Irs-2 coordinates Igf-1 receptor-mediated β-cell development and peripheral insulin signalling

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1 Irs-2 coordintes Igf-1 receptor-medited β-cell development nd peripherl insulin signlling Dominic J. Withers 1,2 *, Deorh J. Burks 1 *, Hether H. Towery 1, Shri L. Altmuro 1, Crrie L. Flint 1 & Morris F. White 1 *These uthors contriuted eqully to this work. Insulin receptor sustrtes (Irs proteins) medite the pleiotropic effects of insulin nd Igf-1 (insulin-like growth fctor-1), including regultion of glucose homeostsis nd cell growth nd survivl. We intercrossed mice heterozygous for two null lleles (Irs1 +/ nd Irs2 +/ ) nd investigted growth nd glucose metolism in mice with vile genotypes. Our experiments reveled tht Irs-1 nd Irs-2 re criticl for emryonic nd post-ntl growth, with Irs-1 hving the predominnt role. By contrst, oth Irs-1 nd Irs-2 function in peripherl crohydrte metolism, ut Irs-2 hs the mjor role in β-cell development nd compenstion for peripherl insulin resistnce. To estlish role for the Igf-1 receptor in β-cells, we intercrossed mice heterozygous for null lleles of Igf1r nd Irs2. Our results revel tht Igf-1 receptors promote β-cell development nd survivl through the Irs-2 signlling pthwy. Thus, Irs-2 integrtes the effects of insulin in peripherl trget tissues with Igf-1 in pncretic β-cells to mintin glucose homeostsis. Introduction Insulin nd Igf-1 ind to distinct cell-surfce receptor tyrosine kinses tht regulte vriety of signlling pthwys controlling metolism, growth nd survivl 1 6. Mice without Igf1r hve retrded growth nd die t irth, wheres mice lcking the insulin receptor disply norml intruterine growth ut die within 72 hours due to severe hyperglycemi nd ketocidosis with hyperinsulinemi 7,8. Mice lcking the pncretic β-cell insulin receptor disply loss of first-phse glucose-stimulted insulin secretion ut do not develop overt dietes 9. The ctivted receptors for insulin nd Igf-1 phosphorylte vrious cellulr sustrtes, including Irs-1 nd Irs-2 (refs 10 12), which integrte the pleiotropic effects of insulin, Igf-1 nd other cytokines on cellulr function. Deletion of Irs1 produces smll, insulin-resistnt mice with nerly norml glucose homeostsis due to compenstory β-cell expnsion In contrst, mice lcking Irs-2 disply nerly norml growth, ut develop dietes 8 10 weeks fter irth ccompnied y reduced β-cell mss nd impired function 13. Here we hve used genetic pproch to define the roles of Irs-1 nd Irs-2 in somtic growth, peripherl crohydrte metolism nd β-cell development nd function. Irs1 +/ nd Irs2 +/ mice were intercrossed to generte progeny with vrious sttes of Irs-1 nd Irs-2 deficiency. To determine the contriutions of the Igf-1 receptor during Irs-2 medited β-cell expnsion, we intercrossed Irs2 +/ mice with Igf1r +/ mice. Our results suggest tht Igf-1 receptors couple to Irs-2 to medite islet development during emryogenesis nd promote β-cell prolifertion nd survivl during post-ntl growth nd in response to peripherl insulin resistnce. Results Chrcteristics of mice lcking Irs-1 nd Irs-2 We intercrossed Irs1 +/ Irs2 +/ mice to produce 185 litters yielding 925 offspring representing 8 of 9 possile genotypes; Southern-lot nlysis conducted t 2 weeks of ge reveled no Irs1 / Irs2 / mice. Irs1 +/ Irs2 / nd Irs1 / Isr2 +/ mice were less frequent thn expected (Irs1 +/ Irs2 /, 2.7% ctul versus 12.5% expected; Irs1 / Isr2 +/, 0.97% ctul versus 12.5% expected). Consequently, mice of other genotypes were orn with n ltered frequency (wild type, 14.3% ctul versus 6.25% expected; Irs1 +/, 18.5% ctul versus 12.5% expected; Irs2 +/, 21.8% ctul versus 12.5% expected; Irs1 /, 4% ctul versus 6.25% expected; Irs2 /, 7.7% ctul versus 6.25% expected; Irs1 +/ Irs2 +/, 30.3% ctul versus 25% expected). A similr distriution of genotypes ws found for 108 emryos (emryonic dy (E) ) from 20 pregnnt femles nd smll numer of ded pups. These results suggest tht Irs1 / Irs2 / emryos re not vile, wheres Irs1 +/ Irs2 / nd Irs1 / Irs2 +/ nimls re vile ut develop with lower thn expected frequency due to wstge efore E16.5. We generted growth curves sed on dily weights from irth to 30 dys of offspring from Irs1 +/ Irs2 +/ intercrosses (Fig. 1). Birth weights for Irs1 +/, Irs1 /, Irs2 +/ nd Irs2 / mice were consistent with previous reports 13,14. Compound heterozygous mice (Irs1 +/ Irs2 +/ ) were 75% the weight of wild-type nimls. Irs1 +/ Irs2 / mice were of similr size to Irs1 / nimls, wheres Irs1 / Irs2 +/ mice were 70 75% smller thn norml littermtes (Fig. 1,). Irs1 / Irs2 +/ mice re mong the smllest vile mice tht hve een generted from vriety of strtegies imed t deleting components of 1 Howrd Hughes Medicl Institute, Joslin Dietes Center, Hrvrd Medicl School, One Joslin Plce, Boston, Msschusetts 02215, USA. 2 Current ddress: Deprtment of Metolic Medicine, Imperil College School of Medicine, Hmmersmith Cmpus, Du Cne Rod, London, W12ONN, UK. Correspondence should e ddressed to M.F.W. (e-mil: morris.white@joslin.hrvrd.edu). 32 nture genetics volume 23 septemer 1999

