Experimental Physiology

Transcription

1 Exp Physiol 1.1 (215) pp Reserh Pper Reserh Pper Vgus nerve ontriutes to metoli syndrome in high-ft diet-fed young nd dult rts Luiz F. Brell, Rosine A. Mirnd, Cludinéi C. S. Frno, Vnder S. Alves, Annd Mlt, Ttine A. S. Rieiro,ClrieGrven, Pulo C. F. Mthisnd Júlio C. de Oliveir Lortory of Seretion Cell Biology, Deprtment of Biotehnology, Genetis nd Cell Biology, Stte University of Mringá, Mringá, Prná,Brzil Experimentl Physiology New Findings Wht is the entrl question of this study? Differentnerve ontriutes periods of life re known for their differentil sensitivity to interventions, nd inresed prsymptheti tivity ffets the development nd mintenne of oesity. Thus, we evluted the involvement of the vgus nerve y performing vgotomy in young or dult rts tht were offered n oesogeni high-ft diet. Wht is the min finding nd its importne? Although the umultion of dipose tissue deresed in oth younger nd older groups, the younger rts showed greter response to the effets of vgotomy in generl. In ddition to the importnt role of the prsymptheti tivity, we suggest tht the vgus nerve ontriutes to the ondition of oesity. Oesity hs eome glol prolem, nd this ondition develops primrily euse of n imlne etween energy intke nd expenditure. The high omplexity involved in the regultion of energy metolism results from severl ftors esides endorine ftors. It hs een suggested tht oesity ould e used y n imlne in the utonomous nervous system, whih ould led to ondition of high prsymptheti tivity in ounterprt to low symptheti tonus. High-ft() diets hve een used to indue oesity in experimentl nimls, nd their use in nimls leds to insulin resistne, hyperinsulinemi nd high prsymptheti tivity, mong other disorders. The im of this work ws to evlute the effets of vgotomy performed t the initition of diet t two different stges of life, wening nd dulthood. The vgotomy redued prsymptheti tivity ( 32 nd 51% in norml ft-fed rts nd 43 nd 55% in diet-fed rts; P <.5) nd ft depots ( 17 nd 33%,onlyindiet-fed rts; P <.5). High-ft diet-fed rts exhiited fsting hyperinsulinemi (fivefold higher in young rts nd threefold higher in older rts; P <.5); however, vgotomy orreted it in younger rts only, nd similr effet ws lso oserved during the gluose tolerne test. The insulin resistne exhiited y the diet-fed groups ws not ltered in the vgotomized rts. We suggest tht the vgus nerve, in ddition to the importnt role of prsymptheti tivity, ontriutes to the ondition of oesity, nd tht non-vgl pthwys my e involved long with the imlned utonomi nervous system. (Resumitted 12 Septemer 214; epted fter revision 5 Novemer 214; first pulished online 8 Novemer 214) Corresponding uthor L. F. Brell: Lortory of Seretion Cell Biology Building H-67, Room 19, Deprtment of Biotehnology, Genetis nd Cell Biology, Stte University of Mringá/UEM, Avenue Colomo, Mringá, Prná, Brzil. Emil: lfrell@gmil.om C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety DOI: /expphysiol

2 58 L. F. Brell nd others Exp Physiol 1.1 (215) pp Introdution Oesity is mjor glol helth prolem, nd its rtes hve persistently risen through the yers. Aording to the World Helth Orgniztion, in 28, more thn 1.4 illion dults were overweight, nd of these, t lest 5 million were oese. Bsilly, oesity results from disrupted lne etween energy input nd energy expenditure. For these people, simple intervention in eting hits nd the ndonment of sedentry lifestyle ould led to weight loss. However, the explntion is not so simple; the lrgest prolem is the high omplexity of energy lne ontrol, whih involves not only endorine ftors ut lso neurl nd geneti kgrounds, mong other ftors. Metoli syndrome will develop nd ontinue in these individuls if nutritionl nd/or physil tivity intervention is not performed; this syndrome enompsses vrious prolems, suh s hypertension, insulin resistne, type 2 dietes nd oesity (Mrtinez, 2; Hslm & Jmes, 25). It hs een proposed tht oese humns nd nimls hve n imlne of the utonomi nervous system nd exhiit high prsymptheti tivity while the symptheti tonus is deresed, depending on the orgn (Bry & York, 1979; Blo et l. 27; Dvy & Orr, 29). Pnreti islets re extensively innervted y vgl holinergi nerves, nd the vgus nerve is responsile for the prsymptheti innervtion, whih is very importnt for the enhnement of gluose-indued insulin seretion (Ahrén, 2). Studies hve shown tht vgotomy is prtly le to lok the development of oesity in humns nd rts (Krl, 1978, 1979; Cox & Powley, 1981; Smith et l. 1983; Blo et l. 2, 27). It is known tht vgus nerve tivity is involved in noli pthwys, inluding n inrese in ell prolifertion, nd vgotomy is le to derese the islet prolifertion in oese ut not len rodents (Edvell & Lindström, 1998). The niml model derived from high-ft () diet feeding is hrterized y peripherl insulin resistne nd fsting hyperinsulinemi, mong severl other fetures. In ddition, these oese nimls show inresed prsymptheti tivity (Llli et l. 28; Brell et l. 212). Moreover, nimls with this model hve deresed entrl sensitivity to mny metoli nd hormonl signls involved in energy homeostsis, prtiulrly insulin nd leptin (Irni et l. 27). One study showed tht diet indues rekdown in the funtionl nd moleulr tivtion of insulin signl-trnsdution pthwys, leding to pro-inflmmtory sttus in the hypothlmus nd resulting in impired norexigeni insulin signlling (De Souz et l. 25). Thus, diets re le to lter mehnisms tht operte in oth the periphery nd the rin, leding to disrupted energy lne nd gluose homeostsis (Woods et l. 24). Given tht inresed prsymptheti tivity ffets the development nd mintenne of oesity, we imed to evlute the involvement of the vgus nerve in the development of oesity y performing vgotomy surgery in young or dult rts nd offering n oesogeni diet simultneously with the surgery. Methods Experimentl nimls nd groups Mle Wistr rts were supplied y the Centrl Animl Fility, Stte University of Mringá nd mintined in the Animl Setor t ontrolled temperture (23 ± 2 C) with 12 h 12 h light drk yle nd with food nd wter d liitum. The Ethis Committee for Experimentl Animls t the Stte University of Mringá pproved the present protool, whih ws written in ordne with federl lws in Brzil. Eight rndom groups were formed, s shown in Fig. 1. Two groups were vgotomized (Vg) t 21 dys of ge, nd two other groups were vgotomized t 6 dys of ge. Equivlent groups were formed with shm-vgotomized (-Vg) rts. After the surgery, the groups were ssigned to et norml-ft () or high-ft () diet until 9 or 12 dys of ge, for totl of 6 dys of -diet feeding. Thus, we hd -9 nd Vg-9, -9 nd Vg-9 groups for the younger rts nd -12 nd Vg-12, -12 nd Vg-12 groups for the older rts. The diet (ft omposition mostly from lrd) ws reported previously (Brell et l. 212), nd its omposition is shown in Tle 1. Mesurement of ody weight (BW) gin nd food onsumption Throughout the experimentl protool, the rts were weighed every 2 dys, nd the food onsumption ws reorded. The food onsumption ws mesured y weighing the remining food, sutrting tht mount from the rtion offered to the nimls the dy efore, nd dividing y the numer of nimls in the ges (Brell et l. 212). As the energeti vlues of the diets were different (3.81 versus kl g 1 for versus ), the vlues in grms were onverted into energy vlues. The re under the urve (AUC) ws lulted for oth BW nd food onsumption. Additionlly, the energy vlues were expressed reltive to 1 g of BW. Bilterl sudiphrgmti vgotomy For the vgotomy, the rts were nesthetized with xylzine nd ketmine [vi n I.M. injetion of.6 nd 3 mg (1 g BW) 1, respetively] nd pled in dorsl reumeny. A 2 m ventrl mid-line skin inision, nd susequently n dominl musle inision, ws mde with its rnil terminus t the level of the xiphoid proess. The oesophgus ws exposed, nd the intestines nd liver C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

3 Exp Physiol 1.1 (215) pp Prsymptheti system nd high-ft diet 59 Tle 1. Components of high-ft diet nd stndrd how Ingredients [g (kg diet) 1 ] Stndrd how ( diet) High-ft () diet Csein 2 2 Surose 1 1 Cornstrh Dextrinted strh Lrd 312 Soyen oil 4 4 Cellulose 5 5 Minerl mix (AIN-93) Vitmin mix (AIN-93) 1 1 L-Cystine Choline itrtrte were retrted. With the id of mgnifying glss, the vgus nerves running long the nterior nd posterior surfes of the oesophgus were gently setioned with fine mirodissetion sissors. The dominl musle inision ws losed with sorle sutures, nd the skin ws losed with non-sorle sutures. After the surgery, the nimls were housed individully for 5 dys. For the shm opertion, the dominl skin nd musle of the nimls were opened nd susequently losed without perturtion of the vgus nerve. In order to verify the effetiveness of vgotomy, when the nimls were killed their stomhs were removed, nd the stomh/bw rtio ws lulted. No deths resulted from the surgeries (Blo et l. 27). All the vgotomized nd shm-vgotomized nimls reovered well from the surgery,withslightimptonthebw.afterthesurgery, the nimls from oth groups te smll mounts of food, whih inresed up to the fourth dy, when their food intke reovered to norml levels. Intrvenous gluose tolerne test (ivgtt) A group of nimls nesthetized s desried in the previous susetion reeived silione nnul (pretreted with heprin t 5 IU ml 1 of.9% sline to prevent lood lots) implnted in the right jugulr vein nd fixed t the k of the nek. One dy fter the surgery, the rts were fsted for 12 h prior to reeiving n infusion of gluose [1 g (kg BW) 1 ] through the nnul without nesthesi. Blood smples were olleted immeditely efore the gluose lod (time zero/sl) nd t 5, 15, 3 nd 45 min fter the gluose lod. The plsm otined from the lood smples ws stored t 2 C until mesurement of the gluose onentrtion y the gluose oxidse method (Bergmeyer & Gwehn, 1974) using ommeril kits (Gliose-PP, Gold Anlis R,BeloHorizonte,Brzil). The insulin onentrtions were determined using n 125 I-lelled insulin rdioimmunossy (PerkinElmer R, Boston, MA, USA) with humn insulin s the stndrd nd n ntiody ginst rt insulin (Sott et l. 1981). The Groups -Vg -9 Lttion Norml-ft diet -9 Lttion High-ft diet Vg Vg-9 Vg-9 Lttion Lttion Norml-ft diet High-ft diet -Vg Lttion Lttion Norml-ft diet Norml-ft diet Vg Norml-ft diet High-ft diet Vg-12 Vg-12 Lttion Lttion Norml-ft diet Norml-ft diet Norml-ft diet High-ft diet Wening 6 dys old 9 dys old 12 dys old Figure 1. Sheme of the experimentl group formtion Arevitions:, norml ft;, high ft;, shm vgotomy; nd Vg, vgotomy. C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

