Differences in metabolic and mitogenic signalling of insulin glargine and insulin aspart B10 in rats

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1 Dietologi (13) 5:1 13 DOI 1.17/s z ARTICLE Differenes in metoli nd mitogeni signlling of insulin glrgine nd insulin sprt B1 in rts N. Tenngels & S. Welte & M. Hofmnn & P. Brenk & R. Shmidt & U. Werner Reeived: Deemer 1 /Aepted: 5 April 13 /Pulished online: My 13 # Springer-Verlg Berlin Heidelerg 13 Astrt Aims/hypothesis In vitro, insulin glrgine (A1Gly,B31Arg, B3Arg humn insulin) hs n insulin reeptor () profile similr to tht of humn insulin, ut slightly higher ffinity for the IGF-1 reeptor (). Insulin sprt B1 (B1Asp humn insulin) (AspB1), the only insulin nlogue with proven rinogeni tivity, hs greter ffinity for nd, nd prolonged oupny time. The phrmologil nd signlling profile of therpeuti nd suprphrmologil doses of glrgine were nlysed in different tissues of rts, nd ompred with humn insulin nd AspB1. Methods Mle Wistr rts were injeted with humn insulin or insulin nlogue t doses of 1 to U/kg, nd the effets on lood gluose nd the phosphoryltion sttus of,, Akt nd extrellulr signl-regulted protein kinse 1/ in musle, ft, liver nd hert smples were investigted. Results Glrgine, AspB1 nd humn insulin lowered lood gluose, with the onset of tion delyed with glrgine. Glrgine tretment resulted in phosphoryltion levels of nd Akt tht were omprle with those hieved with humn insulin, lthough delyed in time in some tissues. AspB1 tretment resulted in t lest twofold higher phosphoryltion levels nd signifintly longer durtion of nd Akt phosphoryltion in most tissues. None of the insulin tretments resulted in detetle phosphoryltion in Eletroni supplementry mteril The online version of this rtile (doi:1.17/s z) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. N. Tenngels () : S. Welte : M. Hofmnn : P. Brenk : R. Shmidt : U. Werner Snofi-Aventis Deutshlnd GmH, Building H1, Room 31, Industrieprk Hoehst, 59 Frnkfurt m Min, Germny e-mil: norert.tenngels@snofi.om musle or hert tissue, wheres intrvenous injetion of IGF-1 inresed phosphoryltion. Conlusions/interprettion The signlling pttern of AspB1 in vivo is distintly different from tht of humn insulin nd insulin glrgine, nd might hllenge the notion tht tivtion of plys role in the oserved rinogeni effet of AspB1. Keywords Akt. Asp [B1] insulin. Humn insulin. IGF-1 reeptor. Insulin glrgine. Insulin reeptor. Reeptor phosphoryltion Arevitions AspB1 Insulin sprt B1 (B1Asp humn insulin) ERK Extrellulr signl-regulted protein kinse IGF-1 reeptor Insulin reeptor Introdution Insulin nlogues in the tretment of ptients with type 1 or type dietes hve een shown to e more effiient, reproduile nd onvenient thn regulr insulin [1]. Due to either sequene or seondry struturl modifitions, nlogues my differ from insulin with respet to metoli poteny, stility, or onset nd durtion of tion. Although these hnges were introdued to lter the time tion profile of the respetive insulin nlogues, they my lso led to n ltered tivtion profile of the insulin reeptor () nd (or) IGF-1 reeptor () signlling pthwys, nd my hnge metoli or mitogeni responses []. A reful investigtion of ute nd long-term effets of insulin nlogues hs een mjor reserh fous. The insulin nlogue insulin sprt B1 (B1Asp humn insulin) (AspB1) ws withdrwn from linil development

2 Dietologi (13) 5: due to higher inidene of rest ner in rts [3]. AspB1 differs from humn insulin y the sustitution of histidine y sprtte in position 1 of the B hin. In vitro, AspB1 displys higher ffinity towrds the nd, prolonged oupny time t the nd higher prolifertion rte in mmmlin ell lines. Although the mehnism y whih AspB1 exerts its mitogeni effet is not ler, it is still ontended tht the nlogue s greter ffinity to the might e t lest in prt responsile [ 7]. This hs led to the generl elief tht insulin nlogues with inresed ffinity in vitro might per se exert n inresed growthpromoting tivity in vivo. However, in vitro studies nnot e diretly pplied to the in vivo sitution. First, the ffinity of the endogenous lignd IGF-1 for is t lest 1-fold greter thn tht of insulin or insulin nlogues. Thus, IGF-1 ompetes with insulin for ouption. Seond, in vitro studies use suprphysiologil (nnomolr) onentrtions vs piomolr onentrtions in vivo. Third, the plsm nd tissue onentrtion nd distriution of insulin nlogues re different in vivo thn in vitro. Fourth, insulin nlogues undergo iotrnsformtion in vivo, neessitting nlysis of the tive metolites. Insulin glrgine (A1Gly,B31Arg,B3Arg humn insulin) is long-ting humn