HORMONAL REGULATION OF HEPATIC AND PANCREATIC ISLET GLUCOKINASE mRNA LEVEL

竞争性聚合酶链式反应测定大鼠胰岛葡萄糖激酶mRNA由于其在分离的大鼠胰岛总RNA中所占比例太少，不能用Northern印迹分析来衡量葡萄糖激酶mRNA的去除量，因此采用竞争性聚合酶链式反应的方法。在预培养24小时后，分离的大鼠胰岛在含有2.2、5.5或16.7 mM葡萄糖的RPMI(+10%FCS)中再培养24小时。为探讨不同激素对胰岛葡萄糖激酶基因表达的影响，在培养液中加入地塞米松(1uM)、三碘甲状腺原氨酸(10uM)、胰高血糖素(1uM)或胰岛素(1uM)。当葡萄糖浓度为16.7 mM时，胰岛葡萄糖激酶基因表达水平高于2.2 mM或5.5 mM葡萄糖时。地塞米松、T3和胰高血糖素显著抑制胰岛葡萄糖激酶mRNA的表达，而胰岛素治疗不改变胰岛葡萄糖激酶mRNA水平。在5.5 mM和16.7 mM葡萄糖中，环-AMP类似物(二丁基cAMP)显著降低葡萄糖激酶的mRNA水平，而环GMP类似物(8-bromo cGMP)则不改变葡萄糖激酶的mRNA水平。此外，口服降糖药(格列本脲)后6h，葡萄糖激酶mRNA水平升高，而葡萄糖激酶mRNA水平在24小时降至基础值。这些结果表明，不同激素和环核苷酸对胰岛葡萄糖激酶基因表达的调节机制与肝细胞不同。

Measurement of islet glucokinase mRNA by competitive PCRSince 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 mRNAAfter 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.