malonyl-coenzyme-a and alpha-glycerophosphoric-acid

Glucose metabolism promotes insulin secretion in β-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes β-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the β-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in β-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in β-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters.

Topics: Adenosine Triphosphate; Animals; Cell Line; Cholesterol Esters; Dihydroxyacetone Phosphate; Dose-Response Relationship, Drug; Fatty Acids; Glucose; Glycerophosphates; Glycogen; Insulin-Secreting Cells; Male; Malonyl Coenzyme A; Rats; Rats, Wistar; Signal Transduction; Triglycerides

The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

Topics: Acetyl Coenzyme A; Acetyl-CoA Carboxylase; Amino Acid Sequence; Fatty Acids; Fungal Proteins; Gene Expression Regulation, Fungal; Glycerol-3-Phosphate Dehydrogenase (NAD+); Glycerophosphates; Lipid Metabolism; Lipomyces; Malonyl Coenzyme A; Metabolic Engineering; Phylogeny; Real-Time Polymerase Chain Reaction; Saccharomyces cerevisiae; Sequence Alignment; Transgenes

The regulation of proinsulin biosynthesis in pancreatic beta-cells is vital for maintaining optimal insulin stores for glucose-induced insulin release. The majority of nutrient fuels that induce insulin release also stimulate proinsulin biosynthesis, but since insulin exocytosis and proinsulin synthesis involve different cellular mechanisms, a point of divergence in the respective metabolic stimulus-response coupling pathways must exist. A parallel examination of the metabolic regulation of proinsulin biosynthesis and insulin secretion was undertaken in the same beta-cells. In MIN6 cells, glucose-induced proinsulin biosynthesis and insulin release shared a requirement for glycolysis to generate stimulus-coupling signals. Pyruvate stimulated both proinsulin synthesis (threshold 0.13-0.2 mM) and insulin release (threshold 0.2-0.3 mM) in MIN6 cells, which was eliminated by an inhibitor of pyruvate transport (1 mM alpha-cyano-4-hydroxycinnamate). A combination of alpha-oxoisohexanoate and glutamine also stimulated proinsulin biosynthesis and insulin release in MIN6 cells, which, together with the effect of pyruvate, indicated that anaplerosis was necessary for instigating secondary metabolic stimulus-coupling signals in the beta-cell. A consequence of increased anaplerosis in beta-cells is a marked increase in malonyl-CoA, which in turn inhibits beta-oxidation and elevates cytosolic fatty acyl-CoA levels. In the beta-cell, long-chain fatty acyl moieties have been strongly implicated as metabolic stimulus-coupling signals for regulating insulin exocytosis. Indeed, it was found in MIN6 cells and isolated rat pancreatic islets that exogenous oleate, palmitate and 2-bromopalmitate all markedly potentiated glucose-induced insulin release. However, in the very same beta-cells, these fatty acids in contrast inhibited glucose-induced proinsulin biosynthesis. This implies that neither fatty acyl moieties nor beta-oxidation are required for the metabolic stimulus-response coupling pathway specific for proinsulin biosynthesis, and represent an early point of divergence of the two signalling pathways for metabolic regulation of proinsulin biosynthesis and insulin release. Therefore alternative metabolic stimulus-coupling factors for the specific control of proinsulin biosynthesis at the translational level were considered. One possibility examined was an increase in glycerophosphate shuttle activity and change in cytosolic redox state of the beta-cell, as reflected by ch

Topics: Animals; Cell Line; Dihydroxyacetone Phosphate; Drug Synergism; Fatty Acids; Glucose; Glycerophosphates; Glycolysis; Insulin; Insulin Secretion; Islets of Langerhans; Keto Acids; Lactic Acid; Male; Malonyl Coenzyme A; Mitochondria; Proinsulin; Pyruvic Acid; Rats; Rats, Sprague-Dawley

