Combined deletion of SCD1 from adipose tissue and liver does not protect

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1 Comined deletion of SCD1 from dipose tissue nd liver does not protect mice from oesity Mtthew T. Flowers 1, Lcmouh de 1, Mggie S. Strle 2 nd Jmes M. Ntmi 1,2,* Deprtments of 1 Biochemistry nd 2 Nutritionl Sciences, University of Wisconsin-Mdison, 433 Bcock Drive, Mdison, Wisconsin 5376 Running Title: Deletion of dipose nd liver SCD1 does not prevent oesity *Correspondence to e ddressed to: Jmes M. Ntmi University of Wisconsin-Mdison Deprtment of Biochemistry 433 Bcock Drive Mdison, WI 5376 Tel: Fx: e-mil: ntmi@iochem.wisc.edu revitions used: dipose SCD1 knockout (); gouti ( y /); ntisense oligonucleotide (SO); diet-induced oesity (DIO); ftty cid (F); glol SCD1 knockout (GKO); liver SCD1 knockout (); liver/dipose SCD1 knockout (L); monounsturted ftty cids (MUF); non-gouti (/); Scd1 flox/flox (); skin SCD1 knockout (SKO); steroyl-co desturse-1 (SCD1), sterol regultory element inding protein-1c (SREBP1c), triglycerides (TG) 1

2 strct Steroyl-Co desturse 1 (SCD1) ctlyzes the synthesis of monounsturted ftty cids (MUF) from sturted F nd mice with whole-ody or skin-specific deletion of SCD1 re resistnt to oesity. Here, we show tht mice lcking SCD1 in dipose nd/or liver re not protected from either genetic- (gouti; y /) or diet-induced oesity (DIO) despite roust reduction in SCD1 MUF products in oth sucutneous nd epididyml white dipose tissue. dipose SCD1 deletion hd no effect on glucose or insulin tolernce, or heptic triglyceride (TG) ccumultion. Interestingly, lck of SCD1 from liver lowered the MUF levels of dipose tissue, nd vice vers, s reflected y the chnges in F composition. Simultneous deletion of SCD1 from liver nd dipose resulted in synergistic lowering of tissue MUF levels, especilly in the y / model in which glucose tolernce ws lso improved. Lstly, we found tht liver nd plsm TG show nerly identicl genotype-dependent differences in F composition indicting tht F composition of plsm TG is predictive for heptic SCD1 ctivity nd TG F composition. The current study suggests tht SCD1 deletion from dipose nd/or liver is insufficient to elicit protection from oesity, ut supports the existence of extensive lipid crosstlk etween liver nd dipose tissue. Supplementry key words: steroyl-co, monounsturted ftty cid, desturse, desturtion index, olete, plmitolete, ftty cid composition, gouti, triglyceride, Cre-lox 2

3 Introduction Steroyl-Co desturse 1 (SCD1) ctlyzes the desturtion of sturted ftty cids (F), primrily plmitte (16:) nd sterte (18:), into the cis-monounsturted ftty cid (MUF) products plmitolete (16:1n7) nd olete (18:1n9), respectively. The metolic role for SCD1 hs een extensively explored in mice with nturlly occurring Scd1 muttions nd those with glol deletion of Scd1 (GKO mice) (1-3). These mice disply remrkle hypermetolic phenotype tht protects them from oesity, insulin resistnce nd heptic stetosis. To determine which tissue or tissues re primrily responsile for these metolic chnges, we hve employed the Cre-lox system to explore the tissue-specific contriutions of SCD1. We previously found tht mice with liver-specific deletion of Scd1 ( mice) re protected from high-crohydrte, ut not high-ft diet-induced oesity (DIO), unlike GKO mice tht re resistnt to oth high-ft nd high-crohydrte DIO (4). This indictes tht inhiition of liver SCD1 lone is insufficient to elicit the hypermetolism nd incresed energy expenditure necessry to compenste for the incresed energy intke ssocited with high-ft feeding. The reduced high-crohydrte diet-induced diposity in nd GKO mice ws ssocited with lock in crohydrte-induced increses in heptic sterol regultory element inding protein-1c (SREBP-1c) proteolytic processing, expression of F synthesis genes nd heptic triglyceride (TG) ccumultion. We recently reported tht mice with skin-specific deletion of Scd1 (SKO mice) recpitulted the hypermetolic phenotype oserved in GKO mice, indicting tht the skin is mjor contriutor to the ltered energy metolism oserved in GKO mice (5). In contrst, SKO mice hd norml crohydrte-induced increse in SREBP-1c mturtion nd F synthesis genes. These heptic oservtions highlight tht not ll of the phenotypes of the SCD1 GKO mice cn e ttriuted to SCD1 deletion in the skin. 3

