Abstract
The INS-r3-GK27 insulinoma cells are endowed with artificially inducible glucokinase under control of the reverse tetracycline-dependent transcriptional activator. Moderate induction of glucokinase has been shown to result in proportionate increases in glycolytic flux and in potentiation of glucose effects on insulin secretion and pyruvate kinase gene expression. In cells with 20-fold overexpression of glucokinase, however, glucose activation of secretion and gene expression was severely impaired. Measurements of the glycolytic flux in cells with 7- and 21-fold increases in glucokinase activity and determination of the flux control coefficient of this enzyme showed that control of glycolysis at the glucokinase step was lost in the cells at the higher level of overexpression. Challenging the cells with glucose above 6 mM resulted in massive accumulation of glucose 6-phosphate and caused a rapid and sustained depletion of cellular ATP, in contrast with the glucose-induced rise in ATP in cells with wild-type glucokinase levels. Loss of cell viability ensued upon prolonged culture in high glucose. In summary, in insulinoma beta cells strongly overexpressing glucokinase, an imbalance between glucose phosphorylation and turnover of glucose 6-phosphate resulted in acute glucose intolerance due to trapping of cellular orthophosphate in dead-end product and severe paralysis of energy metabolism.
Highlights
The major function of the endocrine beta cell in the pancreas is to sense variations of the glucose concentration in the extracellular fluid and to increase its rate of insulin secretion when the sugar concentration rises above the normoglycemic level of 5– 6 mM, or to curtail secretion when glucose falls
A regulatory role of glucokinase in this process was suggested by our earlier finding of a leftward shift in glucose dose response in insulinoma INS-r3-GK27 beta cells overexpressing glucokinase 3– 8-fold above basal level after doxycycline treatment [8]
We have investigated the glucose effect on pyruvate kinase mRNA in cells at higher levels of overexpressed glucokinase activity
Summary
Along the entire glycolytic pathway in the beta cell is an important issue, because the rate of glycolysis appears to be the major determinant for physiological processes such as insulin secretion [4, 5] or the transcriptional regulation of specific genes. The experiments have demonstrated that moderate overexpression of glucokinase results in proportional increase in glycolytic flux and in potentiation of glucose-induced insulin secretion and stimulation of gene expression [8]. These results were in apparent conflict with the study of Becker et al [9], who overexpressed glucokinase at very high levels in cultured pancreatic islets using adenovirusmediated gene transduction but failed to observe parallel effects on glycolysis or insulin release. In an attempt to understand the conflicting conclusions of the two studies, we have examined the metabolic and physiologic consequences of forced overexpression of glucokinase at levels comparable to those in the adenovirus-transduced islets using the tetracycline-inducible INS-r3-GK27 cells
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