4 6 L. F. Brell nd others Exp Physiol 1.1 (215) pp insulin intr- nd interssy vrition oeffiients were 9.8 nd 12.2%, respetively. The inferior nd superior detetion limits were.6 nd 1 ng ml 1, respetively. The glyemi ( Glyemi ) nd insulinemi inrements ( Insulinemi ) were otined y sutrting the fsting plsm gluose nd insulin levels t eh time point during the ivgtt. The totl Glyemi nd Insulinemi inreses were lulted y using the glyemi nd/or insulinemi AUC for the 45 min of the ivgtt. Intrperitonel insulin tolerne test (ipitt) As desried in the previous susetion, the sme method of lood olletion used for the ivgtt ws used to perform the ipitt. Another th of rts hd nnul implnted ndwerefstedfor6hpriortonipitt[1u(kg BW) 1 ]. The sorption rte of gluose y the tissue [the rte onstnt for the dispperne of plsm gluose (K itt ) ws lulted using the formul.693/(t ½ ), whih ws desried previously; Lundek, 1962]. The plsm gluose hlf-life (t ½ ) ws lulted from the slope of the lest-squres nlysis of the plsm gluose onentrtions during the liner phse of deline. Eletril tivity of the vgus nerve A th of nimls from eh experimentl group, fsted for 12 h, were nesthetized with thiopentl vi intrperitonel injetion [45 mg (kg BW) 1 ] nd ws then mnipulted s previously desried (de Oliveir et l. 211; Brell et l. 212). Briefly, in the nterior ervil region, the nerve undle of the superior rnh of the left vgus ws severed from the rotid rtery lose to the trhe under dissetion mirosope. A pir of silver reording eletrodes (.6 mm dimeter) ws pled under the nerve, whih ws overed with silione oil to prevent dehydrtion. The eletrode ws onneted to n eletroni devie (Bio-Amplifitor; Insight Equipmentos, Rieirão Preto, Brzil) tht mplified the eletril signl prior to filtering the frequenies lower thn 1 khz nd higher thn 8 khz. The signl output ws quired using Insight softwre (Insight Equipmentos) nd stored on omputer. The nimls were pled in Frdy ge to void ny eletromgneti interferene during the dt-quisition period. Nerve tivity ws nlysed s the numer of spikes during 5 s period. The spikes were hrterized y depolriztion tht surpssed. After stiliztion of the the signl (2 min), 2 reording periods of 15 s eh were rndomly hosen from eh rt to ssess the numer of spikes. The verge numer of spikes ws used s the nerve firing rte for eh rt. Assessment of oesity At 9 or 12 dys of ge, the rts were nesthetized y n intrperitonel injetion of thiopentl [45 mg (kg BW) 1 ] nd killed y ervil dislotion. The periepididyml nd retroperitonel ft pds were removed nd weighed. The mss of these tissues ws used s simple nd relile method to estimte the ody ft umultion in the rodents (Rogers & We, 198). Oesity ws lso ssessed using the BW nd nso-nl length to lulte the Lee index, whih orresponds to the following rtio: BW ⅓ (in grms) nso-nl length (in entimetres; Bernrdis & Ptterson, 1968). Sttistil nlyses Dt re given s the mens ± SEM nd were sujeted to two-wy ANOVA [vriles: D, diet; S, surgery; nd the letter I stnds for intertion of oth vriles], followed y Tukey s post ho test. Vlues of P <.5 were onsidered sttistilly signifint. All tests were performed using GrphPd Prism version 5. for Windows (GrphPd Softwre In., Sn Diego, CA, USA). Results Biometri nd iohemil prmeters The dt from the 9-dy-old rts re shown in the Tle 2, while those of the 12-dy-old nimls re presented in Tle 3. The -9 rts hd inresed BW ompred with tht of -9 rts (P <.5); however, the BW ws not ltered y vgotomy in either group. The sme pttern ws oserved in the older nimls (P <.5). The urves of BW progression re shown in Fig. 2A nd B. The food intke, expressed in kiloglories, deresed y 14% in oth the shm nd the vgotomized 9 groups ompred with the 9 groups (P <.5). In the older groups, redution in lori intke ws oserved only when ompring the -12 group with the -12 group (P <.5). The Lee index inresed in response to the diet, nd no hnges resulted from the vgotomy. The stomh weights, mesure of the effiy of vgotomy, of ll the vgotomized groups inresed ompred with those of the shm-vgotomized groups (P <.5). The ft tissue umultion is shown in Fig. 3A nd B. Irrespetive of ge or ft depot, the diet-fed nimls exhiited inresed ft tissue umultion ompred with tht of the diet-fed rts (n inrese of 153% in the periepididyml ft pd nd 222% in the retroperitonel ft pd when ompring -9 with -9 nd n inrese of 72% in the periepididyml ft pd nd 78% in the retroperitonel ft pd when ompring -12 with -12; P <.5). For oth ge groups, vgotomy C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

5 Exp Physiol 1.1 (215) pp Prsymptheti system nd high-ft diet 61 Tle 2. Effet of erly vgotomy nd high-ft diet on 9-dy-old rts diet diet Sttistil nlysis Prmeter -9 Vg-9-9 Vg-9 D S I BW (g) 361 ± ± ± ± 12.6 F = 66.2 P <.1 Food intke [kl(1 g 1152 ± ± ± ± 14.7 F = 48.1 BW) 1 ; AUC] P <.1 Lee index 36.4 ± ± ± ± 1.84 F = 36.5 P <.1 Stom [g (1 g) 1 ].421 ±.9.48 ± ±.4.39 ±.13 F = 58.8 P <.1 F = 1.2 n.s. F =.22 n.s. F = 1.99 n.s. F =.5 n.s. F =.79 n.s. F = 5.28 P <.5 F = 27.2 F =.23 n.s. P <.1 Dt re given s men vlues ± SEM, 1 15 nimls per group. Arevitions: AUC, re under urve; BW, finl ody weight; diet, high-ft diet; diet, stndrd how; -9, shm-vgotomised rts fed diet; -9, shm-vgotomised rts fed diet; Stom, stomh weight; Vg-9, vgotomised rts fed diet; nd Vg-9, vgotomised rts fed diet. Vriles: D, diet; S, surgery; nd I, intertion of oth vriles. Vlues within row with different supersript letters re signifintly different from eh other (P <.5). Tle 3. Effet of lte vgotomy nd high-ft diet on 12-dy-old rts diet diet Sttistil nlysis Prmeter -12 Vg Vg-12 