2 rticle Fig. 1 Growth chrcteristics of progeny of Irs1 +/ Irs2 +/ intercross., Growth curves showing men weight±s.e.m. of nimls of the vrious genotypes on C57Bl/6 129SV ckground t the indicted ges (dys). Dt re from 170 litters with t lest 4 nimls per genotype., A 30-dy-old wild-type niml with n Irs1 / Irs2 +/ littermte. growth fctor signlling pthwys, nd demonstrte tht single copy of Irs2 promotes viility for t lest one yer. Metolic consequences of Irs protein deletion At 2 3 weeks of ge, Irs1 +/ Irs2 / mice hd fsting lood sugrs in excess of mg/dl nd thus were dietic. By 4 weeks of ge, the lood glucose vlues exceeded 500 mg/dl s mice displyed progressive polyuri (without ketonuri), polydyspsi nd rrely survived eyond 5 weeks of ge. This progression to the dietic phenotype ws quicker thn tht of Irs2 / mice, in which overt dietes did not usully present until fter 10 weeks c e d f of ge (Fig. 2). In comprison, Irs1 +/ Irs2 +/ nd Irs1 / Irs2 +/ mice hd norml fsting lood glucose t four weeks of ge (Fig. 2). Glucose tolernce tests performed on four-week-old mice reveled tht Irs1 +/ Irs2 / mice were glucose intolernt, wheres Irs2 +/, Irs1 +/ Irs2 +/ nd Irs1 / Irs2 +/ mice displyed only mild glucose intolernce (Fig. 2). At four weeks, oth Irs1 / nd Irs2 / mice hd elevted fsting insulin levels, consistent with compensted peripherl insulin resistnce s previously reported 13, nd Irs1 +/ Irs2 +/ mice were lso hyperinsulinemic. Similrly, Irs1 / Irs2 +/ mice displyed fsting hyperinsulinemi, which mintined nerly Fig. 2 Metolic chrcteristics of progeny of Irs1 +/ Irs2 +/ intercross., After 15-h overnight fst, we determined lood glucose levels on 4- week-old nimls. Results re men vlues±s.e.m. for t lest six nimls of ech genotype., Glucose tolernce tests were performed fter intrperitonel loding with 2 g D-glucose per kg ody weight on nimls fsted overnight for 15 h (wild type, filled circles; Irs2 +/, filled squres; Irs1 +/ Irs2 +/, open tringles; Irs1 / Irs2 +/, filled tringles; Irs1 +/ Irs2 /, open circles). c, Serum insulin levels were mesured y rdioimmunossy on 4-week-old nesthetized nimls fter 15-h overnight fst. d, After 15-h overnight fst, lood glucose levels were determined on 6-month-old nimls. e, Glucose tolernce tests were performed fter intrperitonel loding with 2 g D-glucose per kg ody weight on fsted 6-month-old nimls (wild type, filled circles; Irs2 +/, open tringles; Irs1 +/ Irs2 +/, filled tringles; Irs1 / Irs2 +/, open circles). f, After 15- h overnight fst, serum insulin levels were mesured y rdioimmunossy on 6-month-old nesthetized nimls. f, Results re the men vlues±s.e.m. for t lest four nimls of ech genotype. nture genetics volume 23 septemer

3 c e g d f h Fig. 3 Insulin signlling in skeletl muscle nd liver of progeny of n Irs1 +/ Irs2 +/ intercross. Expression nd tyrosine phosphoryltion of the insulin receptor β-suunit in skeletl muscle () nd liver () of nimls of the indicted genotypes. Superntnts of muscle nd liver homogentes contining totl protein from untreted nd insulin (Ins)-treted 4 8-weekold mice were immunoprecipitted with nti- IRβ ntiody nd lotted for either ntiphosphotyrosine (PY) or IRβ (IR). Expression of Irs-1 nd Irs-2 in skeletl muscle (c) nd liver (d) of nimls is shown. Superntnts of muscle nd liver homogentes of totl protein (2 mg) were immunoprecipitted with either Irs-1 or Irs-2 ntiody nd lotted with the sme ntiody. d, Results re representtive of three nimls of ech genotype. Tyrosine phosphoryltion of Irs-1 nd Irs-2 in skeletl muscle (e) nd liver (f) is shown. Superntnts of muscle nd liver homogentes contining totl protein (2 mg) from untreted nd insulin-treted 4 8-week-old mice were immunoprecipitted with Irs-1 or Irs-2 ntiody lotted for ntiphosphotyrosine (PY). Immunolots re representtive of dt from three nimls per genotype. Insulin-stimulted PI-3K ctivtion in muscle (g) nd liver (h) is shown. Superntnts of muscle nd liver homogentes contining totl protein (2 mg) from untreted (lck rs) nd insulintreted (grey rs) 4 8-week-old mice were immunoprecipitted (IP) in duplicte with the indicted ntiody (Irs-1 or Irs-2); immunoprecipittes were ssyed in vitro for PI-3 kinse ctivity. Dt re the men vlues±s.e.m. of two independent experiments nd represent dt from t lest four nimls of ech genotype. norml glucose tolernce ginst peripherl insulin resistnce (Fig. 2c). By contrst, Irs1 +/ Irs2 / mice hd lower fsting insulin levels thn wild-type nimls (Fig. 2c). Insulin levels in rndomly fed Irs1 +/ Irs2 / mice were lower thn those seen in wild-type mice, demonstrting the presence of reltive insulin insufficiency (dt not shown). By these criteri, Irs2 +/ mice hd no insulin resistnce t this ge s reported 13. Irs1 / Irs2 +/ mice mintined norml fsting lood sugrs when followed up to six months of ge (Fig. 2d). These mice displyed impired glucose tolernce t erly time points during glucose tolernce test, with glucose concentrtions returning to norml y 120 minutes (Fig. 2e). Similr nlysis of Irs2 +/ nd Irs1 +/ Irs2 +/ mice t six months showed tht glucose tolernce hd deteriorted, wheres fsting lood sugrs remined norml (Fig. 2e). At six months of ge, Irs2 +/ mice displyed fsting hyperinsulinemi nd Irs1 +/ Irs2 +/ mice hd slightly higher insulin levels consistent with n gerelted development of insulin resistnce (Fig. 2f). Irs1 / Irs2 +/ mice were lso hyperinsulinemic t six months of ge, demonstrting tht single copy of Irs2 ws sufficient to mintin pproprite β-cell compenstion in the presence of insulin resistnce (Fig. 2f). Insulin signlling pthwys in Irs-deficient mice We exmined insulin-stimulted tyrosine phosphoryltion nd PI-3 kinse ctivity in skeletl muscle nd liver. Signlling events in Irs1 +/ Irs2 / mice were omitted ecuse hyperglycemi nd their short lifespn mde meningful nlysis of genetic mnipultion of these prmeters impossile. Insulin receptor levels nd ctivtion s ssessed y expression nd tyrosine utophosphoryltion of the β-suunit were comprle in liver nd skeletl muscle in ll genotypes studied t four months of ge (Fig. 3,). Expression of Irs-1 or Irs-2 ws reduced (50%) or undetectle, consistent with the genotype of the individul mice; there ws no evidence for compenstory overexpression (Fig. 3c,d, nd dt not shown). Irs-2 tyrosine phosphoryltion ws reduced in liver nd muscle of Irs1 +/+ Irs2 +/ mice, consistent with reduced expression, wheres Irs-1 tyrosine phosphoryltion ws comprle to tht of controls (Fig. 3e,f). By contrst, sl tyrosine phosphoryltion of oth Irs-1 nd Irs-2 ws elevted in liver of Irs1 +/ Irs2 +/ mice (Fig. 3e,f). Elevted Irs-2 tyrosine phosphoryltion ws lso detected in liver of Irs1 / Irs2 +/ mice (Fig. 3f). We ssessed insulin-stimulted PI-3 kinse ctivity in Irs-1 nd Irs-2 immunoprecipittes from oth liver nd muscle t 34 nture genetics volume 23 septemer 1999

4 rticle c d i e f g h j Fig. 4 Islet morphology nd β-cell nlysis in progeny of Irs1 +/ Irs2 +/ intercross. h, Immunostining of insulin in β-cells of pncres sections from four-week-old mice. Permeilized, prffin-emedded pncres sections (5 µm) were incuted with monoclonl nti-insulin ntiodies. Stining ws reveled y fluoroscein nti-mouse ntiodies. Imges of representtive fields were cptured t mgnifiction of 10. i,j, Quntittion of β-cell re in four-week nd four-month-old mice. Pncres sections immunostined for insulin were viewed using Zeiss Axiovert Microscope nd video cmer. Results re men vlues±s.e.m. of t lest four nimls of ech genotype. four months of ge. In Irs2 +/ mice there ws reduction in Irs- 2 ssocited PI-3 kinse ctivity on insulin stimultion in the liver, wheres the ctivity in muscle ws nerly norml compred with wild-type controls (Fig. 3g,h). By contrst, Irs- 1 ssocited PI-3 kinse ctivity decresed slightly in muscle of Irs2 +/ mice, despite the norml expression of Irs- 1 nd norml insulin receptor function (Fig. 3g,h). In Irs1 / Irs2 +/ mice, insulin-stimulted Irs-2 ssocited PI-3 kinse ctivity ws lrgely preserved, especilly in skeletl muscle (Fig. 3g). In Irs1 +/ Irs2 +/ mice, however, there ws less of n increse in Irs-1 ssocited PI-3 kinse ctivity in insulin-stimulted muscle nd liver (Fig. 3g,h) nd in Irs- 2 ssocited PI-3 kinse ctivity in muscle (Fig. 3g). This reduced increse in insulin-stimulted PI-3 kinse ctivity in Irs1 +/ Irs2 +/ mice is due in prt to elevted sl ctivity of the enzyme in