Yerba mate extract (Ilex paraguariensis)attenuates both central and peripheral
inflammatory effects of diet-induced obesity in rats
Gustavo D.Pimentel,Fábio S.Lira,JoséC.Rosa,Aline V.Caris,Fernanda Pinheiro,Eliane B.Ribeiro,
Cláudia M.Oller do Nascimento,Lila M.Oyama ⁎
Departamento de Fisiologia,Disciplina de Fisiologia da Nutrição,Universidade Federal de São Paulo (UNIFESP/EPM),São Paulo/SP,MA:04023–062,Brazil
Received 29July 2011;received in revid form 21April 2012;accepted 30April 2012
Abstract
To clarify the effects of natural dietary components on the metabolic conquences of obesity,we examined the effects of yerba mate extract Ilex paraguariensis on both central and peripheral inflammatory effects of diet-induced obesity and correlated the hypothalamic tumor necrosis factor (TNF)-αlevel with adipo depot weight.Wistar rats were divided into four groups:a control group (CTL)fed with chow diet,a cond group fed with chow diet plus yerba mate extract (CTL+E),a third gro
up fed with a high-fat diet rich in saturated fatty acids (HFD)and a fourth group fed with HFD plus yerba mate extract (HFD +E).Enzyme-linked immunosorbent assay,Western blotting,colorimetric method and treatment by gavage were utilized as materials and methods.The HFD groups showed a significant increa in food intake (kcal),body weight,adipo tissue and leptin level in comparison to CTL and CTL+E.HFD leads to increa of both central and peripheral inflammatory effects,and deregulation of insulin pathway.In addition,yerba mate extract intake blunted the proinflammatory effects of diet-induced obesity in rats by reducing the phosphorylation of hypothalamic IKK and NF κBp65expression and increasing the phosphorylation of I κB α,the expression of adiponectin receptor-1and conquently the amount of IRS-2.Moreover,the increa in interleukin (IL)-6levels in the liver and muscle and of the IL-10/TNF-αratio in groups that received yerba mate extract showed the anti-inflammatory effects of this natural substance.Taken together,our data suggest that the u of yerba mate extract may be uful for reducing low-grade obesity-associated inflammation.©2012Elvier Inc.All rights rerved.
Keywords:Yerba mate extract;Diet-induced obesity;High-fat diet;Inflammation;Cytokines;Insulin resistance;Rat
1.Introduction
脱生
The increa in prevalence of obesity-associated dias such as type 2diabetes mellitus,dyslipidemia and cardiovascular dia ems to be intimately related to low-grade in flammation [1–4].Moreover,veral studies have shown that the intake of a high-fat diet rich in saturated fatty acids increas in flammation and insulin resistance as well as rum leptin and adipo mass [5–9].
红豆功效Some of the mechanisms involved in obesity-associated in flam-mation are linked with the first pha of defen against in fl,the activation of nuclear factor kappa B (NF-κB)and the resulting increa in proin flammatory cytokines such as tumor necrosis factor (TNF)-αand interleukin (IL)-1β.The involvement of toll-like receptors (TLRs)at this stage is also notorious [10–14].TLR4is considered part of the “front line ”of the immune system against pathogens and is responsible for stimulating adaptive immunity and resolving in flammation [10,11,13,15].
In flammatory process can also be activated by the mammalian target of rapamycin (mTOR)pathway.Moreover,the activation of NF-
κB,TLR4and mTOR results in attenuation of insulin signaling and insulin resistance in veral tissues [7,16–19].
Acting as cellular energy nsors,adenosine-monophosphate-activated protein kina (AMPK)and adiponectin act in peripheral tissues and the central nervous system by modulating food intake.Conquently,the inhibition of hypothalamic AMPK activity and an increa in adiponectin levels reduce food intake [20–25].In peripheral tissues,the activation of AMPK and adiponectin favors gluco uptake and reduces level of reduction of proin flammatory cytokines [26–28].
