Abstract
The recently established pancreatic beta-cell line MIN6 retains the ability to secrete insulin in response to physiological glucose concentrations. To investigate the role of glucose transport and phosphorylation in glucose-stimulated insulin secretion by beta-cells, MIN6 cells were stably transfected with a rabbit GLUT1 glucose transporter cDNA or a rat hexokinase I cDNA cloned in an expression vector. Overexpression of GLUT1 increased 3-O-methylglucose uptake, but did not alter either glucose utilization or glucose-stimulated insulin secretion. In contrast, clones overexpressing hexokinase I exhibited enhanced glucose-stimulated insulin secretion at glucose concentrations below 10 mM with a concomitant increase in glucose utilization. Maximal insulin secretion as well as the maximal rate of glucose utilization were not altered in these clones. Insulin secretion stimulated by 2-ketoisocaproate, a non-glucose secretagogue, was not affected by hexokinase I expression. These results strongly suggest that the glucose phosphorylating step, but not glucose transport step, regulates glucose-stimulated insulin secretion by modulating the glycolytic rate in the beta-cell.
Highlights
If p-cells sense the concentrationof glucose through its mecose-stimulated insulin secretionby p-cells, MIN6 cells tabolites, therate of glucose metabolism must vary in proporwere stably transfected with a rabbit GLUTl glucose tion tophysiological range of glucose concentrations
We recently demonstrated that the characteristicsof glucose transport and metabolism by MING cells closely resemble those of isolated islets and thusconcluded that thiscell line is a suitable model for studying glucose-stimulated insulin secretion in pancreatic p-cells (Ishihara et al.,1993)
To investigate the role of glucose transport and phosphorylation in the glucose sensing by p-cells, we have produced MING cell transfectants overexpressing a low K, glucose transporter, GLUT1, or a low K, glucose phosphorylating enzyme, hexokinase I
Summary
Overexpression of GLUTl Glucose lkansporterin MIN6 Cells-TheDNA construct for the expression of the GLUTl glucose transporter was transfected intoMIN6 cells. Labeled M , 50,000protein was immunoprecipitated with antibody against the C-terminal of the GLUTl glucose transporter from clone G1-8 but notfrom clone C1-6 (Fig. 2 B ). The uptake of 0.5 m~ 3-O-methyl-~-glucosewas increased 2-fold i n clone G1-8 as compared with that in clone C1-6 (Fig. 3A).The observation that transport activity does not increase in parallel withincreased GLUTl expression is attributable to the presence of another isoform, GLUT2, which has transport activity. Cell surface glycoproteins of cloneC1-6 (0) and clone G1-8 (0)were labeled with galactose oxidase and NaB3H, in intact cells and subsequently immunoprecipitated with the affinity-purified anti-GLUT1 antibody.
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