Feasibility of Pathways for Transfer of Acyl Groups from Mitochondria to the Cytosol to Form Short Chain Acyl-CoAs in the Pancreatic Beta Cell

Abstract

The mitochondria of pancreatic beta cells are believed to convert insulin secretagogues into products that are translocated to the cytosol where they participate in insulin secretion. We studied the hypothesis that short chain acyl-CoA (SC-CoAs) might be some of these products by discerning the pathways of SC-CoA formation in beta cells. Insulin secretagogues acutely stimulated 1.5-5-fold increases in acetoacetyl-CoA, succinyl-CoA, malonyl-CoA, hydroxymethylglutaryl-CoA (HMG-CoA), and acetyl-CoA in INS-1 832/13 cells as judged from liquid chromatography-tandem mass spectrometry measurements. Studies of 12 relevant enzymes in rat and human pancreatic islets and INS-1 832/13 cells showed the feasibility of at least two redundant pathways, one involving acetoacetate and the other citrate, for the synthesis SC-CoAs from secretagogue carbon in mitochondria and the transfer of their acyl groups to the cytosol where the acyl groups are converted to SC-CoAs. Knockdown of two key cytosolic enzymes in INS-1 832/13 cells with short hairpin RNA supported the proposed scheme. Lowering ATP citrate lyase 88% did not inhibit glucose-induced insulin release indicating citrate is not the only carrier of acyl groups to the cytosol. However, lowering acetoacetyl-CoA synthetase 80% partially inhibited glucose-induced insulin release indicating formation of SC-CoAs from acetoacetate in the cytosol is important for insulin secretion. The results indicate beta cells possess enzyme pathways that can incorporate carbon from glucose into acetyl-CoA, acetoacetyl-CoA, and succinyl-CoA and carbon from leucine into these three SC-CoAs plus HMG-CoA in their mitochondria and enzymes that can form acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, and HMG-CoA in their cytosol.

The role of rapid lipogenesis in insulin secretion: Insulin secretagogues acutely alter lipid composition of INS-1 832/13 cells

“…INS-1 832/13 cells (passage number 10-15) were cultivated as monolayers on 150 mm tissue culture plates in INS-1 medium (RPMI tissue culture medium (contains 11.1 mM glucose) supplemented with 10% fetal bovine serum, 10 mM Hepes buffer, 1 mM pyruvate and 50 μM β-mercaptoethanol) [23] and penicillin (100 units/ml) and gentamycin (100 μg/ml) [12,13]. Twenty-two hours before experiments were performed the glucose concentration in the tissue culture medium was reduced to 5 mM.…”

“…The rate of pyruvate carboxylation correlates with the glucose concentration applied to pancreatic islets and thus is correlated with the rate of insulin secretion [5]. 13 C-NMR isoprotomer studies of glucose metabolism in clonal cell lines have also shown a correlation between insulin secretion and pyruvate flux through the pyruvate carboxylase reaction [8,9]. The purpose of anaplerosis in the beta cell is different from that in many other tissues which possess a high level of pyruvate carboxylase.…”

“…In a search for products of anaplerosis, we uncovered preliminary evidence to suggest that lipids are one of the numerous possible products of secretagogue metabolism in the beta cell. This idea came in part from the fact that certain lipid precursors, that is various short chain acyl-CoAs, are increased by insulin secretagogues in beta cells and the beta cell possesses several pathways for the transfer of acyl groups from the mitochondria to the cytosol, where lipid synthesis would take place [12,13]. It has also been demonstrated that glucose carbon in acutely stimulated beta cells is incorporated into material extractable with organic solvents [14,15], consistent with the idea that secretagogue carbon is incorporated into lipid.…”

“…It has also been demonstrated that glucose carbon in acutely stimulated beta cells is incorporated into material extractable with organic solvents [14,15], consistent with the idea that secretagogue carbon is incorporated into lipid. One of the short chain acyl-CoAs that has been frequently shown to be increased by insulin secretagogues is malonyl-CoA [12][13][14][15][16][17]. Malonyl-CoA supplies the two-carbon units for fatty acid synthesis.…”

“…Malonyl-CoA supplies the two-carbon units for fatty acid synthesis. In some studies, HMGCoA was increased by insulin secretagogues [12,13] and HMG-CoA is a precursor of cholesterol.…”