The consumption of foods and beverages rich in phenolic compounds plays an important role in preventing chronic dias [29–35].Although the biological effects of yerba mate (Ilex paraguariensis )have been studied and its antiobesity effects discusd,only speci fic tissues have been considered,rather than the whole animal [32,34,35].Moreover,the molecular mechanisms underlying its antiobesity effects have not been totally clari fied.In order to shed light on the effects of natural dietary components on the metabolic conquences of obesity,we examined the effects of supplementation of yerba mate extract on the central and peripheral in flammatory effects of diet-induced obesity and correlated the hypothalamic TNF-αlevels with adipo depot weight.
Available online at
Journal of Nutritional Biochemistry xx (2012)xxx –
相亲失败
xxx
⁎Corresponding author.Fax:+551155739525.E-mail address:lmoyama@unifesp.br (L.M.Oyama).
0955-2863/$-e front matter ©2012Elvier Inc.All rights rerved.dx.doi/10.1016/j.jnutbio.2012.04.016
2.Methods
2.1.Animals
The Experimental Rearch Committee of the Federal University of Sao Paulo approved all procedures for the care of the animals ud in this study(protocol no. 2009/1962).Rats were kept under controlled conditions of light(12-h:12-h light–dark cycle with lights on )and temperat
ure(22°C±1°C)at60%±5%humidity and with free access to food and water.
A total of29male Wistar rats aged8weeks and weighing between200and250g were randomly distributed into four groups.They were houd in groups of three to four per cage,with a chow diet(2.8kcal/g,15%kcal from fat,Nuvilab,Brazil)or high-fat diet(4.0 kcal/g,45%kcal from lard fat,Sadia S/A,Brazil)and water ad libitum.The control group (CTL,n=7)was fed with a chow diet plus intragastric administration(gavage)of water. The yerba mate extract group(CTL+E,n=7)was fed with a chow diet plus intragastric administration of yerba mate extract(Leão Jr,Paraná,PR,Brazil)diluted in water.The high-fat group(HFD,n=8)was fed with a diet rich in saturated fatty acids plus intragastric administration of water.The fourth group of rats was fed with HFD plus intragastric administration of yerba mate extract(HFD+E,n=7).Yerba mate extract was given daily at the same time(during the light period)for2months(during the third and fourth month of age),with the administration of0.01g of yerba mate extract/day in the third month(100μl of a solution of0.1g/ml)and0.02g of yerba mate extract/day in the fourth month.The volumes and concentrations were utilized in a previous study[33]. The same volume of water was offered to control groups fed with chow or high-fat diet.
2.2.Diets
HFDs included20%cain(w/w),10%sucro(w/w)and butylated hydroxyto-luene at a rate of0.02%(w/w)of additional oil.The rats received the above-mentioned diets for8weeks.
The fatty acid composition of the chow and high-fat diet diets was determined as previously described[4]and is shown in Table1.The data showed a high percentage of polyunsaturated fatty acids(PUFAs)(C18:2n6)[mainly linoleic acid(LA)],monoun-saturated fatty acids(C18:1n9)(mainly oleic acids)and saturated fatty acids(C16:0) (mainly palmitic acid);the high-fat diet contained high percentages of n-7 monounsaturated(C18:1n7),PUFAs(C18:2n6LA)and saturated fatty acids(C18:0) (mainly stearic acids).
2.3.Lipid profile measurements
Approximately15–18h after oral gavage with the yerba mate extract and after a 12-h fast,the animals were euthanized by decapitation,blood was collected,and rum samples were collected after allowing the blood to clot on ice.Serum was stored frozen at−80°C for analysis.Labtest kits were ud to asss fasting total cholesterol,high-density lipoprotein(HDL-c)and triacylglycerol(TG)levels.The samples were analyzed using an enzymatic method.Low-density lipoprotein(LDL-c)and very low density lipoprotein(VLDL-c)were calculated according to the Friedewald equation[(LDL-c=total cholesterol−(HDL-c)−(TG/5)and(VLDL=TG/5)][36].
2.4.Serum hormone levels
Serum fasting insulin and leptin levels were quantified using enzyme-linked immunosorbent assay(ELISA).The kits were obtained from Millipore Corp.(Bedford, MA,USA).
