Brown adipose tissue activity controls triglyceride clearance

Transcription

1 Supplementry informtion Brown dipose tissue ctivity s triglyceride clernce Alexnder Brtelt 1, Oliver T. Bruns 2, Rudolph Reimer 2, Heinz Hohenerg 2, Hrld Ittrich 3, Kersten Peldschus 3, Michel G. Kul 3, Ulrich I. Tromsdorf 4, Horst Weller 4, Christin Wurisch 5, Alexnder Eychmüller 5, Philip L.S.M. Gordts 6, Frnz Rinninger 7, Kroline Bruegelmnn 1,Brr Freund 1, Peter Nielsen 1, Mrtin Merkel 1,8 nd Joerg Heeren 1 1 Institute of Biochemistry nd Moleculr Biology II: Moleculr Cell Biology, University Medicl Center Hmurg-Eppendorf, Mrtinistrsse 52, D-2246 Hmurg, Germny; 2 Deprtment of Electron Microscopy nd Micro Technology, Heinrich-Pette Institute, Mrtinistrsse 52, D-2251 Hmurg, Germny; 3 Deprtment of Dignostic nd Interventionl Rdiology, University Medicl Center Hmurg-Eppendorf, Mrtinistrsse 52, D-2246 Hmurg, Germny; 4 Institute of Physicl Chemistry, University of Hmurg, Grindelllee 117, D-2146 Hmurg, Germny; 5 Physicl Chemistry, TU Dresden, Bergstrsse 66, D-162 Dresden, Germny; 6 Deprtment of Humn Genetics, University of Leuven, Herestrt 49, B-3 Leuven, Belgium; 7 III. Deprtment of Internl Medicine University Medicl Center Hmurg-Eppendorf, Mrtinistrsse 52, D-2246 Hmurg, Germny; 8 Asklepios Clinic St. Georg, Deprtment of Internl Medicine, Lohmuehlenstrsse, D-299 Hmurg, Germny Nture Medicine doi:1.138/nm.2297

2 2 cholesterol [mg/dl] TRL HDL FPLC frction Figure S1. Cold exposure remodels TRL metolism during the postprndil phse, Two hours fter n oil gvge, plsm lipoproteins of nd exposed FVB mice were seprted y fst performnce liquid chromtogrphy (FPLC). Similr to triglycerides (compre Fig. 1d) pronounced reduction in triglyceride-rich lipoprotein (TRL)-cholesterol levels ws oserved., Plsm from mice two hours fter n oil gvge ppers turid due to the scttering of TRL while plsm from -exposed mice is indistinguishle from fsted mice. Nture Medicine doi:1.138/nm.2297

3 3 H-rdioctivity [cpm] time [min] fter gvge Figure S2. Levels of triolein-derived 3 H-rdioctivity in nd -exposed mice fter n orl ft gvge One, two nd four hours fter n oil gvge 3 H-rdioctivity ws determined in plsm of nd -exposed FVB mice. In the postprndil pek phse, rdioctivity is significntly lower in -exposed mice compred to s. Notly, 3 H-rdioctivity corresponds to plsm triglyceride levels in ut not in mice. Men vlues +/- s.e.m. with n=4 (p<.5; p<.1; p<.1). Nture Medicine doi:1.138/nm.2297

4 7 HDL 6 3 H-rdioctivity cpm [cpm] TRL l:olete degrded olete FPLC frction Figure S3. In mice, 3 H-rdioctivity cn only e found in ftty cid degrdtion products Two hours fter n oil gvge plsm lipoproteins of nd -exposed FVB mice were seprted y FPLC nd 3 H- rdioctivity ws mesured in ech frction. In mice, 3 H- rdioctivity cn only e found in smll molecule ftty cid degrdtion products such s such s H 2 O, cylcrnitins nd others wheres in mice 3 H-rdioctivity cn e found in TRL, HDL, ound to lumin (l) or degrded. Nture Medicine doi:1.138/nm.2297

