Effect of Inhibition of Angiotensin-Converting Enzyme and/or Neutral Endopeptidase on Neuropathy in High-Fat-Fed C57Bl/6J Mice

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1 Effect of Inhiition of Angiotensin-Converting Enzyme nd/or Neutrl Endopeptidse on Neuropthy in High-Ft-Fed C7Bl/J Mice The Hrvrd community hs mde this rticle openly ville. Plese shre how this ccess enefits you. Your story mtters. Cittion Pulished Version Accessed Citle Link Terms of Use Coppey, Lwrence, Bo Lu, Crig Gerrd, nd Mrk A. Yorek. 01. Effect of inhiition of ngiotensin-converting enzyme nd/or neutrl endopeptidse on neuropthy in high-ft-fed C7Bl/J mice. Journl of Oesity 01:30. doi:.11/01/30 June, 01 1:1:7 PM EDT This rticle ws downloded from Hrvrd University's DASH repository, nd is mde ville under the terms nd conditions pplicle to Other Posted Mteril, s set forth t (Article egins on next pge)

2 Hindwi Pulishing Corportion Journl of Oesity Volume 01, Article ID 30, pges doi:.11/01/30 Reserch Article Effect of Inhiition of Angiotensin-Converting Enzyme nd/or Neutrl Endopeptidse on Neuropthy in High-Ft-Fed C7Bl/J Mice Lwrence Coppey, 1, Bo Lu, 3 Crig Gerrd, 3 nd Mrk A. Yorek 1, 1 Deprtment of Veterns Affirs Iow City Helth Cre System, Iow City, IA, USA Deprtment of Internl Medicine, University of Iow, Iow City, IA, USA 3 In Sue Perlmutter Lortory, Children s Hospitl, Deprtment of Peditrics nd Medicine, Hrvrd Medicl School, Boston, MA 011, USA Correspondence should e ddressed to Mrk A. Yorek, mrk-yorek@uiow.edu Received 1 Ferury 01; Revised 1 August 01; Accepted August 01 Acdemic Editor: Jck A. Ynovski Copyright 01 Lwrence Coppey et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. We hve demonstrted tht treting diet-induced oese (DIO) mice with the vsopeptidse inhiitor ileptril improved neurl function. Vsopeptidse inhiitors lock ngiotensin-converting enzyme (ACE) nd neutrl endopeptidse (NEP) ctivity. We propose tht incresed ctivity of ACE nd NEP contriutes to pthophysiology of DIO. To ddress this issue C7Bl/J mice or mice deficient in NEP were fed high-ft diet nd treted with ileptril, enlpril, ACE inhiitor, or cndoxtril, NEP inhiitor, using oth prevention nd intervention protocols. Endpoints included glucose utiliztion nd neurl function determintion. In the prevention study glucose tolernce ws impired in DIO C7Bl/J mice nd improved with ileptril or enlpril. Sensory nerve conduction velocity, therml nociception, nd intrepiderml nerve fier density were impired in DIO C7Bl/J mice nd improved with ileptril or cndoxtril. In the intervention study only enlpril improved glucose tolernce. Sensory nerve conduction velocity nd intrepiderml nerve fier density were improved y ll three tretments, wheres therml nociception ws improved y ileptril or cndoxtril. In NEP-deficient mice DIO impired glucose utiliztion nd this ws improved with enlpril. Nerve function ws not impired y DIO in NEP-deficient mice. These studies suggest tht ACE nd NEP ply role in pthophysiology ssocited with DIO. 1. Introduction Previously we reported tht deletion of neutrl endopeptidse (NEP) provides protection from oesity- nd dietesinduced neurl complictions [1]. We hve lso shown tht treting oese nd streptozotocin-dietic mice with the vsopeptidse inhiitor ileptril prevented neurl complictions including slowing of nerve conduction velocity, therml hypolgesi, nd decresed intrepiderml nerve fier density []. Vsopeptidse inhiitors re drugs tht simultneously inhiit NEP nd ngiotensin-converting enzyme (ACE) ctivity [3]. Recent studies hve shown incresed expression of ngiotensin-ii-forming enzymes in dipose tissue nd incresed ctivity of the renin-ngiotensin system eing implicted in the development of insulin resistnce nd type dietes []. NEP is found in mny tissues including vsculr nd renl tissue nd its ctivity is incresed y ftty cids nd glucose in humn microvsculr cells [ 9]. In the peripherl nervous system NEP is locted in Schwnn cell memrnes surrounding dorsl root gnglion cells nd nerve fiers [, 11]. NEP degrdes mny vso- nd neuroctive peptides including ntriuretic peptides, drenomedullin, rdykinin, nd clcitonin-gene-relted peptide [1, 13]. Therefore, inhiition of ACE nd NEP ctivity would e expected to improve vsculr nd neurl function. In this regrd we hve demonstrted tht treting type 1 nd type dieticrtsswellsgeneticrtmodelofoesitywith ileptril improves vsculr nd neurl dysfunction [1 1].

