( PDF ) Consumo de dieta hiperpalatável, alterações metabólicas e comportamentais: um modelo de obesidade em ratos e suas conseqüências

Download PDF

ads:

UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL

DEPARTAMENTO DE BIOQUÍMICA

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS -

BIOQUÍMICA

CONSUMO DE DIETA HIPERPALATÁVEL, ALTERAÇÕES METABÓLICAS E

COMPORTAMENTAIS: UM MODELO DE OBESIDADE EM RATOS E SUAS

CONSEQUÊNCIAS

CAROLINA GUERINI DE SOUZA

Orientador :

Marcos Luiz Santos Perry

Co-orientador :

Luis Valmor Cruz Portela

Dissertação apresentada ao curso de Pós-Graduação em Ciências Biológicas-

Bioquímica da Universidade Federal do Rio Grande do Sul (UFRGS) como pré-

requisito parcial para obtenção do título de mestre em Bioquímica

Porto Alegre

2007

ads:

Livros Grátis

http://www.livrosgratis.com.br

Milhares de livros grátis para download.

“Tudo posso Naquele que me fortalece”

Filipenses 4:13

ads:

III

AGRADECIMENTOS

Inicialmente agradeço a Deus pela sua bondade em me conceder as oportunidades

recebidas até hoje e pelas mesmas fazerem de mim uma pessoa privilegiada.

Ao professor Perry, que me deu a oportunidade de ser sua aluna mesmo ainda

quando pouco me conhecia; por todo conhecimento transmitido diariamente e pela

sabedoria de ensinar não só bioquímica, mas sobre todas as demais ciências que a

compõem. Pela ajuda nos momentos de dúvidas e ignorância e por ser um verdadeiro

mestre.

Ao Roska, meu maior incentivador, que me fez não desistir das coisas que

acreditava, contribuindo científica e humanamente em minha formação e que muito mais

do que um co-orientador, tornou-se um grande amigo.

Ao professor Diogo, que me abriu as portas do departamento de bioquímica e que

me acolheu em seu grupo, de maneira especial e única.

À Júlia, amiga querida e parceira de bons e maus momentos. Pela amizade sincera e

parceria profissional diárias.

À Ana Elisa, que desde a iniciação científica contribuiu não só com sua amizade,

mas com sua inteligência e solicitude em ajudar, fundamentais para minha formação.

Ao Jean, Giordano e Alexandre, pessoas com as quais aprendi muito e que foram

fundamentais para construção deste trabalho.

À Andréa, bolsista de iniciação científica, por todo apoio e boa vontade em

trabalhar comigo.

À minha irmã Débora, que tanto científica quanto emocionalmente, teve uma

participação importante em minha formação.

Aos amigos do lab 27 Adriano, Daniel, Cíntia, Suzéli, Dé e Betina, pela amizade,

pela convivência e pelo ambiente amigável que proporcionaram.

Aos amigos do lab 26 e 28, que me acolheram, me ouviram, me ajudaram e sempre

me trataram como parte de seu grupo.

Ao professor Sarkis, pelo material fornecido para trabalharmos e pela boa vontade

de colaborar tecnicamente.

Aos meus queridos pais, Olcei e Ângela, pelo exemplo, pelo amor, pela educação,

por tudo que me deram e por tudo que tenho, pois sem eles nada seria possível.

Ao Pablo, meu amado, meu amigo, meu parceiro, pela paciência extrema, pelo

apoio incondicional e pela sua presença, que me torna meus dias muito melhores.

Ao departamento de bioquímica e funcionários, pelo ensino e pelo ambiente de

apoio em todas as intercorrências do mestrado.

Ao CNPq, pelo fornecimento da minha bolsa, que foi decisiva para que pudesse

haver dedicação à este trabalho.

SUMÁRIO

RESUMO ........................................................................................................................ VI

ABSTRACT ...................................................................................................................VII

LISTA DE ABREVIATURAS.......................................................................................VIII

1. INTRODUÇÃO ............................................................................................................. 1

1.1.Obesidade e dieta hiperpalatável .................................................................................. 1

1.2. Resistência à insulina e Diabete Mellitus tipo 2........................................................... 2

1.3. Doenças cardiovasculares............................................................................................ 4

1.4. Alimentos palatáveis, obesidade e comportamento ...................................................... 6

1.5. OBJETIVOS............................................................................................................... 8

1.5.1. Objetivo geral...........................................................................................................8

1.5.2. Objetivos específicos................................................................................................ 8

2 – ARTIGOS.................................................................................................................... 9

2.1 – CAPÍTULO 1............................................................................................................ 9

2.2 – CAPÍTULO 2.......................................................................................................... 29

3. DISCUSSÃO............................................................................................................... 51

4. REFERÊNCIAS........................................................................................................... 57

RESUMO

A obesidade é a maior síndrome do século XXI, alcançando proporções epidêmicas

em todo mundo. Muitas são as causas para o seu desenvolvimento, porém a mais

importante, provavelmente, é o consumo aumentado e a grande disponibilidade de

alimentos altamente palatáveis, ricos em açúcar e gordura. A obesidade e o consumo destes

alimentos são associados ao aparecimento de doenças como hipertensão, diabetes tipo 2 e

doenças cardiovasculares. A resistência à insulina, a diminuição dos níveis de adiponectina

e do óxido nítrico circulantes são fatores de riscos importantes para o surgimento de

doenças cardiovasculares. Além disso, a diminuição dos níveis séricos de adenosina

também está associada com o aparecimento de doenças cardiovasculares e que esta

diminuição pode ser influenciada por alguns fatores ambientais. A hidrólise dos

nucleotídeos da adenina, ATP, ADP e AMP, é uma das formas de manter os níveis de

adenosina circulantes e por isso a atividade das enzimas ectonucleotidases, que regulam

esta hidrólise, é importante para homeostase cardiovascular. Em face disto, tivemos por

objetivo verificar a atividade destas enzimas em um modelo de indução de obesidade por

meio de dieta hiperpalatável, além de verificar as demais alterações na composição corporal

e no metabolismo da glicose, dos lipídios séricos, nos níveis de insulina, adiponectina e de

óxido nítrico, focando nos possíveis efeitos dessas alterações no sistema caridiovascular.

Nossos resultados indicaram que a obesidade induzida pela dieta promove um perfil de

alterações semelhante a diabete mellitus tipo 2, acompanhada de diminuição dos níveis de

óxido nítrico e de diminuição acentuada da atividade das ectonucleotidases, promovendo

um ambiente pró-aterogênico, mesmo sem alteração dos níveis de insulina e adiponectina.

Além das alterações no metabolismo, a obesidade e o consumo de alimentos altamente

palatáveis está associado com alterações cognitivas e comportamentais, como ansiedade,

depressão e memória. Sabe-se também que estes alimentos geram um padrão de adição nos

centros neurais, que pode estar relacionado com doenças psiquiátricas como transtornos de

humor e ansiedade, e muitos estudos populacionais mostram que o estresse oxidativo

também pode ser associado à estes transtornos. Em indivíduos intolerantes à glicose, a

hiperglicemia é a maior fonte de produção de radicais livres e em função disto, decidimos

testar nossos animais submetidos à dieta hiperpalatável para avaliar possíveis alterações

comportamentais e após os testes, medir o estresse oxidativo no córtex frontal e hipocampo

destes animais. Nossos resultados mostraram que os animais que receberam a dieta

hiperpalatável são mais ansiosos dos que receberam a dieta controle, porém sem alterações

de locomoção ou memória. Além disso, os mesmos apresentam maior dano oxidativo nas

proteínas do córtex frontal, uma área muito ligada ao comportamento emocional de

ansiedade e medo, mas sem alterações no hipocampo. Em função do exposto, propomos

que além de promover alterações no metabolismo da glicose e aumentar o risco de doenças

cardiovasculares, a obesidade promovida pelo consumo de uma dieta hiperpalatável

aumenta a ansiedade e a oxidação de proteínas no córtex frontal, afetando também o

sistema nervoso central e o comportamento emocional.

VII

ABSTRACT

Obesity is the major syndrome of 21st century, reaching epidemic proportions

worldwide. There are many causes for obesity but the most important is probably the

overeating and the ready availability of food rich in fat and sugar. Obesity and the

consumption of this type of food are associated to hypertension, type 2 diabetes and

cardiovascular diseases as consequences. Insulin resistance, decreased adiponectin levels

and nitric oxide circulating are strong risk factors to development of cardiovascular

diseases. Besides, decreased of serum adenosine levels are also associated to cardiovascular

diseases and those leveles can be affected by environmental factors. Adenine nucleotides

hydrolysis ATP, ADP and AMP is one way to keep the circulating adenosine levels by

ectonucleotidases activity, which regulates this hydrolysis. For that reason, this enzymes

activity is important for cardiovascular homeostases. Based on this, the aim of our study

was to verify the ectonucleotidases activity in a model of obesity induction through a

highly palatable diet consumption. We also intent to verify another alterations in body

composition, glucose metabolism, serum lipids, insulin, adiponectin and nitric oxide levels,

focusing in possible effects of these alterations in cardiovascular system. Our results

showed that obesity induced by this diet provokes alterations type 2 diabetes like, reduced

levels of nitric oxide and accentuated decreased of ectonucleotidases activity, promoting a

vascular pro-atherogenic environment, even without alterations in insulin and adiponectin

levels. Besides metabolic alterations, obesity and the consumption of palatable foods are

associated to cognition and behavioral alterations as anxiety, depression and memory

capacity. Is well known that in brain circuits palatable foods promote an addiction profile,

which may be related to psychiatry disorders like mood and anxiety. Populational studies

showed that oxidative stress also can be associated to these alterations. As glucose

intolerace is the main source of free radicals production in glucose intolerant individuals,

we decided to test our animals submitted to a highly palatable diet to search for possible

behavioral alterations. After behavioral tests, we decided to measure oxidative stress in

frontal cortex and hippocampus of the same animals. This other part of our results showed

that the animals submitted to a highly palatable diet are more anxious than the animals

submitted to a standard diet, but there were no alterations in locomotion and memory

capacity. The same animals also presented higher oxidative protein damage in frontal

cortex, an important brain structure involved in behavioral regulation and is part of several

well-defined anxiety and fear-related circuits in the forebrain, however no alterations was

observed in the hippocampus. Therefore, we propose that beyond alterations in glucose

metabolism and increasing cardiovascular diseases risk, obesity induced by consumption of

a highly palatable diet increases anxiety and frontal cortex protein oxidation, also affecting

central nervous system and emotional behavioral.

VIII

LISTA DE ABREVIATURAS

ATP – adenosina trifosfato

ADP – adenosina difosfato

AMP – adenosina monofosfato

TAG – triacilglicerol

NO – óxido nítrico

RL – radicais livres

SM – Síndrome Metabólica

DM2 – Diabete Mellitus tipo 2

ENOS – óxido nítrico sintase endotelial

IRS1- substrato receptor de insulina 1

1. INTRODUÇÃO

1.1.Obesidade e dieta hiperpalatável

A obesidade pode ser considerada uma das principais síndromes do século XXI,

estando envolvida na etiologia de uma série de doenças como resistência à insulina,

diabetes, hipertensão e cardiopatias (Unger and Orci, 2001; Dube et al, 2005). Durante o

século XX uma mudança nos padrões de disponibilidade de calorias tomou conta de países

ocidentais. Contribuem para isso, os avanços tecnológicos do mundo moderno, que

tornaram o estilo de vida sedentário, e o maior acesso aos alimentos, que possibilita uma

maior ingestão calórica diária. Freqüentemente, muitos desses alimentos não são os mais

nutritivos e saudáveis, mas sim, os mais “palatáveis” e baratos (Chakravarthy and Booth,

2004). Embora o alto consumo de uma dieta rica em lipídios, carboidratos simples e a

diminuição da atividade física seja facilmente capaz de induzir a obesidade, fatores

genéticos também precisam ser levados em conta, mesmo não justificando os níveis

epidêmicos de obesos na população mundial (Froguel et al., 2000; Saper et al., 2002).

