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.
Abstract. Overexpression of regucalcin has been shown to enhance glucose utilization and lipid production in the cloned rat hepatoma H4-II-E cells in vitro, and it induces insulin resistance. The effect of regucalcin on the gene expression of insulin signaling-related proteins was investigated in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin in vitro. The hepatoma cells (wild-type) and stable regucalcin/ pCXN2-transfected cells (transfectants) 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, 48, or 72 h in a medium containing either vehicle or insulin (10 -9 -10 -7 M) with or without supplementation of glucose (10, 25, or 50 mg/ml of medium). The expression of rat insulin receptor (Insr), phosphatidylinositol 3-kinase (PI3K), glucose transporter 2 (GLUT 2), or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNAs was examined using reverse transcription-polymerase chain reaction analysis with specific primers. GLUT 2 mRNA expression was significantly increased in the transfectants, while Insr, PI3K, and G3PDH mRNA levels were not significantly changed in the transfectants. Culture with insulin (10 -8 or 10 -7 M) caused a significant increase in PI3K mRNA levels in wild-type cells cultured for 24 or 48 h, while it had no effect on Insr mRNA levels. The supplementation of glucose (10, 25, or 50 mg/ml) caused a significant increase in Insr and PI3K mRNA levels in wild-type cells. The effect of insulin or glucose supplementation on these gene expression levels was not seen in the transfectants. The combination of insulin (10 -7 M) and glucose (50 mg/ml) caused a significant increase in Ins and PI3K mRNA levels in wild-type cells. Such an effect was not seen in the transfectants. Culture with insulin or glucose supplementation failed to have a significant effect on GLUT2 and G3PDH mRNA levels in the wild-type cells or transfectants. This study demonstrates that overexpression of regucalcin suppresses the enhancing effect of insulin or glucose on the gene expression of insulin signalingrelated proteins in the cloned rat hepatoma H4-II-E cells.
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