insulin nlogue with n tivity profile very losely mimiking the nturl physiologil profile of sl endogenous insulin seretion. Glrgine differs from humn insulin y sustitution of sprgine y glyine in position 1 of the A hin nd y roxyterminl extension of the B hin y two rginine residues. These hnges use shift in the isoeletri point from ph 5. to.7. Following dministrtion s ler solution of ph, insulin glrgine preipittes t the injetion site euse of its low soluility t physiologil ph levels. The prolonged lood gluose-lowering tivity of insulin glrgine my result from the susequent slow dissolution of the miropreipitte on the sis of low dissoition rte. The dissolution proess is followed y rpid proteolyti degrdtion of prent glrgine, leding to solule metolites s demonstrted in metolism studies in humns, rts nd dogs [ 1]. The two min metolites of insulin glrgine, M1 ([GlyA1]insulin) nd M ([GlyA1,des- ThrB3]insulin) re formed y the sequentil removl of the two rginines from the roxy-terminus of the B hin nd dditionl demintion of threonine in position B3. In plsm, the prinipl irulting ompound is the metolite M1, the exposure of whih inreses s the dose of dministered insulin glrgine inreses [11]. Insulin glrgine hs n in vitro signlling nd metoli profile omprle to tht of humn insulin, ut displys slightly greter ffinity in vitro [, 5, 7]. However, in yer rinogeniity studies, no differene ws oserved in the inidene of mmmry tumours in mie nd rts ompred with ontrols or nimls treted with NPH insulin [1], finding tht n e ttriuted to the phrmodynmi effet of M1, whih hs in vitro metoli nd mitogeni profiles omprle with humn insulin [7]. The im of this study ws to nlyse the time tion profile of glrgine in responsive tissues of rts with respet to phrmologil nd signlling vriles nd to ompre tht profile to those of humn insulin nd AspB1, using therpeuti s well s suprphrmologil doses. We lso investigted the effet of humn insulin, glrgine nd AspB1 on the phosphoryltion of nd ompred it with the effet of IGF-1. Methods Animls Mle Wistr rts (HsdCp:WU) were otined from Chrles River, Sulzfeld, Germny. The nimls were housed in Mrolon ges (1, m ; Ehret, Emmendingen, Germny) on virtully dust-free wood grnulte edding, enrihed with nestling mteril, how stik nd hide tues (n =3 per ge). Animl housing onditions were stndrdised (± C, 55% ±1% reltive humidity, light yle from : to 1: hours) nd stndrd rodent pellet diet (R/M-H 153; ssniff Spezildiäten, Soest, Germny) ws given until study strt. Studies were performed with rts t to 1 weeks of ge, fter limtistion for t lest 1 week. Free ess to tp wter ws mintined t ll times. The nimls were rndomised to five to six rts per group nd deprived of food h efore the strt of n experiment. Injetions All injetion solutions were freshly prepred. To hieve finl doses, regulr humn insulin nd AspB1 were dissolved in.9% (wt/vol.) sline, while glrgine ws dissolved in solution mthing the glrgine pleo t ph. All test drugs nd pleo were dministered s single suutneous injetion. Study 1 In the first study, rts (n=5 ) were injeted with 1 U/kg ( nmol/kg) of humn insulin, glrgine, AspB1 or.9% sline (ontrol group). Blood smples for gluose nd insulin nlyses were tken t time point nd t vrious time points up to 1 min fter the injetion. Blood gluose ws determined enzymtilly from 5 μl of til tip whole lood hemolysed with 5 μl hemolyste (hemolysis regent H, Gluose Hexokinse Fluid 5+1; Hengler Anlytik, Steinh, Germny). Quntifition

3 1 Dietologi (13) 5:1 13 ws with Gluo-qunt Gluose/HK kit (Rohe Dignostis, Penzerg, Germny) using Bekmn Coulter AU (Bekmn Coulter, Krefeld, Germny) or Rohe/Hithi 91 Chemistry Anlyzer (Rohe Dignostis, Mnnheim, Germny). Serum insulin ws determined y humn insulin immunossy (ELISA ; Merodi, Uppsl, Sweden) unless otherwise stted. The mount of insulin glrgine, M1 nd M in plsm ws determined y immunoffinity extrtion followed y liquid hromtogrphy tndem mss spetrometry s desried y Bolli et l [11]. Smples of skeletl (lf) musle, liver, dominl dipose tissue nd hert were removed t the sme time points for nlysis of, Akt (lso known s protein kinse B) nd extrellulr signl-regulted protein kinse (ERK)1/ phosphoryltion. Study In seond study, rts (n=5) were injeted with 1.5, 5 or U/kg humn insulin, glrgine or AspB1 to determine whether these high doses led to n inrese in phosphoryltion. Smples of lf musle, liver, dominl dipose tissue nd hert were removed fter min. As ontrol intended to demonstrte n IGF-1 effet on downstrem signlling, rts were injeted with nmol/kg or with or 13 nmol/kg des[1-3] IGF-1, nd lf