Os alimentos hiperpalatáveis são capazes de estimular o consumo alimentar e

promover mudanças no metabolismo por alterarem a razão fisiológica entre moléculas

orexígenas/ anorexígenas, muitas das quais estão relacionadas com a utilização da glicose, a

oxidação dos ácidos graxos, o gasto energético, além do próprio mecanismo de saciedade

em si, as quais são bases da obesidade e de patologias relacionadas (Erlanson-Albertsson,

2005). Como os mecanismos de homeostasia que tentam controlar as conseqüências da

“superalimentação” foram incapazes de compensá-la, em função do aumento crônico no

balanço calórico, as doenças relacionadas à superalimentação tornaram-se incrivelmente

prevalentes (Neel, 1999).

Em indivíduos obesos alterações como intolerância à glicose, resistência periférica à

insulina, hipertensão, dislipidemias e aterosclerose estão freqüentemente presentes como

comorbidades (Takahashi et al., 2007; Wassink et al., 2007). O conjunto destes fatores é

denominado Síndrome Metabólica (SM) e na maior parte dos casos, esta evolui para o

Diabete Mellitus tipo 2 ou para doença cardiovascular ou, em alguns casos para ambos

(Naderali et al., 2004). Portanto, o tipo de dieta pode ser considerado um fator determinante

para a formação do percentual de gordura bem como para o aparecimento ou a prevenção

das patologias associadas à obesidade (Cnop et al., 2003).

1.2. Resistência à insulina e Diabete Mellitus tipo 2

O aumento de gordura corporal, especialmente da gordura visceral, promove o

aumento de ácidos graxos livres e acúmulo dos mesmos em tecidos que não sejam o

adiposo, como fígado, músculo esquelético e até mesmo nos vasos sangüíneos (Arner et al.,

2005; Yudkin et al., 2005). Ao acúmulo de ácidos graxos livres em tecidos não adiposos foi

atribuído o termo de lipotoxicidade e acredita-se que esta é a causa em comum das

complicações da obesidade, resistência à insulina, diabetes tipo 2 e doenças

cardiovasculares (Unger and Orci, 2001). Esses ácidos graxos acumulados inibem o

metabolismo de carboidratos nestes tecidos via competição de substrato e assim

impossibilitam as ações fisiológicas da insulina, causando resistência periférica à mesma,

promovendo hiperglicemia (Yudkin et al., 2005). À hiperglicemia promovida e à suas

conseqüências foi dado o nome de glicotoxicidade, caracterizadas pela alta capacidade de

produção de radicais livres e alteração na estrutura e função de proteínas, lipídios, citocinas

e até mesmo do DNA das células lesadas, sendo as células endoteliais dos capilares , as da

retina, dos glomérulos renais e dos nervos periféricos as mais suscetíveis à lesões

(Brownlee, 2005). O quadro de resistência periférica à insulina, por glicotoxicidade e/ou

lipotoxicidade, acrescido da diminuição da secreção pancreática deste hormônio caracteriza

a instalação da diabete mellitus tipo 2 (DM2) (Mc Garry, 2002). O DM2 tem por

conseqüências hipertensão, dislipidemias, esteatose hepática, complicações

cardiovasculares e ateroscleróticas, retinopatia, nefropatia, neuropatia periférica e até

mesmo desordens psiquiátricas (Pinhas-Hamiel and Zeitler, 2007).

O aumento do tecido adiposo é um fator determinante para todas estas alterações

pois além de desregular o metabolismo da glicose e dos ácidos graxos por meio da

lipotoxicidade e da glicotoxicidade, gerando resistência à insulina, ainda promove

alterações nas citocinas secretadas pelo mesmo. O tecido adiposo não é mais visto

atualmente somente por sua função energética, mas sim como um órgão ativo em secreções

que regulam desde à sensibilidade à insulina até o controle do peso corporal, saciedade e

resposta inflamatória (Prins, 2002). Dentre as citocinas secretadas por este tecido, as mais

relacionadas com a síndrome metabólica e com o DM2 são a adiponectina e a leptina, por

exercerem ações pró-insulina e também por aumentarem a oxidação de lipídios (Park et al.,

2003; Gil-Campos et al., 2004). Os níveis de adiponectina tem sido utilizados atualmente

como preditores da síndrome metabólica e de doenças cardiovasculares, em função de seu

papel antiaterogênico, protetor do sistema cardiovascular e por estarem diminuídos com o

aumento da gordura corporal (Rothenbacher et al., 2005).

Além de levar ao acúmulo de lipídios no fígado e no músculo, a inabilidade da

insulina também está associada à diminuição de uma vasodilatação-dependente do

endotélio vascular, em resposta a vários vasodilatadores afetados pela obesidade, inclusive

a própria insulina. A diminuição desta resposta é um dos fatores que viabiliza disfunções

endoteliais em indivíduos insulino-resistentes ou diabéticos (Yudkin et al., 2005).

1.3. Doenças cardiovasculares

Embora os estudos tenham mostrado que as causas clássicas para doenças

cardiovasculares (tabagismo, hipertensão e dislipidemia) tenham diminuído nos últimos 25

anos, o aumento da obesidade, do DM2 e da resistência à insulina manteve a prevalência

destas doenças aumentada como antes (Smith, 2007). A aterosclerose é a causa de morte de

¾ dos pacientes que vão à óbito por doenças cardiovasculares (Cheng, 2005).

Os mecanismos fisiopatológicos que aumentam o risco de doenças cardiovasculares

em indivíduos obesos e com resistência à insulina são a hipertensão, os estados pró-

inflamatório e pró-trombótico, dislipidemias, além da hiperglicemia, que gera metabólitos

pró-oxidantes (Rader, 2007). Estes fatores associados promovem disfunção endotelial, seja

por maior resposta inflamatória, agregação plaquetária, oxidação de LDL, vasoconstrição

ou todos juntos.

A resistência à insulina afeta também o endotélio vascular por diminuir a produção

de óxido nítrico, que atua como vasodilatador endógeno. A ativação da enzima óxido

nítrico sintase (e-NOS) ocorre por meio da ativação da cascata insulínica e em resposta à

mesma, ocorre diminuição dos níveis de endotelina, que atua como vasoconstrictor (Yudkin

et al., 2005). O aumento na ingestão calórica também afeta esse sistema, uma vez que em

situações de ingestão excessiva de calorias é formado um depósito de gordura local nos

vasos sangüíneos, com funções vasoregulatórias específicas, secretando citocinas e

regulando as funções deste endotélio, desde atividade enzimática até a transdução de sinais

(Mazurek et al., 2003).

Recentemente a diminuição dos níveis de adenosina vem sendo muito associada à

um maior risco de doenças cardiovasculares, principalmente por desempenhar um efeito

vasodilatador e de melhora do fluxo sangüíneo durante situações de hipóxia (Berne, 1963).

Além disso, ela inibe a agregação plaquetária, a proliferação de células musculares lisas e a

adesão de neutrófilos ao endotélio vascular, sendo todos estes fatores associados a

fisiopatologia de doenças cardiovasculares (Mubgawa et al., 1996; Ralevick and Burnstock,

2003). Sua liberação na corrente sangüínea pode ocorrer por secreção celular ou pela

degradação dos nucleotídeos da adenina extracelulares ATP, ADP E AMP (Latini e Pedata,

2001). Esses nucleotídeos podem ser degradados por nucleotidases, que estão localizadas

em superfícies celulares e solúveis nos meios extracelulares, sendo o endotélio vascular um

de seus principais sítios de localização (Sarkis et al., 1995; Zimmermann et al., 2001).

Alguns fatores ambientais podem diminuir a atividade destas enzimas, como o uso de

imunosupressores e a hiperhomocisteinemia, fatores estes independentes para doenças

cardiovasculares (Chen et al., 2002; Koymada et al., 1996). Em função disto, tem-se

postulado que controle das atividades dessas nucleotidases são importantes para impedir

processos de formação de trombos vasculares pela formação e manutenção do equilíbrio

dos níveis da adenosina, mantendo sua homestase no sistema vascular.

1.4. Alimentos palatáveis, obesidade e comportamento

A obesidade pelo consumo de alimentos palatáveis, tem sido investigada não só por

suas alterações metabólicas, mas também por alterações comportamentais apresentadas por

indivíduos acima do peso. Estudos experimentais têm focado suas investigações em

mecanismos moleculares e alterações comportamentais específicas por consumo de dietas

ricas em açúcar e gordura, promovendo alterações nas células neuronais (Molteni et al,

2002, 2004).

A obesidade por consumo de alimentos altamente palatáveis está associada com

mecanismos de recompensa, promovendo uma resposta de adaptação e gerando padrões de

adição nos centros neurais estimulados por ela (Colantuani et al., 2002; Erlanson-

Albertsson, 2005). Estudos populacionais com pacientes sobrepreso e obesos mostraram

que existe uma prevalência maior de doenças psiquiátricas dentre estes, especialmente

ansiedade, depressão e transtornos de humor (Chakravarthy et al., 2004, Simon GE, 2006;

Becker et al., 2001; Teegarden and Bale, 2007).

A ansiedade é uma das desordens psiquiátricas mais prevalentes atualmente

(Gingrich,2005) e recentemente foi associada ao stress oxidativo, por meio de estudos que

mostraram que genes envolvidos na produção de RL e nas defesas antioxidantes poderiam

modular para mais ou para menos o nível de ansiedade em modelos animais, dependendo

do maior ou menor nível de estresse gerado (Hovatta et al., 2005; Berry et al., 2007).

A hiperglicemia presente na obesidade, na resistência à insulina e do DM2 é

responsável por altos níveis de stress oxidativo nestas situações, uma vez que causa

bloqueio da cadeia transportadora de elétrons, gerando elevados níveis de espécies reativas

de nitrogênio e oxigênio (Nishikawa et al., 2000; Brownlee, 2005). Entretanto as razões

pelas quais as desordens psiquiátricas tem prevalência aumentada na obesidade e podem

ocorrer em conseqüência do DM2 ainda são desconhecidas.

1.5. OBJETIVOS

1.5.1. Objetivo geral

Em função das evidências atuais sobre o consumo de alimentos hiperpalatáveis e a

obesidade por eles desencadeada, procuramos :

• avaliar os efeitos de ambos sobre o metabolismo intermediário, especificamente em

relação aos fatores de risco para doenças cardiovasculares;

• verificar a ação destes alimentos e da obesidade gerada em aspectos

comportamentais e parâmetros bioquímicos associados ao estresse oxidativo.

1.5.2. Objetivos específicos

• Avaliar a massa adiposa, tolerância à glicose, perfil lipídico, resistência à insulina,

níveis de insulina, de adiponectina e de óxido nítrico em soro de ratos submetidos à

dieta hiperpalatável;

• Avaliar a atividade das ectonucleotidases por meio da hidrólise de nucleotídeos da

adenina;

• Avaliar o comportamento de ansiedade, locomoção, aprendizado e memória de ratos

submetidos à dieta hiperpalatável;

• Investigar a existência de stress oxidativo em estruturas do sistema nervoso central

de ratos submetidos à dieta hiperpalatável.

2 – ARTIGOS

2.1 – CAPÍTULO 1

EFFECTS OF A HIGHLY PALATABLE DIET ON LIPIDIC METABOLIC

PARAMETERS, ADIPONECTIN LEVELS AND ADENINE NUCLEOTIDES

HYDROLISYS

Em processo de finalização para ser submetido ao Journal of Nutritional Biochemistry

Effects of a highly palatable diet on lipidic metabolic parameters, adiponectin levels

and adenine nucleotides hydrolisys.

