Systemic Insulin-like Growth Factor-1 Reverses Hypoalgesia and Improves Mobility in a Mouse Model of Diabetic Peripheral Neuropathy

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1 originl rtile Systemi Insulin-like Growth Ftor-1 Reverses Hypolgesi nd Improves Moility in Mouse Model of Dieti Peripherl Neuropthy Qiuming Chu 1, Rod Morelnd 1, Nelson S Yew 1, Joseph Foley 1, Roin Ziegler 1 nd Ronld K Sheule 1 1 Genzyme Corportion, Frminghm, Msshusetts, USA Peripherl neuropthy is prtiulrly deilitting omplition of oth type 1 nd type 2 dietes hrterized y sensory nd motor neuron dmge nd deresed irulting levels of insulin-like growth ftor 1 (IGF-1). Quite often, n erly hyperlgesi is followed y hypolgesi nd musle wekness. Hypolgesi n led to signifint moridity for whih there is no urrent tretment. Hyperglyemi, streptozotoin ()-indued rodent models reprodue these symptoms. We investigted whether inresing systemi IGF-1 ould improve neuronl funtion in hyper- nd hypolgesi -treted mie. Inresed irulting levels of IGF-1 were hieved y delivering plsmid or deno-ssoited virl (AAV) vetor ering mouse IGF-1 to the liver. Treting mie in the hyperlgesi stge prevented lter hypolgesi. Treting mie in the hypolgesi stge reversed existing hypolgesi. This ltter effet ould e seen y merely restoring IGF-1 serum levels to normly, whih ws possile to hieve y IGF-1 gene therpy or insulin tretment. Sensory nerve funtionl orretion ws seen to e orrelted with ttenuted Shwnn ell vuoliztion nd demyelintion in peripherl sensory nerve fiers. A further inrese in serum IGF-1 levels with gene therpy lso improved motor funtion, onsistent with the oserved prevention of oth musle trophy nd peripherl motor nerve fier demyelintion. These results suggest tht the restortion of systemi levels of IGF-1 my prove to e highly effetive therpeuti modlity for treting dieti peripherl neuropthy. Reeived 24 Otoer 7; epted 30 April 8; pulished online 10 June 8. doi: /mt INTRODUCTION Dieti peripherl neuropthy (DPN) is prtiulrly deilitting omplition of dietes resulting in sensory nd motor neuron dmge. Up to 50% of dieti ptients hve some degree of DPN. 1 Erly in the disese proess DPN is mnifested s hyperlgesi, ut over time, ptients egin to experiene hypolgesi nd musle wekness. Hypolgesi is one of the mjor ftors tht n led to signifint moridity y predisposing the lower extremities to injury nd ulertion, ultimtely leding to mputtion. 1,2 There re tretment options for pin relief for the erly hyperlgesi stge of DPN, ut there is no tretment for the lter hypolgesi stge. Although intensive insulin therpy to ontrol lood gluose redues the inidene of new linilly deteted neuropthy, dieti ptients n still develop DPN. Intensive insulin therpy lso signifintly inreses the risk of hypoglyemi episodes during sleep. 3 Hypoglyemi n lso ontriute to the development of hypolgesi. 4 Clerly, speifi therpeutis to tret the hypolgesi ssoited with dietes re needed. Growth ftors hve een suggested s therpeutis for DPN 5 nd hve een shown to promote neuronl survivl, stimulte repir of peripherl nerve injury, nd even indue nerve regenertion under dieti onditions. Reominnt nerve growth ftor 6 nd rin-derived neurotrophi ftor 7 hve een evluted linilly, ut hve not shown signifint enefit. Vsulr endothelil growth ftor 8 (VEGF) nd C-peptide 9 hve lso ompleted Phse I linil trils. Insulin-like growth ftor 1 (IGF-1) provides trophi support for neurons of oth the peripherl nd entrl nervous systems. Systemi IGF-1 levels in ptients with dietes hve een shown to e slightly lower thn those of nondieti individuls, nd serum IGF-1 levels in dietes ptients with DPN re lower thn those in dietes ptients without DPN. 10,11 Although the levels of IGF-1 s well s other neurotrophi ftors derese with ge, IGF-1 n upregulte mny of these ftors, inluding neurotrophin-3, pltelet-derived growth ftor, firolst growth ftor, hypoxiinduile ftor-1α, nd VEGF. 12,13 Dietes ptients my lso lose musle mss, nd this ontriutes to defiits in motor funtion. 14 IGF-1 is known to e myotrophi. 15,16 Given these ttriutes of IGF-1, it is importnt to determine whether the restortion of systemi IGF-1 in dietes ptients to norml or higher-thn-norml levels provides therpeuti enefits for the neuropthy ssoited with the disese. Results in rodent models of DPN suggest tht IGF-1 ould e onsidered s potentil therpeuti for DPN. For exmple, reominnt humn IGF-1 ws found to stilize hyperlgesi in the streptozotoin () rt model of DPN. 17 Trnsgeni mie defiient in IGF-1 develop DPN symptoms, nd reominnt humn IGF-1 ws le to restore oth sensory nd motor nerve Correspondene: Ronld K. Sheule, Genzyme Corportion, 31 New York Avenue, Frminghm, Msshusetts 01701, USA. E-mil: ronld.sheule@genzyme.om 10 vol. 16 no. 8, ug. 8

2 ondution veloities in these mie. 18 It should e noted, however, tht serum gluose levels in these mie were norml, nd therefore these results re not neessrily preditive of the effets of IGF-1 in dieti neuropthy. On histologil nlysis, reominnt humn IGF-1 ws lso seen to reverse neuroxonl dystrophy in the rt model of dieti utonomi neuropthy. 19 Although these results suggest tht systemi IGF-1 ould e therpeuti in DPN, it hs not to dte een shown to provide enefit in the hypolgesi stge of DPN, espeilly where ongoing hyperglyemi is ontriuting ftor in the disese proess itself. Our ultimte gol ws to evlute whether inresing systemi IGF-1 in the mouse model would e effetive in treting the lte-stge disese symptoms, nmely, hypolgesi nd musle wekness. We first investigted whether delivering gene ontining mouse IGF-1 during the hyperlgesi stge ould sueed in interrupting disese progression; nd further, whether the resulting systemi protein ould lso prevent the susequent hypolgesi. The results of suh n investigtion would effetively reprodue nd extend erlier studies in the mouse model using IGF-1 protein infusion. 