3 Journl of Oesity However, little informtion is ville out the effect of vsopeptidse inhiitors in niml models of diet-induced oesity. In order to further elucidte the effects of vsopeptidse inhiitors in peripherl nerve dysfunction ssocited with oesity we exmined the effect of diet-induced oesity on nerve conduction velocity nd therml response ltency in the hindpw of C7Bl/J mice nd mice deficient in NEP treted with ileptril, enlpril, ACE inhiitor, or cndoxtril, NEP inhiitor [1, ].. Mterils nd Methods Unless stted otherwise ll chemicls used in these studies were otined from Sigm Chemicl Co. (St. Louis, MO)..1. Animls. C7Bl/JJ wild-type mice were purchsed from Jckson Lortories. Breeding pirs of neutrl endopeptidse-deficient (NEP / )micewereprovidedydrs.lund Gerrd nd re on the C7Bl/J ckground [17]. These mice hve een red nd colony creted t the Veterns Affirs Medicl Center, Iow City, IA. The C7Bl/J nd NEP / mice were ge-mtched for these studies. Deficiency of NEP ctivity ws confirmed in NEP / mice y mesuring the specific ctivity of NEP in kidney homogentes using the method descried y Ayou nd Melzig [1] with modifiction. Activity of NEP in kidney from C7Bl/J nd NEP / mice ws 0.3 ± 0.0 nd 0.0 ± 0.0 mm 7-mido-3- methylcoumrin (AMC)/min/mg protein, respectively (P < versus C7Bl/J y unpired t-test). This test ws performed on ll mice used in these studies in order to confirm tht NEP ws functionlly knocked out in the NEP / mice. Mice were housed in certified niml cre fcility nd food (Hrln Tekld, no. 7001, Mdison, WI) nd wter were provided d liitum. Adequte mesures were tken to minimize pin or discomfort nd ll of the experiments were conducted in ccordnce with interntionl stndrds on niml welfre nd were complint with ll institutionl nd Ntionl Institutes of Helth guidelines for use of nimls (ACURF protocol 109). The study design consisted of prevention nd intervention protocols. For the prevention protocol C7Bl/J nd NEP / mice t 1 weeks of ge were divided into five groups. One group ws fed stndrd chow diet. A second group ws fed high-ft diet contining gm% ft, gm% protein, nd 1 gm% crohydrte (D11; Reserch Diets, New Brunswick, NJ). The primry source of the incresed ft content in the diet ws soyen oil nd lrd. The control nd high-ft diet contined 0. nd 0.3% sodium chloride, respectively. The verge ft content of the control diet ws. gm% (Hrln Tekld, no. 7001, Mdison, WI). The third through fifth groups were fed the high-ft diet contining ileptril (00 mg/kg in the diet), cndoxtril (30 mg/kg in the diet), or enlpril (00 mg/kg in the diet). These diets were prepred y Reserch Diets nd we provided the drugs. We hve found tht these doses provide mximl inhiition of NEP nd/or ACE ctivity in vivo [, 19]. The experimentl period lsted twelve weeks. For the intervention study the sme five groups of C7Bl/J nd NEP / mice t 1 weeks of ge were fed the control diet (group 1) or high-ft diet (groups ) for 1 weeks. Afterwrds, the four groups of high-ft-fed mice were fed high-ft diet with no dditions (group ) or high-ft diet contining ileptril, cndoxtril, or enlpril (groups 3 ) for 1 weeks. The mice on the control diet remined on the control diet... Intrperitonel Glucose Tolernce Test. After n overnight fst mice were injected with sline solution contining g/kg glucose, i.p. Immeditely prior to the glucose injection nd for 10 minutes fterwrds lood smples were tken to mesure circulting glucose levels with the use of glucose-dehydrogense-sed regent strips (Aviv Accu- Chek, Roche, Mnnheim, Germny) []. Blood smples (0. μl) were tken from til vein tht ws lnced once..3. Therml Nociceptive Response. Therml nociceptive response in the hindpw ws mesured using the Hrgreves method with instrumenttion provided y IITC Life Science, Woodlnd Hills, CA (model 390G) s previously descried []. The test ws performed in lind mnner. Therml nociceptive responses were mesured y plcing the mouse in the oservtion chmer on top of the therml testing pprtus nd llowing it to cclimte to the wrmed glss surfce (33 C) nd surroundings for period of 1 min. The moile het source ws mneuvered so tht it ws under the hel of the hindpw nd then ctivted, process tht strts timer nd loclly wrms the glss surfce, when the mouse withdrew its pw, the timer, nd the het source ws turned off []. Following n initil recording, which ws discrded, two mesurements were mde for ech hindpw, with rest period of min etween ech set of mesurements. The men of the mesurements, reported in seconds, ws used s mesure of the therml nociceptive response ltency... Sensory Nerve Conduction Velocity. Mice were nesthetized with Nemutl (7 mg/kg, i.p., Aott Lortories, North Chicgo, IL) nd sensory nerve conduction velocities were determined s previously descried [1, ]. Briefly, sensory nerve conduction velocity ws recorded in the digitl nerve to the second toe y stimulting with squre-wve pulse of 0.0 ms durtion using the smllest intensity current tht resulted in mximl mplitude response (Grss S Stimultor; Grss Medicl Instruments, Quincy, MA). The sensory nerve ction potentil ws recorded ehind the medil mlleolus. The mximl sensory nerve conduction velocity ws clculted y mesuring the ltency to the onset/pek of the initil negtive deflection nd the distnce etween stimulting nd recording electrodes (mesured in millimeters using Vernier cliper). The sensory nerve conduction velocity ws reported in meters per second... Intrepiderml Nerve Fier Density. Immunorective intrepiderml nerve fier profiles were visulized using confoclmicroscopy s previouslydescried []. Biopsies of skin of the right hindpw were fixed, dehydrted, nd emedded in prffin. Sections (7μm) were collected nd immunostined with nti-pgp9. ntiody (rit nti-humn no.