Carolina Guerini de Souza

*, Ana Elisa Böhmer

, Jean Pierre Oses

, Giordano Gübert

Viola

, Alexandre Pastoris Müller

, Débora Guerini de Souza

, Daniel Neumann

Lesczinski

, Luis Valmor Portela

, Marcos Luiz Santos Perry

Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul – UFRGS

* Corresponding author:

Carolina Guerini de Souza

Departamento de Bioquímica, ICBS

Rua Ramiro Barcelos, 2600 anexo

CEP 90035003

Porto Alegre, RS, Brazil

Phone: 55 51 33085551

Fax: 55 51 33085560

Email: carolguerini@hotmail.com

Abstract

Obesity reached epidemic proportion worlwide and is stimulated by the ready

availability of food rich in fat and sugar (highly palatable diet), increasing the risks of

obesity-associated pathologies. Adiponectin, an adipocitokine, is inversily proportional to

body mass index and its serum levels are decreased in obesity and in cardiac patients, as the

same way nitric oxide levels are decreased in these two situations. ATP, ADP,AMP and

adenosine are molecules also linked to cardiovascular health and can be regulated by

soluble and cell surface located ectonucleotidases. Alterations in the phisiologic hydrolysis

of these nucleotides are related to cardiovascular complications. The aim of this study is

evaluate the effects of a highly palatable diet, as a model of obesity induction, in metabolic

parameteres, adiponectin levels and ectonucleotidases activity. Twenty male Wistar rats

received two diferent diets during four months: standard chow (SC) and highly palatable

(HP). Body weight, body fat mass, glucose intolerance, total cholesterol, HDL cholesterol,

serum triacylglycerol (TAG), liver triacylglycerol and free glycerol were higher in HP

group (p<0.05 and p <0.01), although insulin and adiponectin levels were not different

between groups. However, nitric oxide had a trend to be lower in the HP group (p=0.06)

and the hydrolysis of ATP, ADP and AMP were significantly lower in this same group

(p<0.05 and p <0.01). In conclusion, the consumption of a highly palatable promotes subtle

metabolic alterations involving cardiovascular system homeostasis and increasing the risk

of cardiovascular diseases even when conventional diagnosis markers as insulin and

adiponectin are not altered yet.

Key words : Highly Palatable Diet / Obesity / Adiponectin / Type 2 diabetes / Nucleotide

Hydrolysis / Ectonucleotidases / Cardiovascular Disease

Introduction

Obesity can be considered the major syndrome of 21st century and is envolved in

the etiology of several diseases as insulin resistance, hypertension, cardiovascular diseases

and type 2 diabetes [1,2]. The most important cause of obesity is probably over-eating

(coupled with inactivity) which is stimulated by the ready availability of food rich in fat

and sugar [3]. The consumption of palatable foods (highly palatable diet) is capable to

induce these metabolic alterations, being a decisive factor to increase adipose tissue as well

as to developing of obesity-associated pathologies [4,5].

Adipose tissue secretes bioactive peptides, termed 'adipokines', which act locally

and distally through autocrine, paracrine and endocrine effects [6]. Adiponectin, one of

these citokines, is a 244 amino-acid protein synthesized and secreted exclusively by

adipose tissue, displaying major insulin-sensitizing properties in skeletal muscle, liver and

adipose tissue, antiatherogeinc effects and stimulates nitric oxide production in vascular

endothelial cells, promoting vasodilatation [7,8]. Circulating adiponectin levels

concentrations are inversely correlated with body fat mass and these levels are

predominantly determined by visceral fat [9]. Obesity and insulin resistance negatively

correlates with adiponectin concentration but decreased adiponectin was originarally

described in patients with cardiovascular disease and for that reason changes in vascular

system could be attributed to changes in circulating adiponectin concentration [8].

In vascular system extracellular adenine nucleotides ATP, ADP, AMP and its

nucleoside adenosine have several biological roles [10]. Serum ATP can act as a

vasolidatator or a vasoconstrictor, depending of its concentration in blood flow. Besides,

this nucleotide is substrate for ADP and AMP formation, which are platelet aggregants, and

both can be hydrolised to adenosine, a pontent vasodilatator. In the extracellular space these

nucleotides can be hydrolyzed by soluble and cell surface located ectonucleotidases.Thus, it

has been postulated that the activity of these enzymes is essential for the maintenance of

ATP/ADP/AMP/adenosine appropriate levels contributing to vascular homeostasis [11,12].

Exploring the effects of a highly palatable diet in metabolism and vascular system,

the aim of this study is to verify the effects of this diet in metabolic parameters, adiponectin

levels, ectonucleotidases activity and a possible conection between adiponectin levels and

ectonucleotidases activity, acting together as regulators of vascular system.

Matherials and Methods

Animals and Diets

Twenty 60-day old male Wistar rats weighting from 210 to 230 g were

obtained from the Central Animal House of UFGRS Biochemistry Department, Federal

University of Rio Grande do Sul, Brazil. They were maintained under a standard dark-light

cycle (lights on between 7:00 a.m. and 7:00 p.m.), at a room temperature of 22 + 2°C.

Animal care followed the official governmental guidelines in compliance with the

Federation of Brazilian Societies for Experimental Biology and was approved by the Ethics

Committee of the Federal University of Rio Grande do Sul, Brazil.

Rats were divided into two groups: (1) the control group (SC, n=10), which received

standard laboratory rat chow (65 % carbohydrate totally from starch, 25 % protein and 10%

fat); and (2) the highly palatable diet group (HP, n=10), which received a enriched sucrose

diet (65 % carbohydrates being 34 % in condensed milk, 23% from starch and 8 % from

sucrose, 25 % protein, 10 % fat). All animals had free access to food and water. After four

months of diet, they were killed by decaptation and analyses were performed.

Glucose tolerance test

Glucose tolerance test was performed one week before the animals were sacrificed

in each time. A 50 % glucose solution was injected into the animals (i.p, 2 mg/g) after 6h of

starvation [13]. The blood was collected by a small puncture on tail immediately before and

30, 60, and 120 minutes after the injection. At each time, glucose was measured by a

glucosimeter (AccuChek Active, Roche Diagnostics

, USA).

Blood and Tissue preparation

Animals were anesthetized by sodium tiopenthal (40 mg/Kg) and blood samples

were collected by cardiac punction. Visceral and epididimal fat pads (which we will present

as fat mass) were dissected and weighted. The blood was imediatelly collected and

centrifuged at 5000 g by 10 minutes to obtain serum.

Metabolic parameters and nitric oxide determination

Serum metabolic parameters such as total cholesterol, LDL-cholesterol, HDL

cholesterol and triacilglycerol (TAG) were measured by commercial kits (Roche

Diagnostics, Indianapolis, USA). Serum glycerol was used as a marker of lipolysis by

indirect determination of free fatty acids and was measured with Free Glycerol Reagent Kit

from Sigma Chemical Co. (St. Louis, MO, USA).

Hepatic TAG, a marker of insulin resistance, was measure using a 100 mg liver

sample, homogenized in 1:20 saline solution (0.9%) and the assay was performed with an

aliquot of this mixture, by commercial kits (Roche Diagnostics, Indianapolis, USA). Nitric

oxide was determined with Greiss reagent, as described by Miranda et al., 2001 [14].

Measurement of ATP and ADP hydrolysis

ATP and ADP hydrolysis were evaluated using the method described by Oses et

al.(2004) [15]. The reaction mixture containing 3.0mM ATP or ADP as substrate, 1.0-1.5

mg serum protein and 112.5 mM Tris-HCl, pH 8.0, was incubated at 37ºC for 40 minutes in

a final volume of 2.0 mL. The reaction was stopped by the addition of 0.2 mL of 10%

TCA. All samples were centrifiged at 5000 g for 5 min and the supernatant was used for

measuring the amount of inorganic phosphate (Pi) released through a colorimetric assay

[16]. Incubation time and protein concentrations were chosen to ensure the linearity of the

reaction (results not shown). In order to correct non-enzymatic hydrolisys, we performed

controls by adding serum after TCA. All samples were assayed in duplicate. Enzyme

activities were expressed as nmol of Pi released per minute per miligram of protein.

Measurement of AMP hydrolysis

To evaluate the AMP hydrolysis we used a reaction mixture containing 3.0 mM

AMP in 100 mM Tris-HCl, pH 7.5, incubated with 1.0-1.5 mg of serum protein at 37ºC in a

final volume of 0.2 mL. All other procedures were the same as described above of ATP and

ADP hydrolysis.

Measurement of insulin and adiponectin levels

Serum insulin levels were measured by RIA (BioChem ImmunoSystems, Roma,

Italy) and adiponectin was measure by Elisa kit (Linco Research, St Charles, MO, USA).

Protein determination

Protein was measured by the Comassie Blue method using bovine albumin as

standard [17].

Statistical analysis

Data were analyzed using SPSS 10.0 software, with Student’s T test for independent

samples to parametric variables. Results are expressed as mean ± standard deviation.

Results

At the end of treatment, final body weight was different between SC and HP groups

(p<0.01, Figure 1A) and the same is observed in the fat mass (p<0.01, Figure 1B), which

were almost two fold higher in HP group. As we can observe in figure 1A and 1B, the gain

of body weight related to fat mass was higher in HP group than in SC group (fat mass to

body weight ratio : 0.04 in SC group ; 0.06 in HP group).

The glucose tolerance was lower in HP group and the higher glucose levels were

found at 30 and 60 minutes after the injection (p<0.01, Figure 1C), compared to SC

group.The glucose tolerance test was different between groups starting from 30 minutes

after the glucose injection until the end of the test (120 minutes after), once HP group

glucose levels remain statistically different from fasting glucose levels while SC group was

not different (p<0.05, Figure 1C).

Both total cholesterol and HDL cholesterol of HP group were different between

groups, being higher in HP group (p<0.05, Table 1). The serum TAG levels were two fold

higher in HP group (p<0.05, Table 1) and the same was observed in the liver TAG content

of this group (p<0.01, table1) and the free glycerol (p<0.01, Table 1). Altough glucose

tolerance was altered in HP group, serum insulin and adiponectin levels were not different

between the groups (Table 1). Nitric oxide was not statistically different between groups at

four months of diet, although there is a trend to be higher in SC group (p=0.07, Figure 2A).

The hydrolisys of adenine nucleotides was significantly lower in HP group. In HP

group ATP hydrolisys was 33% lower (p<0.01, figure 2B), ADP hydrolysis was 49% lower

(p<0.01, figure 2C) and AMP hydrolysis was 23 % lower (p<0.05, figure 2D) when

compared to SC group .

Discussion

The consumption of a highly palatable diet enriched with sucrose for four

months was able to alter body composition, promoting gain of weight and fat more

vigorously than a standard diet. As the same way, total cholesterol, HDL cholesterol, serum

TAG, hepatic TAG and free glycerol reached elevated levels on the animals of HP group.

The sucrose consumption also caused severe glucose intolerance, once animals submitted to

the diet had higher glucose plasmatic levels than control group, even 120 minutes after the

i.p. glucose injection, which leads to type 2 diabetes instalation, even without changes in

insulin and adiponectin levels between groups until this point. Associated to that, adenine

nucleotides hydrolisys was decreased in HP group, since the diet considerably lowered

ectonucleotidases activity. The trend to decrease nitric oxide levels in this same group

corroborates with the diet’s effect on ectonucleotidases activity.

