ICER-1γ Overexpression Drives Palmitate-mediated Connexin36 Down-regulation in Insulin-secreting Cells

Florent Allagnat,Florian Alonso,David Martin,Amar Abderrahmani,Gérard Waeber,Jacques-Antoine Haefliger

doi:10.1074/jbc.m708181200

Abstract

Channels formed by the gap junction protein connexin36 (Cx36) contribute to the proper control of insulin secretion. We investigated the impact of chronic hyperlipidemia on Cx36 expression in pancreatic beta-cells. Prolonged exposure to the saturated free fatty acid palmitate reduced the expression of Cx36 in several insulin-secreting cell lines and isolated mouse islets. The effect of palmitate was fully blocked upon protein kinase A (PKA) inhibition by H89 and (Rp)-cAMP, indicating that the cAMP/PKA pathway is involved in the control of Cx36 expression. Palmitate treatment led to overexpression of the inducible cAMP early repressor (ICER-1gamma), which bound to a functional cAMP-response element located in the promoter of the CX36 gene. Inhibition of ICER-1gamma overexpression prevented the Cx36 decrease, as well as the palmitate-induced beta-cell secretory dysfunction. Finally, freshly isolated islets from mice undergoing a long term high fat diet expressed reduced Cx36 levels and increased ICER-1gamma levels. Taken together, these data demonstrate that chronic exposure to palmitate inhibits the Cx36 expression through PKA-mediated ICER-1gamma overexpression. This Cx36 down-regulation may contribute to the reduced glucose sensitivity and altered insulin secretion observed during the pre-diabetic stage and in the metabolic syndrome.

Highlights

The fine-tuning of insulin secretion in response to nutrient stimulation relies on a closely coordinated functioning of pancreatic ␤-cells
We report the effects of a prolonged exposure of insulinsecreting cells to various free fatty acids (FFAs) on Cx36 expression, and we document the generation of a selective decrease of Cx36 expression by saturated FFAs
We demonstrated that ICER-1␥ blockade prevented the deleterious effect of palmitate on ␤-cell secretion, providing a mechanistic explanation for the altered ␤-cell function observed after a prolonged exposure to FFA

Summary

EXPERIMENTAL PROCEDURES

Materials—Glucose, palmitate, methyl ester palmitate, stearate, oleate, linoleate, and (Rp)-cAMP were purchased from Sigma, and compound C and H89 were from Calbiochem. All experiments using FFAs were performed in the absence of serum at 5 mM glucose for INS-1E cells and islets, and 11 mM glucose for MIN6-B1 at a final concentration of 1% bovine serum albumin. Transfection assay consisted of a mixture of the luciferase reporter plasmid containing the mouse luciferase gene under control of different fragments of the human CX36 promoter, together with an empty vector (pCDNA3), or a plasmid allowing constitutive expression of ICER-1 [21] or an ICER antisense plasmid [22], and the internal control plasmid pRL-SV40 (Promega). The total amount of hGH produced by transfected cells and the fraction released into the medium during the incubation period were determined by enzyme-linked immunosorbent assay (Roche Diagnostics). Statistical significance was defined at a value of p Ͻ 0.05 (*), p Ͻ 0.01 (**), and p Ͻ 0.001 (***)

RESULTS

Normal chow

ICER AS