5 3 H-rdioctivity [cpm/mg orgn] 4, 3, 2, 1, liver spleen kidney epiwat suwat muscle hert interscpulr suscpulr deep cervicl BAT Figure S4. Orgn distriution of triolein-derived 3 H-rdioctivity two hours fter gvge Two hours fter n olive oil gvge with trcer mounts of 3 H-triolein orgns were hrvested, weighed, lysed nd rdioctivity ws determined y scintilltion. The specific uptke of BAT is the highest solute nd incresed fter exposure. Men vlues +/- s.e.m. with n=4 (p<.5; p<.1; p<.1). Nture Medicine doi:1.138/nm.2297

6 triglycerides [mg/dl] time [min] fter triton WR-1339 injection triglyceride increse [mg/dl] time [min] fter triton WR-1339 injection c triglycerides [mg/dl] fter 12 min FPLC frction d 3 H-glycerol t 12 min [cpm] Figure S5. VLDL production in nd mice Four hours fsted FVB or mice were injected with triton WR-1339 to lock vsculr lipolysis. Next, one minute fter triton WR-1339 injection, 3 H-glycerol ws injected to rdiolel triglycerides produced in the liver nd incorported into VLDL. Blood ws collected t indicted time points. Lipids were isolted y Dole extrction nd rdioctivity ws mesured y scintilltion counting. As expected sl triglyceride levels re significntly lower in mice. However, we neither oserve significnt differences in totl triglycerides during the experiment () nor in triglyceride increse (). Accordingly, 12 min fter triton WR-1339 injection we oserve neither difference in the FPLC lipoprotein profile (c) nor in the mount of rdioleled VLDL (d). These results show tht exposure does not lter VLDL production. Men vlues +/- s.e.m. with n=6 (p<.1; p<.1). Nture Medicine doi:1.138/nm.2297

7 6 triglycerides [mg/dl] time [min] fter triton WR-1339 injection triglyceride increse [mg/dl] time [min] fter triton WR-1339 injection c 4 d 5 triglycerides [mg/dl] fter 24 min 3 H-triolein t 24 min [cpm] FPLC frction Figure S6. Chylomicron production in nd mice Fsted FVB or mice were injected with triton WR-1339 to lock vsculr lipolysis. Next, 1 min fter triton WR-1339, 3 H-triolein in 2 µl olive oil ws given y gvge to determine intestinl chylomicron production. Blood ws collected t indicted time points nd rdioctivity ws mesured y scintilltion counting. As expected sl triglyceride levels re significntly lower in mice. However, we oserve neither significnt differences in totl triglycerides () nor in triglyceride increse (). Consequently, fter 24 min, we oserve neither significnt difference in the FPLC profile (c) nor in rdioleled triolein present in chylomicrons (d). These results demonstrte tht intestinl lipoprotein production is unltered y exposure. Men vlues +/- s.e.m. with n=6 (p<.1). Nture Medicine doi:1.138/nm.2297

8 lipolysis phospholipids triglycerides cholesterol HO cholesterol ester 59 Fe-SPIO 3 H-triolein 3 H-olete Figure S7. Model of 59 Fe-SPIO- 3 H-triolein TRL efore nd fter lipolysis 3 H-olete is relesed fter LPL-medited lipolysis from 3 H-triolein while 59 Fe- SPIO remins within the core of the TRL prticle (see figure S5). The lyout is dpted from Bruns et l Nture Medicine doi:1.138/nm.2297

9 3 H triolein [cpm] TRL untreted 3 H 59 Fe Fe-SPIO [cpm] 3 H triolein [cpm] TRL pre-heprin plsm 3 H 59 Fe Fe-SPIO [cpm] c post-heprin plsm d pre-heprin plsm + ovine LPL TRL l:olete TRL l:olete 3 H triolein [cpm] H 59 Fe Fe-SPIO [cpm] 3 H triolein [cpm] H 59 Fe Fe-SPIO [cpm] Figure S8. 59 Fe-SPIO is core lel while 3 H-olete is relesed only fter LPL-medited hydrolysis Recominnt TRL contining 59 Fe-SPIO nd 3 H-triolein were incuted for 15 min t 37 C, with PBS, with humn pre-heprin plsm contining no lipolytic ctivity c, with humn post-heprin plsm contining lipolytic ctivity nd d, humn pre-heprin plsm supplemented with 1 µg/ml ovine LPL. Then, incuted mixtures were susequently sujected to FPLC nd rdioctivities were determined in ech frction. 3 H-olete is relesed fter LPL-medited lipolysis while 59 Fe- SPIO remins in the TRL frction. This dt clerly show tht 59 Fe-SPIO is non-relesle lel for the core of lipoprotein prticles. Nture Medicine doi:1.138/nm.2297

