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Peter James

Peter James

Professor

Peter James

Metabolomic and proteomic analysis of a clonal insulin-producing beta-cell line (INS-1 832/13).

Author

  • Celine Fernandez
  • Ulrika Krus
  • Elna Hallgard
  • Peter Spégel
  • Cecilia Holm
  • Morten Krogh
  • Kristofer Wårell
  • Peter James
  • Hindrik Mulder

Summary, in English

Metabolites generated from fuel metabolism in pancreatic beta-cells control exocytosis of insulin, a process which fails in type 2 diabetes. To identify and quantify these metabolites, global and unbiased analysis of cellular metabolism is required. To this end, polar metabolites, extracted from the clonal 832/13 beta-cell line cultured at 2.8 and 16.7 mM glucose for 48 h, were derivatized followed by identification and quantification, using gas chromatography (GC) and mass spectrometry (MS). After culture at 16.7 mM glucose for 48 h, 832/13 beta-cells exhibited a phenotype reminiscent of glucotoxicity with decreased content and secretion of insulin. The metabolomic analysis revealed alterations in the levels of 7 metabolites derived from glycolysis, the TCA cycle and pentose phosphate shunt, and 4 amino acids. Principal component analysis of the metabolite data showed two clusters, corresponding to the cells cultured at 2.8 and 16.7 mM glucose, respectively. Concurrent changes in protein expression were analyzed by 2-D gel electrophoresis followed by LC-MS/MS. The identities of 86 spots corresponding to 75 unique proteins that were significantly different in 832/13 beta-cells cultured at 16.7 mM glucose were established. Only 5 of these were found to be metabolic enzymes that could be involved in the metabolomic alterations observed. Anticipated changes in metabolite levels in cells exposed to increased glucose were observed, while changes in enzyme levels were much less profound. This suggests that substrate availability, allosteric regulation, and/or post-translational modifications are more important determinants of metabolite levels than enzyme expression at the protein level.

Department/s

  • Molecular Endocrinology
  • Diabetes - Islet Patophysiology
  • Department of Experimental Medical Science
  • Computational Biology and Biological Physics - Undergoing reorganization
  • Department of Immunotechnology

Publishing year

2008

Language

English

Pages

400-411

Publication/Series

Journal of Proteome Research

Volume

7

Issue

1

Document type

Journal article

Publisher

The American Chemical Society (ACS)

Topic

  • Endocrinology and Diabetes

Status

Published

Research group

  • Molecular Endocrinology
  • Diabetes - Islet Patophysiology

ISBN/ISSN/Other

  • ISSN: 1535-3893