thapsigargin and Hyperglycemia

Metabolic syndrome, which includes hypertension, hyperglycemia, obesity, insulin resistance, and dyslipidemia, has a negative impact on cognitive health. Endoplasmic reticulum (ER) stress is activated during metabolic syndrome, however it is not known which factor associated with metabolic syndrome contributes to this stress. ER stress has been reported to play a role in the development of insulin resistance in peripheral tissues. The role of ER stress in the development of insulin resistance in hippocampal neurons is not known. In the current study, we investigated ER stress in the hippocampus of 3 different mouse models of metabolic syndrome: the C57BL6 mouse on a high fat (HF) diet; apolipoprotein E, leptin, and apolipoprotein B-48 deficient (ApoE 3KO) mice; and the low density lipoprotein receptor, leptin, and apolipoprotein B-48 deficient (LDLR 3KO) mice. We demonstrate that ER stress is activated in the hippocampus of HF mice, and for the first time, in ApoE 3KO mice, but not LDLR 3KO mice. The HF and ApoE 3KO mice are hyperglycemic; however, the LDLR 3KO mice have normal glycemia. This suggests that hyperglycemia may play a role in the activation of ER stress in the hippocampus. Similarly, we also demonstrate that impaired insulin signaling is only present in the HF and ApoE 3KO mice, which suggests that ER stress may play a role in insulin resistance in the hippocampus. To confirm this we pharmacologically induced ER stress with thapsigargin in human hippocampal neurons. We demonstrate for the first time that thapsigargin leads to ER stress and impaired insulin signaling in human hippocampal neurons. Our results may provide a potential mechanism that links metabolic syndrome and cognitive health.

Topics: Animals; Apolipoprotein B-100; Apolipoproteins B; Apolipoproteins E; Diet, High-Fat; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Heat-Shock Proteins; Hippocampus; Humans; Hyperglycemia; Insulin Resistance; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Phenotype; Receptor, Insulin; Receptors, LDL; Signal Transduction; Thapsigargin

Hyperglycemia-induced cardiomyocyte apoptosis contributes to diabetic cardiomyopathy. Glucagon-like peptide-1 (Glp1) receptor (Glp1r) agonists improve cardiac function and survival in response to ischemia-reperfusion and myocardial infarction. The present studies assessed whether Glp1r activation exerts direct cardioprotective effects in response to hyperglycemia. Treatment with the Glp1r agonist Exendin-4 attenuated apoptosis in neonatal rat ventricular cardiomyocytes cultured in high (33 mM) glucose. This protective effect was mimicked by the cAMP inducer forskolin. The Exendin-4 protective effect was blocked by the Glp1r antagonist Exendin(9-39) or the PKA antagonist H-89. Exendin-4 also protected cardiomyocytes from hydrogen peroxide (H2O2)-induced cell death. Cardiomyocyte protection by Exendin-4 was not due to reduced reactive oxygen species levels. Instead, Exendin-4 treatment reduced endoplasmic reticulum (ER) stress, demonstrated by decreased expression of glucose-regulated protein-78 (GRP78) and CCAT/enhancer-binding homologous protein (CHOP). Reduced ER stress was not due to activation of the unfolded protein response, indicating that Exendin-4 directly prevents ER stress. Exendin-4 treatment selectively protected cardiomyocytes from thapsigargin- but not tunicamycin-induced death. This suggests that Exendin-4 attenuates thapsigargin-mediated inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase-2a (SERCA2a). High glucose attenuates SERCA2a function by reducing SERCA2a mRNA and protein levels, but Exendin-4 treatment prevented this reduction. Exendin-4 treatment also enhanced phosphorylation of the SERCA2a regulator phospholamban (PLN), which would be expected to stimulate SERCA2a activity. In sum, Glp1r activation attenuates high glucose-induced cardiomyocyte apoptosis in association with decreased ER stress and markers of enhanced SERCA2a activity. These findings identify a novel mechanism whereby Glp1-based therapies could be used as treatments for diabetic cardiomyopathy.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Cells, Cultured; Colforsin; Diabetic Cardiomyopathies; Endoplasmic Reticulum Stress; Enzyme Activation; Exenatide; Glucagon-Like Peptide-1 Receptor; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Hydrogen Peroxide; Hyperglycemia; Hypoglycemic Agents; Isoquinolines; Membrane Proteins; Myocytes, Cardiac; Oxidative Stress; Peptide Fragments; Peptides; Phosphorylation; Protein Kinase Inhibitors; Rats; Receptors, Glucagon; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Thapsigargin; Transcription Factor CHOP; Tunicamycin; Unfolded Protein Response; Venoms

In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress. Hyperglycemia-induced ER stress has not been studied in adipocyte differentiation and adipokine expression. Taurine has been known to protect the cells against ER stress. This study examined the effect of taurine on ER stress-induced adipocyte differentiation and adipokine expression to explain the therapeutic effect of taurine on diabetes and obesity. To do this, human preadipocytes were differentiated into adipocytes, in the presence or absence of taurine, under ER stress conditions. Changes in adipokine expression in adipocytes stimulated with IL-1β were investigated in the presence or absence of taurine. Human preadipocytes were treated with thapsigargin (10 nM) or high glucose concentrations (100 mM) as ER stress inducers during differentiation into adipocytes. Thapsigargin inhibited the differentiation of adipocytes in a dose-dependent manner, but the high glucose concentration treatment did not. Taurine 100 mM treatment did not block the inhibition of differentiation of preadipcytes into adipocytes. Furthermore, the high glucose concentration treatment inhibited the expression of adiponectin and increased the expression of leptin in human adipocytes. However, taurine treatment did not affect the expression of two adipokines. In conclusion, the therapeutic mechanism of taurine in diabetes and obesity does not appear to occur by alleviating hyperglycemia-mediated ER stress. To clarify the molecular mechanism by which taurine improves diabetic symptoms and obesity in animal models, the protective effect of taurine against hyperglycemia- or overnutrition-mediated ER stress should be further evaluated under various conditions or types of ER stress.

