Insulin-like growth factor-i and more potent variants restore growth of diabetic rats without inducing all characteristic insulin effects

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1 Biochem. J. (1993) 291, (Printed in Gret Britin) 781 Insulin-like growth fctor-i nd more potent vrints restore growth of dibetic rts without inducing ll chrcteristic insulin effects Frnk M. TOMAS,* Spencer E. KNOWLES, Phillip C. OWENS, Colin S. CHANDLER, Geoffrey L. FRANCIS nd F. John BALLARD Coopertive Reserch Centre for Tissue Growth nd Repir, nd CSIRO, Division of Humn Nutrition, Adelide, S.A. 5, Austrli The effects of grded doses of insulin-like growth fctor-i (IGF- I) nd two vrints which bind poorly to IGF-binding proteins were investigted in 16 g streptozotocin-induced dibetic rts. The two vrints were the truncted form, des(i-3)igf-i, nd nother with rginine t residue 3 nd n N-terminl extension, termed LR3-IGF-I. The peptides were infused vi mini-osmotic pumps. Reference groups received either vehicle or insulin (3 i.u. per dy). Tretment led to mrked dose-dependent increse in growth rte nd nitrogen blnce. The highest dose (695 /tg/dy) of IGF-I incresed body weight by g/7 dys, compred with g/7 dys for the vehicle-treted group. The two vrints were times more potent thn IGF-I in restoring growth. The insulin-treted group gined more weight ( g/7 dys), but the dded gin ws ft ( g of ft/kg crcss wet wt., compred with for ll other groups) rther thn protein. All peptides incresed muscle protein-synthesis rtes nd RNA levels by up to 5 %, with IGF- I the lest potent. These high doses of IGFs did not decrese either the glucosuri or the dily excretion rte of N'-methylhistidine (N'-MH). On the other hnd, insulin tretment mrkedly decresed both glucosuri (from to mmol/dy) nd Nr-MH excretion (from to ,tmol/dy per kg). This experiment shows tht, lthough IGF-I nd vrints cn restore growth in dibetic rts, other insulin-dependent metbolic processes in liver, muscle nd dipose tissue re not restored. INTRODUCTION Impired insulin production nd resultnt dibetes induced in young rts by streptozotocin (STZ) tretment is ccompnied by decrese in circulting levels of insulin-like growth fctor-i (IGF-I) nd IGF-I-binding proteins (IGFBPs) in plsm [1-3]. Administrtion of IGF-I to STZ-dibetic rts restores growth, nitrogen blnce nd muscle protein-synthesis rtes, but does not meliorte the hyperglycemi or glucosuri ssocited with the dernged crbohydrte metbolism [4-6]. Acute studies in norml nd dibetic nimls hve indicted tht IGF-I tretment does increse glucose uptke by the peripherl tissues [7,8], but is much less effective in the inhibition of heptic glucose production. Also, the dose of IGF-I used in these cute experiments is reltively high compred with those used in the chronic infusion studies which report growth promotion. Recently, our lbortory hs reported the higher potency of the vrint des(l-3)igf-i in cultured cells [9] nd in STZdibetic rts [6,1]. This incresed potency ppers relted to the decresed binding of the peptide to IGFBPs [11], which presumbly results in higher vilbility to the receptors of trget cells. It ws suggested tht even higher levels of such vrints my decrese glucose production in the liver of dibetic rts by virtue of mintining high unbound locl concentrtions, which would ct through either specific IGF receptors or the insulin receptor. Thus, in ddition to the restortion of protein ccretion nd growth of dibetic rts chieved by the continuous infusion of reltively low doses of des(l-3)igf-i [6], we considered tht the dministrtion of higher doses my lso moderte the defects in crbohydrte metbolism. To nswer this question, nd lso to estblish reltive potencies