musle nd hert smples were tken min fter the injetion or 5 min fter the injetion. All tissue smples were susequently nlysed for, nd Akt phosphoryltion. Des[1-3]IGF-1 is trunted vrint, whih is more potent thn humn IGF-1 due to redued inding to IGF-inding proteins [13]. Study 3 In third study, the effets on nd phosphoryltion in mmmry tissue were exmined in 7- week-old femle Sprgue Dwley rts (Chrles River). The rts (n=3 ) were injeted with 1.5 U/kg humn insulin, glrgine, AspB1 or sline, nd mmmry tissue ws removed t time nd min. Other rts (n=3 ) were injeted intrvenously with 1 mg/kg des[1-3]igf, with skeletl musle nd hert smples eing removed fter 5 min. Anlyses The phosphoryltion of reeptor nd signlling moleules ws ssessed y western lot nlysis s desried y Bus et l [1]. After immunopreipittion using ntiodies direted ginst the et-suunit of the or (Snt Cruz Biotehnology, Snt Cruz, CA, USA), proteins were seprted on SDS-PAGE gels ( 1% (wt/vol.) resolving gel; Invitrogen, Crlsd, CA, USA), trnsferred to polyvinylidene difluoride memrnes (Rohe Applied Siene, Germny) nd loked (Roti-Blok; Crl Roth, Germny) for 1 h. Memrnes were inuted overnight t C with primry ntiody direted ginst phosphotyrosine (Millipore, Germny), or. Memrnes were wshed in TRIS-uffered sline +.1% (vol./vol.) Tween nd inuted with the pproprite seondry horserdish peroxidse-onjugted ntiody (Snt Cruz). Immunoretive nds were visulised with LumiLight (Rohe) nd deteted with hemiluminesene detetion system (Lumi-Imger; Boehringer, Mnnheim, Germny). Phospho-Akt ws determined using phospho-akt ELISA kit (Life Tehnologies, Grnd Islnd, NY, USA). Study pprovl nd sttistil nlysis The niml studies were pproved y the lol Ethis Committee nd were onduted in ordne with the Priniples of Lortory Cre. All dt re presented s mens±sem. Sttistil nlysis ws y one-wy ANOVA followed y Dunnett s test, nd ws performed with sttistis nlysis softwre (Prism; Grph Pd Softwre, Sn Diego, CA USA). Results Effets on lood gluose nd plsm insulin After injetion of 1 U/kg humn insulin or AspB1, lood gluose levels strted to deline immeditely, rehing minimum vlues of.3±.1 mmol/l nd 3.±. mmol/l fter 3 nd min, respetively. Strting seline vlues were similr t 5.9 nd.1 mmol/l (Fig. 1). The gluoselowering tion of 1 U/kg glrgine ws initilly delyed y 3 min ut gluose levels rehed similr level of.1 ±. mmol/l fter 9 min. However, the AUC ws smller with glrgine thn with humn insulin or AspB1, inditing prolonged gluose-lowering tion of this insulin nlogue. With higher doses, lood gluose ws lowered to the sme extent y humn insulin nd insulin nlogues, rehing to 1% of seline without signifint hypoglyemi (dt not shown). As in humns, glrgine ws effetively nd rpidly metolised in rts. At 1 h fter the injetion of 1 U/kg glrgine, 9% of totl insulin ws identified s the M1 metolite (1,7 pmol/l), wheres the prent ompound ws rely detetle nd the M metolite ws elow the level of quntifition (Fig. 1). The M1 metolite lso ounted for 91% (5,1 pmol/l) nd 7% (, 99 pmol/l) of glrgine in plsm fter injetion of 1.5 nd U/kg glrgine, where the totl insulin glrgine onentrtion inluding metolites ws 5, nd 3,1 pmol/l, respetively (eletroni supplementry mteril [ESM] Fig. 1). However, even with suprphrmologil dose of U/kg, the reltive proportion of glrgine prent ompound (1%, 5, pmol/l) nd M (%, 1, pmol/l) remined low.

4 Dietologi (13) 5: Blood gluose (mmol/l) Plsm or serum insulin (pmol/l) 1 3,,5, 1,5 1, Fig. 1 () Time ourse of lood gluose following injetion of 1 U/kg humn insulin (tringles), glrgine (irles) or AspB1 (squres) in - to 1-week-old mle Wistr rts. () Time ourse of plsm glrgine (white irles), metolite M1 (dimonds) nd totl serum immunoretive insulin (rosses) onentrtions fter injetion of 1 U/kg glrgine in rts s ove (). Metolite M ws elow the lower limit of quntifition. Vlues re men±sem (n=); p<.5 vs humn insulin AspB1 Glrgine AspB1 Glrgine AspB1 Glrgine phosphoryltion in musle phosphoryltion in liver phosphoryltion in ft Fig. () Time ourse of phosphoryltion in musle, () liver nd () ft following injetion of 1 U/kg humn insulin (, tringles), glrgine (irles) or AspB1 (squres) in - to 1-week-old mle Wistr rts. Vlues re men±sem (n=5); p<.5, p<.1 nd p<.1 vs humn insulin., phosphotyrosine Phosphoryltion of the nd signlling moleules The time ourse of phosphoryltion in skeletl musle, liver nd ft tissue ws exmined following injetion of 1 U/kg humn insulin, glrgine or AspB1. In musle, the time to pek phosphoryltion with humn insulin nd AspB1 ws rehed fter 15 min, wheres with glrgine it ws delyed until 3 min (Fig. ). AspB1 tretment resulted in t lest twofold higher pek phosphoryltion levels nd signifintly longer durtion of phosphoryltion thn tretment with humn insulin. Pek phosphoryltion with glrgine ws lower thn with AspB1, ut greter thn with humn insulin; the durtion ws lso longer thn with humn insulin. In liver, the time to pek phosphoryltion ws the sme for ll three insulins (Fig. ). Only AspB1 showed greter phosphoryltion nd longer durtion thn humn insulin. In ft tissue, the time to pek phosphoryltion ws lter nd pek phosphoryltion ws greter with humn insulin thn with glrgine or AspB1, while the durtion of phosphoryltion ws longer with glrgine (Fig. ). Suutneous injetion of 1 U/kg glrgine resulted in pek Akt phosphoryltion omprle with tht of 1 U/kg of humn insulin in skeletl musle, liver nd ft tissue (Fig. 3). Time to pek phosphoryltion ws delyed in musle nd ft tissue, ut not in liver. AspB1 indued signifintly greter pek phosphoryltion in musle nd liver, nd lso prolonged the durtion of Akt phosphoryltion in skeletl musle. None of the insulins signifintly inresed ERK1/ phosphoryltion in ny tissue exmined (dt not shown). The effet of inresing suprphrmologil doses of humn insulin, glrgine nd AspB1 on nd Akt phosphoryltion in musle ws exmined min fter injetion, t time when similr gluose-lowering effets were oserved with eh insulin. Eh insulin inresed nd Akt phosphoryltion, with no signifint differene etween them detetle (Fig. ). Comprle results were oserved in hert (dt not shown). Phosphoryltion of It ws of interest to determine whether the injetion of high doses of humn insulin, AspB1 or glrgine led to n inrese in phosphoryltion in skeletl musle. As ontrol to demonstrte n IGF-1 effet on nd downstrem signlling, des[1-3] IGF-1 ws injeted nd the phosphoryltion of, nd Akt ws determined (Fig. 5). Suutneous injetion of

5 13 Dietologi (13) 5:1 13 Akt phosphoryltion in musle Akt phosphoryltion in liver Akt phosphoryltion in ft 15 1 nmol/kg hd no effet on ny phosphoryltion, wheres injetion of or 13 nmol/kg inresed nd Akt phosphoryltion in dose-dependent mnner, ut hd no effet on phosphoryltion. In ontrst, injetion of inresing doses of humn insulin, glrgine or AspB1 hd no effet on musle phosphoryltion fter min t ny dose (Fig. ). The slight inrese in the tyrosine phosphoryltion signl oserved t very high doses my hve een due to ontminting phospho- in the immunopreipitte. Similr results were oserved in hert (dt not shown). To show whether delivery of insulin nlogues resulted in phosphoryltion in musle, 1 U/kg of eh nlogue ws injeted, nd nd phosphoryltion ws investigted fter 5 min (ESM Fig. ). Wheres signifint phosphoryltion of the ws deteted, no phosphoryltion ws mesurle with ny insulin. Totl insulin levels fter injetion rehed 1,± 1,11 pmol/l. The mjority of the insulin present in plsm ws the M1 metolite (%, 9,35 pmol/l), followed y Fig. 3 () Time ourse of Akt phosphoryltion in skeletl musle, () liver nd () ft following injetion of 1 U/kg humn insulin (tringles), glrgine (irles) nd AspB1 (squres) in - to 1-weekold mle Wistr rts. Vlues re men±sem (n=5); p<.5, p<.1 nd p<.1 vs humn insulin phosphoryltion phosphoryltion phosphoryltion M (1%, 1,1 pmol/l) nd prent glrgine (%, 5 pmol/l), inditing rpid metolism in plsm. Phosphoryltion of nd in mmmry tissue phosphoryltion in mmmry tissue ws not signifintly inresed when 7-week-old femle rts were injeted with 1.5 U/kg humn insulin, glrgine or AspB1 (Fig. 7). The inresed phosphoryltion deteted fter AspB1 injetion minly resulted from the presene of phospho- in the immunopreipitte. Only AspB1 signifintly inresed phosphoryltion. Intrvenous injetion of 1 mg/kg des[1-3]igf-1 inresed phosphoryltion 1-fold ut hd no signifint effet on phosphoryltion. Disussion Glrgine AspB1 Glrgine AspB1 Glrgine AspB1 Given tht ptients with dietes often require life-long insulin tretment, it is essentil to exmine ll steps in the tion of n insulin nlogue in vitro nd in vivo, to exlude unwnted effets like growth-promoting tivities [15]. Mlignnt ell growth is often ssoited with errnt signlling of oth isoforms (-A nd -B) nd. The insulin nd IGF reeptors trigger omplex d Akt phosphoryltion e Akt phosphoryltion f Akt phosphoryltion Glrgine AspB1 Glrgine AspB1 Glrgine AspB1 Fig. ( ) Musle nd (d f) Akt phosphoryltion min fter injetion of (, d) 1.5, (, e) 5or(, f) U/kg humn insulin (), glrgine or AspB1 in - to 1-week-old mle Wistr rts. Vlues re men±sem (n=5); p<.5, p<.1 nd p<.1 vs ontrol