D S I BW (g) 4 ± ± ± ± 14 F = 59.1 P <.1 Food intke [kl(1 g 96.5 ± ± ± ± 41.1 F = 13. BW) 1 ; AUC] P <.1 Lee index ± ± ± ± 2.9 F = 2.8 P <.1 Stom [g (1 g) 1 ].47 ± ± ± ±.49 F = 18.8 P <.1 F = 2.34 n.s. F =.14 n.s. F =.12 n.s. F = 43.8 P <.1 F = 1.8 n.s. F =.31 n.s. F =.2 n.s. F =.5 n.s. Dt re given s men vlues ± SEM, 1 15 nimls per group. Arevitions: AUC, re under urve; BW, finl ody weight; diet, high-ft diet; diet, stndrd how; -12, shm-vgotomised rts fed diet; -12, shm-vgotomised rts fed diet; Stom, stomh weight; Vg-12, vgotomised rts fed diet; nd Vg-12, vgotomised rts fed diet. Vriles: D, diet; S, surgery; nd I, intertion of oth vriles. Vlues within row with different supersript letters re signifintly different from eh other (P <.5). ws effetive t promoting derese in ft tissue weight only in the groups tht reeived the diet ( derese of 17% in the periepididyml ft pd nd 23% in the retroperitonel ft pt when ompring Vg-9 with -9 nd derese of 18% in the periepididyml ft pd nd 33% in the retroperitonel ft pt when ompring Vg-12 with -12; P <.5). The gluose nd insulin levels re shown in Fig. 4A nd B, respetively. The younger rts fed diet (-9) exhiited elevted fsting glyemi (+22%) nd insulinemi (fivefold elevtion; P <.5). Although vgotomy did not lter the pttern of glyemi, it ws le to orret the insulinemi to sl or su-sl levels, deresing the insulinemi y 64% in Vg-9 rts nd y 83% in Vg-9 rts (P <.5). In ontrst, in older nimls, the fsting glyemi ws not ltered in either the shm-operted or vgotomized rts (P >.5). Thefsting insulinemi inresed lmost threefold in the -12 rts (P <.5) ompred with the -12 rts, nd it ws not normlized to sl vlues y the vgotomy (P >.5). Gluose homeostsis nd insulin sensitivity As shown in Fig. 5, irrespetive of ge, vgotomy hd no effet on glyemi or on the inrement in insulinemi during the ivgtt in oth ge groups fed diet. Furthermore, vgotomy ws not le to lok the diet-indued hyperglyemi (+45% when ompring -9 with -9 nd +48% when ompring -12 with -12; Fig. 5A nd B; P <.5). Insulinemi ws mesured during the test; t oth ges, the diet-fed rts exhiited inresed insulin levels (eightfold inrese in -9 ompred with -9 nd 83% in -12 ompred with -12; P <.5). In ontrst, vgotomy ws le to lok the diet-indued hyperinsulinemi only in the younger rts C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

6 62 L. F. Brell nd others Exp Physiol 1.1 (215) pp (derese of 77% in Vg-9 ompred with -9; Fig. 5C; P <.5). Irrespetive of either ge or the diet offered, vgotomy hdnoeffetonthegluoseuptkerte(k itt ;Fig.6A nd B) during the ipitt. However, irrespetive of ge, the diet-fed rts showed deresed K itt. A derese of 38% ws oserved in -9 ompred with -9 (Fig. 6A), nd 32% derese ws oserved in -12 ompred with -12 (Fig. 6B; P <.5). Prsymptheti nervous system tivity As expeted, the prsymptheti tivity ws deresed in oth the erly nd the lte vgotomy groups, irrespetive of the diet. In the younger rts, we oserved derese of 32% in Vg-9 nd 43% in Vg-9 ompred with -9 nd -9, respetively (Fig. 7A; P <.5). Likewise, in the older rts, we oserved redution of 51% in Vg-12 nd 55% in Vg-12 ompred with their ontrols, -12 nd -12, respetively (Fig. 7B; P <.5). In ontrst, the diet led to A Body weight (g) B Body weight (g) Vgotomy Vg- Vg Age (dys) Vgotomy - - Vg- Vg Age (dys) Figure 2. Body weight progression The symols represent the men of the 9- (A) nd 12-dy-old rts (B) with 6 12 nimls from eh group, nd the error rs represent SEM. inresed levels of prsymptheti tivity for oth ges of shm-vgotomized rts. We oserved n inrese of 64% in -9 ompred with -9 (Fig. 7A; P <.5) nd 43% in -12 ompred with -12 (Fig. 7B; P <.5). Representtive exmples of nerve dishrges re shown in Fig. 7C. Disussion In this study, we show tht vgus nerve tivity ontriutes to the diet-indued onset of oesity; the ontriution of the vgus ws similr during perintl nd dult periods. However, its tivity plys ritil role in the regultion of insulinemi in the younger nimls. A Peripididyml ft pd mss [g (1 g BW) 1 ] B Retroperitonel ft pd mss [g (1 g BW) 1 ] D: F = 177 P <.1 S: F = 1.99 n.s. l: F = 9.52 P < dys old D: F = 15 P <.1 S: F = 2.64 n.s. l: F = 13.3 P < D: F = 134 P <.1 S: F = 1.5 P <.1 l: F = 1.63 n.s. Vg 12 dys old D: F = 154 P <.1 S: F = 24.6 P <.1 l: F = 11.8 P <.1 Vg 9 dys old 12 dys old Figure 3. Periepididyml nd retroperitonel ft pds The rs represent the men mss of periepididyml (A) nd retroperitonel ft pds (B) otined from 1 15 nimls from eh group. In this nd susequent figures, the open rs represent the shm-vgotomized rts, nd the filled rs represent the vgotomized rts; the diet is indited elow the x-xes, nd error rs represent the SEM. In this nd susequent figures, dt with different letters re signifintly different from eh other, P <.5 etween groups of the sme ge. C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