17 In n ttempt to mke these findings more relevnt to the linil sitution, we then investigted whether inresing systemi IGF-1 during the hypolgesi stge ould ttenute the hypolgesi nd improve motor funtion even in the presene of ongoing hyperglyemi. To our knowledge, the effiy of IGF-1 hs not s yet een demonstrted in this lter stge of DPN in dieti model. In order to eluidte the pthwys through whih IGF-1 ppered to e ffeting funtion, we evluted the effets of inresing doses of insulin in the sme model. Although insulin hd positive effets on the seleted neurologi nd motor end points, these effets were more limited thn ould e otined y inresing serum IGF-1 using gene delivery. Indeed, the mgnitude of the insulin-medited effets ws found to orrelte with the ility of insulin to stimulte inreses in serum IGF-1, implying tht the mjor pthwy involved in these effets is through the IGF-1 reeptor rther thn the insulin reeptor (IR). RESULTS -treted mie develop hyperlgesi t the erly stge followed y hypolgesi in the presene of signifintly deresed systemi IGF-1 levels tretment of mie resulted in severe dieti phenotype with very high lood gluose (>500 mg/dl) within 3 dys nd signifint derese in ody mss ( 6% weight loss within 1 week). Hyperlgesi in these -treted mie ws deteted s erly s 5 dys fter injetion (dt not shown). At dy 8, -treted mie displyed signifintly inresed sensitivity to mehnil stimultion s mesured y von Frey nlysis (3.61 ± 0.58 g s ompred to 5.31 ± 0.3 g for vehile-treted mie; Supplementry Figure S1). The -treted mie were lso more sensitive to therml stimultion with old wter t dy 8, with lteny deresing to just 19 ± 2% of tht in vehile-treted mie (Supplementry Figure S1). Hypolgesi developed fter further 4 weeks, nd ould e demonstrted in terms of response to old wter stimulus t dy 36 (Supplementry Figure S1). At this time point, lteny in the -treted mie hd inresed to 210 ± 30% of tht in vehile-treted mie. Hypolgesi ws further onfirmed t dy 64 with nother therml stimultion ssy, nmely, hot-plte test (Supplementry Figure S1). Lteny in the -treted mie ws 50 ± 3.5 seonds, whih ws signifintly greter thn tht in vehile-treted mie (33 ± 1.6 seonds). Eletrophysiologi reordings lso doumented signifint slowing of mouse til sensory nerve ondution veloity (SNCV) t dy 115 in treted mie (15.2 ± 1.4 m/s) s ompred to vehile-treted mie (28.4 ± 3.2 m/s; Supplementry Figure S1d). Serum IGF-1 levels in -treted mie were signifintly lower thn those in vehile-treted mie (654 ± 35 ng/ml versus 770 ± 17 ng/ml) t dy 28 (Supplementry Figure S1e). These results re onsistent with the effets oserved fter tretment in rts, nd the dt onfirm tht the tretment prdigm used (see Mterils nd Methods) generted severely dieti mouse with redued irulting IGF-1, n erly hyperlgesi stte followed y lter stte of hypolgesi. Erly tretment with n IGF-1 plsmid inreses serum IGF-1 without orreting hyperglyemi In order to investigte whether we ould ffet the erly hyperlgesi stge of the disese proess y inresing irulting IGF-1, -treted mie were treted with n IGF-1 plsmid (see Mterils nd Methods) t dy 9, fter hyperlgesi ws demonstrted. Figure 1 shows tht serum IGF-1 levels in sline-treted mie ( + sline) were signifintly lower thn those in slinetreted, ge-mthed norml mie (vehile + sline). Liver-sed expression from the IGF-1 plsmid resulted in signifint inreses in serum IGF-1 levels, oth in norml mie (vehile + IGF-1) nd in -treted mie ( + IGF-1). Serum IGF-1 levels delined with ge in prllel in ll the groups. As shown in Figure 1, this elevted systemi IGF-1 ws iotive, s evidened y signifint inreses in the ody msses of the dieti mie ( + IGF-1). Serum IGF-1 (ng/ml) Blood gluose (mg/dl) 1, Dy Dy Body weight (g) Dy Vehile + sline Vehile + IGF-1 + sline + IGF-1 Figure 1 Erly tretment with insulin-like growth ftor 1 (IGF-1) inreses serum IGF-1 levels nd mouse ody mss, ut does not orret lood gluose levels. A plsmid DNA vetor expressing mouse IGF-1 (pdc190-smigf-1, 10 µg/mouse) ws injeted hydrodynmilly into the til veins of vehile-treted or streptozotoin ()-treted mie t dy 9 fter the tretment. () Serum IGF-1, () ody mss, nd () lood gluose levels were mesured t the indited time points. The dt re shown s men ± SEM; n = 5 10 nimls/group. Moleulr Therpy vol. 16 no. 8 ug. 8 11

3 Figure 1 shows tht IGF-1 tretment of -treted mie hd smll trnsient effet on lood gluose [whih, in turn, my hve hd trnsient effet on ody weight (Figure 1)], ut lood gluose remined 500 mg/dl for ll -treted mie, inditing severe hyperglyemi. Sustined high lood gluose levels were onfirmed y hemogloin A1C (HA1) levels. Supplementry Figure S2 shows tht HA1 levels in IGF-1-treted dieti mie (10 ± 0.5%) remined extremely high, nd were not signifintly different from those of dieti mie treted with sline (12 ± 0.3%). Tht is, treting mie with IGF-1 during the hyperlgesi stge of the disese proess resulted in signifint weight gin ut hd little effet on hyperglyemi. Erly IGF-1 tretment prevents lter hypolgesi nd improves moility Hot-plte nd rering tivity ssys were used to evlute the long-term effets of erly IGF-1 tretment on sensory nd motor funtions, respetively. Figure 2 shows tht, when ompred with vehile-treted nimls (33 ± 1.6 seonds), untreted dieti mie t dy 64 fter tretment exhiited signifintly slowed response to therml stimulus (50 ± 3.5 seonds). At this sme time point, dieti mie tht hd een treted with systemi IGF-1 (55 dys fter IGF-1 whih ommened t dy 9 fter tretment) hd response time tht ws indistinguishle from tht of nondieti nimls. Figure 2 shows tht tretment lso resulted in signifint long-term derese in the moility of the mie s mesured y rering tivity. Erly tretment with IGF-1 (from dy 9 fter