6 Dietologi (13) 5: IP: IP: phosphoryltion phosphoryltion d Akt phosphoryltion Vehile nmol/kg Vehile nmol/kg 13 nmol/kg Vehile rnge of intrellulr signls for metolism, ell growth nd prolifertion [1]. Their reltive undne ffets intrellulr signlling nd hs importnt onsequenes for tissuespeifi responses to insulin, IGFs nd insulin nlogues 13 nmol/kg nmol/kg nmol/kg 13 nmol/kg nmol/kg nmol/kg 13 nmol/kg nmol/kg nmol/kg Fig. 5 () Western lot nlysis of the phosphoryltion of, nd Akt t min fter the injetion of nmol/kg, or t 5 min fter the injetion of or 13 nmol/kg des[1-3]igf-1 in - to 1-weekold mle Wistr rts. Quntifition of (), () nd (d) Akt. Vlues re men±sem (n=3); p<.5 nd p<.1 vs ontrol. IP, immunopreipitte;, phosphotyrosine IP: 1 U/kg 1.5 U/kg 5 U/kg U/kg Vehile Insulin Glrgine AspB1 Fig. Western lots of musle phosphoryltion min fter injetion of insulins s indited in - to 1-week-old mle Wistr rts., phosphotyrosine IP: phosphoryltion phosphoryltion Sline Sline 1 mg/kg IGF U/kg Insulin 1.5 U/kg Glrgine 1.5 U/kg AspB1 IGF-1 Glrgine AspB1 Sline IGF-1 Glrgine AspB1 Fig. 7 () Western lot nlysis of the phosphoryltion of nd in mmmry tissue t min fter injetion of sline or 1.5 U/kg humn insulin (), glrgine or AspB1, or t 5 min fter injetion of 1 mg/kg des[1-3]igf-1 in 7-week-old femle Sprgue Dwley rts (n=). () Quntifition of nd () phosphoryltion. Vlues re men±sem (n=3); p<.1 vs ontrol. IP, immunopreipitte;, phosphotyrosine [17, 1]. In ddition, it hs een demonstrted tht overexpression of nd in humn rest rinoms llows insulin nd IGF-1 hyrid reeptors to form. These hyrid reeptors eome tyrosine utophosphorylted when rest ner ells re exposed to IGF-1, ut not to insulin, nd lso medite growth in response to IGF-1 [19 ]. As ner ells hve errnt nd signlling ptterns, it is importnt to understnd how insulin nlogues ffet norml nd nerous ells, s this hs implitions for dietes, ner nd ner tretment. It is generlly elieved tht insulin nlogues with higher ffinity thn humn insulin for in vitro hve greter mitogeni tivity in vivo. This elief is sed on AspB1, the only insulin nlogue with proven rinogeniity in rts [3]. Insulin glrgine is lso thought to hve greter mitogeni tivity in vivo, sed on its slightly higher ffinity for in vitro, ut unlike AspB1, glrgine does not hve greter ffinity for the or prolonged oupny time t the [, 5, 7]. It is, however, diffiult to predit results in vivo on the sis of in vitro dt [3],ndinvitrostudiesdonot onlusively support IGFR tivtion s the mehnism of inresed mitogeni tivity. The mitogeni tion of insulin glrgine is inresed in ertin ell lines with high IGFR: rtios [, 7, ], ut other ell lines with high IGFR: rtios do not respond to insulin glrgine tretment with

7 13 Dietologi (13) 5:1 13 inresed prolifertion [, 5, 7,, 5, 9 3]. Moreover, some ell lines respond to AspB1, ut not to other nlogues with inresed prolifertion [35], while ell lines tht do not hve greter ffinity for IGFR hve lso een reported to show inresed prolifertion upon exposure to other insulin nlogues urrently used in therpy [7,, 3]. One wy of exmining mitogeni tivity in vivo is to diretly determine tumour formtion fter hroni tretment with insulin nd (or) insulin nlogues. AspB1 ws withdrwn from linil development due to higher inidene of rest ner in rts fter 1 months [3]. In ontrst, insulin glrgine did not indue higher inidene of mmmry tumours in lifetime rinogeni studies in femle rts nd mie [37], onfirming tht this sl insulin nlogue is unlikely to pose ner risk in humns. Moreover, in mouse model prone to tumour formtion, Ngel et l [3] demonstrted tht tumour formtion did not inrese with insulin glrgine vs NPH insulin tretment fter 1 months. The lne of evidene indites tht no ommerilly ville insulin nlogue hs rinogeni effets in the humn linil setting [1]. The pproh presented here ws to exmine the time ourse of phosphoryltion of the nd, nd the effets on downstrem signlling moleules of insulin nd insulin nlogues in different tissues in rts. Previous studies hve een performed in mie [39, ]. The one rt study tht hs een reported to dte used suprphrmologil doses nd only investigted downstrem signlling [17]. Blood gluose levels dropped immeditely fter the injetion of humn insulin or AspB1, with no differene etween the two insulins. In ontrst, gluose lowering ws delyed with insulin glrgine, s expeted for longting insulin nlogue, where the mode of tion involves preipittion nd susequent slow relese from the tissue depot [1]. As in humns [11, ], the lowering of lood gluose ould e orrelted to the iotrnsformtion of insulin glrgine into the M1 metolite, whih lks the di-rginine residues []. Glrgine prent nd M were not detetle. Consequently M1, nd