Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 3, U CP. (P < 0101) ; U CP 2 m RNA , R T 2PCR
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1 ISSN CN gQ 16 3 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 3, (,, ) (U CP1, 2, 3), R T 2PCR U CP 1, 2, 3 m RNA U CP 1. U CP 2, 3, U CP 2 ; U CP3 U CP1, 3 m RNA (P < 0101) ; U CP 2 m RNA, (P < 0105) ; U CP3 48 h, U CP 2, 3, U CP s U CP s, U CP s U CP3 (P < 0105) U CP s ; U CP s ; U CP s U CP s, 2,,,,, 2, R T 2PCR Q 51 Regula tory Effects of Fa sting and In sulin on Expression of Uncoupling Prote in-1, 2, 3 Genes J IN Jun2hua, ZHAN G T ie2m ei, ZHAN G En2yi,L I Y i (K ey L aboratory of Geriatrics, B eij ing Institute of Geriatrics, B eij ing H osp ital, M inistry of H ealth of P. R. China, B eij ing , China) Abstract In o rder to investigate the effects of fasting (overn igh t and 48 hou rs) and in su lin on exp ression of uncoup ling p ro tein (U CP) 1, 2 and 3 genes, U CP1, 2 and 3 m RNA levels w ere ob2 served by reverse tran scrip tion g g po lym erase chain reaction (R T 2PCR ) assay in in terscapular b row n adipo se tissue (BA T ), periep ididym al w h ite adipo se tissue (W A T ) and gastrocnem iu s of rats. U CP 1 gene w as exp ressed exclu sively in BA T. U CP2 and 3 genes w ere exp ressed in these th ree types of tissues w hereas m RNA levels of U CP2 and U CP3 w ere rich in W A T and in skeletal m u scle respectively. U CP 1 and U CP 3 m RNA levels w ere decreased (P < 0101) in BA T bu t U CP 2 m RNA level w as increased in th ree types of tissues by overn igh t fasting. U CP3 gene exp ression w as no t influenced in W A T and skeletal m u scle by fasting. P ro longing fasting tim e to 48 hou rs, U CP s m RNA levels in all tissues w ere up regu lated to the sam e as o r over con tro l group excep t 3 (A ) 3 3 Tel: (010) , Fax: (010) , E2m ail: tm zhang@cenpok. net,, , : , :
2 3 : U CP2, 3 m RNA levels that w ere decreased in BA T. In su lin up regu lated U CP s gene exp ression in BA T,W A T and skeletalm u scle (P < 0105). U CP s gene exp ression of rats show ed tissue specifici2 ty. T he differen t exp ression of U CP s gene induced by fasting fim e suggests that the function of sub types of U CP s m ay p lay differen t ro les in energy m etabo lism regu lation. T hese resu lts have al2 so p rovided basis fo r fu rther understanding of the relation sh ip betw een diet2therm ogen sis and body w eigh t regu lation, and the association w ith disease and body w eigh t2m etabo lism equ ilib ri2 um. Key words U ncoup ling p ro tein, Fasting, In su lin,o besity, T ype 2 diadetes, R T 2PCR (uncoup ling p ro tein s, U CP s) U CP s,, 1, A T P,, 111 A T P,,U CP s : W istar 14 ( 8 U CP1, (BA T ), RNA TR Izo l [ 1 ] G IBCO, BA T P rom ega U CP2 112 U CP3, : (1) (n= U CP2 U CP3 3), ; (2) (n= 3),, U CP2 U CP3 5 8, ; (3) 73%, U CP1 59% 57%,U CP2 3 8, (1 IU g,,, ), 4 h ; (4) 48 h (n U CP2 3 U CP1 = 4), 8 2 d 8, [ 2, 3 ], (w h ite adipo se tissues, U CP s W A T ) (b row n adipo se tissues, BA T ) ( skeletal U CP2 m u scle, SM ) 100 m g, RNA, 2 [ 4 U CP3 ] U CP s RNA : G IBCO RNA, 100 m g, U CP s RNA 50 g, A 260g A S 18S, RNA U CP s U CP s 9, 200 g ), (n= 4), : U CP1 U CP2 Β2, actin U CP3 [ 1 3 ] U CP s cdna, Cybercyn T ab le 1 U CP s, R T 2PCR: RNA 10 g