of IGF-I vrints, we hve exmined the effects of IGF-I, des(1-3)igf-i, nd further vrint with even lower ffinity for IGFBPs, LR3-IGF-I [12], in rnge of doses on the growth, crcss composition, protein metbolism nd glucosuri in STZ dibetic rts. EXPERIMENTAL Peptides nd chemicls Recombinnt humn IGF-I nd recombinnt des(1-3)igf-i were supplied by Genentech Inc., South Sn Frncisco, CA, U.S.A. Recombinnt LR3-IGF-I, which hs 13-residue N-terminl extension nd Glu-3 replced by Arg in the IGF sequence [12], ws obtined from GroPep Pty. Ltd., Adelide, Austrli. The peptides were dministered to nimls by using mini-osmotic pumps (Alz, Plo Alto, CA, U.S.A.) which delivered.92,ul/h. The peptides were dissolved in.1 M cetic cid to the concentrtions required for the pumps to deliver 44, 111, 278 or 695,ug/dy ccording to the llocted tretment. Pumps for control nimls were filled with.1 M cetic cid. Protmine zinc insulin [Isophne insulin (N.P.H.); 1 units/ml] ws obtined from CSL-Novo Pty. Ltd., North Rocks, N.S.W., Austrli. The insulin ws diluted in vehicle consisting of.2 % N2HPO4 nd 1.6 % glycerol (ph 7.4) before once-dily subcutneous injection. Streptozotocin ws purchsed from Sigm Chemicl Co., St. Louis, MO, U.S.A., nd dissolved in 2 mm sodium citrte buffer immeditely before dministrtion to the nimls. L-fring-2,6-3H(n)]Phenyllnine (2.22 TBq/mmol) ws obtined from Du Pont (Austrli) Ltd., North Ryde, N.S.W., Austrli. Animls Mle Hooded-Wistr rts were obtined from the CSIRO Division of Humn Nutrition colony nd held in cges t 25 C under controlled lighting (12 h-drk/12 h-light cycle). At 14 g body weight, food ws withdrwn for 6-8 h, fter which the rts Abbrevitions used: IGF-I, insulin-like growth fctor I; STZ, streptozotocin; IGFBP, IGF-1-binding protein; NT-MH, NT-methylhistidine. * To whom correspondence should be ddressed.

2 782 F. M. Toms nd others were injected intrperitonelly with 65 mg of STZ/kg body wt. The rts were then trnsferred into individul metbolism cges nd given diet which contined 18 g of csein nd 1.25 g of methionine/kg nd ws free of Nr-methylhistidine (NT-MH) [13]. Only rts showing polydipsi, polyuri, glucosuri nd ner growth stsis were used in the experiment. Experimentl tretments commenced 7 dys fter STZ injection. The protocol for the experiment ws pproved by the Animl Cre nd Ethics Committee of the CSIRO Division of Humn Nutrition, following the Austrlin Code of Prctice for the Cre nd Use of Animls for Scientific Purposes. Experimentl protocol The rts were given free ccess to food nd wter nd were weighed dily throughout the study. Dily urine nd feces collection nd mesurement of food intke commenced 2 dys before the insertion of the osmotic pumps. Excret smples were stored t -2 C until nlysed. The pumps contining peptide were implnted subcutneously in the supr-scpulr region with the rt under ether nesthesi. Body weight verged g t this time. There were 11 groups of 6 rts, comprising control group, n insulin-treted group nd 9 IGF tretment groups to which the rts were rndomly llocted. Control rts received pump contining vehicle only. The insulin-treted group received dily subcutneous injection of insulin (3 i.u./dy per kg body wt.) s well s vehicle pump. IGF-treted groups received either IGF-I t 111, 278 or 695 jug/dy or one of the two vrints t 44, 111 or 278,ug/dy. After 7 dys of tretment, the rts were restrined in n open-weve cloth while solution contining 15 mm phenyllnine, 77 mm NCl nd 5,uCi of L-[ring-2,6-3H]phenyllnine/ml ws injected (1 ml/kg body wt.) vi lterl til vein. Then 15 min lter the rts were stunned nd decpitted, nd trunk blood ws collected into heprinized tube for 1 s. A consistent section of mixed hind-limb muscles (chiefly