PPARgamma Deacetylation in the Restoration of Metabolic Homeostasis

本申请中提出的研究旨在利用肥胖和糖尿病的基本发现来开发治疗这些疾病的新方法。PPARγ是脂肪细胞生物学的主要调节因子，在调节脂质代谢、葡萄糖稳态、炎症和其他反应中也起着至关重要的作用。它是胰岛素致敏的关键靶点。PI发现赖氨酸K268和K293上的PPARγ去乙酰化促进了储存能量的白色脂肪细胞向消耗能量的棕色脂肪细胞的转化，这一过程被称为“褐变”或“北京”。为了确定PPARγ去乙酰化的生理意义，PI产生了模拟去乙酰化的PPARγ K268R/K293R突变敲入小鼠（2KR）。据初步数据报道，2KR小鼠的能量消耗增加，免受肥胖，特别是内脏肥胖的影响。引人注目的是，当2KR小鼠接受噻唑烷二酮（TZDs）治疗时，它们表现出强烈的胰岛素增敏反应，但没有出现限制TZD临床应用的心脏和骨质流失效应。在这个应用中，他提出定义PPARγ去乙酰化，增加北京和预防tzd诱导的骨质流失的两个最引人注目的作用的机制。在Aim 1中，他将通过体外脂肪细胞分化研究确定PPARγ去乙酰化是否以脂肪细胞细胞自主的方式影响脂肪细胞。接下来，他将通过采用脂肪细胞条件敲入模型来剖析2KR的脂肪特异性功能。在Aim 2中，他将以典型的白色脂肪细胞基因Adipsin为靶标，采用ChIP、报告子和启动子下拉试验来鉴定介导2KR作用的乙酰化反应性协同抑制因子。为了证明该方法的可行性，PI表明他们将转录因子Osr1鉴定为乙酰化依赖性PPARγ辅助因子。他将测试2KR突变体是否通过阻止osr1介导的Adipsin表达来抑制白色脂肪程序。在Aim 3中，PI假设2KR突变体通过抑制Adipsin来调节骨转换。他将首先利用骨髓间充质祖细胞的离体分化和Adipsin敲除小鼠，确定Adipsin在成骨和骨重塑中的作用。接下来，他将在2KR小鼠中使用Adipsin获得或丧失功能的研究来确定Adipsin的抑制是否是保护2KR突变体免受tzd诱导的骨质流失的机制。这些研究将通过阐明PPARγ通过去乙酰化的选择性调控，并提供PPARγ激活的胰岛素增敏功能与其副作用分离的机制见解，从而推动该领域的发展。

The research proposed in this application aims to leverage basic discoveries in obesity and diabetes to develop novel approaches for the therapy of these conditions. PPARγ is the master regulator of adipocyte biology and also plays crucial roles in regulating lipid metabolism, glucose homeostasis, inflammation, and other responses. It represents a key target for insulin sensitization. The PI has discovered that deacetylation of PPARγ on lysine K268 and K293 promotes the transformation of energy-storing white adipocytes into energy-dissipating brown-like adipocytes, a process called “browning” or “beiging”. To establish the physiological significance of PPARγ deacetylation, the PI generated deacetylation-mimetic PPARγ K268R/K293R mutant knock-in mice (2KR). As reported in the preliminary data, 2KR mice have increased energy expenditure and are protected from obesity, particularly visceral obesity. Strikingly, when 2KR mice are treated with thiazolidinediones (TZDs), they show a robust insulin-sensitizing response but fail to develop the cardiac and bone loss effects that have limited the clinical utilization of TZD. In this application, he proposes to define the mechanisms underlying the two most compelling effects of PPARγ deacetylation, increased beiging, and prevention of TZD-induced bone loss. In Aim 1, he will determine whether PPARγ deacetylation affects adipocyte beiging in an adipocyte cell-autonomous fashion by ex vivo adipocyte differentiation studies. Next, he will dissect the fat-specific functions of 2KR by employing an adipocyte conditional knock-in model. In Aim 2, using the prototypical white adipocyte gene Adipsin as a target, he will employ ChIP, reporter, and promoter pull-down assays to identify acetylation-responsive co-repressor that mediate the effects of 2KR. To demonstrate the feasibility of the approach, the PI shows that they identified transcription factor Osr1 as an acetylation-dependent PPARγ cofactor. He will test whether the 2KR mutant represses the white adipose program by preventing Osr1-mediated Adipsin expression. In Aim 3, the PI hypothesizes that the 2KR mutant regulates bone turnover by repressing Adipsin. He will first establish the role of Adipsin in osteogenesis and bone remodeling using ex vivo differentiation of bone marrow mesenchymal progenitor cells and Adipsin knockout mice. Next, he will determine whether repression of Adipsin is the mechanism by which the 2KR mutant is protected against TZD-induced bone loss using Adipsin gain- or loss-of-function studies in 2KR mice. The proposed studies will advance the field by elucidating the selective regulation of PPARγ through deacetylation and provide mechanistic insight to dissociate the insulin-sensitizing function of PPARγ activation from its adverse effects.