Transcription & Function of Adipocyte Beta3AR in Obesity

β3肾上腺素能受体是一种G蛋白偶联受体，主要在脂肪组织中表达，介导去甲肾上腺素刺激白色脂肪组织(WAT)的脂肪分解和棕色脂肪组织(BAT)的产热作用。Beta3AR的选择性激动剂可以预防或逆转犬和啮齿动物模型中的肥胖和胰岛素抵抗，目前正在为人类开发药物。除了对脂解的有效刺激外，β3AR激动剂还能促进典型白色脂肪储存库中具有产热活性的棕色脂肪细胞的出现，但这种作用的机制尚不清楚。在我们之前的资助期间完成的目标确立了脂肪细胞中的Beta3AR与GalpHas和Galphai的结构性偶联，分别导致蛋白激酶A(PKA)和MAPK(ERK1/2)途径的激活，并且脂肪细胞特有的Beta3AR的表达在很大程度上受C/EBPalpha控制，我们定义了C/EBP位点。在探讨β-3AR对脂肪细胞中PKA和MAPK通路的调节中，我们最近发现，β-AR以一种PKA依赖的方式激活脂肪细胞中的p38MAPK激酶，而这种调节的重要生理终点之一是诱导棕色脂肪中的主要生热蛋白UCP1。提出了两个主要假设。从PKA到MKK3/6和p38的信号级联涉及rac1/CDC42和混合谱系激酶(MLKs)，p38MAPK通路需要与PKA一起适当地激活和招募转录因子到UCP1启动子，最终终点是产热增加。有三个目标被提出：(1)表征连接脂肪细胞中的BetaARs和p38 MAP激酶的信号转导途径；检验PKA激活rac1/CDC42和MLKs进而直接激活p38MAPK的直接上游激酶MKK3/6的假设；(2)检验p38 MAPK活性的Beta3AR刺激调节脂肪细胞中PPAR依赖的转录过程的假说。我们将测试PPARGamma和/或PGC-1的磷酸化是否是UCP1基因反式激活所必需的步骤，并检验如下假说：(1)通过直接和间接需要PKA来调节CREB和PPARGamma的双重信号级联反应触发UCP1转录；(3)测试孤儿受体RORGamma是脂肪细胞中Beta3AR基因组织特异性表达的第二个关键转录激活子的假设。这一目的的实验还将检验C/EBP蛋白谱的改变有助于肥胖/糖尿病相关性脂肪细胞β3AR和β1AR基因表达下调的假设。

The beta3-adrenergic receptor (beta3AR) is a G protein- coupled receptor (GPCR) expressed predominantly in adipose tissue, where it mediates the effects of noradrenaline-stimulated lipolysis in white adipose tissue (WAT) and thermogenesis in brown adipose tissue (BAT). Selective agonists for the beta3AR can prevent or reverse obesity and insulin resistance in canine and rodent models, and are under pharmaceutical development for humans. In addition to potent stimulation of lipolysis, beta3AR agonists promote the appearance of thermogenically active brown adipocytes in typical white adipose depots but the mechanism responsible for this effect is not understood. Completion of the Aims in our previous funding period established that the beta3AR in adipocytes is constitutively coupled to both Galphas and Galphai, leading to the activation of the protein kinase A (PKA) and MAP kinase (ERK1/2) pathways, respectively, and that the adipocyte-specific expression of beta3AR is controlled in large part by C/EBPalpha and we defined the C/EBP site. In probing the regulation of the PKA and MAPK pathways in adipocytes by beta3AR, we have recently discovered that betaARs activate p38 MAPK kinase in adipocytes in a PKA-dependent manner, and that one of the important physiological endpoints of this regulation is the induction of the main thermogenic protein in brown fat, UCP1. Two main hypotheses are proposed. That the signaling cascade from PKA to MKK3/6 and p38 involves Rac1/cdc42 and the mixed- lineage kinases (MLKs), and that the p38 MAPK pathway is required in tandem with PKA to appropriately activate and recruit transcription factors to the UCP1-promoter, with the ultimate end point being increased thermogenesis. There are three Aims proposed: (1) to characterize the signal transduction pathway linking the betaARs in adipocytes to the p38 MAP kinase; to test the hypothesis that Rac1/Cdc42 and the MLKs are activated by PKA and, in turn, directly activate MKK3/6, the immediate upstream kinase for p38 MAPK; (2) to test the hypothesis that beta3AR- stimulation of p38 MAP kinase activity regulates PPAR-dependent transcriptional processes in adipocytes. We will test whether phosphorylation of PPARgamma and/or PGC-1 are required steps in the transactivation of the UCP1 gene, and test the hypothesis that a dual signaling cascade requiring PKA both directly and indirectly to modulate CREB and PPARgamma triggers UCP1 transcription; (3) to test the hypothesis that the orphan receptor RORgamma is a second critical transcriptional activator for the tissue-specific expression of the beta3AR gene in adipocytes. Experiments in this aim will also test the hypothesis that alterations in the profile of C/EBP proteins contribute to the obesity/diabetes dependent down-regulation of expression of the beta3AR and beta1AR genes in adipocytes.