4 Journl of Oesity 3 Tle 1: Prevention study in C7Bl/ nd NEP / mice. Effect of high ft diet +/ ileptril, cndoxtril, or enlpril on chnge in ody weight, lood glucose, nd epididyml ft mss. Determintion High ft High ft + ileptril High ft + cndoxtril High ft + enlpril C7Bl/ mice (1) (11) (1) (1) () Strt ody weight (g) 7. ± 0..7 ± ± 0..3 ± ± 0. End ody weight (g) 3. ± 0..9 ± ± 0.9,c.3 ± ± 0.,c Blood glucose (mg/dl) 1 ± 7 13 ± 1 ± 1 ± ± Epididyml ft pd (g) 0.31 ± ± ± 0.0,c 1.13 ± ± 0.0,c Ft pd/finl ody weight (%) 0.9 ± ± ± 0.1,c. ± ± 0.07,c NEP / mice (1) (1) (1) (1) (1) Strt ody weight (g). ± 0.. ± 0.. ± 0..1 ± ± 0. End ody weight (g) 9.1 ± 0..1 ± ± 1.1,c 3. ± ± 0.,c Blood glucose (mg/dl) 139 ± 13 ± 1 ± 173 ± 1 13 ± Epididyml ft pd (g) 0. ± ± ± 0.0,c 0.90 ± ± 0.0,c Ft pd/finl ody weight (%) 0.0 ± ± ± 0.07,c.13 ± ± 0.0,c Dt re presented s the men ± SEM. P < 0.0 compred to control; P < 0.0 compred to high ft; c P < 0.0 compred to cndoxtril. Prentheses indicte the numer of experimentl nimls in ech group (this ntiody cross-rects with rt, mouse guine pig, nd other species), AD Serotic, Morpho Sys US Inc., Rleigh, NC) overnight followed y tretment with secondry ntiody Alex Fluor got nti-rit (Invitrogen, Eugene, OR). Profiles were imged using Zeiss 7 confocl microscope with 0x ojective nd were counted y two individul investigtors tht were linded to the smple identity. All immunorective profiles within the epidermis were counted nd normlized to epiderml length [0]. Length of the epidermis ws determined y drwing polyline long the contour of the epidermis nd recording its length in mm. The numer of intrepiderml nerve fier profiles ws reported per mm length... Dt Anlysis. The results re presented s men ± SE. Comprisons etween the groups for ody weight, lood glucose, sensory nerve conduction velocity, therml nociception, nd intrepiderml nerve fier profiles were conducted using one-wy ANOVA nd Bonferroni s test for multiple comprisons (Prism softwre; GrphPd, Sn Diego, CA). A P vlue of less 0.0 ws considered significnt. 3. Results 3.1. Effect of High-Ft Diet nd Tretment on Weight nd Blood Glucose Levels. Presented in Tle 1 re weight nd lood glucose chnges for C7/BlJ nd NEP / mice used in the prevention study. At 1 weeks of ge the eginning ody weights of C7Bl/J nd NEP / mice were similr. When fed high-ft diet C7Bl/J nd NEP / mice oth gined similr mount of weight. Treting the high-ft diet with ileptril or enlpril ut not cndoxtril completely prevented the gin in weight. The mss of the epididyml ft pd ws significntly incresed in high-ft-fed mice nd mice fed the high-ft diet treted with cndoxtril compred to control mice or mice fed the high-ft diet contining ileptril or enlpril. When clculting the epididyml ft pd mss s percent of totl ody weight epididyml ft pd mss ws significntly incresed in high-ft-fed C7Bl/J mice nd mice fed high-ft diet contining cndoxtril compred to control. Treting the high-ft diet with ileptril nd to greter extent enlpril prevented the increse in epididyml ft pd mss when presented s percent of finl ody weight. Similr results were otined with NEP / mice. Nonfsting lood glucose levels were not significntly different etween C7Bl/J nd NEP / mice fed the control or high-ft diets nd not ffected y ileptril, cndoxtril, or enlpril tretment. In the intervention study ll mice fed the high-ft diet for the initil 1 weeks of the experimentl design gined significnt mount of weight (Tle ). When trnsferred to high-ft diet contining ileptril or enlpril for n dditionl 1 weeks C7Bl/J nd NEP / mice lost weight. Mice remining on the high diet or high-ft diet contining cndoxtril continued to gin weight. The epididyml ft mss decresed fter C7Bl/J or NEP / mice, fed diet contining high ft for 1 weeks, were given high-ft diet contining enlpril. The epididyml ft mss lso ws decresed fter NEP / mice ut not C7Bl/J mice were fed high-ft diet treted with ileptril. Treting high-ft-fed C7Bl/J or NEP / mice with cndoxtril did not reduce epididyml ft mss. A similr trend ws oserved when the epididyml ft mss dt ws presented s percentge of finl ody weight. Nonfsting lood glucose levels were not chnged in C7Bl/J or NEP / mice fed control or high-ft diets with or without ileptril, cndoxtril, or enlpril. 3.. Effect of High-Ft Diet nd Tretment on Glucose Tolernce. In the prevention study C7Bl/J (Figure 1) nd NEP / (Figure ) mice fed the high-ft diet or high-ft diet contining cndoxtril hd n impired glucose clernce curve compred to control mice. In contrst, the glucose

