Ob/ob mice are leptin deficient and become hyperphagic,

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1 ORIGINL RTICLE Glyerol-3-Phosphte yltrnsferse 1 Defiieny in o/o Mie Diminishes Hepti Stetosis ut Does Not Protet ginst Insulin Resistne or Oesity ngel. Wendel, 1 Lei O. Li, 1 Yue Li, 1 Gry W. Cline, 2 Gerld I. Shulmn, 2,3 nd Roslind. Colemn 1 OJECTIVE Hepti stetosis is strongly ssoited with insulin resistne, ut usl role hs not een estlished. In o/o mie, sterol regultory element inding protein 1 (SREP1) medites the indution of stetosis y upregulting trget genes, inluding glyerol-3-phosphte yltrnsferse-1 (Gpt1), whih tlyzes the first nd ommitted step in the pthwy of glyerolipid synthesis. We sked whether o/o mie lking Gpt1 would hve redued hepti stetosis nd improved insulin sensitivity. RESERCH DESIGN ND METHODS Hepti lipids, insulin sensitivity, nd hepti insulin signling were ompred in len (Lep /? ), len-gpt1 /, o/o (Lep o/o ), nd o/o- Gpt1 / mie. RESULTS Compred with o/o mie, the lk of Gpt1 in o/o mie redued hepti triylglyerol (TG) nd diylglyerol (DG) ontent 59 nd 74%, respetively, ut inresed yl-co levels. Despite the redution in hepti lipids, fsting gluose nd insulin onentrtions did not improve, nd insulin tolerne remined impired. In oth o/o nd o/o-gpt1 / mie, insulin resistne ws ompnied y elevted hepti protein kinse C-ε tivtion nd lunted insulin-stimulted kt tivtion. CONCLUSIONS These results suggest tht deresing hepti stetosis lone does not improve insulin resistne, nd tht ftors other thn inresed hepti DG nd TG ontriute to hepti insulin resistne in this genetilly oese model. They lso show tht the SREP1-medited indution of hepti stetosis in o/o mie requires Gpt1. Dietes 59: , 21 O/o mie re leptin defiient nd eome hyperphgi, oese, nd insulin resistnt (1). Severe hepti stetosis in o/o mie is seondry to hepti upregultion of sterol regultory element inding protein 1 (SREP1) nd its trget genes tht inrese lipogenesis nd triylglyerol (TG) synthesis (2). mong these trget genes is Gpt1, whih From the 1 Deprtment of Nutrition, University of North Crolin, Chpel Hill, North Crolin; the 2 Deprtments of Internl Mediine nd Cellulr & Moleulr Physiology, Yle University Shool of Mediine, New Hven, Connetiut; nd the 3 Howrd Hughes Medil Institute, Yle University Shool of Mediine, New Hven, Connetiut. Corresponding uthor: Roslind Colemn, rolemn@un.edu. Reeived 16 Septemer 29 nd epted 22 Ferury 21. Pulished hed of print t on 3 Mrh 21. DOI: /d y the merin Dietes ssoition. Reders my use this rtile s long s the work is properly ited, the use is edutionl nd not for profit, nd the work is not ltered. See -n-nd/3./ for detils. The osts of pulition of this rtile were defryed in prt y the pyment of pge hrges. This rtile must therefore e herey mrked dvertisement in ordne with 18 U.S.C. Setion 1734 solely to indite this ft. enodes glyerol-3-phosphte yltrnsferse-1, the mitohondril enzyme tht tlyzes the retion glyerol-3- phosphte long-hin yl-co 3 lysophosphtidi id, the ommitted step in the pthwy of de novo glyerolipid synthesis (3). Four isoforms of glyerol-3- phosphte yltrnsferse (GPT), eh enoded y seprte gene, re le to initite the glyerolipid synthesis (4), ut only Gpt1 is regulted y SREP1 (5). GPT1 tivity nd mrn re highest in tissues with high pity for TG synthesis; in liver, the GPT1 isoform ounts for 3 5% of totl GPT tivity. Hepti GPT1 tivity nd mrn undne re inresed y insulin vi tivtion of liver X reeptor (LXR) nd SREP1 during onditions tht promote TG synthesis (5). Conversely, glugon, fsting, nd streptozotoinindued dietes derese liver GPT1 mrn nd tivity (6). Elevted hepti TG ontent in humns hs een linked to insulin resistne (7 9). In rts inresing hepti TG ontent y denovirus-medited overexpression of GPT1 indued hepti nd peripherl insulin resistne within 5 7 dys (1). Conversely, Gpt1 / mie fed high-ft sfflower oil diet for 3 weeks were proteted from hepti stetosis nd insulin resistne (11). lthough studies strongly suggest tht reduing hepti stetosis improves hepti insulin sensitivity (11 14), it is unlikely tht the umultion of hepti TG itself, rther thn nother glyerolipid metolite suh s diylglyerol (DG), plys usl role in impired hepti insulin signling (15). Furthermore, the role of GPT1 in hepti stetosis hs een questioned euse oth Gpt1 / mie fed high-ft/high-surose diet for 4 months (16) nd n independently derived Gpt1 / mouse strin fed high-ft diet for 3 months (17) developed hepti stetosis nd eme insulin resistnt, lthough oth developed in the ontext of oesity. euse the insulin-sensitizing effets tht our when deleted Gpt1 redues hepti stetosis pper to e highly sensitive to diet omposition nd durtion, we investigted o/o mie to determine whether the sene of Gpt1 would improve hepti stetosis nd insulin resistne ssoited with geneti-indued oesity. Hepti stetosis in o/o mie is ssoited with 2.3-fold inrese in GPT1 tivity nd 5.5-fold inrese in Gpt1 mrn (18). euse 5-dy 9% denovirus-medited knokdown of hepti Gpt1 in o/o mie resulted in 42% redution in hepti TG nd 3% derese in plsm gluose (19), we hypothesized tht totl lk of Gpt1 in o/o mie would prevent oth hepti stetosis nd insulin resistne. dietes.dietesjournls.org DIETES, VOL. 59, JUNE

2 Gpt1 DEFICIENCY IN o/o MICE RESERCH DESIGN ND METHODS Mie. niml protools were pproved y the University of North Crolin t Chpel Hill Institutionl niml Cre nd Use Committee. Mie were housed in pthogen-free rrier fility on 12-h light/drk yle with free ess to wter nd food (Prol 5P76 Isopro 3; 5.4% ft y weight). To generte Gpt1 / mie on n oese kground, Gpt1 / mie (2), whih hve een krossed to C57L/6J mie seven times, were rossed with Lep /o mie (6.V-Lep o /J; The Jkson Lortory, r Hror, ME). fter rossing the doule heterozygotes, Lep /o -Gpt1 / or Lep /o -Gpt1 / mie were then interrossed to generte Lep o/o -Gpt1 / or Lep o/o -Gpt1 / mie nd their respetive len (Lep /? ) littermtes. Mle mie were used in ll experiments. t 16 weeks, totl len nd ft mss ws mesured using n EhoMRI-1 QNMR system (Eho Medil Systems, LLC, Houston, TX). Fsted (12 h) 16-week-old mie were killed y ervil dislotion, nd trunk lood ws olleted. Clotted lood ws entrifuged t 1,5g for 2 min nd ser were stored t 8 C until nlyzed. Tissues were quikly hrvested, weighed, snp-frozen with liquid nitrogen, nd stored t 8 C until nlyzed. Liver setions were fixed with 4% prformldehyde, prffin-emedded, setioned (.5 m), nd stined with hemtoxylin-eosin. Serum hemistries nd lipids. Serum nonesterified ftty ids nd totl holesterol (Wko Dignostis, Rihmond, V), -hydroxyutyrte (Stnio, oerne, TX), nd triylglyerol nd glyerol (free glyerol nd triglyeride regents; Sigm-ldrih, St. Louis, MO) were determined y enzymti, olorimetri ssys. Insulin ws determined y enzyme-linked immunosorent ssy (Rt/Mouse Insulin ELIS; Millipore, illeri, M). GPT tivity. Liver tissue ws homogenized in Medium I (25 mmol/l surose, 1 mmol/l Tris, ph 7.4, 1 mmol/l EDT, 1 mmol/l dithiothreitol) with 1 up-nd-down strokes with Teflon-glss motor-driven homogenizer. Homogentes were entrifuged t 1,g for 1 h to otin totl memrne frtions. Memrne pellets were rehomogenized in Medium I. GPT speifi tivity ws ssyed t room temperture in 2 l retion mixture ontining 75 mmol/l Tris-HCl, ph 7.5, 4 mmol/l MgCl 2, 1 mg/ml S (essentilly ftty id free), 1 mmol/l dithiothreitol, 8 mmol/l NF, 8 mol/l [ 3 H]glyerol 3-phosphte, nd 8 mol/l plmitoyl-co (2). The retion ws initited y dding 1 3 g of memrne protein fter inuting the memrne protein on ie for 15 min in the presene or sene of 2 mmol/l N-ethylmleimide (NEM), whih intivtes GPT isoforms 2, 3, nd 4. NEM-resistnt tivity (GPT1) ws lulted y sutrting NEM-sensitive tivity from totl tivity. Quntittive rel-time RT-PCR. RN ws extrted from liver using Trizol (Invitrogen, Crlsd, C) nd reverse-trnsried with the High-Cpity DN rhive kit (pplied iosystems, Foster City, C). DN ws mplified y rel-time PCR in totl retion volume of 25 l using solute QPCR Syr Green Fluoresein Mix (ThermoSientifi, Wlthm, M). Primer sets re identified in supplementry Tle 1, ville in n online ppendix t