Topics: Acetylcysteine; Adipocytes; Adiponectin; Cell Differentiation; Endoplasmic Reticulum Stress; Humans; Hyperglycemia; Leptin; Taurine; Taurochenodeoxycholic Acid; Thapsigargin

The endoplasmic reticulum (ER) is an important site for protein folding and becomes "stressed" when its capacity to fold proteins is overwhelmed. In response, "unfolded protein response" (UPR) genes are induced, increasing the capacity to fold proteins; if the response is insufficient, then apoptosis ensues. For investigation of whether proteinuria and hyperglycemia induce ER stress in renal epithelial cells, microarray data from biopsies of established diabetic nephropathy (DN) were analyzed. Expression of UPR genes was significantly different in these biopsies than in control kidneys or biopsies of patients with mild DN, suggesting an association between the degree of DN and UPR gene expression. Expression of the transcription factor XBP1 and the ER chaperones HSPA5 and HYOU1 were increased, but the proapoptotic gene DDIT3 was unchanged. These findings were replicated in an independent cohort of patients with established DN by real-time reverse transcriptase-PCR. Immunofluorescence of renal biopsies from patients with DN confirmed the upregulation for HSPA5 and HYOU1 proteins in tubular epithelia. In biopsies of minimal-change disease, the mRNA levels of some ER stress molecules were also induced, but protein expression of HSPA5 and HYOU1 remained significantly lower than that observed in DN. Exposure of renal tubular epithelial cells to albumin and high glucose in vitro enhanced expression of genes involved in ER stress. These observations suggest that in proteinuric diseases, tubular epithelial cells undergo ER stress, which induces an adaptive, protective UPR. Although this may protect the cells from ER stress, persistence of hyperglycemia and proteinuria may eventually lead to apoptosis.

Topics: Albumins; Cell Line; Diabetic Nephropathies; DNA-Binding Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Epithelial Cells; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Hyperglycemia; Kidney Tubules; Molecular Chaperones; Oligonucleotide Array Sequence Analysis; Protein Folding; Proteins; Proteinuria; Regulatory Factor X Transcription Factors; RNA, Messenger; Thapsigargin; Transcription Factors; Tunicamycin; X-Box Binding Protein 1

Hyperglycemia alters cardiac function and often leads to diabetic cardiomyopathy as cardiomyocyte apoptosis causes a hypertrophied heart to deteriorate to dilation and failure. Paradoxically, many short-term animal models of hyperglycemia protect against ischemia-induced damage, including apoptosis, by limiting Ca(2+) overload. We have determined that, like nonexcitable cells, both neonatal and adult cardiomyocytes respond to depletion of sarcoplasmic/endoplasmic reticulum Ca(2+) stores with an influx of extracellular Ca(2+) through channels distinct from voltage-gated Ca(2+) channels, a process termed capacitative Ca(2+) entry (CCE). Here, we demonstrate that in neonatal rat cardiomyocytes, hyperglycemia decreased CCE induced by angiotensin II or the Ca(2+)ATPase inhibitor thapsigargin. Hyperglycemia also significantly blunted Ca(2+)-dependent hypertrophic responses by approximately 60%, as well as the Ca(2+)-sensitive nuclear translocation of a chimeric protein bearing the nuclear localization signal of a nuclear factor of activated T-cells transcription factor. The attenuation of CCE by hyperglycemia was prevented by azaserine, an inhibitor of hexosamine biosynthesis, and partially by inhibitors of oxidative stress. This complements previous work showing that increasing hexosamine metabolites in neonatal cardiomyocytes also inhibited CCE. The inhibition of CCE by hyperglycemia thus provides a likely explanation for the transition to diabetic cardiomyopathy as well as to the protection afforded to injury after ischemia/reperfusion in diabetic models.

Topics: Animals; Animals, Newborn; Biological Transport; Calcium; Calcium Channels; Cardiomegaly; Cell Nucleus; DNA-Binding Proteins; Glucose; Hexosamines; Hyperglycemia; Myocytes, Cardiac; NFATC Transcription Factors; Nuclear Proteins; Osmolar Concentration; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum; Thapsigargin; Transcription Factors

Concentrations of free cytoplasmic Ca2+ in rat aortic smooth muscle (RASM) cells were monitored using the ratiometric Ca2+ indicator fura 2-acetoxymethyl ester (AM). In RASM cells cultured in 5 mM Glc, incubation with angiotensin II, ATP, or thapsigargin [a selective inhibitor of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase] depleted SR Ca2+ stores and initiated a capacitative Ca2+ influx through the plasma membrane. This influx was resistant to verapamil, a selective inhibitor of L-type voltage-gated Ca2+ channels, but was sensitive to SKF-96365, an inhibitor of the receptor-operated Ca2+ entry pathway. RASM cells cultured in 25 mM Glc exhibited a significant decrease in cytoplasmic Ca2+ responses to agonist-induced Ca2+ release from SR stores and to subsequent capacitative Ca2+ entry. In addition, the cytoplasmic response to thapsigargin-induced release of Ca2+ from the SR in hyperglycemic cells peaked more sharply than in control cells and returned to baseline more rapidly. The effects of hyperglycemia were not overcome by myo-inositol supplementation.

Topics: Adenosine Triphosphate; Angiotensin II; Animals; Aorta; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Cells, Cultured; Cytoplasm; Hyperglycemia; Imidazoles; Male; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum; Terpenes; Thapsigargin