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，而脂肪细胞特异性基因产物与胰岛素抵抗密切相关。因此，鉴定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诱变产生了3T3-L1细胞系，这些细胞系失去了向成熟脂肪细胞分化的能力。使用这种方法，我们发现色氨酸羟化酶-1 (TPH1)，一种限制血清素生物合成的酶，是脂肪细胞分化所必需的。虽然在缺乏正常TPH1基因表达的突变3T3L-1细胞系中分化明显减少，但在该细胞系中重建TPH1基因恢复了其分化潜力。血清素可促进成年小鼠3T3-L1细胞和间质血管部分细胞的分化。另一方面，5-羟色胺抑制剂对氯苯丙氨酸和5-羟基色氨酸受体2A型拮抗剂抑制分化。与野生型小鼠相比，TPH-/-小鼠制备的前脂肪细胞分化明显受损。在基础状态下，我们观察到TPH-/-和野生型小鼠的脂肪组织组织学没有差异。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为明显。此外，与野生型小鼠相比，TPH-/-小鼠的葡萄糖耐量受损更为明显。有趣的是，在肥胖组中，我们观察到TPH-/-小鼠的脂肪细胞非常大（140μm ~），而野生型小鼠的脂肪细胞通常肥大（~ 80μm）。我们还在野生型小鼠中观察到pcna阳性的脂肪细胞，而在TPH-/-小鼠中没有，这表明TPH-/-小鼠的脂肪细胞生成受损。因此，脂肪组织中的血清素作为一种自分泌因子，参与脂肪细胞分化。研究结果还表明，血清素是高脂肪饮食对脂肪细胞生理肥大的反应所必需的，也是维持成年小鼠正常葡萄糖耐量所必需的。我们还发现tg相互作用因子和前脑啡肽-1是前脂肪细胞分化所必需的。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。在缺氧和代谢应激条件下，心肌细胞以糖酵解途径作为主要能量来源进行自我适应。为了寻找这种情况下参与的信号转导机制，我们建立了一种流式细胞术测量GLUT4易位的方法，发现CMKKβ和LKB1都能激活心肌细胞中的AMPK。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