Obesity, glucose intolerance and dyslipidemia are the most commom features of

metabolic syndrome, a complex of alterations that frequently leads to type 2 diabetes and/or

cardiovascular diseases [18,19] and is well know that consumption of diets enriched with

fat, sucrose or both is positively associated to the development of this syndrome [20,3],

although the most used models to induction of obesity and type 2 diabetes are high fat

diets. The increase of body fat mass, caused by these diets, also leads to alterations in

adipose tissue functions, including dysregulation of fatty acids metabolism, insulin

resistance and changes in adipocitokines secretion [21,22]. In our study, we used high

amounts of sugar, changing standard carbohydrate composition, without changes in dietary

fat content. Animals feeded with the highly palatable diet developed alterations on body

composition, glucose tolerance, lipid profile and fatty acids utilization, as the same way that

occurs in high fat diets, but without differences in insulin and adiponectin levels. However,

elevated hepatic TAG and free glycerol are indirect markers of insulin resistance, once

these elevations occur by impaired ability of insulin

to suppress endogenous glucose

production and fatty acid oxidation on liver and defect in insulin suppression of lipolysis at

the level of adipocyte [23,24]. Therefore, insulin resistance already exists even without

changes in serum insulin levels, as we can observe in our results.

Besides alterations in fatty acids metabolism and insulin actions, the increase of

body fat mass are related with changes in adiponectin secretion [25,26]. Adiponectin, an

adipocitokine, is the most abundant gene product in adipose tissue and exerts pro-insulin

actions in muscle and liver, antiaterogenic and antiinflamatory functions in vascular

endotelium and stimulate nitric oxide production and secretion [7,27,28]. Although obesity

decreases serum adiponectin levels, we could not observed that in the animals of HP group,

even they became obese. Our data corroborates with Barnea et al. (2006)[29] which also

did not find alterations in serum adiponectin levels in a model of diet-induced obesity and

insulin resistance in mice, although adiponectin and adiponectin receptors mRNA was

decreased in muscle and liver. However, this work showed in the same animals that they

had insulin sensitivity accompanied by impaired activity of adiponectin-related enzymes.

For this reason, we propose with our results that metabolic alterations maybe starts before

adiponectin levels decreases, as the same way occured with insulin parameters. We can

reinforce that with the lower nitric oxide levels in HP group, one of the most expressive

endogenous vasodilatator, which should follow adiponectin levels, once is stimulated by it,

and that did not occur.

The lower activity of ectonucleotidades is another evidence that changes in

metabolism may starts earlier than the most known alterations, based in the decreased

hydrolysis of adenine nucleotides in the HP group. On vascular system, ATP and their

metabolites (includind ADP, AMP and adenosine) are involved in the control of several

biologic processes and these molecules function on blood vessels, as vasoconstrictors or

vasodilatators, is well know [10]. The ectonucleotidases exerts an essential role in blood

flow regulation and in trombogenesis, by converting ATP, ADP and AMP, which stimulate

vasoconstriction and platelets agregation, in adenosine, which exert the opposite functions

[11,12]. Therefore, the homeostasis of ectonucletidases activity is important to avoid

vascular trombosis and atherogenic process through formation and maintenance of

adenosine levels [30,31].

Atherosclerosis is the cause of death of 80% of type 2 diabetic individuals as a

consequence of untreated hyperglicemia and its alterations in adipose tissue, inflamatory

response and vascular homeostasis [32,18]. Here we demonstrated that consumption of a

highly palatable diet leads to body fat mass increase, metabolic alterations and type 2

diabetes in association with lower ectonucleotidases activity, promoting an appropriate

environment to development of cardiovascular diseases, without changes in insulin and

adiponectin levels. We concluded that possibly cardiovascular disease, type 2 diabetes and

obesity complications have more subtle indicators and starts before most known markers

are altered, as conventional diagnosis.

Acknowledgments

This work was supported by grants from Brazilian agencies FAPERGS, CAPES,

CNPq and FINEP research grant "Rede Instituto Brasileiro de Neurociência (IBN-Net)"#

01.06.0842-00.

References

1- Unger RH, Orci L. Diseases of liporegulation : new perspective on obesity and related

disorders. Faseb J. 2001; 15: 312-21.

2- Dube N, Tremblay ML. Involvement of the small protein tyrosine phosphatases TC-PTP

and PTP1B in signal transduction and diseases: From diabetes, obesity to cell cycle, and

cancer. Biochim Biophys Acta 2005; in press.

3- Erlanson-Albertsson C. How palatable food disrupts appetite regulation. Basic Clin

Pharmacol Toxicol. 2005; 97(2): 61-73.

4- Dobbins RL, Szczepaniak LS, Myhill J, Tamura Y, Uchino H, Giacca A, McGarry JD.

The composition of dietary fat directly influences glucose-stimulated insulin secretion in

rats. Diabetes 2002; 51: 1825-33.

5- Holemans K, Caluwaerts S, Poston L, Van Assche FA. Diet-induced obesity in the rat: a

model for gestational diabetes mellitus. Am J Obstet Gynecol. 2004; 190(3):858-65

6- Ronti T, Lupatelli G, Mannarino E. The endocrine function of adipose tissue: an update.

Clin Endocrinol (Oxf) 2006; 64(4):355-65.

7- Gil-Campos M,Canete RR,Gil A. Adiponectin, the missing link in insulin resistance and

obesity. Clin Nutr.2004;23 (5): 963-74.

8- Real, JMF, Castro A, Vázquez G et al. Adiponectin is associated with vascular function

independent of insulin sensitivity. Diab Care 2004; 27(3): 739-44.

9- Park KG, Park KS, Kim MJ et al.Relationship between serum adiponectin and leptin

concentrations and body fat distribution. Diab Res Clin Pract. 2004; 63: 135-42.

10- Frassetto SS,Dias RD,Sarkis JJ. Characterization of an ATP diphosphohydrolase

activity (APYRASE, EC 3.6.1.5) in rat blood platelets. Mol Cell Biochem. 1993; 129(1):

47-55.

11- Kaczmarek E,Koziak K,Sevigny J,Siegel JB,Anrather J,Beaudoin AR,Bach FH,Robson

SC. Identification and characterization of CD39/vascular ATP diphosphohydrolase. J Biol

Chem. 1996; 271(51): 33116-22.

12- Zimmermann H. Ectonucleotidases: some recent developments and note on

nomenclature. Drug Development Research 2001; 52:44-56.

13- Kiraly MA,Bates HE,Yue JT,Goche-Montes D,Fediuc S,Park E,Matthews SG,Vranic

M,Riddell MC. Attenuation of type 2 diabetes mellitus in the male Zucker diabetic fatty rat:

the effects of stress and non-volitional exercise. Metabolism 2007; 56(6): 732-44.

14- Miranda KM, Espey MG, Wink DA. A Rapid, Simple Spectrophotometric Method For

Simultaneous Detection Of Nitrate And Nitrite. Nitric Oxide: Biology And Chemistry.

2001; 5: 62-71.

15- Oses JP, Cardoso CM, Germano RA, Kirst IB Fürstenau CR, et al. Soluble NTPDase :

an additional system of nucleotide hydrolysis in rat blood serum. Life Sci 2004; 74(26):

3275-84.

16- Chan K, Delfert D, Junger KD. A direct colorimetric assay for Ca+2-ATPase acitivity.

Anal Biochem 1986; 157:375-80.

17-Bradford, MM. A rapid and sensitive method for the quantification of microgram

quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976: 72,

18-54.

18- Cheng, T.O. All teas are not created equal : The chinese green tea and cardiovascular

health. Int J Cardiol. 2005; in press.

19- Jeppesen J,Hansen TW,Rasmussen S,Ibsen H,Torp-Pedersen C,Madsbad S. Insulin

resistance, the metabolic syndrome, and risk of incident cardiovascular disease: a

population-based study. J Am Coll Cardiol. 2007; 49(21): 2112-9.

20- Naderali EK,Fatani S,Williams G. Chronic withdrawal of a high-palatable obesity-

inducing diet completely reverses metabolic and vascular abnormalities associated with

dietary-obesity in the rat. Atherosclerosis 2004;172(1):63-9.

21- Yudkin JS,Eringa E,Stehouwer CD. “Vasocrine” signalling from perivascular fat: a

mechanism linking insulin resistance to vascular disease. Lancet 2005;365 (9473):1817-20.

22 - Yu YH, Ginsberg HN. Adipocyte signalling and lipid homeostasis : Sequelae of

insulin-resistant adipose tissue. Circ Res 2005; 96: 1042-52.

23- Utzschneider KM, Kahn SE. The role of insulin resistance in nonalcoholic fatty liver

disease. J Clin Endocrinol Metab 2006;91(12):4753-61

24- Parekh S,Anania FA. Abnormal lipid and glucose metabolism in obesity: implications

for nonalcoholic fatty liver disease. Gastroenterology 2007; 132 (6): 2191-207.

25- Gravila A.; Chan JL.; Yiannakouris N et al. Serum adiponectin levels are inversely

associated with overall and central fat distribuction but are not direclty regulated by acute

fasting or leptin administration in humans : cross-sectional and interventional studies. J

Clin Endocrinol Metabol. 2003; 88(10): 4823-31.

26- Kumada M,Kihara S, Ouchi N et al. Adiponectin specifically increased tissue inhibitor

of metalloproteinases-1 through interleukin-10 expression in human macrophages.

Circulation 2004; 109: 2046-49.

27- Qi Y, Takahashi N, Hileman SM, Patel HR, Berg AH, Pajvani UB, Scherer PE, Ahima

RS. Adiponectin acts in the brain to decrease body weight. Nat Med. 2004;10(5):524-9.

28- Rothenbacher D,Brenner H,Marz W,Koenig W. Adiponectin, risk of coronary heart

disease and correlations with cardiovascular risk markers. Eur Heart J 2005; 26(16):1640-6.

29- Barnea M,Shamay A,Stark AH,Madar Z. A high-fat diet has a tissue-specific effect on

adiponectin and related enzyme expression. Obesity 2006; 14(12): 2145-53.

30- Sarkis JJF, Battastini AMO, Oliveira EM et al. ATP diphosphohydrolases : An

overview. Journal of the Brazilian Association for the Advancement of Science 1995;

47(3):131-36.

31- Gayle RB ,Maliszewski CR,Gimpel SD,Schoenborn MA,Caspary RG,Richards

C,Brasel K,Price V,Drosopoulos JH,Islam N,Alyonycheva TN,Broekman MJ,Marcus AJ.

Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39. Jour Clin

Invest. 1998; 101(9):1851-9.

32 - Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab

2004; 89: 2548-56.

Legends and figures

Figure 1. Characterization of the obesity model : body mass and glucose tolerance test after

four months of a highly palatable diet intake. A) Final body weight (g); B) Final body fat

mass (g); C) Glucose tolerance test : 0 (fasting), 30, 60 and 120 minutes after i.p. injection

of a 50% glucose solution (2mg/g). SC= starndard chow; HP= highly palatable. * = p<0.05;

**=p<0.01. Results are expressed as mean ± standard deviation.

Table 1. Metabolic parameteres of groups after four months of diets consumption. Results

are expressed as mean ± standard deviation.

Figure 2. Measure of NO levels and ectonucleotidases activity in rat serum after four

months of diets consumption. A) Serum NO levels; B) ATP hydrolysis; C) ADP

hydrolysis; D) AMP hydrolysis. B,C, D = ectonucleotidases specific activity (nmol

phosphate/min/protein mg). SC= standard chow; HP= highly palatable. * = p<0.05;

**=p<0.01. Results are expressed as mean ± standard deviation.

Figure 1

Table 1 - Metabolic parameteres of groups after four months of diets consumption.

Groups SC (n =10) HP (n=10)

Total cholesterol (mg/dl)

47.0 ± 9.0 52.0 ± 7.0*

HDL cholesterol (mg/dl)

29.0 ± 5.0 34.0 ± 5.0*

Serum TAG (mg/dl)

67.0 ± 14.0 128.0 ± 70.0*

Liver TAG (mg%)

1.0 ± 0.3 2.0 ±0.3**

Free Glycerol (mg/ml)

0.01 ± 0.002 0.02 ± 0.003**

Insulin (µUI/ml) 85.0 ± 31.0 80.0 ± 26.0

Adiponectin (ng/ml)

43.0 ± 14.0 39.0 ± 7.0

SC= starndard chow; HP= highly palatable. * = p<0.05; **=p<0.01.