10 59 Fe-SPIO [cpm/mg orgn] H-rdioctivity [cpm/mg orgn] Figure S9. Orgn uptke of TRL leled with 59 Fe-SPIO nd 3 H-triolein Orgn distriution 15 min fter injection of () 59 Fe-SPIO nd () triolein-derived 3 H- rdioctivity. Orgns were hrvested, weighed, lysed nd rdioctivity ws determined y scintilltion. After exposure BAT displys specific 59 Fe-SPIO nd 3 H-triolein uptke similr to the liver, the mjor orgn usully responsile for clernce of lipoprotein remnnts. Men vlues +/- s.e.m. with n=4 (p<.5; p<.1; p<.1). Nture Medicine doi:1.138/nm.2297

11 3 H-cholesteryl ether [cpm] C triolein [cpm] time [min] fter injection time [min] fter injection c d 3 H-cholesteryl ether [cpm/mg orgn] C-triolein [cpm/mg orgn] Figure S1. Plsm clernce nd orgn uptke of TRL leled with 3 H-cholesteryl ether nd 14 C-triolein into nd C57BL/6J mice. Plsm clernce of TRL doule-leled with () 3 H-cholesteryl ether (non-relesle core lipid) nd () 14 C-triolein (relesle ftty cid lel) is drmticlly ccelerted in compred to mice. Orgn uptke of (c) 3 H-cholesteryl ether nd (d) 14 C-triolein into BAT nd to certin extent in suwat is strongly incresed in mice wheres liver nd hert uptke re reduced. Tken together, these dt indicte uptke of whole lipoprotein prticles. Men vlues +/- s.e.m. with n=7 (p<.5; p<.1; p<.1 versus wild-type ). Nture Medicine doi:1.138/nm.2297

12 3 H-cholesteryl ether [cpm per totl tissue] C-triolein [cpm per totl tissue] Figure S11. Totl orgn uptke of TRL lelled with 3 H-cholesteryl ether nd 14 C-triolein into nd C57BL/6J mice. Totl orgn uptke of () 3 H-cholesteryl ether (non-relesle core lipid) nd () 14 C-triolein (relesle ftty cid lel) in compred to mice ws clculted from estimted totl orgn weights displyed in Supplementry tle 3. Men vlues +/- s.e.m. with n=7 (p<.5; p<.1; p<.1 versus wild-type ). Nture Medicine doi:1.138/nm.2297

13 2 Ucp1 expression [copy numer GOI / Tp x1 9 ] 1 5 BAT suwat epiwat Figure S12. Ucp1 expression in rown nd white dipose tissues fter exposure Ucp1 mrna expression levels were drmticlly incresed upon exposure in rown dipose tissue (BAT). In sucutneous white dipose tissue (suwat) ut not in epididyml white dipose tissue (epiwat), Ucp1 expression is detectle nd even induced upon exposure. This suggest the presence nd ctivtion of rown dipocytes in suwat ut not (or to lesser extent) in epiwat. Men vlues +/- s.e.m. with n=12 (p<.1). Nture Medicine doi:1.138/nm.2297

14 w/o SPIO nnosomes Supplementry figures interscpulr suscpulr suscpulr deep cervicl with SPIO nnosomes interiscpulr Figure S13. Non-invsive imging of ctivted BAT depots efore nd fter injection of SPIOlelled TRL Coronr T2-weighted imges of mouse efore (upper pnel) nd fter (lower pnel) injection of SPIO-lelled TRL re shown. Next to the liver, interscpulr, suscpulr nd deep cervicl BAT depots pper lck due to the strong negtive contrst upon uptke of SPIO-lelled TRL. Intensities of other orgns such s skeletl muscle nd white dipose tissues remin unchnged. Nture Medicine doi:1.138/nm.2297

