Objective： To investigate the effect of the calcium channel blocker verapamil on adipocyte differentiation and its mechanism of action. Methods： Preadipocytes from 3T3-L1 strain mouse embryos were cultured and differentiated into matured adipocytes in vitro. Verapamil was added to the culture medium in the concentration of 30 μmol/L on Day 0. Cell differentiation was determined by Oil Red O staining and marker gene mRNA expression was evaluated and compared by RT-PCR. The fluo-3/AM probe and laser scanning confocal microscopy were used to measure intracetlular calcium concentrations. Results： （1）The differentiation rate of 3T3-L1 preadipocytes exposed to verapamil was lower than that of untreated cells. （2）Verapamil promoted the retention of pref-1 gene expression. Lipoprotein lipase expression in the verapamil group was significantly lower than that in the control group on Day 4, Day 6 and Day 8 （P 〈 0.05） and resistin expression was significantly lower than that in the control group on Day 6, Day 8 and Day 10 （P 〈 0.05）. Fatty acid synthase expression in the verapamil group was significantly lower than that in the control group from Day 2 （P 〈 0.05）. （3） Intracellular concentrations of calcium [Ca^2＋]i in the verapamil group were significantly decreased compared with those in the control group on Day 2, Day 4 and Day 6 （P 〈 0.05）, while there was no obvious difference between the two groups on Day 0 （P 〉 0.05）. Conclusion： In 3T3-L1 preadipocytes verapamil significantly reduced adipocyte differentiation, down-regulated the mRNA expression of three marker genes for adipocytes differentiation, and prolonged the mRNA expression of an inhibitor of differentiation. The inhibitory effect of verapamil on differentiation may involve its role as a blocker of calcium influx in adipocytes.
Nan GuShi LiuXirong GuoLi FeiXiaoqin PanMei GuoRonghua Chen
Background 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. Methods 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. Results 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 G-C-resistin significantly inhibited the adipogenic differentiation. Conclusion RBP could effectively rescue the promoted differentiation of resistin overxepressed 3T3-L1 preadipocyte.
LIU FengGUO Xi-rongGONG Hai-xiaNI Yu-huiFEI LiPAN Xiao-qinGUO MeiCHEN Rong-hua
