Effect Of NRF1 On Adipocyte Differentiation

Failure of adipocyte differentiation will cause adipose tissue dysfunction,which contributes to many metabolic disorders such as insulin resistance,diabetes and so on.Previous studies in our lab have shown that inorganic pyrophosphatase 1(PPA1)plays an important role in adipocyte differentiation.We found that expression of PPAI was gradually increased during 3T3-LI differentiation.Knockdown PPAl significantly inhibited 3T3-L1 cell differentiation and impaired mitochondrial function.The aim of this study is to further investigate the function and mechanism of PPAI in adipocyte differentiation both in vivo and in vitro.Firstly,Nurf38,the homologous gene of PPAI,was knockdown in the fat body of Drosophila.Results showed that the size of the larvae and the volume of the lipid droplets were significantly decreased in PPAI knockdown groups.The diameters of the lipid droplets analyzed by 3D laser confocal was significantly reduced when compared with controls.Knockdown Nurf38 in the fat body also caused Drosophila mitochondria morphological abnormalities and smaller size.These results suggest that PPAI may affect the differentiation of adipocytes by influencing the morphology and function of mitochondria.At the cellular level,we found that the interference of PPAI significantly decreased the mitochondrial DNA content during cell differentiation,only about 60%of the control group,while the amount of ATP production was reduced to 66%.Immunofluorescence assay showed that only a small amount of PPAI was located in mitochondria,the rest was located in cytoplasm.To further explore the relationship between PPAI expression and mitochondrial function,we analyzed the PPAI promoter region and found that mitochondrial nuclear respiratory factor 1(NRFI)may be involved in the regulation of PPAI transcriptional activity.By Dual-reported luciferase assay and chromatin immunoprecipitation assay(ChIP),we found that NRF1 could bind to the downstream of the PPAI promoter region from 10 bp to 21 bp,and played an important role in regulating the transcriptional activity of the PPA1.ChIP experiments were performed at different time points during cell differentiation,and the results showed that this regulatory effect persisted during 3T3-L1 cell differentiation.In order to clarify whether NRFI affects the adipocytes differentiation by regulating of mitochondrial function,we first detected the expression of NRF1 in the subcutaneous adipose tissue of HFD mice.The results showed that high fat diet could cause mice differentiation disorder,manifested as down-regulation of PPAR-y expression,while the expression level of NRF1 also decreased.Moreover,expression of NRF1 was gradually increased with cell differentiation.After interfering with NRF1,3T3-LI cells showed significant differentiation defect,expressed as the number of mature adipocytes decreased,the lipid droplets volume reduced.In summary,we found that NRF1 regulated the transcriptional activity of PPAI.Interfering with NRF1 in 3T3-L1 cells caused differentiation disorder.This provides a new theoretical basis for the functional study of NRF1 and PPA1,also the effect of mitochondrial damage on adipogenesis differentiation.