15 5 CM 125 I lumin-rdioctivity [cpm/mg] CM+triton WR-1339 CM CM+triton WR-1339 liver spleen kidney epiwat suwat rin muscle hert BAT 25 CM CM+triton WR-1339 Evns Blue mesured t 62 nm CM CM+triton WR-1339 liver spleen kidney epiwat suwat rin muscle hert BAT Figure S14. Endothelil permeility in reltion to lipolysis in nd mice Endothelil permeility in nd mice ws ssessed y injection of 125 I-lumin () nd Evns Blue (). To investigte the role of lipolysis for endothelil permeility, chylomicrons (CM) lone or together with triton WR-1339 to lock lipolysis were co-injected. () Orgn distriution of 125 I-lumin demonstrtes tht exposure increses endothelil permeility in BAT t lest in prt y n increse in lipolysis. () Orgn distriution of Evns Blue more or less resemles the uptke of 125 I-lumin supporting the notion tht exposure increses endothelil permeility in BAT y TRL lipolysis. Men vlues +/- s.e.m. with n=5 (p<.5; p<.1; p<.1) Nture Medicine doi:1.138/nm.2297

16 3 H-rdioctivity [cpm] c Supplementry figures H-rdioctivity [cpm/mg orgn] time fter injection [min] 75 FVB wild-type FVB wild-type Ldlr -/- Ldlr Apoe -/- -/- Apoe Apo5 -/- 5 -/- Apo5 -/ wild-type LRP1-N2 knockin liver hert epiwatsuwat BAT 3 H-rdioctivity [cpm/mg orgn] d 59 Fe-SPIO [cpm/mg orgn] liver hert epiwat suwat BAT liver wild-type LRP1-N2 knockin hert epiwat suwat BAT Figure S15. TRL uptke into ctivted BAT is independent of LDL receptor, LRP1, poe nd poa5. To prepre rdiolelled-poe-null TRL, poe-deficient FVB mice received n olive oil gvge enriched with 3 H-triolein. Two hours fter gvge, plsm ws hrvested nd TRL were isolted y ultrcentrifugtion. () Turnover nd () orgn uptke of 3 H-triolein-TRL ws investigted in exposed LDL receptor (Ldlr -/- ), Apoe -/-, Apo5 -/- knock-out mice nd FVB wild-type mice, respectively. Plsm removl ws significntly impired only in Apo5 -/- mice which is in line with n ccelerted LPL-medited TRL lipolysis oserved in poa5-trnsgenic mice (Merkel et l., 25). However, BAT-medited prticle uptke ws even higher in Apoe -/- nd Apo5 -/- mice while liver uptke ws lower. Orgn uptke of recominnt TRL lelled with 3 H-triolein (c) nd 59 Fe- SPIO (d) ws studied in LRP1 knock-in mice (Roeroeck et l., 25) nd respective wild-type s. LRP1 knockin crry muttion within the C-terminl domin of LRP1 (LRP1-N2 knockin) tht inctivtes endocytosis medited y LRP1. No difference ws oserved in BAT-medited uptke of recominnt TRL. Men vlues +/- s.e.m. with n=6 (p<.5; p<.1; p<.1 versus wild-type s indicted y color code). In summry, these dt indicte tht processing nd inding to ctivted BAT re independent of 1.) the LPL-modulting fctor ApoA5; 2.) the lipoprotein receptors LDL receptor nd LRP1 which medite TRL remnnt uptke into liver cells (Rohlmnn et l 1998, Ltsch et l 29) nd 3.) poe which is the most importnt lignd for TRL uptke into the liver. Nture Medicine doi:1.138/nm.2297

