Quantitative analysis of pancreatic glucokinase gene expression in cultured beta cells by competitive polymerase chain reaction.
Regulation of glucokinase (GK) gene expression in pancreatic beta cells has been poorly investigated, both due to low abundance of the gene and to difficulties in cells isolation. The present study describes the establishment of a competitive RT-PCR method for quantitative analysis of GK gene. The method has been applied to the analysis of GK mRNA expression RIN 1046-38 cells. We have monitored modifications of GK mRNA expression after different periods of time in culture and we have studied the effect induced by dexamethasone (DEX) treatment. We show that the method is very sensitive and requires very low amount of RNA. Data demonstrate that GK mRNA expression in RIN cells is reduced as a function of passages in culture and that the reduction is positively correlated with the decrease of insulin responsiveness observed in high passages cells. DEX treatment inhibits GK mRNA expression in RIN cells in a dose-dependent and time-dependent manner.
Borboni
P
P
University of Rome Tor Vergata, Department of Internal Medicine, Italy.
Porzio
O
O
Magnaterra
R
R
Fusco
A
A
Sesti
G
G
Lauro
R
R
Marlier
L N
LN
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000235:genetics*; D006168:Guinea Pigs; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine
D017931:DNA Primers; D007328:Insulin; D012333:RNA, Messenger; D003907:Dexamethasone; D005941:Glucokinase