EFFECT OF ACETYL-L-CARNITINE ON
VIP-ERGIC NEURONS IN THE JEJUNUM
SUBM UCOUS PLEXUS OF DIABETIC RATS
M arli Aparecida Def ani
1, Jacqueline Nelisis Zanoni
2, M aria Raquel M arçal Nat ali
2,
Robert o Barbosa Bazot t e
3, M arcílio Hubner de M iranda-Net o
2ABSTRACT - The effect of the treatm ent w ith acetyl-L-carnitine (ALC) on neurons releasing the vasoactive intestinal p olyp ep tid e (VIP) of the sub m ucous p lexus in the jejunum of d iab etic rats w as the p urp ose of our investigation. Diabetes (DM) was induced by injecting streptozotocin endovenously (35m g/kg). After sacrificing the anim als, the jejunum w as collected and p rocessed for VIP d etection. Four g roup s w ere used : C (non-d iab etic), CC (non-(non-d iab etic treate(non-d w ith ALC), D ((non-d iab etic), DC ((non-d iab etes treate(non-d w ith ALC). We analyze(non-d the im m unoreactivity and the cellular p rofile of 126 cell b od ies. The treatm ent w ith ALC im p roved som e asp ects of DM. How ever, it p rom oted a sm all increase in the area of neurons from g roup CC, sug g esting a p ossib le neurotrop hic effect. Neurons from g roup s D and DC show ed a larg e increase in their cellular p rofile and im m unoreactivity w hen com p ared to C and CC, sug g esting a larg er concentration of this neurotransm itter w ithin the neurons that p rod uce it. This ob servation constitutes a recurrent find ing in d iab etic anim als, suggesting that ALC d oesnot interfere in the p athop hysiological m echanism s that unchain a higher p rod uction and /or neurotransm itter accum ulation and increase the p rofile of the VIP-erg ic neurons.
KEY WORDS: acetyl-L-carnitine, sub m ucous p lexus, VIP, d iab etes, jejunum , rat.
Efeito da acetil-L-carnitina sobre neurônios Vip-érgicos do plexo submucoso do jejuno de ratos diabéticos
RESUMO - Investig am os o efeito d a acetil-L-carnitina (ALC) sob re os neurônios q ue exp ressam o p ep tíd eo intestinal vasoativo (VIP) d o p lexo sub m ucoso no jejuno d e ratos d iab éticos. O d iab etes (DM) foi ind uzid o p ela adm inistração endovenosa de estreptozootocina (35m g/kg). Após o sacrifício dos anim ais, o jejuno foi coletado e p ro cessad o p ara a d et ecção d e VIP. Ut ilizo u -se q u at ro g ru p o s: C (n ão d iab ét ico s), CC (n ão d iab ét ico s su p le m e n t a d o s co m ALC), D (d ia b é t ico s) e DC (d ia b é t ico s su p le m e n t a d o s co m ALC). An a liso u -se a im unoreativid ad e e o p erfil celular d e 126 corp os celulares. O tratam ento com ALC m elhorou alg uns asp ectos d o DM. Porém , p rom oveu p eq ueno aum ento na área d os neurônios d o g rup o CC, ind icand o p ossível efeito n e u ro t ró fico . Ne u rô n io s d o s g ru p o s D e DC a p re se n t a ra m g ra n d e a u m e n t o d o p e rfil ce lu la r e n a im unoreatividade em relação a C e CC, sugerindo m aior concentração deste neurotransm issor nestes neurônios. Esta ob servação é constante em anim ais d iab éticos, sug erind o q ue a ALC não interfere nos m ecanism os fisiop atológ icos q ue d esencad eiam a m aior p rod ução e/ou acúm ulo d e neurotransm issor e aum ento d o p erfil d os neurônios VIP-érg icos.
PALAVRAS-CHAVE: acetil-L-carnitina, p lexo sub m ucoso, VIP, d iab etes, jejuno, rato.
State University of Maringá (UEM), Maringá PR, Brazil: 1Professor at Departm ent of Sciences; 2Professor at Departm ent of Morphophysiological
Sciences; 3Professor at Dep artm ent of Pharm acy and Pharm acology. This stud y was sup p orted b y fund s from CAPES.
Received 5 Decem b er 2002, received in fin al fo rm 10 Ju ly 2003. Accep t ed 21 Ju ly 2003.
