AQP7 Involved In Ginsenoside-Rb1-Mediated Anti-obestiy Through The Activation Of PPAR-? Pathway

BackgroundObesity has reached epidemic proportions and created serious public health problems in the world.And the obesity population is growing exponentially every year.Obesity causes an increasingly serious impact on the physical and mental state of people,including cardiovascular diseases(CVDs),endocrine dyscrasia,metabolic disorders and neurodegeneration.And among which CVDs is closely related with obesity.Epidemiological and clinical studies have clearly demonstrated a strong link between obesity and the risk factors of CVDs,such as dyslipidemia,hypertension and glucose intolerance,and atherosclerosis.Obesity is defned by excessive body mass index and the accumulation of adipose tissue.Adipose tissue consists of a large number of adipocyte that laden with triglyceride.Masses of intracellular triglyceride accumulation caused an increase in volume and physiologic derangement of adipocyte,leading to a series of related diseses.Currently available antiobesity drugs have limited efficacy and/or safety concerns,and developing safe and effective medicinal agents is badly in need.Despite our failure to control the high prevalence of obesity,we now have a better understanding of its pathophysiology.Aquaporins(AQPs)is an intrinsic membrane protein that forms a “pore” on the cell membrane and controls the flow of water or glycerol,and at least 13 AQPs have been identified.Numerous studies have evidenced a key role of AQPs in adipose tissue biology.And among them AQP7 plays an important role in the transport of water and glycerol in the cell membrane,especially in adipose tissue.AQP7 is closely related to the occurrence and development of obesity,and AQP7 gene knockout mice develop obesity,lipid metabolism disorder and insulin resistance.Recent study showed that PPAR gamma,a peroxides proliferation activated receptor,was involved in the regulation of AQP7.There is a peroxidase proliferator reaction component(PPRE)located in the promoter region of AQPap gene,and the m RNA and protein levels of AQP7 were posilitively correlated with PPAR.Thus,therapeutic strategies targeting the PPAR?/AQP7 pathway may provide a potential approach for better manegement of obesity.Panax ginseng,a traditional herbal medicine,has been widely used to treat various diseases in Eastern Asia for more than 4000 years.As the most abundant active component isolated from panax ginseng,ginsenoside Rb1(Rb1)has shown great potential in antiobesity effects.However,whether ginsenoside Rb1 can regulate the expression of AQP7 through PPAR? to promote lipid transport and reduce body weight remains unclear.Objective(1)To identify the role of ginsenoside Rb1 in obesity;(2)To determine the effect of ginsenoside Rb1 on lipid transport in mature 3T3-L1 adipocytess.(3)To explore whether the anti-obesity effect of Rb1 is achieved through PPAR? and AQP7 pathway.MethodsAnimalsA total of 90 C57BL/6 mice(male,4-5 weeks old)were obtained from Charles River Laboratories Animal Technology Company(Beijing,China).Mice were randomly divided into two groups to receive different diets after one week of adaptive feeding.Part of mice were given normal diet with 5% fat(NC,n=30),and the rest were fed with high-fat diet with 60 % fat(HFD,n=60).All animal experiments are in accordance with the animal management regulations of the ministry of health of China.