Irs-1 nd Irs-2 immunoprecipittes from muscle nd liver. These results suggest there is complex interply etween Irs-1 nd Irs-2 in the pproprite regultion of PI-3 kinse ctivtion in oth liver nd muscle. Islet morphology in mice lcking Irs proteins We hve previously implicted Irs-2 dependent signlling pthwys in the β-cell compenstory response to insulin resistnce, possily y regulting differentition, prolifertion or survivl of β-cells. Therefore, the rpid onset of dietes in Irs1 +/ Irs2 / mice, ut not in Irs1 / Irs2 +/ mice, prompted us to nlyse islet morphology. β-cell re in multiple pncres c d e f g Fig. 5 Islet morphology nd β-cell mss in Igf1r / mice. c, Pncres sections (5 µm) from neworn pups (post-ntl dy 0.5) were stined with hemtoxylin nd eosin nd photogrphed t mgnifiction of 20. d f, Pncres sections otined from E18 emryos were co-immunostined for insulin nd glucgon. Insulin-positive cells were reveled y fluorescein-lelled secondry ntiodies (green); glucgon-contining cells were detected y rhodmine-lelled secondry ntiodies (red). Imges were collected seprtely t mgnifiction of 20 nd superimposed. g, - nd β-cell res in emryos nd neworns were quntitted from pncres co-immunostined for insulin, glucgon nd mylse. Results re the men vlues±s.e.m. for t lest three nimls of ech genotype. Grey rs, -cell numers; lck rs, β-cell numers. nture genetics volume 23 septemer

5 Fig. 6 Weight nd metolic chrcteristics of progeny of Igf1r +/ Irs2 +/ intercross., Men weights±s.e.m. of 30-dy-old mice. Dt re from ten litters with t lest four nimls per genotype., After 15-h overnight fst, lood glucose levels were determined on 4-weekold nimls. Results re men vlues±s.e.m. for t lest six nimls of ech genotype. c, Glucose tolernce tests were performed fter intrperitonel loding with 2 g D-glucose per kg ody weight on fsted 4-week-old nimls of the indicted genotypes. d, Serum insulin levels were mesured y rdioimmunossy on 4- week-old nesthetized nimls fter 15-h overnight fst. e, After 15-h overnight fst, lood glucose levels were determined on 4-month-old nimls of the indicted genotype. f, Glucose tolernce tests were performed fter intrperitonel loding with 2 g D-glucose per kg ody weight on fsted 4-month-old nimls of the indicted genotypes. g, Serum insulin levels were mesured y rdioimmunossy on 4- month-old nesthetized nimls fter 15-h overnight fst. c g, Results re men vlues±s.e.m. for t lest four nimls of ech genotype. c d cross-sections from 4-week-old Irs1 +/ Irs2 / mice ws reduced more thn 90% (Fig. 4,h,i). Anlysis of Irs1 +/ Irs2 / mice t E18.5 (when islet morphology is initilly estlished) reveled reduced β-cell numer, confirming tht deletion of Irs-2 impired β-cell neogenesis nd prolifertion during development (wild type, cells versus Irs1 +/ Irs2 /, cells, n=2). These findings re consistent with the reltive hypoinsulinemi seen in these nimls nd explin provisionlly their rpid development of dietes. Irs1 / Irs2 +/ mice displyed insulin resistnce nd somtic growth retrdtion, ut reltively norml islet morphology (Fig. 4g). Compred with wild-type mice, the verge β-cell re in Irs1 / Irs2 +/ mice ws incresed 1.8-fold t 4 weeks nd 4 months of ge (Fig. 4,g,i,j). These findings suggest tht Irs-2 dependent signlling pthwys, ut not Irs-1 pthwys, sustin norml islet development nd pproprite compenstory responses during peripherl insulin resistnce. Exmintion of islets from Irs1 +/+ Irs2 +/ mice, which disply little peripherl insulin resistnce, reveled 25% nd 36% reduction in β-cell re t 4 weeks nd 4 months of ge, respectively (Fig. 4c,i,h). By contrst, β-cell re in Irs1 +/ Irs2 +/ mice, which disply peripherl insulin resistnce, ws norml t 4 weeks of ge nd elevted 1.7-fold t 4 months of ge, reching levels similr to those oserved in Irs1 / Irs2 +/+ mice (Fig.,4f,i,j). Thus, expression of Irs-2 my e essentil for β-cell expnsion during the compenstory response to peripherl insulin resistnce. β-cell development in Igf1r / mice To further chrcterize the signls tht ctivte Irs-2 pthwys in β-cell function, we nlysed islet morphology nd glucose homeostsis in Igf1r / mice or mice heterozygous for Igf1r nd lcking Irs2. As previously reported, deletion of the Igf-1 receptor cuses neontl deth within minutes of irth, proly due to respirtory filure, which precludes detiled metolic nlysis 7. Close monitoring of severl litters t irth reveled elevted lood glucose levels in Igf1r / pups (Igf1r /, 262±12 mg/ml versus wild type, 62.8±2.8 