PPARgamma Deacetylation in the Restoration of Metabolic Homeostasis

这项申请中提出的研究旨在利用肥胖症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者，也在调节脂肪代谢、葡萄糖稳态、炎症和其他反应中发挥关键作用。它是胰岛素增敏的一个关键靶点。PI发现，PPARγ在赖氨酸K268和K293上的脱乙酰化促进了储能的白色脂肪细胞向耗能的棕色样脂肪细胞的转变，这一过程被称为“褐化”或“褐变”。为了确定PPARγ脱乙酰化的生理意义，用PI构建了模拟脱乙酰化的PPARγK268R/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，并免受肥胖，特别是内脏肥胖的保护。引人注目的是，当2KR小鼠接受噻唑烷二酮(TZD)治疗时，它们表现出强大的胰岛素敏化反应，但没有出现限制TZD临床应用的心脏和骨骼丢失效应。在这项申请中，他建议确定PPARγ脱乙酰化、增加褐化和防止TZD导致的骨丢失这两个最引人注目的作用背后的机制。在目标1中，他将通过体外脂肪细胞分化研究来确定PPARγ去乙酰化是否影响脂肪细胞以脂肪细胞自主方式发光。接下来，他将通过使用脂肪细胞条件性敲入模型来剖析2KR的脂肪特有功能。在目标2中，以典型的白色脂肪细胞基因Adipsin为靶点，他将使用芯片、报告和启动子下拉试验来寻找介导2KR效应的乙酰化反应共抑制因子。为了证明这种方法的可行性，PI表明他们鉴定了转录因子Osr1是一种乙酰化依赖的PPARγ辅因子。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。在目标3中，PI假设2KR突变体通过抑制Adipsin来调节骨转换。他将首先通过骨髓间充质祖细胞和Adipsin基因敲除小鼠的体外分化来确定Adipsin在成骨和骨重建中的作用。接下来，他将通过在2KR小鼠身上进行的Adipsin获得或功能丧失研究，确定抑制Adipsin是否是2KR突变体防止TZD导致的骨丢失的机制。这些研究将通过阐明PPARγ通过脱乙酰基的选择性调节来推动这一领域的发展，并为分离PPARγ激活的胰岛素增敏功能与其不良影响提供机制上的见解。

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.