Relative role of free fatty acids on expression of miR-335 in human matured adipocytes / 中华实用儿科临床杂志

Objective To explore the role of free fatty acids(FFA) on expression of miR-335 in human matured adipocytes.Methods In order to induce differentiation,confluent human pre-adipocytes (day 0) were subsequently cultured in serum-free PAM containing 50 nmol/L insulin,100 nmol/L dexamethasone,0.5 mmol/L 3-isobutyl1-methylxanthine,and 100 μmol/L rosiglitazone.Then human matured adipocytes (day 16) were treated with 1 mmol/L FFA cocktail composed of lauric acid,myristic acid,linoleic acid,oleic acid,and arachidonic acids for 4,8 and 24 hours.Meanwhile,untreated cells were collected as control group.Total RNA from these adipocytes were extracted and the levels of miR-335 expression were evaluated by real-time PCR.Results The expression of miR-335 at 4,8 and 24 hours showed no statistical significance when compared to 0 hour in untreated matured adipocytes (all P ＞ 0.05).The relative expression of miR-335 after the intervention of FFA in human matured adipocytes were 9.03 ± 0.31,9.85 ±2.41 and 11.23 ± 0.62,respectively at 4,8 and 24 hours when used with snRU6 for normalization,and there was statistical signi-ficance compared with 0 hour in control group (all P ＜ 0.05).The levels of miR-335 were 4.73 ± 0.60,5.38 ± 1.25 and 4.57 ±0.52 at the same time point when used with miR-103 for normalization,and there was statistical significance compared with 0 hour in control group (all P ＜ 0.05).Conclusions FFA exert a positive effect on the miR-335 expression in human matured adipocytes,which provide the basis for the further study about the role of miR-335 in human adipocytes.

Resistin Binding Peptide Stimulates Basal Insulin Secretion of RINm5F Insulinoma Cells / 实用儿科临床杂志

Objective A resistin binding peptide (RBP) was selected by phage display in our previous work. Studies had shown that RBP could antagonize the role of resistin on the lipid metabolism and endocrine function of adipose tissue, but whether RBP affects the insulin secretion of pancreatic cells is still unknown. The aim of this study is to assess the effect of RBP on basal insulin secretion in RINm5F insulinoma cells. Methods The cell viability was measured by 3-[4,5-dimethyhhiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay. The supernatants were assayed for insulin content by enzyme linked immunosorbent assay (ELISA). Reverse transcriptase-PCR assay and Western blotting were used to determine the expression of glucose transporter 2 (GLUT2) involved in insulin secretion. Cytosolic Ca2+, the trigger of insulin exocytosis, was analyzed with the fluorescent probe FURA-3/AM. Results RBP did no effect on the cell viability with a concentration of 10-8-10-12mol/L of 2 hours intervention. But it stimulated basal insulin secretion of RINm5F cells, accompanied by up-regulated increased expression of GLUT2 and elevated concentration of cytosolic Ca2+. Conclusion RBP could stimulate basal insulin secretion without affecting the cell viability.

Overexpression of resistin affect 3T3-L1 adipocyte lipid metabolism / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the effect of resistin overexpression on 3T3-L1 adipocyte lipid and glucose metabolism.</p><p><b>METHODS</b>Expression vector for rat resistin gene was constructed and transfected into 3T3-L1 adipocytes. Cell differentiation and lipid accumulation was determined by Oil Red O staining. Differentiation marker genes (pref-1, C/EBPalpha, FAS) and glucose transporter 4 (GLUT4) gene mRNA expressions were evaluated by reverse transcription-PCR (RT-PCR). Triglyceride (TG) and free fatty acids (FFAs) in adipocytes were measured by colorimetric kit.</p><p><b>RESULTS</b>(1) In resistin-overexpressed adipocytes, the lipid droplets presented at the second day which was earlier than the control cells. (2) The expression of C/EBPalpha and FAS genes in resistin-overexpressed adipocytes were up-regulated and the pref-1 was down-regulated compared with that of the control cells. (3) In resistin-overexpressed adipocytes, cellular TG and FFAs levels were significantly increased (P<0.05). (4) There was no difference in the expression of GLUT4 gene between 3T3-L1 adipocytes and resistin-overexpressed adipocytes (P> 0.05).</p><p><b>CONCLUSION</b>Overexpression of resistin can affect 3T3-L1 adipocyte lipid metabolism and thereby result in obesity and insulin resistance, but have no effect on GLUT4 gene expression.</p>

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>