tretment) resulted in signifint Rering tivity (ounts/15 min) Lteny (s) 60 * * ** * Vehile + sline + IGF 1 + sline + IGF 1 Figure 2 Erly insulin-like growth ftor 1 (IGF-1) tretment prevents hypolgesi nd improves moility. () Response lteny to therml stimultion (hot-plte) ws determined t dy 55 fter injetion of IGF-1 plsmid DNA (pdna) [dy 64 fter streptozotoin () tretment]. Lteny in the + sline group ws signifintly greter thn tht in the two vehile-treted groups (*P < 0.05). Lteny in -treted mie ws normlized y IGF-1 tretment. () Rering tivity ws monitored t dy 67 fter IGF-1 pdna injetion (dy 76 fter tretment). The tivity in the + sline group ws signifintly lower thn in the two vehile-treted groups (**P < 0.01). The dt re shown s men ± SEM, n = 5 10 nimls/group. ttenution of this moility loss. Tken together, these results suggest tht erly IGF-1 tretment prevents the development of longterm hypolgesi nd motor funtion defiits in dieti mie. These funtionl effets of erly IGF-1 tretment were orroorted y histologi findings s shown, for exmple, in Supplementry Figure S3. When ompred with nondieti mie (Supplementry Figure S3), untreted dieti mie showed vuolted Shwnn ells in the sensory nerve fiers (Supplementry Figure S3) t the time they were killed t dy 130 fter tretment. This vuoliztion ws pprent in dorsl spinl nerves t oth the lumr nd srl levels. In ontrst, Shwnn ell vuoliztion ws signifintly ttenuted in dieti mie tht hd een treted with IGF-1 erly in the disese proess (Supplementry Figure S3). Given tht Shwnn ell integrity is ritil for peripherl nerve myelintion nd funtion, these histologi findings re onsistent with the -medited loss of sensory funtion noted in the dieti nimls, nd the ility of IGF-1 to preserve this funtion. The effets of long-term hyperglyemi in dieti mie on tivity (Figure 2) lso orrelted with effets on skeletl musle mss. For exmple, s Supplementry Figure S4 shows, dieti nimls exhiited signifint long-term defiits in oth len nd ft mss s ompred to vehile-treted ontrols. Erly tretment with IGF-1 resulted in signifint inreses in the len, ut not the ft mss of dieti nimls, onsistent with the known noli effets of IGF-1. 15,16 Consistent with these ody-mss results, Supplementry Figure S5 d shows tht IGF-1 tretment prevented the -indued loss of skeletl musle. Supplementry Figure S5e h demonstrtes histologilly, using the tiilis nterior (TA) musle, tht the skeletl musles of dieti nimls were trophied, i.e., the ross-setionl res of individul fiers were redued, nd tht this trophy ws signifintly ttenuted y the erly IGF-1 tretment. After the onset of dieti hypolgesi, effets on ody mss re medited through the IGF-1 reeptor while effets on lood gluose re medited through the IR In order to follow-up these results otined y treting dieti mie with IGF-1 erly in the disese proess, we next investigted whether systemi IGF-1 tretment would prove to e enefiil if delivered lte in the disese proess, i.e., fter the dieti nimls hd developed demonstrle hypolgesi. For these studies, n deno-ssoited virus serotype 8 (AAV8) vetor ering IGF-1 ws dministered t inresing doses t dy 60 fter to generte sustined lood levels of IGF-1. As shown in Figure 3, tretment led to onsistently lower IGF-1 lood levels over time when ompred with the levels in ontrol mie. Treting these dieti mie with inresing doses of AAV-IGF-1 led to dosedependent inreses in serum IGF-1; dose of DNse resistnt prtiles resulted in essentilly norml levels of IGF-1, while dose led to IGF-1 levels pproximtely twofold higher thn norml. At this lte tretment time point (dy 60) the mie weighed signifintly less thn their ontrol ounterprts, nd tretment with inresing doses of AAV-IGF-1 led to dosedependent inreses in ody mss over time (Figure 3). In omprison with ontrols, the -treted dieti mie were severely 12 vol. 16 no. 8 ug. 8

4 Serum IGF-1 (ng/ml) Blood gluose (mg/dl) 1, Body weight (g) Dy Dy Vehile + sline + sline + IGF1 ( ) + IGF1 ( ) + AAVIGF1 ( ) Serum IGF-1 (ng/ml) Blood gluose (mg/dl) 1, Body weight (g) Dy Dy Vehile + sline + sline + 1P + 2P + 3P Dy Dy Figure 3 Lte insulin-like growth ftor 1 (IGF-1) tretment using n deno-ssoited virl (AAV) vetor expressing mouse IGF-1 hs dose-dependent effets on serum IGF-1, ody mss, nd lood gluose levels in streptozotoin ()-treted mie. Inresing doses of AAV-IGF-1 (3 10 9, , or DNse resistnt prtiles/mouse) were dministered intrvenously t dy 60 fter tretment. The lowest dose of vetor ws le to () normlize serum IGF-1 levels, ut hd no effet on () ody mss or () lood gluose levels. The dt re shown s men ± SEM, n = 5 14 nimls/group. hyperglyemi, with sustined lood gluose levels of ~ mg/dl (Figure 3). The lowest dose of AAV-IGF-1 hd essentilly no effet on these lood gluose levels, while inresing doses of vetor resulted in dose-dependent dereses in lood gluose. These lood gluose results were entirely onsistent with prllel HA1C mesurements (Supplementry Figure S6). Tken together, these dt demonstrte tht, when dministered fter the onset of hypolgesi, the lowest dose of AAV-IGF-1 (whih normlized serum IGF-1 levels) hd essentilly no effet on either weight gin or lood gluose. Higher doses of IGF-1 resulted in proportiontely lrger inreses in ody mss nd more signifint dereses in lood gluose s ompred to sline-treted ontrols. In order to gin insight into the reltive roles plyed y the IGF-1 nd IR pthwys in our model, -treted hypolgesi mie were lso treted with inresing doses of insulin (see Mterils nd Methods). Insulin nd IGF-1 n hve qulittively similr ut quntittively dissimilr effets. Eh inds to oth insulin nd IGF-1 reeptors nd n therey medite similr effets, ut the ffinity of eh for its ognte reeptor is signifintly higher. Insulin n lso stimulte the prodution of IGF Indeed, Figure 4 shows tht insulin pellets given to -treted nimls resulted in sustined dose-dependent inreses in serum IGF-1 levels. A single pellet (1P) normlized the serum IGF-1 levels, while three pellets (3P) effetively douled the levels. The 1P dose ws therefore similr to the dose of AAV-IGF1 in terms of its effet in normlizing IGF-1 levels (Figure 3). Importntly, the ility of insulin to inrese serum IGF-1 pers to e limited, s shown y the ft tht the mximum insulin dose (3P) resulted in serum IGF-1 levels tht were signifintly less thn ould e hieved with higher doses of AAV-IGF-1 (Figure 3). Figure 4 Lte tretment with inresing doses of insulin hs dosedependent effets on serum insulin-like growth ftor 1 (IGF-1), ody weight, nd lood gluose levels in streptozotoin ()-treted mie. Inresing doses of insulin (one, two, or three pellets/mouse) were implnted suutneously t dy 64 fter tretment. The lowest dose of insulin () ws le to normlize serum IGF-1 levels, () hd no signifint effet on ody weight, nd () signifintly redued lood gluose levels. The dt re shown s men ± SEM, n = 5 14 nimls/ group. ***P < ompred to the + sline group. The effets of insulin on ody weight were mrginl (Figure 4), with the lowest dose hving essentilly no effet, nd the highest doses inresing ody weight y <%, somewht less thn the inreses seen with AAV-IGF-1 (Figure 3). The lowest dose of insulin signifintly lowered lood gluose (Figure 4), pproximting the effet of the mid-level dose of AAV-IGF-1. Higher insulin doses resulted in progressively lower lood gluose levels, the highest dose often sueeding in normlizing lood gluose, n effet not hieved y even the highest dose of AAV-IGF-1. In ompring the effets of insulin nd AAV-IGF-1 t doses tht normlized serum IGF-1 levels, nmely 1P nd , respetively, it ws seen tht insulin deresed lood gluose signifintly while IGF-1 did not. This finding suggests tht, t these lowest doses, the effets on lood gluose re eing medited through the IR, for whih insulin is the muh more potent lignd. These dt re lso onsistent with the oservtion tht the effets of dded IGF- 1 or insulin on ody weight re medited y IGF-1, presumly through the IGF-1 reeptor (IGF-1R) rther thn through the IR, IGF-1 eing muh more potent thn insulin in this regrd. Dieti hypolgesi responds mximlly to normlized serum IGF-1 levels The effiy of systemi IGF-1 tretment during hypolgesi ws evluted using hot-plte nd SNCV ssys (see Mterils nd Methods). Just prior to IGF-1 tretment (t dy 60 fter tretment), dieti nimls displyed signifintly greter lteny time in the hot-plte ssy s ompred to ontrols (Figure 5). At dy 99, untreted dieti nimls remined hyposensitive y this mesure. In ontrst, in ll the groups treted with IGF-1, lteny ws restored to norml. Importntly, this ws the se even t the Moleulr Therpy vol. 16 no. 8 ug. 8 13

5 lowest dose of AAV-IGF-1, suffiient merely to normlize serum IGF-1 levels (Figure 3), ut there ws no effet on lood gluose or ody mss (Figure 3 nd ). This sensory nerve response (originting in the foot) ppered to e mximl t normlized IGF-1 serum levels. These sensory effets of IGF-1 were onfirmed with results otined from SNCV mesurements. At dy 115 fter tretment (i.e., 55 dys fter IGF-1 tretment), dieti mie displyed signifintly slowed men ondution veloity s ompred to ontrol mie (15.2 ± 4.1 versus 28.4 ± 3.2 m/s) (Figure 5). As with the hot-plte ssy, normlizing of the serum IGF-1 levels ws suffiient to orret this ondution veloity defiit. In order to rrive t etter understnding of the mehnisti origins of these sensory effets of IGF-1 tretment, lteny nd SNCV were lso mesured in -treted mie s funtion of insulin dose. Low dose insulin (1P), whih normlized serum IGF-1 levels (Figure 4) nd deresed lood gluose signifintly (Figure 4), lso normlized the lteny (Figure 5). Higher insulin doses either hd no further effet in reduing lteny (2P), or hd no effet on the -indued inrese in lteny (3P). These effets of insulin on lteny were onsistent with insulin-medited hnges in ondution veloity. For instne, ondution veloities in dieti nimls were normlized t the middle dose (2P), ut tully reverted to lower vlue t the highest insulin dose (3P) (Figure 5d). Perhps the simplest mehnisti interprettion of these results is tht insulin orretion of the hypolgesi generted in the model is medited y its effets on serum IGF-1 levels. Sensory nerve orretion, monitored in terms of oth hot-plte lteny nd SNCV, ws orreted merely y normlizing serum Lteny (s) SNCV (m/s) D60 D99 ** ** ** Vehile + sline + sline *** *** * * + AAV + AAV + AAV ( ) ( )( ) (1p) * (2p) (3p) Figure 5 Impt of insulin-like growth ftor 1 (IGF-1) nd insulin on the therml sensory response nd til sensory nerve ondution veloity (SNCV) in hypolgesi streptozotoin ()-treted mie. () Sensory funtion s mesured y the hot-plte test ws monitored prior to dministrtion of AAV-IGF-1 (dy 60 fter -tretment, open rs) nd t dy 39 fter AAV-IGF-1 dministrtion (dy 99 fter tretment, filled rs). Lteny in -treted nimls ws normlized y IGF-1 tretment, i.e., the ltenies in IGF-1-treted nimls were not signifintly different from those in the vehile + sline ontrol group. () Til SNCV ws mesured t dy 55 fter AAV-IGF-1 dministrtion (dy 115 fter tretment). IGF-1 t ny of the doses ws le to normlize SNCV in -treted mie. () Effets of inresing doses of insulin on lteny. Lower doses normlized lteny, wheres the highest dose hd no effet on lteny. (d) Effets of inresing doses of insulin on SNCV. Mid-level dose (2P) of insulin normlizes ondution veloity; low nd high doses improve, ut do not normlize. The dt re shown s men ± SEM, n = 5 14 nimls/group. AAV, deno-ssoited virus. d IGF-1 levels with AAV-IGF-1 tretment (Figure 3), n effet essentilly duplited t ll insulin doses (Figure 4). However, it is notle tht insulin (ut not low-dose AAV-IGF-1) lso exerted dose-dependent effets on lood gluose. It is lso notle tht the highest insulin dose (3P), whih resulted in osionl hypoglyemi (Figure 4), ws detrimentl to sensory nerves, in tht it oth filed to improve lteny (Figure 5) nd worsened ondution veloity (Figure 5d). Tken together, these dt re onsistent with the interprettion tht dieti hypolgesi n e ounterted y normlizing serum IGF-1 levels, ut tht doing so using insulin runs the risk of episodes of hypoglyemi, rendering this pproh therpeutilly ounterprodutive. 