CEACAM1 regulates insulin clearance in liver

Transcription

1 CEACAM1 regultes insulin lerne in liver Mtthew N. Poy 1, Yn Yng 1, Khijeh Rezei 1, Mts A. Fernström 1, Arhm D. Lee 2, Yoshiki Kio 3, Snr K. Erikson 4 & Soni M. Njjr 1 Pulishe online: 19 Ferury 2002, DOI: /ng840 We hypothesize tht insulin stimultes phosphoryltion of CEACAM1 whih in turn les to upregultion of reeptor-meite insulin enoytosis n egrtion in the heptoyte. We hve generte trnsgeni mie over-expressing in liver ominnt-negtive, phosphoryltion-efetive S503A-CEACAM1 mutnt. Supporting our hypothesis, we foun tht S503A-CEACAM1 trnsgeni mie evelope hyperinsulinemi resulting from impire insulin lerne. The hyperinsulinemi use seonry insulin resistne with impire gluose tolerne n rnom, ut not fsting, hyperglyemi. Trnsgeni mie evelope viserl iposity with inrese mounts of plsm free ftty is n plsm n hepti triglyeries. These finings suggest mehnism through whih insulin signling regultes insulin sensitivity y moulting hepti insulin lerne. Introution Insulin tivtes ifferent intrellulr signling pthwys y promoting protein phosphoryltion. The insulin reeptor is lign-tivte tyrosine kinse tht tivtes mny intrellulr protein sustrtes. Insulin-reeptor sustrtes (IRSs) hve een shown to influene insulin signling in ifferent tissues y promoting the tivtion of phosphtiylinositol-3 (PI-3) kinse n set of PIP-epenent serine/threonine protein kinses 1. It is likely, however, tht itionl sustrtes re lso require for insulin tion. For exmple, pthwy meite y phosphoryltion of the proto-onogene -l n le to gluose uptke inepenent of PI-3 kinse in response to insulin 2. Similrly, α 2 - Heremns Shmi glyoprotein (α 2 -HSG) inhiits signling through the insulin reeptor kinse 3, s n the plsm ell memrne glyoprotein-1 (PC-1) 4. We showe previously tht the trnsmemrne glyoprotein CEACAM1 is phosphorylte in response to insulin in heptoytes 5. The insulin reeptor kinse phosphoryltes single tyrosine resiue (Tyr488) in the intrellulr omin of CEACAM1. In ition, CEACAM1 is phosphorylte y serine/threonine kinses on Ser503 in the sene of insulin. Sustituting Al for Ser503 effetively olishe CEACAM1 phosphoryltion y either serine/threonine or tyrosine kinses, suggesting tht phosphoryltion on Ser503 is require for susequent tyrosine phosphoryltion y the insulin reeptor kinse. The funtion of CEACAM1 remins elusive. It hs een propose to t s ell hesion moleule 6. It is lso known to suppress tumor growth in ells of epithelil origin 7 10, to ownregulte the mitogeni effets of insulin n to upregulte reeptor-meite insulin enoytosis n egrtion Phosphoryltion of CEACAM1 seems to e require for its effet on ell prolifertion n insulin egrtion 12,13. In vitro stuies suggest tht upon its phosphoryltion y the insulin reeptor kinse, CEACAM1 ins iniretly to the reeptor to unergo internliztion in lthrin-ote vesiles 14 s prt of the insulin enoytosis omplex. Beuse reeptor-meite insulin enoytosis n egrtion in the heptoyte onstitutes the prinipl mehnism of insulin lerne, whih ours mostly in liver 15, we hve propose tht CEACAM1 phosphoryltion moultes hepti insulin lerne, ontriuting to the regultion of systemi insulin onentrtions. Beuse of the presene of two losely relte Cem genes in mouse (s oppose to one gene in rt n humns) 16, we generte mouse moel of funtionl intivtion of Cem1 ering ominnt-negtive, phosphoryltion-efetive CEACAM1 mutnt (from S503A rt) in liver. We report tht over-expression of the S503A minigene restrite to liver results in hyperinsulinemi ue to impire insulin lerne, with seonry hepti insulin resistne n impire gluose tolerne in the liverspeifi S503A CEACAM1 mutnt (L-SACC1) mie. These t inite new mehnisti link etween insulin signling n regultion of peripherl insulin sensitivity. Results Phosphoryltion of CEACAM1 in L-SACC1 mie We hrterize n re to homozygosity mouse line (F0 113) rrying six opies of the rt Cem1 S503A trnsgene. After phosphorylting liver lystes in the presene of [γ- 32 P]ATP, we immunopreipitte proteins with speifi ntioies ginst rt CEACAM1 (α-rcc1; Fig. 