FABIO SANTOS DE LIRA

Transcription

PAPEL ANTI-INFLAMATÓRIO DA ADIPONECTINA E DA
INTERLEUCINA-10 EM MODELOS CLÍNICO E
EXPERIMENTAL DE OBESIDADE
Tese apresentada à Universidade
Federal de São Paulo - Escola
Paulista de Medicina, para obtenção
do Título de doutor em Ciências.
São Paulo
2011
PAPEL ANTI-INFLAMATÓRIO DA ADIPONECTINA E DA
INTERLEUCINA-10 EM MODELOS CLÍNICO E
EXPERIMENTAL DE OBESIDADE
Tese apresentada à Universidade
Federal de São Paulo - Escola
Paulista de Medicina, para obtenção
do Título de doutor em Ciências.
Orientadora: Profa. Dra. Cláudia Maria da Penha Oller do Nascimento
Co-orientadores: Profa. Dra. Lila Missae Oyama
Profa. Dra. Ana Raimunda Dâmaso
São Paulo
2011
Lira, Fabio Santos de
Papel anti-inflamatório da adiponectina e da interleucina-10 em
modelos clínico e experimental de obesidade. / Fabio Santos de Lira. - São Paulo, 2011.
xii, 88f.
Tese (Doutorado) - Universidade Federal de São Paulo. Escola Paulista
de Medicina. Programa de Pós-Graduação em Nutrição.
Título em inglês: Role anti-inflammatory of adiponectin and
interleukin-10 in obesity model clinical and experimental.
1.Obesidade 2 Inflamação. 3 Citocinas. 4. Adiponectina 5. IL-10 6.
Tecido adiposo 7.Terapia interdisciplinar 8. TLRs
UNIVERSIDADE FEDERAL DE SÃO PAULO
Campus São Paulo
PROGRAMA DE PÓS-GRADUAÇÃO EM NUTRIÇÃO
Coordenador do Curso de Pós-graduação: Prof. Dr. Mauro Batista de Morais
Este trabalho foi realizado no Programa de Pós-Graduação em nutrição da Universidade
Federal de São Paulo UNIFESP/EPM, com o apoio financeiro da Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e da Fundação de Apararo à
Pesquisa do Estado de São Paulo (FAPESP).
“Para os amigos Tudo, para os inimigos a Lei.”
Robertão
vi
Dedicatória
À minha mãe Jaci, meu pai Lira, minhas irmãs Andréia e
Luciana e meu irmão Cristiano, por tudo o que fizeram por mim.
À minha esposa Sabrina, pela compreensão e amor.
Ao meu pequeno Bruno Kenji, razão do meu viver.
Aos meus sobrinhos Pedro Augusto, Felipe Gabriel e Letícia, por
alegrar nossas vidas.
À meu sogro Akio, minha sogra Fumiko e minha cunhada Silvia,
por todo carinho.
vii
AGRADECIMENTOS
A Professora Dra. Cláudia Maria Oller do Nascimento, pela paciência, amizade, mais que isso, por ter
acreditado no meu potencial.
A Professora Dra. Lila Missae Oyama, pelos ensinamentos e amizade.
A Professora Dra Marília Seelaender, que sempre me apoiou, tendo enorme contribuição sobre minha
formação acadêmica, serei eternamento grato.
A Professora Dra Ana Damaso e todo seu grupo, pelo carinhoso acolhimento. Sou muito grato pela
oportunidade.
Aos Professores Marco Túlio de Mello e Ronaldo Vagner Thomatilei dos Santos, pela oportunidade de
realizar meu pós-doutorado no CEPE, sou muito grato.
A Emília Ribeiro, minha eterna madrinha, obrigada por tudo.
Aos meus amigos do Laboratório de Fisiologia da Nutrição, a “velha guarda” José Cesar (Zeca),
Gustavo Pimentel (Gú), Claudio Alexandre (Claudião), Karina Barros, Ricardo Eguchi (Ric), Vinícius
Martins (Vini), João Felipe Mota, Cristiane Oliveira, Daniela Martins, Valter Tadeu e a “nova
geração” Rachel De Laquila (Rachelzinha), Gabriel Honorato, Mayara Moreno (Demoninho).
As queridas, Ana Lúcia, Carmozinha e Fabiola, sou muito feliz por ter conhecido todas vocês.
Aos amigos, Alex Shimura Yamashita, Nelo Eidy Zanchi, Daniel Venancio (Cabeça), Wilton Darlens
dos Santos, Eivor Martins Junior, Gabriela Chamusca, Valéria Panissa, Pedro Lorena Bezerra,
Leandro Ribeiro Marques, Cássio Couto Moraes, Rodrigo Fermino, Mario Filho.
Aos amigos do Laboratório de Lípides da USP, Renata Silvério (Tchutchuca), Robson (Bibi), Daniela
Caetano (Dani), Luiz Carnevali (Gringo), Felipe Donatto, Michele Joana, Rodrigo Xavier, Miguel
Luiz Batista Jr.
Aos Professores: Erico Chagas Caperuto, Marco Carlos Uchida, Luciana Pisani, Marcela Meneguello
Coutinho, Emer Suavinho Ferro, Alison Colquhoun, Antonio Hebert Lancha Junior. Ao Prof. GG (in
memorinam).
Ao pessoal do Laboratório das Profa Dra Eliane Beraldi Ribeiro, Vera Lúcia Flor Silveira, Ana Lydia.
A todos aqueles, professores, funcionários da Unifesp, pessoal da xérox, que de certa forma,
participaram de nossa formação nesses anos. Não ousaria sequer citar um por um, pois foram tantos e
não quero cometer uma gafe, que neste momento seria imperdoável.
viii
SUMÁRIO
Dedicatória
vi
Agradecimentos
vii
Sumário
viii
Listas
ix-xi
Resumo
xii
1 INTRODUÇÃO
1-2
2 REVISÃO DE LITERATURA
3
2.1 Obesidade
3-4
2.2 Tecido adiposo e processo inflamatório
4-10
2.3 Adipocinas anti-inflamatórias
10-15
3 Objetivos
16
3.1 Objetivo Geral
17
4. MATERIAIS E MÉTODOS
18
4.1 Estudo clínico:
18-23
4.2 Estudo Experimental
23-26
5. APRESENTAÇÃO DOS RESULTADOS E DISCUSSÃO
27
5.1 Manuscrito 1
28-36
5.2 Manuscrito 2
37-55
5.3 Manuscrito 3
56-77
6. CONSIDERAÇÕES FINAIS
78
7. REFERÊNCIAS BIBLIOGRÁFICAS
79-89
ix
Lista de Figuras
Figura 1. Percentual da mudança do biótipo da população adolescente no Brasil.
Figura 2. Recrutamento de macrófagos para tecido adiposo. “Início do ciclo vicioso”.
Figura 3. Visão esquemática do possível mecanismo relacionado à Microbiota intestinal e à obesidade.
Figura 4. Ciclo Vicioso da manutenção da inflamação na obesidade.
Figura 5. Ação anti-inflamatória da IL-10.
Figura 6. Ação celular da resposta anti-inflamatória da adiponectina.
Figura 7. Desenho experimental da Intervenção Interdisciplinar
x
Lista de Abreviaturas e Símbolos
AdipoR1
Receptor de Adiponectina 1
AdipoR2
Receptor de Adiponectina 2
AG
Ácido Graxo
AMPK
Adenosina Monofosfato Quinase
ATGL
Lipase do Triacilglicerol do Adiposo
CREBP
Proteína Ligada ao Elemento Responsivo ao Carboidrato
DM2
Diabetes Mellitus 2
FAIJ
Fator Adiposo induzido pelo Jejum
gp130
Glicoproteína 130
LHS
Lipase Hormônio Sensível
IkB
inibidor do NF-κB
Ikkβ
inibidor Quinase do NFkB sub-unidade beta
IL-10
Interleucina 10
IL-10R
Receptor de IL-10
IL-1ra
Receptor Antagonista de IL-1
IL-1β
Interleucina 1β
IL-6
Interleucina 6
IL-8
Interleucina 8
IMC
Índice de Massa Corporal
IRAK
Receptor da IL-1 associada à Kinase
IRS-1
Substrato do Receptor de Insulina 1
JAK
Janus Quinase
LPL
Lipase de Lipoproteína
xi
LPS
Lipopolissacarídeos
MCP-1
Proteína Quimioatraente de Monócito 1
mRNA
Ácido Ribonucléico mensageiro
MYD88
Myeloid differentiation primary response gene (88)
NF-κB
Fator de transcrição Nuclear kappa B
NK
Matadoras Natural
PPER
Elemento Responsivo ao PPAR
PPAR
Receptor Ativado de Proliferador de Peroxissomo
RTNFI
Receptor do Fator de Necrose Tumoral-α tipo I
RTNFII
Receptor do Fator de Necrose Tumoral-α tipo II
SOCS-3
Sinal de Supressão de Citocina 3
SREBP
Proteína Ligada ao Elemento Responsivo ao Esterol
STAT
Transdutor de Sinal e Ativador Transcripcional
TACE
Enzima Convertora de TNF-α
TLR-2
Toll Like Receptor 2
TLR-4
Toll Like Receptor 4
TNF
Fator de Necrose Tumoral
TRADD
Domínio de proteína de Morte, associado ao receptor de TNF-α tipo 1
TRAF1
Fator associado ao receptor do TNF 1
TRAF2
TRAF6
OMS
Organização Mundial da Saúde
xii
RESUMO
Objetivo: Verificar os efeitos anti-inflamatórios da adiponectina e da interleucina 10 em modelos
clínico e experimental de obesidade. O estudo foi dividido em duas etapas: clínico com adolescentes
obesos submetidos à terapia de redução de peso interdisciplinar por 1 ano; e experimental in vitro,
com adipócitos 3T3-L1. No estudo clínico, foram sujeitos do estudo 18 adolescentes (7 meninos e 11
meninas, idade 15 1,7 anos, índice de massa corporal (IMC) acima do Percentil 95. Os adolescentes
participaram do programa de terapia interdisciplinar por 1 ano. Parâmetros antropométricos e
bioquímicos séricos foram analisados antes e após a terapia. Nesta etapa do projeto observamos que, a
redução da gordura visceral, assim como das adipocinas pró-inflamatórias no soro foram
acompanhadas pelo aumento da adiponectina e da interleucina 10. Outro fator analisado foi a
concentração sérica de endotoxina e resistência à ação da insulina. Pudemos observar que a melhora
do quadro da resistência à ação da insulina foi acompanhada pela redução da endotoxina sérica após a
terapia interdisciplinar. A redução da endotoxina correlacionou-se com aumento da adiponectina
sérica. Tais alterações sugeriram que essas adipocinas anti-inflamatórias (adiponectina e interleucina
10) podem estar envolvidas nos processos anti-inflamatórios induzidos pelo programa de terapia
interdisciplinar, e com a melhora do quadro inflamatório destes adolescentes obesos.
