Roles Of HSPA12A In Obesity And White Adipose Tissue Browning

Background and objectiveObesity is a global health burden with an increasing prevalence.Obesity is closely associated with diabetes,hypertension,hyperlipidemia,pancreatic cancer and other diseases,and therefore is a major health threat.Adipose tissues consist of white adipose tissue(WAT)and brown adipose tissue(BAT).WAT stores surplus energy as triglyceride.Excessive accumulation of WAT leads to overweight and obesity,as well as metabolic diseases.By contrast,BAT dissipates excess energy as heat by uncoupling respiratory chain,which benefits thermogenic adaptation and metabolism.However,distribution of BAT is diminished during postnatal aging.Recently,a subset of inducible brown-like adipocytes,known as beige adipocytes,is found in WAT.Beige adipocytes could be generated by a process termed as browning of WAT.Browning of WAT has been reported to confer numerous benefits in reversing obesity,and has been considered as a potential therapeutic measure for obesity.Heat shock protein A12A(HSPA12A),initially cloned in 2003,is a new member of HSP70 family,.Our previous research showed that HSPA12 A regulated non-alcoholic fatty liver disease,suggesting possible involvement of HSPA12 A in lipid metabolism.However,the expression and distribution of HSPA12 A in adipose tissue and the role of HSPA12 A in physiology and pathophysiological activity of adipose tissues are still unclear.The aim of this study was to preliminarily explore the roles of HSPA12 A on obesity and browning of WAT,providing preliminary data for further study and clinical translation.Methods 1.Expression profile of HSPA12 A in different tissues.HSPA12 A protein levels in different tissues of 8-week-old wild type male mice were quantified by western blot.2.Correlation between the expression level of human WAT HSPA12 A and BMI.The HSPA12 A m RNA levels in subcutaneous WATs of obese patients and normal-weight patients were examined by RT-PCR.The correlation between the level of HSPA12 A m RNA and BMI was analyzed by linear regression.3.Construction and identification of Hspa12 a knockout mice.The Hspa12 a knockout mice models were established by Cre-Lox P gene recombination system.PCR and western blot were used to identify whether the Hspa12 a knockout mice were constructed successfully.4.Effect of Hspa12 a knockout on high-fat-diet-induced obesity.4-week-old male wild-type(WT)and Hspa12 a knockout(Hspa12a-/-)mice were fed with HFD(high-fat diet)for 14 weeks.Body weight was measured to analyze the effect of Hspa12a-/-on HFD induced obesity.5.Effect of low temperature on the expression of HSPA12 A in WAT.The mice were exposed to room temperature(RT,25?)or low temperature(4?,LT)for 5 days.Inguinal WATs were collected for UCP1 immunohistochemical staining to verify whether WAT was browned.Cytoplasmic,nuclear and total protein were extracted to investigate the expression of HSPA12 A by western blot.Frozen sections were subjected to immunofluorescence staining for HSPA12 A.6.Effect of Hspa12a-/-on cold-induced WAT browning.Male 8-week-old Hspa12a-/-mice and WT littermates were housed at 4°C for 5 days.The inguinal WATs were collected to examine protein levels of UCP-1 and PGC-1? by immunohistochemical staining and Western blot.7.Effect of Hspa12a-/-on thermogenic adaptation.8-week-old WT and Hspa12a-/-male mice were housed at 4°C for 5 days.The rectal temperatures were recorded at the indicated time points.Results 1.Though lower than brain,adipose tissues including BAT and WAT(inguinal WAT,visceral WAT and perirenal WAT)showed higher expression levels of HSPA12 A than heart,liver,spleen,lung,pancreas,skeletal muscle,and bone.2.HSPA12 A m RNA level in abdominal subcutaneous adipose tissue of obese patients was significantly higher than that of normal-weight counterparts,and was positively correlated with BMI.3.The Hspa12a-/-mice were successfully constructed according to the results of PCR and Western blot.4.HFD significantly increased the body weights of WT mice.However,Hspa12a-/-mice showed no significant difference in body weight between the normal diet group and the HFD group.More importantly,Hspa12a-/-mice showed less body weight gain than WT mice after HFD feeding.5.Cold-induced browning of the inguinal WAT was confirmed as the increase of UCP1 expression.Elevated nuclear and total HSPA12 A protein levels were detected in inguinal WAT of LT-exposed mice compared to RT controls,although the cytosolic HSPA12 A protein level was unchanged.6.Cold exposure increased the protein levels of UCP-1 and PGC-1? in the inguinal WAT of both genotypes,when compared to their respective RT controls.Notably,the cold-induced increase of UCP1 and PGC-1? expression was markedly enhanced in Hspa12a-/-inguinal WAT compared to WT controls.7.After exposure to cold ambience at 4?,the body temperatures were markedly decreased in both WT and Hspa12a-/-mice.However,the cold-induced drop of body temperature was ameliorated in Hspa12a-/-mice compared to WT controls.Conclusion Hspa12 a knockout inhibits the HFD-induced obesity,promotes cold induced-WAT browning.Inhibition of WAT HSPA12 A may provide a viable strategy for the therapy of obesity and metabolic diseases.