2) n mouse BGP, the rt CEA- CAM1 homolog (Fig. 2). In ontrst to the wil type (Fig. 2, lnes 3 n 4), insulin file to stimulte CEACAM1 phosphoryltion in homozygous L-SACC1 (L-SACC1 homo ) mie (Fig. 2, lnes 1 n 2). Overexpressing this mutnt i not signifintly 1 Deprtments of Phrmology n Therpeutis, n 2 Physil Therpy, Meil College of Ohio, 3035 Arlington Avenue, HSi Builing Room 270, Toleo, Ohio 43614, USA. 3 Seon Deprtment of Internl Meiine, Koe University Shool of Meiine, Kusunoki-ho, Chuo-ku, Koe, Jpn. 4 Deprtment of Meiine, Sn Frniso n the Veterns Affirs Meil Center, University of Cliforni, Sn Frniso, Cliforni, USA. Corresponene shoul e resse to S.M.N. (e-mil: snjjr@mo.eu). 270 nture genetis volume 30 mrh 2002

2 erese phosphoryltion of either the insulin reeptor (Fig. 2, lnes 1 n 2) or IRS-2 (Fig. 2e, lnes 1 n 2), mjor sustrte of the insulin reeptor kinse in liver. These results inite tht olishing CEACAM1 phosphoryltion in liver oes not interfere with phosphoryltion of other sustrtes of the insulin reeptor. It is thus possile tht IRS-epenent signling pthwys n the insulin tions they meite re not ltere in L-SACC1 mie. The S503A trnsgene uses hyperinsulinemi L-SACC1 homo mie showe inrese oy weight (Fig. 3; P<0.05) n viserl iposity t ll ges exmine (P<0.05; Fig. 3). By ontrst, hemizygous (L-SACC1 hemi ) mie were not ifferent from wiltype mie, onsistent with gene-osge effet (Fig. 3). Liver, kiney n musle funtions were unltere (Tle 1). L-SACC1 homo mie t two n eight months showe % inrese in plsm insulin levels ompre with ge-mthe wiltype mie (Fig. 4[i]). Insulin lerne, mesure s fsting molr C-peptie/insulin rtio, ws signifintly reue in L-SACC1 homo mie (Fig. 4[iii]). Moreover, the mount of plsm resiul [ 125 I]-insulin t min fter til injetion ws % higher in L-SACC1 thn wiltype nimls (Fig. 4). Insulin internliztion ws erese y pproximtely 50% (P<0.05) in heptoytes of L-SACC1 homo mie (Fig. 4). In ition, insulin-inue internliztion of insulin reeptors n CEA- CAM1, s mesure y the loss of iotin-lele surfe memrne proteins 14, ws reue in L-SACC1 ompre with wiltype nimls (insulin reeptor: 49.4% in L-SACC1 versus 82.1% in wiltype; CEACAM1: 13.7% in L-SACC1 versus 41.3 in wiltype; Fig. 4). Beuse CEACAM1 unergoes internliztion with the insulin reeptor s prt of the insulin enoytosis omplex 14, these finings inite tht reeptor-meite insulin internliztion n egrtion were erese in L-SACC1 mie. Tht C-peptie levels in L-SACC1 homo mie were milly inrese (Fig. 4[ii]; P<0.05) ws further supporte y unltere β-ell mss (1.611 ± in L-SACC1 versus ± 0.75 in wiltype, P>0.05). Tken together, these finings suggest tht the primry normlity of insulin metolism in L-SACC1 mie is impire insulin lerne rther thn inrese insulin seretion. To ress whether hyperinsulinemi ws primry effet of the trnsgene or seonry to insulin resistne, we exmine insulin sensitivity y intrperitonel insulin tolerne. After insulin injetion, gluose levels erese to similr extent in L-SACC1 homo n ontrols (wiltype n L-SACC1 hemi mie) t two n eight months of ge (Fig. 4e). This suggests tht peripherl tissues mintine insulin sensitivity in L-SACC1 homo mie. In ontrol mie, gluose levels returne to norml within three hours, wheres in L- SACC1 homo they remine suppresse. These results support the possiility tht injete insulin ws lere less effiiently. Seonry insulin resistne in L-SACC1 mie L-SACC1 homo mie evelope mil rnom hyperglyemi t 2 8 months (Fig. 5), initing insulin resistne in these mie. Fsting gluose levels were unltere, however, t two months (Fig. 5; 113 ± 37 in L-SACC1 homo versus 133 ± 41 in wiltype, P>0.05) or ten months (Fig. 5; 143 ± 19 in L-SACC1 homo versus 124 ± 29 in wiltype, P>0.05). Moreover, L-SACC1 homo mie were gluose intolernt (Fig. 5). The isrepny etween their sensitivity to insulin in n insulin tolerne test n their gluose intolerne proly hs severl uses. First, the lrge oses of insulin use in the insulin tolerne test re suffiient to overome the mil egree of peripherl insulin resistne. Seon, the egree of hypoglyemi