Friedman J E, Sun Y, Ishizuka T, Farrell C J, McCormack S E, Herron L M, Hakimi P, Lechner P, Yun J S
Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935, USA.
J Biol Chem. 1997 Dec 12;272(50):31475-81. doi: 10.1074/jbc.272.50.31475.
The molecular mechanisms underlying increased hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription and gluconeogenesis in type II diabetes are largely unknown. To examine the involvement of glucocorticoids and the cis-acting insulin response sequence (IRS, -416/-407) in the genetically obese db/db mouse model, we generated crosses between C57BL/KsJ-db/+ mice and transgenic mice that express -460 or -2000 base pairs of the rat PEPCK gene promoter containing an intact or mutated IRS, linked to a reporter gene. Transgenic mice expressing the intact PEPCK(460)-CRP (C-reactive protein) transgene bred to near homozygosity at the db locus were obese, hyperinsulinemic, and developed fasting hyperglycemia (389 +/- 26 mg/100 ml) between 4 and 10 weeks of age. Levels of CRP reporter gene expression were increased 2-fold despite severe hyperinsulinemia compared with non-diabetic non-obese transgenic mice. Reporter gene expression was also increased 2-fold in transgenic obese diabetic db/db mice bearing a mutation in the IRS, -2000(IRS)-hGx, compared with non-obese non-diabetic transgenic 2000(IRS)-hGx mice. Treatment of obese diabetic db/db transgenic mice with the glucocorticoid receptor blocker RU 486 decreased plasma glucose by 50% and reduced PEPCK, GLUT2, glucose-6-phosphatase, tyrosine aminotransferase, CRP, and hGx reporter gene expression to levels similar to those of non-obese normoglycemic transgenic mice. Taken together, these results establish that -460 bp of 5'-flanking sequence is sufficient to mediate the induction of PEPCK gene transcription in genetically obese db/db mice during the development of hyperglycemia. The results further demonstrate that the mechanism underlying increased expression of gluconeogenic enzymes in the db/db mouse requires the action of glucocorticoids and occurs independently of factors acting through the PEPCK IRS (-416/-407) promoter binding site.
II型糖尿病中肝脏磷酸烯醇丙酮酸羧激酶（PEPCK）基因转录增加和糖异生的分子机制在很大程度上尚不清楚。为了研究糖皮质激素和顺式作用胰岛素反应序列（IRS，-416/-407）在遗传性肥胖db/db小鼠模型中的作用，我们使C57BL/KsJ-db/+小鼠与转基因小鼠杂交，后者表达含有完整或突变IRS的大鼠PEPCK基因启动子的-460或-2000碱基对，并与一个报告基因相连。表达完整PEPCK(460)-CRP（C反应蛋白）转基因且在db位点繁殖至近纯合子的转基因小鼠肥胖、高胰岛素血症，并在4至10周龄时出现空腹高血糖（389±26mg/100ml）。尽管与非糖尿病非肥胖转基因小鼠相比存在严重的高胰岛素血症，但CRP报告基因表达水平仍增加了2倍。与非肥胖非糖尿病转基因2000(IRS)-hGx小鼠相比，携带IRS突变-2000(IRS)-hGx的转基因肥胖糖尿病db/db小鼠中报告基因表达也增加了2倍。用糖皮质激素受体阻滞剂RU 486治疗肥胖糖尿病db/db转基因小鼠可使血糖降低50%，并使PEPCK、GLUT2、葡萄糖-6-磷酸酶、酪氨酸转氨酶、CRP和hGx报告基因表达降至与非肥胖血糖正常转基因小鼠相似的水平。综上所述，这些结果表明5'侧翼序列的-460bp足以介导遗传性肥胖db/db小鼠在高血糖发展过程中PEPCK基因转录的诱导。结果进一步证明，db/db小鼠中糖异生酶表达增加的机制需要糖皮质激素的作用，且独立于通过PEPCK IRS（-416/-407）启动子结合位点起作用的因子。