17 ody core temperture [ C] A B C D C57BL/6 Cd36 -/- Figure S16. Body temperture is drsticlly reduced in CD36-deficient mice fter exposure Two individul CD36-deficient mice nd two respective wildtype littermtes were exposed to 4 C for 12 hours including fsting period in the lst 4 hours. Body core temperture ws reduced to 26.1 C (mouse C) nd 23.8 C (mouse D) in Cd36 -/- mice compred to 35.2 C (mouse A) nd 35.5 C (mouse B) in wildtypes. The reduced ody temperture is ssocited with low locomotor ctivity nd noticele shivering (see lso supplementry movies 5 nd 6). Nture Medicine doi:1.138/nm.2297

18 3H rdioctivity [cpm] 3H rdioctivity [cpm] 1 min fter injection 4 lumin:olete 3 TRL 2 wild-type CD frctions 2 min fter injection 3 lumin:olete 2 TRL wild-type 1 CD frctions c 3H rdioctivity [cpm] min fter injection TRL lumin:olete frctions wild-type CD36 Figure S17. Clernce of TRL s well s ftty cids is delyed in Cd36 -/- mice Pooled plsm smples fter injection of recominnt TRL lelled with 3 H-triolein were seprted y FPLC t time point 1 min (), 2 min () nd 5 min (c). After 1 min () more TRL nd more 3 H-olete ound to lumin re present in the lood in Cd36 -/- mice. 3 H-counts decresed oth in TRL nd lumin frctions over time ( nd c) while TRL nd 3 H-olete lumin clernce ws delyed in Cd36 -/- mice. Nture Medicine doi:1.138/nm.2297

19 4 mrna fold expression [.u.] len len oese oese mrna expression [copy numer GOI / TBPx1 9 ] len len oese oese Ftp1 Ftp3 Ftp4 Cd36 Figure S18. BAT expression pttern is not ltered in mice with diet-induced oesity () Genes importnt for thermogenesis (Pprgc1 nd Ucp1) were incresed upon exposure in len nd insulin-resistnt mice. Genes importnt for lipolytic processing such s Gpihp1 nd Lpl were lso induced upon -exposure in len nd oese mice. Compred to ftty cid trnsporters Ftp1, Ftp3 nd Ftp4 only Cd36 is induced upon -exposure. Men vlues +/- s.e.m. with n=4. No significnt differences were detected etween len nd oese mice. () Compring the totl copy numers in reltion to the housekeeper Tp y quntittive TqMn nlysis, Cd36 mrna undnce is much higher thn mrna levels of Ftp1, Ftp3 nd Ftp4 suggesting tht Cd36 is the most importnt ftty cid trnsporter in BAT. Nture Medicine doi:1.138/nm.2297

20 n.s weight loss [g] weight loss [%] 5 1 len oese len oese Figure S19. Body weight loss fter exposure in len nd oese C57BL/6 mice Age-mtched mle C57BL/6 mice were fed norml chow diet (len) or to provoke diet-induced oesity lrd-sed, dietogenic high-ft diet (oese). Oese mice lose significntly more weight thn len s fter exposure. () However, compring the weight loss in percentge of ody weight, similr reduction in ody weight induced y exposure ws oserved. (ech circle represents n individul mouse; n=1 per group; p<.5; p<.1; p<.1 ). Nture Medicine doi:1.138/nm.2297

21 lood glucose [mg/dl] len len oese oese time fter gvge [min] Figure S2. Glucose tolernce is improved fter exposure Mice received 1 g/kg glucose y orl gvge. Control oese mice displyed glucose intolernce compred to len s ut oese mice clered glucose much more rpidly compred to oese mice. Men vlues +/- s.e.m. with n=5 (p<.5; p<.1; p<.1 versus s indicted y color code). Nture Medicine doi:1.138/nm.2297

22 14 C-deoxyglucose [cpm/mg orgn] len len oese oese 25 liver spleen kidney epiwat suwat muscle hert BAT rin 14 C-deoxyglucose [cpm per totl orgn] len len oese oese liver spleen kidney epiwatsuwatmuscle hert BAT rin Figure S21. BAT ctivtion strongly increses glucose uptke in len nd insulinresistnt oese C57BL/6 mice 6 min fter gvge orgns were removed, lysed in solvle nd 14 C-desoxyglucose ws determined. () specific nd () totl (clculted from Supplementry tles 3 nd 4) glucose uptke into BAT is strongly stimulted fter BAT ctivtion in len nd oese mice. This increse is significntly higher in oese compred to len s. Dt re presented s men +/- s.e.m; n=4 for HFD mice, n=5 for len mice. (p<.5; p<.1; p<.1 versus s indicted y color code). Nture Medicine doi:1.138/nm.2297