mg/dl, n=4). e g f In the developing mouse pncres, endocrine-positive cells occur t E9.5, form interstitil clusters djcent to the ductl epitheli t dy 14.5 nd proliferte nd reorgnize to form mture islets during the remining 4 dys of gesttion 17. Hemtoxylin nd eosin stining of pncretic sections of Igf1r / mice reveled tht morphologicl development of the exocrine tissue nd orgniztion into cini ws norml t irth (Fig. 5 c). In ddition, exocrine pncres of Igf1r / mice stined positively for mylse (dt not shown), confirming functionl development of this tissue. Compred with wildtype emryos, immunohistochemicl nlysis of pncres sections from Igf1r +/ or Igf1r / mice reveled 50% or 85% reduction of oth insulin- nd glucgon-positive cells, respectively, t E16, E18.5 nd P0.5 (Fig. 5d g). Moreover, -cells nd β-cells of Igf1r / pncres filed to orgnize into sphericl structures chrcteristic of mturing islets (Fig. 5d g). Even t irth, islets in these nimls were detected s strings of disorgnized cells, lcking typicl islet morphology. Thus, like Irs-2, Igf1r hs criticl role in the development of norml, functionl islets. Phenotypes in progeny of the Igf1r +/ Irs2 +/ intercross To further chrcterize the effect of the Igf-1 receptor on glucose homeostsis, we intercrossed Igf1r +/ Irs2 +/ mice. At nture genetics volume 23 septemer 1999

6 rticle g c d e f dys of ge, Igf1r +/, Igf1r +/ Irs2 +/ nd Igf1r +/ Irs2 / mice hd nerly norml weights compred with wild-type nimls (Fig. 6). At 2 weeks, Igf1r +/ Irs2 / mice hd fsting lood glucose in excess of mg/dl; t 4 weeks, lood glucose levels exceeded 500 mg/dl (Fig. 6). Similr to Irs1 +/ Irs2 / nimls, these mice developed polyuri, polydypsi, weight loss nd rrely survived eyond five weeks of ge. At this ge, fsting lood glucose ws norml in Igf1r +/ nd Igf1r +/ Irs2 +/ mice (Fig. 6). Igf1r +/ Irs2 / mice were glucose intolernt, wheres Igf1r +/ Irs2 +/ mice hd mild impirment of glucose disposl; Igf1r +/ mice did not differ from wild-type nimls (Fig. 6c). Igf1r +/ Irs2 / mice were hypoinsulinemic fter fsting compred with wild-type nimls, wheres Igf1r +/ Irs2 +/ mice hd mild hyperinsulinemi nd Igf1r +/ mice were slightly hypoinsulinemic (Fig. 6d). Exmintion of these prmeters t four months of ge reveled tht Igf1r +/ mice hd norml fsting lood glucose levels, wheres glucose tolernce nd fsting hyperinsulinemi developed in Igf1r +/ Irs2 +/ mice (Fig. 6e g). Igf1r +/ Irs2 / mice displyed reduction in β-cell re, with insulin-positive cells representing less thn 2% of those seen in wild-type nimls (Fig. 7,d). This reduction in β-cells ws more pronounced thn the 50 60% reduction oserved in Irs2 / mouse islets (Fig. 7e,f). Glucgon-positive cells in Igf1r +/ Irs2 / mice were reduced y only 50%, demonstrting tht defective Igf1r Irs-2 signlling hs more severe consequences for β-cell development nd mintennce (Fig. 7f). Additionlly, nlysis of Igf1r +/ nd Igf1r +/ Irs2 +/ mice reveled compromised β-cell mss, with 30 50% reduction in insulin-positive cell re in these nimls (Fig. 7,c,f). Thus, these oservtions suggest tht Igf1r Irs-2 signlling pthwys re criticl for β-cell prolifertion nd function. Fig. 7 Islet morphology in progeny of Igf1r +/ Irs2 +/ intercross. e, Pncres sections from mice of the indicted genotypes were co-immunostined for insulin nd glucgon. Insulin stining ws detected y fluorescein-lelled nti-mouse ntiodies (green) nd glucgon-positive cells were visulized y rhodmine-lelled secondry ntiodies (red). Sections (5 µm) were photogrphed individully t 20 mgnifiction nd superimposed. f, Quntittion of - nd β-cell re in four-week-old mice. Results re men vlues±s.e.m. for t lest four nimls of ech genotype. Grey rs, -cell re; lck rs, β- cell re. g, Anlysis of poptosis in four-week-old mice. Fluorescent DNA frgmenttion ssys nd immunostining for BAD nd glucgon were performed on pncres sections from mice of the indicted genotypes. DNA frgmenttion ws detected y Cy3-lelling (yellow), BAD stining ws detected y fluorescein-lelled nti-rit ntiodies (green) nd glucgon-positive cells were visulized y rhodmine-lelled secondry ntiodies (red). Sections (5 µm) were photogrphed individully t 20 mgnifiction nd superimposed. Role of Igf1r Irs-2 signlling pthwy in β-cell survivl Insulin-like growth fctors prevent induced poptosis in vriety of cell types 18. In ddition, IRS-dependent pthwys hve een shown to medite the nti-poptotic effects of IGF-1 (ref. 19). As the determinnts of β-cell mss re thought to involve