Goodison S, Kenna S, Ashcroft S J
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, U.K.
Biochem J. 1992 Jul 15;285 ( Pt 2)(Pt 2):563-8. doi: 10.1042/bj2850563.
Northern-blot analysis was used to demonstrate that an increase in extracellular glucose concentration increased the content of preproinsulin mRNA 2.3-fold in the beta-cell line HIT T15. A probe for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase was used as a control. Mannoheptulose blocked this effect of glucose. A stimulatory effect on preproinsulin mRNA levels was also observed in response to mannose and to 4-methyl-2-oxopentanoate. However, galactose and arginine were ineffective. Glucagon, forskolin and dibutyryl cyclic AMP also elicited an increase in HIT-cell preproinsulin mRNA. The ability of the 5' upstream region of the preproinsulin gene to mediate the effect of glucose and other metabolites on transcription was studied by using a bacterial reporter gene technique. HIT cells were transfected with a plasmid, pOK1, containing the upstream region of the rat insulin-1 gene (-345 to +1) linked to chloramphenicol acetyltransferase (CAT). Co-transfection with a plasmid pRSV beta-gal containing beta-galactosidase driven by the Rous sarcoma virus promoter was used as a control for the efficiency of transfection; expression of CAT activity in transfected HIT cells was normalized by reference to expression of beta-galactosidase. Glucose caused a dose-dependent increase in expression of CAT activity, with a half-maximal effect at 5.5 mM and a maximum response of 4-fold. Mannoheptulose blocked this effect of glucose. Other metabolites (mannose, 4-methyl-2-oxopentanoate and leucine plus glutamine) were also able to increase insulin promoter-driven CAT expression, but galactose and arginine were ineffective. The stimulatory effect of glucose on CAT expression was not blocked by verapamil and was inhibited by increasing extracellular Ca2+ from 0.4 to 5 mM. Both dibutyryl cyclic AMP and forskolin caused an increase in insulin promoter-driven gene expression in the presence of 1 mM-glucose, but neither agent further increased the level of expression occurring in the presence of a maximally stimulating glucose concentration. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also increased insulin promoter-driven CAT expression in the presence of 1 mM-, but not 11 mM-glucose. Staurosporine blocked the stimulatory effect not only of PMA but also of glucose and of dibutyryl cyclic AMP. We conclude that the 5' upstream region of the insulin gene contains sequences responsible for mediating the stimulatory effect of glucose on insulin-gene transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
采用Northern印迹分析来证明，细胞外葡萄糖浓度的升高可使β细胞系HIT T15中的胰岛素原前体mRNA含量增加2.3倍。将组成性表达的甘油醛-3-磷酸脱氢酶的探针用作对照。甘露庚酮糖可阻断葡萄糖的这一作用。对甘露糖和4-甲基-2-氧代戊酸的反应也观察到对胰岛素原前体mRNA水平有刺激作用。然而，半乳糖和精氨酸无效。胰高血糖素、福斯可林和二丁酰环磷腺苷也可引起HIT细胞胰岛素原前体mRNA增加。通过使用细菌报告基因技术研究了胰岛素原基因5'上游区域介导葡萄糖和其他代谢物对转录作用的能力。用含有大鼠胰岛素-1基因上游区域（-345至+1）与氯霉素乙酰转移酶（CAT）连接的质粒pOK1转染HIT细胞。将与含有由劳氏肉瘤病毒启动子驱动的β-半乳糖苷酶的质粒pRSVβ-gal共转染用作转染效率的对照；通过参考β-半乳糖苷酶的表达来标准化转染的HIT细胞中CAT活性的表达。葡萄糖导致CAT活性表达呈剂量依赖性增加，在5.5 mM时达到半数最大效应，最大反应为4倍。甘露庚酮糖阻断了葡萄糖的这一作用。其他代谢物（甘露糖、4-甲基-2-氧代戊酸以及亮氨酸加谷氨酰胺）也能够增加胰岛素启动子驱动的CAT表达，但半乳糖和精氨酸无效。葡萄糖对CAT表达的刺激作用未被维拉帕米阻断，且在细胞外Ca2+从0.4 mM增加到5 mM时受到抑制。在存在1 mM葡萄糖的情况下，二丁酰环磷腺苷和福斯可林均导致胰岛素启动子驱动的基因表达增加，但这两种试剂均未进一步增加在最大刺激葡萄糖浓度存在时所出现的表达水平。佛波酯佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）在存在1 mM而非11 mM葡萄糖的情况下也增加胰岛素启动子驱动的CAT表达。星形孢菌素不仅阻断PMA的刺激作用，还阻断葡萄糖和二丁酰环磷腺苷的刺激作用。我们得出结论，胰岛素基因的5'上游区域包含负责介导葡萄糖对胰岛素基因转录刺激作用的序列。（摘要截短于400字）
