title: Glutaredoxin-1 mediates NADPH-dependent stimulation of calcium-dependent insulin secretion.
abstract: Nicotinamide adenine dinucleotide phosphate (NADPH) enhances Ca(2+)-induced exocytosis in pancreatic beta-cells, an effect suggested to involve the cytosolic redox protein glutaredoxin-1 (GRX-1). We here detail the role of GRX-1 in NADPH-stimulated beta-cell exocytosis and glucose-stimulated insulin secretion. Silencing of GRX-1 by RNA interference reduced glucose-stimulated insulin secretion in both clonal INS-1 832/13 cells and primary rat islets. GRX-1 silencing did not affect cell viability or the intracellular redox environment, suggesting that GRX-1 regulates the exocytotic machinery by a local action. By contrast, knockdown of the related protein thioredoxin-1 (TRX-1) was ineffective. Confocal immunocytochemistry revealed that GRX-1 locates to the cell periphery, whereas TRX-1 expression is uniform. These data suggest that the distinct subcellular localizations of TRX-1 and GRX-1 result in differences in substrate specificities and actions on insulin secretion. Single-cell exocytosis was likewise suppressed by GRX-1 knockdown in both rat beta-cells and clonal 832/13 cells, whereas after overexpression exocytosis increased by approximately 40%. Intracellular addition of NADPH (0.1 mm) stimulated Ca(2+)-evoked exocytosis in both cell types. Interestingly, the stimulatory action of NADPH on the exocytotic machinery coincided with an approximately 30% inhibition in whole-cell Ca(2+) currents. After GRX-1 silencing, NADPH failed to amplify insulin release but still inhibited Ca(2+) currents in 832/13 cells. In conclusion, NADPH stimulates the exocytotic machinery in pancreatic beta-cells. This effect is mediated by the NADPH acceptor protein GRX-1 by a local redox reaction that accelerates beta-cell exocytosis and, in turn, insulin secretion.
authors.affiliation: Department of Clinical Sciences, Islet Pathophysiology, Lund University, Clinical Research Centre, Malmö, Sweden. thomas.reinbothe@med.lu.se
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Reinbothe
authors.forename: Rosita
authors.initials: R
authors.lastname: Ivarsson
authors.forename: Dai-Qing
authors.initials: DQ
authors.lastname: Li
authors.forename: Omid
authors.initials: O
authors.lastname: Niazi
authors.forename: Xingjun
authors.initials: X
authors.lastname: Jing
authors.forename: Enming
authors.initials: E
authors.lastname: Zhang
authors.forename: Lena
authors.initials: L
authors.lastname: Stenson
authors.forename: Ulrika
authors.initials: U
authors.lastname: Bryborn
authors.forename: Erik
authors.initials: E
authors.lastname: Renström
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D054477:Glutaredoxins / Q000378:metabolism*; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013879:Thioredoxins / Q000378:metabolism
chemical_list: C516007:Glrx protein, rat; D054477:Glutaredoxins; D007328:Insulin; D013879:Thioredoxins; D009249:NADP; D005947:Glucose; D002118:Calcium