ourring uring n insulin tolerne test epens lrgely on insulin-epenent gluose uptke tht ours primrily in skeletl musle, wheres hyperglyemi following n intrperitonel gluose injetion in roents isproportiontely epens on gluose utiliztion in liver. Thus, the preition from these experiments woul e tht L-SACC1 hs seonry hepti with Fig. 1 Genertion of L-SACC1 mie., Suloning of Apo A-1 promoter upstrem of Cem1 rt minigene ering Ser Al muttion on 503. The muttion rete new PstI site (*) without hnging the reing frme of the rt mutnt protein., Dominnt-negtive effet of rt S503A CEACAM1. SV40-trnsforme mouse heptoytes were trnsfete with rt S503A CEACAM1 mutnt (lnes 3, 4) or left untrnsfete (UT; lnes 1, 2). Internliztion of the insulin reeptor ws mesure s the loss of the iotin-lele α-suunit of the insulin reeptor (IR-α) from the surfe in the presene (even lnes) versus sene (o lnes) of insulin., Southern lot of HinIII/PstI-igeste genomi DNA from niml tils, using [ 32 P]-lele Apo A-1 promoter/s503a Cem1 minigene s proe. In the representtive gel, ns erive from the trnsgene were etete in the F0 mouse 113 in ition to the ns erive from the enogenous gene tht is lso etete in wiltype (WT) nimls., Western lot of liver-speifi expression of S503A CEACAM1 proteins. Plsm memrne proteins from livers n smll intestines of F1 progeny n WT mie were prepre y etergent extrtion n prtil purifition on letin hromtogrphy. Equl mounts (300 mg) of glyoproteins were immunopreipitte with R2-6, polylonl rt CEACAM1 ntioy tht ross-rets with the mouse protein. After nlysis on 7% SDS PAGE, proteins were immunolotte with α-295, polylonl rt CEACAM1 ntioy, n etete y ECL. nture genetis volume 30 mrh

3 insignifint musle insulin resistne. In ft, insulin inue omprle rise in the rte of gluose uptke in soleus musle of 2-month wiltype n L-SACC1 mie t sumximl onentrtions (1.2 nm; 5.20 ± 0.98 in wiltype versus 3.73 ± 0.54 nmol g 1 min 1 in L-SACC1, P<0.05; Fig. 5). Similr results were otine in epitrohleris musles of mie t two n six months (t not shown). In ition, gluose trnsporter-4 (GLUT4) mrna levels were not ltere in soleus musle of L-SACC1 mie (Fig. 5). Moreover, the triglyerie ontent in soleus musle of 8-month L-SACC1 mie ws norml (66 ± 16 in L-SACC1 mie versus 45 ± 9 mg mg 1 protein in wiltype, P>0.05). These finings inite tht L-SACC1 mie o not evelop signifint musle insulin resistne. The reeptor numer in heptoytes ws reue y hlf in L-SACC1 homo mie t two n eight months (pproximtely 2.4 ± in L-SACC1 versus pproximtely 5.62 ± reeptors per ell in wiltype, P<0.05). Moreover, the ffinity of insulin ining (K ) ws virtully unhnge (4.97 ± in L-SACC1 versus 6.55 ± in wiltype, P>0.05). Stey-stte mrna enoing rte-ontrol enzyme for hepti gluoneogenesis, phosphoenolpyruvte roxykinse (PEPCK), ws onsistently signifintly higher (y pproximtely %) in homozygous L-SACC1 t ll ges exmine (Fig. 5e). This suggests tht primry hyperinsulinemi use seonry hepti insulin resistne in L-SACC1 mie. In ontrst, mrna enoing rte-ontrol enzyme in glyogenolysis, glyogen 6-phosphtse (G-6-Pse), ws 35 70% lower in L-SACC1 homo thn in wiltype mie (Fig. 5e). The erese in G-6-Pse oul ounter-regulte the positive effet of elevte PEPCK on hepti gluose proution. This is onsistent with onition of hepti insulin resistne seonry to hyperinsulinemi without signifint inrese in fsting gluose levels n evelopment of ietes in L-SACC1 mie. e Fig. 2 Speifi ltertion of hepti CEACAM1 phosphoryltion in L-SACC1 mie. e, Glyoproteins from liver lystes of wiltype n L-SACC1 homozygous mie t 2 mo were letin-purifie n phosphorylte in the presene ( ) or sene (,e) of [γ- 32 P]ATP. Proteins were then immunopreipitte (Ip) with ntioies ginst mouse iliry glyoprotein (α-mbgp, the mouse CEA- CAM1 homolog; ); rt CEACAM1 (α-rcc1; ); IR-β suunit (IR-β; ) n IRS-2 (e). Phosphoryltion of proteins ws etete y utoriogrphy ( ) n immunolotting (I) with α-phosphotyrosine ntioy (α-ptyr; -e). To ount for the mount of rt CEACAM1 in the immunopellets, lot ws reproe with α-rcc1 (). Tle 1 Biohemistry of norml