25 The orretion of sensory funtion is orrelted with normlizing the serum IGF-1 levels using either AAV-IGF-1 or insulin rther thn with the ontrol of lood gluose levels, therey suggesting mjor role for the IGF-1R, rther thn the IR in this orretion. Findings from histologi exmintion of dorsl spinl nerves t the lumr nd srl levels were onsistent with the effets of tretment on sensory nerve funtion, s shown in Figure 6. In omprison with the pperne of sensory nerve fiers in norml nimls (Figure 6 nd ), fiers from dieti mie showed vuolted Shwnn ells (Figure 6) nd disrupted myelin sheths (Figure 6d). In ontrst, fiers from dieti mie tht hd een treted with IGF-1 fter developing hypolgesi demonstrted essentilly norml Shwnn ell (Figure 6e) nd myelin (Figure 6f) morphology. These enefiil effets were dosedependent, with 7/7 nimls demonstrting norml morphology t dose of DNse resistnt prtiles, 6/6 t , nd 3/5 t Insulin n lso improve morphology nd myelintion (Figure 6g nd h). However, insulin-medited improvements were notly less omplete thn those otined with IGF-1. These insulin-medited effets ppered to orrelte with the level of serum IGF-1 hieved, gin suggesting tht they re medited y the IGF-1 reeptor. Tken together, these results indite tht treting severely dieti mie with systemi IGF-1 n tully reverse existing hypolgesi. This effet is ttriutle, t lest in prt, to its enefiil effets on Shwnn ells. Importntly, these effets on sensory funtion were mximl when serum IGF-1 levels were merely d Figure 6 Insulin-like growth ftor 1 (IGF-1) tretment ttenutes the histopthologi hnges in the dorsl spinl nerves t the lumr nd srl levels. Setions were proessed s desried in the Mterils nd Methods. Mgnifition = 60. The top pnels (,,e,g) were stined with hemtoxylin nd eosin, the ottom pnels (,d,f,h) were stined for myelin with Luxol fst lue nd hemtoxylin. ( nd ) Vehile + sline, ( nd d) streptozotoin () + sline, (e nd f) + AAV-IGF-1 ( DNse resistnt prtiles/mouse), (g nd h) (3P). Imges shown re representtive of six to eight nimls/group. AAV, deno-ssoited virus. e f g h 14 vol. 16 no. 8 ug. 8

6 Rering tivity (ounts/15 min) Len mss * * ** * d e d Ft mss *** Vehile + sline + sline + AAV + AAV + AAV ( ) ( ) ( ) (1P) (2P) (3P) Figure 7 Insulin-like growth ftor 1 (IGF-1) ut not insulin hs signifint dose-dependent effets on rering tivity nd len ody mss. With ( ) IGF-1 or (d f) insulin tretment, ( nd d) rering tivity, ( nd e) len mss, nd ( nd f) ft mss were mesured t dy 100 fter streptozotoin ()-tretment s desried in Mterils nd Methods. The dt re shown s men ± SEM, n = 5 11 nimls/ group. AAV, deno-ssoited virus. normlized, i.e., n inrese of ~100 ng/ml over IGF-1 levels in dieti mie. Morphologi normliztion ppered to require suprnorml serum IGF-1 levels, whih ould e hieved to muh greter degree with AAV-IGF-1 thn with insulin. Impired motor funtion responds mximlly t suprnorml IGF-1 levels As mesure of motor funtion, rering tivity ws signifintly deresed in dieti mie t dy 100 s ompred to ontrols (Figure 7). Wheres orretion of sensory funtion ws found to our even t the lowest dose of AAV-IGF-1 (see erlier text), defets in rering tivity responded only to higher doses of AAV- IGF-1, i.e., doses t whih serum IGF-1 ws sustined t suprnorml levels (Figure 3). At the highest serum IGF-1 levels, rering tivity ws restored to normly. These effets of IGF-1 on motor funtion were mirrored y its effets on ody weight. The dieti ondition resulted in signifint dereses in oth len mss (Figure 7) nd ft mss (Figure 7). In prllel with the dose-dependent effets of IGF-1 on rering tivity, Figure 7 shows tht the low dose of IGF-1 hd only miniml effet on len mss, while the highest dose restored len mss to t lest norml levels. The effets of IGF-1 were restrited to len mss, nd ft mss ws not ffeted signifintly even t the highest dose of IGF-1 (Figure 7). Insulin lso hd effets on motor funtion nd ody weight, ut even t the highest dose the effets were somewht limited s ompred to the effets of IGF-1 (Figure 7d f). These dt re onsistent with effets medited y insulin s limited ility to inrese serum IGF-1 (Figure 4), rther thn with the effets of insulin per se. The effets of insulin, s shown in Figure 7d f mth those expeted of the intermedite levels of IGF-1 indued y insulin (Figure 7 ). Therefore the pprent noli effets of insulin seen in Figure 7e nd f re likely f Figure 8 Insulin-like growth ftor 1 (IGF-1) tretment of hypolgesi streptozotoin ()-treted mie prevents demyelintion in the ventrl spinl nerves. Setions t the lumr nd srl levels were proessed t dy 130 fter tretment s desried in Mterils nd Methods. Mgnifition = 60. () vehile + sline. () + sline. () + AAV-IGF-1 ( DNse resistnt prtiles/mouse). (d) (3P). Imges shown re representtive of six to eight nimls/group. AAV, deno-ssoited virus. medited y insulin-indued IGF-1 ting through the IGF-1 reeptor, nd not y medition through the IR or y the effets of insulin on gluose levels. In order to onfirm nd extend these pprent effets of IGF-1 on skeletl musle, the TA musle ws exmined in more detil. At the highest dose of AAV-IGF-1, the TA mss of dieti nimls ws restored to norml, onsistent with the effets of IGF-1 in these nimls s mesured y len mss (Supplementry Figure S7). This IGF-1-medited inrese in TA mss