36 6 2007 ( ) JOU RNAL O F ZH EJ IAN G UN IV ERS IT Y (M ED ICAL SC IEN CES) V o l 36 N o 6 2007 h ttp: www. journals. zju. edu. cngm ed Β 1, 1, 1, 1, 1, 1, 2 ( 1. 2., 100029) [ ] : 2N 2 (N 2acetyl2l2cysteine, NA C) Β : 59 8 SD (N C ) (H F ) + NA C (NA C ) 20, (1) (M DA ) (GSH ) ; (2), ; (3), Β ; (4) PCR 21 ( IR S21) 22 ( IR S22) 22 (Glu t22) mrna : (1)H F M DA N C, GSH N C, NA C ; (2)H F (G IR ) N C (P < 0101), NA C G IR (P < 0101) ; H F (GS IS), NA C ; (3) H F IR S21 IR S22 Glu t22mrna 4213% 2811% 2219% ( P < 0105 ) ; NA C IR S21 IR S22 Glu t22 mrna H F 4012% 3012%, 1911% (P < 0105) :,, NA C [ ] ; g ; g ; N 2 ; Β ; PCR [ ] R 587. 1 [ ] A [ ] 100829292 (2007) 0620575206 N-acetyl- l-cyste ine im proves function of islet Β cell in hyperl ip idem ic rats and its m echan ism W AN G B ing, L I Hong2liang, YAN G W en2ying, et al (D ep a rtm en t of E nd ocrinology, Ch ina J ap an F riend sh ip H osp ita l, P ek ing U n ion M ed ica l Colleg e, B eij ing 100029, Ch ina) [Abstract ] Objective: To investigate the effect of N 2acetyl2l2cysteine (NA C ) on islet Β cell function in hyperlip idem ic rats and its m echan ism. M ethods: F ifty2n ine m ale SD rats of 8 w eek o ld w ere random ly divided in to 3 group s: no rm al diet group (N C, n= 20), h igh fat diet group (H F, n= 20) and NA C treated group (NA C, n= 19,NA C 300 m g kg - 1 d - 1 and h igh fat diet). A t the : 2007205208 : 2007207211 : (30640081). : (1977- ),,, ; E2m ail: w angbing 000@ sina. com. cn. : (1946- ),,,, ; Em ail: w fl921@yahoo. com. cn
576 ( ) 36 end of 20 w eek s, fasting serum in su lin ( In s), gluco se (Glu), m alonaldehyde (M DA ) and reduced glu tath ione (GSH ) w ere determ ined in p lasm a and pancreas tissue. T he gluco se infu sion rate (G IR ) w as m easu red by euglycem ic hyperin su linem ia clamp to evaluate the peripheral in su lin resistance. Pancreatic islets w ere iso lated and sub jected to a perifu sion m edium con tain ing 3. 3 mmo1gl gluco se fo r 15 m in, fo llow ed by 16. 7 mmo1gl gluco se fo r 30 m in, in su lin con ten t of perifu sion m edium w as m easu red by R IA. T he exp ression s of IR S21, IR S22, Glu t22 gene in islets w ere detected by real tim e PCR. Results: (1) T he in su lin, gluco se and M DA concen tration in H F group w ere h igher than tho se in N C group, bu t GSH levels in p lasm a and pancreas w ere low er. NA C in terven tion cou ld reverse these effectṡ ( 2) T he G IR w as decreased sign ifican tly in H F group compared w ith N C group [ (5. 25 1. 2) vs (13. 56 1. 7)m g m in - 1 kg - 1, P < 0. 01 ], NA C in terven tion reversed these effect: G IR [ (9. 28 1. 50) vs (5. 25 1. 2)m g m in - 1 kg - 1, P < 0. 01 ]. (3) 16. 7 mmo lgl gluco se increased the in su lin secretion in the islet cells of the th ree group s, bu t the peak w as low er in H F group. NA C in terven tion reversed these effectṡ (4) T he gene exp ression of IR S21 w as sign ifican tly decreased by 42. 3% in H F group (P < 0. 05), and the exp ression s of IR S22 and Glu t22 w ere decreased by 28. 1% and 22. 9% (P < 0. 05) compared w ith N C group. In con trast, the exp ression s of IR S21, IR S22, Glu t22 in NA C group increased by 40. 2%, 30. 2% and 19. 