Esteban Nicolas Gurzov

Glucagon-Like Peptide-1 Agonists Protect Pancreatic β-Cells From Lipotoxic Endoplasmic Reticulum Stress Through Upregulation of BiP and JunB

OBJECTIVE Chronic exposure of pancreatic β-cells to saturated free fatty acids (FFAs) causes endoplasmic reticulum (ER) stress and apoptosis and may contribute to β-cell loss in type 2 diabetes. Here, we evaluated the molecular mechanisms involved in the protection of β-cells from lipotoxic ER stress by glucagon-like peptide (GLP)-1 agonists utilized in the treatment of type 2 diabetes. RESEARCH DESIGN AND METHODS INS-1E or fluorescence-activated cell sorter–purified primary rat β-cells were exposed to oleate or palmitate with or without the GLP-1 agonist exendin-4 or forskolin. Cyclopiazonic acid was used as a synthetic ER stressor, while the activating transcription factor 4–C/EBP homologous protein branch was selectively activated with salubrinal. The ER stress signaling pathways modulated by GLP-1 agonists were studied by real-time PCR and Western blot. Knockdown by RNA interference was used to identify mediators of the antiapoptotic GLP-1 effects in the ER stress response and downstream mitochondrial cell death mechanisms. RESULTS Exendin-4 and forskolin protected β-cells against FFAs via the induction of the ER chaperone BiP and the antiapoptotic protein JunB that mediate β-cell survival under lipotoxic conditions. On the other hand, exendin-4 and forskolin protected against synthetic ER stressors by inactivating caspase 12 and upregulating Bcl-2 and X-chromosome–linked inhibitor of apoptosis protein that inhibit mitochondrial apoptosis. CONCLUSIONS These observations suggest that GLP-1 agonists increase in a context-dependent way the β-cell defense mechanisms against different pathways involved in ER stress–induced apoptosis. The identification of the pathways modulated by GLP-1 agonists allows for targeted approaches to alleviate β-cell ER stress in diabetes.

Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction

Diabetes is a metabolic disease affecting nearly 300 million individuals worldwide. Both types of diabetes (1 and 2) are characterized by loss of functional pancreatic β-cell mass causing different degrees of insulin deficiency. The Bcl-2 family has a double-edged effect in diabetes. These proteins are crucial controllers of the mitochondrial pathway of β-cell apoptosis induced by pro-inflammatory cytokines or lipotoxicity. In parallel, some Bcl-2 members also regulate glucose metabolism and β-cell function. In this review, we describe the role of Bcl-2 proteins in β-cell homeostasis and death. We focus on how these proteins interact, their contribution to the crosstalk between endoplasmic reticulum stress and mitochondrial permeabilization, their context-dependent usage following different pro-apoptotic stimuli, and their role in β-cell physiology.

Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis.

Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic β-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1β+IFN-γ and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in β-cell apoptosis. Chemical ER stress-induced DP5 upregulation is JNK/c-Jun-dependent. DP5 activation by cytokines also involves JNK/c-Jun phosphorylation and is antagonized by JunB. Interestingly, cytokine-inducted DP5 expression precedes ER stress: mitochondrial release of cytochrome c and ER stress are actually a consequence of enhanced DP5 activation by cytokine-mediated nitric oxide formation. Our findings show that DP5 is central for β-cell apoptosis after different stimuli, and that it can act up- and downstream of ER stress. These observations contribute to solve two important questions, namely the mechanism by which IL-1β+IFN-γ induce β-cell death and the nature of the downstream signals by which ER stress ‘convinces’ β-cells to trigger apoptosis.

p53 up-regulated modulator of apoptosis (PUMA) activation contributes to pancreatic beta-cell apoptosis induced by proinflammatory cytokines and endoplasmic reticulum stress.