is proly ttriutle to the prevention of the trophy tht is seen in the musle fiers of untreted dieti mie, s doumented y histologi exmintion of fier size (Supplementry Figure S8). Consistent with the effets of IGF-1 nd insulin on len mss, insulin, even t the highest dose, ws less effetive in preventing fier trophy thn AAV-IGF-1. It ws found from histologil exmintion tht s ompred to ontrol mie (Figure 8) ventrl spinl ord motor nerve fiers of the dieti mie hd undergone signifint demyelintion y dy 130 fter tretment (Figure 8). It ws shown tht this demyelintion ould e signifintly ttenuted y AAV-IGF-1 tretment during the hypolgesi stge of the disese (Figure 8). This enefiil effet ws dose dependent, with 7/7 mie showing ler improvement t the highest dose, 5/6 t , nd 3/5 t DNse resistnt prtiles/mouse. Insulin ws lso shown to improve motor neuron morphology, ut only three of six mie showed ler improvement t the highest dose. Overll, the mgnitude nd extent of motor neuron morphologil improvement were onsistent with the serum levels of IGF-1 generted y AAV-IGF-1 or insulin. Tken together, these dt support the notion tht, even fter stte of hypolgesi hs een estlished in severly dieti niml, the restortion of serum IGF-1 levels to normly n reverse sensory ut not motor funtion defiits. Suprnorml serum levels of IGF-1 n prevent skeletl musle loss nd further improve motor nerve struture, resulting in preservtion of motor funtion. DISCUSSION The overll gol of these studies ws to evlute the effiy of systemi IGF-1 in rogting the peripherl neuropthy symptoms Moleulr Therpy vol. 16 no. 8 ug. 8 15

7 typilly ssoited with dietes. For this purpose we delivered gene vetors (plsmid nd AAV) to the liver to provide ontinuous supply of dditionl IGF-1 into the irultion. We hve shown tht this strtegy provides enefit in the mouse not only when initited in the erly, hyperlgesi stge of the disese proess ut, surprisingly, even when it is egun in the lter, hypolgesi stge of neuropthy. Our demonstrtion tht inresing the serum IGF-1 levels erly in the disese proess n prevent the lter development of hypolgesi is onsistent with, nd signifintly extends, n erlier oservtion in the rt model tht infusing IGF-1 during the hyperlgesi stge stilizes the degree of hyperlgesi. 17 Our demonstrtion tht inresing the systemi IGF-1 levels fter the onset of hypolgesi n reverse neuronl defiits is onsistent with, nd onsiderly extends, oservtions mde in IGF-1 trnsgeni mie in whih infusing IGF-1 protein reversed nerve ondution veloity defiits. It is importnt to note tht the trnsgeni mouse model did not hve the dded omplition of ongoing severe hyperglyemi, whih is present in oth the model used here nd in humn DPN. Therefore, the results of our study re unique in showing tht neurologil defiits hving their origins in hyperglyemi stte n nonetheless e orreted y IGF-1, even in the presene of ongoing hyperglyemi. Importntly, y using vrile dosing we hve found tht the neurologil omponents of the disese in the mouse respond mximlly nd n e normlized y merely restoring IGF-1 lood levels to normly, even fter frnk hypolgesi hs een demonstrted; skeletl musle fetures of the disese, however, pper to respond etter to suprnorml (pproximtely twofold ove norml) levels of irulting IGF-1. Tht is, the neuronl nd musulr symptoms respond differentilly to the irulting IGF-1 levels. By using inresing doses of insulin in the sme model, we demonstrted tht insulin ould lso orret the hypolgesi ssoited with lte-stge DPN. Interestingly, the degree of orretion y insulin ppered to orrelte with its ility to inrese serum IGF-1 levels. For exmple, dose of insulin tht ws suffiient to normlize serum IGF-1 hd effets on the sensory omponents of DPN (s ssessed y therml nd ondution veloity ssys) tht were equivlent to those of n IGF-1 gene sed pproh tht lso normlized serum IGF-1. Importntly, however, the ility of insulin to orret the motor nd musle defiits ssoited with DPN in this model (s mesured y rering tivity, len mss, nd musle histology) ws reltively limited s ompred to the impt of gene-sed IGF-1 delivery on these prmeters. This outome is entirely onsistent with wht we hve found to e the reltively limited ility of insulin to inrese serum IGF-1 levels s ompred to the IGF-1 gene delivery pproh. The finding tht the neurologil symptoms n e normlized in the hypolgesi stge of the disese proess y restoring irulting IGF-1 levels to normly y either IGF-1 gene delivery or insulin, even in the presene of ongoing severe hyperglyemi, implies tht these effets re likely medited through the IGF-1 reeptor (IGFR) nd not through the IR. Although IGF-1 n ind to oth the IGFR nd the IR, our oservtion of neurologil enefit in the sene of effets on lood gluose lso suggests tht these effets re medited y the higher ffinity intertion of IGF-1 with the IGFR rther thn y its lower ffinity intertion with the IR. An sene of effets of the inresed irulting IGF-1 on lood levels of C-peptide (Supplementry Figure S9) nd VEGF lso supports the rgument tht these effets re medited y IGF-1 itself, rther thn through trophi effets on remining pnreti β-ells 26 or y indiret effets on other growth ftors. 12,13 Although the mehnisms resulting in DPN re unler, Shwnn ells re known to ply very importnt role. 27 Hyperglyemi omined with impired neurotrophi support my use Shwnn ell mitohondril dysfuntion. 28 This dysfuntion, in turn, ould mnifest s vuoliztion, nd use trophy or loss of the nerve fiers tht re normlly myelinted y the Shwnn ells. Dysfuntion of myelinted nerve fiers (suh s Aδ-type fiers) n e deteted y hnges in ondution veloity. 