5 Journl of Oesity Tle : Intervention study in C7Bl/ nd NEP / mice. Effect of high ft diet +/ ileptril, cndoxtril, or enlpril on chnge in ody weight, lood glucose, nd epididyml ft mss. Determintion High ft High ft + ileptril High ft + cndoxtril High ft + enlpril C7Bl/ mice (3) (3) (1) (11) (1) Strt Body Weight (g) 7.3 ± ± 0.. ± ± ± 0. 1 week Weight (g) 31. ± ± 0.. ± 1.. ± 1.. ± 1.3 End Body Weight (g) 3. ± ± 1.1,d 3.3 ± 1.,. ± 1.,d 33.7 ± 0.9,d Blood glucose (mg/dl) 170 ± 9 1 ± 11 1 ± 9 19 ± ± Epididyml ft pd (g) 0. ± ± ± ± ± 0.0 Ft pd/finl ody weight (%) 1.3 ± ± 0.11,c.3 ± 0.1, 1.7 ± ± 0.1 c NEP / mice (9) () (9) () () Strt Body Weight (g). ± ± ± 1.. ± 1.1. ± 0. 1 week Weight (g).0 ± ± ± ± ± 1. End Body Weight (g) 30. ± ± 1.9,d 9. ± ± 1.,d 30. ± 1.0 Blood glucose (mg/dl) 11 ± 7 17 ± ± 13 ± 1 ± 13 Epididyml ft pd (g) 0.3 ± ± ± ± ± 0.0 Ft pd/finl ody weight (%) 0.73 ± ± ± ± ± 0.19 Dt re presented s the men ± SEM. P < 0.0 compred to control; P < 0.0 compred to high ft; c P < 0.0 compred to ileptril; d P < 0.0 compred to 1 week nimls. Prentheses indicte the numer of experimentl nimls in ech group. clernce curve ws ner norml in C7Bl/J nd NEP / mice fed high-ft diet contining ileptril or enlpril. In the intervention study feeding C7Bl/J (Figure 3) or NEP / (Figure ) mice for weeks high-ft diet cused n impired glucose clernce curve. Treting high-ft-fed C7Bl/J or NEP / mice with enlpril for the lst 1 weeks of the -week period prtilly corrected glucose utiliztion. Treting NEP / mice with ileptril lso prtilly corrected glucose utiliztion (Figure ). Treting C7Bl/J mice with ileptril did not improve glucose utiliztion nor did treting C7Bl/J or NEP / with cndoxtril Effect of High-Ft Diet on Sensory Nerve Conduction Velocity, Therml Nociception, nd Intrepiderml Nerve Fier Profile Density. Dt in Figure (left) demonstrte tht sensory nerve conduction velocity ws significntly decresed in high-ft-fed C7Bl/J mice compred to control mice nd tht this ws prevented y treting high-ft-fed mice with Ileptril or cndoxtril using prevention protocol. Dt in Figure (center) demonstrte tht therml nociception ws significntly decresed in C7Bl/J mice fter 1 weeks of high-ft diet compred to control mice s indicted y n increse in pw withdrwl ltency. Tretment with ileptril or cndoxtril nd to lesser extent enlpril for the 1-week period prevented therml hypolgesi in highft-fed C7Bl/J mice. Dt in Figure (right) demonstrte tht the numer of intrepiderml nerve fier profiles ws significntly decresed in high-ft-fed C7Bl/J mice nd tht tretment of these mice with ileptril or cndoxtril ut not enlpril for 1 weeks prevented the significnt decrese. Sensory nerve conduction velocity, therml nociception, nd intrepiderml nerve fier density were not impired in NEP / mice fed high-ft diet (Figure, left, center, nd right, resp.). We lso determined the effect of treting high-ft-fed C7Bl/J nd NEP / mice using n intervention protocol (Figures 7 nd, resp.). In this study design high-ft-fed mice were untreted for 1 weeks followed y 1 weeks of high-ft diet with or without tretment. Feeding C7Bl/J mice high-ft diet for weeks cused significnt decrese in sensory nerve conduction velocity (Figure 7, left). Treting these mice for the lst 1 weeks of the -week period with ileptril, cndoxtril, or enlpril reversed the slowing of sensory nerve conduction velocity. The decrese in therml nociception in high-ft-fed C7Bl/J mice ws reversed y treting mice with ileptril or cndoxtril ut not enlpril (Figure 7, center). Treting high-ft-fed C7Bl/J mice with ileptril, cndoxtril, or enlpril reversed the decrese in intrepiderml nerve fier profiles (Figure 7, right). Feeding NEP / mice high-ft diet for weeks with or without tretment hd no effect on sensory nerve conduction velocity, therml nociception, or intrepiderml nerve fier density (Figure, left, center, nd right, resp.).. Discussion The min findings from these studies were tht dietinduced oesity cused significnt weight gin nd impired glucose utiliztion in C7Bl/J nd NEP / mice tht ws improved when the mice were treted with ileptril or enlpril using prevention protocol. Using the intervention protocol enlpril ut not ileptril tretment ws effective improving glucose utiliztion y C7Bl/J mice. Treting C7Bl/J or NEP / mice with cndoxtril did not improve

6 Journl of Oesity Blood glucose (mg/dl) Blood glucose (mg/dl) Time (min) HF HF/ileptril HF/cnd HF/enl Figure 1: Plsm glucose concentrtion curve during intrperitonel glucose tolernce test (IPGTT) for C7Bl/J mice fed norml or high-ft-contining diet with or without ileptril, cndoxtril, or enlpril for 1 weeks (prevention protocol). Dt re the men ± SEM in mm. Fsting sl lood glucose levels were 11 ±, 13 ± 9, 13 ±, 17 ±, nd 11 ± 7 mg/dl for control, high ft, high ft + ileptril, high ft + cndoxtril, nd high ft + enlpril, respectively. (closed circles), high ft (open circles), high ft + ileptril (closed innervted tringle), high ft + cndoxtril (open tringle), nd high ft + enlpril (closed rectngle). The re under the curve (AUC) ws significntly different P<0.01 for high-ft-fed mice versus control nd highft-fed mice treted with cndoxtril versus control. There ws no significnt difference for AUC etween mice fed high-ft diet contining ileptril or enlpril versus control. The numer of mice in ech group ws the sme s shown in Tle Time (min) HF HF/ileptril HF/cnd HF/enl Figure : Plsm glucose concentrtion curve during intrperitonel glucose tolernce test (IPGTT) for NEP / mice fed norml or high-ft-contining diet with or without ileptril, cndoxtril, or enlpril for 1 weeks (prevention protocol). Dt re the men ± SEM in mm. Fsting sl lood glucose levels were 113 ±, 11 ± 3, 10 ±, 1 ±, nd 119 ± mg/dl for control, high ft, high ft + ileptril, high ft + cndoxtril, nd high ft + enlpril, respectively. (closed circles), high ft (open circles), high