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PPAR-? activation restores pancreatic islet SERCA2 levels and prevents ?-cell dysfunction under conditions of hyperglycemic and cytokine stress.

ABSTRACT: The maintenance of intracellular Ca(2+) homeostasis in the pancreatic ?-cell is closely regulated by activity of the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) pump. Our data demonstrate a loss of ?-cell SERCA2b expression in several models of type 2 diabetes including islets from db/db mice and cadaveric diabetic human islets. Treatment of 832/13 rat INS-1-derived cells with 25 mm glucose and the proinflammatory cytokine IL-1? led to a similar loss of SERCA2b expression, which was prevented by treatment with the peroxisome proliferator-activated receptor (PPAR)-? agonist, pioglitazone. Pioglitazone was able to also protect against hyperglycemia and cytokine-induced elevations in cytosolic Ca(2+) levels, insulin-secretory defects, and cell death. To determine whether PPAR-? was a direct transcriptional regulator of the SERCA2 gene, luciferase assays were performed and showed that a -259 bp region is sufficient to confer PPAR-? transactivation; EMSA and chromatin immunoprecipitation experiments confirmed that PPAR-? directly binds a PPAR response element in this proximal region. We next sought to characterize the mechanisms by which SERCA2b was down-regulated. INS-1 cells were exposed to high glucose and IL-1? in time course experiments. Within 2 h of exposure, activation of cyclin-dependent kinase 5 (CDK5) was observed and correlated with increased serine-273 phosphorylation of PPAR-? and loss of SERCA2 protein expression, findings that were prevented by pioglitazone and roscovitine, a pharmacological inhibitor of CDK5. We conclude that pioglitazone modulates SERCA2b expression through direct transcriptional regulation of the gene and indirectly through prevention of CDK5-induced phosphorylation of PPAR-?.

SUBMITTER: Kono T

PROVIDER: S-EPMC3275161 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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PPAR-γ activation restores pancreatic islet SERCA2 levels and prevents β-cell dysfunction under conditions of hyperglycemic and cytokine stress.

Kono Tatsuyoshi T Ahn Geonyoung G Moss Dan R DR Gann Liann L Zarain-Herzberg Angel A Nishiki Yurika Y Fueger Patrick T PT Ogihara Takeshi T Evans-Molina Carmella C

Molecular endocrinology (Baltimore, Md.) 20120112 2

The maintenance of intracellular Ca(2+) homeostasis in the pancreatic β-cell is closely regulated by activity of the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) pump. Our data demonstrate a loss of β-cell SERCA2b expression in several models of type 2 diabetes including islets from db/db mice and cadaveric diabetic human islets. Treatment of 832/13 rat INS-1-derived cells with 25 mm glucose and the proinflammatory cytokine IL-1β led to a similar loss of SERCA2b expression, which was preven ...[more]

PMID: 22240811

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Project description:Background/aimsWe recently reported increased phosphorylation (at S536) of the p65 subunit of NFκB (Rel A) in pancreatic beta (INS-1 832/13) cells following exposure to hyperglycemic (HG) conditions. We also demonstrated that HG-induced S536 phosphorylation of p65 is downstream to the regulatory effects of CARD9 since deletion of CARD9 expression significantly attenuated HG-induced S536 phosphorylation of p65 in beta cells. The overall objective of the current investigation is to identify putative mechanisms underlying HG-induced phosphorylation of p65 in islet beta cells following exposure to HG conditions.MethodsINS-1 832/13 cells were incubated in low glucose (LG; 2.5 mM) or high glucose (HG; 20 mM) containing media for 24 hours in the absence or presence of small molecule inhibitors of G protein prenylation and activation. Non-nuclear and nuclear fractions were isolated from INS-1 832/13 cells using a commercially available (NE-PER) kit. Degree of S536 phosphorylation of the p65 subunit was quantified by western blotting and densitometry.ResultsHG-induced p65 phosphorylation was significantly attenuated by inhibitors of protein prenylation (e.g., simvastatin and L-788,123). Pharmacological inhibition of Tiam1-Rac1 (e.g., NSC23766) and Vav2-Rac1 (e.g., Ehop-016) signaling pathways exerted minimal effects on HG-induced p65 phosphorylation. However, EHT-1864, a small molecule compound, which binds to Rac1 thereby preventing GDP/GTP exchange, markedly suppressed HG-induced p65 phosphorylation, suggesting that Rac1 activation is requisite for HG-mediated p65 phosphorylation. Lastly, EHT-1864 significantly inhibited nuclear association of STAT3, but not total p65, in INS-1 832/13 cells exposed to HG conditions.ConclusionActivation of Rac1, a step downstream to HG-induced activation of CARD9, might represent a requisite signaling step in the cascade of events leading to HG-induced S536 phosphorylation of p65 and nuclear association of STAT3 in pancreatic beta cells. Data from these investigations further affirm the role(s) of Rac1 as a mediator of metabolic stress- induced dysfunction of the islet beta cell.

Dataset Information

PPAR-? activation restores pancreatic islet SERCA2 levels and prevents ?-cell dysfunction under conditions of hyperglycemic and cytokine stress.

Publications

PPAR-γ activation restores pancreatic islet SERCA2 levels and prevents β-cell dysfunction under conditions of hyperglycemic and cytokine stress.

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