To gain insight into the regulation of pancreatic beta-cell mitochondrial metabolism, the direct effects on respiration of different mitochondrial substrates, variations in the ATP/ADP ratio and free Ca2+ were examined using isolated mitochondria and permeabilized clonal pancreatic beta-cells (HIT). Respiration from pyruvate was high and not influenced by Ca2+ in State 3 or under various redox states and fixed values of the ATP/ADP ratio; nevertheless, high Ca2+ elevated pyridine nucleotide fluorescence, indicating activation of pyruvate dehydrogenase by Ca2+. Furthermore, in the presence of pyruvate, elevated Ca2+ stimulated CO2 production from pyruvate, increased citrate production and efflux from the mitochondria and inhibited CO2 production from palmitate. The latter observation suggests that beta-cell fatty acid oxidation is not regulated exclusively by malonyl-CoA but also by the mitochondrial redox state. alpha-Glycerophosphate (alpha-GP) oxidation was Ca(2+)-dependent with a half-maximal rate observed at around 300 nM Ca2+. We have recently demonstrated that increases in respiration precede increases in Ca2+ in glucose-stimulated clonal pancreatic beta-cells (HIT), indicating that Ca2+ is not responsible for the initial stimulation of respiration [Civelek, Deeney, Kubik, Schultz, Tornheim and Corkey (1996) Biochem. J. 315, 1015-1019]. It is suggested that respiration is stimulated by increased substrate (alpha-GP and pyruvate) supply together with oscillatory increases in ADP [Nilsson, Schultz, Berggren, Corkey and Tornheim (1996) Biochem. J. 314, 91-94]. The rise in Ca2+, which in itself may not significantly increase net respiration, could have the important functions of (1) activating the alpha-GP shuttle, to maintain an oxidized cytosol and high glycolytic flux; (2) activating pyruvate dehydrogenase, and indirectly pyruvate carboxylase, to sustain production of citrate and hence the putative signal coupling factors, malonyl-CoA and acyl-CoA; and (3) increasing mitochondrial redox state to implement the switch from fatty acid to pyruvate oxidation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Clone Cells; Egtazic Acid; Glycerophosphates; Insulinoma; Islets of Langerhans; Ketoglutaric Acids; Kinetics; Malonyl Coenzyme A; Mitochondria; Oxidation-Reduction; Oxygen Consumption; Pancreatic Neoplasms; Pyruvate Dehydrogenase Complex; Rats

The requirement for a normal insulin response in mediating the starved-to-refed transition, with respect to the partitioning of hepatic fatty acids between beta-oxidation and esterification to glycerol, was studied. Diabetic rats were starved for 24 h and refed ad libitum for various periods of time. There was no increase in plasma insulin in response to the meal. However, the fatty acid oxidation:esterification ratio was very rapidly decreased from the starved to the fed value, most of the transition being achieved within the first hour of refeeding. There was a 2 h lag in the response of hepatic malonyl-CoA concentration, such that this rapid switch from oxidation to esterification could not be explained on the basis of changes in the absolute concentration of this inhibitor of carnitine palmitoyltransferase I (CPT I). Hepatic pyruvate and lactate concentrations both increased by several-fold upon refeeding and peaked after 1 h and 3 h, respectively. The hepatic lactate:pyruvate ratio increased 3.2-fold during the first 3 h of refeeding, suggesting that the cytosolic NAD(+)-NADH couple became much more highly reduced during the lag-period between the onset of inhibition of flux of fatty acids towards oxidation and the rise in malonyl-CoA concentration. This may be indicative of a lowering of intracellular pH, which would amplify greatly the sensitivity of CPT I to the inhibitor. In view of the very rapid and high food intake by these diabetic rats, the possibility is also considered that portal concentrations of amino acids and other metabolites could give rise to an increase in liver cell-volume that would inhibit CPT I acutely by an as yet unknown mechanism [M. Guzman, G. Velasco, J. Castro and V. A. Zammit (1994) FEBS Lett. 344, 239-241].

Topics: Animals; Carnitine O-Palmitoyltransferase; Cholesterol Esters; Diabetes Mellitus, Experimental; Esterification; Fatty Acids; Fatty Acids, Nonesterified; Female; Food; Glycerophosphates; Insulin; Lactates; Lactic Acid; Liver; Malonyl Coenzyme A; Mitochondria, Liver; Oxidation-Reduction; Pyruvates; Pyruvic Acid; Rats; Rats, Wistar; Starvation