ft + ileptril (closed innervted tringle), high ft + cndoxtril (open tringle), nd high ft + enlpril (closed rectngle). The re under the curve (AUC) ws significntly different P < 0.01 nd P<0.0 for high-ft-fed mice versus control nd high-ft-fed mice treted with cndoxtril versus control, respectively. There ws no significnt difference for AUC etween mice fed high-ft diet contining ileptril or enlpril versus control. The numer of mice in ech group ws the sme s shown in Tle 1. weight gin or glucose utiliztion. These dt suggest tht inhiition of ACE nd not NEP ctivity ws responsile for improving weight gin nd glucose utiliztion in highft-fed mice. Diet-induced oesity lso cused slowing of sensory nerve conduction velocity, therml hypolgesi, nd decrese in epiderml nerve fier density in the pw of the hindlim in C7Bl/J mice ut not NEP / mice. The impirment in sensory nerve function endpoints in C7Bl/J mice fed high-ft diet ws prevented/improved when the mice were treted with ileptril or cndoxtril nd to much lesser extent enlpril using either the prevention or intervention protocol. These dt suggest tht incresed NEP ctivity/expression ut not ACE ctivity contriutes to dietinduced oesity-relted deficits in sensory nerve function. Previous studies hve shown tht diet-induced oesity in rodent models cn e prevented y ACE inhiitors nd ngiotensin II receptor lockers [1 ]. In ddition, dietinduced weight gin nd ft mss re reduced, energy expenditure incresed, nd glucose tolernce improved in mice lcking ACE or the ngiotensin II type 1 receptor [, ]. The mechnisms proposed for the improvement in oesity nd glucose tolernce with tretment of rodent models with ACE inhiitors re incresed energy expenditure, liver nd dipose tissue metolic modultion, lower concentrtion of leptin, improved insulin signling, nd incresed glucose nd ftty cid utiliztion y muscle [1 30]. In study compring the effects of Rmipril, n ACE inhiitor, to ileptril in JCR:LA-cp rts, n oese, insulin-resistnt, hyperinsulinemic, normoglycemic model, it ws found tht oth compounds reduced the surge of plsm insulin in mel tolernce test y out 0% ut ileptril ws more eneficil in improving vsculr rectivity [31]. In our study we found tht enlpril trended to e more effective thn ileptril preventing/reducing ft pd mss. This could e ecuse enlpril t the dosge used ws more effective ACE inhiitor thn ileptril in trget tissues. In nother study using oese Zucker rts it ws found tht dul inhiition of ACE nd NEP improved insulin-medited glucose disposl more effectively thn monotherpy nd this effect ws linked to incresed ctivtion of the kininnitric oxide pthwy [3]. In similr independent study it ws found tht Omptrilt, vsopeptidse inhiitor, induced insulin sensitiztion nd incresed myocrdil glucose uptke in oese Zucker rts nd tht the effect of Omptrilt ws greter thn Rmipril in prt due to stimultion of the B receptor [33]. Lter this group reported tht tretment

7 Journl of Oesity Blood glucose (mg/dl) Blood glucose (mg/dl) Time (min) HF HF/ileptril HF/cnd HF/enl Time (min) HF HF/ileptril HF/cnd HF/enl Figure 3: Plsm glucose concentrtion curve during intrperitonel glucose tolernce test (IPGTT) for C7Bl/J mice fed norml or high-ft-contining diet for weeks nd the high-ft diet with or without ileptril, cndoxtril, or enlpril for the lst 1 weeks (intervention protocol). Dt re the men ± SEM in mm. Fsting sl lood glucose levels were 111 ±, 1 ±, 177 ± 11, 17 ±, nd 13 ± mg/dl for control, high ft, high ft + ileptril, high ft + cndoxtril, nd high ft + enlpril, respectively. (closed circles), high ft (open circles), high ft + ileptril (closed innervted tringle), high ft + cndoxtril (open tringle), nd high ft + enlpril (closed rectngle). The re under the curve (AUC) ws significntly different P < 0.0 for high-ft-fed mice versus control nd high-ft-fed mice treted with ileptril versus control. There ws no significnt difference for AUC etween mice fed high-ft diet contining cndoxtril or enlpril versus control. The numer of mice in ech group ws the sme s shown in Tle. Figure : Plsm glucose concentrtion curve during intrperitonel glucose tolernce test (IPGTT) for NEP / mice fed norml or high-ft-contining diet for weeks nd the high-ft diet with or without ileptril, cndoxtril, or enlpril for the lst 1 weeks (intervention protocol). Dt re the men ± SEM in mm. Fsting sl lood glucose levels were 1 ± 7, 131 ±, 13 ±, 10 ±, nd 130 ± 7 mg/dl for control, high ft, high ft + ileptril, high ft + cndoxtril, nd high ft + enlpril, respectively. (closed circles), high ft (open circles), high ft + ileptril (closed innervted tringle), high ft + cndoxtril (open tringle), nd high ft + enlpril (closed rectngle). The re under the curve (AUC) ws significntly different P < 0.01 for high-ft-fed mice versus control nd high ft-fed-mice treted with cndoxtril versus control. There ws no significnt difference for AUC etween mice fed high ft-diet contining ileptril or enlpril versus control. The numer of mice in ech group ws the sme s shown in Tle. of oese Zucker rts with vsopeptidse inhiitor incresed muscle glucose uptke independent of insulin signling [3]. In two of these studies protection of rdykinin from