaction between Bad and Bcl-xL, limiting cytochrome c release and caspase-3 activation. Addition of Compound C to inhibit AMPK reduced Bad phosphorylation and prevented the beneficial effects of AMPK against TNF-alpha-induced cytotoxicity. CONCLUSION: Our data demonstrate that although DEX and MET are used as anti-inflammatory agents or insulin sensitizers, respectively, their common property to phosphorylate AMPK promotes cardiomyocyte cell survival through its regulation of Bad and the mitochondrial apoptotic mechanism.

Rheb activates protein synthesis and growth in adult rat ventricular cardiomyocytes.

The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of protein synthesis, growth and proliferation in mammalian cells, is implicated in the development of cardiac hypertrophy. Ras homolog enriched in brain (Rheb) positively regulates mTORC1. We have studied whether Rheb is sufficient to activate mTOR signaling and promote protein synthesis and cardiac hypertrophy in adult rat ventricular cardiomyocytes (ARVC). Rheb was overexpressed via an adenoviral vector in isolated ARVC. Overexpression of Rheb in ARVC activated mTORC1 signaling, several components of the translational machinery and stimulated protein synthesis. Our direct visualization approach to determine ARVC size revealed that overexpression of Rheb also induced cell growth and indeed did so to similar extent to the hypertrophic agent, phenylephrine (PE). Despite potent activation of mTORC1 signaling, overexpression of Rheb did not induce expression of the cardiac hypertrophic marker mRNAs for brain natriuretic peptide and atrial natriuretic factor, while PE treatment did markedly increase their expression. All the effects of Rheb were blocked by rapamycin, confirming their dependence on mTORC1 signaling. Our findings reveal that Rheb itself can activate both protein synthesis and cell growth in ARVC and demonstrate the key role played by mTORC1 in the growth of cardiomyocytes.

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.

A simplified model for mitochondrial ATP production.

Most of the adenosine triphosphate (ATP) synthesized during glucose metabolism is produced in the mitochondria through oxidative phosphorylation. This is a complex reaction powered by the proton gradient across the mitochondrial inner membrane, which is generated by mitochondrial respiration. A detailed model of this reaction, which includes dynamic equations for the key mitochondrial variables, was developed earlier by Magnus and Keizer. However, this model is extraordinarily complicated. We develop a simpler model that captures the behavior of the original model but is easier to use and to understand. We then use it to investigate the mitochondrial responses to glycolytic and calcium input. We use the model to explain experimental observations of the opposite effects of raising cytosolic Ca(2+)in low and high glucose, and to predict the effects of a mutation in the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (Nnt) in pancreatic beta-cells.

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 beta-cells. In the present study, we report altered Ca2+ homeostasis and reduced responsiveness to insulin, but not glucose, in Cd38-/- beta-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 beta-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 beta-cells.

Reduced expression of the insulin receptor in mouse insulinoma (MIN6) cells reveals multiple roles of insulin signaling in gene expression, proliferation, insulin content, and secretion.

The role of insulin signaling in pancreatic beta cells has become increasingly apparent. Stably transformed insulinoma cell lines (MIN6) were created with small interfering RNA resulting in the reduction of insulin receptor (IR) expression up to 80% (insulin receptor knockdown, IRKDDelta80). Functionally perturbed IR signaling was confirmed with the absence of insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation. Additionally, Akt phosphorylation was reduced and responded poorly to glucose stimulation. Gene expression profiling revealed that reduced IR expression was associated with alterations in expression of >1,500 genes with diverse functions. IRKD cells exhibited low rate of proliferation due to delay in transition from G0/G1 to S phase, whereas susceptibility to apoptosis did not differ from that of control cells. Insulin content was reduced in proportion to the reduction of IR. IRKD cells maintained glucose responsiveness as measured by NADPH generation, whereas Ca2+ responses and insulin secretion were enhanced. IRKDDelta80 and control cells were treated with glucose (25 mm) or insulin (100 nm) for 45 min, and gene expression profiles were assessed. Transcriptional activation of several hundred early response genes common to both glucose and insulin stimulation was observed in control cells. In IRKDDelta80 cells, insulin failed to activate any genes as anticipated. Importantly, glucose stimulation of gene expression in IRKDDelta80 cells showed that most genes previously activated by glucose were no longer activated, suggesting a major autocrine/paracrine effect of insulin on glucose-regulated gene expression. On the other hand, there were a number of glucose-regulated genes in the IRKDDelta80 cells that were not previously observed in control cells, suggesting a feedback regulation of insulin signaling on glucose-regulated gene expression. These results demonstrate important roles of the insulin receptor in islet beta cell gene expression and function and may serve to elucidate molecular defects in animal models with diminished beta cell insulin signaling.