Berberine Promotes Brown Remodeling And Thermogenesis Of Adipose Tissue By Activating AMPK/SIRT1-deacetylated PPARγ

Background:Obesity has become a worldwide problem,threatening human health and increasing the risk of many chronic diseases.Obesity is a chronic state of positive energy balance,leading to excessive accumulation of adipose tissue.How to remedy this state of excess energy has become the focus of many studies about obesity.With the discovery of brown adipose tissue(BAT)in adults,the role of adipose tissue in regulating energy metabolism has been reconsidered.White adipose tissue(WAT)is the main part for energy storage in mammals,while BAT can cause non-shivering thermogenesis through mitochondrial uncoupling protein 1(UCP1)for energy expenditure.Under certain stimulatory conditions,WAT can undergo browning remodeling.They also highly expressed UCP1 like BAT,which promotes energy expenditure.Currently,promoting white adipose tissue browning and increasing brown adipose tissue thermogenesis are considered as potential strategies to treat obesity.PPARγis a key regulator of the differentiation of white and brown adipocytes,and it is highly expressed in WAT and BAT.PPARγcan regulate adipogenesis,differentiation,and related functions in WAT and BAT,which is conducive to improving insulin resistance and reducing the occurrence of obesity-related complications.However,PPARγwith these unique benefits is shadowed by the risk of side effects,such as weight gain,bone loss,fluid retention,and congestive heart failure,which prevents its clinical application.Therefore,more researches have focused on the protein modifications of PPARγto exploit its positive effects on metabolism.PPARγcan be regulated by post-translational modifications,including phosphorylation,acetylation,sumoylation,and ubiquitination Five acetylated lysine residues were identified at positions K98,K107,K218,K268,and K293.Acetylation of PPARγmight concur with phosphorylation by cyclin dependent like kinase5(CDK5),which results in decreasing insulin sensitivity.K268ac and K293ac on PPARγare deacetylated by SIRT1 in a ligand-dependent manner.Deacetylation of PPARγcan promote WAT browning,thereby increasing insulin sensitivity and improving metabolic abnormalities.Under different stimulatory conditions,the deacetylation of PPARγcan selectively promote the expressions of brown adipocyte specific gene or white adipose specific gene.This suggested that selective deacetylated PPARγmay be a regulatory switch for the conversion of WAT to BAT.Berberine(BBR)is an active product isolated from the medicinal plant RhizomaCoptidis,which was used in the treatment of diarrhea.It has received increasing attention for a variety of metabolic benefits in recent years.BBR can inhibit WAT differentiation and accumulation,and increase adipose tissue thermogenesis.Berberine has been proven to be an activator of AMPK,which is responsible for triggering glucose uptake,reducing liver gluconeogenesis,and improving insulin sensitivity.AMPK can enhance SIRT1activity by increasing cellular NAD~+level,which deacetylates downstream SIRT1 targets and modulate their activity.SIRT1 has important effects on glucose homeostasis,insulin sensitivity,and adipogenic regulation via its deacetylase activity.SIRT1-dependent deacetylation of PPARγis a form of selective regulation of PPARγinvolved in brown remodeling.Therefore,we speculate BBR that enhances the AMPK-SIRT1 pathway can act as a selective PPARγactivator to promote adipose tissue remodeling and thermogenesis.Objective:To investigate the effects and underlying mechanism of BBR on adipose tissue remodeling and thermogenesis.Methods:1.Part 1:We constructed a high-fat fed obese mouse model and gave different doses of berberine by gavage.Changes in body weight and food intake of the mice in each group were monitored.Serological tests for relevant indicators,oral glucose tolerance,and insulin tolerance test are performed.The metabolic cage was used to measure the velocity of oxygen consumption,carbon dioxide production,and energy expenditure.The subcutaneous WAT,visceral WAT,and scapular BAT were weighed to compare their contents in each group.Micro-CT was applied to determine the distribution of WAT and BAT in the interscapular region.Cold exposure test was performed to estimate the thermogenic capability of each group.H&E staining and immunohistochemistry were used to detect the size of lipid droplets and the expression of UCP1.RT-PCR was performed to determine the expression of genes related to thermogenesis and lipid metabolism.2.Part 2:The function and mechanism of berberine on WAT browning and BAT thermogenesis were investigated by 3T3-L1 white adipocytes as well as HIB-1b brown adipocytes.Mito-Tracker staining of mitochondria was used to clarify the effect of BBR on the number of mitochondria in WAT and BAT.Transmission electron microscopy was used to observe the effect of berberine on lipid droplets and mitochondria in 3T3-L1 white adipocytes.Western blot was used to detect the expression of SIRT1 and thermogenesis-related protein UCP1 after BBR intervention