Apoptosis pathways underlying adipogenesis

描述(由申请人提供)： 肥胖症作为一个重大的公共卫生问题和主要的可预防的死亡原因继续升级。遗传、环境、行为和社会经济因素会导致超重和肥胖。脂肪组织中前脂肪细胞向成熟脂肪细胞的增殖和分化(脂肪生成)增加是肥胖的核心。以往的研究主要集中在维持脂肪细胞表型的转录调控上。然而，促进原始间充质前体细胞决定脂肪表型的潜在分子机制仍不清楚。我们的长期目标是阐明脂肪形成的细胞和分子机制。为了实现这一目标，我们的目标是确定凋亡基因的选择性剪接如何控制前脂肪细胞的分化和成熟。已有文献表明，前脂肪细胞会发生凋亡，而成熟脂肪细胞不会这样做。根据初步数据，我们的中心假设是，通过选择性剪接控制终末脂肪细胞分化，参与凋亡的基因表达从促凋亡途径明显转变为促生存途径，最终导致肥胖的脂肪数量和质量增加。这项研究的意义在于，一旦我们建立了导致脂肪细胞存活增加的脂肪形成的分子机制，我们就可以操纵细胞凋亡途径的靶点，设计出新的创新方法来预防或逆转体重增加和肥胖。我们提出了三个具体目标。在目标1中，我们将阐明终末脂肪细胞分化是否伴随着凋亡基因的选择性表达。与成熟脂肪细胞不同，前脂肪细胞会发生凋亡。我们的数据表明，在脂肪细胞分化的第4-6天，Bcl2和Bclx，caspase9和PKC4的交替剪接变异体的表达改变为有利于生存的变异体。通过过表达和基因敲除实验，我们将阐明这些基因、脂肪生成和细胞凋亡之间的联系。目的2旨在确定信号激酶PKC4II作为抗成脂介质的作用。PKC4II，一种丝氨酸/苏氨酸蛋白激酶，促进细胞存活。白藜芦醇和姜黄素等天然化合物具有抗脂肪生成和促进脂肪细胞凋亡的活性。我们的数据显示，它们抑制了PKC4II的表达。我们的数据进一步表明，PKC4II是Bcl2介导的生存途径上游的信号激酶。我们建议确定细胞内的靶点，它是脂肪细胞中促进生存的途径的中心，可能被调节以诱导抗成脂作用。在目标3中，将评估这一假说的生理学相关性，确定这些凋亡基因在饮食诱导的肥胖小鼠模型(C57BL/6)中的表达水平，这些肥胖小鼠喂食高脂饮食并补充天然化合物或PKC4II特异性抑制剂。通过显示凋亡基因的选择性剪接调节前脂肪细胞的分化和成熟，拟议的研究将建立一个新的脂肪生成范式，并揭示肥胖治疗的新靶点。

DESCRIPTION (provided by applicant): Obesity continues to escalate as a significant public health problem and as the leading preventable cause of death. Genetic, environmental, behavioral, and socioeconomic factors cause excess weight gain and obesity. Increased proliferation and differentiation of pre-adipocytes to mature adipocytes (adipogenesis) within the fat tissues are central to obesity. Previous studies focused extensively on the transcriptional controls which maintain the adipocyte phenotype. However, the underlying molecular mechanisms that promote determination of primitive mesenchymal precursor cells to the adipose phenotype are still unknown. Our long-term goal is to elucidate the cellular and molecular mechanisms underlying adipogenesis. In pursuit of this goal, our objective is to determine how alternative splicing of apoptotic genes govern the differentiation and maturation of pre-adipocytes. It is well documented that pre-adipocytes undergo apoptosis while mature adipocytes fail to do so. Based on preliminary data, our central hypothesis is that there is a distinct shift in the expression of genes involved in apoptosis from a pro-apoptotic to a pro-survival pathway via alternative splicing controlling terminal adipocyte differentiation which ultimately increases the adipose number and mass leading to obesity. The significance of the proposed research is that once we establish the molecular mechanisms of adipogenesis which result in increased survival of adipocytes, we can manipulate targets of the apoptosis pathway to devise new and innovative approaches to prevent or reverse weight gain and obesity. We propose three specific aims. In Aim 1, we will elucidate whether terminal adipocyte differentiation is accompanied by alternative expression of apoptosis genes. Pre-adipocytes undergo apoptosis unlike mature adipocytes. Our data demonstrate that during adipocyte differentiation between days 4-6, expression of Bcl2 and Bcl-x, caspase 9 and PKC4 alternatively spliced variants change to its pro-survival variants. Using overexpression and knockdown experiments, we will elucidate the link between these genes, adipogenesis and apoptosis. Aim 2 is designed to determine the role of the signaling kinase PKC4II as an anti-adipogenic mediator. PKC4II, a serine/threonine protein kinase promotes cell survival. Natural compounds such as resveratrol and curcumin exhibit anti-adipogenic activities and promote apoptosis in adipocytes. Our data shows that they inhibit PKC4II expression. Our data further points to PKC4II as the signaling kinase upstream of Bcl2-mediated survival pathway. We propose to identify the intracellular target which is central to pro-survival pathways in adipocytes that may be modulated to induce anti-adipogenic effects. In Aim 3 the physiological relevance of this hypothesis will be assessed determining the expression levels of these apoptosis genes in diet-induced obese mouse model (C57BL/6) fed a high-fat diet complemented with the natural compounds or a PKC4II-specific inhibitor. By showing that alternative splicing of apoptotic genes modulate differentiation and maturation of pre-adipocytes, the proposed studies will establish a new paradigm for adipogenesis and reveal new targets for treatment of obesity.