4 Interestingly, mice intrperitonelly injected with Scd1-trgeted ntisense oligonucleotides (SO) re lso protected from the development of high-ft DIO nd insulin resistnce (6, 7). This SO therpy potently reduced SCD1 expression nd ctivity in the liver nd dipose tissue without ffecting SCD1 expression in the muscle or skin. This suggests tht inhiition of SCD1 in the liver nd dipose tissue elicits oesity resistnce vi mechnism tht is physiologiclly distinct from tht cused y skin SCD1 deletion. Since mice re not protected from high-ft DIO (4), we hypothesized tht deletion of dipose SCD1 ( mice), or comined deletion of liver nd dipose SCD1 (L mice), would elicit oesity resistnce nd other improved metolic prmeters previously oserved in SCD1 GKO mice nd those treted with SO. dipose SCD1 ws deleted from oth white nd rown dipose tissue y introducing the P2-Cre llele into our SCD1 flox/flox mice. Surprisingly, nd L mice were not protected from oesity despite roust reductions in dipose MUF content. These results indicte tht deletion or inhiition of SCD1 in the white nd rown dipose tissue, in the presence or sence of liver SCD1 ctivity, hs miniml effect on whole-ody energy expenditure. Our study lso revels tht dipose-derived F impct the F composition of liver, nd vice vers. Lstly, we report tht the MUF composition of plsm TG is strikingly similr to tht of liver TG nd cn potentilly e used s predictor of heptic SCD ctivity. 4

5 Mterils nd methods nimls, Genotyping nd Diets. FBP4(P2)-Cre mice were otined from Brr Khn t Beth Isrel Deconess Medicl Center (8) nd ckcrossed to C57BL/6J for t lest 1 genertions. To generte mice, we crossed C57BL/6J Scd1 flox/flox () mice (4), which trgets exon 3 of the Scd1 gene, with mice heterozygous for P2-Cre to generte compound heterozygous (Scd1 flox/+ ; P2-Cre/+) mice. Mle Scd1 flox/+ ;P2-Cre/+ mice were susequently mted with femle mice to generte Scd1 flox/flox ;P2-Cre/+ mice (). For litter expnsion, mle mice were red with either femle mice to produce or mice, or with femle Scd1 flox/flox ;lumin-cre/+ () mice (4) to produce,, or Scd1 flox/flox ;P2-Cre/+;lumin-Cre/+ (L) mice. lterntively, mle L mice were red with femle mice to generte,,, or L mice. To generte conditionl Scd1-deficient mice lso contining the yellow gouti ( y /) llele, we red mle or mice with femle C57BL/6J y / mice (Jckson Ls stock #21). The F1 mle y / Scd1 flox/+ ;lumin-cre/+ or y / Scd1 flox/+ ;P2-Cre/+ mice were then red with femle non-gouti (/) mice to generte y /, y / nd y / mice. For litter expnsion, mle y / or y / were red with femle / mice. lterntively, mle y / mice were red with femle / mice to generte y /, y /, y / nd y / L mice. Mice were mintined on 12 hr light/drk cycle with free ccess to food nd wter. Breeders were fed Purin 515 nd pups were wened t 3 wks of ge nd susequently fed cerel grin-sed, low-ft stndrd diet (SD; Purin 58). For DIO studies, mle mice were individully cged t 8 wks of ge nd fed either Purin 58 or high-ft diet (Reserch Diets RD12492; 6% kcl ft) for n dditionl 18 wks. For y /-induced oesity studies, mle mice 5

6 were individully cged t 4 wks of ge nd mintined on Purin 58 until 26 wks of ge. nimls were fsted for 4 hr nd scrificed y n overdose of isoflurne nesthesi. The genotyping protocol for distinguishing the Scd1 flox nd wild type Scd1 lleles hs een descried previously (4, 9). For reeding strtegies involving only one Cre trnsgene, we used generic Cre-recominse genotyping strtegy ville t the Jckson Lortories wesite (jxmice.jx.org). For L reeding schemes, we designed genotyping primers specific to either lumin-cre or P2-Cre. lumin-cre ws mplified using primer l-f (5 GC TGC GG CT TC TC 3 ) nd Cre-R (5 GTG CG CT TGC TGT CC TT 3 ) with 54 C nneling temperture. P2-Cre ws mplified using P2-F (5 TG TC TGG CCC CC TTG G 3 ) nd Cre-R with 51 C nneling temperture. ll in vivo experimentl procedures were performed in ccordnce with Ntionl Institutes of Helth Guidelines for the Cre nd Use of nimls nd were pproved y the Institutionl niml Cre nd Use Committee t the University of Wisconsin-Mdison. Immunolot nlysis of SCD1 Liver nd white dipose microsomes were prepred y sequentil centrifugtion. Tissues were first homogenized t 1 mg tissue/ml uffer in.1 M potssium phosphte uffer (ph=7.2) supplemented with 1 μg/ml leupeptin nd 1 mm PMSF. The homogente ws centrifuged t 1, x g for 15 min t 4 C. The superntnt ws susequently centrifuged t 1, x g for 1 h t 4 C. The pellet ws rinsed once nd resuspended in protese inhiitor free.1 M phosphte uffer. Brown dipose tissue ws homogenized t 1 mg tissue/ml in lysis uffer contining 1 mm PMSF, 1 μg/ml leupeptin, 5 μg/ml pepsttin nd 2 μg/ml protinin. SCD1 immunolotting ws performed on 1 μg of protein using polyclonl SCD1 ntiody (Snt Cruz; sc-14719). 6

