Study On The Role And Mechanism Of Nuciferine In Preventing Obesity And Losing Weigh

Part 1The effects and underlying mechanisms of nuciferine on preventing high-fat diet-induced obesityObjectives:Obesity and its related complications are major threats that endanger human health.Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)-mediated fat synthesis and decomposition in liver and adipose tissue is closely related to the occurrence and development of obesity.The traditional Chinese medicine,lotus leaf,has been reported with anti-tumor and anti-inflammatory effects.One previous research reported that the water extract of lotus leaf reduced white adipose tissue(WAT)mass of mice fed with high-fat diet(HFD).Nuciferine(Nuci)is the main active component of lotus leaf,but whether or not Nuci possess the ability of preventing obesity and the underlying mechanism are not clear.Thus,we aimed to explore the effect of Nuci on preventing obesity in HFD-fed mice,as well as the role of AMPK pathway in the process.Methods:This study involves animal and cell experiments.The animal experiments consist of the following two parts:1.Exploration of the effective dosage and way of Nuci administration in HFDfed mice.The experiment consists of two parts.In the first part,different doses of Nuci dissolved in sodium carboxymethylcellulose solution were opted to treat HFD-fed mice by gavage for 8 weeks.HFD-fed C57BL/6J male mice were divided into 4 groups randomly(n=3～4 in each group):the HFD-fed control group(Cont),the low-dose Nuci group(10 mg/kg,Nuci-L),the medium-dose Nuci group(20 mg/kg,Nuci-M),and the high-dose Nuci group(30 mg/kg,Nuci-H).The effects of Nuci on body weight,food intake,amount of WAT,serum indicators for glycolipid metabolism were measured.In the second part,different doses of Nuci were mixed into HFD to treat HFDmice for 7 weeks.Similar to the first part,the HFD-fed male mice were randomly divided into four groups(n=3 in each group):HFD control group(Cont),low-dose Nuci group(0.06%,Nuci-L),medium-dose Nuci group(0.12%,Nuci-M)and high-dose Nuci group(0.24%,Nuci-H).The effect of Nuci on body weight,food intake,amount of WAT,serum indicators for glycolipid metabolism were measured.2.The effects of Nuci on HFD-fed mice:Male C57BL/6J male mice were divided into 4 groups randomly(n=12 in each group):normal diet-fed control group(ND),high-fat diet-fed control group(HFD),high-fat diet-fed Nuci group(HFD-Nuci)and high-fat diet-fed liraglutide group(HFDLira).The Nuci group was given 0.10%Nuci mixed into the HFD,and the HFD-Lira group was given subcutaneous injection of 200 μg/kg/d liraglutide for a total of 12 weeks.The effects of Nuci on body weight,food intake,energy expenditure,amount of WAT,serum levels of adipokines and other indicators for glycolipid metabolism were measured.The phosphorylation of AMPK,mRNA expressions of adipokines and key enzymes involved in fat synthesis and catabolism were determined in liver tissue and adipose tissue.The cell experiments include the following three parts:1.3T3-L1 preadipocytesThe effect of various doses of Nuci administration for different durations on the proliferation of 3T3-L1 preadipocytes was observed.In the meantime,the 3T3-L1 preadipocytes were induced differentiation along with Nuci.Effects of Nuci on intracellular lipid content and the expressions of transcription factors during early(3 days),middle(6 days)and late stage(9 days)differentiation were observed.In fully differentiated adipocytes,different concentrations of Nuci were used to treat for different time,and the effects of Nuci on AMPK phosphorylation,and mRNA levels of adipokines,key enzymes involved in fat synthesis and catabolism were determined.Next,cells were pretreated with AMPK-specific inhibitors,and whether the beneficial effects of Nuci were reversed by AMPK inhibition was observed.In addition,3T3-L1 preadipocytes were transfected with a luciferase reporter gene plasmid containing fatty acid synthase(FAS)promoter,and the effect of Nuci on