Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells

Abstract

OBJECTIVEProlonged exposure of pancreatic β-cells to simultaneously elevated levels of fatty acids and glucose (glucolipotoxicity) impairs insulin gene transcription. However, the intracellular signaling pathways mediating these effects are mostly unknown. This study aimed to ascertain the role of extracellular-regulated kinases (ERKs)1/2, protein kinase B (PKB), and Per-Arnt-Sim kinase (PASK) in palmitate inhibition of insulin gene expression in pancreatic β-cells.RESEARCH DESIGN AND METHODSMIN6 cells and isolated rat islets were cultured in the presence of elevated glucose, with or without palmitate or ceramide. ERK1/2 phosphorylation, PKB phosphorylation, and PASK expression were examined by immunoblotting and real-time PCR. The role of these kinases in insulin gene expression was assessed using pharmacological and molecular approaches.RESULTSExposure of MIN6 cells and islets to elevated glucose induced ERK1/2 and PKB phosphorylation, which was further enhanced by palmitate. Inhibition of ERK1/2, but not of PKB, partially prevented the inhibition of insulin gene expression in the presence of palmitate or ceramide. Glucose-induced expression of PASK mRNA and protein levels was reduced in the presence of palmitate. Overexpression of wild-type PASK increased insulin and pancreatic duodenal homeobox-1 gene expression in MIN6 cells and rat islets incubated with glucose and palmitate, whereas overexpression of a kinase-dead PASK mutant in rat islets decreased expression of insulin and pancreatic duodenal homeobox-1 and increased C/EBPβ expression.CONCLUSIONSBoth the PASK and ERK1/2 signaling pathways mediate palmitate inhibition of insulin gene expression. These findings identify PASK as a novel mediator of glucolipotoxicity on the insulin gene in pancreatic β-cells.

Per-arnt-sim (PAS) domain kinase (PASK) as a regulator of glucagon secretion

“…The latter is consistent with in vitro work in rodent islets by Fontes et al [20] showing reduced PASK levels in response to chronic exposure to palmitate. In that study, upregulation of PASK ameliorated the effects of palmitate to reduce insulin gene expression.…”

“…Conversely, AMPK activation upon falling cellular energy levels is suggested to augment glucagon secretion [17]. The da Silva Xavier study focuses on the per-arnt-sim (PAS) domain kinase (PASK), another serine/threonine kinase [18] implicated in the sensing of cellular energy homeostasis, the control of insulin gene expression [19] and the modulation of insulin gene expression under pathophysiological cell culture conditions [20]. However, while Pask -/-mice displayed reduced plasma insulin responses to glucose they showed normal glucose [21,22] and insulin [21] tolerance.…”

Per-Arnt-Sim Kinase Regulates Pancreatic Duodenal Homeobox-1 Protein Stability via Phosphorylation of Glycogen Synthase Kinase 3β in Pancreatic β-Cells

“…Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Glucose stimulation in MIN6 cells was performed as reported previously (24). Following HIT-T15 transfection, medium was changed to 1 mM glucose for 24 h and to 0.1 mM glucose 0.5% BSA for an additional 2 h, after which cells were stimulated in 0.1 mM, 1.5 mM, or 5 mM glucose.…”

“…5 energy homeostasis (22). Glucose induces PASK gene and protein expression in both isolated rat islets and insulin-secreting MIN6 cells (23,24). At basal glucose levels, overexpression of WT PASK increased both insulin and PDX-1 expression, whereas overexpression of a kinase-dead (KD) mutant of PASK acting as a dominant negative blocked glucose-induced insulin gene expression as well as PDX-1 protein expression (23,24), suggesting that PASK regulates the insulin gene via PDX-1.…”

Protein Kinases and Pancreatic Islet Function

“…Furthermore, inhibition of insulin gene expression by palmitate was reversed by PASK overexpression in MIN6 cells (71), suggesting a role for PASK in protection against lipotoxicity. Thus, PASK appears to exert a protective effect in mature cells and aberrant PASK expression and function may play a role in the development of diabetes.…”