No estudo in vitro, analisamos os mecanismos intracelulares da resposta inflamatória em células
adiposas 3T3-L1, estimuladas com lipopolissacarídeo (LPS), na ausência ou presença da adiponectina
e interleucina 10, isoladas e associadas, a fim de elucidar os efeitos anti-inflamatórios da adiponectina
e interleucina 10. Para tanto, avaliamos a secreção de IL-6 e a cascata de sinalização dos Toll Like
Receptors (TLR-2, TLR-4, MyD88 e TRAF6), assim como o Fator Nuclear kappa B e sua ligação com
DNA. Observamos que, adipócitos 3T3-L1 tratados por 24h mostraram elevada concentração de IL-6
no meio de cultura, assim como, aumento da cascata de sinalização da via do NF-κB e da expressão
protéica do IL-6R, TLR-4, MyD88, TRAF6. A adiponectina e IL-10 inibiram o aumento na
concentração de IL-6, bem como a ligação do NF-κB com DNA. Tomados em conjunto, nossos
resultados tanto clínico quanto experimental fornecem evidência de que a adiponectina e IL-10 têm
importante papel na resposta anti-inflamatória, inibindo a via de sinalização do NF-κB e
consequentemente reduzindo as adipocinas pró-inflamatórias. Corroboram com a ideia de que tais
adipocinas podem ser excelentes estratégias para o tratamento do estado inflamatório na obesidade.
Palavras-Chave: Obesidade; Inflamação; Citocinas; Adiponectina; IL-10; Tecido adiposo; Terapia
Interdisciplinar; TLRs.
1
1. Introdução
A obesidade é um problema de saúde pública sendo considerada uma epidemia mundial (Gruen
et al, 2007). As expectativas são que a prevalência desta doença crescerá nos próximos anos, devido o
aumento do número de pessoas com estilo de vida sedentário e consumo alimentar inadequado. De
acordo com a Organização Mundial da Saúde (OMS), a obesidade e o sobrepeso alcançarão a marca
de 1,6 bilhões em adultos (idade maior que 15 anos) em 2015.
A obesidade atualmente é caracterizada por um quadro inflamatório crônico associado a
aumento plasmático de: endotoxina (como lipopolissacarídeos), ácidos graxos saturados (Kueht et al,
2009; Kashyap et al, 2009) e citocinas pró-inflamatórias (Hukshorn et al, 2004) envolvidos no
desenvolvimento de morbidades como diabetes mellitus, hipertensão, dislipidemias e síndrome
metabólica (Gruen et al, 2007).
O tecido adiposo é um importante órgão secretor de adipocinas pró e anti-inflamatórias. O
fator de necrose tumoral alfa (TNF-α) e a interleucina-6 (IL-6), importantes marcadores inflamatórios,
estimulam a produção de diversas proteínas e citocinas pró-inflamatórias, em diferentes tipos celulares
via ativação do fator nuclear κB (NF-κB), (Haas et al, 2008; Turnbull, Rivier, 1999). Da mesma forma
que as endotoxinas (lipopolissacarideos - LPS) e os ácidos graxos saturados o fazem via ativação de
TLR-4. Diversos estudos apontam que a adição de LPS no meio de cultura de adipócitos 3T3-L1, ativa
o NF-κB, elevando a expressão gênica de adipocinas pró-inflamatórias, e que esta resposta é
favorecida pelos TLR-2 e TLR-4 (Lin et al, 2000; Ajuwon, Spurlock, 2005; Suganami et al, 2007).
Vários relatos da literatura referem que o TNF-α e a IL-6 prejudicam a cascata de sinalização de
insulina aumentando a resistência à ação desse hormônio em diversos tecidos, como no músculo
esquelético, tecido adiposo e fígado (Hotamisligil et al, 1994; Feinstein et al, 1993; Del Aguila et al,
1999). Adicionalmente, a IL-6 tem efeitos pró-inflamatórios, como aumento da produção de proteínas
de fase aguda pelos hepatócitos, aumento da maturação e atividade de linfócitos B, macrófagos,
monócitos e células NK, além de aumentar a expressão de IL-1β e TNF-α nestas células.
A concentração de IL-6 no tecido adiposo é, aproximadamente, 50 vezes maior do que sua
concentração plasmática (Sopasakis et al, 2004) e este tecido contribui com aproximadamente 30% da
IL-6 presente no sangue (Mohamed-Ali et al, 1997).
Juge-Aubry et al (2005) demonstraram que o tecido adiposo é uma fonte importante de IL-10, e
sua secreção apresenta-se aumentada em indivíduos obesos quando comparados com eutróficos.
Estudos têm demonstrado que a IL-10 tem sua produção aumentada no tecido adiposo em processos
inflamatórios, câncer e exercício agudo (Coppack, 2001; Lira et al, 2009a; Rosa et al, 2009b), como
uma reação do organismo na tentativa de minimizar o processo inflamatório nessas condições
(Daftarian et al, 1996). Essa linha de raciocínio sugere que a IL-10 atuaria como um mecanismo de
2
retroalimentação negativa ao excesso de adipocinas pró-inflamatórias, como por exemplo, o TNF(Daftarian et al, 1996; Lira et al, 2009b).
A adiponectina, outra adipocina anti-inflamatória, tem efeito sistêmico, e sua concentração
plasmática está inversamente relacionada com a massa de tecido adiposo e ao IMC (Gil-Campos et al,
2004). Esta adipocina aumenta a sensibilidade à insulina, no músculo esquelético e no tecido adiposo
(Lara-Castro et al, 2007; Gil Campos et al, 2004); tem efeitos anti-inflamatórios sobre as células do
sistema imunológico (Wulster-Radcliffe, 2004), e reduz a formação de placa de ateroma. Portanto, a
diminuição da concentração de adiponectina no plasma, pode estar associada com aumento do quadro
inflamatório, com a diminuição na sensibilidade à insulina e problemas cardiovasculares,
frequentemente observados em obesos (Schober et al, 2007).
Tomados em conjunto, esses estudos sugerem um papel de suma importância da IL-10 e da
adiponectina em doenças inflamatórias crônicas, principalmente devido ao seu efeito modulador da
síntese e secreção do TNF-
e IL-6, desta forma, essas adipocinas vem ganhando um papel de
destaque como possibilidade terapêutica em indivíduos obesos que apresentam o quadro inflamatório
crônico.
Frente a essas informações da literatura, levantamos a hipótese de que a redução das adipocinas
anti-inflamatórias, IL-10 e adiponectina, que ocorre em indivíduos obesos, teria papel relevante no
desencadear do processo inflamatório no tecido adiposo destes indivíduos.
Para verificar esta hipótese analisamos o perfil de citocinas e endotoxina em adolescentes
obesos antes e após terapia interdisciplinar para o tratamento da obesidade e correlacionamos as
citocinas pró-inflamatórias com a concentração plasmática de adiponectina e IL-10. O tratamento
interdisciplinar utilizado no presente estudo vem sendo eficiente na redução da prevalência da
síndrome metabólica, da esteatose hepática não alcoólica, da compulsão alimentar, contribuindo para a
melhoria na qualidade de vida destes adolescentes obesos (Caranti et al, 2007; De Piano et al, 2007;
Carnier et al, 2008; Caranti et al, 2008).
Adicionalmente, realizamos estudo in vitro em adipócitos 3T3-L1 com objetivo de investigar os
efeitos anti-inflamatórios da adiponectina e da IL-10, sobre a via de sinalização à resposta inflamatória
intracelular, em especial sobre o TLR-4 e NF-κB estimulada com LPS.
3
2. Revisão de Literatura
2.1 Obesidade
A obesidade representa grave problema de saúde pública, que afeta tanto países desenvolvidos
quanto em desenvolvimento (OMS, 2010). Estudos epidemiológicos indicam que aproximadamente
65% da população dos Estados Unidos apresentam excesso de peso, sendo que 35% desses indivíduos
são classificados como sobrepeso (25 ≤ IMC ≤ 30 kg/m2) e 30% são considerados obesos (IMC ≥ 30
kg/m2) (Hedley et al, 2004).
Segundo a Pesquisa de Orçamentos Familiares 2008-2009 realizada pelo Instituto Brasileiro de
Geografia e Estatística o aumento de peso em adolescentes de 10 a 19 anos foi contínuo nos últimos
34 anos (IBGE, 2010). Neste período, a prevalência de excesso de peso (IMC ≥ 25 kg/m2) na
população adolescente brasileira passou de 3,7% para 21,7% no sexo masculino e 7,6% para 19% para
o sexo feminino. Já os casos de obesidade (IMC ≥ 30 kg/m2), entre adolescentes, passaram de 0,4%
para 5,9% no sexo masculino, e de 0,7% para 4,0% no sexo feminino (IBGE, 2010) (Figura 1).
Quadro este extremamente preocupante, pois, estima-se que indivíduos obesos apresentam
aumento de 50 a 100% do risco de mortalidade, fato explicado pela associação entre excesso de peso e
gordura abdominal com doenças crônicas não transmissíveis, como hipertensão arterial, dislipidemia,
diabetes mellitus tipo 2 (DM2) e certos tipos de câncer. O excesso de peso e a obesidade resultam da
interação de diversos fatores, como genéticos, metabólicos, comportamentais e ambientais (Ryan,
Kushner, 2010).
No Brasil, vários estudos de base populacional têm mostrado que fatores sócio-demográficos e
comportamentais estão associados ao aumento do peso corporal. Entretanto, ainda existem poucos
estudos acerca do papel desses determinantes na distribuição da gordura corporal. Em geral, os riscos
de desenvolver obesidade abdominal aumentam com a idade e diminuem com a maior escolaridade.
Adicionalmente, observa-se associação entre obesidade abdominal e menopausa (Ronque et al, 2005;
Campos et al, 2006), tornando-se primordial o desenvolvimento de terapias, tanto relacionadas a
mudança de comportamento, como farmacológicas, em especial na adolescência visando reduzir o
impacto do envelhecimento no desenvolvimento de obesidade e doenças a ela associadas.
A obesidade é uma condição heterogênea com relação à distribuição regional da adiposidade:
obesidade visceral refere-se à acumulação de gordura nos depósitos omental e mesentérico, enquanto a
obesidade periférica geralmente refere-se ao aumento de gordura nos depósitos subcutâneos e está
menos relacionada com o desenvolvimento de comorbidades (Cao et al, 2008). As diferenças
funcionais entre os adipócitos viscerais e subcutâneos, tanto no que concerne aos aspectos metabólicos
como secretórios, podem estar relacionadas com sua localização anatômica (Pond, 1996; Hocking et
al, 2010).