7 Glucose nd insulin tolernce tests. For glucose tolernce tests, mice were fsted for four hours nd susequently injected i.p. with 1% dextrose t dose of 1 g/kg ody weight. Blood ws smpled y retrooritl puncture t, 2, 4, 9 nd 18 min post-injection. For insulin tolernce tests, non-fsted mice were injected i.p. with.75 U/kg ody weight of humn insulin (Novo Nordisk). Blood ws collected y retrooritl puncture t, 15, 3, 45 nd 6 min postinjection. Plsm glucose ws nlyzed using colorimetric glucose oxidse method. Tissue nd plsm lipid nlysis. Lipids were extrcted from pproximtely 3 mg of tissue or 1 μl of plsm lipids using modifiction of the Folch procedure (1). Smples were homogenized in 3 ml of CHCl 3 :MeOH (2:1) contining 1 mg/1 ml utylted hydroxytoluene. Heptdecnoic cid ws dded s n internl stndrd to correct for smple loss. Next, 1 ml of cidified sline (.1 N HCl,.9% NCl) ws dded. fter vigorous vortexing nd centrifugtion, the orgnic lyer ws isolted, dried under nitrogen gs nd sujected to TLC on silic gel-6 pltes (EMD Chemicls) in heptne/isopropyl ether/glcil cetic cid (6/4/3, v/v/v). The nds corresponding to stndrds were scrped nd trnsferred to screw cp glss tues contining pentdecnoic cid s n internl stndrd to control for trnsmethyltion efficiency. F were trnsmethylted in the presence of 14% oron trifluoride in methnol. The resulting methyl esters were extrcted with hexne nd nlyzed y gs liquid chromtogrphy s previously descried (11). Totl lipid contents were clculted from individul F content in ech frction. Rel-time quntittive PCR. Totl RN ws extrcted with TRI regent (Moleculr Reserch), treted with Turo DNse (mion), nd reverse trnscried using Multiscrie reverse trnscriptse (pplied Biosystems). Rel-time quntittive PCR ws performed on n BI StepOne Plus instrument using gene-specific primers nd Power SYBR Green mster mix 7

8 (pplied Biosystems). Quntifiction of given genes is expressed s mrn level normlized to housekeeping gene (rp) using the ΔΔCt method. Primer sequences re ville upon request. Dt nd sttisticl nlysis. Dt re expressed s men + SEM nd were compred y one- or two-wy NOV followed y Tukey s Post-hoc test in GrphPd Prism. Comprisons with P<.5 were considered significnt. 8

9 Results To generte mice deficient in dipose Scd1 (), we introduced the P2-Cre trnsgene into our Scd1 flox/flox () colony (4, 5). We lso generted mice with liver-specific deletion of Scd1 () tht expressed the lumin-cre trnsgene (4), nd crossed these with the mice to otin mice with simultneous deletion of Scd1 from oth the liver nd dipose (L). nd L mice were orn t the expected frequency nd were indistinguishle from mice. Importntly, nd L mice do not disply the closed eye fissures, dry skin nd lopeci previously oserved in glol (GKO) nd skin-specific (SKO) Scd1-deficient mice (5). Immunolot nlysis confirmed the deletion of Scd1 from white nd rown dipose tissue of nd L mice, nd from liver of nd L mice (Figure 1). Furthermore, F composition nlysis of TG from oth sucutneous dipose (Figure 1B-E) nd epididyml dipose (Supplementry Figure I) tissue reflects mrked decrese in the undnce of the SCD products plmitolete (16:1n7) nd olete (18:1n9) nd n increse in the SCD sustrtes plmitte (16:) nd sterte (18:) in nd L mice. Effect of dipose Scd1 deletion on oesity nd glucose tolernce To test whether dipose nd/or liver deletion of Scd1 influences the development of oesity, we used oth dietry (high-ft DIO) nd geneticlly-induced ( y / llele) oesity models compred to / mice fed stndrd diet. The high-ft DIO model utilizes hypercloric, semi-purified diet, which is highly undnt in the SCD product olete. Despite the mple MUF content of this high-ft diet, we hve previously shown tht oth GKO nd SKO mice, ut not mice, re remrkly resistnt to oesity on this diet due to hypermetolic phenotype (4, 5). y / mice hve uiquitous expression of the gouti protein, cusing leptin resistnce, incresed food intke nd decresed energy expenditure (2, 12). Thus, the y / model 9

10 llows for oesity-induction y feeding stndrd diet. We found no significnt difference in ody weight, epididyml or sucutneous white dipose weights due to dipose nd/or liver SCD1 deletion in either the DIO or y / models (Figure 2, C, D). dditionlly, food intke ws unffected (Figure 2E,F). Liver weight ws not significntly ffected in / mice fed stndrd or high-ft diet; however, liver weight ws reduced y loss of liver Scd1 in y / nd y / L compred to counterprts (Figure 2B). Fsting glucose s well s glucose nd insulin tolernce were not significntly ffected y dipose Scd1 deletion in / mice fed stndrd or high-ft diet (Figure 3-C). However, liver SCD1 deletion in oth y / L nd y / improved glucose tolernce (Figure 3D). Heptic SCD1 ctivity influences dipose F composition Liver nd dipose tissue TG levels nd their F composition cn e influenced directly y severl sources. First, dietry F, primrily in the form of TG-rich chylomicrons, cn e tken up into tissues vi lipoprotein lipse nd lipoprotein receptor-medited mechnisms. Second, cells cn de novo synthesize F from cetyl-co, nd the rte of de novo synthesis in liver is especilly responsive to dietry nutrients nd hormones such s glucose nd insulin, respectively (13). nd finlly, there is significnt lipid crosstlk etween the dipose nd liver vi the reciprocl exchnge of dipose-derived free F nd heptic derived TG-rich VLDL. Importntly, intrcellulr sturted F (plmitte nd sterte), regrdless of their origin, cn e converted to MUF vi SCD1. Liver SCD1 influenced dipose stores of olete nd the 18:1n9/18: rtio, ut not stores of plmitolete or 16:1n7/16: rtio, in y / mice, ut not / mice (Fig. 1 nd Supp. Fig. I). This suggests tht under conditions of high heptic lipogenesis, liver-derived TG contriute greter mount to dipose TG stores thn during conditions of low heptic lipogenesis. dditionlly, 1