Figure 2

2.2 – CAPÍTULO 2

HIGHLY PALATABLE DIET CONSUMPTION INCREASES PROTEIN OXIDATION

IN RAT FRONTAL CORTEX AND ANXIETY LIKE BEHAVIOR

Publicado na Revista Life Sciences

Highly palatable diet consumption increases protein oxidation in rat frontal cortex

and anxiety like behavior

C.G. Souza

*, J.D. Moreira

, I.R. Siqueira

b,c

, A.G. Pereira

D.K. Rieger

, D. O. Souza

L.V. Portela

, T.M. Souza

, M.L.S. Perry

Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul – UFRGS

Programa de Pós Graduação em Ciências Biológicas-Neurociências, RS, Brazil

Departamento de Farmacologia, Universidade Federal do Rio Grande do Sul, Porto

Alegre, RS, Brazil

* Corresponding author:

Carolina Guerini de Souza

Departamento de Bioquímica, ICBS

Rua Ramiro Barcelos, 2600 anexo

CEP 90035003

Porto Alegre, RS, Brazil

Phone: 55 51 33165551

Fax: 55 51 33165560

Email: carolguerini@hotmail.com

Abstract

Obesity is frequently associated with consumption of high amounts of sugar and/or

fat. Studies have demonstrated a high prevalence of overweigh and obesity associated or

not with increase rates of psychiatry disorders; in particular mood and anxiety disorders.

Recent works have demonstrated an association between specific genes involved in

oxidative stress metabolism and anxiety-like behavior. The aim of this study was to

investigate the effect of a highly palatable diet enriched with sucrose in body fat mass

composition, anxiety behavior and brain oxidative status. Twenty male wistar rats received

two diferent diets during four months: standard chow (SC) and highly palatable (HP).

Metabolic parameters, behavioral tests and oxidative stress status were evaluated. Body fat

mass, insulin sensitivity and glucose tolerance was altered in the HP group (p<0.01). The

same group spends less time in light compartment and had a lower risk assessment

behavior (p<0.05) but no differences was observed in the open field test habituation

(p>0.05). Protein degradation, DCF and TBARS levels was not different in the

hippocampus between groups however there was higher levels of protein degration in

frontal cortex of HP groups (p<0.05), although DCF and TBARS levels don’t differ from

SC group (p>0.05). In conclusion, our data suggest that the consumption of HP diet leads to

an obese phenotype, increases protein oxidation in frontal cortex and appears to induce

anxiety like behavior in rats.

Key words: obesity / highly palatable diet / anxiety / oxidative stress / frontal cortex

Introduction

In modern societies the consumption of high fat and high sugar diets as well as a

sedentary lifestyle has been associated with metabolic derangements and impairment of

brain function (Schrawen et al., 2000; Unger et al., 2001; Molteni et al., 2002, 2004;

McGarry, 2002). Accordingly, in human studies overweigh and obesity maybe associated

with increase rates of psychiatry disorders; in particular mood and anxiety disorders

(Chakravarthy et al., 2004, Simon GE, 2006; Becker et al., 2001; Teegarden and Bale,

2007). Also, experimental studies have focusing on the molecular and behavioral response

underlying the specific effects of high fat and sugar diet on neural cells (Molteni et al,

2002, 2004).

Colantuani et al., (2002) demonstrated that continuous excessive sugar consumption

leads to an addiction profile and increases anxiety behavior when rats are deprivated from

sugar, whereas when animals are refeeding they overeating. In addition to this altered

behavior induced by excessive sugar intake, elevated level of plasmatic glucose has been

considered the main source of free radicals production in glucose intolerant individuals

(Brownlee, 2005).

The imbalance between high cellular levels of reactive oxygen species (ROS) in

relation to cellular antioxidant defenses, namely oxidative stress, may be involved in the

pathogenesis of several brain diseases. If not effectively removed ROS may cause oxidative

cell injury (Gutteridge and Halliwell, 2000; Halliwell and Gutteridge, 2000). Protein

damage, lipid peroxidation and energy failure caused by oxidative stress are frequently

reported alterations that could affect neurons and consequently the brain functioning (Guix

et al., 2005; Moncada and Bolanos, 2006; Calabrese et al., 2006). Recently, a new insight

emerged from the contradictory works of Hovatta et al. (2005) and Berry et al., (2007) in

which they suggests a linking between different genes involved in oxidative stress

metabolism and anxiety like behavior.

Up to our knowledge, the association between the consumption of a highly palatable

diet sucrose enriched (HP), anxiety and brain oxidative damage has not yet been studied.

Thus, the aim of this study was to investigate the effect of a HP diet in anxiety behavior and

brain oxidative status.

Material and Methods

Chemicals

All reagents were of analytical grade. Thiobarbituric acid was obtained from Merck

(Rio de Janeiro, Brazil), while 2’-7’-dichlorofluorescein diacetate, sodium dodecylsulfate,

trichloroacetic acid and phenyl methyl sulfonyl fluoride were purchased from Sigma

Chemical Co. (St. Louis, MO, USA).

Animals and Diets

Twenty 60-day old male Wistar rats weighting from 210 to 220 g were obtained

from the Central Animal House of our Department. They were maintained under a standard

dark-light cycle (lights on between 7:00 a.m. and 7:00 p.m.), at a room temperature of 22 +

2°C. Animal care followed the official governmental guidelines in compliance with the

Federation of Brazilian Societies for Experimental Biology and was approved by the Ethics

Committee of the Federal University of Rio Grande do Sul, Brazil.

Rats were divided into two groups: (1) the control group (SC, n=10), which received

standard laboratory rat chow (50 % carbohydrate, from starch, 22 % protein and 4 % fat);

and (2) the highly palatable diet group (HP, n=10), which received a enriched sucrose diet

(65 % carbohydrates being 34 % in condensed milk and 8 % from sucrose), 25 % protein,

10 % fat). All animals had free access to food and water.

Behavioral tests

After four months animals were allowed to explore an open field (two 5-min

sessions on successive days) and a light-dark arena (one 5-min session). Behavioral testing

was carried out in a special room with constant temperature (21 + 2º C) and light conditions

(60-W light), except for the light-dark exploration task (see details below). Before the

sessions, animals were allowed to adapt to the experimental room for at least 1 h. All tasks

were performed between 09:30 to 12:00 AM.

Open-field exploration

In two successive days, the rats were gently placed in the corner of a 40 x 50 x 60-

cm box, the floor of which was divided into 3 x 4 cm squares, and left free to explore it for

5 min. The number of crossings from one square to another, groomings and rearings was

counted. Latency to leave the first square was also measured.

Light-dark exploration task

The light-dark exploration task consisted of a 40 x 50 x 60-cm box divided equally

into two compartments that were connected by a small opening (10.0 cm x 7.5 cm). The

light compartment was illuminated under a 60-W light. The dark one received only part of

the room illumination (at 20 W). The floor of each compartment was divided into 3 x 2 cm

squares. Rodents are nocturnal animals preferring darker areas, and the decrease in the

exploratory activity in the light area is taken as a measure of anxiety. Animals were gently

placed in the corner of the light compartment and left free to explore it for 5 min. The

following parameters were recorded: (1) the frequency of crossings and rearings from one

square to another within the light or in the dark compartment; (2) the number of entries into

the light compartment, defined as placing the four paws into this compartment; and (3) the

total time spent in the light compartment. In addition, the risk assessment behavior index

(RA, i.e. the number of investigations of the light compartment by placing some but not all

paws) was recorded. After each trial, the apparatus was cleaned with an ethanol solution

(70 %).

Glucose tolerance test

Glucose tolerance test was performed two days before the animals were sacrificed.

A 50 %-glucose solution was injected into the animals (i.p, 2 mg/kg) after 6h of starvation.

The blood was collected by a small puncture on tail immediately before and 30, 60, and 120

minutes after the injection. At each time, glucose was measured by a glucosimeter

(AccuChek Active, Roche Diagnostics

, USA).

Tissue preparation and oxidative stress damage evaluation

One week after the behavioral tests, the rats were killed by decapitation. Visceral

and epididimal fat pads were dissected and weighted. Cerebral frontal cortices and

hippocampi were dissected out and immediately stored at -70°C until biochemical

measurements, when they were homogenized in 10 volumes of ice-cold phosphate buffer

(0.1 M, pH 7.4) containing 140 mM of KCl, 1 mM of ethylenediaminetetraacetic acid, and

1 mM of phenyl methyl sulfonyl fluoride. The homogenate was centrifuged at 960 g for 10

min and the supernatant was used for all subsequent analysis. All steps were carried out at

4°C.

In order to verify a possible impact of obesity and glucose levels in brain oxidative

stress, we evaluated oxidative damage to proteins and lipids in the hippocampus and frontal

cortex. The levels of oxidized fluorescent derivative (DCF), thiobarbituric acid-reactive

substances (TBARS) and the protein residues of tyrosine and tryptophan were quantified in

these structures, with all data expressed as percentages of the control group.

Free radical levels

An aliquot of the sample was incubated with 2’- 7’- dichlorofluorescein diacetate

(100 µM) at 37°C for 30 min. The formation of DCF was monitored at excitation and

emission wavelengths of 488 and 525 nm, respectively, using a fluorescence

spectrophotometer (Hitachi F-2000; Hitachi, Tokyo, Japan). The formation of reactive

oxygen species was quantified by using a DCF standard curve and results were expressed

as picomoles of DCF formed per milligram of protein (Sriram et al., 1997).

Lipid peroxidation assay (TBARS)

Sample aliquots were incubated with 10 % trichloroacetic acid and 0.67 %

thiobarbituric acid. The mixture was heated on a boiling water bath for 30 min, an equal

volume of n-butanol was added, and the final mixture was centrifuged. The organic phase

was collected for fluorescence measurements at excitation and emission wavelengths of

515 and 553 nm, respectively (Bromont et al., 1989). We used 1,1,3,3-tetramethoxypropane

as the standard. TBARS levels are expressed as picomoles of malondialdehyde per

milligram of protein.

Degradation of protein tyrosine residues and tryptophan residues

Sodium dodecylsulfate was added to sample aliquots (final concentration 0.1%).

The tyrosine residues within solubilized proteins were determined fluorometrically at

excitation and emission wavelengths of 277 and 320 nm, respectively (Gusow et al., 2002).

Sodium dodecylsulfate was added to sample aliquots (final concentration 0.1%).

Tryptophan residues within solubilized proteins were determined fluorometrically at

excitation and emission wavelengths of 280 and 345 nm, respectively (Bondy, 1996).

The determination of tyrosine and tryptophan residues is used as a tool for evaluates

oxidative stress in proteins.

Protein assay

Total protein concentration was determined according to the method described by

Lowry (1951) with bovine serum albumin as the standard.

Statistical analysis

Data were analyzed using SPSS 10.0 software. Body weight, glucose tolerance and

open field habituation test results were analyzed by repeated-measures ANOVA and post-

hoc Tukey’s test. The Student’s t test was used for body fat mass, light dark test and

analyses of oxidative stress parameters. The level of statistical significance was set at 95 (p

< 0.05). Results are expressed as mean ± standard error (S.E).

Results

Body Composition and Glucose Tolerance Test

There was no difference between groups in body weight (p>0.05) [F (1.18) = 2.79;

p = 0.112]. However, visceral and epididimal fat mass in the HP group was higher than in

the SC group (9 ± 3 vs. 5±1 and 7 ± 3 vs. 4 ± 1, respectively; p < 0.001), indicating that the

HP diet increases the gain of fat mass (Table 1).