Dra. M . A. Def ani - Depart ment of Sciences, (UEM ) - Avenida Reit or Zef erino Vaz s/n Jardim Universit ário - 87360-000 Goioerê PR - Brasil. E-mail: mad@visaonet .com.br.
Diab et es m ellit u s (DM) m ain ch ro n ic co m p lica-t io n s are, am o n g o lica-t h ers, vascu lar ch an g es an d p eri-p h eral n eu ro eri-p at h ies, w h ich lead t o g ast ro in t est in al d ysfunctions. Several hyp otheses have b een rep orted concerning the m ain causes for the orig in of the d ia-b etic neurop athy. Recent rep orts have d escriia-b ed that t h e carn it in e ab n o rm al m et ab o lism m ay b e o n e o f t h e reaso n s t h at t rig g ers n eu ral d am ag e, asso ciat ed t o so rb it o l accu m u lat io n , fru ct o se an d m io in o sit o l
d ep let io n in t h e n ervo u s cells1,2. DM m ay h ave a d
ifferen t iat ed effect u n d er t h e d ififferen t en t eric n eu -ro act ive su b st an ces. St u d ies h ave sh o w n d ifferen t resp o n ses o f t h e ad ren erg ic, sero t o n erg ic an d p ep t id erg ic n erves an d o f t h e en t eric p lexu s in st rep -t o zo -t o cin -d iab e-t ic ra-t s3,4.
t h e re g u la t io n o f t h e in t e st in a l m o t ilit y. It is a n im p o rt a n t n o n a d ren erg ic, n o n ch o lin erg ic n eu ro t ran sm it t er (NANC) o f t h e p erip h eral n erves, p ro vo -kin g t h e relaxat io n o f m an y t yp es o f vascu lar an d n o n -vascu lar sm o o t h m u scles in m an y p art s o f t h e gastrointestinal tract. The anal p rojection of VIP-ergic neurons in the sm all and large intestine is com p atib le with the suggestion that it acts under the descending in h ib it io n in t h e p erist alt ic reflex. VIP is fo u n d in a g reat n u m b er o f n eu ro n s o f t h e su b m u co u s p lexu s a n d it ra rely o ccu rs in n eu ro n s o f t h e m yen t eric plexus5. In the subm ucous neurons, it acts controlling
t h e secret io n o f w at er an d elect ro lyt es o f t h e in t est in al m u co u s m em b ran e an d su ch alest eraest io n s im -p licat e in d iso rd ers o f t h e d ig est ive syst em6, su ch as
t h e syn d ro m e o f t h e d iab et ic d iarrh ea7. DM affect s
t h e level an d secret io n o f t h e en t eric VIP m o st likely d u e t o ab n o rm alit ies in t h e syn t h esis m ech an ism , lib erat io n an d /o r d eg rad at io n o f VIP8. An o t h er co n
-seq u en ce o f DM is t h e red u ct io n o f t h e free p lasm a ca rn it in e a n d t h e in crea se o f t h e est er ca rn it in e, w h ich can lead t o t h e accu m u lat io n o f lo n g ch ain fatty acid s, thus d isturb ing the stab ility and the funct io n o f m em b ran es. Besid es funct h e p revio u sly m en funct io -ned functions, carnitine is also essential for the lip id ic o xid at io n an d syn t h esis, b ein g in vo lved in t h e lib e-rat io n an d st o ckp ilin g o f m et ab o lic en erg y 9,10.
Rats w ith d iab etes m ellitus p resent a d ysfunction o f t h e en t eric in n ervat io n an d a carn it in e d eficien cy. Th erefo re, w e st u d ied t h e effect o f t h e su p p lem en t at io n w it h acet ylLcarn it in e (ALC) o n t h e su b p o -p u la t io n o f VIP-erg ic n eu ro n s o f t h e su b m u co u s p lexus of the jejunum of strep tozotocin d iab etic rats.