4
O tecido adiposo visceral possui macrófagos residentes, os quais produzem várias moléculas
pró-inflamatórias, como TNF-α e IL-6 e menores quantidades de moléculas anti-inflamatórias, tais
como adiponectina e interleucina-10 (Dandona et al, 1998; Trayhurn, Wood, 2005). Este perfil próinflamatório está associado à resistência à ação da insulina e desempenha papel importante na
patogênese da disfunção endotelial e aterosclerose subsequente (Hamdy et al, 2006).
Este estado pró-inflamatório descrito em indivíduos obesos está principalmente relacionado
com o acúmulo de gordura visceral e não da gordura subcutânea, sendo importante para a verificação
do risco de desenvolvimento de doenças, a análise destes depósitos, o que torna o IMC um índice
pouco acurado na pesquisa clínica.
Thorne et al (2002), relataram que a remoção de tecido adiposo visceral por omentectomia
(retirada do tecido adiposo omental) resultou na diminuição nas concentrações de glicose e de insulina
em 11 homens e 14 mulheres (IMC >35kg/m2), em contraste, a remoção de tecido adiposo subcutâneo,
por método de lipoaspiração nem sempre resultaram em melhorias no metabolismo da glicose ou de
lipídios (Klein et al, 2004).
2.2 Tecido adiposo e processo inflamatório
Na década de 1990 ampliou-se o conhecimento científico sobre a função do tecido adiposo. Até
então, os pesquisadores reconheciam o papel fundamental do tecido adiposo como o principal tecido
armazenador de energia no organismo. No entanto, com a descoberta da leptina em 1994, citocina
5
produzida e liberada pelo tecido adiposo, este tecido passou também a ser considerado um “órgão
endócrino” (Pelleymounter et al, 1995).
Essa descoberta foi de suma importância, dando início aos estudos sobre a atividade secretora
do tecido adiposo. Atualmente, sabemos que o tecido adiposo secreta peptídeos bioativos, chamados
de “adipocinas”, que agem de maneira autócrina, parácrina e endócrina, participando da regulação da
ingestão alimentar e do balanço energético, atuando no sistema imunológico, na sensibilidade à
insulina, na angiogênese, na regulação da pressão arterial e do metabolismo lipídico (Trayhurn,
Beattie, 2001).
O aumento na concentração plasmática do TNF-α e IL-6 estão envolvidos no desenvolvimento
da resistência à insulina encontrada na obesidade (Ronti et al, 2006). Inversamente, a adiponectina,
que tem propriedades antidiabéticas e antiaterogênicas, apresenta sua concentração plasmática
reduzida (Bueno et al, 2008).
A denominação de TNF-α foi derivada da ação deste peptídeo sobre células tumorais,
provocando a necrose desse tipo celular. Esta citocina foi primeiramente descrita em 1975 e
denominada de caquexina. Devido à potente ação contra as células tumorais, posteriormente passou a
ser conhecida como TNF-α (Vercammen et al, 1998).
Potente citocina pró-inflamatória produzida em diversos tipos celulares, o TNF-α age de
maneira autócrina, parácrina e induz, no tecido adiposo e em outras células, a expressão de várias
citocinas inflamatórias, como IL-6, IL-1, IL-8, proteína quimioatraente de monócitos 1 (MCP-1),
dentre outras e reduz a expressão e secreção de adiponectina (Peeraully et al, 2004).
A ação fisiológica do TNF-α depende de sua ligação aos seus receptores. Foram até hoje
descritos dois tipos de receptores, o tipo I (TNF-RI, p55) e o tipo II (TNF-RII, p75) (Bazzoni, Beutler,
1996). Esses receptores que são proteínas transmembrana, possuem a porção extracelular dos dois
tipos de receptores para TNF-α, exibindo arquitetura bem similar, mas os domínios intracelulares são
bem diferentes (Lewis et al, 1991).
A ativação de ambos os receptores pelo TNF-α aumenta a atividade do NF-κB, no entanto, por
vias diferentes. O TNF-RII ativa esse fator nuclear via transdução de sinal pela ação das enzimas
TRAF1 e TRAF2 (Fator associado ao receptor do TNF 1 e 2), enquanto o TNF-RI age recrutando a
enzima TRADD (Domínio de proteína de Morte, associado ao receptor de TNF-α tipo 1) induzindo
uma via de transdução de sinalização diferente (Darnay, Aggarwal, 1997).
A contribuição do TNF- α, produzido pelo tecido adiposo, para a elevação plasmática desta
citocina, presente na obesidade, tem sido bastante discutida, e seu aumento correlaciona-se
positivamente com surgimento do quadro da resistência à insulina (Bulló et al, 2003).
6
Outra adipocina que se encontra elevada em indivíduos obesos é a IL-6. A IL-6 é uma
glicoproteína produzida por uma grande variedade de tipos celulares (Turnbull, Rivier, 1999). A
primeira ação descrita foi o estímulo do crescimento e diferenciação dos linfócitos B (Curfs et al,
1997).
Hoje, sabe-se que a IL-6 é capaz de produzir as mais diversas ações em diferentes tecidos. Seu
aumento no plasma é capaz de elevar a resposta de fase aguda a estímulos agressores (Turnbull,
Rivier, 1999); alterar a homeostase energética (Fischer, 2006); induzir a liberação do hormônio
adrenocorticotrófico, febre, anorexia e fadiga (Robson, 2003).
A IL-6 age via um receptor complexo que contém pelo menos uma subunidade da proteína
transdutora de sinal gp130. Liga-se ao seu receptor na célula alvo e o complexo IL-6-receptor associase a proteína transmembrana gp130, permitindo a transdução de sinal (Taga, Kishimoto 1997; Derouet
et al, 2004). Desta forma, como todas as células até hoje estudadas que expressam a proteína gp130
em seus domínios transmembrana, pode-se inferir que a IL-6 atue em todas ou na maioria das células
do organismo (Althoff et al, 2000; Althoff et al, 2001).
Atualmente sabe-se que indivíduos obesos apresentam um quadro inflamatório crônico de
baixo grau, que é caracterizada pelo aumento sistêmico da proteína C reativa e citocinas próinflamatórias (Petersen, Pedersen, 2005). Este processo inflamatório tem origem em diversas
modificações, tanto diretamente no tecido adiposo como sistêmicas.
A origem do quadro inflamatório no tecido adiposo de indivíduos obesos pode ser explicado,
pelo menos em parte, pelo aumento da presença de macrófagos infiltrados, e a instalação do ciclo
vicioso da produção de citocinas e fatores pró-inflamatórios, na tentativa de restaurar a homeostase do
tecido (Wellen, Hotamisligil, 2003), conforme esquematizado na Figura 2.
7
Alterações no tamanho dos adipócitos e do depósito do tecido adiposo causam mudanças
físicas na área circundante e na atividade parácrina do adipócito. Por exemplo, no início da hipertrofia
dos adipócitos, estes começam, paulatinamente, a elevar a secreção de TNF-α, o que estimula a
produção da MCP-1 pelos preadipócitos e pelas células endoteliais. A MCP-1 é uma quimiocina que
recruta macrófagos para o tecido adiposo. Durante este processo ocorre modificação na secreção de
outras adipocinas, tais como: elevação na leptina e redução na adiponectina. Este quadro favorece o
processo inflamatório, pois, a leptina também estimula a migração de macrófagos para o tecido
adiposo, e a redução de adiponectina promove a adesão de macrófagos nas células endoteliais.
Outros fatores como danos físicos no endotélio e hipóxia, causados por mudanças do tamanho
exarcebado das células adiposas, e danos oxidativos resultantes de um ambiente cada vez mais
lipolítico, também desencadeiam um ambiente propício para a infiltração de macrófagos, semelhante
ao observado na aterosclerose.
Todas estas modificações no tecido adiposo levam a um ciclo vicioso de recrutamento de
macrófagos, aumento na produção de citocinas pró-inflamatórias e comprometimento da função dos
adipócitos, como descrito na revisão de Wellen, Hotamisligil (2003).
Em relação às modificações sistêmicas, alguns pesquisadores postulam que indivíduos obesos
apresentam aumento de endotoxinas circulantes, provenientes da translocação da microbiota intestinal,
tem papel importante no desenvolvimento do quadro inflamatório em diferentes tecidos, e instalação
da resistência à ação da insulina (Creely et al, 2007; Tsukumo et al, 2009).
8
Lipopolissacarídeo, também conhecido como endotoxina, é um potente indutor de inflamação,
provocando elevada produção de TNF-α, IL-6, e reduzindo a produção de adiponectina. Em
circunstâncias normais, apenas pequenas quantidades de endotoxina são translocadas do lúmen
intestinal para a circulação sistêmica, e essa pequena parcela que consegue atravessar é absorvida
rapidamente, e removida por monócitos, células de Kupffer no fígado. No entanto, novas evidências
indicam que em doenças crônicas, tais como a obesidade, ocorre elevação na produção e
estravazamento de endotoxina para a corrente sanguínea, podendo desencadear um estado de
resistência à insulina na obesidade (Cani et al, 2007; Creely et al, 2007), conforme exemplificado na
Figura 3.
Creely et al (2007), demonstraram aumento nas concentrações circulantes de endotoxina em
mulheres portadoras de diabetes mellitus do tipo 2. Neste mesmo trabalho, os autores verificaram que
o LPS elevou expressão protéica de TLR-2 e a secreção de IL-6 e TNF-α por adipócitos isolados do
tecido adiposo subcutâneo.
O LPS e ácidos saturados agem sobre receptores da família Toll Like Receptor, em particular o
TLR-4, ativando a via de sinalização do NF-κB, favorecendo a expressão gênica das adipocinas
proinflamatórias (Takeuchi, Akira, 2001; Lee et al, 2001).
9
De acordo com estudos recentes, a expressão gênica do TLR-2 e TLR-4 está aumentada no
tecido adiposo de camundongos tornados obesos e diabéticos do tipo 2, pela ingestão de dieta
hiperlipídica. Os autores sugerem que, em parte, o aumento dos TLRs seria responsável pela ativação
da resposta inflamatória no tecido adiposo, pois utilizando camundongos trangênicos que expressam
pouco TLR-4, quando tratados com dieta hiperlipídica, não apresentaram este quadro inflamatório e a
ativação do NF-κB neste tecido foi reduzida (Tsukumo et al, 2007).