(Document no.55,2001)and the Guidelines from the national institutes of health conformed to the NIH guidelines(NIH Publication No.85-23,revised 1996).Experiment in miceThe C57BL/6 male mice were randomly divided into six groups after the mice in the HFD group having obesity.The groups were given general food and saline(Chow group)(n=15),general food and Rb1(Chow+Rb1 group)(n=15),and other groups were given high-fat diet and saline(HFD group)(n=15),high-fat diet and Rb1(HFD+Rb1 group)(n=15)(HFD+Rb1),high-fat diet with GW9662 and Rb1(HFD+GW9662+Rb1 group)(n=15),and high-fat diet with DMSO and Rb1(HFD+DMSO+Rb1 group)(n=15)respectively.The treatment concentration of ginsenoside Rb1(B21050.shyuanye,China)was 40mg/Kg,the concentration of GW9662(M6191,sigma US)was 1mg/Kg,and the concentration of DMSO(D8371,solarbio Beijing,China)was 1%.All animal drugs were administered by intraperitoneal injection and the drug was treated for 5 weeks.Body weights were monitored weekly.Intraperitoneal glucose tolerance tests(IPGTT)were conducted at weeks 4 after drug administration.After five-week treatment,mice were sacrificed.Subsequently,subcutaneous adipose tissue and epididymalvisceral adipose tissue were harvested and weighed,and the weight of fat mass divided by the body weight of mice was the body fat rate(BFR).Blood was collected and stored in-80?,and part of organs stored with 4% paraformaldehyde,while the other part was stored in-80 for subsequent experiments.Serum Biochemical analysis and glucose tolerance testsAn IPGTT was conducted at weeks 4 after drug administration.The blood glucose contents were measured from the tail vein of mice at 0,15,30,60 and 120 min after 2g/Kg glucose administration.Blood biochemical test was performed to detect blood lipid(CHO,TG,HDL,LDL,and FFA),blood glucose and liver(AST,ALT)function.Immunohistochemistry and H&E staining analysisThe adipose tissue,which was stored in 4% paraformaldehyde,was buried in paraffin,then the paraffin wax-embedded epididymal adipose tissue prepared from paraffin-embedded samples(5 mm thickness)were stained with H&E and immunohistologically stained,Immunohistochemical staining was were stained with AQP7(AB15568.Merck-Millipore,Germany)and PPAR?,followed by further secondary antibodies(pv-6002.ZSJB-BIO,China)using standard techniques.The positive shadow and cell diameter were analyzed using image-pro Plus 6.0 software(IPP 6.0,Media Cybernetics;Rockville,MD,United States).Cell Culture and TreatmentMouse 3T3-L1 preadipocytes were purchased from ATCC in the United States.The cells were grown in high glucose DMEM with 10% fetal bovine serum at 37°C in a saturated humidity atmosphere of 5% CO 2.The classic cocktail method was used to induce the 3T3-L1 preadipocytes.When more than 90% of the 3T3-L1 preadipocytes were induced into mature 3T3-L1 adipocytes with accumulated lipid droplets in the cytoplasm,it can be used for further study.The optimum concentration was determined by treating with different concentrations of Rb1 for 24 hours.To address the role of PPAR?,the cells were divided into control group,Rb1 group,GW9662+Rb1 group and DMSO+Rb1 group.The dose of GW9662 was 5m M,and the dose of DMSO was 0.01%.The optimum dose of Rb1 was 40?M.Oil Red O Staining and Triglyceride AssayThe lipid droplets in the cytoplasm were measured using Oil Red O.The culture supernatants were collected for detecting the content of free triglyceride by ELISA kit(E1003,applygen,China).Western Blot AnalysisThe total protein of 3T3-l1 adipocytes and mouse epididymal adipose tissue were extracted by total protein extraction kit(P1250,applygen,China).The protein expression levels of AQP7 and PPAR? were detected.The protein were separated by 10% SDS-PAGE gel electrophoresis and then transferred to 0.45?m PVDF membranes(Bio-Rad).Membranes were blocked in 5% skimmed milk powder,then incubated overnight at 4°C with primary antibodies of Aquaporin 7(AB15568,Merck-Millipore,Germany),PPAR?(ab45036,Abcam,UK),p-PPAR?(Ser112)(abs130911a,Absin,China)and Tubulin-?