29 Disruption of unmyelinted nerve fiers (suh s C- nd Aβ-type fiers) usully ours erly in DPN nd is often present without eletrophysiologi evidene of nerve dmge. It mnifests initilly s hyperlgesi in response to therml nd physil (suh s von Frey) stimultions, nd is then followed y hypolgesi. The moleulr nd ellulr mehnisms leding to the effets of IGF-1 on DPN re lrgely unknown. Shwnn ells express IGF-1 reeptors, nd their tivtion y IGF-1 hs een shown to promote myelintion. 30 IGF-1 hs een shown to protet ginst Shwnn ell dysfuntion in vitro in the presene of high gluose. 31 Our ttriution of insulin effets to its ility to inrese serum IGF-1, nd the susequent tivtion of IGFR, is entirely onsistent with previous oservtions on the reltive ilities of IGF-1 nd insulin to promote myelintion nd the pprent noninvolvement of insulin signling in this proess. 33 Our results lso demonstrte tht systemi IGF-1 n restore sensory nerve funtion (therml hypolgesi), nd ttenute Shwnn ell vuoliztion nd demyelintion of peripherl nerves loted in the lumr nd srl spinl ord regions, t the juntion of the entrl nd peripherl nervous systems. 36 These results imply tht irulting IGF-1 my ess not only the peripherl regions ut lso the spinl ord regions to protet or even restore funtion nd morphology of these unmyelinted nd myelinted nerve fiers. Given the onstrints of the mouse model, these results my imply tht the sensory omplitions of DPN n e orreted y restoring irulting IGF-1 levels to normly, i.e., without orreting hyperglyemi. While normlizing irulting IGF-1 levels is suffiient to orret the neurologi defiits of hyperglyemi-indued hypolgesi, higher irulting IGF-1 levels lso hve enefiil effets on other orgn systems, suh s the skeletl musle nd kidney (dt not shown). Although insulin n lso orret the sensory neuropthy ssoited with the hypolgesi stge of this proess in the mouse, we show tht the therpeuti window for insulin is reltively nrrow s ompred to tht of IGF-1. This is euse the doses of insulin required for produing therpeuti effets on motor funtion nd skeletl musle lso used intermittent hypoglyemi, whih tully exerted sensory neuropthy. In dietes ptients, n insulin-sed prdigm to tret DPN would pose n inresed risk of hypoglyemi. In ontrst, using IGF-1 s therpeuti gent (delivered either from gene-sed pproh or infused s protein) would pper to hve muh wider sfety window, sine inresing serum IGF-1 twofold in the -treted mouse hd enefiil effets on oth sensory nd motor funtion without the risk of hypoglyemi vol. 16 no. 8 ug. 8

8 In summry, these results show tht, in dieti niml model, merely restoring the depressed irulting IGF-1 levels to normly n hve signifint enefiil effets nd reverse existing hypolgesi, even in the presene of ongoing hyperglyemi. The results therefore provide proof-of-onept tht systemi IGF-1, provided y either gene or protein therpeuti, or y gents tht n serve to upregulte IGF-1 (suh s testosterone) 37 my provide therpeuti enefit in DPN. Mterils nd Methods Plsmid nd AAV vetors. The plsmid vetor ontins the heptoyterestrited DC190 (pdc190) expression ssette desried erlier. 38 Briefly, pdc190 ontins humn serum lumin promoter (nuleotides 486 to +) to whih re ppended two opies of the humn prothromin enhner (nuleotides 9 to 860). This ssette ws inorported into the CpG-redued kone nd the syntheti mouse IGF-1(E) DNA ws inserted to otin pdc190-smigf-1. The IGF-1 expression ssette ws loned into the previrl plsmid paav/sp70 ( derivtive of pav1) 39 within the AAV2 inverted terminl repet sequenes. A frgment of the humn α-1-ntitrypsin intron ws inluded s stuffer sequene to mke the size of the vetor similr to the wild-type AAV2 genome. The DC190- IGF-1 vetor DNA ws pkged into AAV8 psids using stndrd triple trnsfetion protool. The AAV8 pseudotyped vetor ws purified y iodixnol grdient entrifugtion followed y ion exhnge hromtogrphy over HiTrp Q HP Columns (GE Helthre Bio-Siene, Pistwy, NJ). A rel-time TqMn PCR ssy (ABI PRISM 7700) (Applied Biosystems, Foster City, CA) with primers designed to mplify the vetor-speifi ovine growth hormone polydenyltion sequene ws used to determine the onentrtion of virus prtiles ontining genomes. This onentrtion is expressed s DNse resistnt prtiles per ml. Animl studies. Six- to seven-week-old mle C57BL/6N mie were purhsed from Toni (Germntown, NY) nd were limted for 1 week prior to the study. The mie were housed t n AAALAC-redited fility in ordne with the guidelines estlished y the Ntionl Reserh Counil. All experiments were onduted under n pproved Institutionl Animl Cre nd Use Committee protool. The DPN model ws produed with n (Sigm-Aldrih, St. Louis, MO) regime tht ltes the insulin produing β-ells of the pnres nd results in severe dieti ondition. The solution ws freshly mde in 0.1 mol/l sodium itrte uffer (ph 4.5), nd injeted (intrperitonelly) t 170 mg/kg ( µl/ g mouse). Only mie with lood gluose levels 350 mg/dl within 3 dys of tretment were seleted for the study. The plsmid pdc190-smigf-1 ws delivered y hydrodynmi proedure desried erlier. 41,42 Briefly, 10 µg of plsmid per g mouse ws injeted in 2 ml of sline within 4 5 seonds through til vein. AAV8-smIGF-1 ws injeted intrvenously s slow olus in µl of sline t doses desried in the Figures. Inresing doses of insulin were delivered y implnting one, two, or three 7-dy relese insulin pellets (LinShin Cnd, Toronto, Ontrio, Cnd) suutneously t dy 64 fter tretment. Blood gluose ws monitored pproximtely one to two times per week, nd when signifint inrese ws deteted pellets were repled. Blood ws olleted from the oritl venous plexus of the mie under nesthesi (2 3% isoflurne) using miro-hemtorit pillry tues into whole-lood (EDTA ontining) or serum-seprting tues. Whole-lood tues were stored t 4 C witing ssy for HA1C within 24 hours. Serum ws seprted y entrifuging the smples t 10,000g for 10 minutes, nd then stored t 80 C witing ssy for mouse IGF-1, CPII, nd VEGF. Sensory nd motor funtionl ssys. All funtionl