Author:

Keywords:

Animals, Citric Acid, Cycloheximide, Flavin-Adenine Dinucleotide, Flow Cytometry, Glucose, Glycerolphosphate Dehydrogenase, Islets of Langerhans, L-Lactate Dehydrogenase, Malates, Male, Models, Chemical, Oxidation-Reduction, Protein Synthesis Inhibitors, Pyruvate Carboxylase, Pyruvic Acid, Rats, Rats, Wistar, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, RAT PANCREATIC-ISLETS, INSULIN RELEASE, B-CELLS, PYRUVATE-DEHYDROGENASE, GENE-EXPRESSION, MALONYL-COA, A-CELLS, CARBOXYLASE, SECRETION, GLUCOKINASE, 03 Chemical Sciences, 06 Biological Sciences, 11 Medical and Health Sciences, 31 Biological sciences, 32 Biomedical and clinical sciences, 34 Chemical sciences

Abstract:

Previous studies in rat islets have suggested that anaplerosis plays an important role in the regulation of pancreatic beta cell function and growth. However, the relative contribution of islet beta cells versus non-beta cells to glucose-regulated anaplerosis is not known. Furthermore, the fate of glucose carbon entering the Krebs cycle of islet cells remains to be determined. The present study has examined the anaplerosis of glucose carbon in purified rat beta cells using specific 14C-labeled glucose tracers. Between 5 and 20 mM glucose, the oxidative production of CO2 from [3,4-14C]glucose represented close to 100% of the total glucose utilization by the cells. Anaplerosis, quantified as the difference between 14CO2 production from [3,4-14C]glucose and [6-14C]glucose, was strongly influenced by glucose, particularly between 5 and 10 mM. The dose dependence of glucose-induced insulin secretion correlated with the accumulation of citrate and malate in beta(INS-1) cells. All glucose carbon that was not oxidized to CO2 was recovered from the cells after extraction in trichloroacetic acid. This indirectly indicates that lactate output is minimal in beta cells. From the effect of cycloheximide upon the incorporation of 14C-glucose into the acid-precipitable fraction, it could be calculated that 25% of glucose carbon entering the Krebs cycle via anaplerosis is channeled into protein synthesis. In contrast, non-beta cells (approximately 80% glucagon-producing alpha cells) exhibited rates of glucose oxidation that were (1)/(3) to (1)/(6) those of the total glucose utilization and no detectable anaplerosis from glucose carbon. This difference between the two cell types was associated with a 7-fold higher expression of the anaplerotic enzyme pyruvate carboxylase in beta cells, as well as a 4-fold lower ratio of lactate dehydrogenase to FAD-linked glycerol phosphate dehydrogenase in beta cells versus alpha cells. Finally, glucose caused a dose-dependent suppression of the activity of the pentose phosphate pathway in beta cells. In conclusion, rat beta cells metabolize glucose essentially via aerobic glycolysis, whereas glycolysis in alpha cells is largely anaerobic. The results support the view that anaplerosis is an essential pathway implicated in beta cell activation by glucose.