not glrgine itself, is the primry driver of the phrmodynmi effet nd the longting time tion profile oserved with insulin glrgine tretment [11, ]. The proteses responsile for this tivity pper to e independent of the speies investigted [3]. M1 is the mjor tive metolite even t dose of U/kg, suggesting tht the protese system involved hs high pity. However, t this high dose, glrgine n e found in the irultion, inditing tht sturtion of the system n our. Pek nd Akt phosphoryltion levels indued y insulin glrgine were generlly omprle with those hieved with humn insulin, lthough in some tissues the effets of insulin glrgine were delyed nd (or) prolonged in time. Similr differenes hve een desried y Agouni et l [39], possily refleting differenes in phrmokineti nd/or phrmodynmi properties ross tissues. The omprle pek phosphoryltion of insulin glrgine vs humn insulin reflets the omprle tivity of M1 vs humn insulin nd supports the onlusion tht insulin glrgine ehves like humn insulin in terms of signlling. In ontrst, nd Akt phosphoryltion ws inresed nd prolonged with AspB1, most strikingly in musle nd liver. These results re omptile with the greter ffinity of the for AspB1 nd with the prolonged signlling of the when exposed to AspB1 in vitro [, 7]. Interestingly, t higher doses (1.5 nd U/kg), the differenes in pek phosphoryltion of the nd Akt oserved etween AspB1, glrgine nd insulin were no longer detetle, pprently demonstrting the sturtion of pek phosphoryltion under these high-dose onditions. Hvid et l hve reported tht 1U/kgresultedinomprlepekphosphoryltionof Akt over 15 min [17, 1]. Consequently, therpeuti dose of 1 U/kg seems to reflet differenes in the ffinity of AspB1, insulin nd insulin glrgine to the in vitro [7]. The presene nd tivtion of the in musle, hert nd mmmry tissue ws demonstrted y intrvenous injetion of high dose of IGF-1 (13 nmol/kg), wheres nmol/kg IGF-1 injeted ws unle to generte detetle reeptor utophosphoryltion. A similr result ws reported in mouse hert musle, where the injetion of 13 nmol/kg IGF-1 resulted in strong phosphoryltion of the reeptor, wheres no signl ould e deteted fter injetion of therpeuti nmol/kg dose []. The tight ontrol of phosphoryltion ws lso demonstrted y Hvid et l [17], who reported tht injetion of suprphysiologil dose ( nmol/kg) of IGF-1 in rts inresed Akt phosphoryltion in liver, olon nd mmmry glnd of Sprgue Dwley rts. In greement with reltively poor response to IGF-1, Lee et l reported tht Akt nd ERK phosphoryltion ourred in mouse mmmry glnd tissue only fter lrge olus til vein injetion [5]. Although it hs een demonstrted tht the lrge olus n result in the mjority of the IGF-1 eing in irultion [], it should e noted tht prt of the dose ould e ound to IGF-inding proteins, whih would limit the free onentrtion nd explin redued response [5]. In ny se, we used des[1-3]igf-1, whih lks the N-terminl tripeptide Gly-Pro-Glu nd hs inresed poteny due to redued inding to most of the IGF-inding proteins [13]. Thus injetion of des[1-3]igf-1 should diretly llow hrteristion of the tissue response. Neither humn insulin nor insulin nlogues generted signifint signls in these tissues t doses up to U/kg. The sme holds true when the nlogues were dministered nd the phosphoryltion pttern ws studied fter 5 min. Importntly, nimls tht reeived n insulin glrgine injetion of U/kg showed free serum insulin

8 Dietologi (13) 5: M1 level of nmol/l, whih is 3-fold elow the reported ffinity of M1 to the [7]. In the sme study, the mesured onentrtions of prent glrgine nd M were 5 nmol/l nd nmol/l, respetively, with the totl insulin onentrtion rehing out 3 nmol/l. Assuming similr onentrtions were hieved with AspB1 t dose of U/kg, the lk of phosphoryltion would e remrkle, sine this onentrtion should e le to inrese IGF-1 phosphoryltion in vitro [7]. The tendeny for high dose of AspB1 to show n inrese in the tyrosine phosphoryltion signl of the immunopreipitte my e refletion of phosphorylted ontmintion due to opreipittion of y the ntiody used in these studies. It my lso reflet suunits preipitted s nd/or IGFR hyrids. Hyrid reeptors hve een deteted in numer of tissues inluding humn skeletl nd hert musle, nd dipose tissue [, 7]. Glrgine itself might hve higher ffinity for hyrid reeptors in vitro [, 7], ut M1 is like humn insulin nd thus no signl ws oserved in vivo. In summry, AspB1 tretment resulted in lood gluose profile omprle to tht of insulin. The phosphoryltion of signlling moleules ws inresed nd (or) prolonged in most tissues, whih resemles in vitro findings. The glyemi tion of insulin