脂肪细胞分化过程中影响PAI-1基因转录表达的Dex和C/EBPs顺式调控元件的分析(英)

在研究胰岛素(Ins)、地塞米松(Dex)和甲基异丁基黄嘌呤(Mix)对脂肪细胞分化过程中PAI-1基因表达的影响基础上,为进一步探讨Ins、Dex调控PAI-1基因转录表达的调控机制,应用DNA重组技术,构建含萤光素酶（luciferase）报告基因和PAI-1启动子不同长度片段的嵌合质粒,转染3T3-L1前脂肪细胞并测定报告基因荧光素酶的活性.结果表明,小鼠PAI-1基因起动子－690至－850碱基序列之间有一个Dex的正调控元件.用计算机软件进行分析发现：Dex顺式元件位于PAI-1启动子的－750至－770碱基序列.其组成为：5′ GGTAACCTCTGTTCTCAT 3′.同时还发现在PAI-1启动子的－720至－740碱基序列中,存在一个C/EBPs的结合元件5′CCAAT3′并用凝胶电泳迁移实验对这些元件进行了鉴定.表明Dex正是通过激活转录因子（糖皮质激素受体,GR）和C/EBPα一起与各自的顺式元件结合来促进PAI-1基因的表达.     

The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc.

The visceral musculature of the Drosophila midgut consists of an inner layer of circular and an outer layer of longitudinal muscles. Here, we show that the circular muscles are organised as binucleate syncytia that persist through metamorphosis. At stage 11, prior to the onset of the fusion processes, we detected two classes of myoblasts within the visceral trunk mesoderm. One class expresses the founder-cell marker rP298-LacZ in a one- to two-cells-wide strip along the ventralmost part of the visceral mesoderm, whereas the adjacent two to three cell rows are characterised by the expression of Sticks-and-stones (SNS). During the process of cell fusion at stage 12 SNS expression decreases within the newly formed syncytia that spread out dorsally over the midgut. At both margins of the visceral band several cells remain unfused and continue to express SNS. Additional rP298-LacZ-expressing cells arise from the posterior tip of the mesoderm, migrate anteriorly and eventually fuse with the remaining SNS-expressing cells, generating the longitudinal muscles. Thus, although previous studies proposed a separate primordium for the longitudinal musculature located at the posteriormost part of the mesoderm anlage, our cell lineage analyses as well as our morphological observations reveal that a second population of cells originates from the trunk mesoderm. Mutations of genes that are involved in somatic myoblast fusion, such as sns, dumbfounded (duf) or myoblast city (mbc), also cause severe defects within the visceral musculature. The circular muscles are highly unorganised while the longitudinal muscles are almost absent. Thus the fusion process seems to be essential for a proper visceral myogenesis. Our results provide strong evidence that the founder-cell hypothesis also applies to visceral myogenesis, employing the same genetic components as are used in the somatic myoblast fusion processes.     

Continuous Changes in Triacylglycerol Molecular Species Composition by Fatty Acids in Porcine Adipocytes

It has been demonstrated that the composition of molecular species of adipose tissue triacylglycerols (TGs) from farm animals are not equally synthesized and that some molecular species are preferentially synthesized. The objective of the present study was to determine whether exogenous fatty acids (FAs) would affect the TG composition. To this end, the composition of TG molecular species stored in porcine adipocytes differentiated with several long-chain FAs was analyzed by gas chromatography. The addition of each FA for 6 d increased TG molecular species having two or three added FAs. However, the molecular species compositions at 15 d after the addition of each FA resembled those of cells with no added FAs. Moreover, some common molecular species in all experimental cells increased, as well as cells with no added FAs. It was concluded that the addition of FAs increases the contents of specific molecular species, but does not affect the synthetic processes of individual TG molecular species.     

Flow cytometric analysis of porcine preadipocytes.

In this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal-vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD-1 cells surface antigen was greater in cultures supplemented with 2% pig serum and 10 nM dexamethasone than in other media. In cultures, GH, TNF-alpha and TGF-beta each inhibited lipid deposition, whereas TNF-alpha and TGF-beta, but not GH, inhibited AD-1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF-alpha and TGF-beta was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to the study of adipocyte development.     

Glutamine Links Obesity to Inflammation in Human White Adipose Tissue

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Structural Basis for Glycerol Efflux and Selectivity of Human Aquaporin 7

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Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice.Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.     

Vitamin D limits inflammation-linked microRNA expression in adipocytes in vitro and in vivo: A new mechanism for the regulation of inflammation by vitamin D

Inflammation of adipose tissue is believed to be a contributing factor to many chronic diseases associated with obesity. Vitamin D (VD) is now known to limit this metabolic inflammation by decreasing inflammatory marker expression and leukocyte infiltration in adipose tissue. In this study, we investigated the impact of VD on microRNA (miR) expression in inflammatory conditions in human and mouse adipocytes, using high-throughput methodology (miRNA PCR arrays). Firstly, we identified three miRs (miR-146a, miR-150, and miR-155) positively regulated by TNFα in human adipocytes. Interestingly, the expression of these miRs was strongly prevented by 1,25(OH)₂D preincubation. These results were partly confirmed in 3T3-L1 adipocytes (for miR-146a and miR-150). The ability of VD to control the expression of these miRs was confirmed in diet-induced obese mice: the levels of the three miRs were increased following high fat (HF) diet in epididymal white adipose tissue and reduced in HF diet fed mice supplemented with VD. The involvement of NF-κB signaling in the induction of these miRs was confirmed in vitro and in vivo using aP2-p65 transgenic mice. Finally, the ability of VD to deactivate NF-κB signaling, via p65 and IκB phosphorylation inhibition in murine adipocyte, was observed and could constitute a driving molecular mechanism. This study demonstrated for the first time that VD modulates the expression of miRs in adipocytes in vitro and in adipose tissue in vivo through its impact on NF-κB signaling pathway, which could represent a new mechanism of regulation of inflammation by VD.