M ETHOD
Animal procedures - Male Wist ar rat s (Rat t us norvegi-cus) w eig hing 300-400 g w ere em p loyed . In ord er to ind u-ce t h e d iab et es, t h e rat s w ere kep t fast in g fo r 14 h o u rs an d t h en st rep t o zo t o cin (35m g /Kg /b o d y w eig h t , Sig m a, USA) d isso lved in 10 m M b u ffer cit rat e, p H 4.5, w as in jec-t ed i.v. No n -d ia b ejec-t ic ra jec-t s w ere u sed a s co n jec-t ro l g ro u p s. Th e st rep t o zo t o cin in ject io n resu lt ed in a d iab et ic syn d ro -m e w it h rap id w eig h t lo ss an d p o lyd ip sia. Th e rat s w ere d ivid ed into 4 g roup s: non d iab etic (g roup C), non d iab etic t rea t ed w it h ALC (g ro u p CC), d ia b et ic (g ro u p D), a n d d iab etic treated w ith ALC (g roup DC). The initial b od y w ei-g h t o f ei-g ro u p s C, CC, D an d DC w as 362.0 ± 17.9 ei-g , 328.4 ± 6.3 g , 361.6 ± 2.4 an d 367.0 ± 8.1 g resp ect ively. ALC w as ad m in ist ered fo r 15 w eeks fro m t h e o n set o f t h e d ia-b et es ia-b y t h e ad d it io n o f ALC (SPfarm a - São Pau lo -BR) in w at er (200 m g /kg , p rep ared d aily). Th e an im als w ere kep t in in d ivid u al cag es in an en viro n m en t w it h p h o t o p erio d (6:00 - 18:00/light) and controlled tem p erature (24 ± 2º C). The anim als received water and Nuvital® balanced lab chow
ad libit um. Th e w a t er in t a ke w a s m o n it o red b iw eekly, d uring the whole exp erim ent. Fifteen weeks after the onset o f DM, t h e rat s w ere an est h et ized in t rap erit o n eally w it h tiop ental (40m g /kg /b od y weig ht). The b lood was collected b y h ea rt p u n ct u re t o a ssess g lycem ia11 an d h em o g lo b in12.
M orphomet ric and immunohist ochemical analyses
-Aft er a n a b d o m in a l in cisio n , t h e jeju n u m seg m en t s w ere co llect ed , w ash ed in a p h o sp h at e b u ffer (PBS 0.01M, p H 7.4), and fixed w ith Zam b oni’s liq uid for 18 hours at 4º C13.
Th e seg m en t s w ere p ro cessed in a cco rd a n ce w it h t h e im -m u n o h ist o ch e-m ical t ech n iq u e fo r w h o le--m o u n t p rep ara-tions14 aim ing at d etecting VIPs p resence in the sub m ucous
p lexu s. So o n a ft erw a rd s, t h e seg m en t s w ere o p en a lo n g t h e m esen t eric b o rd er, w ash ed an d d eh yd rat ed , cleared in xylo l, re-h yd rat ed an d lat er o n kep t in PBS 0.01M, p H 7.4. Th e sam p les w ere t h en red u ced w it h t h e h elp o f a circu lar cu t t er an d t h e m u co sa an d m u scu lar layers w ere dissected under stereom icroscopy. The isolated subm ucous la yer w a s in cu b a t ed w it h a n t i-VIP p o lyclo n a l a n t ib o d y 1:200 fo r 16 h o u rs (Pen n in su la Lab s, USA). Th e sam p les w ere w ash ed in PBS an d t h en in cu b at ed w it h seco n d ary a n tib od y conjug ated w ith fluorescein (FITC) (Penninsula Lab s, USA) 1:100, for 1 h, at room tem p erature, und er agi-tation. In the control sam p les, the p rim ary antib od y was sub stituted b y goat serum . The whole-m ount p rep arations were m ounted with glycerol. The im m unofluorescence was analyzed in trinocular b iological op tical m icroscop e, 40 X lens, eq uip p ed with filters for im m unofluorescence (FITC) and an IPPWIN-DCAM im age taking kit. The im ages were t a ke n b y a h ig h -re so lu t io n ca m e ra , t ra n sm it t e d t o m icrocom p uter and record ed in com p act d isc.