comintion of new islet formtion nd prolifertion of pre-existing islets lnced y developmentlly regulted β- cell poptosis 20,21, we exmined islets from the offspring of Igf1r +/ Irs2 +/ mice for the presence of poptosis nd expression of the pro-poptotic protein BAD (ref. 22). Incresed numers of poptotic cells were present in the islets of Irs2 / nd Igf1r +/ Irs2 / mice compred with wild-type nimls (Fig. 7g). There ws lso incresed expression of BAD in the islets of these nimls (Fig. 7g). Apoptotic nuclei nd BAD-positive cells coloclized inside the outer ring glucgon-positive cells (Fig. 7g). These findings suggest tht incresed poptosis might underlie the β-cell filure in these mice. Discussion Irs-1 nd Irs-2 medite the effects of insulin nd Igf-1 on emryonic development, post-ntl somtic growth nd glucose homeostsis. No emryos (16.5 dys or older) null for oth genes hve een detected in our studies, suggesting tht Irs-1 nd Irs-2 re criticl for emryonic development. Irs-1 hs predominnt role in somtic growth, s deletion of Irs1 reduces emryonic nd neontl growth y 40%, wheres deletion of Irs2 reduces growth y 10%. Irs1 +/ Irs2 / mice re pproximtely 60% the size of wild-type nimls, wheres Irs1 / Irs2 +/ mice re only 30% the size of controls, implicting Irs-1 s the principl element y which Igf-1 medites somtic growth. Moreover, our studies indicte tht Irs-2 my prticipte in somtic growth, ut cnnot fully replce Irs-1. These findings nture genetics volume 23 septemer

7 Fig. 8 A model depicting the centrl role of Irs-2 signlling pthwys in the mintennce of norml glucose homeostsis. Igf1r couples to Irs-2 in the pncretic islet to medite β-cell development, prolifertion nd survivl. Irs-2 medited pthwys re lso required for β-cell compenstion in response to peripherl insulin resistnce. In insulin trget tissues, oth Irs-1 nd Irs-2 prticipte in mediting insulin ction on cellulr function. Our results suggest tht ltertions in the lnce of these proteins in peripherl tissues my led to insulin resistnce. re consistent with in vitro oservtions tht Irs-2 fils to fully reconstitute the mitogenic effects of Igf-1 in Irs1 / firolsts or 32D cells 23. Although Irs-1 hs predominnt role in somtic growth nd deletion of either Irs-1 or Irs-2 cuses peripherl insulin resistnce, our dt suggest tht Irs-2 dependent mechnisms my e more criticl in glucose homeostsis. Indeed, insulin my regulte crohydrte metolism in the liver vi Irs-2 rther thn Irs-1 (ref. 24). Additionlly, Irs-2, downstrem of the Igf-1 receptor, is criticl for the development nd mintennce of pproprite β-cell mss. Thus, Irs-2 signlling pthwys hve evolved s common element controlling insulin production nd glucose homeostsis. This hypothesis is highlighted y the finding tht Irs1 / Irs2 +/ mice develop mild glucose intolernce due to compensted peripherl insulin resistnce, wheres Irs1 +/ Irs2 / nd Igf1r +/ Irs2 / mice die from dietes y six weeks of ge due to β-cell insufficiency. The distinct physiologicl functions of Irs-1 nd Irs-2 might e explined prtilly y tissue-specific differences in expression of these proteins; however, oth Irs-1 nd Irs-2 re expressed in most tissues, including liver, muscle nd oth rodent islets nd mouse β-cells 12, Thus, it is unlikely tht the solute expression of either of these proteins in given tissue provides complete explntion for their physiologicl differences. By contrst, the presence of the KRLB domin in Irs-2, which inds the regultory loop of the insulin receptor, might direct iologicl specificity 28,29. It is possile tht direct cross-tlk etween Irs-1 nd Irs-2 my e required to ppropritely regulte pthwys of insulin nd Igf-1 ction. Our oservtions suggest complex interply etween Irs-1 nd Irs-2 in the regultion of downstrem signlling in liver nd muscle; ltertions in the lnce of these signlling molecules my contriute significntly to pthogenesis of peripherl insulin resistnce. The extrcellulr lignds nd intrcellulr signlling pthwys involved in islet development, growth nd survivl re poorly chrcterized, ut these signls re ultimtely trnsmitted to hierrchy of trnscription fctors which regulte differentition nd mintennce of β-cells Our results suggest tht the Igf1r Irs-2 signlling pthwy my prtilly regulte these processes. Endocrine cells develop poorly in Igf1r / emryos nd fil to form typicl islets. By contrst, islets form in Irs2 / mice, ut β-cell mss is reduced t irth nd remins indequte to compenste for peripherl insulin resistnce. Moreover, the