23 3 H-triolein [cpm/mg orgn] len len oese oese liver spleen kidney epiwat suwat muscle hert BAT rin 4 3H triolein [cpm per totl orgn] liver spleen kidney epiwat suwat muscle hert BAT rin Figure S22. BAT ctivtion strongly increses olete uptke in len nd insulinresistnt oese C57BL/6 mice 6 min fter gvge orgns were removed, lysed in solvle nd 3 H-triolein ws determined. () specific nd () totl (clculted from Supplementry tles 3 nd 4) uptke into BAT is strongly stimulted fter BAT ctivtion in len nd oese mice. The increse in specific BAT uptke is independently of insulin levels nd insulin resistnce. The BAT of oese mice is igger in mss rendering the totl uptke higher. Dt re presented s men +/- s.e.m.; n=4 for HFD mice, n=5 for len mice (p<.5; p<.1; p<.1 versus s indicted y color code). Nture Medicine doi:1.138/nm.2297

24 Glut4 copies per housekeeper Tp Glut1 copies per housekeeper Tp muscle hert epiwat suwat BAT muscle hert epiwat suwat BAT Figure S23. Cold exposure increses the expression of glucose trnsporters GLUT4 nd GLUT1 in BAT of C57BL/6J mice In line with erlier studies (Gspretti et l J Physiol 23) we oserve tht exposure increses the expression of () GLUT4 nd () GLUT1 in BAT. Although the effect did not rech sttisticl significnce, the chnges in hert tissue re comprle to those in BAT. The comined increse in GLUT4 nd GLUT1 cn explin incresed uptke of postprndil glucose in tissues with incresed work lod, hert nd BAT. Dt re presented s men +/- s.e.m; n=4 (p<.5; p<.1; p<.1 versus s indicted y color code). Nture Medicine doi:1.138/nm.2297

25 BAT P-S len oese hert P-S len oese Figure S24. Cold exposure increses phosphoryltion of riosoml S6 protein in BAT nd hert of len nd oese C57BL/6J mice BAT nd hert from nd len nd oese mice were hrvested, totl protein isolted nd sujected to Western lotting. As judged y phosphoryltion of riosoml protein S6, direct down-strem trget of the insulin-akt-signling cscde, we oserve more insulin ction fter exposure which is even more pronounced in oese nimls under nd conditions. Nture Medicine doi:1.138/nm.2297

26 59 Fe-SPIO [cpm] c H rdioctivity x ody weight [cpm x g] len len oese oese time fter injection [min] d len len oese oese time fter injection [min] 59 Fe-SPIO [cpm x g] 59 Fe-SPIO x ody weight [cpm x g] len len oese oese time fter injection [min] len len oese oese liver hert epiwat suwat BAT NS Figure S25. Cold exposure ccelertes plsm kinetics nd uptke of recominnt TRL in len s well s in insulin-resistnt oese C57BL/6 mice 3 H-triolein nd 59 Fe-SPIO were emedded into the core of recominnt TRL nd turnover kinetics were studied (compre figure 4). () Plsm clernce of core prticles ws ccelerted in len (circles) nd oese (tringles) - exposed mice. Incresed plsm removl of 59 Fe-SPIO () nd 3 H- triolein (c) remins highly significnt when the dt re corrected for ody weight. (d) The ccelerted clernce is cused y strong increse in TRL uptke into ctivted BAT in len nd oese C57BL/6 mice. Men vlues +/- s.e.m. with n=5 (p<.5; p<.1; p<.1 versus s indicted y color code). Nture Medicine doi:1.138/nm.2297