Type 1 diabetes is an autoimmune disorder characterized by chronic inflammation and pancreatic β-cell loss. Here, we demonstrate that the proinflammatory cytokine interleukin-1β, combined with interferon-γ, induces the expression of the Bcl-2 homology 3 (BH3)-only activator PUMA (p53 up-regulated modulator of apoptosis) in β-cells. Transcriptional activation of PUMA is regulated by nuclear factor-κB and endoplasmic reticulum stress but is independent of p53. PUMA activation leads to mitochondrial Bax translocation, cytochrome c release, and caspase-3 cleavage resulting in β-cell demise. The antiapoptotic Bcl-XL protein is localized mainly at the mitochondria of the β-cells and antagonizes PUMA action, but Bcl-XL is inactivated by the BH3-only sensitizer DP5/Hrk in cytokine-exposed β-cells. Moreover, a pharmacological mimic of the BH3-only sensitizer Bad, which inhibits Bcl-XL and Bcl-2, induces PUMA-dependent β-cell death and potentiates cytokine-induced apoptosis. Our data support a hierarchical activation of BH3-only proteins controlling the intrinsic pathway of β-cell apoptosis in the context of inflammation and type 1 diabetes.

STAT1 is a master regulator of pancreatic beta-cell apoptosis and islet inflammation.

Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.

Cytokines Tumor Necrosis Factor-α and Interferon-γ Induce Pancreatic β-Cell Apoptosis through STAT1-mediated Bim Protein Activation

Type 1 diabetes is characterized by local inflammation (insulitis) in the pancreatic islets causing β-cell loss. The mitochondrial pathway of apoptosis is regulated by the balance and interaction between Bcl-2 members. Here we clarify the molecular mechanism of β-cell death triggered by the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The combination of TNF-α + IFN-γ induced DP5, p53 up-regulated modulator of apoptosis (PUMA), and Bim expression in human islets and rodent β-cells. DP5 and PUMA inactivation by RNA interference partially protected against TNF-α + IFN-γ-induced β-cell apoptosis. DP5 knock-out mice had increased β-cell area, and isolated islets from these mice were resistant to cytokine exposure. Bim expression was transcriptionally regulated by STAT1, and its activation triggered cleavage of caspases. Silencing of Bim protected rodent and human β-cells to a large extent against TNF-α + IFN-γ, indicating a major role of this BH3-only activator protein in the mechanism of apoptosis. Our data support a highly regulated and context-dependent modulation of specific Bcl-2 members controlling the mitochondrial pathway of β-cell apoptosis during insulitis.

PTPN2, a Candidate Gene for Type 1 Diabetes, Modulates Pancreatic β-Cell Apoptosis via Regulation of the BH3-Only Protein Bim

OBJECTIVE Genome-wide association studies allowed the identification of several associations between specific loci and type 1 diabetes (T1D). However, the mechanisms by which most candidate genes predispose to T1D remain unclear. We presently evaluated the mechanisms by which PTPN2, a candidate gene for T1D, modulates β-cell apoptosis after exposure to type I and II interferons (IFNs), cytokines that contribute to β-cell loss in early T1D. RESEARCH DESIGN AND METHODS Small interfering RNAs were used to inhibit PTPN2, STAT1, Bim, and Jun NH2-terminal kinase 1 (JNK1) expression. Cell death was assessed by Hoechst and propidium iodide staining. BAX translocation, Bim phosphorylation, cytochrome c release, and caspases 9 and 3 activation were measured by Western blot or immunofluorescence. RESULTS PTPN2 knockdown exacerbated type I IFN–induced apoptosis in INS-1E, primary rat, and human β-cells. PTPN2 silencing and exposure to type I and II IFNs induced BAX translocation to the mitochondria, cytochrome c release, and caspase 3 activation. There was also an increase in Bim phosphorylation that was at least in part regulated by JNK1. Of note, both Bim and JNK1 knockdown protected β-cells against IFN-induced apoptosis in PTPN2-silenced cells. CONCLUSIONS The present findings suggest that local IFN production may interact with a genetic factor (PTPN2) to induce aberrant proapoptotic activity of the BH3-only protein Bim, resulting in increased β-cell apoptosis via JNK activation and the intrinsic apoptotic pathway. This is the first indication of a direct interaction between a candidate gene for T1D and the activation of a specific downstream proapoptotic pathway in β-cells.