liver, kiney n musle in homozygous L-SACC1 mie WT L-SACC1 homo Liver funtion Asprtte minotrnsferse (µm) ± ± 52.3 Alnine minotrnsferse (µm) 48.7 ± ± 20.9 Alkline phosphtse (µm) 48.0 ± ± 21.5 Biliruin: totl (mg/l) 0.18 ± ± 0.09 Biliruin: iret (mg/l) 0.09 ± ± 0.05 Cholesterol: totl (mg/l) 98.5 ± ± 9.83 Cholesterol: HDL (mg/l) 83.2 ± ± 7.20 Musle funtion: Cretine kinse (µm) ± ± Kiney funtion: Bloo ure nitrogen (mg/l) 27.5 ± ± 3.90 The levels of severl enzymes n of holesterol, iliruin n loo ure nitrogen were etermine in fsting wiltype (WT) n homozygous (homo) L-SACC1 mie t 8 mo. All ssys were rrie out in triplite. Vlues re expresse s men ± s.e.m.. P>0.05 versus WT mie in ll etermintions. Unltere pnreti β-ell funtion in L-SACC1 mie To exmine pnreti β-ell funtion, we mesure insulin seretion in response to gluose. Clultion of the re uner the urve (Fig. 6) revele tht the first-phse insulin response to gluose ws not ltere (P>0.05) in 10-month L-SACC1 homo mie (4.680 ± u.) ompre with ontrols (wiltype: ± n L-SACC1 hemi : ± u.). Similr results were otine in mie t two months (t not shown). This suggests tht hyperinsulinemi n hepti insulin resistne i not signifintly impir β-ell funtion in L-SACC1 homo mie, in greement with no evelopment of ietes in these mie. Altere ft metolism in L-SACC1 mie Beuse elevte FFA levels influene hepti utoregultion of gluose proution, perhps y stimulting gluoneogenesis n ownregulting glyogenolysis in mie 17, we exmine fsting plsm FFA levels in L-SACC1 homo mie. Plsm FFA were signifintly elevte in these mie (Tle 2; P<0.05). This ws ssoite with inrese plsm (Tle 2) n hepti triglyerie ontent (18 ± 1 in L-SACC1 mie versus 10 ± 3 mg mg 1 protein in wiltype, P<0.05). Disussion We hve emonstrte tht over-expression of phosphoryltionefetive Cem1 trnsgene in mouse liver ts in ominntnegtive mnner to inhiit CEACAM1 funtion n results in hyperinsulinemi without ltering liver funtion or insulin-signling through other pthwys. This is in mrke ontrst to mie ering liver-speifi ltion of the insulin-reeptor gene (LIRKO) tht hve ltere liver funtion n IRS-epenent signling pthwys 18. Hyperinsulinemi in L-SACC1 mie is onsistent with the hypothesis tht CEACAM1 is prt of omplex of proteins require for internliztion of the insulin-reeptor omplex 11,12,14,19. Tht hyperinsulinemi in L-SACC1 mie is primrily ue to erese hepti lerne is supporte y severl finings: (i) the inrese in insulin levels is not prllele y proportionl inrese in C-peptie levels (ii) β-ell mss is not ommensurte to the extent of hyperinsulinemi (iii) L-SACC1 mie re more sensitive to exogenously ministere insulin thn nontrnsgeni littermtes n (iv) L-SACC1 mie evelope intr-ominl viserl iposity n elevte plsm FFA, whih re ommonly ssoite with impire insulin lerne Other finings support the hypothesis tht rnom hyperglyemi n impire gluose tolerne in L-SACC1 mie re proly ue to hepti insulin resistne: (i) erese numers n internliztion of insulin reeptors with erese internliztion of insulin n CEACAM1 in primry heptoytes erive 272 nture genetis volume 30 mrh 2002

4 from L-SACC1 mie (ii) elevte PEPCK mrna levels (iii) norml insulin-epenent gluose uptke in isolte musle, suggesting tht hyperglyemi in L-SACC1 mie is ue to inrese hepti gluose proution rther thn impire musle gluose uptke n (iv) elevte plsm FFA in L-SACC1 mie. This woul ontriute to hepti insulin resistne y stimulting hepti synthesis n output of gluose 26 n triglyeries 27. The phenotype of L-SACC1 mie is onsistent with the seonry hepti insulin resistne oserve in reipients of pnres trnsplnts, in whom systemi elivery of insulin is ssoite with hyperinsulinemi n erese first-pss insulin lerne Fig. 3 Growth urves of ge-mthe wiltype n L-SACC1 mie., Wiltype (WT), L-SACC1 homo n L-SACC1 hemi mie were weighe t 2 12 mo. Vlues re expresse s men ± s.. from six litters n represent 20 mie per genotype. P<0.05 for L-SACC1 homo versus WT, n P>0.05 for L-SACC1 hemi versus WT., Viserl ipose tissues from the ominl region were ollete from t lest 15 L-SACC1 homo n WT mie n weighe n mesure