11 the % composition of plmitte, plmitolete, sterte nd olete in dipose TG ws significntly ffected y loss of dipose SCD1 in the sucutneous ft, ut not epididyml ft, of / mice. Upon introgression of the y / llele, oth ft depots were significntly influenced y loss of SCD1 from liver or dipose. These ft-depot specific chnges suggest tht the F composition of ft depots sptilly-oriented closer to the liver, such s viscerl or epididyml ft, is influenced to greter extent y liver-derived F, especilly under sl lipogenic conditions. side from 16- nd 18-cron sturted F nd MUF, the other predominnt F found in dipose is linoleic cid (18:2n6), which comprises pproximtely 25% of dipose F. In / mice, loss of dipose nd/or liver SCD1 hd no significnt effect on the percent composition of 18:2n6 in epididyml or sucutneous ft (dt not shown). Loss of dipose SCD1 in y / mice hd lso no effect on the undnce of 18:2n6 (Supplementry Figure II). Interestingly, loss of liver SCD1 in oth y / nd y / L mice cused elevted levels of 18:2n6 in the epididyml nd sucutneous dipose stores (Supp. Fig. II). To further explore the reltionship etween liver SCD1 nd dipose 18:2n6 levels, we looked t 18:2n6 levels in liver TG nd plsm TG. In / mice, 18:2n6 levels in liver were non-significntly elevted y pproximtely 12% in oth nd L mice compred to nd (~28.7% in nd L compred to 25.5% in nd ), ut plsm 18:2n6 levels were unchnged. However, oth liver nd plsm 18:2n6 levels were incresed in y / nd y / L compred to y / nd y / mice (Supp. Fig. II). Thus, lck of heptic SCD1, when comined with the y / llele, leds to drmtic decrese in 18:1n9 vilility nd concomitnt increse in 18:2n6 enrichment of these liver nd plsm TG, which susequently influence the F composition of the dipose stores. Effect of liver nd dipose SCD1 on heptic nd TG composition 11

12 lthough y / nd y / L mice were not protected from oesity, their liver weights were significntly lower thn the y / nd y / (Fig. 2). We mesured heptic TG levels in oth y / nd / mice. Compred to their / counterprts, y / mice hd significntly elevted heptic TG levels (Figure 4). However, this y /-effect ws severely lunted y loss of liver SCD1 in oth y / nd y / L mice. Consistent with this oservtion, the percent composition of plmitte, plmitolete, sterte nd olete in heptic TG were ll significntly ffected y loss of heptic SCD1 in oth the y / nd y / L mice. (Figures 4 nd 5). Comined loss of liver nd dipose SCD1 lowered heptic TG, the 16:1n7/16: rtio nd the 18:1n9/18: rtio in high-ft fed L mice s well. (Supplementry Figure III). Deletion of liver nd/or dipose SCD1 did not influence heptic TG levels in the / mice nd hd more modest effect on the F composition. Thus, the heptic rte of F synthesis is n importnt determinnt for the ccumultion of SCD sustrtes in the sence of SCD1. In y / mice, the heptic 16:1n7/16: nd 18:1n9/18: rtios were predominntly determined y liver SCD1. nlysis of the desturtion indices of heptic lipids lso reveled prtil role of dipose SCD1 in determining the F composition of the liver. In oth y / nd / mice, the liver TG 16:1n7/16: rtio ws independently nd dditively influenced y loss of liver nd dipose SCD1 (Fig. 4). dipose deletion of SCD1 in y / mice lso reduced the heptic 18:1n9/18: rtio. However, dipose-specific deletion of SCD1 did not protect mice from high-ft DIO- or y /-induced heptic TG ccumultion (Fig. 4 nd Supp. Fig. III). Plsm TG F composition prllels heptic TG F composition In the fsted stte, plsm TG re predominntly trnsported y liver-derived VLDL, which otin their neutrl lipid core during the pre-secretory intrcellulr ssemly of these 12

13 prticles in heptocytes. Therefore, we compred the liver nd plsm TG mss, desturtion indices nd F compositions in oth y / nd / mice with liver nd/or dipose deletion of SCD1 (Fig. 4 nd 5). Similr to the pttern oserved for liver TG mss, plsm TG mss in / mice ws not ffected y loss of dipose nd/or liver SCD1 ut loss of liver SCD1 in y / nd y / L led to reduction of plsm TG levels. Strikingly, the desturtion indices (Fig. 4) nd F composition (Fig. 5) for liver nd plsm TG showed the sme genotype-dependent chnges, suggesting tht the F composition of plsm TG is predictive of oth liver TG F composition nd SCD1 ctivity. 13