in 3T3-L1,HIB-1b cells,and WAT and BAT of obese mice.Oil red O staining was performed to clarify the role of SIRT1 in adipose tissue thermogenesis,lipid metabolism,and lipid accumulation mediated by berberine.Immunofluorescence staining was performed to analyze the effect of BBR on the co-localization of SIRT1 and PPARγ.IP assays were applied to further investigate the effect of BBR on the interaction of SIRT1 and PPARγ.In addition,anti-acetylated lysine antibody and anti-PPARγantibody were used to detect the acetylation levels of PPARγthrough IP assay.3T3-L1 and HIB-1b cell lines with knockdown SIRT1 expression were constructed by lentiviral infection.Sirt1(-/-)MEF cells were obtained from Sirt1(+/-)mice.The SIRT1plasmid and SIRT1 mutant(363HY)plasmid were transfected into SIRT1 knockdown3T3-L1 cells to evaluate the regulatory effect of SIRT1 on the deacetylation level of PPARγ.3.Part 3:The expression of P-AMPK and AMPK in subcutaneous WAT and BAT of high-fat fed mice treated with different doses of BBR was detected.3T3-L1 and HIB-1b cells were induced to differentiate,and their expression of P-AMPK and AMPK was detected by Western blot.3T3-L1 cells were pretreated with BBR,AICAR(AMPK activator),and Compound C(AMPK inhibitor),and their expression of UCP1 was detected by Western blot.Wild-type and SIRT1 knockdown 3T3-L1 cells were pretreated with BBR and AICAR,and their expression of P-AMPK,SIRT1,and UCP1 was detected by Western blot.AMPK knockdown 3T3-L1 cell lines were constructed by lentiviral infection to evaluate the effect of AMPK on PPARγdeacetylation.BBR,AICAR(AMPK activator),and Compound C(AMPK inhibitor)were applied to pretreat 3T3-L1 cells and SIRT1activity was evaluated by NAD~+/NADH.Western blot was applied to evaluate the expression of SIRT1 and UCP1.In Wild-type and SIRT1 knockdown 3T3-L1 cell lines,AICAR and BBR treatments were given and Western blot was applied to evaluate the expression of P-AMPK,SIRT1,and UCP1.AMPK knockdown 3T3-L1 cell line was constructed by lentivirus infection to evaluate the effect of AMPK on PPARγdeacetylation.SIRT1 knockdown 3T3-L1 cell line was constructed by lentiviral infection to evaluate the role of SIRT1 in the regulation of PPARγdeacetylation by AMPK.Western blot,immunofluorescence,and immunohistochemical images were processed by Photoshop 2020.Gray values of Western blots were measured by image J(Ver 1.8.0)software.Relative expression quantity in Real-time quantitative PCR was calculated by 2-ΔΔCt formula.Bar charts were made by Graphpad Prism(Ver 7.0.4)software.All statistical analyses were performed by SPSS(IBM)software,and the results were presented as mean±standard error.The statistical methods were one-way ANOVA(followed by Bonferroni or Dunnet’s post-hoc tests as appropriate)and independent sample t-test analysis.P<0.05 was considered statistically significant.Results:1.Part 1:By constructing a high-fat fed obesity model,we found that BBR dose-dependently reduced high-fat-induced weight gain,alleviated insulin resistance,and relieved dyslipidemia due to high-fat diet,including plasma total cholesterol,triglyceride,and low-density lipoprotein cholesterol.There was no significant difference in energy intake among the HF,BBR-25,and BBR-100 groups.Thus we hypothesized that the metabolic benefits exhibited by BBR on a high-fat diet were not due to reduced energy expenditure by suppressing appetite,but rather by increasing energy expenditure.Metabolic cage data showed that oxygen consumption,carbon dioxide production,and energy expenditure were higher in the groups treated with BBR than in the group with HF alone.The ratio of inguinal adipose tissue and visceral adipose tissue to body weight was increased significantly in HF group than NCD group.When HF mice received BBR treatment,the inguinal adipose tissue/body weight ratio dropped back to nearly the NCD level.The ratio of visceral adipose tissue to body weight decreased significantly in BBR-100 group than HF group,while it was still higher than that of NCD group.There was no significant difference in the ratio of visceral adipose tissue between BBR-25 group and HF group,and both of them were significantly higher than NCD group.With BBR treatment,the ratio of brown adipose tissue was significantly higher than that of HF group.In order to observe the adipose tissue distribution more intuitively,Micro-CT scan and 3D reconstruction of the interscapular region were performed.These results further confirmed that BBR can increase the content of BAT and reduce the content of subcutaneous WAT.Adipose tissue browning is characterized by adaptive thermogenesis to maintain core temperature under cold conditions.In cool exposure test,BBR-100 exhibited outstanding ability of adaptive thermoregulation than HF group.In HE staining,the lipid droplets of the BAT and inguinal white adipose tissue(I-WAT)both showed smaller in BBR treated groups than HF group in dose-dependently.Immunohistochemical analysis of adipose tissues revealed that UCP1 of I-WAT and BAT expression significantly increased in BBR treated groups than HF group.Consistent with the