gstrocnemius, biceps femoris nd plntris) ws rpidly excised nd frozen in tongs precooled in liquid N2. The pelt nd viscerl orgns were then removed nd weighed before being discrded. The remining crcss ws frozen for lter nlysis. Becuse of cging nd other constrints the experiment ws done in three stges, ech with two rts per tretment group. Anlyticl methods The nitrogen contents of food, feces, urine nd dried crcss were mesured by the Dums procedure by using Crlo Erb NA15 Nitrogen Anlyser (Miln, Itly). Urinry N'-MH ws mesured by continuous-flow nlysis [14] fter initil hydrolysis nd ion-exchnge chromtogrphy [15]. Muscle totl RNA contents were mesured s described by Munro nd Fleck [16]. Rtes of muscle protein synthesis were determined essentilly s described by Grlick et l. [17] nd expressed s frctionl rte (K.; % of protein pool synthesized/dy) nd s rte per unit of RNA (g of protein synthesized/dy per g of RNA). The rtionle for the use of the 'flooding-dose' method for mesurement of protein synthesis nd vlidtion for its use in muscle hve been presented elsewhere [17,18]. Urinry glucose ws mesured by glucose oxidse method with continuous-flow nlyser (Sklr Anlyticl, Bred, The Netherlnds). Plsm glucose levels were mesured with glucose test strips (BM-Test-Glycemie 2-8 glucose; Boehringer Mnnheim Austrli Pty. Ltd., Sydney, Austrli) red with blood glucose monitor ('Omniscn', Newcrest Dignostics Ltd., Hong Kong). IGF-I ws mesured in cid/ethnol extrcts of plsm [6], nd IGFBPs were nlysed in untreted plsm by the ligndblot procedure [1]. Sttistics Vlues re presented s mens with the S.E.M. indicted. Tretment effects were initilly ssessed by using two-wy nlysis of vrince, prtitioning vrince due to stges nd tretments. Where the tretment effect ws significnt (P <.5) (NS, not significnt), multiple comprisons mong mens were mde by the Student-Newmn-Keul's test nd comprisons with the vehicle control group were mde by Dunnett's test. Dose-responses nd reltive potencies were exmined by regression nlysis (Systt 5.; Systt Inc., Evnston, IL, U.S.A.), log dose being used s the independent vrible nd growth fctor nd stge s ctegoricl vribles. The vehicle group ws not included in nlyses where log dose ws used. RESULTS Growth responses nd food Intke Ech of the growth fctors incresed growth rte in dose-dependent mnner between tht of the vehicle group ( g/dy) nd the insulin-treted group ( g/dy) (Figure 1). An increse in growth rte ws obvious from dy 1 of tretment with growth fctors (see inset, Figure 1) nd ws sustined throughout the tretment period. The vrints were fold more potent thn ws IGF-I (P <.1), with LR3-IGF-I consistently more potent thn des(l-3)igf-i (P =.1, NS). Except for the insulin-treted group, food intkes remined virtully constnt throughout the experiment. Averged over the 7 tretment dys, they rnged from 31.1 to 34.1 g/dy, with only prtil compenstion for chnges in body size due to differentil growth rtes between tretment groups. Thus the dily food consumption verged over those groups receiving the low, intermedite nd high dose of growth fctor ws 1.84, 1.78 nd 1.71 g/kg body wt. [S.E. of difference (SED) =.37; P <.1] respectively. Administrtion of insulin cused substntil 7 Z 6 > 5 t-, g 4 D._ 3 2 m 2 Q 1 O 4 L J, e Growth-fctor dose (jg/dy) Figure 1 Dose-response for the chnge In body weight of groups of STZdibetic rts receiving Infusions of vehicle (), IGF-I (@), des(1-3)igf-i (A), LR3-IGF-I (-), or dily subcutneous insulin Injection (O), over 7 dys The inset shows the growth curve for the vehicle- nd insulin-treted rts nd those groups receiving 278 jig of IGF-l, des(1-3)igf-1 nd LR3-IGF-1/dy. Ech point is the men of six rts, with S.E.M. indicted by verticl brs.

