br Introduction In addition to ATP production an

Introduction In addition to ATP production, an important role of mitochondria in the pancreatic beta cell is anaplerosis, which is the net synthesis of citric Dronedarone cycle intermediates that are exported to the cytosol where they are converted to numerous other metabolites that stimulate and/or support insulin secretion. Among these cytosolic products derived from mitochondrial metabolites are short chain acyl-CoAs, such as acetyl-CoA, malonyl-CoA, acetoacetyl-CoA and HMG-CoA [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Short chain acyl-CoAs are produced inside mitochondria, but cells are unable to transport them across the mitochondrial inner membrane. In order for a cell to provide short chain acyl-CoAs to the cytosol, they are converted to metabolites in the mitochondria that can be exported to the cytosol where they are converted back to short chain acyl-CoAs. During glucose-stimulated insulin secretion in pancreatic beta cells, including human pancreatic islets, short chain acyl-CoAs can be derived from pyruvate in mitochondria by two different pathways. Pyruvate can be metabolized through pyruvate carboxylase to oxaloacetate and through pyruvate dehydrogenase to form acetyl-CoA. Oxaloacetate and acetyl-CoA can combine to form citrate in the citrate synthase reaction in the mitochondria. Citrate is then exported out of the mitochondria to the cytosol where ATP citrate lyase (ACLY) catalyzes citrate's conversion into both oxaloacetate and acetyl-CoA [5], [6]. Cytosolic acetyl-CoA can be carboxylated by acetyl-CoA carboxylase (ACC) to form malonyl-CoA, which can itself act as a signal or be used for fatty acid synthesis. Cytosolic acetyl-CoA can also be converted to acetoacetyl-CoA and HMG-CoA. In a second biosynthesis pathway, the acetoacetate pathway, that also originates in mitochondria, pyruvate can be converted to acetyl-CoA catalyzed by pyruvate dehydrogenase in the mitochondria. Two acetyl-CoA molecules can combine to form acetoacetyl-CoA. Acetoacetyl-CoA can be converted into acetoacetate by succinyl-CoA:3-ketoacid-CoA transferase (SCOT) and transported out of the mitochondria to the cytosol where it is converted to acetoacetyl-CoA by acetoacetyl-CoA synthetase [9], [10], [11], [12], [13], [14], [15], [16]. Cytosolic acetoacetyl-CoA can then be converted to cytosolic acetyl-CoA and malonyl-CoA and HMG-CoA as well as fatty acids. There are several reports that knockdown of ATP citrate lyase with siRNA technology in beta cells does not impair glucose-stimulated insulin release [15], [17] while the inhibition of SCOT [15] or acetoacetyl-CoA synthetase [15] does lower insulin release suggesting that the formation of short chain acyl-CoAs through acetoacetate is important during insulin secretion. α-Ketoisocaproic acid (KIC), which stimulates insulin release by itself, is used experimentally to study insulin secretion. KIC is the first intermediate in leucine metabolism. Leucine is the only other normal physiologic stimulator of insulin secretion besides glucose. Both KIC and leucine form substantial amounts of acetoacetate that can exit the mitochondria and increase the levels of short chain fatty acyl-CoAs in the cytosol [14]. To understand the relative contribution of the pathway involving ATP citrate lyase versus the acetoacetate pathway in generating cytosolic short chain acyl-CoAs in insulin secretion, we used 13C-labeled glucose and KIC and monitored 13C flux into short chain acyl-CoAs in the INS-1 832/13 cell line, a cell line of homogeneous pancreatic beta cells in which we decreased ATP citrate lyase enzyme activity. We lowered ATP citrate lyase enzyme activity with hydroxycitrate, an inhibitor of with ATP citrate lyase or used an ATP citrate lyase knockdown cell line generated from INS-1 832/13 cells using shRNA technology [15]. Flux of 13C glucose into fatty acids was also monitored as a final end product of cytosolic acetyl-CoA and malonyl-CoA.
Materials and methods