A transmissão do sinal mediado pela ligação do LPS com o TLR-4 constitui um fenômeno
altamente complexo e variado, mediado através de reações envolvendo fosforilação e ubiquitinação de
proteínas alvo. Por exemplo, ocorre ativação da proteína MyD88, que por sua vez ativa o complexo
IRAK (Receptor da IL-1 associada à Kinase)-TRAF 6 (Fator associado ao receptor do TNF 6), esta
última pertence à classe das ubiquitina ligases (E3 ligases) e parece ser essencial para o
desacoplamento do NF-κB da sua proteína inibidora (Iκ-B). O NF-κB, uma vez liberado, migra para o
núcleo ligando-se ao DNA, iniciando a amplificação gênica das proteínas relacionadas à inflamação
(Takeda, Akira, 2004).
O tecido adiposo expressa esse receptor, que pode ser ativado tanto por LPS, como por ácidos
graxos livres (Shi et al, 2006). A ativação desse receptor nos adipócitos aumenta a expressão e
liberação de citocinas via ativação do NF-κB (Schaeffler et al, 2009). A elevação persistente de ácidos
graxos livres no plasma, como ocorre muitas vezes no indivíduo obeso, pode aumentar a secreção de
citocinas via TLR-4 (Francaux, 2009), (Figura 4).
Até o momento, este texto descreve as principais alterações que ocorrem na obesidade que
desencadeiam elevação de adipocinas pró-inflamatórias e suas conseqüências. A seguir, trataremos das
adipocinas anti-inflamatórias, adiponectina e IL-10 e a relação delas com a obesidade.
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2.3 Adipocinas anti-inflamatórias
Como dito anteriormente, é bem estabelecido o aumento na produção de adipocinas próinflamatórias no tecido adiposo de indivíduos obesos e, a participação destas adipocinas na indução de
resistência à insulina e outras comorbidades associadas à obesidade. Pesquisadores da área têm
desenvolvido estudos utilizando diferentes estratégias na tentativa de estabelecer protocolos para
minimizar e/ou restaurar tais alterações (Peraldi et al, 1997; Polak et al, 2006).
Em 1991, Fiorentino e colegas observaram que um fator produzido por células T ativadas foi
capaz de inibir a produção de citocinas pró-inflamatórias. Este fator foi nomeado de interleucina 10
(IL-10).
Estudos têm demonstrado que a IL-10 tem sua produção aumentada no tecido adiposo em
processos inflamatórios (Coppack, 2001; Esposito et al, 2003; Lira et al, 2009), e sugerem que a IL-10
atuaria como um mecanismo de retroalimentação negativa ao excesso de adipocinas pró-inflamatórias,
como por exemplo, o TNF- (Daftarian et al, 1996; Lira et al, 2009b).
Juge-Aubry et al (2005) demonstraram que o tecido adiposo é uma fonte importante de IL-10,
e sua secreção está aumentada em indivíduos obesos quando comparados com eutróficos. A produção
da IL-10 no tecido adiposo de indivíduos obesos é estimulada por LPS e /ou TNF-α, os autores
sugerem, também, que este aumento seja um mecanismo de retroalimentação, na tentativa de
minimizar os efeitos deletérios causados pelo LPS e/ou TNF-α.
11
A IL-10 atua, em uma variedade de tipos celulares, inibindo a produção de várias citocinas
pró-inflamatórias, tais como: TNF- , IL-1 e IL-6, e estimulando a sua própria produção (Moore et al,
1993). Estes efeitos são desencadeados quando a IL-10, liga-se ao seu receptor (IL-10R), ativa a via
JAK-STAT, especificamente a JAK1 e STAT3 em macrófagos (Riley et al, 1999), e essa ativação é
mediada pela SOCS3, reduzindo a atividade quinase do IKK e, portanto, a ativação do NF-κB
(Murray, 2007), (Figura 5).
Esta adipocina, também, inibe a geração de espécies reativas do oxigênio e aumenta a
liberação dos receptores solúveis do TNF (TNFRs), os quais podem antagonizar os efeitos do TNF(Ferrari, 1995; Nozaki et al, 1998).
Estudo recente mostrou que a sensibilidade à insulina no músculo esquelético foi maior em
camundongos que super-expressavam IL-10 no músculo esquelético, quando comparados com
camundongos controle, após tratamento com dieta rica em gordura (Hong et al, 2009).
Atualmente, utiliza-se a relação IL-10/TNF-α como marcador do estado inflamatório, pois se
considera essa razão mais importante na avaliação do quadro inflamatório do que a concentração
isolada de cada uma dessas citocinas. Redução nessa razão é correlacionada com pior prognóstico e
diminuição na expectativa de vida de pessoas que possuem diferentes morbidades (Kaur et al, 2006;
Leonidou et al, 2007). Além disso, essa razão é um importante marcador de esteatose hepática,
mostrando forte correlação negativa (Hashem et al, 2008).
12
Além da IL-10, outra adipocina envolvida na resposta anti-inflamatória em adipócitos, capaz
de amenizar os efeitos deletérios induzidos pelas adipocinas pró-inflamatórias, é a adiponectina
(Yamaguchi et al, 2005) .
A adiponectina é o produto da transcrição do gene apM1, sendo a mais abundante proteína
secretada pelo tecido adiposo em humanos (Maeda et al, 1996, Arita et al, 1999). A regulação
transcricional do gene da adiponectina envolve um conjunto de fatores de transcrição. Os promotores
da adiponectina contêm sítios de ligação da proteína ligada ao elemento responsivo ao esterol
(Proteína Ligada ao Elemento Responsivo ao Esterol: SREBP), receptor ativado de proliferador de
peroxissomo (PPAR), e da proteína ligada ao elemento responsivo ao carboidrato (Proteína Ligada ao
Elemento Responsivo ao Carboidrato: CREBP) (Seo et al, 2004). Pode apresentar-se na forma longa
ou globular, no entanto, quase toda adiponectina parece existir na forma longa no plasma. Fruebis et al
(2001), relataram entretanto que uma pequena quantidade de adiponectina globular foi detectada em
plasma humano.
Até o momento, três receptores de adiponectina foram identificados, AdipoR 1, AdipoR2, e
mais recentemente, a T-caderina. AdipoR1 e AdipoR2 são receptores com sete domínios
transmembrana. O AdipoR1 exibe expressão elevada no músculo esquelético, tanto em humanos
quanto em camundongos. Em contraste, o AdipoR2 é expressa no fígado do rato, e no fígado e
13
músculo esquelético humano. A T-caderina, embora seja um receptor truncado que não tem o domínio
intracelular necessário para a transdução de sinal, pode participar da cascata de sinalização
intracelular, competindo com AdipoR1 e AdipoR2 (Brochu-Gaudreau et al, 2010). A ação da resposta
anti-inflamatória da adiponectina está exemplificada na Figura 6.
Esta adipocina aumenta a sensibilidade à insulina e tem efeitos anti-inflamatórios e
antiaterogênicas (Diez, Iglesias 2003). Diminuição das concentrações de adiponectina sérica tem sido
observada em indivíduos com resistência à insulina, obesidade, diabetes tipo 2 e doença cardíaca (Diez
e Iglesias 2003, Hotta et al, 2000). As concentrações séricas de adiponectina são inversamente
correlacionadas com o índice de adiposidade central, pressão arterial, glicemia de jejum, resistência à
insulina e concentrações séricas de insulina (Yamamoto et al, 2002). Tem sido demonstrado que
adiponectina reduz a produção hepática de glicose e a concentração de triacilglicerol no músculo
esquelético, assim melhorando a sensibilidade à insulina (Prins, 2002).
Salmenniemi et al (2005), verificaram que hipoadiponectinemia está relacionada com diversas
características da síndrome metabólica (aumento da glicemia de jejum, triacilglicerol, obesidade
abdominal e diminuição do HDL-colesterol) e alta concentração de citocinas inflamatórias (IL-6, IL-1,
e Proteína C-reativa).
A adiponectina inibe a via de sinalização dos TLRs e do NF-κB bloqueando a resposta
inflamatória em linhagem celular de macrófagos (RAW264) (Yamaguchi et al, 2005).
14
Estudando os mecanismos envolvidos na resposta anti-inflamatória da adiponectina em
macrófagos, o grupo da Profa Laura Nagy da Universidade de Ohio (EUA) verificou que a
adiponectina, primeiramente, ativa a via inflamatória, aumentando de maneira significativa a
concentração do TNF-α via ativação do NF-κB. A seguir, observou que o aumento exacerbado do
TNF-α estimulou a produção da IL-10, predominando desta maneira a resposta anti-inflamatória. Estes
resultados sugerem, portanto, que a ação anti-inflamatória da adiponectina possa ser dependente da IL10. Corroborando com está inferência, o mesmo grupo verificou que a adição da adiponectina, em
cultura de macrófagos, estimulados com LPS, não inibiu o aumento de TNF-α na presença de
anticorpo bloqueando a ação da IL-10 (Park et al, 2007).
Diversos estudos apontam que a adição de LPS no meio de cultura de adipócitos 3T3-L1 ativa
o NF-κB, elevando a expressão gênica de adipocinas pró-inflamatórias, e que esta resposta é
favorecida pelos TLR-2 e TLR-4 (Lin et al, 2000; Ajuwon, Spurlock, 2005; Suganami et al, 2007).
Ajuwon e Spurlock (2005) demonstram que células 3T3-L1 estimuladas com LPS, quando incubadas
com adiponectina, diminuíram a ativação do NF-κB, reduzindo a produção de adipocinas
proinflamatórias, paralelamente, os autores observaram aumento do fator transcricional PPAR-γ,
conhecido como desencadeador de resposta anti-inflamatória. Tal fator, também, pode estar envolvido
nos efeitos anti-inflamatórios causados pela adiponectina.
A obesidade é uma doença multifatorial, e deve ser tratada por longo prazo. O tratamento da
obesidade pode ser feito com intervenções em diferentes aspectos, incluindo diminuição na ingestão
calórica e aumento do gasto energético imposto pelo exercício físico. O tratamento de longo prazo
com dietas restritivas e capaz de diminuir as citocinas pró-inflamatórias no sangue, assim como a
hiperleptinemia, reduzindo os riscos das morbidades associadas, como hipertensão, diabetes,
dislipidemias e doenças cardiovasculares (Hukshorn et al, 2004). No entanto, os estudos mostram que
as intervenções isoladas, embora muitas vezes eficientes à primeira vista, não são muito efetivas após
período prolongado, quando os indivíduos não mudam efetivamente o estilo de vida (Lawlor,
Chatuverdi, 2006). Desta feita, a literatura nos leva a crer que as intervenções interdisciplinares para o
tratamento da obesidade que contenham orientação médica, nutricional, programa de exercício físico e
acompanhamento psicológico parecem ser mais indicadas para tratar a obesidade, pois visam à
mudança do estilo de vida de forma ampla, aumentando a aderência e a permanencia nos novos
hábitos (Curioni, Lourenço, 2005; Snethen et al, 2006).