tests were performed fter the nimls hd limted to the test room for minutes (lso see Supplementry Mterils nd Methods). Therml sensory nerve funtion ws monitored using Hot-Plte Anlgesi Meter (Columus Instruments, Columus, OH) t 50 C. A single niml ws pled on the hot-plte nd the time tken for noieptive response (shking or liking hind pw) ws mesured; this durtion ws reorded s its lteny to respond. For nimls tht did not respond within 60-seond utoff time, lteny ws defined s 60 seonds. We seleted 50 C euse pilot experiments showed tht the differene in lteny to the het stimulus etween -treted nd ontrol nimls t lter stges of the disese proess (hypolgesi stge) ws more signifint t 50 C thn t 55 C, the stndrd temperture setting for lteny experiments in mie (dt not shown). Nerve ondution veloity ws reorded using n MP150 System with AqKnowledge softwre from Biop Systems (Golet, CA). The mie were nesthetized with isoflurne, nd the SNCV of the udl nerve ws determined s desried. 43 Briefly, stimulting eletrode ws pled t the se of the til nd referene eletrode ws pled 5 mm distl to it. Reording eletrodes were pled on the til 1 nd 2 m distl with respet to the stimulting eletrode. The udl nerve ws stimulted with single squre wve pulse, 0.1 ms in durtion nd 4 volt in intensity. The ltenies of the potentils deteted t the two reording sites fter nerve stimultion were determined (pek to pek), nd the til SNCV ws lulted ordingly (in meters/seond). The entire proedure required <15 minutes. Rering is when the niml rises on its hind legs with its forelegs off the ground. This mesure of motor funtion ws determined s desried erlier. 44 Briefly, individul mie were pled in Plexigls ges surrounded y photoems to pture tivity (Opto-Miro Animl Ativity System; Columus Instruments, Columus, OH). The rering tivity of the mouse (s mesured y reks in photoems t n pproprite height ove the floor of the ge) ws reorded for 15 minutes nd quntified in 5-minute ins. Histology. The mie were killed t the end of the respetive studies, 130 dys fter tretment. The spine t the lumr (L4 6) nd srl (S1 2) levels, nd the TA musle were fixed in 10% neutrl uffer fix solution, emedded in prffin (the spine ws delified efore emedding), nd setioned (3 nd 5 µm for the spine, nd 5 µm for TA) on mirotome using stndrd tehniques. Slides were stined using stndrd proedures, with hemtoxylin nd eosin, or Luxol fst lue nd hemtoxylin for the spine, nd hemtoxylin nd eosin for TA. Luxol fst lue ws used for stining myelin. Quntittive ssys. Serum IGF-1, CPII, nd VEGF were nlyzed using ommerilly ville enzyme-linked immunosorent ssy kits. The rt/mouse IGF-1 enzyme-linked immunosorent ssy kit ws from IDS (Tyne nd Wer, UK), the mouse C-Peptide II enzyme-linked immunosorent ssy kit from Alpo (Slem, NH), nd the mouse VEGF enzymelinked immunosorent ssy kit from R&D Systems (Minnepolis, MN). Blood smples (~ µl) were olleted periodilly from til veins of mie for determining lood gluose using n AuChek Compt Meter (Rohe Dignostis, Indinpolis, IN). HA1C ws nlyzed using HA1C test system (Metrik, Sunnyvle, CA). Len nd ft mss were mesured using the EhoMRI Whole Body Composition Anlyzer (Eho Medil Systems, LLC, Houston, TX). Sttistis/error nlysis. Results ompring two groups were nlyzed using the two-tiled unpired Student s t-test; those ompring multiple groups were nlyzed using one-wy nlysis of vrine followed y Newmn Keuls test. The vlues shown represent mens, nd error rs represent SEM. Asterisks re used to denote results with signifint differenes t the levels of *P < 0.05, **P < 0.01, nd ***P < Aknowledgments We express our thnks to Jonthn Fidler nd Mihel Zho for ssistne with the tivity ssys nd Geoff Akit for help with the nerve ondution veloity experiments. We lso thnk Rihrd Sidmn Moleulr Therpy vol. 16 no. 8 ug. 8 17

9 (Deprtment of Neurology, Hrvrd Medil Shool, Boston, MA) for disussions nd nlyses of Shwnn ell morphology, Elizeth Hutto, Mihel Hwes, Sue Ryn nd Emily Yndl for ssistne with histologi nlyses, nd the Deprtments of Comprtive Mediine nd Vetor Prodution. Finlly, we knowledge Seng H. Cheng, Donn Armentno, nd Cynthi Areeny for helpful disussions. Supplementry mteril Figure S1. tretment results in erly hyper- nd lte hypolgesi with signifintly deresed systemi IGF-1 levels in mie. Figure S2. Erly tretment with IGF-1 does not orret HA1C in mie. Figure S3. Erly IGF-1 tretment prevents Shwnn ell vuoliztion in dorsl spinl nerves. Figure S4. Erly IGF-1 tretment redues dietes-indued loss of len mss. Figure S5. Erly IGF-1 tretment ttenutes dietes-indued skeletl musle loss. Figure S6. IGF-1 tretment t the lowest AAV-IGF-1 dose during hypolgesi hs no effet on HA1C in -treted dieti mie. Figure S7. IGF-1 tretment during hypolgesi prevents skeletl musle loss. Figure S8. IGF-1 tretment during hypolgesi prevents skeletl musle trophy. Figure S9. IGF-1 tretment during hypolgesi does not inrese serum C-peptide levels. Mterils nd Methods. Referenes 1. Gordois, A, Suffhm, P, Sherer, A, Oglesy, A nd Toin, JA (3). The helth re osts of dieti peripherl neuropthy in the US. Dietes Cre 26: Rthur, HM nd Boulton, AJ (7). The neuropthi dieti foot. Nt Clin Prt Endorinol Met 3: Frier, BM (4). Moridity of hypoglyemi in type 1 dietes. Dietes Res Clin Prt 65: S47 S Mohseni, S (1). Hypoglyemi neuropthy. At Neuropthol 102: Leinninger, GM, Vinent, AM nd Feldmn, EL (4).The role of growth ftors in dieti peripherl neuropthy. J Peripher Nerv Syst 9: Apfel, SC, Shwrtz, S, Adornto, BT, Freemn, R, Biton, V, Rendell, M et l. (0). Effiy nd sfety of reominnt humn nerve growth ftor in ptients with dieti polyneuropthy: rndomized ontrolled tril. rhngf Clinil Investigtor Group. JAMA 284: Wellmer, A, Misr, VP, Shrief, MK, Kopelmn, PG nd Annd, P (1). 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