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- Fernandez, Celine, et al.
(författare)
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Metabolomic and proteomic analysis of a clonal insulin-producing beta-cell line (INS-1 832/13).
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:1, s. 400-411
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Krus, Ulrika, et al.
(författare)
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Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in the ATP:ADP ratio in rat pancreatic islets.
- 2006
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 49:7, s. 1578-1586
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Tidskriftsartikel (refereegranskat)abstract
- AIMS/HYPOTHESIS: The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion. METHODS: Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K(+) channel (K(ATP)-channel) conductance. RESULTS: We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca(2+)-induced exocytosis and K(ATP)-channel conductance in single cells, were unaffected. Insulin secretion induced by alpha-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K(ATP)-independent actions of fuel secretagogues are operational, as well as the first and K(ATP)-dependent phase. CONCLUSIONS/INTERPRETATION: Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.
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| 5. |
- Krus, Ulrika
(författare)
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Coupling mechanisms of insulin secretion - roles of mitochondrial metabolism and cAMP.
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type 2 diabetes is a disease that increases tremendously in the western world. It is characterized by insulin resistance and defects in insulin secretion. Insulin resistance is tightly linked to obesity, and usually precedes the onset of type 2 diabetes. When insulin resistance develops, the pancreatic beta-cells compensate by in¬creasing their secretion of insulin, causing hyperinsulinemia. This state may not be a major risk factor per se; in fact, many people develop severe insulin resistance and hyperinsulinemia without ever acquiring diabetes. However, beta-cells in some individuals, perhaps genetically predisposed, are unable to increase their secretion sufficiently to meet the new requirements. This leads to hyperglycemia, the main hallmark of diabetes. To be able to treat diabetes, the defects in the beta-cells causing impaired insulin secretion must be elucidated. The aim of this thesis was to investigate the mechanisms of insulin secretion, and especially what couples glucose stimulation of the beta-cell to insulin secretion. We have found that anaplerosis via pyruvate carboxylase is essential for both phases of glucose-stimulated insulin secretion, presumably via generation of an increased ATP/ADP ratio. Further, we discovered that expression of PDK1 is upregulated in INS-1 832/13 cells cultured at high concentrations of glucose, and that knock-down of PDK1 enhances insulin secretion. Both these findings prove that mitochondrial metabolism is important for insulin secretion, and points to the involvement of pyruvate cycling. We have also showed that PKA signaling is stimulated by glucose, and that inhibition of PKA decreases glucose-stimulated insulin secretion in INS-1 832/13 cells.
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