s perentge of totl oy weight. Vlues re expresse s men ± s..; P<0.05 for L-SACC1 versus WT. in liver 28,29. Although hyperinsulinemi in L-SACC1 mie is similr to tht of LIRKO mie, the mehnism of its evelopment iffers in these two moels. In L-SACC1 mie, hyperinsulinemi resulte minly from impire insulin lerne n, in turn, use seonry hepti insulin resistne, wheres in the LIRKO mouse, primry hepti insulin resistne use impire insulin lerne, insulin hyperseretion n extreme hyperinsulinemi 18. Nevertheless, neither mouse moel of hepti insulin resistne evelope ietes, supporting the notion tht itionl tissues must e involve for the evelopment of overt type 2 ietes. e Fig. 4 Hyperinsulinemi in L-SACC1 mie is ue to impire insulin lerne., Plsm insulin (i) n C-peptie (ii) levels were mesure in whole venous loo rwn etween 11:00 m n 1:00 pm from 2-mo n 8-mo ge-mthe wiltype (WT) n L-SACC1 homo mie fste overnight. Insulin lerne ws lulte s the molr rtio of C-peptie to insulin (iii). Dt represent t lest three sets of experiments with t lest 30 mie of eh genotype. Vlues re expresse s men ± s... Asterisks inite sttistilly signifint ifferene (P<0.05 y one ftor ANOVA)., Metoli lerne of [ 125 I]-insulin injete in til vein ws mesure in 8-mo WT (open squres) n L-SACC1 homo mie (lose irles). Vlues re expresse s men ± s.e.m. of the perent of the mount of plsm insulin t 7.5 min from eight mie of eh genotype., [ 125 I]-insulin internliztion in primry heptoytes erive from ge-mthe WT (open squres) n L-SACC1 mie (lose irles) ws mesure. After ining, [ 125 I]-insulin ws llowe to internlize t 37 C for 0 90 min, s inite on the horizontl xis. Internlize lign ws plotte on the vertil xis s perent of speifilly oun lign. Vlues re expresse s men ± s.. from triplite experiments rrie out on t lest three ge-mthe mie of eh genotype., Primry heptoytes erive from WT n L-SACC1 homo mie were trete with uffer (, lnes) or insulin (+, lnes) efore the leling of ell-surfe proteins with iotin. Cells were lyse n the proteins immunopreipitte with polylonl ntioies ginst mouse CEACAM1 (CC1) n the α-suunit of the insulin reeptor (IR-α) prior to nlysis y 7% SDS PAGE, immunolotting with HRP-lele streptviin n etetion y ECL. The mount of CC1 n IR-α internlize in response to insulin ws lulte s the ifferene in the mount of iotin-lele proteins efore n fter insulin tretment reltive to the mount of iotin-lele proteins in the sene of insulin (grph). Experiments were repete on t lest three mie for eh genotype. e, Insulin tolerne test. Gluose levels were mesure in venous loo from ge-mthe wiltype (WT, open squres), L-SACC1 hemi (open irles) n L-SACC1 homo (lose irles) L- mie injete i.p. with insulin (0.125 U kg 1 ) n fste overnight. Dt represent the men ± s.. of 24 mie from eh genotype. *Sttistilly signifint ifferene (P<0.05), WT versus L-SACC1 hemi. nture genetis volume 30 mrh

5 Fig. 5 Seonry insulin resistne in L-SACC1 mie., Rnom gluose levels were mesure in venous loo rwn t 10:00 m from 15 nesthetize ge-mthe mie. Dt re the men of two etermintions per mouse rrie out seven ys prt t the sme time perio. Vlues re expresse s men ± s... Asterisks enote sttistilly signifint ifferene (P<0.05) etween L-SACC1 homo mie n ontrols (WT n L- SACC1 hemi )., Gluose tolerne test. Whole-loo gluose ws etermine t min fter gluose injetion (2 mg kg 1 ) of gemthe 2 mo n 10 mo WT n L-SACC1 homo mie fste overnight. Vlues re expresse s men ± s.. for t lest 12 mie in eh group., Insulin-epenent 2-eoxy-Dgluose (2-DG) uptke ws mesure in insulin-trete (1.2 nm) isolte soleus musles from gemthe 2-mo mie tht h een fste. Vlues re expresse s men ± s.. of eight smples for eh mouse genotype.,e, Poly (A) mrna ws purifie from soleus musle () n from livers (e) of ge-mthe WT n L-SACC1 homo mie t 2 8 mo. Results re representtive of t lest four mie from eh genotype t eh ge. e Hyperinsulinemi in L-SACC1 mie i not prllel the extent of the metoli ltertion with hyperglyemi, suggesting tht itionl mehnisms unerlie insulin resistne. The ssoition of hepti insulin resistne with elevtion