US 2005O2397O6A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0239706A1 Backhed et al. (43) Pub. Date: Oct. 27, 2005 (54) MODULATION OF FIAF AND THE Related U.S. Application Data GASTRONTESTINAL MICROBOTAASA MEANS TO CONTROL ENERGY STORAGE (63) Continuation-in-part of application No. 10/432,819, INA SUBJECT filed on Oct. 31, 2003. (75) Inventors: Fredrik Backhed, St. Louis, MO (US); (60) Provisional application No. 60/591.313, filed on Jul. John Rawls, St. Louis, MO (US); 27, 2004. Justin Sonnenburg, St. Louis, MO (US); Lora V. Hooper, Coppell, TX Publication Classification (US); Jeffrey I. Gordon, St. Louis, MO (US) (51) Int. Cl. ................................................. A61K 38/17 (52) U.S. Cl. ................................................................ 514/12 Correspondence Address: POLSINELL SHALTON WELTE (57) ABSTRACT SUELTHAUS PC. 700 W. 47TH STREET The invention provides compositions and methods to modu SUTE 1000 late fat Storage and weight loSS in a Subject. In certain KANSAS CITY, MO 64112-1802 (US) aspects of the invention, fat storage (adiposity) and weight (73) Assignee: Washington University in St. Louis loSS is modulated by altering the Subject's gastrointestinal microbiota population. In other aspects of the invention, fat (21) Appl. No.: 11/080,755 Storage and weight loSS is modulated by altering the amount of or the activity of the protein, fasting-induced adipocyte (22) Filed: Mar. 15, 2005 factor, in the Subject. B 1 2 5 10 2 5 a 4 WAT Heart 2 ? g3 52 5 al - O &sCS ésO C G D 2 F. 80 g5 150 3560in see a C- t sq 100 o 40 5 so 3 as). We 8.3 kb Neo 2320 & S. S. s' probe 3. ++ +- -- s SSSSSS in a 7 anis Fiafiafgenotype See of 4 + v. Patent Application Publication Oct. 27, 2005 Sheet 1 of 41 US 2005/0239706A1 Fig. 1 Epithelium Mesenchyme Before capture Before capture Patent Application Publication Oct. 27, 2005 Sheet 2 of 41 US 2005/0239706A1 Fig. 2 spr2a OOO 8O SOO 40. 2O Colipase 8O SO O O 1.5 1.O O cS isN tS isN tS isN. vius crypt se epithelium chyne Patent Application Publication Oct. 27, 2005 Sheet 3 of 41 US 2005/0239706A1 Fig. 3 lactase O OB OS OA O2 Golipase angiogenin-3 O 2 OO V 50 Patent Application Publication Oct. 27, 2005 Sheet 4 of 41 US 2005/0239706A1 Figure 4: Angiogenin-4 and -3 nucleotide sequence alignment angiogeniu- l -gaacrracaceaasaccorsiclecassascacacagorasacriticariccaeill 2. t s angiogeniu-3 Cuscicarecscacisco Stecca'ssacACSAASCUAACACAccCCs Consensus agott adao Og aggadootgtotodaggagdao agotaged to to go to g 6. Bl 9. ill angiogenin- Sec.ScGSCCGCSA CAAccCAS analogerlin-3 TSGAGSAAGonggccaccTLSSAATCGEAAG start Casersus ttggaggaaagatggoopagotttggaatu stgttgaaagagats atgagtega st 2 lil s lsi augiogenil- s (CPS angiogenit-3 exact sect CC sease NCAGS-Ace ecCACNGCCF Cascal Coasess otttgttgttggtott tutg gttctg. ittgat oatge actatggotoag at l 2D 2. 2. angiogenin-s gaa-...--AGGTACGAAAAATTCCTACGICAGCACIAGATGCCAASCCAAAGGGCCGGGac angiogenin-3 AACTACASGAAAAAAccreacticaGCACAARSCCAGCCACCSCCGSGAE Consensus aggtao aaaattgot to aggagtatatgcaa.go Gala godggga 2. as s 2. 2B 2. anoriocrenit-4Ai is Cussless al 3. 32. 33 3. 35. angiogenil anogenin-5 a Salf Yu is a WAY is v Y WAY Wor Consensus aaoacotttat datg tado agaa aadata go oatatgttgga agaa gigaag 35 37 3. 