7 Exp Physiol 1.1 (215) pp Prsymptheti system nd high-ft diet 63 It is well estlished tht diets re effetive t promoting inreses in BW nd ft pds in rts nd humns (Mnn, 22; Brell et l. 212). In the present work, we show inresed BW nd sustntil umultion of ft tissue in the diet-fed groups t oth ges, s we demonstrted previously (Brell et l. 212). Although vgotomy did not hnge the finl BW, it ws effetive t prtly loking the umultion of ft tissue indued y the diet, ut not in the rts fed diet. In nother study, surgil vgotomy performed fter indution of oesity ws prtly le to lok the fetures of oesity in nother oese rt model (Blo et l. 27). An ge-ssoited oesity study hs shown tht A Fsting gluose onentrtions (mg dl 1 ) B Fsting insulin onentrtions (ng ml 1 ) D: F = 35.1 P <.1 S: F = 1.59 n.s. l: F =.21 n.s. 9 dys old 12 dys old D: F = 28.5 P <.1 S: F = 28.2 P <.1 l: F = 17.3 P <.1 D: F =.1 n.s. S: F = 2. n.s. l: F =.9 n.s. D: F = 57.8 P <.1 S: F =.13 n.s. l: F =.69 n.s. 9 dys old 12 dys old SH Vg Vg Figure 4. Fsting glyemi nd insulinemi The rs represent the men of glyemi (A) nd insulinemi (B) otined from 1 15 nimls for eh group. the long-term effet of vgotomy ws deresed BW nd ftpdsinnimlsfeddiet(sternset l. 212). Although vgotomy uses hnges in gstrointestinl tivityndthusltersfoodintkenddigestiveehviour (Bernstein & Goehler, 1983), we verified tht the surgery did not ffet the results, s shown y the food intke dt. Defferenttion studies with psiin showed tht vgl fires re not involved in the long-term regultion of food intke (vn de Wll et l. 25). We lso showed tht the - rts ingested fewer lories thn did the - rts. The food nd lorie intke dt re very ontroversil nd re reported in severl studies (Leiowitz et l. 24; Borst & Conover, 25; Brell et l. 212); we should onsider the vritions in the dietry omposition nd the durtions of the diets, s well s the different niml strins used throughout the studies, whih ould led to vried results. Nevertheless, some uthors hve limed tht rts typilly prefer diets nd gin more weight even if the energeti intke is not high (Slfni, 21). Additionlly, lrge mount of lipids in the diet is rguly one of the most influentil ftors for the indution nd mintenne of oesity (Rz et l. 25). The elevted gluose levels in the 9 rts nd the lk of hnges in the older nimls (12 rts) my e due to the different timing of introdution of the diet, euse under- or overnutrition during ritil periods of life, suh s during gesttion, lttion nd dolesene, my led to vrious metoli disturnes in the offspring lter life (Hles & Brker, 21; Bteson et l. 24; Vikers et l. 25; vn Aeelen et l. 212). As we hve reently shown, the sme diet offered in either peripuertl life or dulthood indued milder ltertions in the metolism of rts tht reeived the diet only during dult life ompred with those tht reeived the diet eginning t wening (Brell et l. 212). This informtion indites tht the metolism of the 12 rts, whih reeived the diet immeditedly fter the dolesent period, hnges in severl wys tht re mrkers for metoli syndrome; however, it llows us to suggest tht they n support this lori lod etter thn younger nimls. We found tht insulinemi ws elevted in the rts ut with different mgnitudes for the two ges. The rts tht reeived immeditely fter wening hd higher insulinemi ompred with tht of the older group tht reeived fter the dolesent period; moreover, the insulin levels of Vg-9 rts were normlized to levels similr to those of the non-operted 9 rts. In ontrst, no differenes were found in the insulin levels of oth the Vg-12 nd the Vg-12 rts ompred with the respetive non-operted ontrol nimls. It is evident tht vgotomy loked the hyperinsulinemi promoted y the diet in the younger rts, ut it is importnt to note tht this loking effet ws effetive in the rts only when the vgotomy ws pplied during the erly stge of life. For vrious gluose onentrtions, islets from oese nimls C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

8 64 L. F. Brell nd others Exp Physiol 1.1 (215) pp A Δ Gluose onentrtions (mg dl 1 ) B Δ Gluose onentrtions (mg dl 1 ) C Δ Insulin onentrtions (ng ml 1 ) D Δ Insulin onentrtions (ng ml 1 ) Δ Gluose onentrtions [mg dl 1 (45 min) 1 ;AUC] D: F = 17.5 P <.1 S: F =.5 n.s. l: F =.4 n.s Time (min) Δ Gluose onentrtions [mg dl 1 (45 min) 1 ;AUC] Time (min) Δ Insulin onentrtions [ng ml 1 (45 min) 1 ;AUC] D: F = 17.4 P <.1 S: F =.32 n.s. l: F =.3 n.s. D: F = 7.9 P <.1 S: F = 44.4 P <.1 l: F = 33.9 P < Time (min) Δ Insulin onentrtions [ng ml 1 (45 min) 1 ;AUC] Time (min) 4 2 D: F = 17.5 P <.5 S: F = 3.11 n.s. l: F = 1.93 n.s. Vg -9 Vg-9-9 Vg-9 Vg -12 Vg Vg-12 Vg -9 Vg-9-9 Vg-9 Vg -12 Vg Vg-12 Figure 5. Plsm gluose nd insulin onentrtions from the intrvenous gluose tolerne test (ivgtt) of 9- (A nd C) nd 12-dy-old nimls (B nd D) hve previously een shown to relese more insulin thn relesed y islets from their len littermtes. This high seretion pttern is supported y inresed pnreti holinergi stimultion, whih is etter oserved when the prsymptheti system tivity is mesured indiretly or diretly (Chen et l. 1993; Somprin et l. 29). Given tht hyperinsulinemi is one of the ftors involved in metoli syndrome, vgotomy ould led to deresed nd normlized insulin seretion in the islets of oese nimls, leding to levels omprle to those of len nimls, whih were previously reorded using rts with hypothlmi oesity (Blo et l. 22). Vgotomy ws not effetive t reduing the fsting lood insulin levels of the older nimls. It is not esy to explin the lk of derese in the fsting insulin levels in the Vg-12 rts nd the deresed umultion of dipose tissue in this group euse lipolysis/lipogenesis is primrily driven y the tions of insulin in this tissue (Fryn, 22; Dyk, 29). However, in this se, the utonomi nervous system, through possile symptheti hypertivity, should e the determinnt tht dereses ft umultion. In the present study, we lso show tht peripherl