FAS promoter activity was observed.2.Primary murine mature adipocytes Mature adipocytes from epididymal adipose tissue(eWAT)of mice in the ND,HFD,HFD-Nuci group were isolated and cultured,and the effect of Nuci on lipid content in the primary cultured mouse mature adipocytes of each group were observed.3.Adipose-Derived Stem Cell(ADSC)derived from human visceral adipose tissue:ADSC cells derived from the visceral adipose tissue of simple obesity patients were isolated and cultured.The effect of Nuci on the expressions of adipokines and key enzymes involved in fat synthesis and catabolism were measured.Fully differentiated human ADSC cells were also pretreated with AMPK-specific inhibitors,and whether the beneficial effects of Nuci were reversed by AMPK inhibition was observed.4.HepG2 cellsHepG2 cells were directly treated with Nuci or Nuci with pretreatment of 0.4 mM palmitic acid(PA)to generate cell model of nonalcoholic steatohepatitis).The effects of Nuci on the expressions of adipokines and key enzymes involved in fat synthesis and catabolism were measured.Next,cells were pretreated with AMPK-specific inhibitors to observe whether the beneficial effects of Nuci were reversed due to the inhibition of AMPK.In addition,HepG2 cells were transfected with luciferase reporter gene plasmids containing FAS and hormone sensitive lipase(HSL)promoters,respectively,and the effect of Nuci on the activities of FAS/HSL promoters in HepG2 cells were observed.Results:The results of the animal experiments are as follows:1.Exploration of the effective dosage of Nuci and the way of administration in HFD-fed mice.In the first part of the experiment,four groups of mice(n=3～4 in each group),including the Cont group,the Nuci-L group(10 mg/kg),the Nuci-M group(20 mg/kg)and the Nuci-H group(30 mg/kg)were treated with Nuci of different doses by gavage for 8 weeks.Nuci was dissolved in carboxymethyl cellulose sodium.There was no significant difference in body weight,food intake,the amount of WAT and liver between the groups.In addition,Nuci had little effect on blood glucose at each time point and overall blood glucose AUC both in the intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT).In terms of indicators for glycolipid metabolism,serum free fatty acid(FFA)levels in the Nuci-H group was 1.54 times higher than that in the Cont group(1060.7±612.2 mmol/L vs.1628.6±642.8 mmol/L).L,P<0.05),however,other indicators were of no difference between the groups.In the second part of the experiment,four groups of mice(n=3 in each group),including the Cont group,the Nuci-L group(0.06%),the Nuci-M group(0.12%)and the Nuci-H group(0.24%)were fed with HFD mixed with Nuci of different doses for 8 weeks.The body weight of mice in the Nuci-H group were significantly reduced compared with the Cont group mice after 2 weeks of intervention.In addition,the body weight of mice in the Nuci-M and Nuci-L groups were significantly reduced after 4 or 5 weeks of intervention,respectively(P<0.05).Compared with the Cont group,all three doses of Nuci significantly reduced total amount of WAT(P<0.05)in HFD-fed mice.In IPGTT and IPITT,compared to the Cont group,the blood glucose of the mice in the Nuci-M group was significantly reduced at 60 min(P<0.05),and the blood glucose AUC of the Nuci-M group trended to decrease.The serum indicators for glycolipid metabolism showed that the serum total cholesterol(TC)and triglyceride(TG)levels of the Nuci-M group mice tended to decrease.In addition,compared with the Cont group,the fasting blood glucose(FBG)of the Nuci-L and Nuci-M groups decreased by 30.7%and 29.2%,respectively,suggesting that supplementation of 0.12%and 0.24%Nuci can improve glycolipid metabolism in HFD-fed mice.However,it is worth noting that the levels of serum aspartate aminotransferase(AST)in mice treated with different doses of Nuci increased as the concentrations of Nuci increases.Compared with the Cont group,the serum AST levels in the Nuci-L,Nuci-M and Nuci-H groups were increased by 