effect of Irs2 disruption is more profound in Igf1r +/ mice, suggesting n importnt role for the Igf1r Irs-2 signlling pthwy. Although Igf1r / emryos re reduced in size, the overll developmentl dely in ossifiction nd neuronl development is only out 1 2 dys, suggesting tht the defects in β-cell development re specific effect of the genetic lesion rther thn glol developmentl retrdtion 7,35. In ddition, incresed numers of poptotic cells nd incresed expression of the pro-poptotic protein BAD in the islets of Irs2 / nd Igf1r +/ Irs2 / mice suggest tht Igf1r Irs-2 pthwys might protect β-cells from poptosis. Thus, decresed survivl represents one mechnism underlying the β-cell defect in these nimls. The reltion etween β-cell function nd peripherl tissues in the development of dietes is well estlished 36. Our genetic models reinforce this concept nd emphsize β-cell dysfunction s criticl determinnt in the development of dietes. We hve developed mouse models of dietes in which the disese ensues s consequence of fundmentl defect in β-cell development: Irs2 /, Irs1 +/ Irs2 / nd Igf1r +/ Irs2 / mice re orn with insufficient β-cells to mintin proper glucose homeostsis. Moreover, sed on our nlysis of these mice t 4 6 weeks, they do not possess the mechnisms to generte new β-cells or to sustin survivl of existing β-cells. Therefore, when the β-cell defect comines with peripherl insulin resistnce, insulin-producing cells urn out within few weeks nd overt dietes ensues. Thus, these mouse models emphsize the fundmentl necessity of Igf1r Irs-2 signlling in the development nd mintennce β-cell function (Fig. 8). We hve lso generted models tht emphsize the interply etween peripherl insulin resistnce nd the β-cell response. Irs1 +/ Irs2 +/ nd Irs1 / Irs2 +/ mice re orn with reltively norml β-cell mss. Due to the reduced expression of Irs-1 nd Irs-2 in peripherl tissues, these len nimls re insulin-resistnt nd ecome mildly glucose intolernt with ge; however, β-cell compenstion is roust nd dietes does not develop. Therefore, these findings demonstrte tht prtil defects in oth the β-cell nd peripherl tissues underlie ge-dependent development of dietes. Thus, Irs1 +/ Irs2 +/ nd Irs1 / Irs2 +/ mice represent polygenic models tht my resemle non- 38 nture genetics volume 23 septemer 1999

8 rticle insulin dependent dietes mellitus (NIDDM), in which the comintion of two mild defects in insulin signlling produces insulin resistnce nd progression to dietes. Our results demonstrte tht the Irs protein signlling system, nd in prticulr Irs-2, hs role in integrting crohydrte metolism in peripherl tissues with β-cell function. The chrcteristics of the mouse models developed from our studies my enle identifiction of the moleculr defects tht determine dietes in humns, prticulrly t the level of the β-cell itself. These models suggest tht lesions in the Irs-2 signlling pthwy re mjor fctor in oth β-cell dysfunction nd peripherl insulin resistnce. To dte, genetic nlysis of dietic popultions hs found no significnt polymorphisms in Irs2 (ref. 12); however, our oservtions suggest tht other mechnisms such s reduced expression of Irs-2 or the IGF-1 receptor, prticulrly in the β-cell, my predispose individuls to dietes. Our models provide unique tools to identify nd chrcterize β-cell growth nd survivl pthwys nd present opportunities to link signlling pthwys to the regultion of trnscription fctors tht promote β-cell development nd function. Methods Genertion of Irs1 +/ Irs2 +/ mice. We generted Irs1 +/ nd Irs2 +/ mice using descried gene trgeting strtegies 13. Both mouse lines were mintined on mixed C57Bl/6 129Sv genetic ckground to fcilitte comprtive nlysis. To otin mice tht re compound heterozygotes for null lleles of Irs1 nd Irs2, we intercrossed Irs1 +/ with Irs2 +/ nimls. Irs1 +/ Irs2 +/ mice were vile nd otined with the expected mendelin frequency (12.5%). Irs1 +/ Irs2 +/ mice were fertile nd intercrossed to otin progeny of ll comintions of deletion of Irs1 nd Irs2. We genotyped nimls t 1 2 weeks y Southern lot on genomic DNA otined from til smples s descried 13. Genertion of Igf1r / nd Igf1r +/ Irs2 +/ mice. The genertion nd genotyping of Igf1r / mice hs een descried 7. To otin Igf1r +/ Irs2 +/ mice, we intercrossed Irs2 +/ nd Igf1r +/ mice. Igf1r +/ Irs2 +/ mice were vile nd otined with expected mendelin frequency (12.5%). Igf1r +/ Irs2 +/ mice were fertile nd intercrossed to otin progeny of ll comintions of deletion