insulin resistne ws displyed in oth the young nd the dult groups tht exhiited low K itt vlues t oth ges. In ddition, vgotomy did not lok this pttern, inditing tht the surgery lone, whih ws performed prior to the oesogeni diet tretment, ws not le to inhiit the onset of insulin resistne indued y the diet, either in the erlier or lter developmentl stge of life. Our dt re in greement with previous study using nother experimentl model of metoli dysfuntion. The insulin resistne indued y syntheti gluoortioid, dexmethsone, whih is ssoited with high fsting glyemi nd insulinemi, ws found in 9-dy-old rts, even when the vgotomy ws performed prior to the hroni tretment with the syntheti gluoortioid (Angelini et l. 21). Confirming previous studies, we report sustntil stressful effet on the metolism of diet-indued oese rts; this effet leds to impired regultion of gluose nd insulin homeostsis (Simonsson & Ahrén, 1998; Doins et l. 22; Prk et l. 27). In ddition to the gluose-indued insulin seretion, the response to other seretgogues, suh s rhol nd glugon-like peptide-1, is hnged in the diet-indued model of oesity, whih exhiits higher sensitivity to these The symols represent the men glyemi (A nd B) nd insulinemi (C nd D) from 6 1 nimls from eh group, nd the error rs represent the SEM. The inset t the top right of eh pnel shows the re under the urve lulted from the entire ivgtt. The rs represent the mens from 6 7 rts from eh group. C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

9 Exp Physiol 1.1 (215) pp Prsymptheti system nd high-ft diet 65 moleules nd results in hyperinsulinemi (Simonsson & Ahrén, 1998). Furthermore, it hs reently een shown tht high expression of hem oxygense-1, stress-responsive enzyme displying pivotl ntioxidnt nd nti-inflmmtory tivity, hs no protetive effet ginst the diet-indued onset of oesity nd its metoli disturnes (Hung et l. 213). Likewise, we lso show in the present work tht vgotomy does not disply protetive effets tht hnge this metoli impirment, irrespetive of ge; however, the young vgotomized rts tht reeived the diet mintined their gluose levels t the expense of less insulin relese, wheres the Vg-12 group showed no differene in the gluose nd insulin levels ompred with those of -12 rts during the ivgtt. These dt orroorte the ft tht prsymptheti pthwys re importnt ut not prepondernt over other pthwys; the dt indite effiy regrding the redution of postprndil gluose levels with less prsymptheti tonus tht is evident in the Vg-9 rts. This hnge n e used y redued holinergi insulinotrophi tion A K itt (% min 1 ) B K itt (% min 1 ) D: F = 19.9 P <.1 S: F =.12 n.s. l: F =.17 n.s. D: F = 26.9 P <.1 S: F = 1.5 n.s. l: F =.17 n.s. Vg Vg Figure 6. Rte onstnt for plsm gluose dispperne (K itt ) of 9- (A) nd 12-dy-old nimls (B) All groups were sujeted to n intrperitonel insulin tolerne test s desried in the Methods, nd the rte onstnt for plsm gluose dispperne (K itt )ws lulted. The rs represent the men K itt, whih ws otined from 6 1 nimls from eh group. through less etylholine relese nd redued inding to musrini reeptors nd/or ltered signl trnsdution in pnreti β-ells. Interestingly, it is known tht hyperglyemi vgotomized nimls show inresed expression of musrini reeptor 3 (Angelini et l. 21). It hs lredy een shown tht vgotomized mie exhiit redued gluose tolerne nd, indeed, tht holinergi innervtion plys n importnt role in severl spets of metoli regultion (Edvell & Lindström, 1998). Blo nd o-workers (27) reported tht vgotomy ws le to improve gluose tolerne nd insulin sensitivity in oese rts (Blo et l. 27); however, in ontrst to our study, their study used the vgotomy intervention in rts whose oesity hd lredy een estlished; in our study, the vgotomy ws performed immeditely efore the oesogeni diet ws offered. It ws shown tht gluoortioid-treted vgotomized rts hve ltered protein expression of musrini reeptor sutype M 3 in pnreti islets; moreover, when stimulted with gluose nd rhol, n etylholine nlogue, the ells of either vgotomized or gluoortioid-treted vgotomized rts serete greter mounts of insulin (Angelini et l. 21). Therefore, these ltertions indite tht vgotomy indues islet dpttions, suh s higher sensitivity to these seretgogues, leding to inresed seretory responses. It is possile tht these dpttions re needed, llowing the islets to hndle the deresed prsymptheti tonus. Interestingly, the young vgotomized rts (from oth diet groups) hd deresed fsting insulinemi nd mintined their normlized plsm gluose onentrtions ompred with those of the respetive ontrol rts without hnges in the insulin sensitivity. We suggest tht this finding is relted to n ltered expression of insulin-indued gluose uptke signlling omponents, whih ould led to norml (or lmost norml) response to insulin in these vgotomized rts, even for low hormonl levels. Although this ltered expression hs lredy een shown in diet-fed rts, s the inresed tivtion of the CAP (Cl-ssoited protein)/cl (Csits -linege lymphom)/c3g (gunine nuleotide exhnge ftor) pthwy in dipose tissue (Prd et l. 26), it remins to e demonstrted in vgotomized rts. Our group hs lredy shown tht diet-fed oese rts present disruption of utonomi lne (Brell et l. 212). This imlne is lso present in predieti monosodium L-glutmte (MSG)-oese mie (Somprin et l. 29). Confirming these dt, the present study uses diret mesurement of the vgus nerve to show tht diet-fed rts hve higher vgus nerve tivity thn tht of len nimls. As expeted, ll the vgotomized rts showed deresed levels of vgus nerve tivity. Furthermore, the vgotomized diet-fed rts, irrespetive of the developmentl stge of life t whih the vgotomy ws performed, hd deresed C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

