Palmitate Inhibits Insulin Gene Expression by Altering PDX-1 Nuclear Localization and Reducing MafA Expression in Isolated Rat Islets of Langerhans

Abstract

Abnormalities in lipid metabolism have been proposed as contributing factors to both defective insulin secretion from the pancreatic beta cell and peripheral insulin resistance in type 2 diabetes. Previously, we have shown that prolonged exposure of isolated rat islets of Langerhans to excessive fatty acid levels impairs insulin gene transcription. This study was designed to assess whether palmitate alters the expression and binding activity of the key regulatory factors pancreas-duodenum homeobox-1 (PDX-1), MafA, and Beta2, which respectively bind to the A3, C1, and E1 elements in the proximal region of the insulin promoter. Nuclear extracts of isolated rat islets cultured with 0.5 mM palmitate exhibited reduced binding activity to the A3 and C1 elements but not the E1 element. Palmitate did not affect the overall expression of PDX-1 but reduced its nuclear localization. In contrast, palmitate blocked the stimulation of MafA mRNA and protein expression by glucose. Combined adenovirus-mediated overexpression of PDX-1 and MafA in islets completely prevented the inhibition of insulin gene expression by palmitate. These results demonstrate that prolonged exposure of islets to palmitate inhibits insulin gene transcription by impairing nuclear localization of PDX-1 and cellular expression of MafA.The prevalence of diabetes mellitus is increasing dramatically in Western countries, in part because of the increase in obesity. Type 2 diabetes mellitus, the most frequent form of the disease, is characterized by defective insulin secretion from the pancreatic beta cells and peripheral insulin resistance. According to the lipotoxicity hypothesis, abnormalities in lipid metabolism contribute to both defects (1) and in particular to the inexorable decline of beta cell function observed during the course of the disease (2). However, the mechanisms of lipotoxicity in the beta cell remain largely unknown.In vitro, prolonged exposure to excessive concentrations of fatty acids inhibits glucose-stimulated insulin secretion (3-7) and insulin gene expression (8 -11). Previous studies in our laboratory have shown that deleterious effects of fatty acids appear mediated by distinct mechanisms; whereas inhibition of insulin secretion is observed after culture with palmitate, oleate, and other fatty acids (7), insulin gene expression is only affected by palmitate and is mediated via de novo synthesis of ceramide (11). In isolated rat islets, we have shown that palmitate markedly blunts the activation by glucose of an insulin promoter reporter construct, indicating a transcriptional mode of action (11). However, the mechanisms by which palmitate affects the insulin promoter are unknown.Both beta cell-specific expression and metabolic regulation of the insulin gene are conferred by a highly conserved region lying ϳ340 bp upstream of the transcription initiation site that constitutes the promoter/enhancer region (12-14). The main glucose-responsive elements on the insulin promoter are the highly conserved A3 (15), C1 (16), and E1 (16) sites,...

Islet beta cell failure in the 60% pancreatectomised obese hyperlipidaemic Zucker fatty rat: severe dysfunction with altered glycerolipid metabolism without steatosis or a falling beta cell mass

“…A lowered proinsulin mRNA level is a common feature observed in beta cell studies with combination of high glucose and excess NEFA [16]; here, in fact, proinsulin mRNA levels were 50% reduced in the ZF-Px islets. However, this is often assumed to imply a reduced rate of proinsulin production.…”

“…On the other hand, in vitro and whole-animal studies have reported a direct inhibitory effect of excess NEFA, such as occurs with insulin resistance, on glucose-mediated insulin secretion [15], proinsulin biosynthesis [15,16] and beta cell survival [17,18]. Also in vitro studies have shown a synergistic toxic effect of high glucose and saturated NEFA, a process termed glucolipotoxicity, leading to beta cell dysfunction and death [19,20].…”

Inhibition of MafA transcriptional activity and human insulin gene transcription by interleukin-1β and mitogen-activated protein kinase kinase kinase in pancreatic islet beta cells

“…In mice lacking MafA, reduced insulin 1 and 2 gene transcription, glucose intolerance and development of diabetes mellitus was observed [12]. At least at the beta cell level the transcriptional activity of MafA seems to be regulated by either changes in MafA synthesis or by an enhanced degradation of this protein [11,16,26,48,49]. However, in the present study overproduction of MEKK1 did not decrease the amount of MafA produced in JEG cells (data not shown), arguing against a MEKK1-induced degradation of MafA.…”

A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis

“…Based upon these studies, several mechanisms have been postulated: (a) increased β cell apoptosis via and Bcl-2), with resulting loss of downregulation of anti-apoptotic genes (Bcl XL functional cell mass [56,67], (b) loss of activity of key Pdx1 target genes whose products are involved in glucose-stimulated insulin transcription and secretion (including Glut2, glucokinase, MafA, Nkx6.1, insulin) [57,63,[68][69][70][71][72][73][74][75][76], and (d) loss of new β cell formation/regeneration [64,77]; in this regard, studies suggest that the action of glucagon-like peptide 1 (GLP1) in enhancing β cell growth and formation in the adult may rely upon activation of Pdx1 in β cells and potential precursor cell types, such as those residing within ducts [74,[78][79][80][81][82].…”

“…Although some reports in the literature have described physiologic circumstances where the distribution of Pdx1 might be cytoplasmic, it is believed that cytoplasmic sequestration may represent more a mechanism to attenuate the nuclear action of Pdx1 under physiologic or pathologic conditions, rather than to promote a specific cytoplasmic function. For example, the negative effect of fatty acids on β cell function may be related to their eventual sequestration of Pdx1 in the cytoplasm [76], whereas the positive effect of glucose on insulin transcription may be a result of its enhancement of Pdx1nuclear translocation [108]. A basic amino acid sequence with the homeodomain of Pdx1, RRMKWKK, is believed to function as the nuclear targeting sequence [109].…”