Th ro u g h t h e Im ag e-Pro -Plu s 3.0.1 so ft w are o f im ag e analysis, we m easured the area (µm2) of 126 cellular b od ies
o f im m u n o react ive VIPerg ic n eu ro n s (VIPIR) in each st u -d ie-d g ro u p . We u se-d t h e m ean valu e an -d t h e st an -d ar-d d eviat io n o f t h e m easu res o f t h e 126 cell b o d ies o f VIP-IR n eu ro n s fro m t h e co n t ro l (C) t o cla ssify t h e n eu ro n s in sm all, m ed iu m an d larg e. We co n sid ered as sm all t h o se n eu ro n s w h o se valu es w ere sim ilar o r in ferio r t o t h e d iffe-rence b etween the m ean value and the stand ard d eviation. La rg e n eu ro n s w ere t h o se w h o se m ea n va lu es w ere sim i-la r o r h ig h er t h a n t h e su m b et w een t h e m ea n a n d t h e stand ard d eviation. We called m ed ium neurons those who-se m ea n va lu es w ere b et w een t h ewho-se va lu es.
St at ist ical analysis - Th e d at a relat ed t o b o d y w eig h t , w at er in t ake, g lycem ia an d b lo o d g lu co se w ere assessed t h ro u g h va ria n ce a n a lysis a n d Tu key t est t o co m p a re t h e m ean . As t h e VIP-IR n eu ro n s d id n o t p resen t a n o rm al d is-t rib u is-t io n in is-t h eir cellu lar b o d y area, w e em p lo yed is-t h e va-rian ce an alysis an d t est “t ” o f St u d en t t o co m p are t h e m ean s. Th e an alyses w ere p erfo rm ed w it h t h e So ft w are Prism 2.01. Th e ch i-sq u are t est w as ap p lied (χ2) t o an alyze
RESULTS
Est ablishing t he diabet es and biochemical
para-met ers - St rep t o zo t o cin p ro m o t ed t h e DM ch
arac-terized by polydipsia and hyperglycem ia (Table 1) and b y t h e sm allest w eig h t g ain in relat io n t o t h e n o n -d iab et ic rat s. Th e g ro u p s C an -d CC p resen t e-d , o n average, a final body weight of 472 ± 23.9g and 452.6 ± 13.1g resp ectively, while the rats from group s D and DC p resented , on average, a final b od y weight of 316.6 ± 3.5g and 336.6 ± 14.1g resp ectively. There-fore, accord ing to these d ata, the results p resented
by the groups D and DC revealed significant statistical d iffe re n ce s w h e n co m p a re d t o t h e n o n -d ia b e t ic group s resp ectively (C and CC) (p < 0.05).
We observed that the ALC-supplem entation redu-ced the glycem ia for group DC. However, there was no significant difference (p> 0.05) between this group (DC) and group D. The anim als from groups D and DC presented high levels of blood glucose in relation to groups C and CC. The ALC-supplem entation did not alter the blood glucose levels in the diabetic anim als from group DC in relation to group D (p> 0.05).
M orphologic analysis - We o b served VIP-IR cell
b o d ies in t h e su b m u co u s p lexu s o f t h e jeju n u m in t h e 4 an alyzed g ro u p s. Gro u p D p resen t ed in t en se im m u n o react ivit y in t h e n ervo u s fib ers an d cellu lar b o d ies. Ho w ever, t h e g ro u p CC an d g ro u p DC p re-sen t ed t h e h ig h est im m u n o react ivit y (Fig 1).
Th e VIP-IR cell b o d ies area averag es varied fro m 306.2 µm2 (g ro u p C) t o 535.5 µm2 (g ro u p DC) (Tab le
2). Th e area o f cell b o d ies fro m g ro u p s D an d DC in creased sig n ifican t ly in relat io n t o g ro u p s C an d CC. The ALC-sup p lem entation p rom oted an increase in t h e area o f t h e VIP-IR cell b o d ies fro m g ro u p CC in relat io n t o g ro u p C (p < 0.05). Th e area o f n eu ro n s fro m g ro u p DC w as larg er t h an t h o se fro m g ro u p D. Ho w ever, t h is d ifferen ce w as n o t sig n ifican t . Table 1. Glycemia (GLI), blood glucose (HbG), w at er consumpt ion
(CDA) of non-diabet ic animals (C), non-diabet ic animals t reat ed w it h acet yl-L-carnit ine (CC), diabet ic animals (D) and diabet ic t reat ed w it h acet yl-L carnit ine (DC). The result s are expressed as mean ± st andard deviat ion. n = 5 rat s per group .