glrgine ws (slightly) retrded ompred with insulin. Insulin glrgine is rpidly nd effetively metolised to M1 under therpeuti nd high-dose onditions, nd the phosphoryltion of signlling moleules in tissues ws generlly omprle to tht of insulin, ut retrded in time in some tissues. phosphoryltion ould not e deteted in severl tissues upon exposure to insulin glrgine or AspB1, even t high dose nd different routes of dministrtion. We onlude tht in rts AspB1 hs different signlling profile to tht of insulin nd insulin glrgine, nd tht the slightly elevted tivity of AspB1 in vitro did not trnslte into phosphoryltion in vivo. Consequently, we hypothesise tht the greter mitogeni effet of AspB1 is most likely to e sed on its ltered profile in vivo. It is therefore tempting to speulte tht the greter mitogeni effets of insulin nd insulin nlogues re solely sed on the growth-promoting tivity of the itself, nd tht tivtion y insulin nlogues my e less relevnt under therpeuti onditions thn previously disussed. Aknowledgements We thnk M. Funke, D. Hrtmnn nd C. Jung for tehnil ssistne. Editoril support ws provided y T. Clus of PPSI ( PAREXEL ompny) nd ws funded y Snofi. Funding Dulity of interest The study ws funded y Snofi. All uthors re employees of Snofi. Contriution sttement NT nd UW mde sustntil ontriutions to the oneption nd design of the study, to the nlysis nd interprettion of the dt, to the drfting nd revising of the mnusript, nd lso pproved the version to e pulished. SW, MH, PB nd RS ontriuted sustntilly to the quisition of dt, ritilly reviewed the mnusript nd pproved the version to e pulished. Referenes 1. Rossetti P, Porellti F, Fnelli CG, Perriello G, Torlone E, Bolli GB () Superiority of insulin nlogues versus humn insulin in the tretment of dietes mellitus. Arh Physiol Biohem 11:3 1. Le Roith D (7) Insulin glrgine nd reeptor-medited signlling: linil implitions in treting type dietes. Dietes Met Res Rev 3: Hnsen BF, Kurtzhls P, Jensen AB, Dejgrd A, Russell-Jones D (11) Insulin X1 revisited: super-mitogeni insulin nlogue. Dietologi 5: 31. Bhr M, Kolter T, Seipke G, Ekel J (1997) Growth promoting nd metoli tivity of the humn insulin nlogue [GlyA1, ArgB31, ArgB3]insulin (HOE 91) in musle ells. Eur J Phrmol 3: Berti L, Kellerer M, Bossenmier B, Seffer E, Seipke G, Hring HU (199) The long ting humn insulin nlog HOE 91: hrteristis of insulin signlling in omprison to Asp(B1) nd regulr insulin. Horm Met Res 3: Kurtzhls P, Shffer L, Sorensen A et l () Correltions of reeptor inding nd metoli nd mitogeni potenies of insulin nlogs designed for linil use. Dietes 9: Sommerfeld MR, Muller G, Tshnk G et l (1) In vitro metoli nd mitogeni signling of insulin glrgine nd its metolites. PLoS One 5:e95. Kuerzel GU, Shukl U, Sholtz HE et l (3) Biotrnsformtion of insulin glrgine fter suutneous injetion in helthy sujets. Curr Med Res Opin 19:3 9. Werner U, Shmidt R, Blir E, Renn SM, Tenngels N (1) The moleulr mehnism of insulin glrgine metolism in vivo. Dietes 1(suppl 1):A5, Astrt 1. Tenngels N, Werner U (13) The metoli nd mitogeni properties of sl insulin nlogues. Arh Physiol Biohem 119: Bolli GB, Hhn A, Shmidt R et l (1) Plsm exposure to insulin glrgine nd its metolites M1 nd M fter suutneous injetion of therpeuti nd suprtherpeuti doses of glrgine in sujets with type 1 dietes mellitus. Dietes Cre 35: 3 1. Stmmerger I, Bue A, Durhfeld-Meyer B, Donuuer H, Troshu G () Evlution of the rinogeni potentil of insulin glrgine (LANTUS) in rts nd mie. Int J Toxiol 1: Bllrd FJ, Wlle JC, Frnis GL, Red LC, Toms FM (199) Des(1-3)IGF-I: trunted form of insulin-like growth ftor-i. Int J Biohem Cell Biol : Bus D, Heermeier K, De Hoop M et l () Identifition of novel AS1 splie vrint tht regultes GLUT trnslotion nd gluose-uptke in rt musle ells. Cell Signl : Pilli O, Pnhgnul R (1) Insulin therpies pst, present nd future. Drug Disov Tody : Tniguhi CM, Emnuelli B, Khn CR () Critil nodes in signlling pthwys: insights into insulin tion. Nt Rev Mol Cell Biol 7: Hvid H, Fels JJ, Kirk RK et l (11) In situ phosphoryltion of Akt nd ERK1/ in rt mmmry glnd, olon, nd liver following tretment with humn insulin nd IGF-1. Toxiol Pthol 39:3