O tratamento interdisciplinar para o tratamento de adolescentes obesos, sob a direção da Profa
Dra Ana Dâmaso, que vem sendo desenvolvido na UNIFESP, tem se mostrado eficiente na redução da
prevalência da síndrome metabólica, da esteatose hepática não alcoólica e, da compulsão alimentar
contribuindo para a melhoria na qualidade de vida desses adolescentes (Caranti et al, 2007; Carnier et
al, 2008; Caranti et al, 2008; De Piano et al, 2007; Carnier et al, 2010; de Lima Sanches et al, 2011).
15
Frente aos efeitos benéficos observados por este tratamento interdisciplinar e os relatados
sobre a importância da IL-10 e da adiponectina em doenças inflamatórias crônicas, principalmente
devido aos seus efeitos moduladores da síntese e secreção do TNF- e IL-6 e, levantamos a hipótese
que o tratamento interdisciplinar modifica o perfil de citocinas e endotoxina em adolescentes obesos, e
que isto estaria relacionado à elevação na IL-10 e adiponectina que, via redução da ativação do TLR-4,
inibiria a cascata de sinalização para ativação do NF-κB no tecido adiposo.
16
3. OBJETIVOS
Neste estudo nos propusemos responder as seguintes questões:
Os efeitos benéficos da terapia interdisciplinar para o tratamento da obesidade estão
correlacionados com a elevação das adipocinas anti-inflamatórias (adiponectina e IL10)?
correlacionados com a redução das citocinas pró-inflamatórias (TNF-α e IL-6),
endotoxina e dos depósitos de gordura corporal?
Os efeitos anti-inflamatórios da adiponectina e da IL-10 em adipócitos 3T3-L1 são
dissociados ou interdependentes?
A ação anti-inflamatória, individual ou conjunta, da adiponectina e IL-10 em adipócitos
3T3-L1, é mediada por alterações na via de sinalização do TLR-4 e do NF- κB?
17
3.1 Objetivos específicos
1) Analisar o perfil de citocinas pró-inflamatórias (TNF-α e IL-6) e anti-inflamatórias
(adiponectina e IL-10), e correlacionar com os depósitos de gordura subcutânea e
visceral, antes e após um ano de terapia interdisciplinar em adolescentes obesos.
2) Avaliar a concentração de endotoxina, e correlacionar perfil de citocinas pro e antiinflamatórias, e resistência à ação da insulina, antes e após um ano de terapia
interdisciplinar em adolescentes obesos.
3) Quantificar a produção de IL-6 no meio de cultura, e a expressão protéica das seguintes
proteínas: IL-6R, TLR-2, TLR-4, MYD88 e TRAF6 em células 3T3-L1, na presença de
IL-10, adiponectina e IL-10 mais adiponectina, após estímulo de 24h com LPS.
4) Determinar a interação do fator transcripcional NF-κB: (subunidades NF-κB p50 e NFκB p65) com DNA. Avaliar parâmetros na presença de IL-10, adiponectina e IL-10
mais adiponectina, em células 3T3-L1, após 24h de estimulo com LPS.
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4. MATERIAIS E MÉTODOS
4.1 Modelo Clínico
População
Para o desenvolvimento desta pesquisa, foram recrutados ciquenta e quatro (54) adolescentes (de
ambos os gêneros) obesos do programa de intervenção interdisciplinar do GEO/CEPE UNIFESP,
iniciado em 2004, conforme exemplificado abaixo na Figura 7.
Trinta e nove (39) adolescentes completaram até o final à Intervenção Interdisciplinar. Para o
presente estudo, foram selecionados dezoito (18) adolescentes, os quais exibiram perda de massa
gorda maior que 5%.
19
Critérios de inclusão
Adolescentes com idade entre 13 a 19 anos, pós-púberes, com o IMC acima do Percentil 95
proposto pelas curvas do “Centers for Disease Control and Prevention” (CDC) (2000), estágio puberal
V segundo os critérios de Tanner (Tanner et al, 1976), além da assinatura do termo de consentimento
pelos familiares dos participantes do estudo.
Critérios de não inclusão
Como critérios de não inclusão foram selecionados os seguintes itens: limitações músculoesqueléticas que impeçam a intervenção, doença genética, hormonal ou metabólica, uso crônico de
álcool, usuário de drogas e outras causas de esteatose como hepatite viral B e C, hepatite auto-imune e
doenças metabólicas como hemocromatose.
Este estudo foi realizado de acordo com os princípios da declaração de Helsinki e foi previamente
aprovado pelo Comitê de Ética da Universidade Federal de São Paulo - Escola Paulista de Medicina
sob o número (#0135/04). Os voluntários foram incluídos no estudo após consentimento assinado.
Descrição da Intervenção Interdisciplinar para o tratamento da obesidade
Após os exames diagnósticos, os adolescentes obesos foram submetidos à Intervenção
Interdisciplinar para o tratamento da obesidade, de acordo com o modelo preconizado pelo Grupo de
Estudo da Obesidade (GEO), desenvolvido no Centro de Estudos em Psicobiologia e Exercício
(CEPE), do Departamento de Psicobiologia da UNIFESP/EPM e Programa de Pós Graduação em
Nutrição.
O Protocolo baseia-se em intervenção Interdisciplinar em longo prazo (1 ano), incluindo a triagem,
os exames e o desenvolvimento da pesquisa. Para isto, todos os voluntários tiveram acompanhamento
nutricional e psicológico semanais; clínico (consultas mensais com o endocrinologista); treinamento
físico combinado (exercício aeróbio e treinamento de força).
20
Protocolo de Exercício
Durante 48 semanas de intervenção Interdisciplinar os adolescentes obesos foram submetidos ao
programa de exercícios combinados três vezes por semana, consistindo em 30 minutos de exercícios
aeróbios por sessão de treinamento (bicicleta ergométrica e esteira ergométrica) e treinamento de força
adotando os seguintes exercícios: elevação lateral, supino sentado, puxador costas, remada baixa,
abdominais, hiperextensão lombar, leg press, flexão de joelhos, flexão de panturrilha e rosca direta.
Todos os sujeitos foram familiarizados com o protocolo de treinamento, durante 2 semanas, antes de
iniciarem o programa. Os exercícios aeróbios foram realizados na intensidade do esforço referente ao
limiar ventilatório 1, determinado por análise direta de gases. O treinamento de força foi orientado
seguindo os princípios para controle da carga e do volume do treinamento propostos por Kraemer et al
(2006). A periodização do treinamento de força utilizou o modelo não linear (ondulatório) proposto
por Kraemer et al (1997), desta forma, o protocolo consistiu na alteração semanal da carga, dividido
em semana de cargas altas (5 a 7-repetições máximas, RM) e semana de cargas moderadas (10 a 12RM). Os sujeitos realizaram 18 séries por sessão, distribuídas em 3 séries para cada exercício. O
intervalo entre as séries dependeu da carga adotada na sessão de treinamento, tendo intervalos de 2
minutos para as semanas com cargas altas e intervalos de 1 minuto para as semanas com cargas
moderadas. Os voluntários realizaram o protocolo de treinamento de força após os 30 minutos de
exercício aeróbio.
Avaliação e Intervenção Nutricional
Uma vez por semana os adolescentes receberam aulas de educação nutricional em pequenos
grupos, abrangendo temas como pirâmide alimentar, dietas da moda, rotulagem nutricional, diferentes
tipos de gordura, alimentação de fast food, aprendendo a montar um lanche saudável entre outros.
Ressalta-se que também foram oferecidas consultas nutricionais individuais.
A avaliação alimentar foi realizada no início, e ao término da intervenção por meio do registro
alimentar de três dias não consecutivos, incluindo dois dias da semana e um do final de semana. A
nutricionista orientou os pais e voluntários sobre o preenchimento do registro alimentar de três dias.
21
As porções foram relatadas em termos de medida caseira por referência de um Atlas de porções
alimentares. Os dados alimentares obtidos foram analisados por meio do software Nutwin (UNIFESP,
2002).
Avaliação e Intervenção Psicológica
Para o atendimento psicológico os adolescentes foram divididos em pequenos grupos. A
intervenção psicológica consistiu em dinâmicas, aulas e sessões terapêuticas uma vez por semana,
compreendendo temas como auto-estima, imagem corporal, depressão, ansiedade, transtornos
alimentares, questões familiares, entre outros. Ressalta-se que a intervenção psicológica também
consiste em consultas individuais, conforme a anuência do paciente.
Análises sanguíneas
Parâmetros Bioquímicos:
As análises sanguíneas foram realizadas através de punção periférica da veia do antebraço, após
jejum noturno de 12 horas.
Para dosagem das adipocinas (Adiponectina, IL-6, IL-10 e TNF-α; R&D System, Inc.,
Minneapolis, USA), endotoxina (LONZA Cologne GmbH, Suiça) e insulina (Millipore, 290 Concord
Road, Billerica, MA 01821) foi utilizado o método enzyme-linked immunoabsorbent assay (ELISA)
de captura. Este ensaio foi realizado em amostras de soro, proveniente da situação basal e após 1 ano
de terapia interdisciplinar. Placas com 96 poços foram sensibilizadas com 100 µL de anticorpo
monoclonal anti-humano (Adiponectina, IL-6, IL-10, TNF-α, insulina e endotoxina) (anticorpo de
captura) e incubadas por 2h em temperatura ambiente. Após este período, os poços foram lavados por
3 vezes com tampão para lavagem (0,05% Tween
20 em PBS, pH 7,2 – 7,4). Posteriormente, a placa
foi bloqueada para evitar ligações inespecíficas com 300 µL de solução de bloqueio (1% BSA em
PBS, pH 7,2 – 7,4, 0,2 µm filtrado) e incubada por 1 hora em temperatura ambiente. Findo este prazo,
os poços foram lavados novamente como descritos acima.
22
Após o bloqueio, foram adicionados 100 µL por poço das amostras e dos padrões diluídos
previamente em reagente de diluição (1% BSA em PBS, pH 7,2 – 7,4, 0,2 µm filtrado), e cobertos
com fita adesiva . Em dois poços foram colocados somente o reagente de diluição para caracterização
do branco. A placa foi incubada por 2 horas em temperatura ambiente. Após este período, os poços
foram lavados por 3 vezes com tampão de lavagem (0,05% Tween
20 em PBS, pH 7,2 – 7,4).