dietes.dietesjournls.org/ontent/erly/21/3/2/d9-138/suppl/dc1. Trget gene expression ws normlized to endogenous 18S rrn nd expressed s 2 t reltive to the len-gpt1 / group (21). Tissue lipid metolites. Tissues were homogenized in 1 (wt/vol) old lysis uffer (2 mmol/l Tris se, 5 mmol/l NCl, 25 mmol/l surose, 5 mmol/l NF, 5 mmol/l N 4 P 2 O 7 1 H 2 O, 1% Triton X-1, nd protese inhiitors). Lipids were extrted (22), dried under N 2 gs, nd soluilized in 3:1:1 (vol/vol/vol) tert-utnol, methnol, nd Triton X-1 (23). Tissue lipid extrts were nlyzed for TG y n enzymti ssy (Triglyeride nd Free-Glyerol regents; Sigm-ldrih). Dt re expressed s equivlent triolein onentrtions. DG nd yl-cos were extrted nd mesured y liquid hromtogrphy tndem mss spetrometry (11). For ermides, 5 mg of tissue ws homogenized with 1 ml ie-old methnol. Chloroform (2 ml) nd n internl stndrd (5 nmol C17: ermide; vnti Polr Lipids, In., lster, L) were dded, the smple ws mixed for 15 min, nd wter (.6 ml) ws dded. fter entrifugtion t 4 C, the orgni lyer ws olleted, dried overnight in vuum oven, nd reonstituted with hloroform. Lipid metolites were sujeted to liquid hromtogrphy tndem mss spetrometry nlysis. turo ion spry soure ws interfed with n PI 3 tndem mss spetrometer (pplied iosystems) in onjuntion with two Perkin Elmer 2 Series miropumps nd 2 Series utosmpler (Perkin Elmer, Norwlk, CT). Totl yl-co, ermide, nd DG ontent were expressed s the sum of individul speies. Insulin tolerne tests. t 15 weeks of ge, fter n overnight (12 h) fst, mie were injeted intrperitonelly with insulin (Humulin: R; Eli Lilly, Indinpolis, IN) t either.5 units/kg ody wt (len mie) or 1.5 units/kg ody wt (o/o mie). Gluose in til vein lood ws mesured immeditely efore injetion (time ) nd t 15, 3, 6, 9, nd 12 min fter injetion (One Touh Ultr gluometer; Lifesn, Milpits, C). res of the urves were lulted s the net re ontined etween individul selines (set y the gluose vlue t time ) nd urves using the trpezoidl rule (24). Western lot nlysis. Protein kinse C-ε (PKCε) memrne trnslotion ws determined y immunolotting ytosol nd memrne protein extrts (4 g) with rit nti-pkcε (1:1; Snt Cruz iotehnology, Snt Cruz, C) (25,26). Trnslotion for eh smple ws determined s the rtio of the density of PKCε of the memrne frtion over the density of PKCε of the ytosoli frtion. To evlute insulin signling, fter n overnight (12 h) fst, insulin (2 units/kg ody wt) or PS ws dministered intrperitonelly to 6- or 16-weekold mie. fter 1 min, mie were killed y ervil dislotion, nd livers were exised nd snp-frozen in liquid nitrogen. Tissues were homogenized in lysis uffer (2 mmol/l Tris, 15 mmol/l NCl, 1 mmol/l EDT, 1 mmol/l EGT, 5 mmol/l NF, 2.5 mmol/l N 4 P 2 O 7 1 H 2 O, 1% Triton X-1, nd Complete Mini Protese Inhiitor Coktil; Rohe Dignostis, Indinpolis, IN) nd entrifuged t 15,g for 15 min. Protein (4 g) ws resolved on 1% polyrylmide gels, trnsferred to nitroellulose memrnes, proed for phosphorylted kt (P-kt; Thr38; 1:1,), stripped, nd reproed for totl kt nd -tuulin (Cell Signling Tehnology, Dnvers, M). Mture SREP1 (Novus iologils, LLC, Littleton, CO) nd phosphoenolpyruvte roxykinse (m In., Cmridge, M) protein expression ws determined from livers of mie fsted overnight (12 h) using the sme method. Sttistil nlyses. Dt re expressed s lest squre mens (LSM) SEM. The min nd intertion effets of the o phenotype (len nd o/o) nd Gpt1 genotype (Gpt1 / nd Gpt1 / ) were nlyzed y two-wy NOV using omplete model in the generl liner model (GLM) proedure of the SS (Version 9.2; SS Institute, In., Cry, NC). ody weights over time nd insulin tolerne test urves were nlyzed s repeted mesures. Comprisons of sl versus insulin-stimulted kt expression within group were nlyzed y Student t test. Differenes of P.5 were onsidered signifint. RESULTS The sene of Gpt1 diminished hepti stetosis in o/o mie. Hepti stetosis in o/o mie is medited y SREP1, whih inreses the expression of lipogeni genes (27), inluding Gpt1 (5). To determine the ontriution of Gpt1 to the development of hepti stetosis in o/o mie, we interrossed o/o