RORα在维持胰岛β细胞功能稳态中的作用机制及对2型糖尿病的治疗研究

Recent epidemiological evidence points to an increase in the incidence of diabetes worldwide. Type 2 diabetes mellitus (T2DM) representing ∼85-95% of diabetes cases. T2DM pathogenesis is complex, characterized by genetic predisposition together with metabolic abnormalities, defective insulin secretion and action, and elevated endogenous glucose production.. Evidence from both human and animal studies suggests that T2DM is characterized by decreased functional β-cell mass that cannot adapt insulin secretion to compensate for increasing insulin resistance driving the development of overt T2DM. Significant β-cell failure is now believed to take place at an early stage in disease progression; that is, β-cell function declines sharply before and after the diagnosis of T2DM. β-cell function continues declining progressively despite treatment with antidiabetic medications. Therefore, new therapeutic classes of diabetes medications act to preserve functional β-cells or improve β-cell function, thus potentially altering the course of the disease.. Oxidative stress, an imbalance between oxidative and antioxidative systems of cells and tissues, is a result of over production of oxidative-free radicals and associated reactive oxygen species (ROS). One outcome of excessive levels of ROS is the modification of the structure and function of cellular proteins and lipids, leading to cellular dysfunction including impaired energy metabolism, altered cell signalling and cell cycle control, and overall dysfunctional biological activity, immune activation and inflammation. The main outcomes of dysfunctional redox balance in T2DM are increased insulin resistance and impaired β-cell insulin secretion. Glucose catabolism in β-cell results in ATP generation, which is a primary driver of glucose-stimulated insulin secretion (GSIS). Glucose metabolism also stimulates mitochondrial generation of ROS. The subsequent formation of hydrogen peroxide (H2O2), unless rapidly removed, can suppress β-cell metabolic activity resulting in inhibition of insulin secretion. Rodent pancreatic β-cells have low levels of H2O2-detoxifying and redox-regulating enzymes, e.g. CAT, GPx, and glutathione reductase. . Retinoic acid-related orphan receptor a (RORα) belongs to the nuclear hormone receptor superfamily that regulates diverse target genes associated with metabolic homeostasis. RORa binds to ROR response elements in the promoter of target genes. RORa has been implicated in the regulation of diverse lipid and glucose metabolic pathways in both experimental animals and human patients. Staggerer mice (RORα sg/sg), displaying C-terminal deletions and dysfunction of RORα, show changed metabolic homeostasis through alterations in the expression of a number of genes, such as those encoding FGF21,SRC2, and PPARc. Earlier studies suggested that RORα has a protective function against oxidative stress. The treatment of melatonin, a putative ligand of RORα, reduced the level of oxidative damage mediated by β-amyloid, ceramide, or H2O2.. Our preliminary study showed that the genetic variants in RORα is associated with the risk of T2DM in Chinese, and the level in pancreatic islets is decreased significantly in patients with T2DM and in db/db mice, an animal model of T2DM. Moreover, the treatment of SR1078, an agonist of RORα, attenuated the level of oxidative damage induced by H2O2 in MIN6 cell line. The specific aims of the current project are (1) To investigate the effect of RORα on the β-cell function; (2) To clarify the underlying mechanisms on the regulation of ROS in pancreatic β-cell regulating; (3) To test whether or expression of RORα protects β-cell function and attenuates the development of diabetes in db/db mice.

胰岛β细胞功能障碍是T2DM胰岛素抵抗和疾病发展的核心因素，β细胞的氧化应激是导致其功能降低的重要原因。我们前期发现RORα影响T2DM发生，其表达在T2DM胰岛β细胞中降低，其激动剂可显著减弱H2O2对MIN6胰岛细胞的损伤。据此假设：RORα通过调控氧化应激影响β细胞功能，在β细胞中高表达RORα可改善氧化应激引起的损伤，减轻T2DM。本项目将（1）应用腺相关病毒rAAV8-Cas9特异敲除C57小鼠β细胞RORα基因，阐明RORα对β细胞功能的影响；（2）通过转基因rAAV8-RORα增加db/db T2DM小鼠β细胞RORα水平，探究RORα对β细胞功能改善及T2DM治疗效果；（3）在原代胰岛细胞及MIN6细胞中，高表达或敲除RORα基因，研究RORα对氧化应激的调控。该项目将阐明RORα对胰岛β细胞的影响及作用机制，认识其在T2DM发生中的新功能，为T2DM预防和治疗提供新靶点。

虽然胰岛素抵抗是2型糖尿病（T2DM）重要的病理生理特征，但胰岛β细胞功能障碍是导致T2DM发展的核心因素。由高血糖引起的氧化应激是导致胰岛β细胞功能缺损的重要原因。因此，如果能减弱或消除β细胞的氧化应激，则可保护胰岛β细胞的正常生理功能，将可能成为治疗T2DM的有效手段。该项目应用C57BL/6小鼠、db/db糖尿病小鼠和MIN6胰岛β细胞系，通过在胰岛β细胞特异性高表达或敲除维甲酸相关孤核受体α（RORα）基因等策略，研究了RORa对胰岛β细胞功能的影响及作用机制。发现Rora基因敲除小鼠血糖水平（包括空腹和自由进食状态）显著升高，β细胞分泌胰岛素的能力降低，但外周胰岛素敏感性不受影响。Rora基因敲除小鼠的胰岛面积和的密度显著降低，胰岛β增殖能力显著减弱，但不影响β细胞的凋亡。此外，RORa缺失导致胰岛β细胞在葡萄糖刺激下胰岛素分泌（GSIS）能力损伤，其可能与线粒体损伤、葡萄糖转运蛋白2（GLUT2）的表达以及β细胞对葡糖糖的吸收降低相关。5-胆甾烯-3β-醇硫酸酯盐（CS）是公认的RORa激动剂。我们发现CS能改善链脲佐菌素（STZ）诱导的糖尿病小鼠的糖尿病表型，即改善糖尿病小鼠多饮、多食、体重减轻以及缓解小鼠高血糖和高血脂表型。在分离的胰岛和β 细胞系中，发现CS可保护糖毒性和/或脂毒性对β细胞的损伤，提高β细胞的存活率、抑制细胞凋亡、并改善胰岛素分泌功能。该项目明确了RORα对胰岛β细胞功能的影响，并解决了其作用的分子机制。在此基础上，发现激动RORa可提高β细胞在应激条件下的存活率，部分恢复β细胞的胰岛素分泌功能，显著缓解糖尿病表型。