degrdtion y NEP ws found to improve insulin ction [3, 33]. Interestingly, it hs een shown tht ntriuretic peptides promote muscle mitochondril iogenesis nd ft oxidtion s to prevent oesity nd glucose intolernce [3]. The ntriuretic peptides re lso degrded y NEP [3]. Becuse NEP is expressed in skeletl muscle in reltively lrge mounts nd eing locted on the cell surfce, NEP is le to hydrolyze peptides in the vicinity of their receptors therey neutrlizing their ioctivity [3, 3]. However, inhiition of ACE my lso led to protection of rdykinin levels y inhiiting kininse-ii-medited degrdtion of this nonpeptide [37]. Henriksen et l. [3] reported tht ACE inhiition y cptopril improved glucose trnsport in insulin-resistnt muscle of the oese Zucker rt. They ttriuted the improvement to modultion of insulin ction y rdykinin medited through B receptors nd y n increse in nitric oxide production. Since rdykinin nd ntriuretic peptides my hve role in enhncing insulin ction nd regulting glucose nd ftty cid metolism y muscle protecting their ioctive function y preventing degrdtion through inhiition of ACE nd/or NEP my e therpeutic pproch for tretment of oesity nd insulin resistnce [3, 3, 3]. We hve previously shown tht treting dietic rts with enlpril improved vsculr nd nerve endpoints ut enlpril ws less effective in rts fed high-ft diet [19, 3, 39, 0]. In contrst, we found tht treting oese or dietic rts with ileptril ws more effective thn enlpril in improving peripherl nerve dysfunction [19, 3, 0]. This ws ttriuted to e due to improvement in vsculr function. We hve shown tht treting dietic or dietinduced oese rodents with ileptril improves vsodiltion of lood vessels tht provide circultion to the scitic nerve y reducing oxidtive stress nd preventing degrdtion of vsoctive peptides y NEP including clcitonin gene-relted peptide nd C-type ntriuretic peptide therey mintining endoneuril lood flow nd preventing ischemi [3]. Since NEP degrdes oth clcitonin gene-relted peptide nd sustnce P, peptides importnt in pin signling pthwys,

8 Journl of Oesity SNCV (m/s) 0 1 Therml nociception (s) 1 IENF (profiles/mm) 1 1 C7BI/ prevention High ft High ft + ileptril High ft + cndoxtril High ft + enlpril Figure : Sensory nerve conduction velocity, therml nociception, nd intrepiderml nerve fier profiles for C7Bl/J mice fed norml or high-ft-contining diet with or without ileptril, cndoxtril, or enlpril for 1 weeks (prevention protocol). (open r), high ft (right htch r), high ft + ileptril (left htch r), high ft + cndoxtril (cross htch r), nd high ft + enlpril (closed r). Dt re the men ± SEM. The numer of mice in ech group ws the sme s shown in Tle 1.,P<0.0 versus control;, P<0.0 versus high-ft-fed mice SNCV (m/s) 0 1 Therml nociception (s) 3 IENF (profiles/mm) NEP / prevention High ft High ft + ileptril High ft + cndoxtril High ft + enlpril Figure : Sensory nerve conduction velocity, therml nociception, nd intrepiderml nerve fier profiles for NEP / mice fed norml or high-ft-contining diet with or without ileptril, cndoxtril, or enlpril for 1 weeks (prevention protocol). (open r), high ft (right htch r), high ft + ileptril (left htch r), high ft + cndoxtril (cross htch r), nd high ft + enlpril (closed r). Dt re the men ± SEM. The numer of mice in ech group ws the sme s shown in Tle 1.,P<0.0 versus control;, P<0.0 versus high-ft-fed mice.

9 Journl of Oesity SNCV (m/s) 0 1 Therml nociception (s) IENF (profiles/mm) 1 C7BI/ intervention High ft High ft + ileptril High ft + cndoxtril High ft + enlpril Figure 7: Sensory nerve conduction velocity, therml nociception, nd intrepiderml nerve fier profiles for C7Bl/J mice fed norml or high-ft-contining diet for weeks with or without ileptril, cndoxtril, or enlpril for the lst 1 weeks (intervention protocol). (open r), high ft (right htch r), high ft + ileptril (left htch r), high ft + cndoxtril (cross htch r), nd high ft + enlpril (closed r). Dt re the men ± SEM. The numer of mice in ech group ws the sme s shown in Tle., P<0.0 versus control;, P<0.0 versus high-ft-fed mice SNCV (m/s) 0 1 Therml nociception (s) 3 IENF (profiles/mm) NEP / prevention High ft High ft + ileptril High ft + cndoxtril High ft + enlpril Figure : Sensory nerve conduction velocity, therml nociception, nd intrepiderml nerve fier profiles for NEP / mice fed norml or high-ft-contining diet for weeks with or without ileptril, cndoxtril, or enlpril for the lst 1 weeks (intervention protocol). (open r), high ft (right htch r), high ft + ileptril (left htch r), high ft + cndoxtril (cross htch r), nd high ft + enlpril (closed r). Dt re the men ± SEM. The numer of mice in ech group ws the sme s shown in Tle.,P<0.0 versus control;, P<0.0 versus high-ft-fed mice.

