Glucose‐dependent regulation of the l ‐pyruvate kinase gene in a hepatoma cell line is independent of insulin and cyclic AMP

Abstract

Hepatocyte-like mhAT3F cells have been derived from the hepatoma of a transgenic mouse expressing the SV40 large T antigen under the control of the antithrombin III gene regulatory region (Antoine, B., Levrat, F., Vallet, V., Berbar, T., Cartier, N., Dubois, N., Briand, P., and Kahn, A. (1992) Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice. Exp. Cell. Res. 200, 175-185; F. Levrat et al., unpublished results). In these cells, the L-PK gene is transcriptionally activated by glucose, as it is in vivo and in cultured hepatocytes. However, in contrast to the L-PK gene regulation in the liver and isolated hepatocytes, the glucose responsiveness does not require insulin and is not blocked by cyclic AMP. In mhAT3F cells, the insensitivity to insulin might be due to the replacement of insulin-dependent glucokinase by insulin-independent hexokinases able to phosphorylate glucose in the absence of the hormone. The glucose-dependent activation of the L-PK gene is delayed, requires ongoing protein synthesis, and is mediated by the same glucose response element as in vivo and in isolated hepatocytes. These results suggest that the glucose-dependent signaling pathway responsible for the transcriptional activation of glycolytic and lipogenic genes requires glucose phosphorylation, a phenomenon that is insulin-dependent in the liver but insulin-independent in cultured hepatoma cells. Nevertheless, the action of glucose 6-phosphate is most likely indirect.

Transcriptional control of genes that regulate glycolysis and gluconeogenesis in adult liver

“…An activating region located around -3 kb is required for full promoter activity [79,88]. This region corresponds to a liver-specific DNAase I-hypersensitive site (HSS-2) [85] [93]. Since Foufelle et al [94] demonstrated that glucose must be converted into glucose 6-phosphate by a hexokinase to exert its action on gene transcription, Axel Kahn's group proposed that insulin is required for the production of glucokinase in hepatocytes while hepatoma cells may constitutively express hexokinases, thus bypassing the need for insulin [95].…”

“…While cycloheximide may block the insulin induction of glucokinase, it also inhibits a step downwards in the pathway of glucose stimulation of transcription. Indeed, Lefranqois-Martinez et al [93] showed that in their hepatoma cells the insulin-independent glucose stimulation of L promoter activity could still be blocked by cycloheximide. Insulin and glucose also stabilize the mRNA [96].…”

Role of the GLUT 2 Glucose Transporter in the Response of the L-type Pyruvate Kinase Gene to Glucose in Liver-derived Cells

“…In these three glucose-sensitive tissues, glucose transport and phosphorylation are performed by various isoforms of glucose transporters and hexokinases, respectively. Glucose phosphorylation to glucose 6-phosphate, required for glucose action on the transcriptional machinery (2)(3)(4)(5)(6), is mainly mediated by hexokinase (HK) 1 II in adipocytes and by hexokinase IV (or glucokinase) in the liver and ␤ cells; expression of the glucokinase gene is constitutive in the pancreas and insulin-dependent in the liver due to the existence of two different tissue-specific alternative promoters (7). However, glucokinase is replaced by other insulin-independent hexokinase isoforms (mainly HK I) in cultured hepatoma cell lines (2,8,9).…”

“…Glucose phosphorylation to glucose 6-phosphate, required for glucose action on the transcriptional machinery (2)(3)(4)(5)(6), is mainly mediated by hexokinase (HK) 1 II in adipocytes and by hexokinase IV (or glucokinase) in the liver and ␤ cells; expression of the glucokinase gene is constitutive in the pancreas and insulin-dependent in the liver due to the existence of two different tissue-specific alternative promoters (7). However, glucokinase is replaced by other insulin-independent hexokinase isoforms (mainly HK I) in cultured hepatoma cell lines (2,8,9). Tissues whose function is regulated by glucose also possess particular glucose transporters, GLUT 4 in adipocytes and GLUT 2 in tissues secreting glucose into the blood (liver, small intestine, and proximal tubular cells of the kidney) as well as GLUT 2 in the ␤ cells of the islets of Langerhans (10,11).…”

“…In most hepatoma cell lines in culture, glucose responsiveness of glucose-dependent genes is lost (15) even when these cells remain well differentiated (2). In fact, expression of glucose-responsive genes is generally constitutive in these cells in which glucokinase is replaced by HK I and GLUT 2 is replaced by GLUT 1, another glucose transporter isoform expressed in most cells not specialized in metabolism regulation, e.g.…”