糖尿病下调肝视黄醇脱氢酶和上调视黄醛脱氢酶活性与表达机制及其在肝胰岛素抵抗和糖尿病中作用

Our previous report showed that diabetes downregualted hepatic retinol dehydrogenase (ADHs/RDHs) but upregulated retinaldehyde dehydrogenase (RALDHs) activities and expressions, leading to significant decreases in concentration of hepatic retinaldehyde and significant increases in concentration of hepatic retinoic acid in rats. Here, we used both the induced diabetic animals and spontaneously diabetic animals: 1) to investigate the real mechanisms that diabetes decreased hepatic ADHs/RDHs and increased RALDHs activities and expressions, as well as their contribution to unbalance levels of hepatic retinaldehyde and retinoic acid in liver; 2) to study effects of alterations in levels of hepatic retinoic acid and retinaldehyde on glucose metabolism and lipid metabolism, as well as expressions of corresponding targeted proteins and mRNAs such as PEPCK and SREBP-1c in diabetic animals. The results would be further verified using RALDH1a1-/- mice. 3) to document effects of retinaldehyde and retinoic acid on hepatic glucose metabolism and lipid metabolism and expression of the corresponding proteins and which insulin signaling pathways were involved in glucose metabolism and lipid metabolism mediated by retinaldehyde and retinoic acid in primary rat/human hepatocytes and HepG2 cells, which would be further verified using gene silencing and transfection of RALDH1. Above results would support the our hypothesis, i.e “Diabetes downregualted ADHs/RDHs but upregulated RALDHs activities and expressions, leading to unbalance of hepatic retinaldehyde and retinoic acid, which impaired hepatic glucose metabolism and lipid metabolism, in turn, aggravated hepatic insulin residence and diabetes”. The findings would highlight roles of unbalance levels of hepatic retinal and retinoic acid in liver insulin resistance and diabetes, as well as give gudainces for clinical medication of retinoids and therapeutic target of RALDH1.

鉴于糖尿病下调肝视黄醇脱氢酶(ADHs/RDHs)和上调视黄醛脱氢酶(RALDHs)活性与表达，导致肝视黄醛浓度降低和视黄酸浓度升高事实，在文献和前期基础上用诱导性和自发性糖尿病鼠，以肝为靶器官，结合基因敲除动物，研究1)糖尿病下调肝ADHs/RDHs和上调RALDHs活性和表达机制及其对视黄醛和视黄酸浓度失衡贡献；2)肝视黄醛和视黄酸浓度失衡与PEPCK等肝糖代谢和SREBP-1c等脂肪代谢相关蛋白表达关系；3)视黄醛和视黄酸对肝细胞糖和脂肪代谢及相关蛋白表达影响，用RALDH1基因沉默和转染验证。验证“糖尿病下调肝ADHs/RDHs和上调RALDHs活性，导致视黄醛和视黄酸浓度失衡，损伤肝糖代谢和脂肪代谢，加重肝胰岛素抵抗和糖尿病”推论。其成果对于阐明糖尿病引起肝视黄醛与视黄酸浓度失衡机制及其在肝胰岛素抵抗和糖尿病中作用，维生素A类药物合理使用和RALDH1作为药物潜在靶点有重要意义。

糖尿病和肥胖状态下肝组织视黄醛浓度显著降低，视黄酸浓度显著增加，升高的视黄醛脱氢酶（Retinaldehyde dehydrogenases, RALDHs）水平是导致体内视黄醛和视黄酸等维生素A类物质紊乱的主要原因，然而其生理学意义不清。我们的研究发现视黄醛和视黄酸均可上调PCK和G-6-P等基因表达，但在RALDH抑制剂存在下这种协同作用抵消，视黄醛的诱导作用是通过转化为视黄酸实施的，而视黄醛本身则呈现拮抗视黄酸作用。糖尿病状态下肝脏中视黄醛水平降低和视黄酸水平升高共同导致磷酸烯醇式丙酮酸羧激酶1（Phosphoenolpyruvate carboxykinase 1, PCK1）表达增加，肝脏糖异生增强。视黄醛本身调控PCK1蛋白表达的作用可能与视黄酸相反。.进一步的研究发现糖尿病诱导肝脏中RALDHs活性与表达，导致肝脏中视黄醛水平降低，视黄醛以及视黄醛联合柠檬醛给药改善糖尿病小鼠的空腹血糖、口服葡萄糖和丙酮酸耐量，逆转糖尿病诱导的PCK1和G-6-P表达增加。并用在体沉默RALDH1基因验证。在人肝细胞中，视黄醛显著抑制PCK1和G-6-P表达，沉默RALDH1基因加强视黄醛对PCK1和G-6-P表达的抑制。荧光素酶报告基因分析显示视黄醛是RXRα的抑制剂以及RARα的部分激动剂，视黄醛通过抑制RXRα/DR1介导的PCK1和G-6-P基因表达的调控。糖尿病状态下肝脏中降低的视黄醛水平导致PCK1和G-6-P表达增加，肝脏糖异生增加并恶化糖尿病，外源性补充视黄醛可以改善糖尿病症状。.我们的研究还发现视黄酸给药剂量依赖性的降低糖尿病大鼠对葡萄糖刺激的胰岛素分泌。促进肝和胰腺组织中脂质与胰岛素合成相关基因表达，伴随着胰腺和肝脏受损加重。视黄酸剂量依赖性抑制INS-1细胞葡萄糖刺激的胰岛素分泌。视黄酸升高INS-1细胞中SREBP-1c与UCP2表达，伴随着ATP合成的减少。沉默细胞中SREBP-1c或使用SREBP-1c抑制剂，可以逆转视黄酸增加UCP2的表达、减少ATP 合成和抑制胰岛素分泌的作用。沉默UCP2基因可以逆转视黄酸对ATP合成和胰岛素分泌的抑制作用。综上所述，视黄酸增加糖尿病大鼠胰腺的脂质和胰岛素合成，但降低葡萄糖刺激的胰岛素分泌，并进一步加重糖尿病症状。视黄酸可能通过激活RXR/SREBP-1c/UCP2通路进而抑制胰岛细胞的胰岛素分泌。