Effect Of Angiotensin â…¡ On Proliferation And Differetiation Of 3T3-L1 Preadipocyte

The primary reason of obesity is hypertrophy and hyperplasia in adipocyte. Studieson proliferation and differentiation of preadipocyte will reveal the physiological andpathophysiological mechanisms underlying adipose tissue development. RAS is apeptidergic system with endocrine characteristics. As the main effector peptide of theRAS, Angâ…¡is involves in control of obesity and other correlative disease. However,paradoxical results about the effects of Angâ…¡were reported. 3T3-L1 preadipocyte wasused for culture in vitro model in this study. The effect of Angâ…¡on proliferation anddifferentiation in 3T3-L1 preadipocyte is investigated by serum or serum starvationmethod, Oil Red O staining, spectrophotometry and reverse transcription- polymerasechain reaction (RT-PCR) in this article.1. The biological characteristic of growth and differentiation in 3T3-L1To draw the growth curve though cell counting by trypanblau staining, the resultindicated that growth of cells is extremely vigorous in DMEM containing 10% new borncalf serum, 37℃,5%CO₂, the exponential phase of growth began from the second day andplatform phase was achieved in the fifth day. In microscope, the global lipid droplet canbe observed on the fourth day in 3T3-L1 after induction. It became single lipid dropletfrom the ring's shape finally. Differentiation of 3T3-L1 was induced by Cocktail method.Intracytoplasmic lipids were quantitated by Oil Red O staining and GPDH activity wasassayed spectrophotometrically in cytosolic extracts of adipocytes by measuring theoxidation rate of NADH. The result indicated that intracytoplasmic lipids were increasedwith the differentiation time increased, and increased quickly after six days whendifferentiation medium was added. GPDH activity appeared the same regularity whichincreased quickly after four days when differentiation medium was added. The maximumvalue of GPDH activity was achieved till eight days and maintained the high level. 2. Effect of Angâ…¡on proliferation of 3T3-L1The proliferation of 3T3-L1 was assayed by MTT with serum and starvation method.The result indicated that Angâ…¡has no significant influence on proleferation of 3T3-L1 at0.01μmol/L,0.1μmol/L,1μmol/L,10μmol/L(P＞0.05). It suggested that Angâ…¡has nosignificant influence on proliferation of 3T3-L1.3. Effect of Angâ…¡on differentiation of 3T3-L1 preadipocyteWe investigated changes of intracytoplasmic lipids content and GPDH activity aftertreated with Angâ…¡(1μmol/L,100 nmol/L,10 nmol/L,1 nmol/L,0.1 nmol/L) accordingto its physiological concentration. AngII significantly increased lipid content at 100nmol/L, as well 10 nmol/L, and GPDH activity at 100 nmol/L over control levels by 62%,21%, 64% after 8 d. These results suggested that Angâ…¡promoted the differentiation of3T3-L1 preadipocyte.4. Effect of Angâ…¡on the expression of transcription factors PPARγ,C/EBPα,SREBPlc mRNA during 3T3-L1 preadipocyted differentiationRT-PCR assays showed that Angâ…¡(100 nmol/L) increased the expression of PPARγ,C/EBPα,SREBPlc mRNA over control levels by 48%, 50%, 43% after 6 d, consistentwith rise in lipids content and GPDH activity upon Angâ…¡treatment. These findingssuggested that molecule regulation mechanisms of the positive role of Angâ…¡in 3T3-L1differentiation related to upregulation of expression of the PPARγ,C/EBPα,SREBPlcmRNA as adipogenic transcription factors.