3 396 16, U CP1 Β2actin o ligo (dt ), U CP2 U CP3 3 (an ti2sen se), g RNA M 2M L v 80 U, 75 l, 37 2 h, 5140 mu gl ), 48 h cdna ; PCR 410 l, 64% (P < 0105) 83% (P < 300 nm o lgl, T aq DNA 0101), 215 U, 50 l, PCR 92 (T ab le 2) 45 s, 55 1 m in, 72 1 m in, 35 Table 2 P lasm a gluco se and insulin levels in four group s of Table 1 DNA sequences of U CP1,U CP2,U CP3 and Β2actin gene p rim ers N am e P rim ers L ength of PCR p roducts U CP1 5 2GT GA GT TCGA CAA CT TCC GAA GT G23 Sense 620 bp 5 2CA T GA GGTCA TA T GTCA C CA GCTC23 U CP2 5 2AA CA GT TCTA CA CCAA GG GC23 Sense 479 bp 5 2A GCA T GGTAA GGGCA CA G T G23 U CP3 5 2GGA CTA CCA CCT GCTCA C T G23 Sense 302 bp 5 2CCCGTAA CA TCTGGA CTT T 23 Β2actin 5 2T T GTAA CCAA CT GGGA CG A TA T GG23 Sense 650 bp 5 2GA TCTTGA TCTTCA TGGT GCTA GG23 (P < 0101) (T ab le 2) 212 (2413 rats Gluco se (mmo lgl ) Insulin (mu gl ) Standard fed (contro l) (n= 3) overnigh t fasting (n= 3) overnigh t fasting + insulin (n= 4) 48 h fasting (n= 4) Compared w ith contro l. 3 P < P < PCR U CP1, 2, 3 cdna, U CP1 H ind g 505, 115 bp U CP2 Sm a g 335, 145 bp U CP3 H p a g 219, 83 bp R T 2PCR 2%, (F ig. 1), PCR : PCR, 2% DNA m RNA : Im agem aster VD S 310 ( ) R T 2PCR Β2actin R T 2PCR A 100%, A g A Β2actin m RNA : ; : x θ s, t 2 F ig. 1 Restriction endonuclease m ap of U CP s PCR p rod UCP1 m RNA 48 h U CP1 ( (F ig. 2A ) mm o lgl ) 55% 73% 15%, U CP1 m RNA ucts 1, 21U CP2 PCR p roduct digested w ithout o r w ith Sm a g ; 31PCR m arker; 4, 5. U CP3 PCR p roduct digested w ithout o r w ith H p a g ; 6, 7. U CP1 PCR p roduct digested w ithout o r w ith H ind g
4 3 : % (P < 0101), 48 h (P < 0101), ; U CP1 m RNA, U CP2 84% 99% (P < m RNA (P < 0105), 0101) (F ig. 2B ) 215 UCP2 m RNA 216 UCP3 m RNA U CP2 U CP3, U CP2 m RNA, U CP3 m RNA (F ig. 2A ), U CP2 m RNA, 48 h U CP3, 48h, U CP2 m RNA, U CP3 63% 65% (P < 0101) ; m RNA (P < 0105), U CP2 m RNA ( U CP3 25% 18% (P <, ) 0105),, 48 h U CP2 m RNA (P < 0105), 48 h 23% 28% (P < 0105), ; ; U CP2 m RNA 26% (P < 0101),, U CP3 m RNA, 48 h U CP3 m RNA U CP2 m RNA 34% 28% (F ig. 2C) (F ig. 2A ), U CP3, U CP3, 17% 19% (P < 0101) (F ig. 2D ) F ig. 2 U CP sgβ2actin mrna ratio of WA T, BA T and m uscle in rats of four group s w h ich are fed standard diet (contro l), overnight fasting (O F), overnight fasting w ith insulin adm inistration (O F+ Ins) and fasting fo r 48 hours (48hF) (A )Rep resentative bands of U CP s by RT2PCR; (B)U CP1gΒ2actin mrna ratio; (C)U CP2gΒ2actin mrna ratio; (D )U CP3gΒ2actin mrna ratio 3 P < 0105, 3 3 P < 0101, compared w ith contro l; + P < 0101, compared w ith O F
5 ,, U CP2 A T P, U CP2 U CP3 m RNA U CP2 [ 6 U CP3 m RNA ] (U CP1, 2, 3) U CP s, 40%, U CP s U CP3 U CP 3 U CP s m RNA, U CP 2 m RNA 311 UCPs U CP2 U CP3 m RNA 2 5 [ 7, U CP s ] [ 2, 3 ] U CP s U CP 3 U CP s, U CP 3 m RNA 312 UCPs U CP s, U CP s : 1) U CP s U CP ) U CP1 U CP2 3 U CP1 2 3 m RNA ;, U CP s U CP s,, U CP s U CP s U CP s, U CP s U CP s U CP1 2 U CP3, U CP2 3, 313 UCPs [ 8, ] U CP2 3 ; U CP2 3, m RNA U CP s U CP1 m RNA, [ 5 ], L ep tin ( ) [ 9 ], U CP2 U CP3, U CP s U CP2 3 U CP1,, U CP s,, U CP s, U CP s
