thapsigargin and Diabetes-Mellitus

Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca. MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca. Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca. These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca

Topics: Animals; Apoptosis; Calcium; Cell Line; Diabetes Mellitus; Endoplasmic Reticulum Stress; Focal Adhesion Kinase 1; Focal Adhesion Kinase 2; Glucose; Insulin-Secreting Cells; Lupanes; Mice; Phosphorylation; Thapsigargin; Triterpenes; Tyrosine

Beta-cell death and dysfunction are involved in the development of type 1 and 2 diabetes. ER-stress impairs beta-cells function resulting in pro-apoptotic stimuli that promote cell death. Hence, the identification of protective mechanisms in response to ER-stress could lead to novel therapeutic targets and insight in the pathology of these diseases. Here, we report the identification of proteins involved in dysregulated pathways upon thapsigargin treatment of MIN6 cells. Utilizing quantitative proteomics we identified upregulation of proteins involved in protein folding, unfolded protein response, redox homeostasis, proteasome processes associated with endoplasmic reticulum and downregulation of TCA cycle, cellular respiration, lipid metabolism and ribosome assembly processes associated to mitochondria and eukaryotic initiation translation factor components. Subsequently, pro-inflammatory cytokine treatment was performed to mimic pathological changes observed in beta-cells during diabetes. Cytokines induced ER stress and impaired mitochondrial function in beta-cells corroborating the results obtained with the proteomic approach. HSPB1 levels are increased by prolactin on pancreatic beta-cells and this protein is a key factor for cytoprotection although its role has not been fully elucidated. Here we show that while up-regulation of HSPB1 was able to restore the mitochondrial dysfunction induced by beta-cells' exposure to inflammatory cytokines, silencing of this chaperone abrogated the beneficial effects promoted by PRL. Taken together, our results outline the importance of HSPB1 to mitigate beta-cell dysfunction. Further studies are needed to elucidate its role in diabetes.

Topics: Animals; Apoptosis; Cell Death; Cell Line; Cell Respiration; Cytokines; Diabetes Mellitus; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Insulin-Secreting Cells; Mice; Mitochondria; Molecular Chaperones; Proteomics; Thapsigargin

Endoplasmic reticulum (ER) stress is associated with the development of diabetes. The present study sought to investigate the effect of Liraglutide, a glucagon like peptide 1 analogue, on ER stress in β-cells. We found that Liraglutide protected the pancreatic INS-1 cells from thapsigargin-induced ER stress and the ER stress associated cell apoptosis, mainly by suppressing the PERK and IRE1 pathways. We further tested the effects of Liraglutide in the Akita mouse, an ER-stress induced type 1 diabetes model. After administration of Liraglutide for 8weeks, p-eIF2α and p-JNK were significantly decreased in the pancreas of the Akita mouse, while the treatment showed no significant impact on the levels of insulin of INS-cells. Taken together, our findings suggest that Liraglutide may protect pancreatic cells from ER stress and its related cell death.

Topics: Animals; Cell Death; Cytoprotection; Diabetes Mellitus; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Glucagon-Like Peptide 1; Insulin-Secreting Cells; JNK Mitogen-Activated Protein Kinases; Liraglutide; Male; Mice; Mice, Inbred C57BL; Phosphorylation; Rats; Signal Transduction; Thapsigargin

In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues.

Topics: Abdominal Fat; Adipocytes; Animals; Carrier Proteins; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Fatty Acids; Homeostasis; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Lipase; Lipolysis; Male; MAP Kinase Signaling System; Obesity; p38 Mitogen-Activated Protein Kinases; Perilipin-1; Phosphoproteins; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Thapsigargin; Tunicamycin

Blood cells from subjects with hypertension and/or diabetes mellitus have been successfully studied in the past to gain insight into pathological alterations of several signal transduction pathways. Diabetes mellitus is also considered to be a disease of abnormal cellular Ca2+ metabolism, as metabolic derangements of Ca2+ transport have been noticed both in the prediabetic state and as a consequence of hyperglycemia and oxidative stress. In this report, we used peripheral blood lymphocytes from type 2 diabetes patients and control subjects to study and delineate different mechanisms of Ca2+ turnover that determine the level of cytosolic calcium (Ca(i)). While demonstrating the specific Ca2+ turnover alterations, we suggest that insights into the pathophysiology of diabetic complications originating from signal transduction defects could be conveniently studied using blood cell types such as lymphocytes and that such studies could lead to the identification of new molecular drug targets.

Topics: Calcium; Calcium Signaling; Cytosol; Diabetes Mellitus; Dose-Response Relationship, Drug; Lymphocytes; Models, Biological; Thapsigargin

D-Myo-inositol (3,4,5,6) tetrakisphosphate [Ins(3,4,5,6)P(4)] or phosphatidylinositol 3-kinase (PI 3-kinase) activity acts to inhibit calcium-dependent chloride secretion in T84 colonic epithelial cells. To further distinguish between the contributions of these two signaling pathways to the inhibition of secretion, we studied effects of insulin, because the insulin receptor links to PI 3-kinase but not to pathways postulated to generate Ins(3,4,5,6)P(4). Chloride secretion across T84 cell monolayers was studied in Ussing chambers. Activation of PI 3-kinase was assessed by Western blotting. Basolateral, but not apical, addition of insulin inhibited carbachol- and thapsigargin-induced chloride secretion in a time- and concentration-dependent fashion. Insulin-like growth factor-I (IGF-I) had similar effects. Insulin had no effect on Ins(3,4,5,6)P(4) levels, and the inhibitory effects of insulin and IGF-I on chloride secretion were fully reversed by the PI 3-kinase inhibitors wortmannin and LY-294002. Western blot analysis showed that both insulin and IGF-I recruited the 85-kDa regulatory and 110-kDa catalytic subunits of PI 3-kinase to anti-phosphotyrosine immunoprecipitates. In conclusion, insulin and IGF-I act to inhibit calcium-dependent chloride secretion through a PI 3-kinase-dependent pathway. Because insulin is released in a pulsatile fashion postprandially and IGF-I levels are elevated in pathological settings, our findings may have physiological and/or pathophysiological significance.

Topics: Androstadienes; Carbachol; Cells, Cultured; Chlorides; Cholinergic Agonists; Chromones; Colon; Diabetes Mellitus; Diarrhea; Enzyme Inhibitors; Humans; Hypoglycemic Agents; Insulin; Insulin-Like Growth Factor I; Intestinal Mucosa; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Thapsigargin; Tyrosine; Wortmannin