nd Gpt1 / mie. Compred with len littermtes, totl hepti GPT speifi tivity in o/o mie ws 33% higher, nd oth GPT1 tivity (NEM-resistnt GPT; Fig. 1) nd mrn expression (Fig. 1) were 2.2-fold higher. In o/o mie lking Gpt1 (o/o-gpt1 / ), totl GPT tivity ws redued to levels oserved in len mie, nd GPT1 tivity ws diminished. The sene of Gpt1 indued 28% inrese in NEM-sensitive GPT tivity (GPT2, GPT3, nd GPT4) in o/o mie (Fig. 1). This inrese in NEM-sensitive GPT tivity ws likely due to inreses in oth Gpt-3 nd -4 expression, lthough only the inrese of Gpt3 mrn expression in o/o mie ws signifint (supplementry Fig. 1 nd C). Colletively, the dt suggest tht elevted GPT1 tivity is required for the inrese in TG umultion in o/o liver (28). Furthermore, the slight inrese in tivity nd messge of GPT3 nd GPT4 did not ompenste for the derese in totl GPT tivity in Gpt1 / mie. s previously reported (28), o/o mie developed grossly stetoti livers with mrostetosis nd mirostetosis tht weighed four times more thn livers from len littermtes (Tle 1) nd umulted nerly five times s muh TG (Fig. 2 nd ). In o/o-gpt1 / mie, the lk of Gpt1 redued liver weight y 28% nd hepti TG ontent y 59%, evidened y the sene of lrge lipid droplets. In o/o livers, the lipogeni genes ftty id synthse (Fsn) nd steroyl-co desturse 1 (Sd1) were inresed 4- nd 6.4-fold, respetively. However, neither Fsn nd Sd1 nor trnsription ftors tht regulte lipogenesis, SREP1 (Sref1) nd ChREP (Mlxipl), were ffeted y the lk of Gpt1 (supplemen DIETES, VOL. 59, JUNE 21 dietes.dietesjournls.org

3 .. WENDEL ND SSOCITES GPT S (nmol/min/mg protein) Gpt1 +/+ Gpt1 -/- Gpt1 mrn (reltive expression) len o/o Totl. len o/o len o/o len o/o NEM-R NEM-S FIG. 1. : Totl, NEM-resistnt (NEM-R), nd NEM-sensitive (NEM-S) GPT speifi tivities. Liver totl prtiulte frtions (n 3 4) were ssyed for GPT tivity in the presene or sene of 2 mmol/l NEM s desried in the RESERCH DESIGN ND METHODS setion. : Hepti gene expression of Gpt1 ws determined y quntittive RT-PCR nd expressed s 2 t reltive to the endogenous ontrol 18S rrn nd the len-gpt1 / group (n 4). Len (Lep /? ) nd o/o (Lep o/o ). Dt re LSM SE. Signifint differenes (P <.5) re denoted y different letters. S, speifi tivity. try Fig. 2). Similrly, protein expression of the mture form of SREP1 ws not ffeted y the sene of Gpt1 (supplementry Fig. 2). The expression of genes relted to -oxidtion ws not ltered (supplementry Fig. 2C). Comined, these dt suggest tht Gpt1 ppered to e mjor SREP1 trget gene using hepti stetosis in o/o mie. Lk of Gpt did not protet o/o mie from insulin resistne. euse n ute knokdown of Gpt1 dereses plsm gluose in o/o mie (19), we then sked whether the elevted fsting gluose nd insulin levels or the insulin resistne tht is normlly oserved in o/o mie might e improved y sustined derese in hepti TG. t 16 weeks of ge, ompred with len mie, o/o mie hd 1.7- nd 16.2-fold higher serum fsting gluose nd insulin onentrtions, respetively (Fig. 2C nd D). The sene of Gpt1 did not redue gluose or insulin; on the ontrry, oth were higher in o/o-gpt1 / mie thn in o/o mie. The elevted gluose levels in the sene of Gpt1 my e explined prtly y n inrese in hepti gluose-6-phosphte (G6p) mrn expression, key enzyme in gluoneogenesis. However, mrn nd protein expression of phosphoenolpyruvte roxykinse ws not ltered (supplementry Fig. 2D nd E). In ddition to not improving fsting gluose nd insulin, the sene of Gpt1 did not ffet insulin sensitivity in either len or o/o mie (Fig. 2E nd F). euse hepti stetosis nd insulin resistne re progressive disorders tht likely ffeted multiple tissues in 16-week-old o/o mie, we exmined younger mie to determine whether they might e more insulin sensitive. t 6 weeks, livers from o/o mie were lredy stetoti nd ontined 1.7-fold more hepti TG thn livers from len mie (supplementry Fig. 3). The sene of Gpt1 diminished hepti TG y 68 nd 66% in young len nd o/o mie, respetively, nd effetively normlized the ontent of hepti TG. Despite the norml hepti TG ontent nd lowered fsting gluose onentrtions in young