| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
|
|---|
| Research Abstract |
Measurement of islet glucokinase mRNA by competitive PCR
Since it is too small amounts in total RNA extracted from isolated rat pancreatic islets to measure the abandance of glucokinase mRNA by Northern blot analysis, competitive PCR method was applied. This method enabled us to measure the level of glucokinase mRNA using as small as 0.2 ug of total RNA.
Mechanism of regulation of islet glucokinase mRNA
After 24 hs of preculture, isolated rat islats were cultured for additional 24 hs in RPMI medium (+10% FCS) with 2.2,5.5, or 16.7 mM glucose. In order to investigate the effects of verious hormones on the abandance of islet glucokinase mRNA, dexamethason (1uM), tri-iodothyronine (10uM), Glucagon (1uM), or insulin (1uM) was added in the culture medium. Islet glucokinase mRNA level was higher in the presence of 16.7 mM glucose than at 2.2 or 5.5 mM glucose. While administration of dexamethason, T3, and glucagon significantly inhibited the abandance of islet glucokinase mRNA, insulin treatment did not change glucokinase mRNA level. Cyclic-AMP analogue (dibutytyl cAMP) significantly reduced glucokinase mRNA level in 5.5 and 16.7 mM glucose although cyclic GMP analogue (8-bromo cGMP) produced no change in glucokinase mRNA. Furthermore, glucokinase mRNA increased at 6 hs after administration of oral hypoglycemic agent (glibenclamide), whereas glucokinase mRNA level decreased to basal values at 24 hs. These results suggest that various hormones and cyclic nucleotides regulate glucokinase mRNA level at pancreatic islets by the different mechanism from at hepatocytes.
|