Additionally, after four months HP diet triggered changes in the glucose

homeostasis, which is supported by an impaired glucose tolerance (p<0.001)[F (3,54) =

8.80; p=0.0000] (Figure 1A) and also by increased liver triacylglycerol (TAG)

concentration (HP, 1.9 ± 0.3 vs SC, 0.9 ± 0.3 mg%; p<0.01, Table 1), an indirect parameter

for demonstrate overall insulin resistance (Burget et al., 2006).

Behavioral Parameters

In the open field habituation test, there was no difference between groups in the

number of crossings, rearings, and in the latency for leaving the first square (p>0.05)[F

(1,18)= 0.35, 0.18, and 0.20, respectively, p = 0.56] (Fig. 1B). In the light-dark task, the HP

group spent less time in the light compartment than the SC group (p<0.05, Fig. 1C) and the

locomotion of this group was lower in the same compartment (p<0.05, Fig. 1D). The HP

group also had a trend to decrease the locomotion behavior on dark compartment (p= 0.07;

Fig. 1E) and higher risk assessment behavior index than controls (p < 0.01; Fig.1F).

Oxidative stress parameters

There was no difference between groups in hippocampus regarding the content of

tyrosine and tryptophan, DCF levels, and TBARS production (data not shown).

A significant lower content of tyrosine and tryptophan was observed in HP

compared to control group indicating the presence of high protein damage induced by diet

(p<0.05, Fig. 2A). There was no difference between groups in DCF and TBARS levels

(p>0.05, Fig.2B and Fig. 2C, respectively).

Discussion

The results presented here show that animals submitted to highly palatable diet

enriched wit sucrose (HP) had glucose intolerance, increased in insulin resistance and fat

body composition, despite no differences on total body weight. Taking together these

findings suggest that HP rats have obese like phenotype and impairment in glucose

metabolism. Furthermore, HP diet increased anxiety like behavior and oxidative damage in

proteins of the frontal cortex.

In the dark-light choice task, animals submitted to HP diet spent less time in the

light compartment and, at the same time, they showed a higher number of refusals for

entering the light compartment, which according to Gingrich (2005) and Hovatta et al.,

(2005) is a well-accepted feature of anxious behavior. Open field task was used to evaluate

the locomotion capacity of animals, plus anxiety like behavior as a complementary measure

of the light dark task. The anxiety like-behavior appears to be unrelated to any gross motor

alteration, since the locomotion in the open field task was normal.

Considerable evidence has been accumulated to demonstrate that feeding behavior

is closely related to emotions. Many obese patients tend to eat more when they are

emotionally tense or depressed or simply bored (Vaswani et al.,1983). Additionally, obese

patients frequently have psychiatric co-morbidities. Although depressive symptoms are

frequently attributed to obese patients, the associations between anxiety disorders and

obesity often go unrecognized. In an obese population of UK, Fifty-six percent of patients

met the minimum criteria for an anxiety disorder and forty-eight percent met the minimum

criteria for depression (Tuthill et al., 2006).

Chronic elevation of glucose levels is one metabolic alteration triggered by this diet

consumption is reported as the main source of free radical production in glucose intolerance

situations. An overload of glucose blocks the flux of electrons transport chain, generating

elevated levels of reactive nitrogen and oxygen species (Nishikawa et al., 2000; Brownlee,

2005). Recent data from several reports indicate that these free radicals are involved in the

biochemical mechanisms underlying neuropsychiatric disorders in human (Ozcan et al.,

2004). The free radicals usually affect biomolecules, such as, proteins, lipids and DNA, and

mitochondria (Griffiths, 2000; Murray et al., 2003).

The HP group had decreased tyrosine and tryptophan residues in frontal cortex

proteins, an important brain structure involved in behavioral regulation, and together with

hippocampus, amygdala and hypothalamus, which are limbic regions, form part of several

well-defined anxiety and fear-related circuits in the forebrain (Singewald et al., 2003;

Hovatta et al., 2005). The formation of reactive nitrogen and oxygen species can alter

protein conformation through reactions with aminoacids residues, being tyrosine and

tryptophan some of the most susceptible aminoacids because they have lowest potentials

reduction (Alvarez and Radi, 2003). Oxidation of proteins is a very common consequence

of oxidative stress, leading to changes in structure and function of these molecules (Alvarez

et al., 1999).

It has long been established that genetic contributions increase the vulnerability to

anxiety disorders, but the precise genes involved are unknown. In a recent work, Hovatta et

al., (2005) proposes that, in mouse brain, overexpression of genes related to antioxidant

enzymatic defenses (glyoxalase 1 and glutathione reductase 1) increases anxiety behavior

and that these genes have a causal role in the genesis of anxiety. Conversely, Berry et al

(2007) showed that deletion of gene p66

Shc

, which regulates reactive oxygen species

metabolism and apoptosis, reduced pain sensitivity and anxiety, which is in line with our

findings regarding ROS and anxiety. It is possible that genetic variability may at least in

part responsible for these inter-species and interindividual differences. Moreover, it has

been demonstrated that a high palatable diet was able to influence gene expression of

proteins involved in the mechanisms underlying neuroplasticity in hipocampus (Molteni et

al., 2002). Thus, the influence of high palatable diet on genes related to oxidative stress and

anxiety like behavior seems to be an interesting field for future investigations.

Considering the limitations of our methodological approach, and the many brain

systems involved in anxiety disorders this study was not able to establish a causal effect

among HP diet and/or behavioral and biochemical parameters. Additional oxidative stress,

molecular and behavioral parameters could be useful in order to search for more robust

associations.

In conclusion, our data suggest that the consumption of high sucrose diet leads to an

obese phenotype, increases protein oxidation in frontal cortex and appears to induce anxiety

like behavior in rats.

Acknowledgments

This work was supported by grants from Brazilian agencies FAPERGS, CAPES,

CNPq and FINEP research grant "Rede Instituto Brasileiro de Neurociência (IBN-Net)" #

01.06.0842-00.

References

Alvarez, B., Ferrer-Sueta, G., Freeman, B.A., Radi, R.,1999. Kinetics of peroxynitrite

reaction with amino acids and human serum albumin. The Journal of Biological Chemistry

274, 842-848 .

Alvarez, B., Radi, R.2003. Peroxynitrite reactivity with amino acids and proteins. Amino

Acids 25(3-4), 295-311.

Becker, E.S., Margraf, J., Turke, V., Soeder, U., Neumer, S., 2001. Obesity and mental

illness in a representative sample of young women. International Journal of Obesity and

Related Metabolic Disorders 25(1), S5-9.

Berry, A.,Capone, F.,Giorgio, M.,Pelicci, P.G.,de Kloet, E.R.,Alleva, E.,Minghetti,

L.,Cirulli, F., 2007. Deletion of the life span determinant p66Shc prevents age-dependent

increases in emotionality and pain sensitivity in mice. Experimental Gerontology 42(1-2),

37-45.

Bondy, S.C., 1996. Evaluation of free radical-initiated oxidant events within the nervous

system. In: Perez-Polo JR, Editor. Methods in Neuroscience. Volume 30. San Diego:

Academic Press, 243-259.

Bromont, C., Marie, C., Bralet, J.,1989. Increased lipid peroxidation in vulnerable brain

regions after transient forebrain ischemia in rats. Stroke 20, 918 - 924.

Brownlee, M.,2005. The pathobiology of diabetic complications. Diabetes 54.

Burgert, T.S., Taksali, S.E., Dziura, J., Goodman, T.R., Yeckel, C.W., Papademetris, X.,

Constable, R.T., Weiss, R., Tamborlane, W.V., Savoye, M., Seyal, A.A., Caprio, S., 2006.

Alanine aminotransferase levels and fatty liver in childhood obesity: associations with

insulin resistance, adiponectin, and visceral fat. The Journal of Clinical Endocrinology and

Metabolism 91(11), 4287-4294.

Calabrese, V., Butterfield, D.A., Scapagnini, G., Stella, A.M., Maines, M.D., 2006. Redox

regulation of heat shock protein expression by signaling involving nitric oxide and carbon

monoxide: relevance to brain aging, neurodegenerative disorders, and longevity.

Antioxidants & Redox Signaling 8(3-4), 444-477.

Chakravarthy, M.V., Booth, F.W., 2004. Eating, exercise, and Thrifty genotypes:

connecting the dots toward an evolutionary understanding of modern chronic diseases.

Journal of Applied Physiology 96, 3-10.

Colantuoni, C., Rada, P., McCarthy, J., Patten, C., Avena, N.M., Chadeayne, A., Hoebel,

B.G., 2002. Evidence that intermittent, excessive sugar intake causes endogenous opioid

dependence. Obesity Research 10 (6), 478-488.

Gingrich, J.A., 2005. Oxidative stress is the new stress. Nature Medicine 11(12), 1281-

1282.

Griffiths, H.R., 2000. Antioxidants and protein oxidation. Free Radical Research 33, S47-

58.

Guix, F.X., Uribesalgo, I., Coma, M., Munoz, F.J., 2005. The physiology and

pathophysiology of nitric oxide in the brain. Progress in Neurobiology 76 (2), 126-152.

Gusow, K., Szabelski, M., Rzeska, A., Karolczak, J., Sulowska, H., Wiczk, W., 2002.

Photophysical properties of tyrosine at low pH range. Chemical Physics Letters 362, 519-

526.

Gutteridge, J.M., Halliwell, B., 2000. Free radicals and antioxidants in the year 2000. A

historical look to the future. Annals of the New York Academy of Sciences 899, 136-147.

Halliwell, B., Gutteridge, J.M.C., 2000. Free Radicals in Biology and Medicine. 3ed,

Clarendon, Oxford.

Hovatta, I., Tennant, R.S., Helton, R., Marr, R.A., Singer, O., Redwine, J.M., Ellison, J.A.,

Schadt, E.E., Verma, I.M., Lockhart, D.J., Barlow, C., 2005. Glyoxalase 1 and glutathione

reductase 1 regulate anxiety in mice. Nature 438(7068), 662-666 .

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with

the Folin phenol reagent. The Journal of Biological Chemistry 193, 265-275.

McGarry, J.D., 2002. Dysregulation of fatty acid metabolism in the etiology of type 2

diabetes. Diabetes 51, 7-18.

Molteni, R., Barnard, A R.J., Ying, A Z., Robertsa, A C. K., Mez-Pinilla, F.G., 2002. A

high-fat, refined sugar diet reduces hippocampal brain-derived neurotrophic factor,

neuronal plasticity and learning. Neuroscience 112(4), 803-814.

Molteni, R., Wu, A A., Vaynman, A S., Ying, A Z., Barnarda, R. J., Mez-Pinilla, F.G.,

2004. Exercise reverses the harmful effects of consumption of a high-fat diet on synaptic

and behavioral plasticity associated to the action of brain-derived neurotrophic factor.

Neuroscience 123, 429-440.

Moncada, S., Bolanos, J.P.,2006. Nitric oxide, cell bioenergetics and neurodegeneration.

Journal of Neurochemistry 97(6), 1676-1689.

Murray, J., Taylor, S.W., Zhang, B., Ghosh, S.S. , Capaldi, R.A.,2003. Oxidative damage

to mitochondrial complex I due to peroxynitrite.The Journal of Biological Chemistry

278(39), 37223-37230.

Nishikawa, T., Edelstein, D., Du, X.L., Yamagishi, S., Matsumura, T., Kaneda, Y., Yorek,

M.A., Beebe, D., Oates, P.J., Hammes, H.P., Giardino, I., Brownlee, M., 2000.

Normalizing mitochondrial superoxide production blocks three pathways of

hyperglycaemic damage. Nature 404, 787-790.

Ozcan, M.E., Gulec, M., Ozerol, E., Polat, R., Akyol, O., 2004. Antioxidant enzyme

activities and oxidative stress in affective disorders. International Clinical

Psychopharmacology 19(2), 89-95.