27 Supplementry tles wild-type wild-type Cd36 -/- Cd36 -/- triglycerides [mg/dl] cholesterol [mg/dl] free ftty cids [mg/dl] Supplementry tle 1: Plsm lipids of wild-type nd Cd36 -/- efore nd fter exposure Next to nimls t 22 C CD36-deficient mice nd wild-type littermtes were exposed 6 h including 4 h fsting t 4 C. In Cd36 -/- mice nd wild-type littermtes triglycerides nd cholesterol levels decrese while free ftty cids rise fter exposure. Compred to wild-types the increse in free ftty levels in plsm of Cd36 -/- in nd even more in mice underlines the importnce of this receptor for lipid uptke. Nture Medicine doi:1.138/nm.2297

28 Supplementry tles len len oese oese triglycerides [mg/dl] cholesterol [mg/dl] glucose [mg/dl] insulin [ng/ml] HOMA [mg 2 /dl 2 ] diponectin [µg/ml] leptin [pg/ml] Supplementry tle 2. Metolic plsm prmeters in len nd oese mice. In this diet-induced oesity model, triglycerides re low nd cholesterol levels elevted significntly. As expected the HFD leds to hyperglycemi nd hyperinsulinemi which is in line with the oserved insulin resistnce (compre Supplementry Fig. 2). Leptin is mrkedly incresed in HFD-fed mice, ut strongly decreses oth in oese nd len mice fter exposure. Adiponectin remins unchnged or lower respectively. Nture Medicine doi:1.138/nm.2297

29 Supplementry tles tissue [g] [%] totl ody 27 1 liver spleen.85.3 kidney epiwat suwat.8 3 muscle hert BAT Supplementry tle 3: Len tissue weight estimtions underlying totl orgn uptke clcultions Arevtions: epiwat: epididyml white dipose tissue, suwat: sucutneous white dipose tissue, BAT: rown dipose tissue. To clculte from specific uptke in [cpm/mg] totl orgn uptke, totl tissue weights were clculted from tissue percentges of totl len C57BL/6J ody weight estimtions. Nture Medicine doi:1.138/nm.2297

30 Supplementry tles tissue [g] [%] totl ody 42 1 liver spleen.85.2 kidney epiwat suwat (inguinl) muscle hert.18.4 BAT Supplementry tle 4: Oese tissue weight estimtions underlying totl orgn uptke clcultions Arevtions: epiwat: epididyml white dipose tissue, suwat: sucutneous white dipose tissue, BAT: rown dipose tissue. To clculte from specific uptke in [cpm/mg] totl orgn uptke, totl tissue weights were clculted from tissue percentges of totl oese C57BL/6J ody weight estimtions. Nture Medicine doi:1.138/nm.2297

31 Supplementry tles FoV [mm] mtrix slices / stcks thickness [mm] FA [ ] TR [ms] TE [ms] echoes NSA dynmic intervl [s] Totl scn time [min] T2-weighted coronl GRE T2-weighted xil GRE T2 -weighted dynmic 1 xil GRE / N/A 3: / N/A 5: / :16 Supplementry tle 5: Detiled prmeters of the MRI-sequences pplied in the experiments Arevtions: Grdient echo sequence (GRE), Field of View (FoV), excittion flip ngle (FA), Repetition Time (TR), Echo Time (TE), Numer of Signl Averges (NSA). The mesurements were done in the imging-mode t 37 C. 1 For ccelertion of the dynmic mesurements without lost of sptil resolution the key-hole technique (etween the first nd lst imge only the low sptil frequencies of the originl full, high-resolution dt set re repetedly mesured) ws pplied. The key-hole fctor ws 32%. Nture Medicine doi:1.138/nm.2297

