Combined high-fat diet and sustained high sucrose consumption promotes NAFLD in a murine model

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1 ORIGINAL ARTICLE July-August, Vol. 14 No. 4, 215: Combined high-ft diet nd sustined high sucrose consumption promotes NAFLD in murine model Gonzlo Torres-Villlobos,*, Nshl Hmdn-Pérez,* Armndo R. Tovr, Guillermo Ordz-Nv, Brulio Mrtínez-Benítez, Iván Torre-Villlvzo, Sofí Morán-Rmos, Andre Díz-Villseñor, Lili G. Norieg, Mrci Hirirt, Roberto Medin-Sntillán, Mrí del Crmen Cstillo-Hernndez, Nhum Méndez-Sánchez, Misel Uribe, Nimbe Torres * Deprtmento de Cirugí Experimentl, Instituto Ncionl de Ciencis Médics y Nutrición Slvdor Zubirán, Mexico City. Mexico. Deprtmento de Posgrdo e Investigción, Instituto Politécnico Ncionl, Escuel Superior de Medicin, Mexico City. Mexico. Deprtmento de Fisiologí de l Nutrición, Instituto Ncionl de Ciencis Médics y Nutrición Slvdor Zubirán, Mexico City. Mexico. Deprtmento de Ptologí, Instituto Ncionl de Ciencis Médics y Nutrición Slvdor Zubirán, Mexico City. Mexico. Instituto de Fisiologí Celulr, Universidd Ncionl Autónom de México, Mexico City. Mexico. Liver Reserch Unit. Medic Sur Clinic & Foundtion. Mexico City, Mexico. ABSTRACT Bckground. The study of NAFLD in humns hs severl limittions. Using murine models helps to understnd disese pthogenesis. Aim. Evlute the impct of 4 different diets in the production of NAFLD with emphsis on combined high-ft plus sustined high sucrose consumption. Mteril nd methods. Eight week-old mle Wistr rts were divided in four groups nd fed for 9 dys with the following diets: 1) Control chow diet (C); 2) High-ft cholesterol diet (HFC) + 5% sucrose in drinking wter. 3) High-ft cornstrch diet (HFCO) + 5% sucrose in drinking wter. 4) Chow diet + 2% sucrose in drinking wter (HSD). Metbolic chnges, leptin levels, liver histology, heptic nd plsm lipid composition, fsting plsm glucose nd insulin nd liver gene expression of FAS, SREBP-1 nd PPAR-α were evluted. Results. The HFC diet hd the highest grde of stetosis (grde 2 of 3) nd HSD showed lso stetosis (grde 1). Liver weight TG nd cholesterol concentrtions in liver were greter in the HFC diet. There were no incresed levels of iron in the liver. Rts in HFC gined significntly more weight (P <.1). All experimentl groups showed fsting hyperglycemi. HFC hd the highest glucose level (158.5 ± 7 mg/dl) (P <.5). The HSD nd the HFCO diets developed lso hyperglycemi. HSD hd significntly higher fsting hyperinsulinemi. Serum leptin ws higher in the HFC diet (p =.1). In conclusion, the HFC diet with combintion of high ft nd high sucrose is more effective in producing NAFLD compred with high sucrose diet only. Key words. Nonlcoholic ftty liver disese. Obesity. Animl model. Diet. INTRODUCTION Correspondence nd reprint request: Nimbe Torres, M.D. Deprtmento de Fisiologí de l Nutrición, Instituto Ncionl de Ciencis Médics y Nutrición Slvdor Zubirán, Mexico City, 14. E-mil: nimbet@quetzl.innsz.mx Mnuscript received: September 17, 214. Mnuscript ccepted: December 14, 214. Non-lcoholic ftty liver disese (NAFLD) is n importnt cuse of liver disese nd is emerging s the most common chronic liver condition in the Western World. 1 It my progress to non-lcoholic stetoheptitis (NASH), fibrosis, cirrhosis nd occsionlly to heptocellulr crcinom. 2 Despite the high prevlence of NAFLD nd its ssocited consequences, the underlying etiologic fctors tht determine disese progression re poorly understood. 