in FFAs n inrese viserl iposity provies potentil mehnism to explin this pprent isrepny. Our moel supports the hypothesis tht erese insulin lerne in the presene of inrese ominl iposity n irulting FFA provies n lterntive ompenstory mehnism of peripherl insulin resistne 30,31. Hypertriglyeriemi in L-SACC1 mie my t lest prtilly e ttriute to hyperinsulinemi 32 n to inrese gluoseinue e novo hepti ftty-i synthesis n lipogenesis. The inrese in plsm triglyeries my, in turn, promote elevtion in plsm FFA. For instne, it is possile tht not ll unesterifie ftty is relese from hyrolysis of triglyeries y lipoprotein lipse re trnsporte into ipoytes; some my remin lumin-orne in the loo. Bse on the Rnle yle 33,34, it is resonle to preit tht hroni stimultion of gluose uptke in musle y hronilly elevte insulin levels woul ompete with FFA uptke in musle n erese FFA oxition n lerne. We therefore hypothesize tht elevtion in plsm FFA in our moel results from inrese triglyerie relese from liver rther thn from inrese lipolysis in ipoytes. This is in greement with oservtions of unltere lipolysis with ttennt eese FFA reesterifition in the peripherl tissues of oese sujets with higher insulin onentrtions thn their len ontrols 35. Alterntively, elevte plsm FFA oul result from inrese e novo tissue lipogenesis in proportion to inrese insulin levels in L-SACC1 mie, s hs een reporte in hyperinsulinemi-euglyemi norml rts 36. Further stuies re require to sertin the nture of the isturne in ft metolism. Notly, the extent of impirment of ft metolism is isproportionte to the extent of insulin resistne. For instne, mie with primry hepti insulin resistne i not evelop elevte FFA espite hving higher insulin levels thn L-SACC1 trnsgenis 18,37. This suggests tht impirment of ft metolism in L-SACC1 mie is relte to the mehnism of their insulin resistne (tht is, seonry to impire insulin lerne rther thn primry geneti efet in insulin reeptor expression). Altere ft metolism my explin the persistene of hyperglyemi, gluose intolerne n insulin resistne in L-SACC1 mie s oppose to LIRKO mie tht regine hepti insulin sensitivity t six months of ge. The inrese in triglyerie ontent in livers of L-SACC1 trnsgenis my lso suggest tht the impirment of ft metolism is seonry to the hnge of hepti insulin sensitivity rought out y the trnsgene, n not to sustrte reistriution s, for instne, in musle-speifi insulin reeptor knokouts 38. The inrese in liver triglyeries my ontriute to hepti insulin resistne tht, in turn, my result from hyperinsulinemi. These stuies emonstrte novel mehnism y whih Fig. 6 Norml pnreti β-ell funtion in L-SACC1 homo mie. We mesure insulin seretion in response to gluose injetions in 10-mo wiltype (WT, open squres), L-SACC1 hemi (open irles) n L-SACC1 homo (open squres) mie tht h een fste. Whole loo ws rwn t inite time points from retrooritl sinuses to etermine plsm insulin level. Dt represent the verge of t lest two sets of experiments using minimum of 12 mie of eh genotype. Vlues re expresse s men ± s.e.m nture genetis volume 30 mrh 2002

6 Tle 2 Elevte levels of free ftty i n triglyeries in plsm of L-SACC1 mie Plsm free ftty is Plsm triglyeries (meq L 1 ) (mg/l) Genotype (n) 2 mo 8 mo 2 mo 8 mo WT (24) ± ± ± ± 4.34 L-SACC1 (21) ± 0.02* ± 0.05* 94.6 ± 9.73* ± 8.80* Fsting plsm FFA n triglyeries were etermine in ge-mthe mie t 2 mo n 8 mo. The numer of mie teste is inite in prentheses. All ssys were rrie out in triplite. Vlues re expresse s men ± s.e.m.. *P<0.05 versus WT mie. CEACAM1-epenent insulin signling in heptoytes is linke to regultion of hepti insulin sensitivity y moulting insulin metolism in liver. They lso provie in vivo eviene of the iret role of insulin lerne in liver in mintining rohyrte n ft homeostsis. Methos Genertion of liver-speifi S503A CEACAM1 mie (L-SACC1). We sulone the 490-p frgment 5 of the trnsltion strt site in genomi DNA of humn polipoprotein A-1 (Apo A-1) 39,40 upstrem of Cem1 DNA (from S503A rt) into n EoRI site 41 in pgem9z. We introue intron 1 of rt Cem1 etween NeI t nt 10 in