Gro u p GLI m g d L Hb G/ % CDA/m L
C 105.4 ± 11.2a 3.9 ± 0.2a 62.13 ± 0.9a
CC 99.8 ± 5.9a 3.9 ± 0.2a 51.1 ± 1.1a
D 344.4 ± 15.2b 6.6 ± 0.2b 173.92 ± 1.6b
DC 286.0 ± 21.1b 6.8 ± 0.3b 164.6 ± 7.6b Mean s fo llo w ed b y d ifferen t let t ers in t h e sam e co lu m n h ave d ifferen t valu es b y Tu key t est (p < 0.05)
Th e n eu ro n s w ere d ivid ed in 3 g ro u p s acco rd in g to their sizes: sm all neurons (with values b elow 171.8
µm2), larg e (w it h valu es ab o ve 440.6 µm2), an d m
e-d iu m (w it h in t erm ee-d iat e valu es).
Th ro u g h t h e t est o f t h e χ2, w e fo u n d sig n ifican t
d ifferences am ong the sm all, m ed ium and large neu-ro n s w h en w e co m p ared t h e 4 in vest ig at ed g neu-ro u p s. Th e sa m e w a s o b served w h en w e co m p a red t h e g ro u p s tw o b y tw o (C x CC; C x D; C x DC; CC x D; CC x DC; D x DC). Fig u re 2 sh o w s t h e relat ive freq u en cy o f VIP-erg ic n eu ro n s, acco rd in g t o t h e classificat io n in t o sm all, m ed iu m an d larg e. We o b served t h at in t h e g ro u p s C an d CC t h ere is a p red o m in an ce o f m ed iu m size n eu ro n s w h ile in t h e g ro u p s D an d DC, t h e p revalen ce is o f larg e n eu ro n s.
DISCUSSION
An in crease (p < 0.05) o f t h e g lycem ia an d b lo o d g lu co se w as o b served in g ro u p D, co n firm in g t h at t h ese an im als h ad t h e d iab et ic syn d ro m e, sim ilar t o
w h at w as o b served b y Zan o n i et al.15. Th e ALC su p
-p lem en t at io n d id n o t act im m ed iat ely in t h e m et a-b olic control of DM, as evid enced a-b y the sim ilar levels o f b lo o d g lu co se b et w een g ro u p s D an d DC. On t h e other hand , althoug h not statistically sig nificant, the g lycem ia level aft er fast in g w as 20% lo w er o n ave-rag e in g ro u p DC w h en co m p ared t o g ro u p D. Th is fact , p lu s t h e o b servat io n t h at rat s fro m g ro u p DC had a sm aller w eig ht loss, m ay ind icate that the sup -p lem en t at io n h el-p ed im -p ro ve t h e carn it in e levels, w h ich a re vit a l fo r t h e lip id ic o xid a t io n a n d syn -thesis9,10. These results are consistent to the fact that
t h e d iab et es lead s t o a red u ct io n in t h e levels o f m i-t o co n d ria l ALC, p ro m o i-t in g a ccu m u la i-t io n o f fa i-t i-t y acid s h in d erin g , t h erefo re, t h e fu n ct io n o f t h e cell b o d y m em b ra n es a n d o f t h e n eu ro n s n ervo u s fi-b ers10. Th e d ep let io n o f ALC, w h ich h ap p en s in DM,
is o n e o f t h e fact o rs t h at co n t rib u t e t o t h e d evelo p -m en t o f t h e d iab et ic n eu ro p at h y2.
