A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor.</p><p><b>METHODS</b>Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide.</p><p><b>RESULTS</b>Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation.</p><p><b>CONCLUSION</b>RBP could effectively rescue the promoted differentiation of resistin overexpressed 3T3-L1 preadipocyte.</p>

Expression of TSG-6 gene during 3T3-L1 preadipocyte differentiation and regulative role of tumor necrosis factor-alpha / 中华儿科杂志

<p><b>OBJECTIVE</b>Tumor necrosis factor alpha-stimulated gene-6 (TSG-6 gene) differentially expressed in adipose tissue of obese and normal human subjects or rats. To explore the relationship between the differential expression of TSG-6 and adipocyte differentiation, adipogenesis and obesity, the present study aimed to investigate the changes of TSG-6 gene expression during 3T3-L1 preadipocyte differentiation and to analyze the regulative role of TNF-alpha on TSG-6 gene expression in matured 3T3-L1 adipocytes.</p><p><b>METHODS</b>3T3-L1 preadipocytes were cultured in vitro and differentiated into the matured adipocytes. TNF-alpha in different concentrations (0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml) was added into the culture medium of fully differentiated adipocytes (day 10) for various times (0.5 h, 2 h, 6 h, 12 h, 24 h). Total RNA from these adipocytes was extracted and the levels of TSG-6 gene mRNA expression were evaluated by RT-PCR.</p><p><b>RESULTS</b>(1) In preadipocytes, the level of TSG-6 gene mRNA expression remained low. In the presence of dexamethasone (Dex), MIX and insulin, with the 3T3-L1 preadipocytes being differentiated into the matured adipocytes, the level of TSG-6 gene mRNA expression was upregulated and reached the higher level in fully differentiated adipocytes. There is a significant difference between any two detected phases in the levels of TSG-6 gene mRNA expression (P < 0.05), except that the levels of TSG-6 gene mRNA expression did not increase obviously on day 0 to day 2, day 3 to day 5, day 4 to day 6 and day 7 to day 10 (P > 0.05). (2) Treatment of day 10 3T3-L1 adipocytes with TNF-alpha of different concentrations (0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml) resulted in a significant decrease in the level of TSG-6 gene mRNA expression. The inhibition effect of TNF-alpha on TSG-6 gene mRNA expression generally tended to be reinforced with the increasing concentrations of TNF-alpha and the elongation of time course, except for the period of 6 - 24 h after the stimulation of 10.0 ng/ml TNF-alpha. When 0.1 ng/ml TNF-alpha was applied, the level of TSG-6 gene expression decreased by 33.73% at 6 h, 97.39% at 12 h. While 1.0 ng/ml TNF-alpha was used, the level of TSG-6 gene expression decreased by 78.68% at 6 h, which remained until 24 h. At a concentration of TNF-alpha up to 10.0 ng/ml, the level of TSG-6 gene expression decreased by 96.27% at 2 h. TSG-6 gene expression was almost fully inhibited.</p><p><b>CONCLUSION</b>(1) TSG-6 gene may be involved in adipocyte differentiation and adipogenesis. (2) TNF-alpha can downregulate the mRNA expression of TSG-6 gene in matured adipocytes. The inhibitory effect of TNF-alpha on TSG-6 gene expression is generally dose-correlated.</p>