1%, respectively than tho se in H F group. Conclusion: NA C can reverse functional diso rder of islet Βcells induced by h igh2fat2diet feeding. T h is an tiox idan t effect m igh t be associated w ith upgrading gene exp ression of in su lin signal tran sduction mo lecu les in islet Β cells. [ Key words ] A cetylcysteine; Islets of langerhan sgcyto l; In su lingsecret; N 2acetyl2l2cysteine; Islet Βcells; R eal2tim e PCR [ J Zhejiang U n iv (M edical Sci), 2007, 36 (6) : 5752580. ] Β 2 ( T 2DM ), T 2DM,,, T 2DM [122 ] N 2 (N 2acetyl2l2cysteine, NA C ),, Β, PCR, NA C 1 1. 1 8 SD 59 (SPF, ), (N C, n = 20) (H F, n= 20) + NA C (NA C, n = 19) N C ( 64% 23% 13%, ), H F ( 20% 21% 66%,, ), NA C NA C 300 m g kg - 1 d - 1 (NA C Sigm a, 115K01261), 20 1. 2, L inco (m alonaldehyde, M DA ) ( reduced
6,. Β 577 glu tath ione, GSH ) 1. 3 3% (50 m ggkg),,,,, 12 mu kg - 1 d - 1, 20% 4. 8 5. 2 mmo lgl, 5 6 (gluco se infu sion rate, G IR ),, 1. 4 (1) 3% (50 m ggkg),,,, 5,,,, 1 m ggm l p 8 m l,,, 6 m l H ank s (2) 38 10 m in, 15 s,, 4 10 m l 4 H ank s 30 m l (3) 600 Λm, 50 m l 1 000 rgm in 4 1 m in,, 4 H ank s,, H ank s 2 15 m l, 1 000 rgm in 4 2 m in, (4) 25% F ico ll 4 m l, 23% 20% 11% F ico ll H ank s 2 m l, 3 000 rgm in, 4 20 m in, 23% 20% 20% 11%, 4 H ank s 50 m l 2 1. 5 m l EP 1. 5 Pharm acia U pp sala 50, : (0 15 m in), 3. 3 mmo lg L, ; (16 45 m in), 16. 7 mmo lgl 30 m in [3 ] 1. 6 PCR T rizo l ( Invitrogen, ) RNA, ; RNA R evert ra A ce2 ΑcDNA (TO YOBO, ) cdna [4 ] AB I R PR ISM 7300 ( AB I B io system ) PCR SYBR R Green PCR M aster M ix TO YOBO ( ) PCR : SYBR R Green R ealtim e PCR M aster M ix 25 Λl, 18 Λl, 1 Λl, 1 Λl, cdna 5 Λl : 95 60 s, 1 ; 95 15 s; 60 15 s; 72 45 s; 40 2 - CT (GA PDH, sen se 5 2T GGGT GT G AA CCA CGA GAA 23, an tisen se 2GGCA T G GA CT GT GGTCA T GA 23, 145 bp; IR S21, sen se 5 2CA CCCA GT T T T TCGA CA CCT 23, an tisen se 5 2GCT T GT TCT T GGA GTCA GCC 23, 284 bp; IR S22, sen se 5 2CTA CCCA CT G A GCCCAA GA G23, an tisen se 5 2CCA GGG A T GAA GCA GGA CTA 23, 152 bp; Glu t22, sen se 5 2TA GCAA CT GGGTCT GCAA T 23, an tisen se 292 bp ) 5 2GTA GTCCTA CA CTCA T G23, 1. 7 SPSS 10. 0 x θ s,, ( ), SN K2q 2 2. 1 20, H F (FBG) (F IN S) N C, NA C H F ( 1) 2. 2 H F M DA N C, GSH N C,NA C ( 2) 2. 3 60 m in [ ( 4. 80
578 ( ) 36 1 Table 1 Comparison of body w eigh t, visceral fat ratio, fasting b lood gluco se and in su lin level in the th ree group s of rats [x θ s o r m edian (range) ] g ( g FBGg F IN Sg g ) g% (mmo l L - 1 ) (m IU L - 1 ) N C 20 624 55 18 4 3. 1 0. 7 4. 86 0. 47 11. 5 (3. 6 19. 4) H F 20 789 91 # 66 14 # 8. 4 1. 7 # 5. 80 0. 57 3 23. 7 (14. 2 33. 4) # NA C 19 680 85 3 47 5 3 6. 9 0. 8 3 5. 03 0. 42 3 13. 6 (9. 8 20. 3) # F 5. 77 22. 190 10. 185 7. 294 6. 956 H F vs N C o r NA C vs H F, 3 P < 0. 05, # P < 0. 01 2 Table 2 Comparison of ox idative stress indexes in p lasm a and pancreas in the th ree group s of rats (x θ s) M DA g (nmo l m l - 1 ) GSH g (m g m l - 1 ) M DA g (mmo l m g - 1 p ro t) GSH g (m g g - 1 p ro t) N C 20 13. 39 3. 41 141. 5 9. 2 0. 94 0. 18 33. 56 3. 01 H F 20 21. 56 4. 82 # 103. 2 8. 9 # 1. 37 0. 17 # 24. 87 2. 65 # NA C 19 17. 23 4. 12 3 121. 6 8. 4 3 1. 21 0. 16 3 29. 62 2. 76 3 F 4. 611 14. 087 5. 892 7. 190 H F vs N C o r NA C vs H F, 3 P < 0. 05, # P < 0. 01 5. 20)mmo lgl ], H F N C G IR [ (13. 56 1. 7) m g m in - 1 kg - 1 vs (5. 25 1. 2)m g m in - 1 kg - 1, P < 0. 01 ] NA C G IR H F [ (9. 28 1. 