1.28,1.47 and 1.58 times,respectively(P<0.05),suggesting that with the increase of concentration,Nuci may have adverse effects on liver functions.Therefore,it may not be appropriate to choose a relatively higher dose for subsequent experiments.2.The effects of Nuci on HFD-fed miceAccording to the results of the exploratory experiments depicted above,we decided to choose mixing Nuci into HFD as the way for administration,and 0.10%Nuci was selected for subsequent experiments.Male C57BL/6J mice were randomly divided into 4 groups(n=12 in each group),including Litt,Cont,Nuci and Lira groups.The experimental results were as follows:(1)Compared with the HFD group,the body weight of the mice in the Nuci group was decreased significantly after the first week,and the average body weight of mice in the Nuci group was decreased by 28.8%compared with the control group after 12 weeks of intervention(P<0.05).Similarly,the mice in the HFD-Lira group also lost 23.0%(P<0.05)of body weight compared with the HFD group after 12 weeks.Food intake did not differ significantly among the groups.In addition,Nuci significantly reduced the amount of WAT compared to the Cont group by 73.8%(P<0.05)but had little effect on brown adipose tissue(BAT)or liver weight;(2)Compared with HFD group,Nuci reduced serum TC,TG and lowdensity lipoprotein cholesterol(LDL-C)levels by 10.6%,55.9%and 30.1%,respectively(P<0.05).In addition,Nuci and liraglutide significantly reduced the FBG level(P<0.05),effectively improved insulin sensitivity,and increased the serum levels of two adipokines,FGF21 and ZAG compared to the Cont group(P<0.05).There were no significant changes in liver function between the groups.(3)Metabolic chamber monitoring showed that Nuci significantly increased the O2 and CO2 consumption in mice.Compared with the HFD group,the energy expenditure(EE)of the mice in the Nuci group increased by 1.41 times in the light cycle and 1.17 times in the dark cycle(P<0.05),but Nuci had little effect on the respiratory exchange rate(RER)and locomotor activity.(4)Histological analysis of liver showed that Nuci and liraglutide significantly improved HFD-induced hepatic steatosis.Further mechanistic study found that the mRNA expressions of lipogenesis-related genes sterol-regulatory element binding protein 1(SREBP1),FAS,and acetyl-CoA carboxylase(ACC)in the liver of mice in HFD group were significantly increased,reaching 9.67-,3.62-and 6.64-fold of those in the ND group(P<0.05),and Nuci and liraglutide could significantly reduce the mRNA levels of these genes(P<0.05).In addition,Nuci significantly increased the mRNA expressions of key enzymes in lipid catabolism including HSL and adipose triglyceride lipase(ATGL),Peroxisome proliferator-activated receptor α(PPARα),carnitine palmitoyltransferase 1α(CPT1α),and adipokines fibroblast growth factor 21(FGF21)and zinc α2 glycoprotein(ZAG)in the liver(P<0.05).Western blotting showed that the level of AMPK phosphorylation in the liver of mice in the Nuci group was increased to 3.38 times than that of the HFD group.(5)Nuci significantly reduced the amount of eWAT,inhibited the expressions of ACC,FAS and SREBP1 in adipose tissue,and promote the expression of HSL,ATGL,uncoupling protein(UCP1)and adipokines FGF21 and ZAG(P<0.05).Western blotting showed that Nuci promoted the phosphorylation of AMPK in eWAT,which was 2.55 times higher than that of the HFD group(P<0.05).The results of the cell experiments are as follows:1.3T3-L1 preadipocytes:The proliferation of 3T3-L1 preadipocytes was inhibited by Nuci in a dose-and time-dependent manner(P<0.05).The levels of intracellular lipid accumulation after 3,6,9 days of differentiation were significantly reduced by 20 μM Nuci,with TG contents being decreased by 47.2%,59.9%and 55.4%compared to the controls,respectively(P<0.05).In addition,5-20 μM Nuci significantly reduced the mRNA expressions of peroxisome proliferator-activated receptor γ(PPARy),CCAAT/enhancer binding protein α(C/EBPα),CCAAT/enhancer binding protein