Establishment of the therapeutic strategy for the cardiovascular complications in Metabolic syndrome by gene trap method

前脂肪细胞向成熟脂肪细胞的分化与脂肪细胞特异性基因产物的表达密切相关，这些基因产物与胰岛素抵抗密切相关。因此，识别分化为成熟脂肪细胞所需的基因是至关重要的。我们使用逆转录病毒插入介导的随机突变产生了3T3-L1细胞系，这些细胞系失去了分化为成熟脂肪细胞的能力。利用这种方法，我们发现色氨酸羟基酶-1(TPH1)是脂肪细胞分化所必需的，它是5-羟色胺生物合成的限速酶。虽然在缺乏正常TPH1基因表达的突变型3T3L-1细胞系中，分化明显减少，但该细胞系中TPH1基因的重组恢复了其分化潜力。5-羟色胺可促进3T3-L1细胞和成年小鼠间质血管部分细胞的分化。另一方面，5-羟色胺SY…更多的合成抑制剂对氯苯丙氨酸和5-羟色氨酸受体2A型拮抗剂抑制分化。与野生型小鼠相比，来自TPH-/-小鼠的前脂肪细胞的分化明显受损。我们观察到，在基础状态下，TPH-/-和野生型小鼠之间的脂肪组织学没有差异。然而，在高脂饮食诱导肥胖后，TPH1-/-小鼠的体重和肠系膜脂肪重量的增加比野生型小鼠更显著。此外，与野生型小鼠相比，TPH-/-小鼠的葡萄糖耐量受损更明显。有趣的是，在肥胖组中，我们在Tph-/-小鼠中观察到极大的(140μm~)脂肪细胞，而在野生型小鼠中，我们观察到通常肥大的(~80μm)脂肪细胞。我们还在野生型小鼠中观察到了增殖细胞核抗原阳性的脂肪细胞，但在TPH-/-小鼠中没有观察到，这表明TPH-/-小鼠脂肪细胞的产生受到了损害。因此，脂肪组织中的5-羟色胺作为一种自分泌因子参与了脂肪细胞的分化。这些结果还表明，5-羟色胺对于高脂饮食下脂肪细胞的生理性肥大以及成年小鼠正常糖耐量的维持是必需的。我们还发现，前脂肪细胞的分化需要甘油三酯相互作用因子和前脑啡肽原-1。我们现在正试图通过靶向这些分子来建立针对肥胖和代谢综合征的治疗策略。在低氧和代谢应激条件下，心肌细胞通过利用糖酵解途径作为主要能量来源来适应自己。为了探讨这种情况下的信号转导机制，我们建立了一种用流式细胞仪检测谷氨酸氨基转移酶4易位的方法，发现心肌细胞中CMKK1和LKB1AMPK都激活了β。较少

The differentiation of preadipocytes into mature adipocytes is closely associated with the expression of adipocyte-specific gene products that are deeply involved in insulin resistance. Therefore, it is critical that we identify the genes required for differentiation into mature adipocytes. We used retrovirus insertion-mediated random mutagenesis to generate 3T3-L1 cell lines which lose their ability to differentiate into mature adipocytes. Using this approach, we discovered that tryptophan hydroxylase-1 (TPH1), a rate-limiting enzyme for the biosynthesis of serotonin, was required for adipocyte differentiation. While differentiation was clearly reduced in a mutant 3T3L-1 cell line that lacked expression of the normal TPH1 gene, reconstitution of the TPH1 gene in this cell line restored its differentiation potential. The administration of serotonin enhanced the differentiation in 3T3-L1 cells and in cells of stromal vascular fraction from adult mice. On the other hand, the serotonin sy … More nthesis inhibitor p-chlorophenylalanine and 5-hydroxy tryptophan receptor type 2A antagonists inhibited differentiation. Differentiation was significantly impaired in preadipocytes prepared from TPH-/- mice compared to those from wild-type mice. We observed no differences in the histology of adipose tissue between TPH-/- and wild-type mice at the basal state. However, after obesity was induced by a high-fat diet, increases in body weight and mesenteric fat weight were more prominent in TPH1-/- mice than in wild-type mice. Furthermore, glucose tolerance was more prominently impaired in TPH-/- mice than in wild-type mice. Interestingly, in obese groups, we observed extremely large (140μm〜) adipocytes in TPH-/- mice, in contrast to the usually hypertrophied (〜80μm) adipocytes in wild-type mice. We also observed PCNA-positive adipocytes in wild-type mice but not in TPH-/- mice, which suggests that adipocyte production is impaired in TPH-/- mice. Thus, serotonin in adipose tissue acts as an autocrine factor and is involved in adipocyte differentiation. The results also suggest that serotonin is required for the physiological hypertrophy of adipocytes in response to a high-fat diet, and for the preservation of normal glucose tolerance in adult mice.We also found TG-interacting factor and preproenkephalin-1 are required for the differentiation of preadiopcytes. We are now trying to establish therapeutic strategies against obesity and metabolic syndrome by targeting these molecules.In hypoxia and metabolic stress condition, cardiomyocytes adapt themselves by utilizing glycolytic pathway for their major energy source. In order to find the signal transduction mechanisms involved in such conditions, we have established a method to measure GLUT4 translocation by flow cytometry and found that both CMKKβ and LKB1 activate AMPK in cardiomyocytes. Less