32 Supplementry methods Preprtion nd leling of TRL We isolted humn TRL from pocii-deficient sujects (pproved y Ärztekmmer Hmurg, Germny). Lipids were extrcted y the method of Folch. A detiled method for the leling of TRL with nnocrystls ws descried recently 4. Briefly, for emedding 1 mg of the lipid extrct (pproximtely 8% triglycerides, 1% cholesterol nd 1% phospholipids) were dissolved in chloroform nd mixed with either hydrophoic superprmgnetic iron-oxide (SPIO) nnocrystls, CdSe/CdS/ZnS-core-shell-shell nnocrystls or with mixture of 59 Fe-oxide nnocrystls (2 KBq/1 mg lipid) (B.F. et l., in preprtion) nd 1.85 MBq per 1 mg lipid [9,1-3 H(N)]-triolein (PerkinElmer), respectively. The solvent ws removed nd TRL were formed in 1 ml PBS y soniction. Potentil ggregtes were removed y filtrtion using 45 nm filter prior to intrvenous injection (2 mg lipid per mouse). For prepring mouse poe-null TRL, Apoe -/- mice received n orl gvge with 2 µl olive oil contining 1.48 MBq [9,1-3 H(N)]-triolein. Two hours fter injection we isolted rdioleled TRL from plsm y ultrcentrifugtion. To produce BODIPY-TRL, we incuted humn dipocytes 36 with fluorescent ftty cid nlogues 4,4-difluoro-5,7- dimethyl-4-or-3,4-diz-s-indcene-3-hexdecnoic cid (BODIPY FL C16, Invitrogen) over night nd hrvested dipocyte TG y Folch extrction. For leling of TRL 1 mg lipid extrct (see ove) ws mixed with 1 mg dipocyte extrct contining BODIPY- TG nd BODIPY-TRL were formed s descried ove. Plsm Prmeters Plsm triglycerides, cholesterol nd free ftty cids were determined using commercil kits (Roche nd Wko). Plsm insulin ws mesured using rt/mouse insulin ssy kit (Chrystl Chem). Adiponectin nd leptin were determined using n ELISA from R&D Systems. For lipoprotein profiling pooled plsm ws seprted y FPLC using S6-superose columns (GE Helthcre) nd rdioctivity nd/or lipid levels were nlyzed in ech frction. Orl glucose nd ft tolernce test were comined y gvge of mixture of liposomes contining 12 KBq Nture Medicine doi:1.138/nm.2297

33 [9,1-3 H(N)]-triolein /g ody weight nd glucose (1 mg/g ody weight) trced with.62 KBq 2-desoxy-D-[ 14 C] -glucose/g ody weight in H 2 O. Totl RNA extrction nd Rel-time quntittive PCR Totl RNA from BAT ws extrcted using the TissueLyser nd RNesy system (Qigen) ccording to the mnufcturer s instructions. DNA contminnts were removed vi column DNse tretment (RNse-Free DNse set, Qigen). Totl RNA (1 µg) ws used for cdna preprtion (High-Cpcity cdna Archive Kit, Applied Biosystems) ccording to the mnufcturer s instructions. Rel-time RT-PCR ws performed nd reltive expression ws clculted y normliztion to selected housekeeper mrna (Tp) y the Ct method s descried previously 38. Assy-on-Demnd primer/proe sets supplied y Applied Biosystems were used (Assy IDs re ville upon request). Endothelil Permeility Control nd -exposed mice were injected with TRL (1 mg TG /per mouse), triton WR s descried ove nd 1 µl Evns Blue (Sigm, 5 mg/ml in PBS) or 125 I-lumin (Sigm, 1mg with 2 KBq per mouse). Rdioleling ws performed using Iodoeds ccording to the mnufcturer s instructions (Pierce). After 6 min mice were nesthetized nd extensively perfused with PBS-heprin (1 U/mL) contining 1% BSA (Sigm). Orgns were hrvested nd extrcted in 1:1 (w/v) dimethylformmide t 37 C for 6 min. OD ws mesured t 62 nm. For determintion of rdioctivity, orgns were gmm-counted nd uptke clculted in counts/mg orgn. Western lotting Snp-frozen tissue smples were lysed in RIPA uffer supplemented with complete mini protese inhiitor cocktil (Roche), 5 mm N 3 VO 4 nd.1% SDS using using the TissueLyser (Qigen). Proteins were seprted on NuPAGE Bis-Tris 4-12% grdient gels (Invitrogen). P-S6 ws detected with Phospho-S6 Riosoml Protein (Ser24/244) Antiody #2215 (Cell Signling) Nture Medicine doi:1.138/nm.2297