Após as lavagens, adicionou 100 µL do anticorpo de detecção (Anticorpo anti-Humano
Adiponectina, IL-6, IL-10, TNF-α, insulina e endotoxina Biotinilado) diluídos previamente em
reagente de diluição (1% BSA em PBS, pH 7,2 – 7,4, 0,2 µm filtrado) na concentração estabelecida,
cobertos com fita adesiva e incubado por 2 horas em temperatura ambiente. Os poços foram
novamente lavados como descrito acima. Posteriormente, adicionou-se 100 µL de Streptoavidina-HRP
(1:250) por poço, que foram cobertos com papel laminado e incubados por 30 minutos, à temperatura
ambiente. Findo este prazo, os poços foram novamente lavados como descrito acima. Posteriormente,
a solução de substrato (mistura dos reagentes de cores A - H2O2 e B - Tetrametilbenzidina) foi
adicionada na diluição de 1:1 por poço, seguido de incubação por 30 minutos à temperatura ambiente,
evitando-se contato direto da placa com a luz. A reação foi interrompida com 50 µL de H2SO4 30%
por poço sob agitação lenta. A leitura foi feita em leitor de ELISA (Power Wave, Bio-tek), utilizandose filtro de 450 nm.
Para dosagem da glicemia de jejum, foi utilizado Kit Glicose PAP Liquiform (Labtest Diagnostica
S.A), por método colorimétrico, sendo a mistura da amostra com reagente incubado por 15 minutos
em temperatura a 37°C. O princípio da reação foi: A glicose oxidase (GOD) catalisa a oxidação da
glicose de acordo com a seguinte reação:
Glicose + O2 + H2O
GOD
Ácido Glucônico + H2O2
O peróxido de hidrogênio formado reage com 4-aminoantipirina e fenol, sob ação catalisadora da
peroxidase, através de uma reação oxidativa de acoplamento formando uma antipirilquininimina
vermelha cuja intensidade de cor é proporcional à concentração de glicose da amostra. A leitura foi
feita em leitor de ELISA (Power Wave, Bio-tek), utilizando-se filtro de 520 nm.
23
Análise estatística do modelo clínico
A distribuição dos dados foi checada pelo teste de variança de Bartlett, e os dados expressos em
média ± desvio padrão. Os valores outliers estatísticos foram identificados utilizando um teste de
Grubbs (GraphPad Software, San Diego, CA) e subseqüente removidos. Todos os dados que
permaneceram foram analisados pelo programa GraphPad Prism (version 5.00). As diferenças entre as
situações para todos os parametros foram analisadas pelo teste “t de student”. O teste de Pearson foi
aplicado para verificar possiveis correlações entre as variaveis. O nível de significância fixado foi de
p<0,05.
4.2 Modelo Experimental
Cultura de células - Adipócitos 3T3-L1
As células 3T3-L1 foram obtidas a partir da American Type Culture Collection e congeladas
em nitrogênio líquido até o dia de uso quando foram então descongeladas à temperatura ambiente. As
células foram mantidas em meio de crescimento contendo os seguintes constituintes: meio Dulbecco´s
Eagle modificado (DMEM) com 25mM de glicose, 1,0mM de piruvato, 4,02 de mM L-alanylglutamina e 10% de soro fetal bovino (Sigma) e cultivadas a 37°C em atmosfera umidificada com 5%
CO2 / 95% ar. O meio de cultura foi trocado a cada quatro dias no máximo até a confluência das
células quando então foi feita a diferenciação destas células utilizando-se, por quatro dias, meio de
diferenciação contendo 0,25µM de dexametasona, 0,5mM de 3-isobutyl-1-methyl-xanthine, 5µg/mL
de insulina (Sigma), penicilina e estreptomicina (1mL para cada 100mL de meio). Após os quatro dias
com o meio de diferenciação, as células foram cultivadas por dez dias em meio de alimentação
contendo DMEM modificado e 5µg/mL insulina.
No décimo dia da diferenciação os adipócitos foram mantidos por 24 horas em meio contendo
0,5% de soro fetal bovino. Após este período os adipócitos foram tratados com meio de incubação
livre de soro fetal bovino acrescido de LPS (100ng/mL), LPS associado à Adiponectina (50 ng/mL),
LPS associado à IL-10 (5 ng/mL) ou LPS associado à Adiponectina mais IL-10 por 24 horas. O meio
24
de incubação e as células foram coletados após 24h após os tratamentos, para análise precisa da
resposta anti-inflamatória.
As doses foram padronizadas em nosso laboratório, e escolhemos a melhor dose frente ao
estímulo inflamatório com LPS. As seguintes doses foram testadas: Adiponectina (10, 50 e
100ng/mL), e IL-10 (5, 10 e 20ng/mL).
Experimentos e coleta de amostras das células
Para determinação da concentração de IL-6 no meio de cultura, o sobrenadante foi coletado e
estocado a –80ºC para posterior análise da adipocina com kit de ELISA comercial (R&D System®).
Western blotting - IL-6R, TLR-2, TLR-4, MyD88 e TRAF6.
As células foram colocadas em 0,5 ml de tampão específico para extratos totais, o tampão de
extração para extrato total foi composto por: Trizma base 100 mM pH 7.5, EDTA 10 mM, SDS 10%,
fluoreto de sódio 100 mM, pirofosfato de sódio 10 mM, ortovanadato de sódio 10 mM. O tampão foi
preparado no dia do experimento e aquecido em banho maria fervente.
As células 3T3-L1 foram rapidamente homogeneizadas com tampão específico usando-se uma
seringa e agulha de calibre 23 gauge. O homogenato foi em seguida fervido por 10 minutos e
centrifugado por 40 minutos a 12000 rpm a 4°C. O sobrenadante foi mantido em gelo e o teor de
proteínas totais determinado pelo método de Bradford (1976).
As amostras foram adicionadas, na proporção de 4:1, do tampão de Laemmli (azul de
bromofenol 0,01%, fosfato de sódio 50mM, glicerol 25%, SDS 1%) contendo 200mM de DTT. O
volume de 100 g de proteína foi submetido à eletroforese em gel de poliacrilamida desnaturante a
10% e submetido à eletroforese. Após separação eletroforética, as amostras foram transferidas para
membrana de nitrocelulose por 2 horas à temperatura ambiente (ou overnight a 4 C) e a membrana foi
25
então bloqueada por 2 horas em 15 ml de solução bloqueadora, composta de solução basal (Trizma
base 10 mM, NaCl 150 mM, Tween 20 50 l/ml) contendo 5% de leite desnaturado. Em seguida, foi
feita a incubação com o anticorpo primário específico, overnight em temperatura ambiente, com o
anticorpo dissolvido em solução basal e BSA 1%. A seguir, a membrana foi incubada por uma hora
com anticorpo secundário associado a peroxidase. O anticorpo secundário constituiu-se sempre de
uma anti-imunoglobulina dirigida contra o animal produtor de anticorpo primário. Após algumas
lavagens com solução basal, a membrana foi revelada por quimioluminescência após adição do
reagente de revelação (ECL da Amersham) e então a membrana foi exposta a filme de raio X. As
bandas de interesse foram identificadas pelo seu padrão de migração eletroforética, por comparação
com padrões de Mr conhecidos e quantificadas por densitometria, utilizando-se o programa Scion
Image.
Ensaio da interação NF-κB-DNA por imunoensaio.
Extração das proteínas nucleares: As células adiposas 3T3-L1 foram coletadas em 0,5 mL de
tampão de extração nuclear. As proteínas nucleares foram extraídas em duas etapas, de acordo com as
instruções do fabricante Panomics (Santa clara, CA). Após esta etapa, a suspensão foi rapidamente
centrifugada para precipitação dos núcleos. Em seguida, o sedimento foi ressuspenso em 60 µL de
tampão C (Hepes 20mM; MgCL2 1,5mM; DTT 0,5mM; PMSF 0,2mM; NaCL 420mM; glicerol 25%)
e incubados durante 20 minutos em gelo. Após este intervalo, as amostras foram centrifugadas (14.000
rpm) e a quantidade de proteína total determinada pelo método de BSA.
Ativação do NF B(p50 e 65): Amostras provenientes da extração das proteínas nucleares (510 µg/proteína por poço) foram plaqueadas em placas com 96 poços contendo sítios de ligação
específicos para moléculas ativadas NF Bp50 (EK 1110, Panomics, Inc.) e 65 (EK1120, Panomics,
Inc.) (oligonucleotídeos biotinilados). Esses oligonucleotídeos foram então imobilizados através da
adição de streptavidin coated. Desta forma, NF
Bp50 e 65 ligados ao oligonucleotídeo foram
detectados por anticorpo direto contra essas moléculas e após adição de anticorpo secundário
26
conjugado com peroxidase (horseradish), o que provera sensibilidade colorimétrica. Os níveis dos
complexos DNA – proteínas formadas foram quantificados por espectrofotometria a 450 nm.
Análise estatística do modelo experimental
A análise estatística foi realizada para todos os valores mensurados no estudo. A análise
comparativa dos dados quantitativos foi apresentada utilizando o ANOVA de um caminho, seguido de
post-teste de Tukey. Quando necessário utilizamos o teste “t de student“ não pareado. Para todas as
análises o valor de p menor que 0,05 foi considerado estatisticamente significante.
27
5. Resultados
Os resultados desta tese estão apresentados na forma de 3 trabalhos científicos:
Visceral fat decreased by long-term interdisciplinary lifestyle therapy correlated positively with
interleukin-6 and tumor necrosis factor-alpha and negatively with adiponectin levels in obese
adolescents. Lira FS, Rosa JC, Dos Santos RV, Venancio DP, Carnier J, Sanches PD, do Nascimento
CM, de Piano A, Tock L, Tufik S, de Mello MT, Dâmaso AR, Oyama LM. Metabolism. 2011
Mar;60(3):359-65. Epub 2010 Mar 31.
Decreases endotoxin levels and improves insulin resistance in obese adolescents after long-term
interdisciplinary therapy. Lira FS, Rosa JC, Pimentel GD, Santos RV, Carnier J, Sanches PD, do
Nascimento CM, de Piano A, Tock L, Tufik S, de Mello MT, Oyama LM, Dâmaso AR. (submetido).
Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-κB pathway in
3T3-L1 adipocytes. Lira FS; Rosa JC; Pimentel GD; Seelaender M; Damaso AR, Oyama LM and
Oller do Nascimento C. (submetido).
28
5.1 Manuscrito 1
Visceral fat decreased by long-term interdisciplinary lifestyle therapy correlated positively with
interleukin-6 and tumor necrosis factor-alpha and negatively with adiponectin levels in obese
adolescents. Lira FS, Rosa JC, Dos Santos RV, Venancio DP, Carnier J, Sanches PD, do Nascimento
CM, de Piano A, Tock L, Tufik S, de Mello MT, Dâmaso AR, Oyama LM. Metabolism. 2011
Mar;60(3):359-65. Epub 2010 Mar 31.
