| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
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
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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
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