immunohistochemistry results,Western blot showed UCP1 expression significantly increased in IWAT and BAT of BBR treated groups than HF group.The m RNA levels of thermogenesis gene and fatty acid oxidation gene in I-WAT and BAT of mice treated with BBR were significantly higher than those in mice treated with HF alone.2.Part 2:To investigate whether BBR has the ability to improve the thermogenesis in vitro,the effects of BBR treatment were assessed in 3T3-L1 white adipocytes and HIB1B brown adipocytes.We stained 3T3-L1 and HIB1B with Mito-Tracker Red,which is a red-fluorescent dye that stains mitochondrial mass.The results revealed that BBR-treated adipocytes exhibited obviously strong Red stain in the cytoplasm,which means more mitochondrial mass.Transmission electron microscopy showed that 3T3-L1 cells treated with BBR exhibited the characteristics of brown-like adipocytes that increased mitochondrial density and smaller lipid droplets.We next elucidated whether SIRT1 is necessary for BBR to regulate the brown remodeling of adipose tissue.Consistent with the data in mice,Western blot showed SIRT1 and UCP1 expression of 3T3-L1 and HIB1B cells with BBR treatment was increased in a dose-dependent manner,but not PPARγ.BBR treatment and overexpression of SIRT1 both significantly increased the UCP1 expression.In contrast,we blocked the SIRT1 in 3T3-L1 and HIB1B cells with lentivirus,which almost prevents the total increased expression in UCP1 that was originally induced by BBR.The m RNA levels of thermogenesis gene and fatty acid oxidation gene were increased by BBR,but not in sh SIRT1 cells,indicating that BBR-induced brown remodeling was dampened by SIRT1 knockdown.Strikingly,the reduction of lipid accumulation in white adipocytes caused by BBR was largely blocked in 3T3-L1 cell lines stably expressing sh RNA-targeting SIRT1as assessed by Oil Red O staining.Taken together,these data indicate that BBR regulates adipocyte brown remodeling dependent on SIRT1.Using immunofluorescence,SIRT1 and PPARγwere found to be intensively colocalized at the perinuclear region.In SIRT1 overexpression 3T3-L1 cells,we detected SIRT1 and PPARγrespectively in PPARγand SIRT1 immunoprecipitates from cell lysates increased in cells with BBR treatment,which further proved that BBR caused more binding of SIRT1 and PPARγ.3T3-L1 and HIB1b cells treated with BBR after differentiation,and then assayed for acetylation of PPARγvia IP assay with anti-acetylated lysine antibody and anti-PPARγantibody.Our results showed that when cells were treated with BBR,the level of acetylated PPARγwas significantly decreased.In other words,BBR increased the deacetylation modification of PPARγ,which is consistent with our speculation.To further elucidate whether SIRT1 is the deacetylase that is responsible for PPARγdeacetylation,Wild-type and SIRT1 knockdown 3T3-L1 and HIB1b cells were respectively treated by BBR.The results showed that SIRT1 knockdown resulted in the disappearance of the changes in acetylation level of PPARγinduced by BBR treatment.Sirt1-/-MEF cells further corroborated this idea,which indicated that BBR increased the deacetylation modification of PPARγdepended on SIRT1 activation.Then,we further transfected Wild-type-SIRT1,sh SIRT1,and SIRT1 mutant(363HY)plasmids into 3T3-L1 cells treated with BBR.It can be inferred from the results that BBR significantly increased the PPARγdeacetylation by Wild-type-SIRT1,but not a catalytically inactive SIRT1 mutant(H363Y).3.Part 3:We examined the expression of AMPK in response to BBR treatment in IWAT and BAT by Western blot.The results showed that P-AMPK expression of IWAT and BAT of mice with BBR treatment was increased in a dose-dependent manner than those of mice with HF,but not AMPK.This indicates that BBR can activate AMPK both in subcutaneous WAT and BAT.Mature white adipocytes were pre-treated with BBR,AICAR,and Compound C.Our study showed that BBR and AICAR increased the activity of SIRT1and the expression of SIRT1 and UCP1.Pre-treatment with Compound C diminished the BBR-induced expression of SIRT1 and UCP1.The degrees of activation of UCP1 by BBR and AICAR were significantly attenuated by SIRT1 knockdown.To further elucidate whether BBR regulates PPARγdeacetylation via AMPK,we constructed a 3T3-L1 cell line with knockdown AMPK expression using lentivirus.Wild-type and sh AMPK 3T3-L1cells were treated with BBR.We found that the changes in acetylation levels of PPARγinduced by BBR were significantly blocked in sh AMPK 3T3-L1 cells.Administration of AICAR stimulation in the sh SIRT1 cell line revealed that the effect of AICAR on PPARγdeacetylation was found to be inhibited.From this,we conclude that AMPK regulates the level of PPARγdeacetylation dependent on SIRT1.Conclusion:Berberine promotes the brown phenotype of WAT and the thermogenic activity of BAT,which increases basal metabolism,and ameliorates weight gain and metabolic abnormalities in obese mice due to high-fat diet.BBR increases the level of deacetylation of PPARγby activating the AMPK/SIRT1 pathway,which promotes brown remodeling of WAT and thermogenesis of BAT.