10 66 L. F. Brell nd others Exp Physiol 1.1 (215) pp vgus nerve tivity tht ws omprle to the levels shown y oth groups fed with the diet. These low levels of prsymptheti tivity might explin the deresed fsting insulinemi exhiited y the younger vgotomized rts tht ws not oserved in the older groups. Vgl stimultion is involved in the potentition of insulin seretion when gluose onentrtions re high nd medites the erly inrese in the insulin relese fter the presenttion of food. Severl models of oesity exhiit generl inrese in prsymptheti tivity, whih in turn leds to inresed levels of irulting insulin. The inresed insulin levels ugment the tivity of noli Numer of spikes per 5 s A D: F = 15.2 P <.1 S: F = 2.6 P <.1 l: F = 2.99 n.s. Vg Numer of spikes per 5 s B D: F = 5.94 P <.5 S: F = 28.8 P <.1 l: F = 1.23 n.s. Vg C 5-9 dys old 5-12 dys old Vg- 5 Vg Vg- 5 Vg Figure 7. Vgus nerve eletril tivity of 9- nd 12-dy-old nimls Vgus nerve eletril tivity of 9- (A) nd 12-dy-old nimls (B). The rs represent the men spike numers tht were otined from 7 15 nimls for eh group. Representtive reords of nerve dishrges for eh niml group (C). C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

11 Exp Physiol 1.1 (215) pp Prsymptheti system nd high-ft diet 67 pthwys, suh s the inresed lipogenesis tht is involved in the development of oesity (Kneto et l. 1974; Bry & York, 1979; Bry, 1991; Ahrén, 2). We hve previously shown tht offering diet during the puertl period n led to more drsti effets thn offering the sme diet only during dulthood (Brell et l. 212). Therefore, we might suggest tht ny intervention, suh s vgotomy, in the erly developmentl stges of life is more effetive t hieving metoli effets euse these erly periods re hrterized y neuroendorine development nd rin mturtion, nd ny intervention in this phse n led to different onsequenes during dult life. In summry, the effet of vgotomy ws oserved s deresed umultion of dipose tissue in oth younger nd older groups. Moreover, only the younger vgotomized rts exhiited low plsm insulin onentrtions in oth the fsting nd the fed stte. Although it is not esy to explin the deresed vgus nerve tivity ssoited with norml gluose levels without inresed peripherl insulin sensitivity, in the older rts, the diet my indue impired modultion, suh s intrellulr ell signlling tht is greter thn the expeted enefiil effets of vgotomy. In ddition, it is known tht the prsymptheti nervous system plys n importnt role in the regultion of gluose nd insulin for homeostsis, whih llows us to suggest tht some other non-vgl pthwy might e involved in this regultion, possiility tht remins to e studied. Referenes Ahrén B (2). Autonomi regultion of islet hormone seretion implitions for helth nd disese. Dietologi 43, Angelini N, Rfho A, Boshero AC & Bosqueiro JR (21). Involvement of the holinergi pthwy in gluoortioid-indued hyperinsulinemi in rts. Dietes Res Clin Prt 87, Blo SL, Bonfleur ML, Crneiro EM, Amrl ME, Filiputti E & Mthis PC (22). Prsymptheti tivity hnges insulin response to gluose nd neurotrnsmitters. Dietes Met 28, 3S13 3S17; disussion 3S18 3S112. Blo SL, Grssiolli S, Rieiro RA, Bonfleur ML, Grven C, Brito Mdo N, Andrezzi AE, Mthis PC & Torrezn R (27). Ft storge is prtilly dependent on vgl tivity nd insulin seretion of hypothlmi oese rt. Endorine 31, Blo SL, Mthis PC, Bonfleur ML, Alves, Siroti FJ, Monteiro OG, Rieiro FB & Souz AC (2). Vgotomy redues oesity in MSG-treted rts. Res Commun Mol Pthol Phrmol 18, Brell LF, de Oliveir JC, Brno RC, Cmrgo RL, Gomes RM, Mendes FC, Mirnd RA, Grven C, Torrezn R, Grssiolli S & de Freits Mthis PC (212). Erly exposure to high-ft diet hs more drsti onsequenes on metolism ompred with exposure during dulthood in rts. Horm Met Res 44, Bteson P, Brker D, Clutton-Brok T, De D, D Udine B, Foley RA, Glukmn P, Godfrey K, Kirkwood T, Lhr MM, MNmr J, Metlfe NB, Monghn P, Spener HG & Sultn SE (24). Developmentl plstiity nd humn helth. Nture 43, Bergmeyer HU & Gwehn K (1974). Methods of Enzymti Anlysis. Verlg Chemie, Weinheim; Ademi Press, New York. Bernrdis LL & Ptterson BD (1968). Correltion etween Lee index nd rss ft ontent in wenling nd dults femle rts with hypothlmi lesions. J Endorinol 4, Bernstein IL & Goehler LE (1983). Vgotomy produes lerned food versions in the rt. Behv Neurosi 97, Borst SE & Conover CF (25). High-ft diet indues inresed tissue expression of T-α. Life Si 77, Bry GA (1991). Oesity, disorder of nutrient prtitioning: the MONA LISA hypothesis. JNutr121, Bry GA & York DA (1979). Hypothlmi nd geneti oesity in experimentl nimls: n utonomi nd endorine hypothesis. Physiol Rev 59, Chen NG, Tssv TM & Romsos DR (1993). Threshold for gluose-stimulted insulin seretion in pnreti islets of genetilly oese (o/o) mie is normlly low. JNutr123, Cox JE & Powley TL (1981). Prior vgotomy loks VMH oesity in pir-fed rts. Am J Physiol Endorinol Met 24, E573 E583. Dvy KP & Orr JS (29). Symptheti nervous system ehvior in humn oesity. Neurosi Bioehv Rev 33, de Oliveir JC, Somprin DX, Andrezzi AE, Brno