Effect Of Neuropepitide Y On Proliferation And Differentiation Of3T3-L1Preadipocyte And The Related Machanism

ObjectiveTo observe the effects of Neuropeptide Y (NPY) on the proliferation and the differentiation of3T3-L1preadipocytes and investigate the possible mechanism of NPY on the differentiation of3T3-L1preadipocytes..Methods1. Cultivation, differentiation and identification of3T3-L1preadipocytes:3T3-L1cells were in vitro recovered, cultured and passaged. Cells were routinely induced to differentiate by the hormonal cocktail,containing0.5mmol/1IBMX,1umol/1dexamethasone and5ug/ml insulin. Oil red staining was used to identify the fully differentiated adipocytes.2.3T3-L1preadipocytes were cultured for24hours in the presence of NPY with different concentrations(10-7M、10-8M、10-9M、10-10M、10-11M、10-12M、10-13M、10-14M、10-15M). MTT assay was applied to evaluate the cell growth.3.3T3-L1preadipocytes were induced to differentiation in both treated groups containing NPY with different concentrations (10-7M、10-9M、10-11M) and control groups (without NPY). Effects of NPY on the lipid synthesis during3T3-L1preadipocytes differentiation were detected by Oil red O staining analysis, triglycerides accumulation determination and lipid count.4. PPARy and DLK-1mRNA expression were detected by RT-PCR and RT-FQ PCR while C/EBP a and PPARy protein expression were detected by Western Blot when adipocytes were induced to differentiation for8days.5. Protein expression of ERK,p-ERK,P38,p-P38,JNK,p-JNK were measured by Western Blot when3T3-L1preadipocytes NPY-treated were induced to differentiation for8days.Results1.3T3-L1preadipocytes were induced into mature adipocytes by hormonal cocktail successfully. Oil red O staining was used to clarified matural adipocytes.2.After3T3-L1preadipocytes were cultured for24hours in the presence of NPY with different concentrations, we found that physiological dose and even low dose of NPY could promote proliferation of the3T3-L1preadipocytes while high dose of NPY (10-7Mn10-8M)could inhibit proliferation of preadipocytes.3. Both Oil red O staining and triglycerides accumulation determination revealed that the formation of lipid droplets especially the size of the lipids drolets was significantly increased by the high dose of NPY, while physiological dose had no effects on preadipocyte differentiation.4. RT-PCR showed that NPY could inhibit expression of DLK-1mRNA and high dose of NPY could increase expression of PPARy mRNA. Western blot results suggested that high dose of NPY could improve expression of PPARγ、C/EBP a protein significantly.5. High dose of NPY induced3T3-L1differentiation with effects of inhibition of phosphorylation of ERK1/2protein,promotion of P38protein expression and decrease of JNK protein expression,which could be inhibited by insulin.Conclution1. Physiological dose and even low dose of NPY could promote preadipocyte proliferation, while high dose (10-/M)could inhibit cell proliferation.2. High concentration of NPY (≥10-9M) could promote differentiation of3T3-L1preadipocyte,with enlarged size of lipid droplet,while physiological dose and even low dose of NPY had no effects on preadipocyte differentiation.3. NPY induced adipocyte differentiation was in associatied with changes of MAPK pathway.