3 Insulin-like growth fctor vrints in dibetic rts *. 18.,. 16 r- ' r, 1 c 8 - O 6 l + 4 Figure 2 tretment 'A Growth-fctor dose (jug/dy) Dose-response curves for cumulfive nitrogen retention during Symbols nd tretments re given in Figure 1 legend. T j/;/'+ decrese in dily food intke to n verge of g, or g/kg body wt. (P <.1). Nitrogen blnce The nitrogen retention over the tretment period is n indictor of somtic growth. Cumultive nitrogen blnces incresed in dose-relted mnner during IGF tretment (P <.1, Figure 2), with IGF-I bout 2-3 times less potent thn the two vrints (P <.1). However, unlike the response pttern for growth rte, insulin tretment ws not more effective thn the highest dose of IGFs. Thus the insulin-treted group retined mg of nitrogen over 7 dys, compred with mg for the group receiving 278,tg of LR3-IGF-I/dy. Glucose excretion Averge glucose excretion ws not decresed by growth-fctor tretment (Figure 3). However, there ws smll but sttisticlly significnt dose effect of the growth fctors evident in 1% EE " o 3 2 E 1 o -1 & -2 x _3 ID -3 s -5 &1 6 r- -7 I -8 ) r- -9 (- () Tretment Time (dys) (b) ( Growth-fctor dose (jug/dy) Figure 3 () Dily glucose excretion by rts receiving either infusions of vehicle, IGF-1, des(1-3)igf-l nd LR3-IGF-I t 278 1tg/dy or dily subcutneous insuhn injecton t 1.25 mg/dy per kg, nd (b) doseresponses for the chnge In the rte of glucose excretion by grmups of STZdibetic rts receiving Infusions of vehicle, IGF-1, des(1-3)igf-1, LR3-IGF-l or subcutneous dily insulin injection over 7 dys Symbols nd tretments re given in Figure 1 legend. lowering ofglucose excretion, from n verge of 97. mmol/dy t the lowest dose of ech to 89.2 mmol/dy t the highest dose (P <.2). The dose effect is illustrted by compring the chnge in verge glucose excretion rte from the initil 2-dy control collections fter tretment (Figure 3b). LR3-IGF-I tended to be Tble 1 Muscle protein nd RNA contents nd protein-synthesis rtes fter 7 dys trtment of STZ-induced dibetic rts Vlues re mens+s.e.m. for six nimls in ech group, with the peptide doses (,ug/dy) shown in prentheses: *P<.5, **P<.1, ***P<.1 versus vehicle-treted control rts. Protein synthesis Protein content RNA content (g of protein/dy Tretment group (mg/g wet wt. of muscle) (/tg/g of protein) (Ks, %/dy)t per g of RNA) Vehicle Insulin IGF-I () (225) (695) Des(1-3)IGF-I (44) LR3-IGF-l (44) SED t Ks, proportion of protein pool synthesized per dy *** ** *** *** * *** *

4 784 F. M. Toms nd others Tble 2 Molr nd frctionl excretlon rte (KmH) of NT-MH nd concentrtions in plsm nd in muscle intrcellulr wter fter 6-7 dys tretment of STZ-induced dibetic rts Vlues re mens+ S.E.M. for six nimls in ech group, with the peptide doses (4ug/dy) shown in prentheses. KMH (proportion of crcss NT-MH pool excreted per dy) is clculted from verge Nr-MH excretion on dys 6 nd 7 nd the crcss N'-MH content. *P <.5, **P <.1, ***P <.1 versus vehicle-treted control rts. Urine NT-MH KMH Plsm N -MH Muscle NT-MH Tretment group (,umol/dy per kg) (%/dy) (mm) (mm)t Vehicle () Insulin (225) IGF-I (695) Des(1-3)IGF-l (44) LR3-IGF-I (44) SED * * * t Concentrtion in muscle wter ws estimted s [(nmol/g of muscle) -.2(nmol/ml of plsm)]/.6. It ws ssumed tht muscle contins 2 9 of extrcellulr nd 6 9 of intrcellulr wter per kg wet wt. nd tht the interstitil spce (15 g/kg) is t equilibrium with plsm. No other corrections were mde. the most potent of the three IGF peptides for lowering of glucose excretion, with the effect more pronounced when glucose excretion ws expressed per unit body weight or food intke. Nonetheless, insulin tretment ws very much more effective thn the growth fctors nd decresed glucose excretion to mmol/dy, less thn 4 % of tht in the other groups by the lst dy of