3 The study of NAFLD with humn tissue hs severl limittions becuse the occurrence nd progression of NAFLD requires long period of time, it is difficult to obtining tissue smples, there is limited bility to define cuses nd effects nd ethicl limittions exists. 4 Rodent biologicl processes re similr to those in mn nd using murine models help us to understnd the disese pthogenesis. Up to the present time, no niml model completely reproduces the histopthology nd pthophysiology of humn NAFLD/NASH. The use of niml models with deletion or over-expression of single gene, my not mimic the etiology of humn disese becuse NAFLD is complex pthology with mny influencing fctors. Also,

2 541 Combined high-ft diet nd sustined high sucrose consumption promotes NAFLD in murine model., 215; 14 (4): experimentl diets ssocited with development of NAFLD my not completely simulte the humn diets, but they re theoreticlly desirble for their similrity to the humn condition. The rodent model should exhibit lso metbolic bnormlities such s obesity, insulin resistnce, fsting hyperglycemi, dyslipidemi nd ltered dipokine profile. Thus the more of the previously mentioned metbolic criteri re met, the more intrinsiclly useful the model becomes. 5 A chronic high-ft diet or sustined high fructose consumption diet hve been used s models of the metbolic syndrome with histologicl fetures of mild-moderte NAFLD/NASH. 6 A problem with the high-ft diet, is tht rodents my dpt to high-ft feeding nd become resistnt to obesity development, nd/or other metbolic bnormlities. 7 The combintion of high-ft diet nd sustined high sucrose consumption my resemblnce modern humn diet. There hve been repots tht NAFLD is ssocited with heptic iron overlod, indicting tht iron-induced oxidtive stress my be relted to NAFLD pthology. 8 The ims of the present work were to chrcterize nd compre liver histology, heptic nd plsm lipid composition, iron liver content, fsting plsm glucose nd insulin chnges, leptin levels nd liver gene expression of ftty cid synthse (FAS), Sterol regultory element-binding protein-1 (SREBP-1) nd peroxisome prolifertor-ctivted receptor lph (PPAR-α) in 4 different types of diets with emphsis on combined high-ft plus sustined high sucrose consumption. MATERIAL AND METHODS The niml protocol ws pproved by the Animl Committee of the Ntionl Institute of Medicl Sciences nd Nutrition, ccording to the Guide for the Cre nd Use of Lbortory Animls. Animls Eight week-old mle Wistr rts weighing n verge of 22 g were obtined from the Experimentl Reserch Deprtment nd Animl Cre Fcility t the Instituto Ncionl de Ciencis Médics y Nutrición, Mexico City. All nimls were housed in smll individul stinless-steel cges in controlled temperture nd humidity environment with 12-h light/12-h drk cycle nd hd free ccess to wter nd food throughout the study. An importnt spect is tht the rts were essentilly sedentry in smll cges with little opportunity for physicl ctivity, except to et nd drink. After 1-week cclimtion period, the rts were rndomly divided in four groups of five nimls ech group nd fed for 9 dys with one of the following diets: Control chow diet (C) (stndrd rodent chow pellets contining 13.4% ft, 28.5% protein, nd 57.9% crbohydrte by clories; Purin, Richmond, IN). High-ft cholesterol diet (HFC) contining 45% ft (lrd), 2.7% csein protein nd 34.1% crbohydrte with dextrose (29%), sucrose, cellulose nd inulin (1.72%) + 5% sucrose in drinking wter. High-ft cornstrch diet (HFCO) mde with chow diet dded with 2% of lrd, contining 5% ft, 16.5% protein nd 33.5% crbohydrte with strch (25.52%) + 5% sucrose in drinking wter. Chow diet (13.4% ft, 28.5% protein, nd 57.9% crbohydrte) + 2% sucrose in drinking wter (HSD). 