A Nuclear Magnetic Resonance-Based Demonstration of Substantial Oxidative l-Alanine Metabolism and l-Alanine-Enhanced Glucose Metabolism in a Clonal Pancreatic β-Cell Line

Abstract

Early experiments indicated that islet beta-cells substantially metabolized L-alanine but that insulin secretion was largely unaffected by the amino acid. It was subsequently demonstrated using more intricate studies that L-alanine is a strong stimulus to insulin secretion in the presence of glucose in normal rodent islets and beta-cell lines. Using (13)C nuclear magnetic resonance (NMR), we have demonstrated substantial oxidative metabolism of L-alanine by the clonal beta-cell line BRIN-BD11, with time-dependent increases in production of cellular glutamate and aspartate. Stimulatory effects of L-alanine on insulin secretion were attenuated by the inhibition of beta-cell oxidative phosphorylation using oligomycin. Additionally, we detected substantial production of lactate, alanine, and glutamate from glucose (16.7 mmol/l) after 60 min. On addition of 10 mmol/l L-alanine to a stimulus of 16.7 mmol/l glucose, the utilization rate of glucose increased approximately 2.4-fold. L-Alanine dramatically enhanced NMR-measurable aspects of glucose metabolism (both oxidative and nonoxidative). The enhanced rate of entry of glucose-derived pyruvate into the tricarboxylic acid (TCA) cycle in the presence of alanine may have stimulated rates of generation of key metabolites, including ATP, which affect the insulin secretory process. Thus L-alanine metabolism, in addition to the enhancing effect on glucose metabolism, contributes to the stimulatory effects of this amino acid on insulin secretion in vitro.

Insulin secretion profiles are modified by overexpression of glutamate dehydrogenase in pancreatic islets

“…Moreover, GDH overexpression did not modify the glucose-induced increase in cellular glutamate concentrations. It has been shown by NMR spectroscopy in beta-cell lines that glutamate is a major product of glucose metabolism [31,59]. Taken together, these results indicate a prominent contribution of transamination reactions in the generation of glutamate under glucose stimulation, in accordance with a recent report [60].…”

13C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and ?-glutamyl cycle activity in a clonal pancreatic beta-cell line

“…The hypothesis that D-glucose is converted into L-glutamate in the beta cell is controversial. However, we have shown previoustly, using 13 C NMR, that glucose is converted to L-glutamate in the beta cell [24]. This is difficult to reconcile with the common perception that GDH is operating in the direction of L-glutamate→2-oxoglutarate in physiological conditions in the beta cell.…”

Insights into the role of anaplerosis in insulin secretion: a 13C NMR study

“…Although broadly accepted, this mechanism cannot adequately address all the observed insulin secretion anomalies. Consequently, the importance of specific enzymatic pathways to insulin release is still under investigation [5][6][7][8][9][10][11]. Understanding these pathways is an important step towards discovering novel avenues of therapeutic intervention and assisting cellular engineers in tailoring superior beta cell surrogate cell lines.…”