β(C/EBPβ),FAS,ACC,HSL and ATGL in differentiating preadipocytes by 39.2-92.5%compared with the control group(P<0.05).Besides,the mRNA levels of FAS,ACC and SREBP1 were significantly decreased by 22.6-45.2%,and the expression of adipocytokines FGF21 and ZAG were promoted with administration of 20 μM Nuci(P<0.05).Further mechanistic studies showed that 2.5-20 μM Nuci significantly reduced FAS promoter activity,and also significantly promoted AMPK phosphorylation in adipocytes at the protein level.After pretreatment with CC,a selective AMPK inhibitor,the effects of Nuci on reducing fat accumulation,inhibiting fat synthesis,and promoting the expression of key lipolytic genes were abolished.2.Primary murine mature adipocytesCompared with the HFD group,the size of mature adipocytes derived from eWAT of mice in the HFD-Nuci group was significantly reduced.Besides,oil red O staining showed that the intracellular lipid contents in primary mature adipocytes of mice in HFD-Nuci was significantly reduced compared to the Cont group(P<0.05).3.ADSC cells derived from human visceral adipose tissue:In fully differentiated human primary ADSC cells,the intracellular lipid contents was significantly reduced after treatment of Nuci.Further mechanism studies found that Nuci reduced FAS,ACC,and SREBP1,while increasing FGF21 and ZAG mRNA levels(P<0.05).Similar to the results obtained in 3T3-L1 preadipocytes induced mature adipocytes,Nuci significantly promoted AMPK phosphorylation in ADSC induced mature adipocytes,whereas reduced fat accumulation and inhibited lipid synthesis.However,with pretreatment of the selective AMPK inhibitor CC,the beneficial effects of Nuci were canceled.4.HepG2 cells(1)The viability of HepG2 cells did not alter after treatment of 0-20 μM Nuci for 48 h;(2)With the increase of the concentration of Nuci,the lipid contents in the cells gradually decreased.20 μM Nuci reduced lipid content by 35.8%(P<0.05)compared with the controls.Similarly,the intracellular TG content was also significantly decreased(P<0.05);(3)Similar to the results obtained in liver,20 μM Nuci significantly inhibited the expression of SREBP1,FAS and ACC by 27.7%,27.0%and 29.4%respectively(P<0.05).In the meantime,Nuci also promoted the expression of HSL ATGL,PPARa and CPTla(P<0.05),as for adipokines,Nuci remarkably increased the mRNA levels of FGF21 and ZAG to 2.16-fold and 1.61-fold higher than that of the control group,respectively(P<0.05);(4)Further transient transfection of luciferase reporter gene plasmids containing FAS or HSL promoter was used to explore the possible mechanism by which Nuci regulates the expression of FAS and HSL.The results showed that 20 μM Nuci significantly reduced the luciferase activity of the transfected FAS promoter plasmid.In comparison with the control group which was transfected with the vector plasmid,the luciferase activity of FAS promoter was reduced by 44.2%by Nuci,and the luciferase activity of HSL promoter was upregulated to 1.39 times higher than that of the control group(P<0.05);(5)0.4mM PA was used to induce the establishment of a nonalcoholic steatosis cell model.As expected,oil red O staining and TG contents determination showed that PA could significantly increase intracellular lipid content and significantly reduce AMPK phosphorylation in HepG2 cells(P<0.05).However,treatment of 10-20 μM Nuci notably decreased the intracellular lipid content and the increased phosphorylation level of AMPK(P<0.05).Next,cells were pretreated with the selective AMPK inhibitor CC.The results indicated that CC abolished the facilitation of AMPK phosphorylation and beneficial effects including inhibition of lipid accumulation induced by Nuci(P<0.05).Conclusions:1.Nuci possessed the ability of preventing obesity,reducing the amount of adipose tissue,promoting energy consumption,and improving glucolipid metabolism in HFD-fed mice.2.Nuci improved HFD-induced nonalcoholic steatosis and abnormal adipose tissue expansion.The mechanism may be related to its ability