9 High-ft diets were weekly prepred in our lbortory in pellets nd stored t 4 C. Body weight nd food intke were recorded every dy. At dy 3 nd 6, nd fter n overnight fst, blood smple ws collected from the til vein for mesurement of serum biochemicl vribles. At the end of the study (dy 9), body weight ws recorded nd the rts were killed fter n overnight fst. The rts were killed by decpittion fter CO 2 nesthetiztion. Blood ws collected in tubes with gel nd clot ctivtor (BD) nd centrifuged t 1,5 x g for 1 min; serum ws seprted nd stored t -7 C until further nlysis. Liver lipid content nd histologicl nlysis During euthnsi the liver ws removed, weighed nd immeditely frozen nd stored t -8 C. Heptic lipids were extrcted with chloroform methnol using the Folch method. 1 From the extrcts, heptic cholesterol nd TG concentrtions were mesured using commercilly vilble enzymtic colorimetric kits (DiSys Dignostic Systems Interntionl, Holzheim, Germny). Before cryopreservtion, liver tissue smples were tken nd fixed in formlin for hemtoxylin-nd-eosin nd Msson-trichrome stining. A pthologist

3 542 Torres-Villlobos G, et l., 215; 14 (4): blindly clssified the grde of stetosis ccording to Kleiner nd Brunt. 11 Prussin blue stin ws performed to identify iron in the liver. Quntittive rel-time PCR Totl RNA ws extrcted from heptic tissue. 12 After RNA extrction integrity ws confirmed by gel electrophoresis, concentrtion nd purity ws mesured by espectophotometry with Nnodrop ND- 1. cdna synthesis ws crried out with M-MLV reverse trnscriptse nd oligo(dt)12e18 primer (Invitrogen, Crlsbd, CA). mrnas expression ws mesured by rel-time quntittive PCR using Tqmn Universl Mster Mix (Applied Biosystems/ Roche, Brnchburg, NJ). RT-PCR ssys for ech gene were crried out in triplicte using 1 ng of reverse trnscriptse product in 96-well opticl pltes with n ABI Prism 7 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR scheme used ws 5 C for 2 min, 95 C for 1 min, nd then 4 cycles of 95 C for 15 sec followed by 6 C for 1 min. The 18S ribosoml RNA ws used s n invrint control for liver smples ( E). TqMn fluorogenic probes/oligonucleotide primers were obtined from Applied Biosystems for the following genes: ftty cid synthse (FAS; Rn569117_m1), peroxisome prolifertor-ctivted receptor- (PPAR;Rn566193_m1), sterol regultory element binding protein-1 (SREBP-1;Rn _m1). The reltive mounts of mrna were clculted by using the ΔΔCt method with n efficiency djustment ccording to the Pfffl eqution. 13 Serum mesurements Serum glucose ws mesured with the YSI select 27 Biochemistry Anlyzer (YSI Incorported). Triglyceride (TG) nd cholesterol concentrtions were mesured using n enzymtic-photometric ssy kit (DiSys Dignostic Systems). Serum insulin ws nlyzed by rdioinmunossy kit (Linco Reserch Immunossy nd Millipore) ccording to the mnufcturer instructions. Serum leptin ws nlyzed using RAT LEPTIN ELISA KIT 96-Well Plte, Millipore-USA (Ct. #EZRL- 83K). This ssy is Sndwich ELISA bsed, sequentilly, on: Cpture of leptin molecules from smples to the wells of microtiter plte coted with monoclonl nti-leptin ntibody. Wshing of unbound mterils from smples. Binding of second biotinylted monoclonl nti-leptin ntibody to the cptured molecules. Wshing of unbound mterils from smples. Binding of streptvidin-horserdish peroxidse conjugte to the immobilized biotinylted ntibody. Wshing of excess free enzyme conjugtes, nd Quntifiction of immobilized ntibody-enzyme conjugtes by monitoring horserdish peroxidse