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

Abstract

Aims/hypothesis. Pancreatic islet cells and clonal beta-cell lines can metabolise L-glutamine at high rates. The pathway of L-glutamine metabolism has traditionally been described as L-glutamine→L-glutamate→2- oxoglutarate→oxidation in TCA cycle following conversion to pyruvate. Controversially, the metabolism of D-glucose to L-glutamate in beta cells is not widely accepted. However, L-glutamate has been proposed to be a stimulation-secretion coupling factor in glucose-induced insulin secretion. We aimed to investigate the metabolism of glutamine and glucose by using 13C NMR analysis. Methods. BRIN-BD11 cells were incubated in the presence of 16.7 mmol/l [1-13C]glucose, 2 mmol/l [2- 13C]L-glycine or 2 mmol/l [1,2-13C]glutamine in the presence or absence of other amino acids or inhibitors. After an incubation period the cellular metabolites were extracted using a PCA extract procedure. After neutralisation, the extracts were prepared for analysis using 13C-NMR spectroscopy. Results. Using 13C NMR analysis we have shown that L-glutamine could be metabolised in BRIN-BD11 cells via reactions constituting part of the γ-glutamyl cycle producing glutathione. Moderate or high activities of the enzymes required for these pathways of metabolism including glutaminase, γ-glutamyltransferase and γ-glutamylcysteine synthetase were observed. We additionally report significant D-glucose metabolism to L-glutamate. Addition of the aminotransferase inhibitor, aminooxyacetate, attenuated L-glutamate production from D-glucose. Conclusion/interpretation. We propose that L-glutamine metabolism is important in the beta cell for generation of stimulus-secretion coupling factors, precursors of glutathione synthesis and for supplying carbon for oxidation in the TCA cycle. D-glucose, under appropriate conditions, can be converted to L-glutamate via an aminotransferase catalysed step.