Specificity in Beta Cell Expression of l-3-Hydroxyacyl-CoA Dehydrogenase, Short Chain, and Potential Role in Down-regulating Insulin Release

Geert A Martens,Annelies Vervoort,Mark Van De Casteele,Geert Stangé,Karine Hellemans,Hong Vien Van Thi,Frans Schuit,Danieöl Pipeleers

doi:10.1074/jbc.m700083200

Abstract

A loss-of-function mutation of the mitochondrial beta-oxidation enzyme l-3-hydroxyacyl-CoA dehydrogenase, short chain (HADHSC), has been associated with hyperinsulinemic hypoglycemia in man. It is still unclear whether loss of glucose homeostasis in these patients (partly) results from a dysregulation of beta cells. This study examines HADHSC expression in purified rat beta cells and investigates whether its selective suppression elevates insulin release. Beta cells expressed the highest levels of HADHSC mRNA and protein of all examined tissues, including those with high rates of mitochondrial beta-oxidation. On the other hand, beta cells expressed relatively low levels of other beta-oxidation enzymes (acyl-CoA dehydrogenase short, medium, and long chain and acetyl-coenzyme A acyltransferase 2). HADHSC expression was sequence-specifically silenced by RNA interference, and the effects were examined on glucose-stimulated insulin secretion following 48-72 h of suppression. In both rat beta cells and in the beta cell line INS1 832-13, HADHSC silencing resulted in elevated insulin release at low and at high glucose concentrations, which appeared not to be caused by increased rates of glucose metabolism or an inhibition in fatty acid oxidation. These data indicate that the normal beta cell phenotype is characterized by a high expression of HADHSC and a low expression of other beta-oxidation enzymes. Down-regulation of HADHSC causes an elevated secretory activity suggesting that this enzyme protects against inappropriately high insulin levels and hypoglycemia.

Highlights

Introduction of shRNAi Plasmids inINS1 CellsShort hairpin RNA interference plasmids targeting rat HADHSC matched the criteria of both the Dharmacon [14] and Whitehead [15] siRNA design protocols
In this study we examine the role of the mitochondrial ␤-oxidation enzyme L-3-hydroxyacyl-CoA dehydrogenase, short chain (HADHSC),2 in the regulation of glucose-induced insulin release by normal beta cells
HADHSC Suppression in INS1 Cells Does Not Stimulate glucose-stimulated insulin secretion (GSIS) in INS1 cells with a varying degree of HADHSC protein supby Stimulating Glucose Metabolism—We examined whether pression (Table 1)

Summary

EXPERIMENTAL PROCEDURES

Rat INS1 832-13 cells were cultured in 10% fetal calf serum (Hyclone), RPMI 1640 medium, GlutaMAXTM (Invitrogen), supplemented to 10 mM HEPES, 1 mM sodium pyruvate, penicillin/streptomycin, and 50 ␮M ␤-mercaptoethanol (designated as “INS1 culture medium”). FACS-purified rat beta cells were cultured in Ham’s F-10 nutrient mixture (Invitrogen) supplemented with 0.5% BSA (Cohn analog, Sigma), 2 mM glutamine, 10 mM glucose, penicillin (100 units/ml), streptomycin (0.1 mg/ml), 2% fetal calf serum (Hyclone). RNA from other rat tissues and from INS1 832-13 cells (passage 46 –52) was extracted using TRIzol reagent. Expression of target genes were normalized toward ␤-actin (⌬Ct) and expressed versus a chosen calibrator (comparative ⌬⌬Ct method). Relative mRNA levels of HADHSC versus other ␤-oxidation enzymes were calculated using the formula 2Ϫ⌬Ct, where ⌬Ct ϭ (Ct value of HAHSC) Ϫ (Ct of other enzyme), measured in same run

HADHSC Protein Expression Analysis by Western Blotting and Immune Staining

CoA dehydrogenase isoenzymes

RESULTS

Effect of Sustained HADHSC

Findings

DISCUSSION