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Role of Ca2+ in apoptosis evoked by human amylin in pancreatic islet beta-cells.

ABSTRACT: The objective of these studies was to clarify the role of Ca(2+) in the mechanism of death evoked by human amylin (hA) in islet beta-cells. hA forms fibrils in vitro and islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet beta-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 microM) manner. Dying cells underwent shrinkage of the nucleus, with clumping and segregation of chromatin into masses that lay against the nuclear envelope, and internucleosomal DNA fragmentation. These cells therefore show many features of apoptosis, although aspects of the morphology might be characteristic of this particular cell type rather than of a general apoptotic nature. Aurintricarboxylic acid, an inhibitor of both Ca(2+)-dependent and Ca(2+)-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 microM. Direct measurements of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in fura-2 acetoxymethyl ester (AM)-loaded beta-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising [Ca(2+)](i). Modulators of Ca(2+) regulation were tested for their effects on hA-induced beta-cell apoptosis. Ca(2+) ionophore (A23187) and thapsigargin (an inhibitor of endoplasmic reticular Ca(2+)-ATPase activity) by themselves evoked apoptosis accompanied by increased [Ca(2+)](i). Neither the Ca(2+) channel blocker verapamil, the extracellular Ca(2+) chelator EGTA nor the cytosolic Ca(2+) buffer bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid ('BAPTA')/AM protected beta-cells from hA-evoked apoptosis. Prolonged incubation of beta-cells with a lethal dose of hA altered neither the basal [Ca(2+)](i) nor the thapsigargin-induced release of Ca(2+) from intracellular stores. Furthermore, (45)CaCl(2) uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca(2+) homoeostasis do have a significant role in certain forms of beta-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet beta-cells.

SUBMITTER: Bai JZ

PROVIDER: S-EPMC1220523 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Project description:Human amylin-derived oligomers and aggregates are believed to play an important role in the pathogenesis of type II diabetes mellitus (T2DM). In addition to amylin-evoked cell attrition, T2DM is often accompanied by elevated serum copper levels. Although previous studies have shown that human amylin, in the course of its aggregation, produces hydrogen peroxide (H2O2) in solution, and that this process is exacerbated in the presence of copper(ii) ions (Cu(2+)), very little is known about the mechanism of interaction between Cu(2+) and amylin in pancreatic ?-cells, including its pathological significance. Hence, in this study we investigated the mechanism by which Cu(2+) and human amylin catalyze formation of reactive oxygen species (ROS) in cells and in vitro, and examined the modulatory effect of Cu(2+) on amylin aggregation and toxicity in pancreatic rat insulinoma (RIN-m5F) ?-cells. Our results indicate that Cu(2+) interacts with human and rat amylin to form metalo-peptide complexes with low aggregative and oxidative properties. Human and non-amyloidogenic rat amylin produced minute (nM) amounts of H2O2, the accumulation of which was slightly enhanced in the presence of Cu(2+). In a marked contrast to human and rat amylin, and in the presence of the reducing agents glutathione and ascorbate, Cu(2+) produced ?M concentrations of H2O2 surpassing the amylin effect by several fold. The current study shows that human and rat amylin not only produce but also quench H2O2, and that human but not rat amylin significantly decreases the amount of H2O2 in solution produced by Cu(2+) and glutathione. Similarly, human amylin was found to also decrease hydroxyl radical formation elicited by Cu(2+) and glutathione. Furthermore, Cu(2+) mitigated the toxic effect of human amylin by inhibiting activation of pro-apoptotic caspase-3 and stress-kinase signaling pathways in rat pancreatic insulinoma cells in part by stabilizing human amylin in its native conformational state. This sacrificial quenching of metal-catalyzed ROS by human amylin and copper's anti-aggregative and anti-apoptotic properties suggest a novel and protective role for the copper-amylin complex.