This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
Author's personal copy
Available online at www.sciencedirect.com
Metabolism Clinical and Experimental 60 (2011) 359 – 365
www.metabolismjournal.com
Visceral fat decreased by long-term interdisciplinary lifestyle therapy
correlated positively with interleukin-6 and tumor necrosis factor–α and
negatively with adiponectin levels in obese adolescents
Fábio Santos Liraa,⁎, Jose Cesar Rosaa , Ronaldo Vagner dos Santosb , Daniel Paulino Venancioc ,
June Carniera , Priscila de Lima Sanchesa , Claudia Maria Oller do Nascimentoa,d , Aline de Pianoa ,
Lian Tocka , Sergio Tufikc , Marco Túlio de Melloa,c , Ana R. Dâmasoa,b,⁎, Lila Missae Oyamaa,b,⁎
a
Postgraduate Program of Nutrition, Federal University of São Paulo–UNIFESP, São Paulo/SP 04020-060, Brazil
b
Department of Biosciences, Federal University of São Paulo–UNIFESP, São Paulo/SP 04020-060, Brazil
c
Department of Psychobiology, Federal University of São Paulo–UNIFESP, São Paulo/SP 04020-060, Brazil
d
Department of Physiology, Federal University of São Paulo–UNIFESP, São Paulo/SP 04020-060, Brazil
Received 25 November 2009; accepted 16 February 2010
Abstract
The purpose of this study was to assess the level of cytokine expression in correlation with visceral and subcutaneous fat in obese
adolescents admitted to long-term interdisciplinary weight loss therapy. The study was a longitudinal clinical intervention of interdisciplinary
therapy. Adolescents (18, aged 15-19 years) with body mass indexes greater than the 95th percentile were admitted and evaluated at baseline
and again after 1 year of interdisciplinary therapy. Visceral and subcutaneous fat was analyzed by ultrasonography. Blood samples were
collected to analyze tumor necrosis factor–α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and adiponectin concentrations that were
measured by enzyme-linked immunosorbent assay. The most important finding in the present investigation is that the long-term
interdisciplinary lifestyle therapy decreased visceral fat. Positive correlations between IL-6 levels and visceral fat (r = 0.42, P b .02) and
TNF-α levels and visceral fat (r = 0.40, P b .05) were observed. Negative correlations between TNF-α levels and subcutaneous fat (r =
−0.46, P b .01) and adiponectin levels and subcutaneous fat (r = −0.43, P b .03) were also observed. In addition, we found a positive
correlation between TNF-α levels and the visceral to subcutaneous fat ratio (r = 0.42, P b .02) and a negative correlation between adiponectin
level and the visceral to subcutaneous fat ratio (r = −0.69, P b .001). Despite the limitation of sample size, our results indicate that the
observed massive weight loss (mainly visceral fat) was highly correlated with a decreased inflammatory state, suggesting that the
interdisciplinary therapy was effective in decreasing inflammatory markers.
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
Obesity is a heterogeneous condition with respect to the
regional distribution of fat: visceral obesity refers to fat
accumulation within omental and mesenteric fat depots,
whereas peripheral obesity generally refers to subcutaneous
fat accumulation [1]. The functional differences between
⁎ Corresponding authors. Rua Marselhesa no. 535, Vila Clementino,
São Paulo/SP 04020-060, Brazil. Tel.: +55 11 5572 0177; fax: +55 11
55720177.
E-mail addresses: fabioslira@gmail.com (F.S. Lira),
ana.damaso@unifesp.br (A.R. Dâmaso), lmoyama@gmail.com
(L.M. Oyama).
0026-0495/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.metabol.2010.02.017
visceral and subcutaneous adipocytes may be related to their
anatomical location. Visceral adipose tissue and its adipose
tissue resident macrophages produce many proinflammatory
cytokines, such as tumor necrosis factor–α (TNF-α) and
interleukin-6 (IL-6), and less adiponectin and interleukin-10
(IL-10) [2-4]. These cytokine changes induce insulin
resistance and play a major role in the pathogenesis of
endothelial dysfunction and subsequent atherosclerosis [5].
Such differences are also reflected in the contrasting roles
that visceral and subcutaneous adipose tissues play in the
pathogenesis of obesity-related cardiometabolic problems in
both lean and obese individuals [6]. Thorne et al [7] reported
that the removal of visceral adipose tissue by omentectomy
resulted in decreased glucose and insulin levels in humans,
360
F.S. Lira et al. / Metabolism Clinical and Experimental 60 (2011) 359–365
whereas the removal of subcutaneous adipose tissue by
liposuction did not always result in improvements in glucose
metabolism or lipid levels [8,9].
Different strategies are adopted with the intention of
restoring the inflammatory state in obese subjects. Several
studies have shown that diet-induced weight loss significantly
decreases the levels of markers of inflammation, such as TNFα, IL-6, and C-reactive protein [10,11]. On the other hand,
sustained aerobic exercise is recommended both for the
prevention and treatment of several chronic diseases [12-14].
Moreover, endurance training seems to induce an increase in the
secretion of anti-inflammatory cytokines by adipose tissue [15].
Recently, our group has shown that long-term interdisciplinary lifestyle therapy is effective in controlling the
psychologic aspects, nonalcoholic fatty liver disease, body
composition, bone mineral density, and hormonal alterations
[16-21] commonly observed in obese patients. However,
despite these promising results, few studies have addressed
the effects of long-term multidisciplinary intervention on
pro- and anti-inflammatory cytokine levels.
Thus, the aim of this study was to assess the effect of longterm multidisciplinary intervention on visceral and subcutaneous fat loss and cytokine levels in obese adolescents.
2. Material and methods
2.1. Population
Adolescents were invited to participate in 1-year-long
multidisciplinary therapy to promote changes in their
sedentary lifestyle and nutritional habits. The basic requirements for participation were motivation and high attendance
at the therapy sessions.
Fifty-four adolescents were invited to participate in a
1-year-long Interdisciplinary Obesity Program of the Federal
University of São Paulo-Paulista Medical School to promote
changes in their sedentary lifestyle and nutritional habits.
The basic requirements for participation were motivation and
high attendance at the therapy sessions. Thirty-nine adolescents continued until the end of the therapy. For the present
study, 18 obese adolescents who lost more than 5% fat mass
(range sample: 5.4% at 22.50% fat mass) were selected.
These adolescents were evaluated at baseline and after
long-term (1 year) weight loss intervention. This study was
carried out in accordance with the principles of the
Declaration of Helsinki and was formally approved by the
Institutional Ethical Committee (#0135/04). Informed consent was obtained from all subjects and/or their parents, and
agreement of the adolescents and their families to participate
was voluntary.
The ages of the 18 participants ranged from 15 to 19 years
(16.6 ± 1.67 years), and the average body mass index (BMI)
was 37.03 ± 3.78 kg/m2 (7 boys and 11 girls). All
participants met the inclusion criteria of postpubertal stage
V based on the Tanner stages [22] and of obesity (BMI N95th
percentile) according to the Centers for Disease Control and
Prevention reference growth charts (2004). Noninclusion
criteria included identifiable genetic, metabolic, or endocrine
disease or previous drug utilization [23].
2.2. Study protocol and medical screening
Subjects were medically screened, their pubertal stage
was assessed, and their anthropometric measures were
recorded (ie, height, weight, BMI, and body composition).
The endocrinologist completed a clinical interview including
questions to determine eligibility based on inclusion and
exclusion criteria. Blood samples were collected and
analyzed, and an ultrasound (US) was performed. The
procedures were scheduled for the same time of day for all
subjects to remove any influence of diurnal variations.
Thereafter, obese adolescents started the interdisciplinary
weight loss program (described in a later section).
2.3. Anthropometric measurements and body composition
Subjects were weighed on a Filizola scale (São Paulo-SP,
Brazil) while wearing light clothing and no shoes, and
weight was recorded to the nearest 0.1 kg. Height was
measured using a wall-mounted stadiometer (Sanny, São
Paulo-SP, Brazil; model ES 2030) to the nearest 0.5 cm.
Body mass index was calculated as body weight divided by
height squared. Body composition was estimated by
plethysmography using the BOD POD body composition
system (version 1.69; Life Measurement Instruments,
Concord, CA). This is the most advanced technique for
assessing body composition available today. The patented air
displacement plethysmography used by the BOD POD and
PEA POD is similar in principle to hydrostatic (or
“underwater”) weighing. The obvious difference is that air
is more convenient and comfortable than water, such that air
displacement plethysmography provides a much simpler and
safer testing environment, better reliability, and significantly
improved repeatability and accuracy [24].
2.4. Serum analysis
Blood samples were collected in the outpatient clinic at
around 8:00 AM after an overnight fast. Cytokine (TNF-α, IL6, and IL-10) and adiponectin concentrations were measured
using commercially available enzyme-linked immunosorbent
assay kits from eBioscience (San Diego, CA) and R&D
Systems (Minneapolis, MN) according to the manufacturer's
manual.
2.5. Visceral and subcutaneous adiposity measurements
All abdominal ultrasonographic procedures and the
measurements of visceral and subcutaneous fat tissue were
performed by the same physician, who was blinded to the
subjects' assignment group. This physician was a specialist
in imaging diagnostics using a 3.5-MHz multifrequency
transducer (broadband), which reduces the risk of misclassification. The intraexamination coefficient of variation for
US was 0.8%.
361
Table 1
Effect of long-term multidisciplinary lifestyle therapy on body fat and cytokines levels (n = 18)
Age (y)
Body weight (kg)
BMI (kg/m2)
Percentage fat
Fat mass (kg)
Fat-free mass (kg)
VO2max (mL/[kg min])
Visceral fat (cm)
Subcutaneous fat (cm)
Visceral to subcutaneous ratio
Adiponectin (ng/mL)
TNF-α (pg/mL)
IL-6 (pg/mL)
IL-10 (pg/mL)
IL-10/TNF-α ratio
Before
After
% Change
P value
15±1.73
95.03 ± 13.06
34.99 ± 4.00
47.50 ± 6.99
49.6 ± 10.1
55.8 ± 7.49
27.5 ± 6.87
4.19 ± 1.16
3.61 ± 0.47
1.17 ± 0.34
9.70 ± 2.13
22.76 ± 29.67
49.32 ± 34.91
7.86 ± 9.02
0.40 ± 0.37
16 ±0.63
85.59±11.58
31.71 ± 4.00
35.81 ± 9.60
32.9 ± 12.3
58.1 ± 7.71
30.3 ± 8.10
2.13 ± 0.84
2.85 ± 0.77
0.80±0.38
12.98 ± 2.17
20.88 ± 29.22
34.53 ± 21.46
14.74 ± 22.95
0.58 ± 0.65
1 ± 0.51
−11%
−9.3%
−24%
−33%
+4%
+10%
−49%
−21%
−31%
+33%
−8%
−29%
+87%
+43%
.11
.05
.05
b.001
b.001
.19
.01
b.001
.001
.006
b.001
.59
.13
.16
.28
Results are expressed as mean value ± SD.