o/o mie tht lked Gpt1 (supplementry Fig. 3), serum insulin remined elevted (supplementry Fig. 3C) nd insulin sensitivity remined impired (supplementry Fig. 3D nd E). These dt demonstrte tht erlystge hyperinsulinemi nd whole-ody insulin resistne were not improved despite norml ontent of hepti TG. TLE 1 Physiologi prmeters nd fsting serum nlytes Len (Lep /? ) o/o (Lep o/o ) Ftors (P vlue) Gpt1 / Gpt1 / Gpt1 / Gpt1 / Pheno Gpt1 Int Physiologi prmeters Finl ody wt (g) Len mss (% ody wt) Ft mss (% ody wt) Liver weight (g) Serum nlytes Nonesterified ftty ids (mmol/l) Triylglyerol (mmol/l) Glyerol (mmol/l) Totl holesterol (mmol/l) hydroxyutyrte (mmol/l) Finl ody weights (n 12 16) nd ft nd len msses (n 6 13) were mesured t 16 weeks. Serum nlytes (n 4 9) were mesured from ser of mie fsted for 12 h. Vlues represent LSM SE with signifint differenes (P.5) within rows denoted y different letters. Ftors inlude the min effets of phenotype (Pheno; len or o/o) nd Gpt1 nd their intertion (Int). dietes.dietesjournls.org DIETES, VOL. 59, JUNE

4 Gpt1 DEFICIENCY IN o/o MICE len o/o Gpt1 +/+ Gpt1 -/- Totl triylglyerol (mg TG / g liver) Gpt1 +/+ Gpt1 -/- len o/o C Gluose (mmol/l) Gpt1 +/+ Gpt1 -/- len o/o E Gluose (mmol/l) len Gpt1 +/+ Gpt1 -/ Time (min) Net re (mmol/l * min) Gpt1+/+ Gpt1-/- D Insulin (pmol/l) len o/o F Gluose (mmol/l) o/o Gpt1 +/+ Gpt1 -/ Time (min) Net re (mmol/l * min) Gpt1+/+ Gpt1-/- FIG. 2. Oesity-indued hepti stetosis, ut not insulin resistne, ws diminished in Gpt1 / mie. : Representtive liver setions stined with hemtoxylin-eosin from 16-week-old mle mie. Imges re t 1 mgnifition with 1 m indited y the white r in upper left pnel. : Hepti triylglyerol ontent (n 9 1). Fsting (C) gluose nd (D) insulin. Insulin tolerne tests were onduted in 15-week-old mle mie. Len mie (E; n 6 8) were given.5 units insulin/kg ody wt y intrperitonel injetion, nd o/o mie (F; n 7 9) were dministered 1.5 units insulin/kg ody wt. Gluose ws mesured from til vein lood t times indited nd net res of the urves (insets) were lulted s desried in the RESERCH DESIGN ND METHODS setion. Len (Lep /? ) nd o/o (Lep o/o ). Dt re expressed s LSM SE. Signifint differenes (P <.5) re denoted y different letters. ( high-qulity digitl representtion of this figure is ville in the online issue.) Chnges in hepti lipids due to lk of Gpt1. Inreses in the ontent of hepti lipids suh s DG, yl-co, nd ermide hve een proposed to medite hepti insulin resistne (29,3). Compred with len mie, totl hepti DG in o/o liver ws five times higher (Fig. 3), nd when Gpt1 ws sent, DG ontent deresed to norml levels. The mjor DG speies refleted this pttern, with the lrgest differenes pprent in C18:1-C18:1 nd C18:1-C16: (Fig. 3). In Gpt1-null mie, liver yl-co ontent is elevted twofold euse yl-cos re sustrtes for GPT1 (11). lthough phenotype (len or o/o) did not hve n effet on totl hepti yl-co ontent, oth len nd o/o livers defiient in Gpt1 ontined 3.3- nd 5.3-fold more 1324 DIETES, VOL. 59, JUNE 21 dietes.dietesjournls.org

5 .. WENDEL ND SSOCITES Totl diylglyerol len o/o Diylglyerol 18 len-gpt1 +/+ len-gpt1 -/ C18:1-C16: C2:4-C2:5 C18:1-C18:1 C18:1-C18:2 C18:2-C18:2 o/o-gpt1 +/+ o/o-gpt1 -/- C16:-C18:2 C Totl yl-co len o/o D yl-co d C16: C16:1 C18: C18:1 C18:2 C18:3 d E Totl ermide len o/o F Cermide C16: C18: C2: C22: C24:1 C24: FIG. 3. Chnges in hepti lipids due to lk of Gpt1. nd : Totl hepti diylglyerol nd speies. C nd D: Totl yl-co nd speies. E nd F: Totl ermide nd speies (n 6). Lipids were extrted from livers nd mesured s desried in the RESERCH DESIGN ND METHODS setion. Len (Lep /? ) nd o/o (Lep o/o ). Dt re LSM SE. Signifint differenes (P <.5) re denoted y different letters. totl yl-co thn their Gpt1 / ounterprts, respetively (Fig. 3C). ll the reported yl-co speies were inresed, ut the mjor one ffeted ws 18:1-Co, with 3.8- nd 6.