exon 1 n BmHI t nt 313 in exon 2 (ref. 41). We re-sulone the NotI-exise Apo A-1/S503A Cem1 minigene in pdna3 (Fig. 1). As seen with lign-inue reeptor internliztion, ition of insulin erese surfe expression of iotinlele insulin reeptor 14 in untrnsfete SV-40 trnsforme heptoytes (UT; Fig. 1, lnes 2 versus 1). In ontrst, in heptoytes expressing the minigene, internliztion of the insulin reeptor in the presene of insulin ws inhiite (Fig. 1, lnes 4 versus 3), suggesting tht the mutnt is ominnt-negtive. We exise the promoter-minigene-polyenyltion DNA frgment n injete it into the pronulei of single-ell fertilize mouse emryos erive from C57BL/6 FVB mtings. We ientifie four F0 founers y Southern-lot nlysis of genomi DNA from HinIII/PstIigeste til n proing with [ 32 P]-lele promoter/s503a Cem1 minigene (Fig. 1) 41,42. Northern-lot nlysis on poly(a) mrna (Amion; t not shown) n western-lot nlysis using polylonl α- CEACAM1 ntioy tht ross-rets with rt n mouse proteins inite tht the rt trnsprotein ws etete t high levels in liver of trnsgeni F1 mie (Fig. 1) ut not in intestines (Fig. 1) or kineys (t not shown). Animl husnry. We re hemizygous mie (L-SACC1 hemi ) to homozygosity y rother/sister mtings. We onfirme homozygosity y quntittive Southern lot n empirilly y mting homozygous mie (L-SACC1 homo ). Animls were kept in 12-h rk light yle n fe stnr how liitum. All proeures esrie elow were pprove y the Institutionl Animl Cre n Utiliztion Committee. Phosphoryltion. We issete livers from nesthetize nimls n froze them immeitely efore lysis n prtil purifition y letin hromtogrphy 5. After rrying out insulin-inue phosphoryltion in the presene of [γ- 32 P]ATP 5, we immunopreipitte proteins with nti-iliry glyoprotein (BGP, the mouse homolog of rt CEACAM1) or nti-rt CEACAM1 ntioies n nlyze them using SDS PAGE n utoriogrphy. For insulin-reeptor n IRS-2 phosphoryltion, we rrie out the retion in the sene of [γ- 32 P]ATP, immunopreipitte proteins with monolonl ntioies ginst the IR-β suunit or IRS-2, immunolotte with ntiphosphotyrosine ntioies (α-ptyr) n etete proteins y enhne hemiluminesene (ECL; Amershm Phrmi Bioteh). Phenotypi nlysis. We mesure loo gluose levels using gluometer (Au-hek, Boehringer Mnnheim), plsm insulin n C-pepties with rioimmunossys (RIA, Lino), fsting plsm free ftty is (FFA) using the NEFA C kit (Wko) n triglyeries using the Infinity Triglyeries regent (Sigm). We etermine tissue-triglyerie ontent in homogenize tissues y extrting lipi with CHCL 3 :CH 3 OH (2:1, v:v), rying uner N 2, resuspening in ethnol, n mesuring triglyeries levels s the ifferene etween free n totl glyerol 43. Liver, musle n kiney funtions were ssye y Anlytis. Metoli lerne ssy. We fste ge-mthe 8-mo mle mie for 18 h (from 5:00 pm to 12:00 pm the next y) prior to intrvenous til vein injetion with 0.05 µg of humn reominnt [ 125 I]-insulin 44. We ollete venous loo smples (100 µl per mouse) t 7.5, 15, 60 n 90 min fter injetion, ounte 50 ml plsm n mesure resiul insulin in pm ml 1 plsm. Insulin tolerne test. We fste mie for 19 h (from 4:00 pm to 11:00 m the next y) prior to intrperitonel injetion (i.p.) with insulin (0.125 mu g 1 oy weight). We rew venous loo from the retro-oritl sinuses t 0, 30, 60 n 180 min fter insulin injetion to etermine loo gluose levels. Intrperitonel gluose tolerne test n insulin seretion. We injete mie fste overnight with gluose (2 mg kg 1 oy weight) i.p. n rew venous loo from the retro-oritl sinuses t 0, 15, 30, 60 n 120 min fter injetion to etermine gluose levels. We simultneously mesure serete insulin from β-ells in plsm t 2, 7 n 30 min. Morphometri nlysis of pnres. Pnret were setione into 5- mm 3 portions, fixe in 10% formlin-pbs (ph 7.4), ethnol-ehyrte n prffin-emee 37,45. We mounte onseutive setions on glss slies, rehyrte, permeilize in 0.1% Triton X-100 n immunostine with mouse insulin ntioies (Sigm). We systemtilly viewe setions with Nikon Optiphot-2 mirosope (Nikon) n Spot vieo mer (Dignosti Instruments) with umulting imges from eight non-overlpping fiels of mm 2. We exmine t