We ob served an increase in the im m unoreactivity t o VIP in t h e su b m u co u s p lexu s o f t h e jeju n u m in t h e rat s fro m g ro u p D, sim ilar t o w h at w as o b served b y Belai & Bu rn st o ck4 w h o evid en ced in t en sely st
ai-n ed VIP-erg ic ai-n eu ro ai-n s iai-n t h e su b m u co u s p lexu s o f rat s, six an d eig h t w eeks aft er st rep t o zo t o cin -d iab e-tes. Fourteen w eeks after ind ucing the d iab etes w ith alo xan a, t h e levels o f su b st an ce P an d m et io n in e-encep haline w ere red uced w hile the levels of the VIP neurotransm itter increased d rastically16. We can infer
t h at t h e in crease o f t h e im m u n o react ivit y is d irect ly relat ed t o t h e in crease o f n o n -lib erat ed VIP levels in the sub m ucous neurons. Chang es in the sub m ucous VIP-erg ic n eu ro n s resu lt in d isg estive tract d iso rd ers, su ch as t h e d iarrh eic d iab et es, sin ce t h e in crease o f VIP release resu lt s in an in creased w at er an d elet ro -lit es secret io n , so ft en in g t h e fecces. Sim ilar resu lt s w ere o b t ain ed b y Belai et al.17, w h o n o t iced an in
-crease in t h e im m u n o react ivit y o f VIP-erg ic n eu ro n s. We d o b elieve t h at t h e in crease in t h e syn t h esis o f VIP b y the m ienteric neuron in d iab etic rats m ay have a triggering factor in dim inishing the intestinal tonus. Th is w o u ld co n t rib u t e t o t h e d iarrh eic d iab et es ap -p ea ra n ce, sin ce a sm a ller in t est in a l t ra ffic w o u ld en h an ce t h e su scep t ib ilit y t o in t est in al in fect io n s.
The ALC sup p lem entation d id not stop the occur-ren ce o f a larg e in crease in t h e im m u n o react ivit y o f VIP-erg ic n eu ro n s o f t h e su b m u co u s p lexu s o f t h e je ju n u m o f d ia b e t ic ra t s. Th is sh o w s t h a t t h is su b st an ce is less effect ive t h an p o n alrest at , w h ich in t h e st u d ies o f Belaiet al.17 st o p p ed t h e so rb it o l
fo rm at io n b y in h ib it in g t h e en zym e ald o se red u t ase a n d a lso st o p p ed t h e in crea se o f VIP im m u n o re-Table 2. M ean and st andard deviat ion of VIP-IR cell body areas in
animals f rom non-diabet ic (C), non-diabet ic t reat ed w it h acet yl-L-carnit ine (CC), diabet ic (D) and diabet ic t reat ed w it h acet yl-L carnit ine (DC).n = 5 rat s.
Gro u p Mean ± SD
C 306.2 ± 17.7a
CC 371.3 ± 16.4b
D 503.8 ± 15.8c
DC 535.5 ± 11.9c
Mean s fo llo w ed b y d ifferen t let t ers in t h e sam e co lu m n h ave d ifferen t valu es b y t St u d en t t est (p < 0.05)
act ivit y an d g alan in a. Ho w ever, ALC is b en eficial fo r t h e in t eg rit y o f t h e n erve b ecau se it p reserves t h e m ioinositol content without interfering in the activity o f t h e p o lyo l p at h w ay, sin ce it s so rb it o l co n t en t s is not altered in hyperglycem ic rats after treatm ent with ALC. An o t h er p o sit ive asp ect is t h at t h e lip id ic p ero -xid a t io n , w h ich is in t e n sifie d in t h e d ia b e t e s, is p reven t ed w it h ALC1,18. Ho w ever, as w e h ave alread y
m en t io n ed , it d o es n o t red u ce t h e im m u n o reacivit y t o VIP in t h e su b m u co u s p lexu s n eu ro n s.
We o b served t h at t h e area o f t h e cell b o d ies o f VIP-erg ic n eu ro n s in g ro u p CC sh o w ed a sm all b u t sig n ifica n t in crea se w h en co m p a red t o g ro u p C, along with an increase in the im m unoreacivity, which co u ld b e relat ed t o a p o ssib le n eu ro t ro p h ic effect o f t h e carn it in e. Th e exp ressive in crease o f t h e area ve-rified in g ro u p s D an d DC co u ld b e relat ed n o t o n ly to the increm ent of the synthesis m achinery b ut also t o it s accu m u lat io n an d exp ressio n d ifficu lt ies. We o b served an in crease in t h e im m u n o react ivit y t o VIP associated to an increase of the area, thus ind icating t h at t h e accu m u lat io n o f t h is su b st an ce w it h in t h e neurons was sim ilar to what was p reviously ob served in VIP-erg ic neurons of the term inal ileum of d iab etic rat s 15. See et al.19 an alyzed t h e su b m u co u s VIP n eu
-ro n s o f t h e jeju n u m o f rat s aft er ch em ical d en erva-t io n a n d verified erva-t h a erva-t erva-t h e a rea o f erva-t h ese n eu ro n s in creases o n ce t h e m yen t eric an d ext rin sic p u lse is rem o ved fro m t h e jeju n u m . Ho w ever, t h ere is n o ch an g e in t h e p ercen t ag e o f su b m u co u s n eu ro n s exp ressin g VIP. Th e in crease in t h e cellu lar size t h at follows denervation suggests that the extrinsic neural p u lse h as an in h ib it o ry in flu en ce o n t h e su b m u co u s n e rve s sin ce , a lt h o u g h t h e m ye n t e ric a n d t h e su b m u co u s p lexu s are sp at ially ap art , t h ey fo rm an in t e g ra t e d u n it2 0. Th u s , t h e re d u ct io n o f t h e
inhib itory p ulse b y the neurons of the m yenteric p le-xus, which are red uced in the chronic DM21-25 for
neuro n s in t h e su b m u co u s p lexu s, co u ld resu lt in an in -crease in t h e VIP p ro d u ct io n , in creasin g t h e area o f t h e cell b o d ies o f t h e su b m u co u s VIP-erg ic n eu ro n s.