-fold inreses in len- nd o/o-gpt1 / mie, respetively (Fig. 3D). In ddition to TG synthesis nd -oxidtion, yl-cos my lso e direted towrd the synthesis of ermide, nother purported modultor of insulin resistne (31). Compred with len mie, the ontent of ermide in livers from o/o mie ws 25% lower (Fig. 3E), refleted primrily y fewer long-hin ermides, C22: nd C24: (Fig. 3F), even though C16: ermide ws higher. Gpt1 defiieny did not signifintly ffet totl ermide ontent, ut deresed C22: nd C24: ermides y 52% in len mie. Thus, totl ermide ontent ws not ssoited with insulin resistne in this model. Lk of Gpt1 did not restore hepti insulin signling in o/o mie. euse oth DG nd TG deresed mrkedly in Gpt1-defiient liver, we exmined the effet of Gpt1 defiieny on insulin signling. Hepti stetosis dietes.dietesjournls.org DIETES, VOL. 59, JUNE

6 Gpt1 DEFICIENCY IN o/o MICE Gpt1 PKCε memrne ytosol len o/o len o/o +/+ -/- +/+ -/- +/+ -/- +/+ -/- Gpt1 P-kt (Thr 38) totl-kt α-tuulin sl insulin-stim. len o/o len o/o +/+ -/- +/+ -/- +/+ -/- +/+ -/- PKCe memrne/ytosol rtio (reltive expression) Gpt1 +/+ Gpt1 -/- len o/o P-kt / totl-kt (reltive expression) 25 sl insulin * 2 * * * +/+ -/- +/+ -/- len o/o FIG. 4. Hepti insulin signling ws not improved in Gpt1 / mie. : PKC protein expression in ytosoli nd memrne frtions of livers from 16-week-old mie ws determined y Western lot nlysis. rs represent densities of the memrne to ytosoli rtio of PKC expression reltive to the len-gpt1 / mie (n 8). : sl nd insulin-stimulted phosphorylted kt expression. Mie were dministered either 2. units insulin/kg ody wt or PS y intrperitonel injetion for 1 min. rs represent densities of P-kt/totl kt reltive to the len-gpt1 / mie (n 4 5). Representtive lots re shown. Len (Lep /? ) nd o/o (Lep o/o ). Dt re expressed s LSM SE. Signifint differenes (P <.5) mong groups re denoted y different letters; * indites signifint differene (P <.5) etween sl nd insulin-stimulted P-kt/totl kt within group. ontriutes to insulin resistne vi inresed DG tivtion of PKCε nd susequent impirment of the insulin signling pthwy (11). To determine whether the improved hepti lipid profile resued hepti insulin signling, we exmined tivtion of PKCε nd kt. In 16-weekold mie, onomitnt with impired insulin sensitivity, ompred with len mie, o/o mie hd nerly twofold higher hepti PKCε tivtion, mrked y the inresed rtio of memrne to ytosoli PKCε (Fig. 4). Despite hving ontent of hepti DG similr to tht of len mie, PKCε tivtion ws elevted in o/o-gpt1 / liver, lthough to lesser extent thn in o/o mie. t 6 weeks, no differenes in hepti PKC tivtion were deteted mong groups (supplementry Fig. 4). tivted PKCε inds nd intivtes the insulin reeptor kinse, leding to impired downstrem insulin signling, suh s kt tivtion (32). When either 16- or 6-week-old len mie were stimulted with insulin, the mount of phosphorylted kt in liver drmtilly inresed ompred with sl expression (Fig. 4 nd supplementry Fig. 4). In o/o mie, however, the response to insulin ws lunted y t lest 65%, nd this lunted response ws not resued when Gpt1 ws defiient. These dt indite tht, lthough hepti TG nd DG ontent ws onsiderly lower in o/o-gpt1 / mie, hepti insulin signling did not improve. Lk of Gpt1 did not redue geneti oesity. Insulin resistne oth in the liver nd in peripherl tissues suh s musle nd ft n ontriute to whole-ody insulin resistne. lthough GPT1 onstitutes only 1% of totl GPT tivity in dipose tissue, dipose GPT1 speifi tivity is high (5), nd femle Gpt1 / mie hve redued ody nd gondl dipose depot weights (2). We determined whether the sene of Gpt1 would redue oesity in o/o mie. t 16 weeks, o/o mie weighed twie s muh s their len littermtes (Fig. 5; Tle 1). However, the sene of Gpt1 in len or o/o mie did lter ody weights over time or finl ody weights in either mle (Fig. 5; Tle 1) or femle (dt not shown) mie. Compred with len mie, gondl, retroperitonel, nd inguinl white dipose nd suspulr rown dipose tissue depots weighed drmtilly more in the o/o mie, nd were unffeted y the sene of Gpt1 (Fig. 5). Quntittive nuler mgneti resonne nlysis onfirmed tht the Gpt1 defiieny did not hve n effet on totl len or ft mss s perentge of ody weight (Tle 1). Lk of Gpt1 did not improve musle lipids. Oesitymedited inreses