Overexpression of regucalcin suppresses gene expression of insulin signaling-related proteins in cloned rat hepatoma H4-II-E cells: involvement of insulin resistance.
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 signaling-related proteins in the cloned rat hepatoma H4-II-E cells.
Nakashima
Chikage
C
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Yamaguchi
Masayoshi
M
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002265:Carboxylic Ester Hydrolases; D002999:Clone Cells; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D051273:Glucose Transporter Type 2 / Q000235:genetics / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007333:Insulin Resistance; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D002135:Calcium-Binding Proteins; D002265:Carboxylic Ester Hydrolases; D005947:Glucose; D051273:Glucose Transporter Type 2; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D019869:Phosphatidylinositol 3-Kinases; D012333:RNA, Messenger; D011972:Receptor, Insulin; C513325:Rgn protein, rat