The Function Of Macrophage-specific ATP6V0d2 In The Pathological Process Of Obesity

Purpose: The internalization and degradation of triglyceride in macrophages is hindered in obese adipose tissue,which enhances the lipid accumulation and insulin resistance in adipocytes.As a subunit protein of V-ATPase,ATP6V0d2 shows a specific high expression level in macrophage and possibility to participate in lysosomal acidification and membrane fusion,thus is closely related to degradation activities in macrophage.This thesis explores the role of macrophage-specific ATP6V0d2 protein in fat metabolism,development of obesity,and during the pathological process of insulin resistance.Methods: 1)High-fat diet-induced obesity model in wild-type and Atp6v0d2-/-mice was constructed to observe the change of body weight,body fat ratio,blood lipid and blood glucose level;2)BMDMs from obese model mice were isolated and purified.Flow cytometry was used to detect the M1/M2 polarization of wild-type and Atp6v0d2-/-mice macrophages;3)Protein of mouse BMDMs,embryonic fibroblast stem cells and mature adipocytes was extracted.Western blot was used to evaluate the ATP6V0d2 protein expression in these major cell components of adipose tissue;4)BMDMs from obese wild-type and Atp6v0d2-/-mice were isolated and purified,then simulated in an obese adipose tissue-like environment with saturated fatty acid.Oil red O-staining of cells was used to observe the difference in intracellular triglyceride accumulation;5)RNA from wild-type and Atp6v0d2-/-BMDMs stimulated with fatty acid was extracted.RT-PCR assay was used to evaluate the m RNA level of genes involved in lipogenesis and lipolysis;6)RNA from BMDMs before and after fatty acids stimulation was extracted.The expression of Atp6v0d1 and Atp6v0d2 m RNA was detected by RT-PCR;7)Protein of BMDMs from obese and lean wild-type mice was extracted.The ATP6V0d2 protein expression was detected by Western blot.Results: 1)Atp6v0d2-/-obese mice showed a higher rate in body weight gain and higher basal blood glucose level than those of wild-type mice.And the polarization tendency of BMDMs was stronger towards M1 than M2 in Atp6v0d2-/-mice,comparing to wildtype mice.2)In adipose tissue,macrophages showed specific high expression of ATP6V0d2 protein.Fatty acid-stimulated Atp6v0d2-/-macrophages accumulated more lipid droplets than in wild-type,but there was no statistical difference in the transcription level of lipid metabolism-related enzymes.3)Fatty acid stimulation down-regulates the m RNA level of Atp6v0d2 in macrophages.Consistent with this result,long-term administration of high-fat diets inhibited the protein expression of ATP6V0d2 in wild-type mice.Conclusion: In adipose tissue,ATP6V0d2 is mainly expressed in macrophages and participates in the macrophage degradation of triglycerides,but it does not act by regulating the transcription of enzymes involved in lipid metabolism.Deletion of mouse ATP6V0d2 protein suppresses macrophage polarization towards M2 phenotype,accelerates the development of obesity,and increases basal glucose levels.In addition,fatty acids have a negative regulatory effect on the transcription and expression of ATP6V0d2.