of Igf1r nd Irs2. Genotyping of E emryos nd 2-weekold nimls ws performed y Southern lot s descried 7. Metolic studies. Animls were mintined on norml light/drk cycle nd hndled in ccordnce with Joslin Dietes Center Animl Cre nd Use Committee protocols. We determined glucose levels of lood from mouse tils using Glucometer Elite glucometer (Byer). We otined lood for plsm insulin levels y retrooritl leeds on nesthetized mice or y til leeds. Immunorective insulin levels were mesured either y rdioimmunssy using rt insulin (Linco) s stndrd or using n ELISA (CrystlChem) with mouse insulin s stndrd. We performed glucose tolernce tests on nimls fter 15-h overnight fst s descried 13. Immunoprecipittion, western-lot nlysis nd PI-3K ssys. Liver nd muscle tissue lystes were removed nd homogenized t 4 o C s descried 13. The homogentes were llowed to soluilize for 1 h t 4 o C nd clrified y centrifugtion t 15,000 r.p.m. for 30 min. Superntnts contining totl protein (2 mg) were immunoprecipitted with either n nti-irs-2 ntiody (residues of mouse Irs-2), nti-irs-1 ntiody (residues of mouse Irs-1) or nti-ir-β ntiody. Blots were proed with polyclonl nti-irs-1, nti-irs-2, monoclonl nti-phosphotyrosine (clone 4G10) ntiodies nd detected y either 125 I-protein A (ICN Biochemicls) or enhnced chemiluminescence. For PI-3 kinse enzymtic ssys, we injected humn insulin (5 units) s olus into the inferior ven cv of nesthetized mice, nd removed liver, gstrocnemius nd qudriceps muscles t 1, 2.5 nd 3 min fter insulin injection. They were homogenized, soluilized nd superntnts contining totl protein (2 mg) immunoprecipitted for 2 h with nti-irs-2 or nti-irs-1 ntiody s ove. Immune complexes were collected, wshed nd the PI-3 kinse rection performed s descried 13. We quntified 32 P incorportion using Phosphorimger (Moleculr Dynmics). Immunohistochemistry of pncres, quntittion of β-cells nd detection of poptosis. For nlysis of dult pncreses, nimls were killed y overdose of sodium mytl. Ech pncres ws removed, clered of ft nd spleen, weighed nd fixed overnight in Bouin s solution. We emedded tissues in prffin nd mounted consecutive sections (5 µm) on slides. Following re-hydrtion nd permeiliztion (0.1% Triton X- 100), sections were immunostined for -cells using mouse monoclonl nti-glucgon ntiodies (Sigm). β-cells were immunostined using either guine pig nti-insulin (Linco) or mouse nti-insulin ntiodies (Sigm). Detection ws performed using rhodmine nd fluorescein ntiodies (Jckson Immunoreserch). We incuted sections riefly in DAPI (0.01%) to revel totl cell nuclei. BAD stining ws performed with rit polyclonl ntiody (Snt Cruz) with detection s ove. We identified poptotic cells in de-prffinized pncretic sections using fluorescent DNA frgmenttion detection ssy (Oncogene Reserch) nd performed co-locliztion with glucgon nd BAD stining s ove. For nlysis of emryonic pncres, we killed pregnnt mice y dministrtion of n overdose of sodium mytl on dy E16.5 or E18.5. Fetuses were plced in phosphte-uffered sline (PBS) nd the dorsl nd ventrl pncretic uds crefully dissected nd fixed in Bouin s for 3 4 h. Tissue specimens were wshed in PBS nd trnsferred to 10% uffered formlin. Pncres from ech neonte ws lso collected nd fixed y the sme procedure. We immunostined prffin-emedded sections s descried ove. To ssess the morphology nd mss of the exocrine pncres in these emryos, we lso stined these sections with rit nti-mylse ntiody (Sigm). For quntittion of β-cell re, sections were viewed using Zeiss Axiovert S100 TV microscope nd video cmer t mgnifiction of 20. Two sections of ech pncres were covered systemticlly y ccumulting imges from 8 non-overlpping fields of µm 2. Anlyses of β-cell re nd size were performed using Openl imge nlysis softwre (Improvision Imging). This softwre clirted the mgnifiction of ech microgrph. The percentge of cells positive for insulin or glucgon ws clculted nd corrected for pncretic weight. For nlysis of β-cells in emryonic or neontl pncres sections, cell counting rther thn re mesurement ws performed y the Openl softwre. Acknowledgements We thnk A. Efstrtidis for Igf1r +/ mice; B. Chethm for nti-irβ ntiodies; nd J. Mrron for ssistnce in preprtion of this mnuscript. This work ws supported y DK D.J.W. is n MRC (UK) Clinicin Scientist nd D.J.B. ws supported y grnt from the JDFI during portion of these studies. Received 8 April; ccepted 9 July nture genetics volume 23 septemer

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