Chinese Journal of Biochemistry and Molecular Biology . 40 % FAS mgπml ( ) ; FAS

Transcription

1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 19 (3) : ,, ( ) 3 (FAS), FAS FAS 40 % FAS mgπml ( ) ; 0146 mgπml 015 min 50 % 32 min 90 % FAS, mgπ ml FAS NADPH, A, A FAS ; FAS FAS FAS Q55 Inhibition to Fatty Acid Synthase with Extract of Tuber Fleeceflower Root LI Li2chun, WU Xiao2dong, TIAN Wei2xi 3 ( Department of Biology, Graduate School of Chinese Academy of Sciences, Beijing , China) Abstract Fatty acid synthase (FAS) is reported as a potential new therapeutic target for obesity However, there are limited known FAS inhibitors presently It is shown that extract of Chinese medicine tuber fleeceflower root ( TFR) inhibited FAS activity by both strong reversible fast2binding and irreversible slow2 binding inhibitions 40 % ethanol was the optimal solvent for extraction Half inhibition concentration( IC 50 ) of TFR to FAS overall reaction was mgπml calculated on the basis of TFR weight in the extraction The time course of irreversible inhibition by TFR extract was biphasic In the presence of 0 46 mgπml of TFR the inhibition in fast2phase led to over 50 % activity loss which was completed within 30 s while the slow2phase inactivation led to 90 % activity loss within 32 min TFR extract strongly inhibited ketoacyl reduction of FAS with mgπml of IC 50, and weakly inhibited enoyl reduction The assay of inhibition kinetics showed that the inhibition to FAS by extract of TFR was non2competitive against substrate NADPH, nearly competitive against malonyl2coa and un2competitive against acetyl2coa It was speculated that an inhibitor in TFR reacted on malonyl thansacylase of FAS Treatment of TFR extract by oral incubation reduced body weight and food intake of rats significantly The FAS activity in livers of the tested group rats was lower than that of the control : : TelΠFax : ,E2mail : ac cn, Received :October 31,2002 ;Accepted :March 5,2003 Supported by the R & D foundation of Proctor & Gamble Company 3 Corresponding author TelΠFax : ,E2mail ac cn China Academic Journal Electronic Publishing House All rights reserved

2 rats at the end of the animal test These results showed that FAS inhibitor in TFR had a stronger inhibition ability than the known inhibitors Its characteristics of inhibition kinetics were much different from the known inhibitors It is expected to be a new effective FAS inhibitor, and may be of great value for the study of FAS mechanism and the control of obesity Key words fatty acid synthase, inhibition, tuber fleeceflower root, kinetics, fat reducing 2 5 [1 5 ] 111,, A A, ( EC (NADPH) SIGMA ,,fatty acid synthase, (DTT) FAS) 2000 J Hopkins Loftus Science FAS 1 [11 FAS A Tian ] A Y HITACHI 557,HITACHI CRZ0B2 FAS,GL220 [2 4 ] FAS SD [5,6 FAS ] 300 gπ,fas Loftus 2 FAS :C75 : 22 % 15 % (cerulenin) C75 J Hopkins 13 % 15 % 22 % 9 % 2 % 018 %,NaCl 015 % 017 % [7 ] 112 ( EGCG) FAS,, 20 ml [8 C75 ] [9 EGCG ] h rπmin 15 min (tuber fleeceflower root, TFR) - 20 ( Polygonum multiflorum Thunb ) 113 FAS molπl A,10 molπl [10 ], A, 35 molπl NADPH, 1 mmolπl FAS? EDTA, 1 mmolπl DTT, 100 mmolπl ph710 KH 2 PO 4 2 FAS? K 2 HPO 4, g FAS 340 nm FAS, FAS 2 ml [12 ] China Academic Journal Electronic Publishing House All rights reserved

