Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes

AbstractTo explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function

FEM熱応力解析による鋳物の冷却過程における変形予測のための生型の構成方程式の実験的構築とバリデーション

早稲田大学博士(工学)早大学位記番号:新9530doctoral thesi

Lentiviral and Moloney retroviral expression of green fluorescent protein in somatotrophs in vivo.

Previous studies have shown that the locus control region (LCR) and the promoter of the growth hormone (GH) gene can control the expression of GH. Therefore, lenti- and retro-viral vectors with these elements might be useful to monitor the activation of the GH gene and the development of newborn somatotrophs. To test this, we first constructed a lentiviral vector, which expresses green fluorescent protein (GFP) under the control of these elements, and injected them into rat pituitaries in situ and in vivo. The lentiviral vector expressed GFP specifically in the anterior lobe, and nearly all GFP-positive cells were anti-GH immunoreactive. The GFP expression was upregulated by the administration of growth hormone releasing hormone and an IGF-1 receptor blocker. Furthermore, the social isolation stress, which was shown to decrease the GH secretion, decreased the GFP expression. Second, we injected the retroviral vector into neonatal rat pituitaries in vivo. At 30 days postinjection (DPI), almost all GFP-positive cells were anti-GH positive and anti-prolactin negative as the lentiviral expression. However, GFP was transiently expressed by developing lactotrophs at 8 and 16 DPI, suggesting that our vector lacks an element(s) which suppresses the expression. Meanwhile, the retrovirally labeled cells tended to cluster with the cells of same type. An analysis of cell numbers in each cluster revealed some features of cell proliferation. These viral vectors are shown to be useful tools to monitor the activation of the GH gene and the development of somatotrophs

Anti-GH and anti-prolactin double immunostaining.

<p>Pituitary slices were prepared from Mv-GHp-nuGFP-injected rats at 16 DPI, and immunostained with anti-GH (red) and anti-prolactin (blue) antibodies. nuGFP-expressing cells were classified as anti-GH positive (A), prolactin positive (B), or double negative (C). None of the cells were GFP, GH, and prolactin triple positive. Bar, 20 µm. (D) Percentages of GH-positive, prolactin-positive, and double-negative cells at 16 and 30 DPI.</p

In vivo lentiviral expression of GFP.

<p>(A) Typical confocal images of the pituitaries injected with Lv-GHp-nuGFP from four rats. We fixed the pituitaries at 8 DPI and sliced them sagittally. Bar, 0.5 mm. (B–D) Somatotroph-specific expression of GFP in Lv-GHp-nuGFP-injected rat pituitary. The pituitary slices, prepared at 8 DPI, were immunostained with anti-GH (B), anti-prolactin (C), and anti-ACTH (D) antibodies. The immunoreactions were visualized with Alexa-569 (red fluorescence). Note the green fluorescence of GFP in the nuclei and the surrounding red anti-GH immunoreactivity. Most GFP-positive cells were GH positive at 8 DPI. Only a few GFP-expressing cells were GH negative (arrow in B). In contrast, most GFP-positive cells were prolactin and ACTH negative, but only a few were prolactin and ACTH positive (arrows in C and D). Bar, 20 µm. (E) Percentages of GH-, prolactin-, and ACTH-positive cells at 8 and 16 DPI. We analyzed 100 GFP-expressing cells from 2 rats. (F) Lower GFP expression in the GH-negative and prolactin-positive cells. The expression of GFP was analyzed with ImageJ software and was indicated as the arbitrary unit (au) of the software. The expressions of GFP were lower in GH-negative and prolactin-positive cells (n = 10, p<0.00001).</p

Schematic illustration of lenti- and retro-viral vectors.

<p>A. Structure of the Lv-GHp-nuGFP. B. Structure of Mv-GHp-nuGFP and Mv-GHp-GFP. The upstream CMV promoter was used to transcribe the viral genomic RNAs. CMV, cytomegalovirus promoter; LTR, long terminal repeat; <i>ψ</i>, RNA packaging signal; RRE, Rev responsive element; cPPT, central polypurine tract: LCR, locus control region; GHp, promoter of GH gene; GFP, green fluoresent protein gene. NLS, nuclear localization signal; WRRE, woodchuck hepatitis virus posttranscriptional regulatory element; ∇, deletion in the U3 region of the 3′ long terminal repeat.</p

Cluster analysis of GFP-expressing cells.

<p>(A) Homogenous clustering of GH-positive and GH-negative cells. The open columns indicate the theoretical values according to the random clustering model of heterogeneous cells, and the closed columns indicate the results for GH at 30 DPI. Note that the numbers of all-GH-positive and all-GH-negative clusters were higher than those in the random model, and the number of mixed clusters was smaller than that in the random model. (B) Homogenous clustering of prolactin-positive and prolactin-negative cells at 16 DPI. Again, the numbers of all-prolactin-positive and all-prolactin-negative clusters were larger than those in the random model, and that of the mixed clusters was smaller than that in the random model. (C) Increase in cell number in each cluster. The cell number per cluster was significantly increased from 8 to 16 DPI, but the increase from 16 to 30 DPI was not significant (n = 18, 19, and 17, respectively; *, p<0.0000001, ANOVA). (D) A small cell number in the GH-positive clusters. The number of cells in each of the all-GH-positive clusters was significantly smaller than that of the all-negative clusters (n = 13 and 4, respectively; p<0.05).</p