RC, MrtinsAG,GrvenC,GrssiolliS,RinldiW,BrosFB & Mthis PC (211). Metoli imprinting y mternl protein mlnourishment impirs vgl tivity in dult rts. J Neuroendorinol 23, De Souz CT, Arujo EP, Bordin S, Ashimine R, Zollner RL, Boshero AC, Sd MJ & Velloso LA (25). Consumption of ft-rih diet tivtes proinflmmtory response nd indues insulin resistne in the hypothlmus. Endorinology 146, Doins RL, Szzepnik LS, Myhill J, Tmur Y, Uhino H, Gi A & MGrry JD (22). The omposition of dietry ft diretly influenes gluose-stimulted insulin seretion in rts. Dietes 51, Dyk DJ (29). Adipokines s regultors of musle metolism nd insulin sensitivity. Appl Physiol Nutr Met 34, EdvellA&Lindström P (1998). Vgotomy in young oese hyperglyemi mie: effets on syndrome development nd islet prolifertion. Am J Physiol Endorinol Met 274, E134 E139. Fryn KN (22). Adipose tissue s uffer for dily lipid flux. Dietologi 45, Hles CN & Brker DJ (21). The thrifty phenotype hypothesis. Br Med Bull 6, 5 2. Hslm DW & Jmes WP (25). Oesity. Lnet 366, C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety

12 68 L. F. Brell nd others Exp Physiol 1.1 (215) pp Hung JY, Ching MT & Chu LY (213). Adipose overexpression of heme oxygense-1 does not protet ginst high ft diet-indued insulin resistne in mie. PLoS ONE 8, e Irni BG, Dunn-Meynell AA & Levin BE (27). Altered hypothlmi leptin, insulin, nd melnoortin inding ssoited with moderte-ft diet nd predisposition to oesity. Endorinology 148, Kneto A, Miki E & Kosk K (1974). Effets of vgl stimultion on glugon nd insulin seretion. Endorinology 95, Krl JG (1978). Vgotomy for tretment of severe oesity. Lnet 1(859), Krl JG (1979). Vgotomy s tretment for morid oesity. Surg Clin North Am 59, Llli CA, Puli JR, Prd PO, Cintr DE, Ropelle ER, Velloso LA & Sd MJ (28). Sttin modultes insulin signling nd insulin resistne in liver nd musle of rts fed high-ft diet. Metolism 57, Leiowitz SF, Dourmshkin JT, Chng GQ, Hill JO, Gyles EC, Fried SK & Wng J (24). Aute high-ft diet prdigms link glnin to triglyerides nd their trnsport nd metolism in musle. Brin Res 18, Lundek K (1962). Intrvenous gluose tolerne s tool in definition nd dignosis of dietes mellitus. Br Med J 1(5291), Mnn JI (22). Diet nd risk of oronry hert disese nd type2dietes.lnet 36, Mrtinez JA (2). Body-weight regultion: uses of oesity. Pro Nutr So 59, Prk S, Hong SM, Lee JE & Sung SR (27). Exerise improves gluose homeostsis tht hs een impired y high-ft diet y potentiting pnreti β-ell funtion nd mss through IRS2 in dieti rts. JApplPhysiol13, Prd PO, Puli JR, Ropelle ER, Zehin HG, Crvlheir JBC, Velloso LA & Sd MJA (26). Seletive modultion of the CAP/Cl pthwy in the dipose tissue of high ft diet treted rts. FEBS Lett 58, Rz I, Eldor R, Cerne S & frir E (25). Dietes: insulin resistne nd derngements in lipid metolism. Cure through intervention in ft trnsport nd storge. Dietes Met Res Rev 21, Rogers P & We GP (198). Estimtion of ody ft in norml nd oese mie. Br J Nutr 43, Slfni A (21). Psyhoiology of food preferenes. Int J Oes Relt Met Disord 25 (Suppl 5), S13 S16. Somprin DX, Gomes RM, Grssiolli S, Rinldi W, Mrtins AG, de Oliveir JC, Grven C & de Freits Mthis PC (29). Autonomi tivity nd glyemi homeostsis re mintined y preoious nd low intensity trining exerises in MSG-progrmmed oese mie. Endorine 36, Sott AM, Atwter I & Rojs E (1981). A method for the simultneous mesurement of insulin relese nd B ell memrne potentil in single mouse islets of Lngerhns. Dietologi 21, Simonsson E & Ahrén B (1998). Potentited β-ell response to non-gluose stimuli in insulin-resistnt C57BL/6J mie. Eur JPhrmol35, Smith DK, Srfeh J & Howrd L (1983). Trunl vgotomy in hypothlmi oesity. Lnet 1(8337), Sterns AT, Blkrishnn A, Rdmnesh A, Ashley SW, Rhods DB & Tvkkolizdeh A (212). Reltive ontriutions of fferent vgl fiers to resistne to diet-indued oesity. Dig Dis Si 57, vnaeelenaf,elissg,bossuytpm,groeede,vnder Shouw YT, Roseoom TJ & Uiterwl CS (212). Fmine exposure in the young nd the risk of type 2 dietes in dulthood. Dietes 61, vn de Wll EH, Pomp ER, Strue JH, Sheurink AJ & Koolhs JM (25). Defferenttion ffets short-term ut not long-term ontrol of food intke. Physiol Behv 84, Vikers MH, Glukmn PD, Coveny AH, Hofmn PL, Cutfield WS, Gertler A, Breier BH & Hrris M (25). Neontl leptin tretment reverses developmentl progrmming. Endorinology 146, WoodsSC,D AlessioDA,TsoP,RushingPA,CleggDJ,Benoit SC, Gotoh K, Liu M & Seeley RJ (24). Consumption of high-ft diet lters the homeostti regultion of energy lne. Physiol Behv 83, Additionl informtion Competing interests None delred. Author ontriutions L.F.B., R.A.M., P.C.F.M. nd J.C.O. were responsile for the oneption nd design of the study. L.F.B., C.C.S.F., V.S.A., A.M., T.A.S.R. nd C.G. olleted, nlysed nd interpreted the dt. L.F.B., P.C.F.M. nd J.C.O. drfted nd/or ritilly reviewed the mnusript. All uthors pproved the finl version of the mnusript, ll persons designted s uthors qulify for uthorship, nd ll those who qulify for uthorship re listed. Funding This work ws supported y the Brzilin governmentl genies Conselho Nionl de Desenvolvimento Científio e Tenológio (CNPq) nd Coordenção de Aperfeiçomento de Pessol de Nível Superior (CAPES), nd the Prná Siene Foundtion, Fundção Aruári. Aknowledgements We thnk Dr Sndr Blo for tehing us the vgotomy surgery. C 214 The Authors. Experimentl Physiology C 214 The Physiologil Soiety