2.5.TNF-α,IL-6and IL-10protein level determined by ELISA
Following decapitation,the hypothalamus,retroperitoneal and epididymal fat, liver and gastrocnemius muscle were removed,discted,homogenized and centrifuged at12,000g for40min at4°C;the supernatant was saved,and the protein concentration was determined using the BCA assay(Bio-Rad,Hercules,CA,USA),using bovine rum albumin(BSA)as a reference.Quantitative asssment of TNF-α,IL-6and IL-10proteins was carried out via ELISA(DuoSet ELISA,R&D Systems,Minneapolis,MN, USA)following the recommendations of the manufacturer.All samples were run in duplicate,and the mean value was reported.牛肉面做法
2.6.Protein analysis by Western blotting
After euthanasia,the hypothalami were discted and homogenized in1.0ml of solubilization buffer at4°C[1%Triton X-100,100mm Tris–HCl(pH7.4),100mm sodium pyrophosphate,100mm sodiumfluori
de,10mm EDTA,10mm sodium orthovanadate,2.0mm phenylmethylsulfonylfluoride and0.1mg aprotinin/ml]using a Polytron(model713T;Fisatom Equipamentos Científicos,São Paulo,SP/Brazil). Insoluble material was removed by centrifugation for30min at9000g in a70Ti rotor (Beckman,Fullerton,CA,USA)at4°C.The protein concentration of the supernatants was determined using the BCA assay(Bio-Rad,Hercules,CA,USA).Proteins were denatured by boiling(5min)in a Laemmli sample buffer[37]containing100mM DTT and were run on8%,10%or12%sodium dodecyl sulfate polyacrylamide gel electrophoresis in a Bio-Rad miniature slab gel apparatus.
The proteins were electrotransferred from gels to nitrocellulo membranes for ~1.30h/4gels at15V(constant)in a Bio-Rad midry transfer apparatus.Nonspecific protein binding to the nitrocellulo was reduced by preincubation for2h at22°C in blocking buffer(5%nonfat dry milk,10mM Tris,150mM NaCl and0.02%Tween20). The nitrocellulo membranes were incubated overnight at4°C with antibodies against IR,IRS-1,IRS-2,PKC,Akt/PKB,AMPK,FAS,Adipo-R1,TNFα-R1,IL-6-R1,IL10-R1,TLR2, TLR4,MyD88,TRAF6,NFκBp50,NFκBp65,phospho IKK,phospho IκBαand alpha-tubulin obtained from Santa Cruz Biotechnology(Santa Cruz,CA,USA)and mTOR obtained from Cell(Cell Signaling Technology,Inc.,MA,USA)diluted1:1000with blocking buffer supplemented with1%BSA and then washed for30min in blocking buffer without BSA.The blots were subquently i
ncubated with peroxida-conjugated condary antibody for1h at22°C.To evaluate protein loading,the membranes were stripped and reblotted with an anti-alpha-tubulin antibody as appropriate.Specific bands were detected by chemiluminescence,and visualization/ capture was performed by exposure of the membranes to RXfilms.Band intensities were quantified by optical densitometry of developed autoradiographs(Scion Image software,Scion Corporation,Frederick,MD,USA).
2.7.Statistical analysis
孔乙己主题The results were analyzed using the GraphPad Prism statistics software package version5.0for Windows(GraphPad Software,San Diego,CA,USA).The data are expresd as the means±S.E.M.and were distributed normally according to the Table1
Fatty acid composition of the control and high-fat diets
Fatty acids%Total fatty acids
Control diet High-fat diet C14:00.114 1.025
C14:1c0.0250.014
C15:00.0480.011
C15:00.013
C16:013.3720.96
C16:1n70.206 1.878
C16:4n10.053
C17:00.038
C17:00.0910.287
C16:3n40.221
C16:4n10.0530.094
C18:0 2.94510.069
C18:1n735.615购买的英文
工作经历怎么写C18:1n924.128 2.345
C18:1n11 1.189
C18:2n6LA49.45322.811
C18:2n6t0.131
C18:3n60.02
C18:3n40.026
C18:3n3LNA 3.731 1.398
C20:0iso0.4190.032
C18:4n30.0550.138
C20:00.4190.209
C20:1n90.674
C20:2n60.0490.687
C20:4n6AA0.095
C20:3n60.088
C21:00.245
C20:3n30.088
C20:4n30.0940.199
C20:5n3EPA 1.9710.024
C21:5n30.074
C22:00.0420.085
C22:2n60.0410.207
C22:4n60.104
C22:5n60.185
C22:5n30.2190.039
C22:6n3DHA 1.138
C23:0.072
C24:00.3360.04
C24:10.037
Total100100
ΣSFA17.44032.99
ΣMUFA25.58540.526
ΣPUFA56.97326.46
ΣPUFA n-649.63824.233
ΣPUFA n-37.282 1.886
n3/n6ratio 6.81612.848
2G.D.Pimentel et al./Journal of Nutritional Biochemistry xx(2012)xxx–xxx
Kolmogorov –Smirnov test.The data were analyzed using analysis of variance for comparison between four groups.A value of P b .05was considered statistically signi ficant.