in musle TG ontent orrelte strongly with whole-ody insulin resistne (29). Even though GPT1 speifi tivity is very low in musle (5), we exmined lipids in musle to determine whether hnges in musulr lipid ould ffet whole-ody insulin resistne. In gstronemius musle, the ontent of TG nd DG in o/o mie ws 4- nd 2.3-fold higher, respetively (supplementry Fig. 5 nd ). The sene of Gpt1 in len or o/o mie hd no effet on musle TG or DG ontent or on the DG speies present (supplementry Fig. 5C). Musle yl-co nd ermide ontent nd moleulr speies in o/o mie were lso unffeted y the sene of Gpt1 (supplementry Fig. 5D G). The sene of Gpt1 did not lter serum lipids. Corresponding with the development of oesity, serum holesterol nd TG were elevted in o/o mie (33), wheres in Gpt1 / mie, serum holesterol is norml nd serum TG ws redued (11). The sene of Gpt1, 1326 DIETES, VOL. 59, JUNE 21 dietes.dietesjournls.org

7 .. WENDEL ND SSOCITES ody weight (g) dipose mss (g) 7 len-gpt1 +/+ len-gpt1 -/- 6 o/o- Gpt1 +/+ o/o- Gpt1 -/ ge (weeks) len o/o gwt rwt iwt T Gpt1 +/+ Gpt1 -/- len o/o len o/o len o/o FIG. 5. Gpt1 / mie were not resistnt to geneti-indued oesity. : Weight gin of len nd o/o mle mie from 4 to 16 weeks of ge (n 12 17). : dipose depot weights of len nd o/o mle mie t 16 weeks of ge (n 1 13). Gondl white dipose tissue (gwt), retroperitonel (rwt), inguinl (iwt), nd suspulr rown dipose tissue (T) depots were mesured. Len (Lep /? ) nd o/o (Lep o/o ). Dt re LSM SE. Signifint differenes (P <.5) re denoted y different letters. however, did not diminish serum lipids in o/o mie (Tle 1). s reported previously in Gpt1 / mie (16), -hydroxyutyrte ws higher in oth len nd o/o- Gpt1 / mie, lthough this effet ws not sttistilly signifint. DISCUSSION Severl studies hve demonstrted tht reduing hepti TG ontent dereses hepti insulin resistne (9,11 14). In the liver, loss- nd gin-of-funtion studies with Gpt1 support role for hepti stetosis in the development of insulin resistne (4). Gpt1 / mie re proteted from high-ft diet indued hepti stetosis nd insulin resistne (11), wheres rts tht overexpress Gpt1 in liver hve inresed hepti TG umultion nd hepti insulin resistne (1). Here, we show tht the sene of Gpt1 in oth 6- nd 16-week-old o/o mie prevents severe hepti stetosis ut does not protet ginst oesity-ssoited insulin resistne nd impired hepti insulin signling. The sene of Gpt1 redued hepti TG ontent y 59% in o/o mie; however, the TG ontent ws still nerly doule tht of livers from len, insulin-sensitive mie. Similrly, in o/o-srep1 / mie, the hepti TG ontent is less thn hlf tht of o/o mie, ut still lmost twie tht of len mie (2). The redued hepti TG ontent in o/o-srep1 / mie ours simultneously with 7% redution of mrn for hepti Gpt1, diret trget of SREP1 (5). Like our o/o-gpt1 / mie, despite mrked dereses in hepti stetosis, o/o- Srep1 / mie lso remin oese nd insulin resistnt. In o/o mie, the sene of either Srep1 or Gpt1 results in similr dereses in hepti TG. Thus, GPT1 ppers to e responsile for most of SREP1-regulted hepti TG umultion. lthough the hepti TG ontent of 16-week-old o/o- Gpt1 / mie ws similr to levels present in mie with diet-indued insulin resistne (16,34,35), even t 6 weeks, o/o-gpt1 / mie were hyperinsulinemi, insulin resistnt, nd hd impired hepti insulin signling despite the ft tht their hepti TG ontent ws lower thn tht of their insulin-sensitive len littermtes. These results demonstrte tht in o/o mie, preventing hepti stetosis did not protet ginst insulin resistne, whih is onsistent with other studies reporting dissoition etween hepti stetosis nd insulin resistne (2,16,36 38). euse ellulr TG is stored in lipid droplets, nd thus segregted from signling events, it is elieved tht other, perhps relted, lipid metolites interfere with insulin signling pthwys (15,39). lthough the umultion of yl-cos hs een ssoited with insulin resistne (4,41), mie tht overexpress diylglyerol yltrnsferse-2 in liver nd high-ft fed Gpt1 / mie hve inresed hepti yl-co ontent ut do not develop insulin resistne (11,36). Hepti yl-co