3 3 : ml mmolπl KH 2 PO 4 2K 2 HPO 4,pH 710 ; 1 mmolπl EDTA,60 mmolπl,35 molπl NADPH, g FAS d 2 ml 340 nm 2 ml [12 ] 20 d 2 d 114 FAS 11713, A i 2 ml EDTA A o A i ΠA o,, 402, 1 - A i ΠA 0 HITACHI27060 ( Glu) (Cho) ( TG) mgπml FAS, d 1 2 h g FAS, FAS 40 % 2 ml 340 nm [12 ] (10 Π, Π ), mmolπl KH 2 PO 4 2K 2 HPO 4,pH 710 ; 1 mmolπl fenfluramine (011 mgπml ), EDTA, 60 mmolπl, 35 molπl NADPH, ( 011 molπl, 0107 molπl, 1 mmolπl EDTA, 1mmolΠL DTT, ph 718), 1 Sephadex G225 [13 ] min rπmin, 30 min rπmin,60 min,, A t FAS A 0 A t ΠA 0-60,1 - A t ΠA FAS 112 l ( 0103 mgπml) ( ) Fig11A FAS, 40 % 60 % FAS 3,40 % 116 Fig11B FAS 40 % 212 Fig 2 FAS ml 3 (40 % ) China Academic Journal Electronic Publishing House All rights reserved

4 Fig 1 The remaining activity of FAS inhibited by extracts of TFR with different solvents of various polarities (A) The concentration of TFR in the reaction is 0 03 mgπml (B) The concentration of TFR is 0101 mgπml, ( IC 50 ) mgπml,01018 mgπml 0112 mgπml IC % FAS 4 % EGCG FAS IC mgπml [14 ] mgπml [8 ] C75 FAS [8 ] 213 FAS 2,0146 mgπml,015 min FAS 50 %,32 min 90 % Fig13 2, 98 % 912 gπml 40 l 2 ml gπml IC 50 6 gπml, C75 FAS [8 ],0112 mgπml C75 Fig13 C75 Fig 2 Fast2binding inhibition to FAS with different TFR concentrations The data show the remaining activity of FAS by fast2binding inhibition with TFR to ( ) overall reaction, IC 50 is mgπml ; ( ) ketoacyl reduction, IC 50 is mg Πml ; ( ) enoyl reduction, IC 50 is 0 12 mgπml China Academic Journal Electronic Publishing House All rights reserved

5 3 : 301 C75 C75 10 %, FAS mgπ ml C75 2Π5 FAS C75 Fig 4 Lineweaver2Burk plot for the inhibition of reduction of FAS by TFR extract ketoacyl The fixed concentration of ethyl acetoacetate is 0 1 molπl, and the concentration of TFR in the reaction system is : ( ) 0 gπml, ( ) 815 g Πml, ( ) 15 g Πml, ( ) 25 g Πml The velocity was expressed by the concentration (nmolπl) of produced palmitic acid in one minute Fig 3 Time course of slow2binding inhibition of TFR extract and C75 to FAS overall reaction The concentration in mixture is : C75, 0 12 mgπml ; : TFR, 0 46 mgπml, respectively 214 T2 ( P < 0105) fenfluramine Table 1 Influences of TFR on rats body weight, Fig1 4 NADPH FAS, NADPH K is = gπml 2 K ii d gπml T Table 2 fenfluramine Fig 5A Fig 5B A A FAS 9 % T ( P < 0105) A K is = 0 98 gπ Table 2 Influences of TFR on ratsπfood intake ml, K ii = 1814 gπml 20,Fig1 5A A K ii 6 gπml ( fenfluramine) ( ) T2 Table g, fenfluramine 8105 g, Group Water Fenfluramine TFR Average weight change (gπrat) P value (T2test) Group Water Fenfluramine TFR Average food intake (gπrat day) P value (T2test) FAS 20 d ( ) Table China Academic Journal Electronic Publishing House All rights reserved

6 Fig 5 Lineweaver2Burk plot for the inhibition of overall reaction of FAS by TFR extract The fixed concentrations of (A) malonyl CoA, 10 molπl ; NADPH, 35 molπl, (B) acetyl CoA, 5 molπl ; NADPH, 35 molπl, the concentration of TFR in the reaction system is : ( ) 0 gπml, ( ) 2175 gπml ( ) 415 gπml ( ) 515 gπml FAS FAS [2,9 C75 EGCG ] 313, FAS 70 % Table 3 Influence of TFR on ratsπ blood fat and liver FAS activity GluΠmmol L - 1 ChoΠmmol L - 1 TGΠmmol L - 1 Group Water Fenfluramine TFR LDL2CΠmmol L FAS relative activity FAS FAS FAS C75 C75 C75 EGCG FAS IC molπl, mgπml ; 45 s 15 %,60 min 72 % ( EGCG 0 33 mgπ ml) [8 ] EGCG ( ) NADPH EGCG 312 FAS FAS FAS FAS FAS FAS FAS A 2 A A [15 ], A A, A A China Academic Journal Electronic Publishing House All rights reserved