of inhibiting the proliferation and differentiation of preadipocytes,inhibiting intracellular lipid synthesis,promoting lipolysis,fatty acid oxidation and the expression of adipocytokines.3.Nuci reduced lipid contents by activating AMPK signaling pathway.Part 2The effects of Nuci on high-fat diet-induced obesity or genetic obesity and its possible mechanismObjectives:The situation is grim for the prevention and control of obesity.In recent years,the construction of the gut microbiota has been reported to have a major impact on obesity.Nuci is the main component of lotus leaf,a traditional Chinese medicine.Our previous research found that Nuci has the effect of preventing obesity,but whether Nuci exerts beneficial effect on high-fat diet-induced obesity(DIO)or genetic obesity has not been reported yet.Therefore,the purpose of this study was to treat DIO mice and genetically obese ob/ob(spontaneous leptin gene mutation)and db/db mice(spontaneous leptin receptor gene mutation)by Nuci to observe its effects.The intestinal flora and serum metabolomics analysis were performed in ob/ob mice to explore possible mechanisms.Methods:The results of the animal experiments are as follows:1.The effects of Nuci on DIO mice:7-week-old male C57BL/6J mice were adaptively fed for 1 week in prior to the experiment,then randomly divided into three groups and fed with either ND or HFD(to generate diet-induced obesity(DIO)model)for 12 weeks.Next,mice were divided into 3 groups(n=6 in each group):ND control group(ND),HFD control group(HFD),and HFD supplemented with Nuci group(HFD-Nuci).Among them,the high-fat diet lotus Nuci group was given 0.10%1 Nuci in the feed for a total of 6 weeks of intervention.The effects of Nuci on body weight,food intake,amount of WAT,serum indicators for glycolipid metabolism were measured,and leptin sensitivity test was performed.In addition,the expressions of key enzymes involved in fat synthesis and catabolism fatty acid oxidase and the mRNA levels of adipokines in liver and visceral adipose tissue were detected.2.The effects of Nuci on ob/ob mice6-week-old male ob/ob mice were adaptively fed for 1 week and randomly divided into 4 groups(n=7～9 in each group):wild-type control group(Litt),ob/ob control group(Cont),ob/ob Nuci group(Nuci),and ob/ob liraglutide positive control group(Lira).The Nuci group was given 0.10%Nuci mixed in the feed,and the liraglutide group was given subcutaneous injection of 200 μg/kg/d liraglutide.The experiment carried on for 8 weeks.The effects of Nuci on body weight,food intake,amount of WAT,serum indicators for glycolipid metabolism were measured,the serum levels of adipokines and the expressions of key enzymes involved in fat synthesis and catabolism fatty acid oxidase and the mRNA levels of adipokines in liver and visceral adipose tissue.Blood samples were collected for quasi-targeted metabolomic analysis,and cecal contents were harvested for 16S sequencing analysis.3.The effects of Nuci on db/db mice6-week-old male db/db mice were adaptively fed for 1 week,they were fed with ND and were then randomly divided into 3 groups(n=6～8 in each group):wild-type control group(Litt),db/db control group(Cont),db/db Nuci group(Nuci).0.10%lotus leaf alkaloid was added to the diet for the Nuci group and treated for 8 weeks.The effects of Nuci on body weight,food intake,amount of WAT,serum indicators for glycolipid metabolism were measured,and leptin sensitivity test was performed.In addition,the expressions of key enzymes involved in fat synthesis and catabolism fatty acid oxidase and the mRNA levels of adipokines in liver and visceral adipose tissue were detected.Results:1.The effects of Nuci on DIO mice:In DIO mice,treatment of Nuci for 6 weeks significantly reduced body weight gain,inhibited food intake,and decreased liver weight and the amount of WAT(P<0.05).Oral glucose tolerance test(OGTT)found that the blood glucose of Nuci group decreased significantly at 30 and 60 min,the blood glucose AUC was decreased by 20.6%(P<0.05),and its sensitivity was better than IPGTT.The leptin sensitivity of DIO mice was of no significance among the groups.In DIO mice,treatment of Nuci for 8 weeks significantly reduced the serum levels of AST and ALT,reduced the levels of FBG and TC,and increase the level of serum HDL-C(P<0.05).Further studies found that Nuci significantly reduced SREBP1,FAS and ACC mRNA levels in liver and visceral adipose tissue,while increasing glucose transporter(GLUT4)and UCP1 mRNA levels.