the experiment. At the end of the tretment period, plsm glucose levels of insulin-treted rts were mm, less thn hlf the combined verge for ll the other groups ( mm) which did not respond to growth-fctor tretment. Muscle protein synthesis nd brekdown Frctionl synthesis rtes of muscle protein (K., % /dy) were incresed up to 5 % in rts treted with growth fctor compred with the control group (Tble 1). The effect of dose ws highly significnt (P <.1). Although both vrints were on verge more potent thn IGF-I in promoting synthesis, this difference ws only sttisticlly significnt (P <.5) for des(i-3)igf-i. Insulin-treted rts showed significntly higher increment in protein-synthesis rtes thn ws obtined with the growth fctors (P <.5), even though the lst insulin injection ws bout 24 h before the mesurement. Tretment did not ffect muscle protein concentrtion, but tissue RNA levels, expressed reltive to protein content, showed mrked dose-relted increse (P <.1), such tht the rte of protein synthesized per g of RNA remined virtully constnt (Tble 1). N'-MH excretion, n index of totl myofibrillr protein brekdown, ws not chnged from control levels by ny of the IGFs (Tble 2). On the other hnd, insulin tretment led to 2 % decrese in NT-MH excretion (P <.1). The verge rte ofexcretion ofnt-mh over the lst 2 dys of tretment, expressed s frction of the crcss N'-MH pool excreted per dy (KMH, %/dy, equivlent to the frctionl rte of brekdown of myofibrillr protein), tended to be higher thn the control vlue in groups receiving growth fctors (NS; P >.5) but ws significntly lower in the insulin-treted group (P <.2; Tble 2). Free Nr-MH levels in the intrcellulr fluid of muscle tissue re lso n index of the rte of brekdown of myofibrillr protein in mny conditions. The results in Tble 2 show tht intrcellulr levels of N7-MH in both growth-fctor- nd insulin-treted rts were not significntly different. However, plsm Nr-MH levels were higher in the insulin-treted group (P <.5) nd suggest tht both Nr-MH clernce nd production rtes my hve been lower. Crcss composition The proportionl contribution of wter, protein nd ft to the crcss wet weight ws significntly ltered in insulin-treted groups (P <.1) with ft incresed more thn 2-fold (Tble 3). On the other hnd, crcss ft concentrtion significntly declined with incresing doses of ech IGF peptide (P <.5), reflecting lck of growth in ft mss s body weight incresed. Protein concentrtion of the ft-free crcss ws not ffected by IGF or insulin tretment. The bsolute mount of crcss protein ws significntly incresed bove tht of control nimls by the intermedite (P <.5) nd high (P <.1) doses of growth fctors nd by insulin (P <.1; results not shown). IGF-I concentrtions nd IGFBPs The plsm IGF-I concentrtion just before insertion of the osmotic pumps ws pprox. 5,ug/l. IGF-I infusion incresed verge IGF-I levels progressively with incresing dosge, to chieve up to 4-fold those of the vehicle-treted controls. Tretment with des(i-3)igf-i, which hs bout hlf the potency of IGF-I in the rdioimmunossy, showed reltively minor rise in mesured IGF-I levels compred with controls, wheres tretment with LR3-IGF-I, which hs only 1% of the potency of IGF-I in the rdioimmunossy, cused no chnge in ssyed IGF-I levels (results not shown). Insulin injections led to sustined fold increse in circulting IGF-I levels bove the dibetic control rts. The higher-moleculr-mss IGFBPs (presumbly IGFBP-3) in the finl plsm smples were incresed fter tretment with