7 3 : 303 FAS 2 C75 EGCG FAS FAS, 314 ( References) 2 [16 ], 315 FAS, [17 19 ] IC FAS FAS quercetin2 32gluctoside,quercetin232arabinoside, tricin, 32o2galloyl2 procyanidin B phosphatidyl [18 ] FAS,, ( EGCG) FAS IC , FAS FAS,, FAS 1 Bray G A Contemporary Diagnosis and management of obesity Newtown, Pa : Handbooks in Health Care 1998 : Loftus T M, Jaworsky D E, Frehywot GL, Townsend C A, Ronnett G V, Lane M D, Kuhajda F P Reduced food intake and body weight in mice treated with fatty acid synthase inhibition Science, 2000, 288 (30) : Bouchard C Inhibition of food intake by inhibitors of fatty acid synthase N Engl J Med 2000,343(25) : Li L C, Tian W X New evolution : Inhibitors of fatty scid synthase and fat2reducing study Chin Sci Bull, 2002, 47(2) : ( Tian Wei2xi, Dong Yan, Quan Hui, Chen Wen2feng The dependence of fat level of hen on activity of fatty acid synthase in liver on different ages Biochem J), 1996,12 (2) : Chin 6 Li M, Shi Y, Tian W X Factors influencing the levels of fatty acid synthase complex activity in fowl Biochem Mol Biol Int, 1999, 47 (1) : Kuhajda F P, Pizer E S, Li J N, Mani N S, Frehywot GL, Townsend C A Synthesis and antitumor activity of an inhibitor of fatty acid synthase Proc Natl Acad Sci USA, 2000, 97(7) : Wang X Tian W X Green Tea Epigallocatechin Gallate : A natural inhibitor of fatty2acid synthase Biochem Biophys Res Commun,2001, 288 (5) : Kao Y H, Hiipakka R A, Liao S Modulation of endocrine systems and food intake by green tea epigallocatechin gallate Endocrinology, 2000, 141(3) : : (Jiangsu New Mediccal College ed Dictionary of Traditional Chinese Medicine, Shanghai : Shanghai Science and Technology Press),1997 : 11 Tian W X,Hsu R Y,Wang Y S Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthetase of chicken liver J Biol Chem,1985,260(20) : NADPH ( Tian Wei2xi, Jiang Ruo2fan, Wu Hai2bin, Shi Yan2hong, Wang Yi2huang substrate inhibition by NADPH and kinetics of fatty acid synthase from duck liver Chin Biochem J),1994,10 (4) : Penefsky H S Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase J Biol Chem, 1977,252(9) : Vance D, Goldberg I, Mitsuhashi O, Bloch K Inhibition of fatty acid The synthetase by the antibiotic cerulenin Biochem Biophys Res Commun, China Academic Journal Electronic Publishing House All rights reserved

8 , 48(3) : Soulie J M, Sheplock G J, Tian W X, Hsu R Y Transient kinetic studies of fatty acid synthetase J Biol Chem, 1984, 259 (1) : Pharmacological Action of Commonly2Used Traditional Chinese ( Gen Wen2kui, Yang Yu2ying, Li Yu2 sheng, Liang Jian, He Qi2jun The influence of Polygonum multiflorum Thurb Extract on experimental animal blood fat Guangxi Med),1990, 12 (4) : :1992 : ( National Committee of Pharmacopoeia Pharmacopoeia of P R China Beijing : Chemical Industry Publication House), : ( Huang Tai2kang ed Handbook of Composition and Medicine Beijng : Chinese Medical Science and Technology Publication House),1999 : : (Wang Xian2 kai ed Natural Herbs Chemistry Beijing : People Sanitation Press), China Academic Journal Electronic Publishing House All rights reserved