3.Results 3.1.Food intake
A signi ficant increa in absolute food intake (g/week)was obrved from the cond to the eighth week of treatment in the CTL and CTL+E groups in comparison with the HFD and HFD+E groups (Fig.1A).However,the HFD and HFD+E groups showed higher caloric food intake (kcal/week)than the CTL and CTL+E groups (Fig.1).
3.2.Body and adipo depot weight
Body weight was higher in the HFD and HFD+E groups than in the CTL and CTL+E groups.Moreover,it was similar in the CTL and CTL+E groups and the HFD and HFD+E groups (Fig.1B).
Exposure to the HFD caud an increa in the retroperitoneal and epididymal adipo depots in comparison with the control diet.The yerba mate extract did not prevent the increa in adipo depots (Fig.1C,D).The liver and muscle weight did not differ between groups (Fig.1E,F).
3.3.Serum metabolic profile
The rum lipid pro file (total cholesterol,LDL-c,HDL-c,VLDL and triacylglycerols)and rum fasting insulin did not differ between treatments (Fig.2A –F).However,leptin levels were signi ficantly higher in the HFD and HFD+E groups than in the CTL and CTL+E groups (Fig.2G).
3.4.Cytokine profile in the central nervous system and peripheral tissues 3.4.1.TNF-α
石头城是对我国哪座城市的美称
TNF-αlevels were similar in the hypothalamus and retroperito-neal adipo tissue among groups (Fig.3A,B).However,the TNF-αconcentration in the epididymal adipo tissue was higher in the HFD and HFD+E groups than in the CTL and CTL+E groups (Fig.3C).In the liver,the TNF-αlevels were higher only in the CTL+E group when compared to the CTL group (Fig.3D).In the muscle,higher TNF-αlevels were found in the CTL+E group than in the CTL group and in the HFD group in comparison with the CTL group (Fig.3E).
3.4.2.IL-6
IL-6concentrations were similar in the hypothalamus,retroper-itoneal and epididymal adipo tissue and liver in all groups (Fig.3F –I).However,the CTL+E group had higher IL-6levels in the muscle than the CTL group.Moreover,the HFD+E group had higher IL-6expression than the CTL group (Fig.3J).
3.4.3.IL-10
IL-10levels were similar in the hypothalamus,retroperitoneal and epididymal adipo tissue and muscle in all groups (Fig.3K –M and O).However,the CTL+E group had higher IL-10levels in the liver than the CTL group.In addition,the HFD+E group had higher IL-10expression than the HFD group,but the HFD group had lower IL-10concentrations than the CTL group (Fig.3N).
3.4.4.IL-10/TNF-αratio
Calculation of the IL-10/TNF-αratio in order to identify any possible increa in the anti-in flammatory effect of yerba mate extract showed an increa in the ratio in the hypothalamus in the CTL+E group compared with the CTL group (Fig.3P).In both retroperitoneal adipo and liver tissues,the ratio was incread in the HFD+E group compared with the CTL,CTL+E and HFD groups (Fig.3Q,S).In the epididymal adipo tissue,the IL-10/TNF-αwas
Fig.1.Determining the food intake,body,adipo,liver and muscle weight.CTL (n =7):rats fed with a chow diet plus intragastric administration (gavage)of water;CTL+E (n =7):rats fed with a chow diet plus intragastric administration of yerba mate extract;HFD (n =8):rats fed with a high-fat diet rich in saturated fatty acids plus intragastric administration of water;HFD+E (n =7):rats fed with HFD plus intragastric administration of yerba mate extract.Data are expresd in means±S.E.M.*P b .05vs.CT
L or HFD groups;#P b .05vs.CTL or CTL+E.