Project description:The goal of this study was to determine whether recombinant Islet NeoGenesis Associated Protein (rINGAP) and its active core, a pentadecapeptide INGAP104-118 (Ingap-p), protect β cells against cytokine-induced death. INGAP has been shown to induce islet neogenesis in diabetic animals, to stimulate β-cell proliferation and differentiation, and to improve islet survival and function. Importantly, Ingap-p has shown promising results in clinical trials for diabetes (phase I/II). However, the full potential of INGAP and its mechanisms of action remain poorly understood. Using rat insulinoma cells RINm5F and INS-1 treated with interleukin-1β (IL-1β) and interferon-gamma (IFN-γ), we demonstrate here that both rINGAP and Ingap-p inhibit apoptosis, Caspase-3 activation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and explore the related signaling pathways. As expected, IL-1β induced nuclear factor kappa B (NF-κB), p38, and JNK signaling, whereas interferon-gamma (IFN-γ) activated the JAK2/STAT1 pathway and potentiated the IL-1β effects. Both rINGAP and Ingap-p decreased phosphorylation of IKKα/β, IkBα, and p65, although p65 nuclear translocation was not inhibited. rINGAP, used for further analysis, also inhibited STAT3, p38, and JNK activation. Interestingly, all inhibitory effects of rINGAP were observed for the cytokine cocktail, not IL-1β alone, and were roughly equal to reversing the potentiating effects of INFγ. Furthermore, rINGAP had no effect on IL-1β/NF-κB-induced gene expression (e.g., Ccl2, Sod2) but downregulated several IFNγ-stimulated (Irf1, Socs1, Socs3) or IFNγ-potentiated (Nos2) genes. This, however, was observed again only for the cytokine cocktail, not IFNγ alone, and rINGAP did not inhibit the IFNγ-induced JAK2/STAT1 activation. Together, these intriguing results suggest that INGAP does not target either IL-1β or IFNγ individually but rather inhibits the signaling crosstalk between the two, the exact mechanism of which remains to be investigated. In summary, our study characterizes the anti-inflammatory effects of INGAP, both protein and peptide, and suggests a new therapeutic utility for INGAP in the treatment of diabetes.

Project description:Aims/Hypothesis: Galectin 3 appears to play a proinflammatory role in several inflammatory and autoimmune diseases. Also, there is evidence that galectin 3 plays a role in both type-1 and type-2 diabetes. During obesity, hematopoietic cell-derived galectin 3 induces insulin resistance. While the role of galectin 3 expressed in islet-invading immune cells in both type-1 and type-2 diabetes has been studied, the importance of the expression of this molecule on the target pancreatic β cells has not been defined. Methods: To clarify the role of galectin 3 expression in β cells during obesity-induced diabetogenesis, we developed transgenic mice selectively overexpressing galectin 3 in β cells and tested their susceptibility to obesity-induced type-2 diabetes. Obesity was induced with a 16-week high-fat diet regime. Pancreatic β cells were tested for susceptibility to apoptosis induced by non-esterified fatty acids and cytokines as well as parameters of oxidative stress. Results: Our results demonstrated that overexpression of galectin 3 increases β-cell apoptosis in HFD conditions and increases the percentage of proinflammatory F4/80+ macrophages in islets that express galectin 3 and TLR4. In isolated islets, we have shown that galectin 3 overexpression increases cytokine and palmitate-triggered β-cell apoptosis and also increases NO2--induced oxidative stress of β cells. Also, in pancreatic lymph nodes, macrophages were shifted toward a proinflammatory TNF-α-producing phenotype. Conclusions/Interpretation: By complementary in vivo and in vitro approaches, we have shown that galectin 3-overexpression facilitates β-cell damage, enhances cytokine and palmitate-triggered β-cell apoptosis, and increases NO2--induced oxidative stress in β cells. Further, the results suggest that increased expression of galectin 3 in the pancreatic β cells affects the metabolism of glucose and glycoregulation in mice on a high-fat diet, affecting both fasting glycemic values and glycemia after glucose loading.

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Role of Ca2+ in apoptosis evoked by human amylin in pancreatic islet beta-cells.

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