ctivities in the presence of the substrte 3,3,5,5 -tetrmethylbenzidine. The enzyme ctivity ws mesured spectrophotometriclly to 45 nm bsorbnce (Epoch. Biotek-USA). Sttisticl nlyses Results re presented s men ± SEM. Dt tht were not normlly distributed were logrithmiclly trnsformed prior to nlysis. Differences mong experimentl groups were evluted by one-wy ANO- VA followed by Bonferroni s multiple comprison test, nd differences were indicted with different letters in ech figure (>b>c>d). Differences were considered sttisticlly significnt t P <.5. All sttisticl nlyses were conducted using GrphPd Prism 5. (Sn Diego, CA). RESULTS Histologicl nlysis of the liver Histologicl nlysis with hemtoxylin-nd-eosin stining showed: HFC diet hd grde 2 (grde 2/3: 34-66% of heptocytes) microvesiculr nd mcrovesiculr stetosis in zone in 3 in ll rts. The higher-grde of stetosis in the HFC diet, correltes with higher liver weight, higher heptic TG nd cholesterol levels nd lso with high circulting leptin levels showed in this diet. HSD hd grde 1 stetosis (grde 1/3: 5-33% of heptocytes) 11 in ll nimls (Figure 1). No stetosis developed in the HFCO or C diets. Prussin blue stin ws negtive in both heptocytes nd ducts. This shows tht the exmined tissue hve no incresed levels of iron in the liver. Serum nd heptic lipid concentrtions Rts fed with the HFC diet hd serum cholesterol concentrtion (7.78 ± 6.3 mg/dl) tht ws pproxi-

4 543 Combined high-ft diet nd sustined high sucrose consumption promotes NAFLD in murine model., 215; 14 (4): Figure 1. Grde of liver stetosis. Histologic nlysis of livers with hemtoxylin-nd-eosin stining showed higher stetosis in the HFC (grde 2 of 3: 34-66% of heptocytes) but lso stetosis in the HSD (grde 1 of 3: 5-33% of heptocytes). 1 C: control chow diet. HFC: high-ft cholesterol. HFCO: high-ft cornstrch. HSD: high sucrose diet. G: grde. Tble 1. TG, cholesterol nd liver weight. Liver weigh (g) TG (mg/g) 7.75 ± ±.42* 12.8 ± ± 1.1 Cholesterol (mg/g) 21.4 ± ± 1.6** 26.1 ± 3.8* ± 1.3 Liver weight TG nd cholesterol concentrtions were significntly higher in rts with the HFC diet compred with the other groups. * p <.5. ** p <.1. mtely 25% higher thn the C diet (53.23 ± mg/ dl) nd higher thn the other groups. The serum cholesterol level in the HSD ws lso 16% higher (63.58 ± 9.9 mg/dl) tht the C diet. Interestingly, serum cholesterol level in the HFCO (42.43 ± 5.2 mg/dl) diet ws pproximtely 21% lower thn tht of C diet (p =.4). Compred with ll groups the HSD diet developed the highest plsm TG concentrtion ( ± 32 mg/dl) nd it ws more thn twice compred with C diet (74.69 ± 15.2 mg/dl, P =.2). TG serum concentrtions of the HFC ( ± 4.9 mg/dl) diet ws 41% greter thn C nd HFCO ws similr to the C diet (75.41 ± 16.4 mg/dl). Liver weight, TG nd cholesterol concentrtions in liver were significntly higher in rts with the HFC diet compred with the other groups (Tble 1). Heptic gene expression of SREBP-1, FAS nd PPAR-α To study the effect of the different diets on the expression of lipogenic nd ftty cid oxidtion genes, we mesured the mrna of the trnscription fctor SREBP-1 nd its trget gene FAS in the liver (Figure 2A nd 2B). Interestingly, the HSD group expressed significntly higher mounts of SREBP-1 thn the other groups. FAS expression followed similr trend with higher mounts in the HSD diet. In the other groups tested, just the HFC diet produced significnt increse in the expression of SREBP-1 compred with C. The expression of the trnscription fctor PPARα which is involved in ftty cid oxidtion, is shown in figure 2C.

