Overexpression of a modified human malonyl-CoA decarboxylase blocks the glucose-induced increase in malonyl-CoA level but has no impact on insulin secretion in INS-1-derived (832/13) beta-cells

The long-chain acyl-CoA (LC-CoA) model of glucose stimulated insulin secret ion (GSIS) holds that secretion is linked to a glucose induced increase in malonyl-CoA level and accumulation of LC-CoA in the cytosol. We have previo usly tested the validity of this proposal by overexpressing goose malonyl-C oA decarboxylase (MCD) in INS-1 cells, but these studies have been criticiz ed due to: 1) the small insulin secretion response (2-4-fold) of the INS-1 cells used; 2) unknown contribution of the ATP-sensitive K+ (K-ATP) channel -independent pathway of GSIS in INS-1 cells, which has been implicated as t he site at which lipids regulate insulin granule exocytosis; and 3) deletio n of the N-terminal mitochondrial targeting sequence, but not the C-termina l peroxisomal targeting sequence in the goose MCD construct, raising the po ssibility that a significant fraction of the overexpressed enzyme was local ized to peroxisomes. To address these outstanding concerns, INS-1 derived 8 32/13 cells, which exhibit robust K-ATP channel-dependent and -independent pathways of GSIS, were treated with a new adenovirus encoding human MCD lac king both its mitochondrial and peroxisomal targeting sequences (AdCMV-MCD Delta5), resulting in large increases in cytosolic MCD activity. Treatment of 832/13 cells with AdCMV-MCD Delta5 completely blocked the glucose-induce d rise in malonyl-CoA and attenuated the inhibitory effect of glucose on fa tty acid oxidation. However, MCD overexpression had no effect on K-ATP chan nel-dependent or -independent GSIS in 832/13 cells. Furthermore, combined t reatment of 832/13 cells with AdCMV-MCD Delta5 and triacsin C, an inhibitor of long chain acyl CoA synthetase that reduces LC-CoA levels, did not impa ir GSIS. These findings extend our previous observations and are not consis tent with the LC-CoA hypothesis as originally set forth.