Function of 9-cis-retinoic acid

描述（由申请人提供）： 项目主任/主要研究者（最后，第一，中间）：那不勒斯，约瑟夫。L. 本研究的长期目标是确定9-顺式维甲酸（9 cRA）的生理功能和作用机制。9 cRA具有不同的药理作用，这促使了对用于治疗代谢疾病的类似物（rexinoids）的评价。然而，9 cRA尚未在体内肝脏、血清、脑、脂肪等组织中用灵敏的LC/MS/MS检测到，使其状态不确定（autacoid还是药物？）。这里的数据在体内鉴定了9 cRA（仅在胰腺中），表明2-细胞生物合成9 cRA，并且葡萄糖在832/13细胞中诱导顺式-类维生素A还原酶Dhrs 3的mRNA（4倍）并抑制顺式-类维生素A脱氢酶Rdh 5的mRNA（10倍）。我们还表明，9 cRA减少70%的胰岛素分泌从分离的小鼠胰岛在15分钟内，并在2-细胞系832/13通过减少Glut 2和葡萄糖激酶活性，ATP和钙。给药9 cRA损害体内葡萄糖耐量并在葡萄糖耐量试验期间降低血清胰岛素。9 cRA在从禁食到进食状态的过渡中降低，并且葡萄糖给药在15分钟内使体内9 cRA降低>80%。这些数据支持9 cRA和葡萄糖相反地调节葡萄糖刺激的胰岛素分泌的模型，并且指示9 cRA的非基因组作用机制（除了基因组作用机制之外）。从长远来看，9 cRA降低了至少两个对胰腺发育和胰岛素分泌至关重要的基因的表达：Pdx-1和HNF 41。值得注意的是，这表明六种单基因疾病中至少有两种被称为年轻人成熟期发病糖尿病（MODY）是由9 cRA调控的基因缺陷引起的，即Pdx-1和HNF 41的缺陷分别是MODY 4和MODY 1的基础。编码葡萄糖激酶的基因中的缺陷是第三种MODY的基础，即MODY 2：9 cRA调节葡萄糖激酶活性。这些数据验证了9 cRA作为天然存在的类维生素A具有在类维生素A中独特的生理功能，并鉴定了有助于胰岛素分泌的β 2细胞的新组分。本项目将测试9 cRA作为一种自体激素的功能，在低血糖期间减弱胰岛素分泌，以预防低血糖。具体目的是确定：1）9 cRA在葡萄糖刺激的胰岛素分泌的短期调节中的生理功能; 2）减少胰岛素分泌的快速9 cRA作用的机制; 3）9 cRA是否需要RAR用于2-细胞中的非基因组和/或基因组作用; 4）Dhrs 3和Rdh 5在2-细胞9 cRA生物合成中的功能。 目的1将检验以下假设：9 cRA的适度增加使胰腺对葡萄糖脱敏，并且血糖增加克服了9 cRA损害胰岛素分泌的能力。 目的2将验证9 cRA通过Glut 2和GK的翻译后修饰发挥功能的假设。 目的3验证RAR的非基因组和基因组作用介导9 cRA在2-细胞中的作用的假设。 目的4将检验葡萄糖通过Dhrs 3和Rdh 5控制2-细胞中9 cRA的假设。 该项目将有助于对类维生素A作用机制、核激素受体功能、Glut 2和GK调节以及胰岛素分泌的新见解。PHS 398/2590（Rev.06/09）

DESCRIPTION (provided by applicant): Program Director/Principal Investigator (Last, First, Middle): Napoli, Joseph. L. This research has long-term goals to determine 9-cis-retinoic acid (9cRA) physiological function and mechanisms of action. 9cRA has diverse pharmacological actions, which have prompted evaluation of analogs (rexinoids) for treating metabolic disease. Yet, 9cRA has not been detected in vivo in liver, serum, brain, adipose et al. with a sensitive LC/MS/MS assay, leaving its status unsettled (autacoid or drug?). The data here identify 9cRA in vivo (only in pancreas), show that 2-cells biosynthesize 9cRA, and that glucose induces mRNA of the cis-retinoid reductase Dhrs3 (4-fold) and represses mRNA of the cis-retinoid dehydrogenase Rdh5 (10-fold) in 832/13 cells. We also show that 9cRA decreases insulin secretion from isolated mouse islets by 70% within 15 min, and in the 2-cell line 832/13 by reducing Glut2 and glucokinase activities, ATP and calcium. Dosing 9cRA impairs glucose tolerance in vivo and reduces serum insulin during a glucose tolerance test. 9cRA decreases in the transition from the fasted to the fed state, and glucose dosing reduces 9cRA in vivo >80% within 15 min. These data support a model in which 9cRA and glucose work in opposition to regulate glucose-stimulated insulin secretion, and indicate non-genomic mechanisms of action for 9cRA (in addition to genomic mechanisms of action). In the longer term, 9cRA decreases expression of at least two genes crucial for pancreas development and insulin secretion: Pdx-1 and HNF41. Notably, this shows that at least two of the six monogenic diseases known as maturity onset diabetes of the young (MODY) are caused by defects in genes regulated by 9cRA, i.e. defects in Pdx-1 and HNF41 underlie MODY4 and MODY1, respectively. A defect in the gene that encodes glucokinase underlies a third MODY, i.e. MODY 2: 9cRA modulates glucokinase activity. These data validate 9cRA as a naturally occurring retinoid with physiological function(s) unique among retinoids, and identify a novel component of 2-cells that contributes to insulin secretion. This project will test 9cRA function as an autacoid that attenuates insulin secretion during low glucose to prevent hypoglycemia. The specific aims are to determine: 1) physiological function(s) of 9cRA in short-term regulation of glucose stimulated insulin secretion; 2) mechanisms of rapid 9cRA actions that diminish insulin secretion; 3) whether 9cRA requires RAR for non-genomic and/or genomic actions in the 2- cell; 4) functions of Dhrs3 and Rdh5 in 2-cell 9cRA biosynthesis. Aim 1 will test the hypothesis that modest increases in 9cRA desensitize the pancreas to glucose, and that increasing blood glucose overcomes ability of 9cRA to impair insulin secretion. Aim 2 will test the hypothesis that 9cRA functions through post-translational modification of Glut2 and GK. Aim 3 will test the hypothesis that non-genomic and genomic actions of RAR mediate the actions of 9cRA in the 2-cell. Aim 4 will test the hypothesis that glucose controls 9cRA in the 2-cell through Dhrs3 and Rdh5. This project should contribute novel insight into mechanisms of retinoid action, nuclear hormone receptor function, regulation of Glut2 and GK, and insulin secretion. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page