Schrawen, P., Westerterp, K.R., 2000. The role of high- fat diets and physical activity in the

regulation of body weight. The British Journal of Nutrition 84, 417-427.

Simon, G.E., Von Korff, M., Saunders, K., Miglioretti, D.L., Crane, P.K., van Belle,

G.,Kessler, R.C., 2006. Association between obesity and psychiatric disorders in the US

adult population. Archives of General Psychiatry 63 (7), 824-830.

Singewald, N., Salchner, P., Sharp, T, 2003. Induction of c-Fos expression in specific areas

of the fear circuitry in rat forebrain by anxiogenic drugs. Biological Psychiatry 53 (4), 275-

283.

Sriram, K., Pai, K.S., Boyd, M.R., Ravindranath, V., 1997. Evidence for generation of

oxidative stress in brain by MPTP: in vitro and in vivo studies in mice. Brain Research 21,

44 -52.

Teegarden, S.L.,Bale,T.L., 2007. Decreases in dietary preference produce increased

emotionality and risk for dietary relapse. Biological Psychiatry 61(9), 1021-1029.

Tuthill, A.,Slawik, H.,O'Rahilly, S.,Finer, N., 2006. Psychiatric co-morbidities in patients

attending specialist obesity services in the UK. QJM : monthly journal of the Association of

Physicians 99(5), 317-325.

Unger, R.H., Orci, L., 2001. Diseases of liporegulation : new perspective on obesity and

related disorders. The FASEB Journal 15, 312-321.

Vaswani, K., Tejwani, G.A., Mousa, S., 1983. Stress induced differential intake of various

diets and water by rat: the role of the opiate system. Life Science 32, 1983-1996.

Legend for figures

Table 1. Body weight and fat mass. SC= Standard Chow; HP = Highly Palatable.

* = p <0.01

Figure 1. Metabolic and behavioral parameters: A) Glucose tolerance test (GTT), * =

p<0.05; **= p<0.01; B) Open field habituation test (OF), first and second days; C) Light

dark task (LD): time in light compartment; D) Light dark task(LD): locomotion in light

compartment; E) Light dark task(LD): locomotion in dark compartment; F) Light dark task:

risk assessment behavioral index (RAB). SC= Standard Chow; HP= Highly Palatable.

Results are expressed as mean ± standard error. * = p <0.05, ** = p<0.01.

Figure 2. Oxidative stress in the frontal cortex: A) Tyrosine and tryptophan residues levels

(TIR/TRY); B) DCF levels; C) TBARS levels. SC= Standard Chow; HP= Highly Palatable.

Results are expressed as mean ± standard error. * = p<0.05.

Table 1

Groups SC (n =10) HP (n=10)

Initial Body Weight (g)

219.0 ± 18.0 225.0 ± 18.0

Final Body Weight (g)

350.0 ± 35.0 375.0 ± 40.0

Visceral fat mass (g)

5.0 ± 1.0 9.0 ± 3.0*

Epididimal fat mass (g)

4.0 ± 1.0 7.0 ± 3.0*

Liver TAG

(

)

0.9 ± 0.3 1.9 ± 0.3*

Figure 1

1st day 2nd day

Crossings

0 30 60 90 120

100

150

200

250

300

350

GTTA)

Minutes

Serum Glucose

(mg/dL)

SC HP

100

LD - Time in light box

Seconds

SC HP

LD Locomotion in light box

Crossings

SC HP

100

LD - Locomotion in dark box

Crossings

SC HP

LD -Risk assessment behavior

Number of refusal

1st day 2nd day

Crossings

0 30 60 90 120

100

150

200

250

300

350

GTTA)

Minutes

Serum Glucose

(mg/dL)

SC HP

100

LD - Time in light box

Seconds

SC HP

LD Locomotion in light box

Crossings

SC HP

100

LD - Locomotion in dark box

Crossings

SC HP

LD -Risk assessment behavior

Number of refusal

Figure 2

3. DISCUSSÃO

O aumento alarmante da obesidade nos últimos anos é atualmente um problema de

saúde pública mundial. Muitas são as causas envolvidas em sua etiologia, desde alterações

genéticas envolvidas com os genes das melanocortinas e da leptina até a inatividade física,

porém o consumo exagerado de alimentos hiperpalatáveis e sua alta disponibilidade é,

provavelmente, a maior causa dos níveis epidêmicos da obesidade (Cheng 2005, Erlanson-

Albertsson, 2005).

O consumo de alimentos palatáveis estimula no SNC regiões límbicas relacionadas

à sensação de prazer, euforia e recompensa, envolvendo ação de neurotransmissores e

opióides, modulados pela própria alimentação (Colantuani et al., 2002). Esses estímulos

afetam o comportamento alimentar de tal forma que o consumo a longo prazo deste tipo de

alimento pode ser comparado com padrões de adição, devido ao feedback positivo gerado

no sistema de recompensa, chamado “come back for more” (Kelley et al., 2002). A ingestão

excessiva e contínua de açúcar já é descrita como um promotor deste padrão de adição

(Colantuani et al., 2002).

O consumo regular de alimentos palatáveis promove obesidade e por conseqüência,

hiperglicemia e resistência à insulina. Em nossos resultados observamos que os animais

submetidos à dieta por 4 meses desenvolveram hiperglicemia severa, característica de

diabetes tipo 2, acompanhada de aumento do glicerol sérico (um indicador indireto de

ácidos graxos livres no sangue) e esteatose hepática (acúmulo de gordura no fígado). Essas

alterações ocorrem por uma incapacidade da insulina de inibir a síntese endógena de

glicose e a oxidação de ácidos graxos no fígado, bem como de inibir a lipólise à nível de

adipócito (Utzschneider and Kahn, 2006), embora os níveis séricos de insulina não

estivessem alterados em nosso estudo. A sinalização insulínica diminuída ou inexistente

também pode afetar o sistema cardiovascular, uma vez que este hormônio ativa a enzima

óxido nítrico sintase endotelial (eNOS). O óxido nítrico é o mais potente vasodilatador

endógeno e sua síntese ocorre pela via da fosforilação do susbstrato receptor de insulina1

(IRS1) → PI3K→ Akt, que ativa a eNOS. Esta mesma sinalização inibe a produção de

moléculas vasoconstritoras, que geram uma resposta aumentada quando existe resistência

insulínica, caracterizando uma disfunção endotelial (Yudkin et al., 2005). A disfunção

endotelial é um marcador inicial da aterosclerose e muitos são os fatores de risco que

predispõe a esta, podendo antes mesmo disso, causar a própria disfunção endotelial (Cheng,

2005).

Alterações nas citocinas secretadas pelo tecido adiposo também influenciam a

funcionalidade do endotélio. A adiponectina é uma adipocitocina muito atuante no sistema

cardiovascular por diminuir a adesão de monócitos, os fatores de coagulação e promover

vasodilatação por meio de NO, além de sua ação pró-insulina. Não é à toa que pacientes

cardíacos possuem menores níveis circulantes de adiponectina, que tem sua secreção e

função também comprometidas em pacientes obesos, já que o aumento do tecido adiposo é

negativamente correlacionado com os níveis desta adipocitocina (Cnop et al., 2003, Real

2004). Não conseguimos observar isto em nosso trabalho, pois embora os níveis de NO

estivessem diminuídos, o mesmo não ocorreu com a adiponectina, que ainda não havia

sofrido nenhuma alteração sérica. Por esta razão, levantamos a hipótese de que alterações

mais sutis, porém já comprometedoras, possam já estar ocorrendo antes mesmo que

maiores marcadores e moduladores, como adiponectina e insulina séricas, estejam

alterados. Corrobora com isto, a resistência à insulina existente no fígado e no tecido

adiposo mesmo sem hipo ou hiperinsulinismo nos animais. A dislipidemia apresentada, que

é um fator de risco clássico para doenças cardiovasculares, é mais uma alteração

complementar de nossa hipótese.

O sistema cardiovascular é um dos mais afetados pela instalação do DM2 e de suas

comorbidades associadas, uma vez que 80% das mortes de pacientes diabéticos ocorre por

aterosclerose e três quartos destas são por doenças coronarianas (Cheng, 2005). Ainda neste

sistema, o ATP extracelular e seus metabólitos (incluindo ADP, AMP e adenosina) estão

envolvidos no controle de diversos processos biológicos. A importância destes nucleotídeos

e seus derivados na corrente sangüínea atuando em processos de vasodilatação,

vasoconstrição, agregação plaquetária, inflamação e dor é bem estabelecida (Frassetto et

al., 1993; Ravelick e Burnstock, 2003). Sabe-se que o ATP liberado na corrente sangüínea,

dependendo da sua concentração, pode atuar como um vasodilatador ou vasoconstritor.

Além disso, é substrato para formação de ADP, um proagregante plaquetário, e ambos

podem ser hidrolisados até adenosina, um potente vasodilatador. Os nucleotídeos

extracelulares podem ser hidrolisados por várias nucleotidases, que estão localizadas em

superfícies celulares e solúveis nos meios extracelulares, sendo o endotélio vascular um de

seus principais sítios de localização e atuação. Estas enzimas exercem um papel importante

na regulação do fluxo sangüíneo e da trombogênese. As nucleotidases convertem o ADP,

um proagregante, em adenosina, uma molécula antiagregante, o que ajuda a controlar a

agregação plaquetária intravascular (Kaczmarek et al., 1996; Zimmermann et al., 2001).

Portanto, o controle das atividades dessas nucleotidases são importantes para impedir

processos de formação de trombos vasculares pela formação e manutenção do equilíbrio

dos níveis da adenosina (Gayle et al., 1998). A diminuição da hidrólise destes nucleotídeos

em nosso modelo de obesidade e DM2 é um fator importante a ser considerado para o

desenvolvimento da aterosclerose, em concordância com o conjunto de alterações

apresentadas, independente do decréscimo de insulina e adiponectina séricas. O perfil

apresentado por estes animais sugere que possivelmente as doenças cardiovasculares, o

DM2 e a própria obesidade tenham complicações que iniciem antes que alterações de

diagnóstico convencional apareçam.

Paralelamente com esses efeitos no metabolismo intermediário, algumas mudanças

nas funções cerebrais e cognitivas tem sido descritas neste mesmo modelo de obesidade.

Estudos populacionais com indivíduos com sobrepeso e obesidade mostram uma possível

maior incidência de desordens psiquiátricas nos mesmos, especialmente transtornos de

humor e ansiedade (Chakravarhty et al., 2004, Simons, 2006, Teegarden and Bale, 2007).

Em concordância com estas afirmações, observamos nos animais submetidos à dieta

hiperpalatável, um comportamento mais aversivo e menos explorador, alterações estas,

características de ansiedade aumentada em modelos animais (Gingrich,2005). Muitas

evidências científicas mostram que o comportamento alimentar é fortemente ligado à

emoções, indicando que uma grande maioria de indivíduos obesos alcançam critérios

mínimos de ansiedade e depressão apresentando freqüentemente maiores comorbidades

psiquiátricas do que pacientes não-obesos(Vaswani et al., 1983; Tuthill et al., 2006). Os

mecanismos ligando estas duas patologias ainda não foram elucidados, porém o estresse

pode ser um de seus fatores desencadeantes.

