Effect And Mechanism Of PLIN1 Gene Silencing On Lipolysis Of 3T3-L1 Adipocytes

Objective:To investigate the effect on lipolysis of 3T3-L1 adipocytes by blocking perilipin 1gene expression, and to obtain insights into its molecular mechanism for researching biological weight-reducing aid.Methods:1. Three sh RNA recombinant vectors targeting mouse PLIN1 gene and one negative control were designed, synthesized and detected by bacterium liquid PCR reaction and DNA sequencing.2. 3T3-L1 preadipocytes were generally induced to differentiate by the hormonal cocktail. The vectors were also transfected into the 3T3-L1 adipocytes with lipofectamine.3. Bodipy 493/503 staining was used to observe the lipid droplets size, and the cell nucleus were stained with Hoechest 33258. The content of cellular triglyceride(TG) and glycerol were detected by enzymic method to evaluate the lipolysis.4. The Perilipin 1A(PLIN1A), adipose triglyceride lipase(ATGL), hormone-sensitive lipase(HSL) and phosphorylase HSL(p-HSL) were tested by western blot, and the cyclic adenosine monophosphate(c AMP) and protein kinase A(PKA) were measured by enzyme linked immunosorbent assay(ELISA) after transfection. In addition, adipocytes were treated with 10?mol/L isoproterenol(ISO) for 6 hours.Results:1. As the differentiation of the 3T3-L1 preadipocytes, cells became much larger and rounder with massive lipid droplets. After fluorescence staining, lipid droplets were distributed around the nucleus, and on the 8th day of inducing differentiation, the diameter of lipid droplets increased clearly than the 4th day.2. The content of TG increased as the differentiation of the 3T3-L1 preadipocytes.3. The transfection efficiency was more than 40% in all groups two days later,and the three positive vectors could significantly inhibit the protein expression of PLIN1A(P<0. 05).4. Blocking PLIN1 gene expression decreased lipid droplets size and cellular triglyceride level and increased glycerol level. In addition, the level of ATGL and HSL increased significantly(P<0.05), and the level of cellular c AMP and PKA had no obvious change(P>0.05).5. Compared to the negative control group, the protein expression of ATGL increased obviously in ISO+sh-PLIN1 group and sh-PLIN1 group(P<0.05), but with no difference between the two groups(P>0.05). Moreover, glycerol releases increased with decreased TG content, and the leave of HSL, p-HSL, c AMP and PKA increased significantly in ISO+sh-PLIN1 group and ISO group(P<0.05).Conclusions:1. Every recombinant vector was constructed successfully and they were effective to down-regulate PLIN1 protein.2. Blocking PLIN1 gene could promote lipolysis in 3T3-L1 adipocytes by up-regulating the level of ATGL and HSL, and c AMP-PKA signaling pathway had no significant effect on it.3. ISO could activate the c AMP/PKA pathway to promote lipolysis in 3T3-L1 adipocytes by up-regulating the level of HSL and p-HSL, and there was no significant effect of sh-PLIN1 vector on it.