Abstract
Insulin secretion by the beta cell depends on anaplerosis in which insulin secretagogues are metabolized by mitochondria into molecules that are most likely exported to the extramitochondrial space where they have signaling roles. However, very little is known about the products of anaplerosis. We discovered an experimental paradigm that has begun to provide new information about these products. When various intracellular metabolites were applied in combination to overnight-cultured rat or human pancreatic islets or to INS-1 832/13 cells, they interacted synergistically to strongly stimulate insulin release. When these same metabolites were applied individually to these cells, insulin stimulation was poor. Discerning the contributions of the individual compounds to metabolism has begun to allow us to dissect some of the pathways involved in insulin secretion, which was not possible from studying individual secretagogues. Monomethyl succinate (MMS) combined with a barely stimulatory concentration of alpha-ketoisocaproate (KIC) (2 mm) stimulated insulin release in cultured rat islets 18-fold (versus 21-fold for 16.7 mm glucose). MMS plus low glucose (2 mm) or pyruvate (5 mm) gave 11- and 9-fold stimulations. These agents also potentiated MMS-induced insulin release in fresh islets, and KIC plus MMS gave synergistic insulin release in cultured human islets. In INS-1 cells, neither MMS nor KIC (10 mm) was an insulin secretagogue, but when added together KIC (2 mm) and MMS stimulated insulin release 7-fold (versus 12-fold for glucose). In islets and INS-1 cells, conditions that stimulated insulin release caused large relative increases in acetoacetate, which is a precursor of pathways to short chain acyl-CoAs. Liquid chromatography-tandem mass spectrometry measurements of acetyl-CoA, acetoacetyl-CoA, succinyl-CoA, hydroxymethylglutaryl-CoA, and malonyl-CoA confirmed that they were increased by insulin secretagogues. The results suggest a new mechanism of insulin secretion in which anaplerosis increases short chain acyl-CoAs that have roles in insulin exocytosis.
Highlights
MARCH 2, 2007 VOLUME 282 NUMBER 9 cell is especially high
We were unable to discern why cultured islets or INS-1 cells are unable to respond to these individual secretagogues, we did discover that when these nonsecretagogues were combined together in the insulin release test solution, they acutely released an amount of insulin that was almost as large as that stimulated by glucose
When 10 mM methyl succinate was combined with MMS plus low glucose (2 mM) KIC, insulin release was increased 18-fold and was nearly as high as that observed with 16.7 mM
Summary
MARCH 2, 2007 VOLUME 282 NUMBER 9 cell is especially high. For example, glucose, the most potent physiologic insulin secretagogue, stimulates insulin secretion by its metabolism through aerobic glycolysis. In an effort to explain part of the requirement for the high rate of anaplerosis in insulin secretion, we proposed the “succinate mechanism” of insulin release (2, 14) This hypothesis used our own data and data from the literature to suggest why methyl esters of succinic acid have consistently been found to be the only insulinotropic esters of any esters of a citric acid cycle intermediate tested, or the most insulinotropic, depending on the individual study. The citrate can be exported from the mitochondria and used for biosynthetic reactions (Fig. 1, reaction 13 and other reactions) These pathways of methyl succinate metabolism might not be fully operative in pancreatic islets under all conditions or in some types of beta cells, such as the INS-1 cell line. The data suggested that the synergizing insulin stimulants are metabolized by pathways that can increase the cellular concentration of short chain acyl-CoAs
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