title: Reconstitution of glucotoxic HIT-T15 cells with somatostatin transcription factor-1 partially restores insulin promoter activity.
abstract: We have reported that chronic culture of HIT-T15 cells in medium containing supraphysiologic glucose concentrations (11.1 mmol/l) causes a decrease in insulin mRNA levels, insulin content, and insulin release. Furthermore, decreases in insulin gene transcription and binding activity of two essential beta-cell transcription factors, somatostatin transcription factor-1 (STF-1; also known as GSTF, IDX-1, IPF-1, PDX-1, and GSF) and RIPE-3b1 activator, are associated with this glucotoxic effect. In this study, we observed that the loss of RIPE-3b1 occurs much earlier (79% decrease at passage [p]81) than the loss of STF-1 (65% decrease at p104), with abolishment of both factors by p122. Since the STF-1, but not the RIPE-3b1 activator, gene has been cloned, we examined its restorative effects on insulin gene promoter activity after reconstitution with STF-1 cDNA. Basal insulin promoter activities normalized to early (p71-74) passage cells (1.000 +/- 0.069) were 0.4066 +/- 0.093 and 0.142 +/- 0.034 for intermediate (p102-106) and late (p118-122) passage cells, respectively. Early, intermediate, and late passage cells, all chronically cultured in medium containing 11.1 mmol/l glucose, were transfected with STF-1 alone or cotransfected with E2-5, an E-box factor known to be synergistically associated with STF-1. Compared with basal levels, we observed a trend toward an increase in insulin promoter activity in intermediate passage cells with STF-1 transfection (1.43-fold) that became a significant increase when E2-5 was cotransfected (1.78-fold). In late passage cells, transfection of STF-1 alone significantly stimulated a 2.2-fold increase in the insulin promoter activity. Cotransfection of STF-1 and E2-5 in late passage cells stimulated insulin promoter activity 2.8-fold, which was 40% of the activity observed in early passage cells. Control studies in glucotoxic betaTC-6 cells deficient in RIPE-3b1 activator but not STF-1 did not demonstrate an increase in insulin promoter activity after STF-1 transfection. We conclude that loss of RIPE-3b1 activity precedes loss of STF-1 activity in glucotoxic HIT-T15 cells and that defective promoter activity can be partially restored by STF-1 transfection and predict that eventual cloning of the RIPE-3b1 gene will allow cotransfection studies with both factors that will allow full reconstitution of insulin promoter activity.
authors.affiliation: Pacific Northwest Research Foundation, Division of Metabolism, Endocrinology, and Nutrition, University of Washington, Seattle 98122, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Harmon
authors.forename: Y
authors.initials: Y
authors.lastname: Tanaka
authors.forename: L K
authors.initials: LK
authors.lastname: Olson
authors.forename: R P
authors.initials: RP
authors.lastname: Robertson
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D018398:Homeodomain Proteins; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics*; D007515:Islets of Langerhans / Q000502:physiology*; D007700:Kinetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015534:Trans-Activators / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection
chemical_list: D018398:Homeodomain Proteins; D007328:Insulin; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D015500:Chloramphenicol O-Acetyltransferase; D005947:Glucose