3 4. angiogeniu-4. CCITATGGAGAAAACTTCAGAATAASCAATTCTCCCTCCAGATCACGACTTGEACGCAC angiogenin-3 ce accusetta seates Cease Consensus gettatag aaa.ott agaataagaattute tteoag to assactites augao s2. s t 45 3. angiogenia- ICAAGAGGGIGLXCCCLX3GCGTCGAIGCGSGIACCGAgCCTTTAAASATTCAGACATAT angiogen-3 Acciacoscoes seccuccius CAct Costases a gagggittgct giggoto gatga gtag gotstttaaagattittaga atatt El al SO 52 3. creat- graecceeaaga SCCEGGCGNPCCAcacca's Crack GE E-3 Gasco SGAEGSGCGGCCXCCNCCASCANAGIC stop Consensus ttattooitgttgaagatggotggaetgtoca uttegatagtotttitato agbo cigtag E. iogenin- eccleocococcCaesacagogic coccacAcces Willis CASCGCCCCCGCACAGACCASPSPs acceCCCAcces Corpseuss aigiocreli Eli s sy, A., M. War. O Consensus atgaatgttca d tactitta giged tgaattatuttgaaatatattot 55 S. 7. ogeniu-4 ccTCATIATAATGCACASAAAAAAGATATOEXCAAAAMCCATAAAAAAAAAAAAAAAAA EEis CCS is a GCACSAAAAAAAACAAAAACAAAAAAAA----------- sees got atttataatguagagaaataaagatatotaaraa dua aaaaaa 72. 73 7. 75 7. 77 angiogenail- ABAAAAAAA SEQDNO29 aloren-3 --------- SEOD NO30 Cocsetes Patent Application Publication Oct. 27, 2005 Sheet 5 of 41 US 2005/0239706A1 Figure 5: Alignment of mouse angi genin family members l ll 2. 3. 4. angiogenin TiSPCPFFWGYWPTA-IRYKFIREDAKKRDDRC angiogenin ASGPLF,F,GVWFADDSETEFTDARPERDDRYC angiogelin-3 EVESFGSLLIVFLISLDVIPPTLAnNYRYIKFTTEYDAKPTGRDYRYC angiogenin RB ESPGPLFFFTLGVWPPTSDDSKYKFTYEAKPKGRDDRYC Conseals In mappillwflglwvipptland ry kfilthydarpkgridryc 5 5. 7. Bl 9. angiogenin-6 ESSKERKLTSPCKDWTFEGTKKRRICKKGSPYEREISNSPF angiogelin-l ERKRRSLTSPCKDYRTFEG, KSRRAICG.GSPYRNassPFC ESKKRKTSPCKETFESTKRRKACGETRYGFRSNESRF Esi KRRTSFCKDWTFETKRKACGESPYRIRISKRF esmkrkltepckdwateih tk nikai.cg aspyg n ris B to lol lill 2. 3. l SEID angiogelin-6 TTCTBSRGSPPPCGRFKDRWADGEESFS 3 . angiogenin WTTKTGGSFRPFCYRASAGFREWWACEGLUEFDESEFSI. 32 angiogenin-3 vTTCTHKGGSPRPPCOYNAFKDFRYIVLACEDCPVHFDESFISP 33 angiogenir WTTCTERGRSPRPPCRRASKGFRIIIGCEGFWBFDESISP 34 CDs easis wittcth ggsprppc yra k fryiviace gapvhfdesfisp Patent Application Publication Oct. 27, 2005 Sheet 6 of 41 US 2005/0239706A1 Fig. 6 Locations of primers specific for mouse angiogenin family members angiogenin-d angiogenin-3 angiogenin-1 i angiogening? Consensus angiogenin-d angiogenit-3 angiogenin-1. angiogenin RP Consensus O). ll l2 3. 4. angiogenin-4 ACGS RS angiogenin-3 angiogenin-1 : W angiogeninrPSA Consensus 15 l6 l 8. 19 angiogenin-4 angiogenin-3 angiogenil angiogeninkp SS SittigiassissiTTEgg Consensus gaaagtatgatgaagaaaagaaagctaaccto co tigcaaagatgtcaa 20 21 22 23. 24l angiogenin-d angiogenin-3 angiogeninri angiogen?inEP 35 OE. E. s Consensus cacctttatcCatg ceccaagaacaa.catceaeggc.catctgtgga age 2S 25i 27 28 29 angiogenin-d Saayaaaass s s angiogenin-3 T. angiogenin-i alagiotein Siii.55:iii.5iSigEcESi3S2, Consensus a gigaegcc.cttatggag aaactu agaataegcaa tactc ctitccag 30 3. 32. 34 angiogenium-4 E332 says: saxa se:sawaxy's angiogenin-3 angiogenin-1 angiogenirrP Consensus 35 36 3. 