5 Tble 3 Crcss composition of dibefic rts fter 7 dys tretment Insulin-like growth fctor vrints in dibetic rts 785 Vlues re mens + S.E.M. (n = 6) with the peptide dose (/tg/dy) shown in prentheses: *P <.5, **P <.1, ***P <.1 versus vehicle-treted control rts. Protein vlues re nitrogen x Content in crcss (g/kg) Tretment group Wter Protein Ft Residue Vehicle () Insulin (225) "* IGF-I (695) *** Des(1-3)1GF-I (44) * LR3-IGF-I (44) * * SED " "' either insulin or IGF-I s reported previously [6]. Lesser increses were indicted by the lignd blots for rts treted with IGF vrints. DISCUSSION Continuous infusion of IGF-I to dibetic rts produced growth responses in proportion to the infused dose, s previously described by us [6] nd by others [4,5]. We now illustrte tht the dose-response in dibetic rts extends substntilly beyond tht defined previously. The highest dose rtes used here were bout 2.5 times those of the previous report [6] nd induced growth rtes lmost equl to tht observed for the insulin-treted rts. In fct, in terms of somtic growth the highest doses of des(l- 3)IGF-I nd LR3-IGF-I were equipotent with the insulin tretment used here (1.25 mg/dy per kg body wt.) nd stimulted len growth to the norml rnge for these rts. Unlike insulin, the growth fctors did not stimulte ft deposition, nd in fct ft mss did not increse in proportion to the body mss. This difference could be explined by the pucity of type 1 IGF receptors in dipose tissue [19] nd lso rgues ginst the IGF peptides cting vi the insulin receptor to stimulte glucose uptke or other processes in this study. The difference between the reltive responses of body weight nd nitrogen retention for insulin nd the growth fctors cn be explined by their reltive effects on lipid deposition. Despite normliztion of nitrogen blnce nd growth, there ws virtully no effect of growth-fctor tretment on heptic glucose production s ssessed by urinry glucose output. Although there ws n indiction of smll dose effect, glucose excretion continued to rise during tretment in ll except the insulin-treted group. Thus, despite the higher dosge of IGFs nd the higher potency of these vrints for tissue nd orgn growth, the effect on glucose excretion ws similr to tht in our previous study [6]. The highest level of IGF-I infused (695,ug/dy) gve dose rnging from 3.2 to 2.5,ug/min per kg during the course of the infusion. Acute studies hve generlly used even higher doses up to 16.3,ug of IGF-I/min per kg [7,8,2,21]. The reported cute inhibition of heptic glucose production ws dependent on the dose nd chosen model, but ppers to be bout hlf of tht obtined with insulin t dose equipotent for plsm glucose decrese or glucose disposl. The lrge differences in the responsiveness to IGF-I between the vrious rt models my reflect metbolic differences. For exmple, rts mde dibetic with STZ disply mrked insulin resistnce despite incresed binding cpcity of the insulin receptors, implying post-receptor defect [22-25]. These chnges in the insulin-receptor/signlling processes my be importnt, s the lck of heptic type-i IGF-I receptors require IGF-I to ct vi the insulin receptors [26]. One potentilly importnt difference between the cute studies nd our chronic infusion experiment is the possible role of the binding proteins. In cute studies there would be little chnge in the levels of IGFBPs. In contrst we found substntil rise in the levels of IGFBPs in response to the IGF infusions, which would blunt the ultimte increse in the levels of free IGF-I vilble for receptor binding. This rgument does not explin the lck of effect of the vrints on glucose excretion, however, s LR3-IGF-I especilly does not bind to the IGFBPs in the rt [9,1,12]. The explntion my simply be tht extrordinrily high levels of free IGF-I (s my be generted in cute experiments) re needed to produce responses vi the insulin receptor. Thus levels of IGF-I nd vrints required to produce biologicl responses in H35 cells [12], heptom line tht hs bundnt insulin receptors but no IGF-I receptors [27], re bout 1- nd 1-fold