6 3 : U CP s, U CP s, U CP s,, U CP s,, ; [ 10 U CP2 U CP3 ] U CP s, U CP s U CP s Chem, 1998, 273: 5 8, (References) 1 N iso li E, Tonello C, Curruba M O. N erve grow th facto r, Β2 adreno recep to r and uncoup ling p ro tein 1 exp ression in rat brow n fat during po stnatal developm enṫ N eurosci L etṫ 1998, 246: Sh im abukaro M, Zhou Y T, L ee Y. Induction of uncoup ling p ro2 tein 2 mrna by troglitazone in the pancreatic islets of Zucker diabetic fatty rats. B iochem B iop hy s R es Comm un, 1997, 237: V idal2puig A, So lanes G, Grujic D, F lier J S, L ow ellb B. U CP3: A n uncoup ling p ro tein homo logue exp ressed p referentially and abundantly in skeletal m uscle and brow n adipo se tissue. B iochem B iop hy s R es Comm un, 1997, 235: H jeltnes N, Fernstrom M, Zierath J R, Krook A. Regulation of U CP2 and U CP3 by m uscle disuse and physical activity in te2 trap legic subjects. D iabetolog ia, 1999, 42: T eruel T, V alverde A M, N avarro P, Benito M, L o renzo M. In2 hibition of P I 32kinase and ras blocks IGF21 and insulin2induced uncoup ling p ro tein 1 gene exp ression in brow n adipocytes. J Cell P hy siol, 1998, 176: M illet L, V idal H, A ndreelli F, L arrouy D, R iou J P, R icquier D, L aville M, L angin D. Increased uncoup ling p ro tein22 and23 mr2 NA exp ression during fasting in obese and lean hum ans. J C lin Invest, 1997, 100: Bo ss O, Sam ec S, Kuhne F, B ijlenga P, A ssim acopoulous2jean2 net F, Seydoux J, Giacobino J2P,M uzzin P. U ncoup ling p ro tein2 3 exp ression in rodent skeletal m uscle is modulated by food in2 take but no t by changes in environm ental temperature. J B iol 8 W eigle D S, Selfridge L E, Schw artz M W, Seeley R J, Cum 2 m ings D E, H avel P J, Kuijpe J L, Beltrandelrio H. E levated free fatty acids induce uncoup ling p ro tein 3 exp ression in m uscle. A po tential exp lanation fo r the effect of fasting. D iabetes, 1998, 47: ,,,. (Zhang T ie2 M ei, L in Ya2Hong, J in Jun2H ua, Yao W en2h ua. Effects of in2 sulin and food intake on OB gene exp ression in rats. Ch in J D ia2 betes), 1998, 6: Sam ec S, Seydouz J, D ulloo A G. Intero rgan signaling betw een adipo se tissue m etabo lism and skeletal m usule uncoup ling p ro2 tein homo logues. Is there a ro le fo r circulating free fatty acid? D iabetes, 1998, 47:
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Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.
LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.
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Supplementary Table 1. Primers used for RT-qPCR analysis of striatal and nigral tissue.
Supplementary Table 1. Primers used for RT-qPCR analysis of striatal and nigral tissue. Gene Forward Primer (5-3 ) Reverse Primer (5-3 ) Dopaminergic Markers TH CTG GCC ATT GAT GTA CTG GA ACA CAC ATG GGA
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