ontent is norml in o/o mie nd inreses when Gpt1 is sent, ut the extent of insulin resistne does not hnge. Thus, in o/o liver, inresed yl-co ontent itself does not impir insulin signling. Similrly, the sene of GPT1 inreses the ontent of plmitoyl-co, the sustrte for the first step in ermide iosynthesis. However, the sene of Gpt1 did not inrese hepti ermide ontent; thus, ermide did not ontriute to hepti insulin resistne in this model. DG hs een most strongly ssoited with insulin resistne euse it tivtes PKC- in the musle (39,42) nd PKC-ε in the liver (32,43), leding to redutions in downstrem insulin signling. Gpt1 / mie tht re proteted from sfflower oil indued hepti insulin resistne hve redued hepti DG ontent nd PKCε tivtion, onomitnt with improved hepti insulin signling (11); onversely, rts tht overexpress GPT1 in the liver show elevted DG nd PKCε tivtion oupled with hepti insulin resistne (1). In the urrent study, however, despite normlized hepti DG ontent in o/o-gpt1 / mie, hepti insulin signling remined impired. In this study, the derese in DG proly reflets redution in lipid droplet DG ontent rther thn memrne DG (44), euse tivted PKCε ws unffeted. lterntively, signl other thn DG tht is generted during metoli dysfuntion my e responsile for PKC tivtion. Studies linking redued hepti stetosis to improved insulin sensitivity lso report onomitnt dereses in diposity (14,45). euse dipose mss ws not diminished in the o/o-gpt1 / mie, ny improvements in either hepti or whole-ody insulin resistne due to lower hepti lipids ould well e overshdowed y other dietes.dietesjournls.org DIETES, VOL. 59, JUNE

8 Gpt1 DEFICIENCY IN o/o MICE ftors tht interfere with hepti insulin signling (46). Rodents defiient in leptin or the leptin reeptor serete exess ortiosterone (47,48). dministering liver-seletive gluoortioid reeptor ntgonist to leptin reeptor defiient rts inreses gluose disposl nd dereses hepti gluose prodution nd fsting gluose (49), demonstrting tht inresed gluoortioid signling impirs hepti nd peripherl insulin sensitivity. Thus, it is likely tht elevted plsm gluoortioid levels or other oesity-relted ftors my hve previled over ny improvements in insulin sensitivity rising from n improved hepti lipid profile in this model. Fsting serum gluose nd insulin onentrtions were 21 nd 48% higher, respetively, in o/o-gpt1 / mie thn in o/o mie despite similr insulin sensitivity. Similrly, Gpt1 / mie fed high-ft/high-surose diet re less gluose tolernt nd hve 11% higher fsting gluose nd 64% higher insulin levels thn wild-type mie (16), nd o/o mie with liver-speifi Gpt1 knokdown hve twofold inrese in plsm insulin (19). Hyperinsulinemi n e used y -ell hypertrophy nd hyperplsi (5), ut we did not oserve differenes in islet size or numer (dt not shown). Even t 6 weeks, len nd o/o-gpt1 / mie hd 56 nd 27% higher fsting insulin levels, respetively, thn their Gpt1 / ounterprts (supplementry Fig. 3C), suggesting tht Gpt1 / mie re hyperinsulinemi, unrelted to the presene of insulin resistne. In summry, we hve demonstrted tht GPT1 is ritil for the umultion of TG nd DG during the development of hepti stetosis nd is mjor enzyme responsile for the lipogeni effet of SREP1 in o/o mie (2). These results suggest tht deresing hepti stetosis lone does not improve insulin resistne in this genetilly oese model, nd tht ftors other thn inresed hepti DG nd TG ontriute to hepti insulin resistne in o/o mie. CKNOWLEDGMENTS This work ws supported y Ntionl Institutes of Helth (NIH) grnts DK (R..C.), DK-4936, U24 DK-59635, nd P3 DK (G.I.S.); postdotorl fellowships from the NIH (DK-7129;..W.) nd the merin Hert ssoition-mid-tlnti Region (L.O.L.); nd n NIH grnt to the UNC Clinil Nutrition Reserh Unit (P3 DK-5635). No potentil onflits of interest relevnt to this rtile were reported. We thnk Shuli Wng nd Juheng Gong for expert tehnil ssistne. REFERENCES 1. Zhng Y, Proen R, Mffei M, rone M, Leopold L, Friedmn JM: Positionl loning of the mouse oese gene nd its humn homologue. 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