(AF7010,Affinity,US).Secondary antibody(ZSGB-BIO,China)was goat anti-rabbit immunoglobulin G antibody.Statistical AnalysisThe results are expressed as the means ± SEM.Multiple group comparisons were analyzed by ANOVA method followed by Tukey post hoc test,and the significant differences between the mean values of two groups were using unpaired two-tailed Student's t-test.The level of statistical significance was defined as P < 0.05.Results1.Compared with the body weight of the normal diet mice,the high-fat diet mice were 1 to 1.5 times heavier than normal diet mice.The model of simple obesity mouse model was successful.2.Compared with the control group,the body weight of ginsenoside Rb1 treatment obese mice group was significantly decreased(P<0.05),and compared the body weight of HFD + Rb1 group and HFD +Rb1+GW9662 obese mice,The effect of Rb1 on weight loss could be partly reversed by treating with the PPAR gamma specific inhibitor GW9662.3.Compared with the Control group,the body fat rate of obese mice in HFD+Rb1 group was considerably decreased(P<0.01).Compared with Rb1 group,the body fat rate of HFD+GW9662+Rb1 group was significantly increased(P<0.01),and the body fat of the HFD+DMSO+Rb1 group was lower than that of the HFD+GW9662+Rb1 group.4.Glucose tolerance was observed by IPGTT assay.Compared with the Control group,the Rb1 treatment group had better blood glucose regulation ability(P<0.01).After the treatment of GW9662,the ability of regulating blood glucose was considerably reduced(P<0.01).5.The serum levels of free fatty acid in the Rb1 group were significantly lower than other groups(P<0.05),but there was no significant difference in TG,LDL-C,HDL-C,CHO and blood glucose levels.6.Compared with the Control group,the levels of serum AST and ALT in Rb1 group were lower(P<0.05),and the serum levels of AST in GW9662+Rb1 group was higher than Rb1 group(P<0.05),while there are no difference in ATL levels..7.The adipose cell diameter of obese mice in Rb1 treatment group was significantly smaller than that in the Control group(P<0.01)and HFD+GW9662+Rb1 group(P<0.01),the adipose cell diameter of obese mice in HFD+DMSO+Rb1 group was significantly lower than that in HFD+GW9662+Rb1 group(P<0.01).8.The protein expression level of AQP7,PPAR gamma and p-PPAR gamma in HFD+Rb1 group was significantly higher than those in the Control group(P<0.01)and HFD+GW9662+Rb1 group(P<0.01).Compared with HFD+GW9662+Rb1 group and HFD+DMSO+Rb1 group,those protein expression levels were lower than HFD+DMSO+Rb1 group,but there was no significant difference in the levels of activated phosphorylation PPAR gamma(ser112)in the protein of the four groups.9.In vitro experiments,with different concentrations of Rb1,the differentiation and maturation of 3T3-L1 adipocytes were stimulated.Then the optimal concentration of Rb1 was determined as 40umol/L.10.In vitro experiments,the size of lipid droplets of the mature 3T3-L1 adipocytes in Ginsenoside Rb1 treatment group and DMSO+Rb1 treatment group were significantly smaller than control group and GW9662+Rb1 group,and the levels of free triglyceride in the medium of Rb1 group and DMSO+Rb1 group were higher than other two groups.11.Compared with the Control group,ginsenoside Rb1 treatment group promoted the secretion of adiponectin in 3T3-L1 adipocytes and inhibited the secretion of leptin.12.In vitro experiment,the protein expression level of AQP7,PPAR gamma and p-PPAR gamma in Rb1 group was significantly higher than those in the Control group(P<0.01)and GW9662+Rb1 group(P<0.01).Compared with GW9662+Rb1 group and DMSO+Rb1 group,the protein expression levels of the DMSO+Rb1 group were significantly increased(P<0.01).However,there was no significant difference in the levels of p-PPAR gamma/PPAR gamma in the four groups.Conclusions1.Ginsenoside Rb1 significantly reduced the body weight,fat mass weight and the size of adipose cell of obese mice;it also decreased the level of free fatty acid in the serum and protects the liver function of obese mice.2.Ginsenoside Rb1 promotes the excretion of triglyceride in 3T3-L1 adipocytes,improves the endocrine function of 3T3-L1 adipocytes.Ginsenoside Rb1 promotes the secretion of adiponectin,and reduces the secretion of leptin.3.Ginsenoside Rb1 promotes the expression of AQP7 through the PPAR? signaling pathways and finally leading to lipid transport.This study provides new ideas and directions for further exploration and prevention of obesity and related complications.