lest three setions from three nimls per genotype for totl of 18 inepenent setions. We tre n nlyze β-ells in NIH Imge v Dt were expresse s perentge of the totl re ontining insulin-lele ells. Primry heptoytes. We isolte heptoytes y perfusing with 1 mg ml 1 ollgense in L-15 meium ontining 1 mg ml 1 gluose (Gio BRL). We plte ells t ells per well n inute t 37 C in DMEM-FBS-peniillin-streptomyin (Gio BRL). We refe ultures with DMEM fter 2-h tthment perio 46. Lign ining n internliztion. We grew primry heptoytes for 24 h prior to [ 125 I]-insulin (10 pm) ining t 4 C in uffer F (100 mm HEPES, ph 7.4, 120 mm NCl, 1.2 mm MgSO 4, 1 mm EDTA, 15 mm CH 3 COON, 10 mm gluose, 1% BSA). We remove unoun insulin n inute ells t 37 C for 0 90 min efore inuting them in 0.1% BSA-PBS (ph 3.5) for 10 min 11. We ounte the i wsh s surfe-oun, non-internlize insulin n NOH-soluilize ells s internlize ell-ssoite lign. We lulte internlize insulin s perent ell-ssoite per speifilly oun lign (the sum of surfe-oun plus ell-ssoite lign). Biotin leling of surfe memrne proteins. After inution of primry heptoytes in the sene or presene of 100 nm insulin t 37 C for 5 min, we inute ells with iotin s previously esrie 14. Following lysis in 1% Triton-X, proteins were immunopreipitte with monolonl ntioy ginst BGP (mouse homolog of rt CEACAM1), eletrophorese, immunolotte with HRP-onjugte streptviin n etete with ECL. Insulin-reeptor quntifition. We inute primry heptoytes t 37 C in triplite pltes for 24 h efore inuting t 4 C for 5 h in Buffer F supplemente with insulin (0, 1, 5, 10, 100 n 1,000 ng ml 1 ) n [ 125 I]-lele insulin (20 pm; pm ml 1 ). NOH-lyse ells were ounte n the IR numer etermine y Sthr-plot nlysis using the SCAFIT v. 4.7 progrm (Ntionl Institutes of Helth). Gluose uptke in isolte musle. As esrie previously 47,48, we remove soleus musle from hinlims of fste mie 47,48, pre-equilirte it for 1 h in oxygente Kres-Henseleit uffer ontining 2 mm pyruvte, 36 mm mnnitol n 0.1% BSA with or without 1,200 pm insulin, n inute it t 30 C t 95% O 2, 5% CO 2 for 20 min in 2-eoxy-D- nture genetis volume 30 mrh

7 [l,2-3 H]gluose (1 mm), [U- 14 C]mnnitol (39 mm) n 0.1% BSA. Musles were immeitely lotte n frozen in liqui nitrogen, n store t 80 C efore igestion, ounting n mesuring of intrellulr 2-DG umultion in nmol g wet musle 1 min 1. Sttistil proeures. We nlyze t with Sttview softwre (Aus onepts) using one-ftor ANOVA nlysis. P vlues less thn 0.05 were onsiere to e sttisilly signifint. Aknowlegments We thnk D. Aili, S.F. Previs n G.I. Shulmn for ritil reing of the mnusript n for helpful sientifi isussions, n E.M. Ruin n N. Beuhemin for proviing the polipoprotein A-1 promoter DNA n ntimouse BGP ntioies, respetively. We thnk S. Roson, E. Tietz n S. Lilly for ssistne in immunohistohemistry n mirosopy, n T. Di n R. Ruh for ssistne in primry heptoytes. We lso thnk the Ohio University Eison Biotehnology Institute Collortive Trnsgenis, Athens, Ohio for performing DNA miroinjetions s prt of their servie grnt support (to S.M.N.). This work ws hiefly supporte y two grnts from the Ntionl Institutes of Helth Ntionl Institute of Dietes & Digestive & Kiney Diseses (to S.M.N.), n prtilly y Sigm-Tu Reserh (to S.M.N.) n the Deprtment of Veterns Affirs (to S.K.E.). M.N.P. ws prtilly supporte y the Institutionl Pre-otorl Ntionl Reserh Servie Awr fellowship from the Ntionl Institutes of Helth. Competing interests sttement The uthors elre tht they hve no ompeting finnil interests. Reeive 30 Otoer; epte 12 Deemer Bker, J.M. et l. Insulin stimultion of phosphtiylinositol 3-kinse tivity mps to insulin reeptor regions require for enogenous sustrte phosphoryltion. J. Biol. Chem. 267, (1992). 2. Bumnn, C.A. et l. CAP efines seon signlling pthwy require for insulin-stimulte gluose trnsport. Nture 407, (2000). 3. Mthews, S.T. et l. Alph2-HSG, speifi inhiitor of insulin reeptor utophosphoryltion, interts with the insulin reeptor. Mol. Cell. Enorinol. 164, (2000). 4. Mux, B.A. & Golfine, I.D. 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