10 Journl of Oesity 9 it is likely tht locking NEP ctivity or mice deficient in NEP would mintin higher levels of oth of these neuroctive peptides nd perhps e more sensitive to pinful stimuli.. Conclusion Dt from clinicl trils clerly suggest tht ctivtion of the renin-ngiotensin-ldosterone system plys n importnt role in the pthophysiology of the metolic syndrome through interction of wide rnge of physiologicl nd moleculr mechnisms [1]. More recently in humns ctivity of NEP hs een shown to correlte with ody mss index nd mesures of insulin resistnce with incresing ctivity found in sujects with multiple crdiovsculr risk fctors []. In these studies we found tht feeding mice highft diet cuses weight gin, impired glucose tolernce, nd sensory nerve dysfunction. Inhiition of ACE ws effective in reducing weight gin nd improving glucose utiliztion ut generlly noneffective in improving sensory nerve dysfunction. In contrst, inhiition of NEP improved sensory nerve dysfunction nd hd no effect in improving weight gin or glucose utiliztion. Treting high-ft-fed mice with vsopeptidse inhiitor improved weight gin, glucose utiliztion, nd sensory nerve function. Thus, we conclude tht incresed ctivity of NEP is ssocited with sensory nerve dysfunction in n niml model of diet-induced oesity nd tht dul inhiition of ACE nd NEP my e more effective thn monotherpy in reducing insulin resistnce nd the sensory nerve complictions ssocited with metolic syndrome. Acknowledgments This mteril is sed upon work supported y reserch grnt from the Juvenile Dietes Reserch Foundtion, Deprtment of Veterns Affirs, Veterns Helth Administrtion, Office of Reserch nd Development, Biomedicl Lortory Reserch nd Development (BX0010) nd NIH Awrd DK The content of this pper is solely the responsiility of the uthors nd does not necessrily represent the officil views of the grnting gencies. The uthors hve no conflict of interestes to report. The uthors would like to extend their pprecition to Snofi Aventis, MERCK, nd Pfizer for supplying ileptril, enlpril, nd cndoxtril, respectively, for these studies. Snofi Aventis, MERCK, or Pfizer did not hve ny involvement in the preprtion or decision to sumit this pper. References [1] E.P.Dvidson,L.J.Coppey,B.Luetl., Therolesofstreptozotocin neurotoxicity nd neutrl endopeptidse in murine experimentl dietic neuropthy, Experimentl Dietes Reserch, vol. 009, Article ID 3190, 009. [] L. J. Coppey, E. P. Dvidson, B. Lu, C. Gerrd, nd M. A. Yorek, Vsopeptidse inhiitor ileptril (AVE7) prevents oesity- nd dietes-induced neuropthy in C7Bl/J mice, Neurophrmcology, vol. 0, no. -3, pp. 9, 011. [3] M. Weer, Emerging tretments for hypertension: potentil role for vsopeptidse inhiition, Americn Journl of Hypertension, vol. 1, no. 11, prt, pp. 139S 17S, [] A. M. Shrm, Is there rtionle for ngiotensin lockde in the mngement of oesity hypertension? Hypertension, vol., no. 1, pp. 1 19, 00. [] H. Vtter, L. Schilling, P. Schmiedek, nd H. Ehrenreich, Evidence for functionl endothelin-converting enzyme ctivity in isolted rt silr rtery: effect of inhiitors, Journl of Crdiovsculr Phrmcology, vol. 31, no. 1, pp. S S7, 199. [] W. Gonzlez, J. M. Soleilhc, M. C. Fournié-Zluski, B. P. Roques, nd J. B. Michel, Chrcteriztion of neutrl endopeptidse in vsculr cells, modultion of vsoctive peptide levels, Europen Journl of Phrmcology, vol. 3, no. 3, pp , 199. [7] R. M. Edwrds, M. Pullen, nd P. Nmi, Distriution of neutrl endopeptidse ctivity long the rt nd rit nephron, Phrmcology, vol. 9, no. 1, pp. 0, [] F. Eihr, G. Seno Di Mrco, M. A. Julino, nd D. E. Csrini, Neutrl endopeptidse expression in mesngil cells, Journl of the Renin-Angiotensin-Aldosterone System, vol., no., pp. 33, 003. [9] P. Mungmn, M. L. Spenny, R. N. Tmur, nd N. S. Girn, Ftty cids nd glucose increse neutrl endopeptidse ctivity in humn microvsculr endothelil cells, Shock, vol. 19, no., pp. 0 1, 003. [] R. Mtss, A. J. Kenny, nd A. J. Turner, An immunohistochemicl study of endopeptidse-.11 ( enkephlinse ) in the pig nervous system, Neuroscience, vol. 1, no., pp , 19. [11] C. Kioussi, A. Mmlki, K. Jessen, R. Mirsky, L. B. Hersh, nd R. Mtss, Expression of endopeptidse-.11 (common cute lympholstic leukemi ntigen CD) in the scitic nerve of the dult rt fter lesion nd during regenertion, Europen Journl of Neuroscience, vol. 7, no., pp , 199. [1] Q. Pu nd E. L. Schiffrin, Effect of ACE/NEP inhiition on crdic nd vsculr collgen in stroke-prone spontneously hypertensive rts, Americn Journl of Hypertension, vol. 1, no., pp. 7 7, 001. [13] J. S. Flors, Vsopeptidse inhiition: novel pproch to crdiovsculr therpy, Cndin Journl of Crdiology, vol. 1, no., pp , 00. [1] E. P. Dvidson, T. L. Kleinschmidt, C. L. Oltmn, D. D. Lund, nd M. A. Yorek, Tretment of streptozotocin-induced dietic rts with AVE7, vsopeptidse inhiitor: effect on vsculr nd neurl disese, Dietes, vol., no., pp. 3 3, 007. [1] C. L. Oltmn, E. P. Dvidson, L. J. Coppey, T. L. Kleinschmidt, nd M. A. Yorek, Tretment of Zucker dietic ftty rts with AVE7 improves vsculr nd neurl dysfunction, Dietes, Oesity nd Metolism, vol. 11, no. 3, pp. 3 33, 009. [1] E. P. Dvidson, L. J. Coppey, T. L. Kleinschmidt, C. L. Oltmn, nd M. A. Yorek, Vsculr nd neurl dysfunctions in oese Zucker rts: effect of AVE7, Experimentl Dietes Reserch, vol. 009, Article ID 9137, 009. [17] B. Lu, N. P. Gerrd, L. F. Kolkowski Jr. et l., Neutrl endopeptidse modultion of septic shock, Journl of Experimentl Medicine, vol. 11, no., pp. 71 7, 199. [1] S. Ayou nd M. F. Melzig, Induction of neutrl endopeptidse (NEP) ctivity of SK-N-SH cells y nturl compounds from green te, Journl of Phrmcy nd Phrmcology, vol., no., pp. 9 01, 00.