O estresse oxidativo está muito associado aos mecanismos bioquímicos de

desordens psiquiátricas (Ozcan et al., 2004) e trabalhos recentes mostram que a diminuição

da produção de RL é capaz de diminuir a ansiedade em modelos animais (Berry et al.,

2007). A oxidação de proteínas, lipídios e até mesmo do DNA promovida por radicais

livres (RL) ocasiona alteração da estrutura e perda da função destas moléculas e em nosso

trabalho constatamos uma maior oxidação nas proteínas do córtex frontal dos animais

submetidos à dieta, sendo que o córtex frontal é uma importante estrutura envolvida na

regulação comportamental, fazendo parte de circuitos cerebrais relacionados à ansiedade e

medo (Singewald et al., 2003; Hovatta et al., 2005). É importante salientar que este

aumento de ansiedade e de proteínas oxidadas ocorreu em animais hiperglicêmicos e que a

hiperglicemia crônica é a principal fonte de produção de RL em indivíduos intolerantes à

glicose e diabéticos (Brownlee, 2005). Entretanto, considerando os muitos sistemas

envolvidos nas desordens de ansiedade, não é possível ainda estabelecer um efeito causal

entre esta alteração e os fatores associados citados.

Considerando todos os aspectos avaliados, concluímos que a obesidade induzida

pelo consumo de uma dieta hiperpalatável altera parâmetros metabólicos promotores de um

microambiente propício à doenças cardiovasculares, com alterações mais sutis do que as

tradicionalmente investigadas, além de aumentar os níveis de ansiedade e o estresse

oxidativo no SNC, promovendo também mudanças comportamentais, influenciadas

pelo tipo de alimentação adquirida.

4. REFERÊNCIAS

Arner P. Human fat cell lipolysis : biochemistry, regulation and clinical role. Best Pract Res

Clin Endocrinol and Metabol. 19(4): 417-82, 2005.

Becker ES, Margraf J, Turke V et al.. Obesity and mental illness in a representative sample

of young women. International Journal of Obesity and Related Metabolic Disorders 25(1):

S5-9, 2001.

Berne RM. Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood

flow. Am J Physiol 204: 317-22, 1963.

Berry A, Capone F, Giorgio M et al. Deletion of the life span determinant p66Shc prevents

age-dependent increases in emotionality and pain sensitivity in mice. Exp Ger 42(1-2): 37-

45, 2007.

Brownlee M. The pathobiology of diabetic complications. Diab 54, 2005.

Chakravarthy MV, Booth FW. Eating, exercise, and Thrifty genotypes: connecting the dots

toward an evolutionary understanding of modern chronic diseases. Journal of Applied

Physiology 96: 3-10, 2004.

Chen YF, Li PL, Zou AP. Effect of hyperhomocysteinemia on plasma or tissue adenosine

levels and renal function. Circulation 106: 1275-81, 2002.

Cheng TO. All teas are not created equal : The chinese green tea and cardiovascular health.

Int J Cardiol in press, 2005.

Cnop M, Havel PJ, Utzschneider KM. et al. Relationship of adiponectin to body fat

distribution, insulin sensitivity and plasma lipoproteins : evidence for independent roles of

age and sex. Diabetologia 46: 459-69, 2003.

Colantuoni C, Rada P, McCarthy J et al. Evidence that intermittent, excessive sugar intake

causes endogenous opioid dependence. Obesity Research 10 (6): 478-88, 2002.

Dube N, Tremblay ML. Involvement of the small protein tyrosine phosphatases TC-PTP

and PTP1B in signal transduction and diseases: From diabetes, obesity to cell cycle, and

cancer. Biochim Biophys Acta in press, 2005.

Erlanson-Albertsson C. How palatable food disrupts appetite regulation. Basic Clin

Pharmacol Toxicol 97(2): 61-73, 2005.

Frassetto SS, Dias RD, Sarkis JJ. Characterization of an ATP diphosphohydrolase activity

(APYRASE, EC 3.6.1.5) in rat blood platelets. Mol Cell Biochem 129 (1): 47-55,1993.

Froguel P, Guy-Grand B and Clement K. Genetics of obesity: towards the understanding of

a complex syndrome. Presse Med. 29: 564–571, 2000.

Gayle RB, Maliszewski CR,Gimpel SD et al. Inhibition of platelet function by recombinant

soluble ecto-ADPase/CD39. J Clin Invest 101(9): 1851-9,1998.

Gil-Campos M,Canete RR, Gil A. Adiponectin, the missing link in insulin resistance and

obesity. Clin Nutr.23(5): 963-74, 2004.

Gingrich JA. Oxidative stress is the new stress. Nature Medicine 11(12): 1281-82, 2005.

Hovatta I, Tennant RS, Helton R et al. Glyoxalase 1 and glutathione reductase 1 regulate

anxiety in mice. Nature 438(7068): 662-66, 2005.

Kaczmarek E, Koziak K, Sevigny J, Siegel JB et al. Identification and characterization of

CD39/vascular ATP diphosphohydrolase. J Biol Chem 271(51): 33116-22, 1996.

Kelley AE, Bakshi VP, Haber SN et al. Opioid modulation of taste hedonics within the

ventral striatum. Physiol. Behav. 76: 365–77, 2002.

Koyamada N,Miyatake T,Candinas D et al.. Apyrase administration prolongs discordant

xenograft survival. Transplantation 62(12): 1739-43, 1996.

Latini S, Pedata F. Adenosine in central nervous system : release mechanisms and

extracellular concentrations. Neurochem 79(3): 463-84, 2001.

Mazurek T, Zhang LF, Zalewski A, et al. Human epicardial adipose tissue is a source of

inflammatory mediators. Circulation; 108: 2460-66, 2003.

McGarry JD. Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes.

Diab 51: 7-18, 2002.

Molteni R, Barnard ARJ, Ying AZ et al. A high-fat, refined sugar diet reduces hippocampal

brain-derived neurotrophic factor, neuronal plasticity and learning. Neurosc 112(4): 803-14,

2002.

Molteni R, Wu AA, Vaynman AS et al. Exercise reverses the harmful effects of

consumption of a high-fat diet on synaptic and behavioral plasticity associated to the action

of brain-derived neurotrophic factor. Neuros 123: 429-40, 2004.

Mubagwa K,Mullane K,Flameng W. Role of adenosine in the heart and circulation.

Cardiovasc Res 32(5): 797-813, 1996.

Naderali EK,Fatani S,Williams G. Chronic withdrawal of a high-palatable obesity-inducing

diet completely reverses metabolic and vascular abnormalities associated with dietary-

obesity in the rat. Atherosclerosis 172(1):63-9, 2004.

Neel, JV. The “Thritty genotype”. Nutr Rev 57: S2-S9, 1998.

Nishikawa T, Edelstein D, Du XL et al.. Normalizing mitochondrial superoxide production

blocks three pathways of hyperglycaemic damage. Nature 404: 787-90, 2000.

Ozcan ME, Gulec M, Ozerol E et al. Antioxidant enzyme activities and oxidative stress in

affective disorders. Intern Clin Psyc 9(2): 89-95, 2004.

Park KG, Park KS, Kim MJ et al. Relationship between serum adiponectin and leptin

concentrations and body fat distribution. Diab Res Clin Pract.63: 135-42, 2004.

Pinhas-Hamiel O,Zeitler P. Acute and chronic complications of type 2 diabetes mellitus in

children and adolescents. Lancet. 369(9575): 1823-31, 2007.

Prins Jb. Adipose tissue as an endocrine organ. Best Pract Res Clin Endocrinol Metab

16(4):639-51, 2002.

Rader DJ. Effect of insulin resistance, dyslipidemia, and intra-abdominal adiposity on the

development of cardiovascular disease and diabetes mellitus. Am J Med. 120(3): S1: S12-8,

2007.

Ravelic V, Burnstock G. Involvement of purinergic signaling in cardiovascular disease.

Drug News Perspect. 16(3): 133-40, 2003.

Real JMF, Castro A, Vázquez G et al. Adiponectin is associated with vascular function

independent of insulin sensitivity. Diab Care 27(3): 739-44,2004.

Rothenbacher D, Brenner H, März W. Adiponectin, risk of coronary heart disease and

correlations with cardiovascular risk markers. Eur Heart J 26(16):1640-46, 2005.

Saper C, Chou BTC and Elmquist JK. The need to feed: homeostatic and hedonic control of

eating. Neuron. 36: 199– 211, 2002.

Sarkis JJF, Battastini AMO, Oliveira EM et al. ATP diphosphohydrolases : An overview.

Journal of the Brazilian Association for the Advancement of Science 47(3):131-36, 1995.

Simon GE, Von Korff M, Saunders K et al. Association between obesity and psychiatric

disorders in the US adult population. Arch Gen Psyc 63 (7): 824-30, 2006.

Singewald N, Salchner P, Sharp T. Induction of c-Fos expression in specific areas of the

fear circuitry in rat forebrain by anxiogenic drugs. Biol Psyc 53 (4): 275-83, 2003.

Smith, SC. Multiple risk factors for cardiovascular disease and diabetes mellitus. Am J

Med. 120:S3-S11, 2007.

Takahashi K,Bokura H,Kobayashi S,Iijima K,Nagai A,Yamaguchi S. Metabolic syndrome

increases the risk of ischemic stroke in women. Intern Med 46(10): 643-48, 2007.

Teegarden SL, Bale TL. Decreases in dietary preference produce increased emotionality

and risk for dietary relapse. Biological Psychiatry 61(9):1021-29, 2007.

Tuthill A, Slawik H, O'Rahilly S et al. Psychiatric co-morbidities in patients attending

specialist obesity services in the UK. QJM : monthly journal of the Association of

Physicians 99(5): 317-25, 2006.

Unger RH, Orci L. Diseases of liporegulation : new perspective on obesity and related

disorders. Faseb J.15: 312-21, 2001.

Utzschneider KM, Kahn SE. The role of insulin resistance in nonalcoholic fatty liver

disease. J Clin Endocrinol Metab 91(12): 4753-61, 2006.

Vaswani K, Tejwani GA, Mousa S. Stress induced differential intake of various diets and

water by rat: the role of the opiate system. Life Sci 32: 1983-96, 1983.

Wassink AM,Olijhoek JK,Visseren FL. The metabolic syndrome: metabolic changes with

vascular consequences. Eur J Clin Invest 37(1): 8-17, 2007.

Yudkin JS, Eringa E, Stehouwer CD. Vasocrine" signalling from perivascular fat: a

mechanism linking insulin resistance to vascular disease. Lancet 365 (9473):1817-20, 2005.

Zimmermann H. Ectonucleotidases: some recent developments and note on nomenclature.

Drug Development Research 52: 44-56, 2001.

Livros Grátis
( http://www.livrosgratis.com.br )
 
Milhares de Livros para Download:
 
Baixar livros de Administração
Baixar livros de Agronomia
Baixar livros de Arquitetura
Baixar livros de Artes
Baixar livros de Astronomia
Baixar livros de Biologia Geral
Baixar livros de Ciência da Computação
Baixar livros de Ciência da Informação
Baixar livros de Ciência Política
Baixar livros de Ciências da Saúde
Baixar livros de Comunicação
Baixar livros do Conselho Nacional de Educação - CNE
Baixar livros de Defesa civil
Baixar livros de Direito
Baixar livros de Direitos humanos
Baixar livros de Economia
Baixar livros de Economia Doméstica
Baixar livros de Educação
Baixar livros de Educação - Trânsito
Baixar livros de Educação Física
Baixar livros de Engenharia Aeroespacial
Baixar livros de Farmácia
Baixar livros de Filosofia
Baixar livros de Física
Baixar livros de Geociências
Baixar livros de Geografia
Baixar livros de História
Baixar livros de Línguas

Baixar livros de Literatura
Baixar livros de Literatura de Cordel
Baixar livros de Literatura Infantil
Baixar livros de Matemática
Baixar livros de Medicina
Baixar livros de Medicina Veterinária
Baixar livros de Meio Ambiente
Baixar livros de Meteorologia
Baixar Monografias e TCC
Baixar livros Multidisciplinar
Baixar livros de Música
Baixar livros de Psicologia
Baixar livros de Química
Baixar livros de Saúde Coletiva
Baixar livros de Serviço Social
Baixar livros de Sociologia
Baixar livros de Teologia
Baixar livros de Trabalho
Baixar livros de Turismo
 
 