5 544 Torres-Villlobos G, et l., 215; 14 (4): A B C b b 6 2 c bc 4 1 d b b b.5 b 2. mrna (Reltive units) mrna (Reltive units) mrna (Reltive units) Figure 2. A. SREBP-1c expression. B. FAS expression. C. PPAR-α expression. Reltive expression of SREBP-1 (A), FAS (B) nd PPAR-α (C) in liver compring four different diets. Expressions were higher in the HSD group. C: control chow diet. HFC: high-ft cholesterol. HFCO: high-ft cornstrch. HSD: high sucrose diet. Insulin (ng/ml) b b Figure 3. Fsting plsm insulin t dy 9. Fsting plsm insulin level in four different diets. The HSD developed higher fsting hyperinsulinemi thn the other groups. C: control chow diet. HFC: high-ft cholesterol. HFCO: high-ft cornstrch. HSD: high sucrose diet. Leptin (ng/ml) Figure 4. Serum leptin. Hyperleptinemi ws registered in the HFC, HFCO nd HSD groups, however the highest leptin levels were found in HFC diet. C: control chow diet. HFC: high-ft cholesterol. HFCO: high-ft cornstrch. HSD: high sucrose diet. b Weight gin At the beginning of the study, rts from the four experimentl groups were not significntly different. At the end of the study the weight gin in the groups were s follow: C: ± 8.5 g. HFC: ± 14.3 g. HFCO: ± 1.7 g. HSD: 53.6 ± 12 g. Rts in the HFC diet gined significntly more weight (P <.1) compred with the other groups. Glucose nd insulin levels The more of the metbolic bnormlities tht the diet produces, the more intrinsiclly useful the model becomes. Glucose concentrtions in ll groups t dy were <1 mg/dl. By the end of the study ll experimentl groups (HFC, HSD nd HFCO) showed fsting hyperglycemi. The HFC diet hd the highest glucose level (158.5 ± 7 mg/dl) compred with the control group (116 ± 2 mg/dl) (P <.5). The HSD ( ± 11.9 mg/dl) (P <.5) nd the HFCO diet ( ± 3.5 mg/dl) developed lso hyperglycemi compred with the C. The HSD rts developed significntly higher fsting hyperinsulinemi compred with C on dy 9 (Figure 3). HFC nd HFCO diets showed higher fsting hyperinsulinemi but it ws no significnt difference compred to C. Serum leptin concentrtions Rts in the HFC, HFCO nd HSD developed significnt hyperleptinemi compred with C. Serum

6 545 Combined high-ft diet nd sustined high sucrose consumption promotes NAFLD in murine model., 215; 14 (4): leptin levels were higher in the HFC diet compred with the other groups (p =.1) (Figure 4). DISCUSSION Different studies hve shown tht murine biologicl processes re similr to those in humn. When murine models of NAFLD/NASH with either spontneous or induced genetic muttions re used, mny differences re found with the condition encountered t the bedside. 5 The development of dietry obesity models in rts mimicking humn obesity my help understnding its pthology nd evlute new therpeutic strtegies. To pproximte humn disese we tested 3 different diets, two with the combintion of high-ft nd sustined high sucrose consumption nd one with high sucrose only nd compre them with control group. This diets resemblnce modern occidentl diet or cfeteri diet. Different diets hve induced obesity nd ftty liver in rodents, suggesting tht over nutrition with either crbohydrtes, fts or both might ply role in the development of NAFLD. 14 Between these 3 models, the HFC with high sucrose consumption demonstrted higher degree of obesity nd NAFLD. It hs been reported tht the development of metbolic syndrome in mice is fster with high-ft diets, while rts re more susceptible to develop metbolic ltertions with high crbohydrte diets. We chose Wistr rts becuse this specie does not show susceptibility to develop obesity nd dibetes. 