11 Journl of Oesity [19] E.P.Dvidson,L.J.Coppey,B.Dke,ndM.A.Yorek, Effect of tretment of sprgue dwley rts with AVE7, enlpril or cndoxtril on diet induced oesity, Journl of Oesity, vol. 011, Article ID 9, 011. [0] K. K. Beiswenger, N. A. Clcutt, nd A. P. Mizisin, Epiderml nerve fier quntifiction in the ssessment of dietic neuropthy, Act Histochemistry, vol. 1, no., pp. 31 3, 00. [1] E. L. Sntos, K. de Picoli Souz, P. B. Guimrães et l., Effect of ngiotensin converting enzyme inhiitor enlpril on ody weight nd composition in young rts, Interntionl Immunophrmcology, vol., no., pp. 7 3, 00. [] R. S. Weisinger, T. K. Stnley, D. P. Begg, H. S. Weisinger, K. J. Sprk, nd M. Jois, Angiotensin converting enzyme inhiition lowers ody weight nd improves glucose tolernce in C7BL/J mice mintined on high ft diet, Physiology nd Behvior, vol. 9, no. 1-, pp , 009. [3] E. L. Sntos, K. de Picoli Souz, E. D. d Silv et l., Long term tretment with ACE inhiitor enlpril decreses ody weight gin nd increses life spn in rts, Biochemicl Phrmcology, vol. 7, no., pp. 91 9, 009. [] K. Arki, T. Mski, I. Ktsurgi, K. Tnk, T. Kkum, nd H. Yoshimtsu, Telmisrtn prevents oesity nd increses the expression of uncoupling protein 1 in diet-induced oese mice, Hypertension, vol., no. 1, pp. 1 7, 00. [] A. P. Jysooriy, M. L. Mthi, L. L. Wlker et l., Mice lcking ngiotensin-converting enzyme hve incresed energy expenditure, with reduced ft mss nd improved glucose clernce, Proceedings of the Ntionl Acdemy of Sciences of the United Sttes of Americ, vol., no. 1, pp. 31 3, 00. [] R. Kouym, T. Sugnmi, J. Nishid et l., Attenution of diet-induced weight gin nd diposity through incresed energy expenditure in mice lcking ngiotensin II type 1 receptor, Endocrinology, vol. 1, no., pp , 00. [7] M. Nwno, M. Ani, M. Funki et l., Imidpril, n ngiotensin-converting enzyme inhiitor, improves insulin sensitivity y enhncing signl trnsduction vi insulin receptor sustrte proteins nd improving vsculr resistnce in the Zucker ftty rt, Metolism, vol., no., pp. 1 1, [] E. J. Henriksen, S. Jco, H. J. Augustin, nd G. J. Dietze, Glucose trnsport ctivity in insulin-resistnt rt muscle: effects of ngiotensin-converting enzyme inhiitors nd rdykinin ntgonism, Dietes, vol., supplement 1, pp. S1 S1, 199. [9] E. J. Henriksen nd S. Jco, Effects of cptopril on glucose trnsport ctivity in skeletl muscle of oese Zucker rts, Metolism, vol., no., pp. 7 7, 199. [30] K. Sugimoto, L. Kzdová, N. R. Qi et l., Telmisrtn increses ftty cid oxidtion in skeletl muscle through peroxisome prolifertor-ctivted receptor-γ dependent pthwy, Journl of Hypertension, vol., no., pp , 00. [31] J. C. Russell, S. E. Kelly, nd S. Schäfer, Vsopeptidse inhiition improves insulin sensitivity nd endothelil function in the JCR:LA-cp rt, Journl of Crdiovsculr Phrmcology, vol., no., pp., 00. [3] V. Arin, N. Clperon, M. C. Fournié-Zluski, B. P. Roques, nd J. Peyroux, Acute effect of the dul ngiotensinconverting enzyme nd neutrl endopeptidse -11 inhiitor mixnpril on insulin sensitivity in oese Zucker rt, British Journl of Phrmcology, vol. 133, no., pp. 9 0, 001. [33] C. H. Wng, N. Leung, N. Lpointe et l., Vsopeptidse inhiitor omptrilt induces profound insulin sensitiztion nd increses myocrdil glucose uptke in Zucker ftty rts: studies compring vsopeptidse inhiitor, ngiotensinconverting enzyme inhiitor, nd ngiotensin II type I receptor locker, Circultion, vol. 7, no. 1, pp , 003. [3] V.Wong,L.Szeto,K.Uffelmn, I. G. Fntus, nd G. F. Lewis, Enhncement of muscle glucose uptke y the vsopeptidse inhiitor, omptrilt, is independent of insulin signling nd the AMP kinse pthwy, Journl of Endocrinology, vol. 190, no., pp. 1 0, 00. [3] K. Miyshit, H. Itoh, H. Tsujimoto et l., Ntriuretic peptides/cgmp/cgmp-dependent protein kinse cscdes promote muscle mitochondril iogenesis nd prevent oesity, Dietes, vol., no. 1, pp. 0 9, 009. [3] M. A. Yorek, The potentil role of ngiotensin converting enzyme nd vsopeptidse inhiitors in the tretment of dietic neuropthy, Current Drug Trgets, vol. 9, no. 1, pp. 77, 00. [37]H.Y.T.Yng,E.G.Erdös, nd Y. Levin, A dipeptidyl croxypeptidse tht converts ngiotensin I nd inctivtes rdykinin, Biochimic et Biophysic Act, vol. 1, no., pp , [3] E. J. Henriksen, S. Jco, T. R. Kinnick, E. B. Younglood, M. B. Schmit, nd G. J. Dietze, ACE inhiition nd glucose trnsport in insulin-resistnt muscle: roles of rdykinin nd nitric oxide, Americn Journl of Physiology, vol. 77, no. 1, pp. R33 R33, [39] L. J. Coppey, E. P. Dvidson, T. W. Rinehrt et l., ACE inhiitor or ngiotensin II receptor ntgonist ttenutes dietic neuropthy in streptozotocin-induced dietic rts, Dietes, vol., no., pp. 31 3, 00. [0] C. L. Oltmn, E. P. Dvidson, L. J. Coppey, T. L. Kleinschmidt, B. Dke, nd M. A. Yorek, Role of the effect of inhiition of neutrl endopeptidse on vsculr nd neurl complictions in streptozotocin-induced dietic rts, Europen Journl of Phrmcology, vol. 0, no. -3, pp., 011. [1] S. Engeli, Role of the renin-ngiotensin-ldosterone system in the metolic syndrome, Contriutions to Nephrology, vol. 11, pp. 1 13, 00. [] K. F. Stndeven, K. Hess, A. M. Crter et l., Neprilysin, oesity nd the metolic syndrome, Interntionl Journl of Oesity, vol. 3, pp. 31 0, 011.