Mitochondrial Metabolism Sets the Maximal Limit of Fuel-stimulated Insulin Secretion in a Model Pancreatic Beta Cell

Abstract

The precise metabolic steps that couple glucose catabolism to insulin secretion in the pancreatic beta cell are incompletely understood. ATP generated from glycolytic metabolism in the cytosol, from mitochondrial metabolism, and/or from the hydrogen shuttles operating between cytosolic and mitochondrial compartments has been implicated as an important coupling factor. To identify the importance of each of these metabolic pathways, we have compared the fates of four fuel secretagogues (glucose, pyruvate, dihydroxyacetone, and glycerol) in the INS1-E beta cell line. Two of these fuels, dihydroxyacetone and glycerol, are normally ineffective as secretagogues but are enabled by adenovirus-mediated expression of glycerol kinase. Comparison of these two particular fuels allows the effect of redox state on insulin secretion to be evaluated since the phosphorylated products dihydroxyacetone phosphate and glycerol phosphate lie on opposite sides of the NADH-consuming glycerophosphate dehydrogenase reaction. Based upon measurements of glycolytic metabolites, mitochondrial oxidation, mitochondrial matrix calcium, and mitochondrial membrane potential, we find that insulin secretion most tightly correlates with mitochondrial metabolism for each of the four fuels. In the case of glucose stimulation, the high control strength of glucose phosphorylation sets the pace of glucose metabolism and thus the rate of insulin secretion. However, bypassing this reaction with pyruvate, dihydroxyacetone, or glycerol uncovers constraints imposed by mitochondrial metabolism, each of which attains a similar maximal limit of insulin secretion. More specifically, we found that the hyperpolarization of the mitochondrial membrane, related to the proton export from the mitochondrial matrix, correlates well with insulin secretion. Based on these findings, we propose that fuel-stimulated secretion is in fact limited by the inherent thermodynamic constraints of proton gradient formation.The pancreatic islet beta cell secretes insulin in response to several metabolic fuels, and this occurs via the metabolism of the stimulatory agents rather than their interaction with a ATP is generated by both cytosolic and mitochondrial reactions. Cytosolic ATP production comes from two reactions in the distal portion of glycolysis, 3-phosphoglycerate kinase and pyruvate kinase. Mitochondrial ATP is derived in part from hydrogen shuttles, primarily the malate-aspartate (5-7) and glycerophosphate shuttles, (8 -10) which are very active in islet beta cells. Finally, a major portion of ATP production comes from mitochondrial oxidation of glucose-derived pyruvate. There is evidence to support an important role of each of these discrete sources of ATP in the regulation of insulin secretion. Evidence for a role of glycolysis-derived ATP comes from studies in which inhibitors of glycolytic but not mitochondrial ATPproducing reactions inhibited GSIS (11). Evidence for an important role of hydrogen shuttles comes from studies showing near complete impairment of GSIS in islet...

β-Cell Mitochondria Exhibit Membrane Potential Heterogeneity That Can Be Altered by Stimulatory or Toxic Fuel Levels

“…Essential to these processes is mitochondrial energy state, in the form of an electrochemical gradient, commonly termed the mitochondrial membrane potential (⌬⌿). This gradient influences ATP-to-ADP ratio, redox state (3)(4)(5)(6), and reactive oxygen species (ROS), as well as calcium sequestration (2).…”

Mitochondrial regulation of insulin production in rat pancreatic islets

“…**p<0.01 and ***p<0.001 relative to islets at 3.3 mmol/l glucose was measured by microspectrofluorimetry (excitation/emission 490/530 nm, 505 nm long-pass dichroic mirror) 15 min before and after addition of 10 μmol/l carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) [25]. After background subtraction, the rhodamine-123 fluorescence in each islet was normalised to the maximal fluorescence measured in the presence of FCCP [26], and the average relative fluorescence over the 15 min preceding FCCP addition was calculated.…”

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

“…14 C incorporation into FFA and diglycerides was increased to a smaller extent. Pyruvate is a potent stimulant of insulin release in INS-1 cells [12,13,37,38]. 14 C incorporation into lipids in the presence of both concentrations of pyruvate was also increased, but not quite to the extent of 14 C incorporation from glucose (Table 4).…”