Wh e n a n a lyzin g t h e fre q u e n cy o f n e u ro n s acco rd in g t o t h eir size, w e verified t h at t h e g ro u p s (C an d CC) p resen t ed a h ig h er n u m b er o f m ed iu m n eu ro n s w h ile t h e d iab et ic g ro u p s (D an d DC) sh o -w ed a h ig h er n u m b er o f la rg e n eu ro n s. Th a t d e-m o n st rat es t h at t h ere is a sig n ifican t in crease in t h e a rea s o f t h e cell b o d ies o f t h e VIP-erg ic n eu ro n s, m aking m any neurons chang e categ ory, i. e., m aking sm a ll n eu ro n s ch a n g e in t o m ed iu m a n d m ed iu m n eu ro n s in t o larg e o n es. Th e p red o m in an ce o f larg e n eu ro n s w as verified b y Zan o n iet al.21 in t h e cecu m
o f rat s w it h t w o an d eig h t m o n t h s o f DM. We also o b served t h at ALC d o es n o t h ave an y effect o n t h is cat eg o ry ch an g e b y t h e n eu ro n s, sin ce t h e resu lt s b et w een g ro u p s D an d DC w ere sim ilar.
We conclude that ALC prom otes the im provem ent o f so m e asp ect s o f t h e d iab et es su ch as a sm aller b od y weight loss and im p rovem ent in glycem ia levels after fasting; however, regard ing the cellular asp ects, the ALC-sup p lem entation p rom oted a sm all increase in t h e cellu lar p ro file area in t h e n eu ro n s o f g ro u p CC. Th is m ay b e an in d icat ive o f a p o ssib le n eu ro t ro -p h ic effect . Ho w ever, w h en w e co m -p ared g ro u -p s D an d DC w e verified t h at t h e VIP-erg ic n eu ro n s o f t h e su b m u co u s p lexu s o f t h e jeju n u m o f b o t h g ro u p s g o t h ro u g h a larg e in crease in t h eir cell p ro file an d im m u n o reacivit y w h en co m p ared t o t h e co n t ro ls, suggesting a higher concentration of this neurotrans-m it t er in sid e t h e n eu ro n s t h at p ro d u ce it . As t h is fin d in g is a co n st an t in d iab et ic an im als, t h ere is a p ro b ab ilit y t h at t h e ALC w as n o t cap ab le t o in t erfe-re with the p athop hysiological m echanism s that trig-g er t h e h itrig-g h er p ro d u ct io n an d /o r accu m u lat io n o f this neurotransm itter and increase the p rofile of VIP-erg ic n eu ro n s.
Acknow ledgements - We w o u ld like t o t h an k Nat ali Kira Ta m u ra - Ph a rm a ce u t ica l Bio ch e m ist ry - Re g io n a l Aca d e m ica l Ho sp it a l o f Ma rin g á - St a t e Un ive rsit y o f Marin g á; Crist in a Helen a Teles Prad o Freg o n esi - Assist an t Pro fe sso r - UNESP - Pre sid e n t e Pru d e n t e Ca m p u s, Physiotherap y Dep artm ent, Sciences & Technolog y Colleg e (FCT); Angela Alves Pereira - Assistant Professor - UNIOESTE - Bio lo g y Dep art m en t .
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