Abstract
The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of glucose utilization and lipid production was investigated using the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin. The hepatoma cells (wild‐type) and stable regucalcin/pCXN2‐transfected cells (transfectant) were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. Cells with subconfluency were cultured for 24 or 72 h in medium containing either vehicle or insulin (10^−8^ or 10^−7^ M) with or without supplementation of glucose (10, 25, or 50 mg/ml of medium) in the absence of insulin. The production of triglyceride and free fatty acid was significantly increased in transfectants cultured without insulin and glucose supplementation as compared with that of wild‐type cells. The supplementation of glucose (10, 25, or 50 mg/ml) caused a remarkable increase in medium glucose consumption, triglyceride, and free fatty acid productions in transfectants cultured without insulin. The presence of insulin (10^−7^ M) caused a significant increase in medium glucose consumption, triglyceride, and free fatty acid productions in wild‐type cells cultured with glucose supplementation. These increases were significantly prevented in transfectants cultured for 72 h. The expression of acetyl‐CoA carboxylase, HMG‐CoA reductase, glucokinase, pyruvate kinase, and glyceroaldehyde‐3‐phosphate dehydrogenase (G3PDH) mRNAs in wild‐type cells was not significantly changed by culture with or without glucose supplementation in the presence of insulin. These gene expressions were not significantly changed in transfectants. The expression of glucose transporter 2 mRNA was significantly increased in transfectants as compared with that of wild‐type cells. Such an increase was not seen in transfectants cultured in the presence of insulin with or without glucose supplementation. This study demonstrates that overexpression of regucalcin enhances glucose utilization and lipid production in the cloned rat hepatoma H4‐II‐E cells, and that it regulates the effect of insulin. J. Cell. Biochem. 99: 1582–1592, 2006. © 2006 Wiley‐Liss, Inc.