Abstract
The rat insulinoma cell line RIN 1046-38 loses glucose-stimulated insulin secretion as a function of time in culture. We found that the loss of glucose sensing in these cells was correlated with the loss of expression of GLUT-2 and glucokinase. Stable transfection of RIN cells with a plasmid containing the GLUT-2 cDNA conferred glucose-stimulated insulin release in intermediate but not high passage cells, with the near-maximal 3-fold increase occurring at 50 microM glucose. GLUT-2 expressing cells also exhibited a larger response to the combination of 5 mM glucose + 1 microM forskolin than untransfected cells (7.9 versus 1.6-2.7-fold, respectively). GLUT-2 expressing intermediate passage, but not high passage, RIN cells exhibited a 4-fold increase in glucokinase enzymatic activity relative to nonexpressing controls. Glucokinase activity was also increased by transfer of the GLUT-2 gene into intermediate passage RIN cells via recombinant adenovirus. Preincubation of GLUT-2 expressing intermediate passage RIN cells with 2-deoxyglucose to inhibit low Km hexokinases resulted in a glucose-stimulated insulin secretion response that was shifted toward the physiologic range. These studies indicate that GLUT-2 expression confers both a high and low affinity glucose-stimulated insulin secretion response to intermediate passage RIN cells.
Highlights
From the SGifford Laboratories for Diabetes Research and Departments of Biochemistry and Internal Medicine, University of n x a s, Southwestern Medical Center,Dallas, Texas 75235, the Weterans AffairsMedical Center, Dallas, Texas 75216, and the Illlniversity of Massachussetts Medical Center, Worcester,Massachussetts 01655
RNA blothybridization analysis was state insulin, glucokinase, and GLUT-2mRNA levels were evaluated in RIN 1046-38 cells of low or high passage number (17 passages versus 69 passages, respectively).As seen in Fig. 1, insulin mRNA levels were substantially decreased in high passage RIN cells as compared with low passage RIN cells, carried out with 32P-labeledantisense GLUT-2, GLUT-1, glucokinase, while the mature glucokinase and GLUT-2 transcripts were insulin, or p-actin probes [6,7,8]
In addition to establishing thaptassage-dependent disappearanceof glucose-stimulated insulin release iscorrelated with reduced expression of these two gene products,we show that restoration of GLUT-2 expression in intermediate g* 100 s.E
Summary
By a brief washing of the cells with HEPEShicarbonate and exposureto a range of glucose concentrations. Glucokinase activity was discriminated the PCB-7 vectorcontaining the rat GLUT-2cDNA[7,8]or the rat islet from hexokinase activity by performing the reactions in the presence of glucokinase cDNA[3,6].The transfection conditions wereas previously 10m~ glucose6-phosphate.Reactions were allowedto run for 90 min at described foAr tT-2Oins cells [7,8].Stable transfectants were selectedby 37 "Cand terminatedby the addition of 3%methanol in 95% ethanol. Cells werewashed twice with U343, as previously described [22].The glucokinase-primaryantibody phosphate-buffered saline (PBS) and cultured in RPMI 1640 me- complex was visualized on film with horseradish peroxidase goat antidium containing 11m~ glucose for 24-48 h. The efficiency of gene transfer was estimated by incubating RIN cells with similar amounts of the AdCMV-PGal virus [18], which contains a nuclear localizingvariant of the Escherichia coli P-galactosidase gene.
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