5)m g m in - 1 kg - 1 vs (5. 25 1. 2)m g m in - 1 kg - 1, P < 0. 01 ], 1 2. 4 Β (gluco se stim u late in su lin secretion, GS IS) H F, F = 16. 421, P < 0. 01; vs H F, 3 P < 0105, N C [ (64. 5 8. 2)m IU gl vs (28. 5 6. 4)m IU gl, P < 0. 01), GS IS,, 1g 4,, # P < 0. 01 1 F ig. 1 Comparison of gluco se infu sion rate in the th ree group s NA C H F [ (43. 2 7. 3) 4213%, IR S22 Glu t22 28. 1% m IU gl vs ( 64. 5 8. 2) m IU gl, P < 0. 01), 2219% (P < 0. 05) NA C IR S21 GS IS H F 2, 2 mrna H F 40. 2%, IR S22 2. 5 PCR N C Glu t22 3012% 1911% ( P <, H F IR S21 mrna 0105), 3
6,. Β 579, F = 18. 259, P < 0. 01; vs H F, 3 P < 0105, # P < 0. 01 2 F ig. 2 Changes of in su lin secretion du ring perifu sion in the th ree group s of rats H F N C NA C H F, 3 P < 0. 05 3 PCR F ig. 3 Changes of gene exp ression 3, Β, Β,, Β, [5 ], T 2DM Β,, T 2DM,, Β [6 ],, N C, H F, ; NA C, H F, 76. 8%, NA C, Β,,,, H F N C, 16. 7 mmo lg L H F, ; NA C, GS IS H F, NA C GS IS Β, Β Β Kubo ta [7 ], IR S22, Β, Β, : Β IR S21, IR S22 Glu t22mrna, H F IR S21 IR S22 Glu t22 N C 57. 7%, 71. 9% 77. 1%,, NA C IR S21 IR S 22 Glu t22 H F 40. 2%, 30. 2% 19. 1%, Β, Β, NA C, [8 ], Β? Calsson,, GS IS, ( reactive oxygen species, RO S), GSH, [9 ]
580 ( ) 36 M DA, GSH g,, C E, NA C, RO S, NA C, ;,NA C GSH,, GSH, GSH, C E,NA C, RO S,, NA C, M DA, GSH,, Β IR S21 IR S22 GLU T 22 mrna, NA C Β,, T 2DM References: [1 ] M A RCH IOL I R, SCHW E IGER C, L EVAN T ESi G, et al. A ntioxidant vitam ins and p revention of cardiovascular disease: ep idem io logical and clinical trial data [J ]. L ip ids, 2001, 36: S532S63. [2 ] O ZK IL IC A C, CEN G IZ M, O ZA YD IN, A et al. T he ro le of N 2acetylcysteine treatm ent on antioxidative status in patients w ith type II diabetes m ellitus [ J ]. Basic Cl in Physiol Pharmacol, 2006, 17 (4): 2452254. [3 ] WU Yun2hong, L I X iu2jun, L I Hong2liang, et al (,,, ). A study on the m echanism of islet cell insulin resistance in h igh2 fat2diet obese rats [J ]. National M edical Journal of Ch ina ( ), 2005, 85 (27): 19072 1910. (in Ch inese) [4 ] DU R ui2qin, L I Hong2liang, YAN G W eng2ying, et al (,,, ). T he exp ression of insulin signal transduction m o lecules and islet Αcell insulin resistance [J ]. National M edical Journal of Ch ina ( ), 2006, 86 (36): 254222546. (in Ch inese) [5 ] M ULL ER DAN Y GUO CA I HUAN G, ST EPHAN IE AM IEL. Identification of insulin signaling elem ents in hum anβcells autocrine regulation of insulin gene exp ression [ J ]. D iabetes, 2006, (55): 283522842. [6 ] GER ICH J E. T he genetic basis of type 2 diabetes m ellitus: im paired insulin secretion versus im paired insulin sensitivity [ J ]. Endocr Rev, 1998, (19): 4912503. [7 ] KUBO TA N, TOBE K, T ERAU CH I Y, et al. D isrup tion of insulin recep to r substrate22 causes type 2 diabetes because of liver insulin resistance and lack of compensato ry Β2cell hyperp lasia [J ]. D iabetes, 2000, 49: 188021889. [8 ] M A IESE K, M ORHAN S D, CHON G Z Z. O xidative stress bio logy and cell injure during type 1 and type 2 diabetes m ellitus [ J ]. Curr Neurovasc Res, 2007, 4 (1): 63271. [9 ] CA RL SSON C, BOR G L A,W EL SH N. Sodium palm itate induces partial m itochondrial uncoup ling and reactive oxygen species in rat pancreatic islets in v itro [ J ]. Endocr inology, 1999, (140): 342223428. [ ]