9 Animls with the HSD diet developed insulin resistnce; the implictions of peripherl insulin resistnce my be seen in the incresed expression of lipogenic genes in the liver. The weight increse in HSD diet hs been reported in the literture Insulin resistnce plys n importnt roll in the development of NAFLD becuse of the inhibitory effect of this hormone in heptic TG synthesis. The development of ftty liver is influenced by severl fctors such s the increse in de novo synthesis of FFA by the liver with n incresed expression of SREBP-1c probbly by insulin stimultion, nd decrese in the ntilipolytic effect of this hormone on dipose tissue nd the consumption of ftty cids in diet. 18 When there is n increse ccumultion of heptic TG, ftty cids oxidtion is inefficient, nd there is lso n increse in the production nd relese of VLDL in the circultion, expressed by n increment in serum TG. In the HSD we found n increse in de novo lipid synthesis, ftty cid oxidtion nd elevted serum TG concentrtions. From the 1% of FFA reching the liver 6% re produced by lipolytic ctivity in dipose tissue, 1% of dietry intke nd 3% of de novo heptic synthesis. 19,2 The HFC diet rts developed the gretest weight gin, which lso correltes with the greter lipid ccumultion in the heptic tissue. In situtions in which the lipogenic rte is incresed, s consumption of high ft diets or crbohydrtes, hyperglycemi nd hyperinsulinemi re ssocited with chnges in the cellulr metbolism of lipids, decresing oxidtion nd leding to n increse in ftty cid esterifiction incresing heptic TG synthesis like the ones seen in HSD nd HFC diets. Rts in the HSD hd greter levels of serum TG but less heptic lipid concentrtions thn the HFC diet. This is becuse the increse in heptic oxidtion inhibits TG heptic ccumultion, incresing the systemic relese. In the HFC diet oxidtion ws reduced nd greter levels of heptic stetosis were found. The development of severe phenotype of ftty liver disese with necroinflmmtory chnges nd profibrogenic response hs been reported with mice fed with trns fts nd high-fructose. 21 The bsence of necroinflmmtory chnges nd profibrogenic response in our study my be relted to shorter durtion of the diet nd the use of rts insted of mice. Some studies hve demonstrted tht heptic iron overlod my hve pthogenic role in the onset of liver stetosis nd insulin resistnce in erly stges, with lter decrese to level similr to tht in the control group. 8 We did not find heptic iron deposits possibly relted with longer evlution period or different evlution method. CONCLUSION The HFC diet with combintion of high ft nd high sucrose is more effective in producing NAFLD compred with high sucrose diet only. The HFC diet resembles the composition of fst food Western diet nd dministered for 9 dys recpitultes fetures of the metbolic syndrome nd NAFLD. This represents novel smll niml model of NAFLD with high similrities to the humn condition with n increse in intrheptic triglycerides ssocited with the reduction of insulin sensitivity. The results highlight the contribution of different dietry compositions in the development of ftty liver.