(P<0.05)2.The effects of Nuci on ob/ob mice(1)Compared with the control group,Nuci reduced weight gain,decreased the amount of WAT,and improved glucose metabolism in ob/ob mice(P<0.05).Nuci also significantly improved liver function(P<0.05).In terms of lipid metabolism,Nuci reduced the levels of TC,TG and LDL-C by 40.0%,38.0%and 58.9%,respectively(P<0.05),and decreased blood glucose by 33.2%(P<0.05).(2)Histological analysis of liver and eWAT showed that Nuci notably reduced the accumulation of lipid in liver and reduce the area of adipocytes in eWAT of ob/ob mice.(3)The 16S sequencing analysis of intestinal flora showed that Nuci significantly altered the microbiota structure of ob/ob mice and reduce the ratio of Firmicutes to Bacteroidota(2.12±0.48 vs.1.10±0.25,P<0.05)and the relative abundance of Actinobacteriota(2.39±0.91 vs.0.25±0.04,P<0.05),and significantly increased the relative abundance of Verrucomicrobiota.In addition,at genus level,the abundances of Akkermansia,Bacteroides and Blautia were significantly increased to 40.9-2.95-and 3.58-fold to the Cont group(P<0,05).besides,the abundance of Lachnospiraceae_NK4A136_group,Alistipe,Helicobacter and Colidextribacter were significantly decreased by 68.7%,58.7%,67.3%and 52.9%compared with the Cont group(P<0.05);(4)The metabolomics analysis showed that serum metabolites in mice were significantly changed after the intervention of Nuci.Further KEGG signaling pathway enrichment analysis showed that the different metabolites between the two groups mainly concerning insulin resistance,biosynthesis of ubiquinones and other terpenoidquinones and mTOR signaling pathway.(5)We further determined the mRNA levels of key genes involved in insulin resistance and mTOR signaling pathways in liver and visceral adipose tissue.The results showed that the mRNA expression of lipinl,a target gene downstream of mTOR that directly regulates lipid biosynthesis,was significantly reduced by 88.5%and 29.8%in liver and visceral adipose tissue,respectively,after Nuci treatment(P<0.05).In addition,the expression of GLUT2 mRNA in liver was decreased,and the expression of GLUT4 in adipose tissue was increased,suggesting that the insulin resistance of liver and adipose tissue was improved.3.The effects of Nuci on db/db miceAfter 5 weeks of intervention,Nuci significantly reduced body weight gain and inhibited food intake in db/db mice(P<0.05).Histological analysis of liver and eWAT showed that Nuci notably reduced the accumulation of lipid in liver and reduce thearea of adipocytes in eWAT of db/db mice.OGTT and IPITT showed that the glucose metabolism of db/db mice were significantly improved(P<0.05).In addition,in db/db mice,Nuci reduced serum TG and FBG levels by 59.3%and 24.4%,respectively(P<0.05),Further studies found that Nuci significantly reduced mRNA levels of SREBP1,FAS and ACC in liver and visceral WAT(P<0.05),while inhibiting glucose 6 phosphatase(G6Pase)expressions in liver,and increased UCP1 expressions in adipose tissue(P<0.05).Conclusions:1.Nuci has the effects of losing weight,reducing lipid contents and improving glycolipid metabolism in different obese murine models,including DIO,ob/ob and db/db mice.2.The anti-obesity and anti-hyperglycemia effect of Nuci was achieved through inhibiting lipid synthesis and regulating glucose transport in the liver and adipose tissue of obese mice.3.Nuci may exert the beneficial effects by regulating the construction of intestinal flora,and through mediating insulin resistance and mTOR signaling pathway.