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

L Brennan, M Corless, C Hewage, JPG Malthouse, Neville McClenaghan, Peter Flatt, P Newsholme

Research output: Contribution to journalArticle

55 Citations (Scopus)

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 C-13 NMR analysis. Methods. BRIN-BD11 cells were incubated in the presence of 16.7 mmol/l [1-C-13]glucose, 2 mmol/l [2-C-13]L-glycine or 2 mmol/l [1,2-C-13]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 C-13-NMR spectroscopy. Results. Using C-13 NMR analysis we have shown that L-glutamine could be metabolised in BRIN-BD11 cells via reactions constituting part of the gamma-glutamyl cycle producing glutathione. Moderate or high activities of the enzymes required for these pathways of metabolism including glutaminase, gamma-glutamyltransferase and gamma-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.
LanguageEnglish
Pages1512-1521
JournalDiabetologia
Volume46
Issue number11
DOIs
Publication statusPublished - Nov 2003

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Activity Cycles
Insulin-Secreting Cells
Glutamine
Glutamic Acid
Glucose
Cell Line
Islets of Langerhans
Glutathione
Aminooxyacetic Acid
Glutamate-Cysteine Ligase
Glutaminase
Passive Cutaneous Anaphylaxis
gamma-Glutamyltransferase
Transaminases
Pyruvic Acid
Glycine
Magnetic Resonance Spectroscopy
Carbon
Insulin
Amino Acids

Cite this

Brennan, L ; Corless, M ; Hewage, C ; Malthouse, JPG ; McClenaghan, Neville ; Flatt, Peter ; Newsholme, P. / C-13 NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and gamma-glutamyl cycle activity in a clonal pancreatic beta-cell line. In: Diabetologia. 2003 ; Vol. 46, No. 11. pp. 1512-1521.
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title = "C-13 NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and gamma-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 C-13 NMR analysis. Methods. BRIN-BD11 cells were incubated in the presence of 16.7 mmol/l [1-C-13]glucose, 2 mmol/l [2-C-13]L-glycine or 2 mmol/l [1,2-C-13]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 C-13-NMR spectroscopy. Results. Using C-13 NMR analysis we have shown that L-glutamine could be metabolised in BRIN-BD11 cells via reactions constituting part of the gamma-glutamyl cycle producing glutathione. Moderate or high activities of the enzymes required for these pathways of metabolism including glutaminase, gamma-glutamyltransferase and gamma-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.",
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C-13 NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and gamma-glutamyl cycle activity in a clonal pancreatic beta-cell line. / Brennan, L; Corless, M; Hewage, C; Malthouse, JPG; McClenaghan, Neville; Flatt, Peter; Newsholme, P.

In: Diabetologia, Vol. 46, No. 11, 11.2003, p. 1512-1521.

Research output: Contribution to journalArticle

TY - JOUR

T1 - C-13 NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and gamma-glutamyl cycle activity in a clonal pancreatic beta-cell line

AU - Brennan, L

AU - Corless, M

AU - Hewage, C

AU - Malthouse, JPG

AU - McClenaghan, Neville

AU - Flatt, Peter

AU - Newsholme, P

PY - 2003/11

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N2 - 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 C-13 NMR analysis. Methods. BRIN-BD11 cells were incubated in the presence of 16.7 mmol/l [1-C-13]glucose, 2 mmol/l [2-C-13]L-glycine or 2 mmol/l [1,2-C-13]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 C-13-NMR spectroscopy. Results. Using C-13 NMR analysis we have shown that L-glutamine could be metabolised in BRIN-BD11 cells via reactions constituting part of the gamma-glutamyl cycle producing glutathione. Moderate or high activities of the enzymes required for these pathways of metabolism including glutaminase, gamma-glutamyltransferase and gamma-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.

AB - 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 C-13 NMR analysis. Methods. BRIN-BD11 cells were incubated in the presence of 16.7 mmol/l [1-C-13]glucose, 2 mmol/l [2-C-13]L-glycine or 2 mmol/l [1,2-C-13]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 C-13-NMR spectroscopy. Results. Using C-13 NMR analysis we have shown that L-glutamine could be metabolised in BRIN-BD11 cells via reactions constituting part of the gamma-glutamyl cycle producing glutathione. Moderate or high activities of the enzymes required for these pathways of metabolism including glutaminase, gamma-glutamyltransferase and gamma-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.

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DO - 10.1007/s00125-003-1184-7

M3 - Article

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SP - 1512

EP - 1521

JO - Diabetologia

T2 - Diabetologia

JF - Diabetologia

SN - 0012-186X

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