3
G.D.Pimentel et al./Journal of Nutritional Biochemistry xx (2012)xxx –xxx
higher in the CTL+E group than in the CTL group(Fig.3R).In the muscle,inclusion of yerba mate extract in the control diet and the HFD incread the IL-10/TNF-αratio in comparison with the CTL and HFD diets that did not include yerba mate extract(Fig.3T).
3.5.Insulin signaling,mTOR,AMPK,FAS and adiponectin receptor-1 (AdipoR1)expression in the hypothalamus
In order to determine whether the reduction in inflammatory status in both central and peripheral tissue may also improve insulin signaling,we evaluated IR,IRS-1,IRS-2,PKC and Akt/PKB expression and only found changes in IRS-2levels,with an increa in the HFD+E group compared to the HFD group(Fig.4C).IR(Fig.4A),IRS-1 (Fig.4B),PKC(Fig.4D)and Akt/PKB(Fig.4E)levels did not differ between groups.
To clarify the increa in caloric intake in the HFD and HFD+E groups in comparison with the CTL an
d CTL+E groups,we evaluated the hypothalamic expression of some of the proteins related to appetite regulation,including mTOR,AMPK,FAS and AdipoR1.The expression of thefirst three was similar in all groups(Fig.4F–H),but AdipoR1expression was lower in the HFD group compared to the CTL and CTL+E groups,and higher in the HFD+E group than the HFD group(Fig.4I).Therefore,we hypothesize that food intake was modulated by adiponectin.
3.6.Inflammatory signaling in the hypothalamus
Inflammatory signaling was evaluated by examining the expres-sion of cytokine receptors(TNF-α,IL-6and IL-10),NFκBp50,NFκBp65, TLR2,TLR4,TRAF6and MyD88and the phosphorylation of IKK and IκBα.
Consumption of yerba mate extract reverd the increa in phosphorylation of IKK in both the CTL and HFD groups.In addition, the HFD group showed higher pIKK than the CTL and CTL+E groups (Fig.5A).There was also an increa in IκBαphosphorylation in both the CTL+E and HFD+E groups(Fig.5B).
NFκBp65and TLR4expression was higher in the HFD than the CTL and CTL+E groups.Furthermore,yerba mate extract reduces NFκBp65and TLR4expression in the HFD+E group
compared to the HFD group(Fig.5D,F).
TNFα-R1expression was only reduced in the CTL+E group compared to the CTL group(Fig.5I).Moreover,NFκBp50,TLR2, TRAF6,MyD88,IL6-R1and IL10-R1expression was similar among all studied groups(Fig.5C,E,G,H,J,K).
3.7.Relationship between retroperitoneal adipo depot weight and hypothalamic TNF-αlevels
In order to determine whether the retroperitoneal adipo depot was responsible for supplying and possibly increasing the hypotha-lamic TNF-αlevels,we performed a correlation analysis to identify any connection between the two and found that the increa in retroperitoneal adipo depot was responsible for the increa in hypothalamic TNF-αlevels in both the CTL and HFD groups(Fig.6A, C).Surprisingly,the yerba mate extract prevented this association between retroperitoneal adipo tissue weight and hypothalamic TNF-αexpression(Fig.6B,D).
3.8.Relationship between retroperitoneal and epididymal adipo depot weight and rum fasting insulin and leptin levels
With the aim of identifying whether the retroperitoneal adipo depot was responsible for supplying a
nd possibly increasing the rum fasting insulin and leptin concentrations,we performed correlations to determine the possible associations.The data showed a positive correlation between retroperitoneal fat and fasting insulin(r=0.74,P=.05)in the CTL+E group(Supplementary File1B) and epididymal fat with fasting insulin(r=0.81,P=.02)in the CTL
Fig.2.Serum lipid profile and fasting insulin and leptin levels.Data are expresd in means±S.E.M.*P b.05vs.CTL or HFD groups;#P b.05vs.CTL or CTL+E. 4G.D.Pimentel et al./Journal of Nutritional Biochemistry xx(2012)xxx–xxx