Tumor necrosis factor-α and interferon-γ induce pancreatic β-cell apoptosis through STAT1-mediated Bim activation

Type 1 diabetes is characterized by local inflammation (insulitis) in the pancreatic islets causing β-cell loss. The mitochondrial pathway of apoptosis is regulated by the balance and interaction between Bcl-2 members. Here we clarify the molecular mechanism of β-cell death triggered by the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The combination of TNF-α + IFN-γ induced DP5, p53 up-regulated modulator of apoptosis (PUMA), and Bim expression in human islets and rodent β-cells. DP5 and PUMA inactivation by RNA interference partially protected against TNF-α + IFN-γ-induced β-cell apoptosis. DP5 knock-out mice had increased β-cell area, and isolated islets from these mice were resistant to cytokine exposure. Bim expression was transcriptionally regulated by STAT1, and its activation triggered cleavage of caspases. Silencing of Bim protected rodent and human β-cells to a large extent against TNF-α + IFN-γ, indicating a major role of this BH3-only activator protein in the mechanism of apoptosis. Our data support a highly regulated and context-dependent modulation of specific Bcl-2 members controlling the mitochondrial pathway of β-cell apoptosis during insulitis.

Death Protein 5 and p53-Upregulated Modulator of Apoptosis Mediate the Endoplasmic Reticulum Stress–Mitochondrial Dialog Triggering Lipotoxic Rodent and Human β-Cell Apoptosis

Environmental factors such as diets rich in saturated fats contribute to dysfunction and death of pancreatic β-cells in diabetes. Endoplasmic reticulum (ER) stress is elicited in β-cells by saturated fatty acids. Here we show that palmitate-induced β-cell apoptosis is mediated by the intrinsic mitochondrial pathway. By microarray analysis, we identified a palmitate-triggered ER stress gene expression signature and the induction of the BH3-only proteins death protein 5 (DP5) and p53-upregulated modulator of apoptosis (PUMA). Knockdown of either protein reduced cytochrome c release, caspase-3 activation, and apoptosis in rat and human β-cells. DP5 induction depends on inositol-requiring enzyme 1 (IRE1)–dependent c-Jun NH2-terminal kinase and PKR–like ER kinase (PERK)–induced activating transcription factor (ATF3) binding to its promoter. PUMA expression is also PERK/ATF3-dependent, through tribbles 3 (TRB3)–regulated AKT inhibition and FoxO3a activation. DP5−/− mice are protected from high fat diet–induced loss of glucose tolerance and have twofold greater pancreatic β-cell mass. This study elucidates the crosstalk between lipotoxic ER stress and the mitochondrial pathway of apoptosis that causes β-cell death in diabetes.

RNA interference against Hec1 inhibits tumor growth in vivo

Hec1 (highly expressed in cancer) plays an important role in chromosome segregation by interacting with a subset of checkpoint proteins that survey proper chromosome alignment and bipolar spindle attachment. In order to disrupt mitotic progression of tumor cell lines, we have used retrovirus and adenovirus vectors that inhibit Hec1 synthesis. Vector-expressed short hairpin RNAs (shRNAs) caused very efficient depletion of the target protein, cellular arrest and considerable mitotic catastrophe induction 96 h post infection in human cervix-adenocarcinoma (HeLa) and glioblastoma (U-373-MG) cell lines. Furthermore, adenocarcinomas induced in the flanks of nude mice show significant reduction in size compared with control when treated with either Hec1-shRNA retroviruses or adenoviruses. These results indicate that depletion of Hec1 could be used as a new strategy to block the dividing cell, and therefore against cancer.