14 Discussion lthough mice with glol deletion of Scd1 (GKO) disply remrkle oesity resistnce (2-4) nd insulin sensitivity (14), the importnce of dipose SCD1 for whole-ody energy metolism is uncler. SCD1 is highly expressed in oth white nd rown dipose tissue. Previous studies in mice with whole-ody deletion of SCD1 hve documented incresed insulin signling nd uncoupling protein expression in the white nd rown dipose tissue (15-17). Due to the simultneous deletion of Scd1 from ll tissues in GKO mice, it is uncler whether metolic chnges in prticulr tissue re direct effect of locl loss of SCD1 function, or n indirect effect stemming from loss of SCD1 in distl tissues. Tissue-specific deletion using the Cre-lox system relies upon the use of tissue-specific promoter to drive the expression of the Cre-recominse. The FBP4(P2)-Cre trnsgene is highly expressed in oth white nd rown dipose tissue (8, 18). The differentition of predipocytes into mture dipocytes leds to the ctivtion of severl dipose-specific genes, including Fp4 nd Scd1 (19). Therefore, it is likely tht the FBP4-Cre-medited deletion of Scd1 occurs sometime during or immeditely fter dipocyte differentition. It is noteworthy to cknowledge tht P2-Cre expression nd concomitnt deletion of Scd1 in our studies re likely not restricted to dipocytes. The P2-Cre trnsgene hs lso een detected during emryonic development (2), in mcrophges nd mcrophge rich tissues (21, 22), one mrrow (21), crdic nd skeletl muscle, intestine, stomch, pncres nd in the centrl nd peripherl nervous system (23). However, the endogenous P2 gene is predominntly expressed in dipocytes nd the P2-Cre-medited recomintion in these other tissues is likely incomplete due to the lower expression level of Cre. Nonetheless, P2-Cre medited deletion of Scd1 from 14

15 dipocytes nd potentilly other cell types filed to recpitulte the oesity resistnce oserved in GKO mice. We recently reported tht skin-specific deletion of SCD1 elicited hypermetolic phenotype comprle to tht oserved in GKO mice (5). These mice resisted dipocyte hypertrophy nd mintined insulin sensitivity when chllenged with high-ft feeding, nd this ws ssocited with incresed uncoupling protein expression in vriety of peripherl tissues such s white nd rown dipose tissue, muscle nd liver. Thus, ltered skin lipid metolism cn indirectly influence metolic flux nd cell signling in peripherl tissues. However, these dt do not necessrily exclude role for SCD1 in other tissues such s rown or white dipose tissue, ecuse the severity of the cutneous phenotypes in SKO mice my msk the contriutions of SCD1 in these tissues to glol energy homeostsis. Therefore, it is possile tht n extrcutneous mechnism for oesity resistnce lso exists in the GKO mouse. This is supported y studies in mice treted with Scd1-trgeted SO, which re reported to not ffect the skin (6, 7). However, the filure of dipose nd/or liver Scd1 deletion to prevent oesity in either the y / or DIO models strongly suggests tht loss of SCD1 function in these tissues is not the mechnism for oesity resistnce in the GKO mice or SO-treted mice. Furthermore, we did not oserve incresed expression of thermogenic genes (Ucp1, Ucp2, dr2, Dio2, Pprgc1) in the rown dipose tissue of or L mice (Supplementry Figure IV). Intrperitonel injected SOs result in the ccumultion of SOs in severl tissues. In ddition to the reported effects of SCD1 SOs on liver, white dipose nd rown dipose, we speculte tht the SO-medited oesity resistnce is due to dditionl inhiition of Scd1 in cells or tissues not trgeted y the lumin-cre nd P2-Cre. In the cse of the dipose tissue, the SO tretment my e eliciting effects on oth pre-dipocytes nd mture dipocytes, unlike the 15

16 P2-Cre medited recomintion tht requires ctivtion of the P2 promoter during dipocyte differentition. Inhiition of SCD1 during the pre-dipocyte stge my cuse distinct metolic chnges compred to deletion of Scd1 in the lte stges of or post-differentition. lterntively, the SO my e eliciting off trget effects on other genes contriuting to oesity resistnce, unlike the Cre-lox system which is specific to Scd1. Unlike the dult mouse liver, which expresses primrily Scd1, oth Scd1 nd Scd2 re highly expressed in the mouse dipose tissue (24). We found tht the expression of Scd2 in white nd rown dipose tissue ws unffected y deletion of Scd1 vi P2-Cre (Supp. Fig. IV). Therefore, it is lso possile tht the nti-oesity effects of pulished Scd1-trgeted SO (6, 7) re occurring due to the simultneous inhiition of oth Scd1 nd Scd2. This would predict tht there re two SCD-medited mechnisms to oesity resistnce in mice: 1) deletion of Scd1 in skin, or 2) inhiition of Scd1 nd Scd2 from the liver nd dipose. The filure of Scd1 deletion from the liver to protect ginst the y /-induced oesity is somewht surprising in light of our previous oservtions of reduced diposity in mice fed semi-purified high-crohydrte diet (4). Since the y / mice in our study were fed cerel grin-sed high-crohydrte, low-ft diet, we hypothesized tht lck of liver SCD1 would reduce heptic conversion of these dietry crohydrtes into TG, reduced liver VLDL TG secretion nd reduce overll ody diposity. lthough we did oserve reduced heptic MUF nd TG content, this is presumly insufficient to ffect overll energy homeostsis. The leptinresistnt phenotype of the y / mice results in oth incresed food intke nd decresed energy expenditure. Thus, hypermetolic response tht increses whole-ody energy expenditure, such s tht oserved in GKO nd SKO mice (5), my e necessry to comt the positive energy lnce tht exists in the y / nd DIO oesity chllenges. 16