7 546 Torres-Villlobos G, et l., 215; 14 (4): With continuing improvement of niml models, scientists will be ble to developed sufficient knowledge of disese pthogenesis, which will eventully guide to full understnding of humn NAFLD. ABBREVIATIONS C: control chow diet. FAS: ftty cid synthse. HFC: high-ft cholesterol. HFCO: high-ft cornstrch. HSD: high sucrose diet. NAFLD: non-lcoholic ftty liver disese. NASH: non-lcoholic stetoheptitis. PPAR-α: peroxisome prolifertor-ctivted receptor lph. SREBP1: sterol regultory element-binding protein-1. FINANCIAL SUPPORT This reserch ws supported by the Consejo Ncionl de Cienci y Tecnologí (CONACYT). Grnt number: to Gonzlo Torres-Villlobos. ACKNOWLEDGEMENTS The uthors cknowledge Dr. Vicente Snchez- Vlle for support in determining leptin. REFERENCES 1. Lzo M, Clrk JM. The epidemiology of nonlcoholic ftty liver disese: globl perspective. Semin Liver Dis 28; 28: Adms LA, Angulo P, Lindor KD. Nonlcoholic ftty liver disese. CMAJ 25; 172: Cohen JC, Horton JD, Hobbs HH. Humn ftty liver disese: old questions nd new insights. Science 211; 332: Tkhshi Y, Soejim Y, Fukusto T. Animl models of nonlcoholic ftty liver disese/nonlcoholic stetoheptitis. World J Gstroenterol 212; 18: Anstee QM. Animl models in nonlcoholic stetoheptitis reserch: utility nd clinicl trnsltion. Liver Int 211; 31: Cobbold JF, Anstee QM, Goldin RD, Willims HR, Mtthews HC, North BV, Abslom N, et l. Phenotyping murine models of non-lcoholic ftty liver disese through metbolic profiling of intct liver tissue. Clin Sci (Lond) 29; 116: Romesting C, Piquet MA, Bedu E, Rouleu V, Dutresme M, Hourmnd-Ollivier I, Filippi C, et l. Long term highly sturted ft diet does not induce NASH in Wistr rts. Nutr Metb (Lond) 27; 4: Tsuchiy H, Ebt Y, Skbe T, Hm S, Kogure K, Shiot G. High-ft, high-fructose diet induces heptic iron overlod vi hepcidin-independent mechnism prior to the onset of liver stetosis nd insulin resistnce in mice. Metbolism 213; 62(1): Lrque C, Velsco M, Nvrro-Tbleros V, Duhne M, Aguirre J, Gutierrez-Reyes G, Moreno J, et l. Erly endocrine nd moleculr chnges in metbolic syndrome models. IUBMB Life 211; 63: Folch J, Lees M, Slone Stnley GH. A simple method for the isoltion nd purifiction of totl lipides from niml tissues. J Biol Chem 1957; 226: Kleiner DE, Brunt EM. Nonlcoholic ftty liver disese: pthologic ptterns nd biopsy evlution in clinicl reserch. Semin Liver Dis 212; 32: Chomczynski P, Scchi N. Single-step method of RNA isoltion by cid gunidinium thiocynte-phenol-chloroform extrction. Anl Biochem 1987; 162: Pfffl MW. A new mthemticl model for reltive quntifiction in rel-time RT-PCR. Nucleic Acids Res 21; 29: e Koteish A, Diehl AM. Animl models of stetosis. Semin Liver Dis 21; 21: Aguiler AA, Diz GH, Brcelt ML, Guerrero OA, Ros RM. Effects of fish oil on hypertension, plsm lipids, nd tumor necrosis fctor-lph in rts with sucrose-induced metbolic syndrome. J Nutr Biochem 24; 15: El Hfidi M, Cuellr A, Rmirez J, Bnos G. Effect of sucrose ddition to drinking wter, tht induces hypertension in the rts, on liver microsoml Delt9 nd Delt5-desturse ctivities. J Nutr Biochem 21; 12: Oron-Hermn M, Kmri Y, Grossmn E, Yeger G, Peleg E, Sbty Z, Shmiss A, et l. Metbolic syndrome: comprison of the two commonly used niml models. Am J Hypertens 28; 21: Vnni E, Buginesi E, Kotronen A, De Minicis S, Yki-Jrvinen H, Svegliti-Broni G. From the metbolic syndrome to NAFLD or vice vers? Dig Liver Dis 21; 42: Vrel-Rey M, Embde N, Ariz U, Lu SC, Mto JM, Mrtinez-Chntr ML. Non-lcoholic stetoheptitis nd niml models: understnding the humn disese. Int J Biochem Cell Biol 29; 41: Postic C, Girrd J. Contribution of de novo ftty cid synthesis to heptic stetosis nd insulin resistnce: lessons from geneticlly engineered mice. J Clin Invest 28; 118: Tetri LH, Bsrnoglu M, Brunt EM, Yerin LM, Neuschwnder-Tetri BA. Severe NAFLD with heptic necroinflmmtory chnges in mice fed trns fts nd high-fructose corn syrup equivlent. Am J Physiol Gstrointest Liver Physiol 28; 295(5): G987-G995.