higher respectively thn for insulin. The smll but sttisticlly significnt effect of LR3-IGF-I on glucose excretion by the dibetic rts indictes tht this peptide, which binds very poorly to the principl IGFBPs in the rt, my hve lmost reched effective concentrtions for ction vi the insulin receptor. The observed improvement in nitrogen blnce nd somtic growth with growth-fctor tretment could only rise through co-ordinte chnges in protein turnover which led to n incresed positive difference between protein synthesis nd brekdown. Our dt confirm dose-relted increse in muscle protein-synthesis rtes with IGF tretment s reported previously for both dibetic [6] nd dexmethsone-treted [1] rts. The increse could be entirely explined by concomitnt chnges in the concentrtion of RNA in the tissue, in greement with our previous report [6]. In contrst, the Nr-MH-excretion dt indicte tht the IGFs do not decrese the rte of muscle protein

6 786 F. M. Toms nd others brekdown in dibetic rts nd point to nother difference in the ctions of insulin nd IGFs. Both Goodmn [28] nd Pin et l. [29] hve shown tht rtes of muscle protein brekdown re incresed during cute dibetes nd subsequently decresed below norml with chronic dibetes. Our rts received the peptide infusions from 8 to 15 dys fter STZ tretment, which my be trnsition period between cute nd chronic phses, nd in fct the rtes of Nr-MH excretion were close to those expected for non-dibetic rts of equivlent ge nd weight [1]. The contribution of non-skeletl muscle sources of NT-MH, in prticulr gut smooth muscle [28], must be considered when evluting the dt. Reltive gut weight in rts is elevted by dibetes, further incresed by growth-fctor tretment nd returned to norml by insulin tretment [3]. However, gut protein-synthesis rtes re not incresed by IGFs [3-32], pointing to lower brekdown rte (nd Nr-MH relese) to ccount for the growth. Furthermore, the N"-MH-excretion dt re supported by lterntive estimtes of protein brekdown bsed on the dt for protein synthesis nd crcss protein ccretion (results not shown), s well s by the intrcellulr levels of Nr-MH in skeletl muscle, which indicte no mjor effect of the IGFs on Nr-MH relese. On blnce, we believe tht our dt indicte tht IGF-I nd the tested vrints exert little influence on muscle protein brekdown in dibetic rts, t lest where rtes of protein brekdown re ner norml. The filure to produce n insulinlike effect, even t high doses of vrints, my be relted to the continuing high rte of heptic gluconeogenesis nd coincident distortion of the norml mino cid pttern. In conclusion, we hve found tht with the chronic infusion of high dose of IGF-I to dibetic rts there is no evidence of decrese in heptic glucose production, despite mrked increse in somtic growth rte. This ws lso the cse for the more potent vrints of IGF-I tested, which presumbly mintined even higher levels of free peptide, s they do not bind to the principl IGFBPs in the rt. These results differ from those obtined by others from cute studies. Our dt show tht longterm dministrtion of IGFs ppers to induce counter-regultory mechnisms which modify IGF ctions nd indicte tht the prediction of longer-term effects from cute studies my be unrelible. The IGFs lso filed to decrese the rte of excretion of NT-MH or to ffect the rte of crcss ft deposition, which contrst with the effects of insulin. Further work is needed to investigte whether these effects my be relted to the continuing heptic gluconeogenesis or to ltered tissue sensitivities towrds insulin nd IGF. We thnk K. Edson, K. Lymm, A. Collins, S. Mdden, 1. Skene, M. Perce, K. Irvine, M. Conlon nd J. Burgoyne for technicl ssistnce. REFERENCES 1 Phillips, L. S. nd Young, H. S. 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