17 Plmitolete (16:1n7) relesed into circultion from the dipose tissue hs een suggested to ct s eneficil lipokine tht improves metolic derngements including insulin resistnce nd heptic stetosis (25). The comined dipose deficiency of the P2 (FBP4) nd ml1 (FBP5) F inding proteins cused elevted levels of dipose 16:1n7 concomitnt with improved metolic phenotype (25), nd this predicts tht reducing norml levels of dipose 16:1n7 y deleting SCD1 would worsen metolism. However, deletion of dipose SCD1 in our study hd no ffect on ody weight, glucose or insulin tolernce in stndrd diet fed mice, s well s in two models of oesity (DIO nd y /), despite significnt lowering of dipose levels of 16:1n7. Furthermore, simultneous deletion of liver nd dipose SCD1 ctully hd eneficil effect of lowering liver TG levels nd improving glucose tolernce compred to control mice. The F rtios 16:1n7/16: nd 18:1n9/18: in liver TG hve een previously shown in humn studies to correlte very well with the F rtios found in plsm TG (26). In our current study, we found remrkle similrity etween the liver nd plsm TG F compositions tht reflected oth y /- nd SCD1-genotype effects. lthough the liver SCD1 genotype comprised mjority of the effect on the liver nd plsm 18:1n9/18: rtio, we found the 16:1n7/16: to e influenced y oth the liver nd dipose SCD1 genotype. dditionlly, we found the dipose 18:1n9/18: rtio to e influenced y heptic SCD1 in y / mice. Therefore, liver nd dipose F stores cn reciproclly influence the composition of one nother presumly due to the exchnge of liver-derived VLDL TG nd dipose-derived free F. We lso oserved tht the mgnitude of the heptic SCD1 genotype effect on these heptic F rtios ws drmticlly influenced y the presence or sence of the y / llele. Thus, diet nd genetic fctors tht 17

18 predispose n individul to insulin resistnce nd incresed heptic lipogenesis cn ffect the pprent correltion of heptic F rtios with heptic SCD1 levels. 18

19 cknowledgements: This work ws supported y NIH grnt R1-DK62388 (to J.M.N.) M.T.F ws supported y NIH postdoctorl trining grnt T32-DK7665 nd n mericn Hert ssocition postdoctorl fellowship. M.S.S ws supported y NIH predoctorl trining grnt T32-DK

20 References 1. Flowers, M. T., nd J. M. Ntmi. 28. Role of steroyl-coenzyme desturse in regulting lipid metolism. Current Opinion in Lipidology 19: Miyzki, M., H. Smpth, X. Liu, M. T. Flowers, K. Chu,. Dorzyn, nd J. M. Ntmi. 29. Steroyl-Co desturse-1 deficiency ttenutes oesity nd insulin resistnce in leptin-resistnt oese mice. Biochemicl nd Biophysicl Reserch Communictions 38: Ntmi, J. M., M. Miyzki, J. P. Stoehr, H. Ln, C. M. Kendziorski, B. S. Yndell, Y. Song, P. Cohen, J. M. Friedmn, nd. D. ttie. 22. Loss of steroyl-co desturse-1 function protects mice ginst diposity. Proceedings of the Ntionl cdemy of Sciences of the United Sttes of meric 99: Miyzki, M., M. T. Flowers, H. Smpth, K. Chu, C. Otzelerger, X. Liu, nd J. M. Ntmi. 27. Heptic steroyl-co desturse-1 deficiency protects mice from crohydrteinduced diposity nd heptic stetosis. Cell Met 6: Smpth, H., M. T. Flowers, X. Liu, C. M. Pton, R. Sullivn, K. Chu, M. Zho, nd J. M. Ntmi. 29. Skin-specific deletion of steroyl-co desturse-1 lters skin lipid composition nd protects mice from high ft diet-induced oesity. Journl of Biologicl Chemistry 284: Jing, G., Z. Li, F. Liu, K. Ellsworth, Q. Dlls-Yng, M. Wu, J. Ronn, C. Esu, C. Murphy, D. Szlkowski, R. Bergeron, T. Doeer, nd B. B. Zhng. 25. Prevention of oesity in mice y ntisense oligonucleotide inhiitors of steroyl-co desturse-1. J. Clin. Invest. 115: Brown, J. M., S. Chung, J. K. Swyer, C. Degirolmo, H. M. lger, T. Nguyen, X. Zhu, M. N. Duong,. L. Wiley, R. Shh, M.. Dvis, K. Kelley, M. D. Wilson, C. Kent, J. S. Prks, nd L. L. Rudel. 28. Inhiition of steroyl-coenzyme desturse 1 dissocites insulin resistnce nd oesity from therosclerosis. Circultion 118: el, E. D., O. Peroni, J. K. Kim, Y. B. Kim, O. Boss, E. Hdro, T. Minnemnn, G. I. Shulmn, nd B. B. Khn. 21. dipose-selective trgeting of the GLUT4 gene impirs insulin ction in muscle nd liver. Nture 49: Miyzki, M., W. C. Mn, nd J. M. Ntmi. 21. Trgeted disruption of steroyl-co desturse1 gene in mice cuses trophy of seceous nd meiomin glnds nd depletion of wx esters in the eyelid. J. Nutr. 131: Folch, J., M. Lees, nd G. H. Slone Stnley simple method for the isoltion nd purifiction of totl lipides from niml tissues. J. Biol. Chem. 226: Miyzki, M., H. J. Kim, W. C. Mn, nd J. M. Ntmi. 21. Oleoyl-Co is the mjor de novo product of steroyl-co desturse 1 gene isoform nd sustrte for the iosynthesis of the Hrderin glnd 1-lkyl-2,3-dicylglycerol. Journl of Biologicl Chemistry 276: Brsh, G. S., nd M. W. Schwrtz. 22. Genetic pproches to studying energy lnce: perception nd integrtion. Nt Rev Genet 3: Flowers, M. T., nd J. M. Ntmi. 29. Steroyl-Co desturse nd its reltion to highcrohydrte diets nd oesity. Biochimic et Biophysic ct 1791:

21 14. Flowers, J. B., M. E. Rgli, K. L. Schueler, M. T. Flowers, H. Ln, M. P. Keller, J. M. Ntmi, nd. D. ttie. 27. Loss of steroyl-co desturse-1 improves insulin sensitivity in len mice ut worsens dietes in leptin-deficient oese mice. Dietes 56: Liu, X., M. Miyzki, M. T. Flowers, H. Smpth, M. Zho, K. Chu, C. M. Pton, D. S. Joo, nd J. M. Ntmi. 21. Loss of Steroyl-Co desturse-1 ttenutes dipocyte inflmmtion: effects of dipocyte-derived olete. rteriosclerosis, Thromosis, nd Vsculr Biology 3: Rhmn, S. M.,. Dorzyn, S. H. Lee, P. Dorzyn, M. Miyzki, nd J. M. Ntmi. 25. Steroyl-Co desturse 1 deficiency increses insulin signling nd glycogen ccumultion in rown dipose tissue. m J Physiol Endocrinol Met 288: E Lee, S. H.,. Dorzyn, P. Dorzyn, S. M. Rhmn, M. Miyzki, nd J. M. Ntmi. 24. Lck of steroyl-co desturse 1 upregultes sl thermogenesis ut cuses hypothermi in cold environment. Journl of Lipid Reserch 45: He, W., Y. Brk,. Hevener, P. Olson, D. Lio, J. Le, M. Nelson, E. Ong, J. M. Olefsky, nd R. M. Evns. 23. dipose-specific peroxisome prolifertor-ctivted receptor gmm knockout cuses insulin resistnce in ft nd liver ut not in muscle. Proceedings of the Ntionl cdemy of Sciences of the United Sttes of meric 1: Christy, R. J., V. W. Yng, J. M. Ntmi, D. E. Geimn, W. H. Lndschulz,. D. Friedmn, Y. Nkeppu, T. J. Kelly, nd M. D. Lne Differentition-induced gene expression in 3T3-L1 predipocytes: CCT/enhncer inding protein intercts with nd ctivtes the promoters of two dipocyte-specific genes. Genes Dev. 3: Urs, S.,. Hrrington, L. Liw, nd D. Smll. 26. Selective expression of n P2/Ftty cid Binding Protein 4-Cre trnsgene in non-dipogenic tissues during emryonic development. Trnsgenic Res. 15: Mo, J., T. Yng, Z. Gu, W. C. Heird, M. J. Finegold, B. Lee, nd S. J. Wkil. 29. P2- Cre-medited inctivtion of cetyl-co croxylse 1 cuses growth retrdtion nd reduced lipid ccumultion in dipose tissues. Proceedings of the Ntionl cdemy of Sciences of the United Sttes of meric 16: Wng, Z. V., Y. Deng, Q.. Wng, K. Sun, nd P. E. Scherer. 21. Identifiction nd chrcteriztion of promoter cssette conferring dipocyte-specific gene expression. Endocrinology 151: Mrtens, K.,. Bottelergs, nd M. Bes. 21. Ectopic recomintion in the centrl nd peripherl nervous system y P2/FBP4-Cre mice: implictions for metolism reserch. FEBS Letters 584: Kestner, K. H., J. M. Ntmi, T. J. Kelly, Jr., nd M. D. Lne Differentitioninduced gene expression in 3T3-L1 predipocytes. second differentilly expressed gene encoding steroyl-co desturse. J. Biol. Chem. 264: Co, H., K. Gerhold, J. R. Myers, M. M. Wiest, S. M. Wtkins, nd G. S. Hotmisligil. 28. Identifiction of lipokine, lipid hormone linking dipose tissue to systemic metolism. Cell 134: Peter,.,. Cegn, S. Wgner, R. Lehmnn, N. Stefn,. Konigsriner, I. Konigsriner, H. U. Hring, nd E. Schleicher. 29. Heptic lipid composition nd steroyl-coenzyme desturse 1 mrn expression cn e estimted from plsm VLDL ftty cid rtios. Clinicl Chemistry 55:

22 Figure Legends Figure 1: Vlidtion of dipose SCD1 deletion y immunolot nd F composition. ) SCD1 protein expression in liver, epididyml white dipose, sucutneous white dipose, nd rown dipose. (LX), (L), () nd L(L); B,C) F composition of sucutneous ft in / (B) nd y / (C) mice fed stndrd diet were nlyzed y one-wy NOV. D) 16:1n7/16: nd E) 18:1n9/18: desturtion indices for sucutneous ft were nlyzed y two-wy NOV. Different letters indicte significnt differences (p<.5) etween SCD1 genotypes within the / or y / groups. *p<.5 for y / vs. /, for n=5-7 per group. Figure 2: Deletion of dipose nd/or liver SCD1 does not protect mice from oesity. ) Body weight, B) liver weight, C) epididyml ft pd weight nd D) sucutneous ft pd weight were mesured t scrifice in mle mice t 6-months of ge fter eing fed either stndrd diet (SD) for oth / nd y /, or high-ft diet (DIO) for /. Food intke for E) y / nd F) DIO mice were mesured over the course of five to seven dys. y / SD, / SD, nd / DIO groups were ech independently nlyzed y one-wy NOV. *p<.5 vs., n=5 to 11 per group. Figure 3: Effect of SCD1 deletion on glucose nd insulin tolernce. Glucose tolernce tests were performed in ) / mice fed stndrd diet, C) / mice fed high-ft diet (DIO) nd D) y / mice fed stndrd diet. Insulin tolernce tests (B) were done in / mice fed the stndrd diet. Tests were done t 5-6 months of ge. #, p<.5 vs. ; *, p<.5 vs. L; $, p<.5 vs. L, n=5 to 11 per group. 22

23 Figure 4: Effect of SCD1 deletion on liver nd plsm TG mss nd desturtion indices. ) Liver TG mss nd B) plsm TG mss in / nd y / mice fed stndrd diet. C,D) 16:1n7/16: TG desturtion index for liver (C) nd plsm (D). E,F) 18:1n9/18: desturtion index for liver (E) nd plsm (F). Dt were nlyzed y two-wy NOV. Different letters indicte significnt differences (p<.5) etween SCD1 genotypes within the / or y / groups. *p<.5 for y / vs. /, for n=5-7 per group. Figure 5: Effect of SCD1 deletion on liver nd plsm TG F composition. F composition of ) / liver TG, B) / plsm TG, C) y / liver TG nd D) y / plsm TG in mice fed stndrd diet. Dt were nlyzed y one-wy NOV. Different letters indicte significnt differences (p<.5) mongst SCD1 genotypes within the / or y / groups for n=5-7 per group. 23

24 Figure 1 L L LX LX L L L LX L L L LX LX L LX L Sucut. Ft L L L L L Epidid. Ft Brown Ft Liver LX LX LX L L L L L LX LX LX

25 Figure 1B-1E B % Totl D 16:1n7 / 16: / Sucutneous Ft 4 c 3 L 2 1 c 16: 16:1(n7) 18: 18:1(n9)18:1(n7) Sucutneous Ft (16:1n7 / 16:).5.4 L C % Totl E 18:1n9 / 18: y/ Sucuteneous Ft 5 4 c 3 c 2 1 c c c 16: 16:1(n7) 18: 18:1(n9)18:1(n7) Sucutneous Ft (18:1n9 / 18:) L 2 15 * c L 1 5 d. / y/ / y/

26 Figure 2 Body Weight (g) L Finl Body Weight B Tissue Weight (g) L Liver * * / SD y/ SD / DIO / SD y/ SD / DIO C Tissue Weight (g) E Epididyml White dipose L / SD y/ SD / DIO y/ Food Intke D Tissue Weight (g) F Sucutneous White dipose L / SD y/ SD / DIO DIO Food Intke Food Intke (g/dy) Food Intke (g/dy) L L

27 Figure 3 Non-gouti GTT, Stndrd Diet B Non-gouti ITT, Stndrd Diet Glucose (mg/dl) C Glucose (mg/dl) L Time (minutes) Non-gouti GTT, DIO L Glucose (mg/dl) D Glucose (mg/dl) Time (minutes) # L gouti GTT, Stndrd Diet # * * $ L Time (minutes) Time (minutes)

28 Figure 4 mg TG per g tissue Liver TG Mss * * * * L B Plsm TG (mg/dl) Plsm TG Mss *, * L * c,c / y/ / y/ C 16:1n7 / 16: Liver TG 16:1n7 / 16: c / * * c y/ L d D 16:1n7 / 16: Plsm TG 16:1n7 / 16: L *.1 c.5 c c. / y/ E 18:1n9 / 18: Liver TG 18:1n9 / 18: / * * c y/ * L c F 18:1n9 / 18: Plsm TG 18:1n9 / 18: * * / y/ L

29 Figure 5 % Totl C % Totl / Liver TG Ftty cid Composition c c c 16: 16:1(n7) 18: 18:1(n9)18:1(n7) y/ Liver TG Ftty cid Composition c 16: 16:1(n7) 18: 18:1(n9)18:1(n7) L L B % Totl D % Totl / Plsm TG Ftty cid Composition c 16: 16:1(n7) 18: 18:1(n9) 18:1(n7) y/ Plsm TG Ftty cid Composition c 16: 16:1(n7) 18: 18:1(n9)18:1(n7) L L