38 39 angiogenin f - area 3. angiogenin-3 &E. M angiogenin-1 E. angiogenin RP C Casess SEO O NO. angiogenil- 61 angiogenin-3 62 angiogenin-1 63 angiogenin RP Egg 64 Consensus ggcc tigtocacttcgatgegtCttittetcagtcc tag Patent Application Publication Oct. 27, 2005 Sheet 7 of 41 US 2005/0239706A1 Figure 7: Tissue distribution of Angiogenin-4 mRNA qRT-PCR analysis: 6.0 S.O Agarose gel analysis (with Gapdh control) Patent Application Publication Oct. 27, 2005 Sheet 8 of 41 US 2005/0239706A1 Figure 8: Tissue distribution of angiogenin-1 mRNA qRT-PCR analysis: Patent Application Publication Oct. 27, 2005 Sheet 9 of 41 US 2005/0239706A1 Figure 9: Tissue distribution of angiogenin-3 mRNA Quantitative real-time RT-PCR analysis: Patent Application Publication Oct. 27, 2005 Sheet 10 of 41 US 2005/0239706A1 Figure 10: RT-PCR analysis showing absence of angiogenin-related protein expression - as a 2 a SA 2 S 22 R as & R & K SS bp 194 194 118- 18 72- -72 expected amplicon size: 130 bp Note that Gapdh expression levels for each of these tissues are shown in Figure 4 l. distal small 14. brain 2. middle Small S. heart 3. proximal small 16. bladder 4. Squamous 17. skin 5. glandular stomach 18. tranchealthyroid 6. ascending colon 19. pancreas 7. descending colon 20. Salivary gland 8. rect 21. testes 9. Cec 22. prostate 10. kidney 23. ovary 11. liver 24. uterus 12. lung 25. mammary gland 13. spleen w 26. genomic DNA 27. Water Patent Application Publication Oct. 27, 2005 Sheet 11 of 41 US 2005/0239706A1 Figure 11: Microbial regulation of angiogenin-4 expression in the small intestine Pair 1 -0-germ-free ys -- conventionalized8 is asi gs s 3 5 7 9 11 13 15 intestinal segment Pair 2 2.5 w -0-germ-free 2.0 -- conventionalized S. s 15 s 3. 1.0 O. 5 O. O 1 3 5. 7 9 11 13 15 inte tinals gment Patent Application Publication Oct. 27, 2005 Sheet 12 of 41 US 2005/0239706A1 Figure 12 Regulation of angiogenin-4 expression during postnatal development germ-free 11 5 10 15 20. 25 30 days after birth Patent Application Publication Oct. 27, 2005 Sheet 13 of 41 US 2005/0239706A1 Figure 13 Cellular localization of angiogenin-4 expression in small intestige: qRT-PCR analysis feels is lated from the crypt base wild-type CR2-tox176 (lacking Paneth mouse strain Patent Application Publication Oct. 27, 2005 Sheet 14 of 41 US 2005/0239706A1 Figs. 14A-14D <C(%)qe?ÁpOG O (Kep/6)uo?duunsu00MO?O LO<+croÇN•Q.D. Patent Application Publication Oct. 27, 2005 Sheet 15 of 41 US 2005/0239706A1 Figs. 15A-15C eye A 3 15 12 2 2 2 1.0 8 CD cy S c 8 1 0.5 2 4 - (D O O $ S dš -NQ S S OS C C i? CONV-D C B 300 -- GF 100 () r g 8 75 g200 o e CD 50 f 8 100 2 as 25 CD O SS O O 3O 6O 90 120 O 30 6O 90 120 time (min) time (min) Patent Application Publication Oct. 27, 2005 Sheet 16 of 41 US 2005/0239706A1 Figs. 16A-16D rt G in t S15 g D E a g 200 c 10 SS 22 100 9> 5 E3ed & 9 O eso D ChREBP 5 Patent Application Publication Oct. 27, 2005 Sheet 17 of 41 US 2005/0239706A1 Fig. 17A-17F A B 125 $5100 de 2 75 is59 50 E23 25 C o CC cr’ sS. (° 3 4 WA Heart 2. arra g3 S 52 s 5 o s $sCS &sC D 2 F. 80 5 150 gas 60 a g g t sq 100 o 40 6 sol E3 r as c 8.3 kb gi20 O .S. c. X Martinspotte see-H*3. ++ + -i- *S. \s S if 47 gi Fiafgenotype w Souther to Norther to 0-f v- 84 -- - -- Patent Application Publication Oct. 27, 2005 Sheet 18 of 41 US 2005/0239706A1 Fig. 18 increased hepatic u-1 (ChREBPISREBP-1)lipogenesis Processing of dietary Microbial u- polysaccharides colonization of the gut Suppression of u s Fiaf in the gut Triglyceride epithelium LPL activity - adipocytesstorage in Patent Application Publication Oct. 27, 2005 Sheet 19 of 41 US 2005/023970.6 A1 Fig.