9 13 Dietologi (13) 5: Pndini G, Frs F, Mineo R, Si L, Vigneri R, Belfiore A () Insulin/insulin-like growth ftor I hyrid reeptors hve different iologil hrteristis depending on the insulin reeptor isoform involved. J Biol Chem 77: Belfiore A, Frs F, Pndini G, Si L, Vigneri R (9) Insulin reeptor isoforms nd insulin reeptor/insulin-like growth ftor reeptor hyrids in physiology nd disese. Endor Rev 3:5 3. Moxhm CP, Duronio V, Jos S (199) Insulin-like growth ftor I reeptor et-suunit heterogeneity. Evidene for hyrid tetrmers omposed of insulin-like growth ftor I nd insulin reeptor heterodimers. J Biol Chem : Pndini G, Vigneri R, Costntino A et l (1999) Insulin nd insulin-like growth ftor-i (IGF-I) reeptor overexpression in rest ners leds to insulin/igf-i hyrid reeptor overexpression: evidene for seond mehnism of IGF-I signling. Clin Cner Res 5: Soos MA, Field CE, Siddle K (1993) Purified hyrid insulin/ insulin-like growth ftor-i reeptors ind insulin-like growth ftor-i, ut not insulin, with high ffinity. Biohem J 9(Pt ):19 3. Sndow J (9) Growth effets of insulin nd insulin nlogues. Arh Physiol Biohem 115:7 5. Ekrdt K, My C, Koenen M, Ekel J (7) IGF-1 reeptor signlling determines the mitogeni poteny of insulin nlogues in humn smooth musle ells nd firolsts. Dietologi 5: Myer D, Shukl A, Enzmnn H () Prolifertive effets of insulin nlogues on mmmry epithelil ells. Arh Physiol Biohem 11:3. Shukl A, Grisourd J, Ehemnn V, Hermni A, Enzmnn H, Myer D (9) Anlysis of signling pthwys relted to ell prolifertion stimulted y insulin nlogs in humn mmmry epithelil ell lines. Endor Relt Cner 1: Weinstein D, Simon M, Yehezkel E, Lron Z, Werner H (9) Insulin nlogues disply IGF-I-like mitogeni nd nti-poptoti tivities in ultured ner ells. Dietes Met Res Rev 5:1 9. Yehezkel E, Weinstein D, Simon M, Srfstein R, Lron Z, Werner H (1) Long-ting insulin nlogues eliit typil signlling events medited y the insulin reeptor nd insulin-like growth ftor-i reeptor. Dietologi 53: Cirldi TP, Crter L, Seipke G, Mudlir S, Henry RR (1) Effets of the long-ting insulin nlog insulin glrgine on ultured humn skeletl musle ells: omprisons to insulin nd IGF- I. J Clin Endorinol Met : Liefvendhl E, Arnqvist HJ () Mitogeni effet of the insulin nlogue glrgine in mlignnt ells in omprison with insulin nd IGF-I. Horm Met Res : Mussig K, Stiger H, Kntrtzis K, Fritshe A, Knz L, Hring HU (1) Dietes, insulin, insulin nlogues, nd ner. Dtsh Med Wohenshr 135:9 99, rtile in Germn 3. Stiger K, Stiger H, Shweitzer MA et l (5) Insulin nd its nlogue glrgine do not ffet viility nd prolifertion of humn oronry rtery endothelil nd smooth musle ells. Dietologi : Stiger K, Hennige AM, Stiger H, Hring HU, Kellerer M (7) Comprison of the mitogeni poteny of regulr humn insulin nd its nlogue glrgine in norml nd trnsformed humn rest epithelil ells. Horm Met Res 39: Wd T, Azegmi M, Sugiym M, Tsuneki H, Ssok T () Chrteristis of signlling properties medited y long-ting insulin nlogue glrgine nd detemir in trget ells of insulin. Dietes Res Clin Prt 1: Drejer K (199) The iotivity of insulin nlogues from in vitro reeptor inding to in vivo gluose uptke. Dietes Met Rev : Si L, Cssrino MF, Genu M et l (1) Insulin nlogues differently tivte insulin reeptor isoforms nd post-reeptor signlling. Dietologi 53: Stmmerger I, Esserment L (1) Insulin glrgine: reevlution of rodent rinogeniity findings. Int J Toxiol 31: Ngel JM, Stff J, Renner-Muller I et l (1) Insulin glrgine nd NPH insulin inrese to similr degree epithelil ell prolifertion nd errnt rypt foi formtion in olons of dieti mie. Horm Cner 1: Agouni A, Owen C, Czopek A, Mody N, Deliegovi M (1) In vivo differentil effets of fsting, re-feeding, insulin nd insulin stimultion time ourse on insulin signling pthwy omponents in peripherl tissues. Biohem Biophys Res Commun 1: Hennige AM, Lehmnn R, Weigert C et l (5) Insulin glulisine: insulin reeptor signling hrteristis in vivo. Dietes 5: Berhtold H, Hilgenfeld R (1999) Binding of phenol to R insulin hexmers. Biopolymers 51: Luidi P, Porellti F, Rossetti P et l (1) Metolism of insulin glrgine fter repeted dily suutneous injetions in sujets with type dietes mellitus. Dietes Cre 35(): Agin A, Jendidier N, Gsser F, Gruker D, Spin R (7) Glrgine lood iotrnsformtion: in vitro pprisl with humn insulin immunossy. Dietes Met 33:5 1. Iked H, Shiojim I, Ozs Y et l (9) Intertion of myordil insulin reeptor nd IGF reeptor signling in exerise-indued rdi hypertrophy. J Mol Cell Crdiol 7: Lee AV, Tylor ST, Greenll J et l (3) Rpid indution of IGF- signling in norml nd tumor tissue following intrvenous injetion of IGF-I in mie. Horm Met Res 35: Wlton PE, Gopinth R, Burleigh BD, Etherton TD (199) Administrtion of reominnt humn insulin-like growth ftor I to pigs: determintion of irulting hlf-lives nd hromtogrphi profiles. Horm Res 31: Pierre-Eugene C, Pgesy P, Nguyen TT et l (1) Effet of insulin nlogues on insulin/igf1 hyrid reeptors: inresed tivtion y glrgine ut not y its metolites M1 nd M. PLoS One 7:e199