Ultrasound measurements of intraabdominal (“visceral”)
and subcutaneous fat were taken. The US-determined subcutaneous fat was defined as the distance between the skin
and external face of the recto abdominis muscle, and visceral
fat was defined as the distance between the internal face of
the same muscle and the anterior wall of the aorta. Cutoff
points to define visceral obesity by ultrasonographic
parameters were based on previous methodological descriptions by Ribeiro-Filho et al [25].
2.6. Clinical intervention
2.6.1. Dietary program
Energy intake was set at the levels recommended by the
dietary reference intake for subjects with low levels of
physical activity of the same age and sex following a
balanced diet [26]. No drugs or antioxidants were recommended. Once a week, adolescents had a dietetics lesson
providing information on the following: the food pyramid,
diet record assessment, weight loss diets and miracle diets,
food labels, dietetics, fat-free and low-calorie foods, fats
(kinds, sources, and substitute foods), fast food calories and
nutritional composition, good nutritional choices in special
occasions, healthy sandwiches, shakes and products to
promote weight loss, functional foods, and decisions on
food choices. All patients received individual nutritional
consultation during the intervention program.
A 3-day dietary record was collected at the beginning of
the study and again at 12 months into the program. Because
most obese people underreport their food consumption, each
adolescent was asked to record their diet with the help of
their parents [27]. The degree of underreporting may be
substantial; however, this is a validated method to assess
dietary consumption [28]. Portions were measured in terms
of familiar volumes and sizes. The dietician taught
the parents and the adolescents how to record food
consumption. These dietary data were transferred to a
computer by the same dietician, and the nutrient composition
was analyzed by a PC program developed at the Federal
University of São Paulo–Paulista Medical School (Nutwin
software for Windows, 1.5 version, 2002) that used data
from Western and local food tables. In addition, the parents
were encouraged by a dietitian to call if they needed
extra information.
2.6.2. Exercise program
During the 1-year interdisciplinary intervention period,
adolescents followed a personalized aerobic training program that included a 60-minute session completed 3 times a
week (180 min/wk) under the supervision of a sports
therapist. Each program was developed according to the
results of an initial oxygen uptake test for aerobic exercises
(cycle ergometer and treadmill). The intensity was set at a
workload corresponding to a ventilatory threshold of 1
(50%-70% of oxygen uptake test). Adolescents were under
heart rate monitoring during the aerobic sessions. The
exercise program was based on the 2009 recommendations
given by the American College of Sports Medicine [29].
Fig. 1. Correlation of IL-6 levels and visceral fat.
362
Fig. 2. Correlation of TNF-α levels and visceral fat.
2.6.3. Psychologic intervention
Diagnoses of common psychologic problems associated
with obesity, such as depression, disturbances of body
image, anxiety, and decreased self-esteem, were established
by validated questionnaires. During the interdisciplinary
intervention, the adolescents had weekly psychologic
support group sessions. During these sessions, the adolescents discussed the following topics: body image; alimentary
disorders including bulimia, anorexia nervosa, and binge
eating; the signs, symptoms, and health consequences
of these disorders; the relationship between feelings and
food; and family problems such as alcoholism, among other
topics. Individual psychologic therapy was recommended if
individuals were found to have nutritional or behavioral
problems [19].
Fig. 4. Correlation of TNF-α levels and subcutaneous fat.
differences between groups for all parameters were assessed
by a paired Student t test. The Pearson correlation coefficient
was calculated to assess the relationship between variables.
The analysis was carried out with the significance level set
at P b .05.
3. Results
The data distribution was checked by the Bartlett test for
equal variances, and the data are reported as means ± SD.
Statistical outliers within each treatment group were
identified using a Grubbs test (GraphPad Software, San
Diego, CA) and subsequently removed. All remaining data
were analyzed by GraphPad Prism (version 5.00). The
Long-term therapy was effective in reducing body weight
(−11%), BMI (−9.3%), percentage fat (−24%; range before,
32.3%-58.4% and after, 10.2%-55.5%), visceral fat (−49%;
range before, 2.2-6.5 cm and after, 0.8-3.7 cm), and
subcutaneous fat (−21%; range before, 2.6-4.5 cm and
after, 1.6-3.9 cm). Long-term therapy increased fat-free mass
(+4%; range before, 44.1-71.2 kg and after, 44.2-74.9 kg)
and VO2max (+10%; range before, 21.2-34.1 mL/[kg min]
and after, 22.7-38.4 mL/[kg min]). A reduction in TNF-α
(−8%; range before, 4.13-109.45 pg/mL and after, 6.31121.26 pg/mL) and IL-6 (−29%; range before, 9.47-111.41
pg/mL and after, 16.56-96.45 pg/mL) was observed after
long-term therapy, as was an increase in IL-10 (+87%; range
before, 0.16-38.59 pg/mL and after, 0.16-67.13 pg/mL). On
the other hand, the ratio of IL-10 to TNF-α (+43%; range
Fig. 3. Correlation of adiponectin levels and visceral fat.
Fig. 5. Correlation of TNF-α levels and visceral to subcutaneous ratio.
2.7. Statistical analysis
363
before or after therapy. However, there was a tendency for
proinflammatory cytokines levels to be higher in the boys
than in the girls. The visceral fat depot and the ratio of
visceral to subcutaneous fat pads were higher in boys than
girls, and the subcutaneous fat pad was lower in boys than
girls (data not shown).
4. Discussion
Fig. 6. Correlation of TNF-α levels and visceral to subcutaneous ratio.
before, 0.011-1.10 and after, 0.019-2.06 ratio) was increased; but the difference was not statistically significant
(P N .05) (Table 1). Indeed, adiponectin levels were
increased (+33%; range before, 6.12-13.86 μg/mL and
after, 8.43-15.99 μg/mL; P b .0001). These results are
shown in Table 1.
The most important findings in the present investigation
are the observed positive correlation between IL-6 levels
with visceral fat (r = 0.42, P b .02, Fig. 1), TNF-α levels with
visceral fat (r = 0.40, P b .05, Fig. 2), and the negative
correlations between TNF-α and adiponectin levels and
TNF-α and subcutaneous fat (r = −0.46, P b .01, Fig. 3; r =
−0.43, P b .03, Fig. 4). In addition, there was a positive
correlation between TNF-α levels and the ratio of visceral to
subcutaneous fat (r = 0.42, P b .02, Fig. 5) and a negative
correlation between adiponectin levels and the ratio of
visceral to subcutaneous fat (r = −0.69, P b .001, Fig. 6).
Based on the theoretical equation, adiponectin concentration
was a predictive factor of the ratio of visceral to
subcutaneous fat (equation: y = −4.445x + 15.07, r =
0.4809, Fig. 7).
No statistical differences were observed in TNF-α, IL-6,
IL-10, or adiponectin levels between boys and girls either
Fig. 7. Theoretical equation: adiponectin concentration is a predictive factor
to visceral to subcutaneous ratio.
In the present study, we examined the relationship
between circulating IL-6, TNF-α, IL-10, and adiponectin
concentrations and direct measures of visceral and subcutaneous adiposity. The results indicate that IL-6 and TNF-α
levels were positively correlated with visceral fat and
negatively correlated with adiponectin levels. Previous
studies have shown that visceral fat in obese adolescents
correlates with fatty liver, neuroendocrine alterations, and
insulin resistance [17,30]. Our data are consistent with those
of Park et al [31], which showed that circulating IL-6 levels
were significantly associated with visceral adiposity. These
data are also consistent with Fontana et al [32], who reported
in massively obese subjects that plasma IL-6 concentrations
were much higher in the portal vein than in systemic arterial
blood; these results suggest that visceral fat is an important
source of IL-6 production in obese people. Many studies
have shown that visceral obesity is associated with a higher
expression of cytokines than subcutaneous obesity [6,33,34].
Cao et al [1] found a significant increase in TNF-α
expression in omental adipose tissue as compared with
subcutaneous adipose tissue in obese individuals. These data
show that omental TNF-α expression is highly correlated
with insulin sensitivity, and the authors suggest that visceral
fat is associated with a decrease in insulin sensitivity that
could lead to an increased risk of cardiovascular disease. Van
der Poorten et al [33] reported that central obesity, in which
fat mass is predominantly intraabdominal, is more strongly
associated with insulin resistance, dyslipidemia, and atherosclerosis than peripheral obesity, in which fat is predominantly gluteofemoral.
In the present study, long-term interdisciplinary lifestyle
therapy was effective in decreasing visceral fat (49%) and
increasing adiponectin levels (33%). Despite the lack of a
statistically significant difference in the mean levels of TNFα, IL-6, and IL-10 before and after therapy, there was
an observed decrease of 8% and 29% and an increase of
87%, respectively.
A previous study [16] demonstrated that interdisciplinary
lifestyle therapy was efficient in restoring the parameters
associated with reduced visceral fat in obese adolescents with
metabolic syndrome and insulin resistance. Our data suggest
that visceral fat is correlated with a marked inflammatory
state, whereas subcutaneous fat showed the opposite effect. In
fact, TNF-α was significantly negatively correlated with
subcutaneous fat. In addition, when the ratio of visceral to
subcutaneous fat was assessed in relation to cytokine levels,
364
we observed a positive correlation with TNF-α levels and a
negative correlation with adiponectin levels.
Klein et al [8] stated that abdominal liposuction should
not, by itself, be considered a clinical therapy for obesity.
The aspiration of large amounts of subcutaneous abdominal
fat in women with abdominal obesity may have cosmetic
benefits; but the procedure does not significantly improve
insulin sensitivity in the liver, skeletal muscle, or adipose
tissue; serum concentrations of inflammatory markers; or
other risk factors for coronary heart disease. However, Porter
et al [35] demonstrated that, whereas abdominal adiposity
(visceral and subcutaneous fat) is associated with a higher
absolute risk of metabolic and cardiovascular disease,
subcutaneous abdominal fat is not associated with a linear
increase in the prevalence of risk factors among the obese.
Indeed, subcutaneous adipose tissue may actually be a
protective fat depot in obese individuals in the case of high
triglycerides. In their review, Chaston and Dixon [36] related
that preferential loss of visceral fat compared with
subcutaneous fat is greatest with modest weight loss; the
effect is attenuated, and possibly lost completely, with
increasing weight loss.
It has been hypothesized that a reduction in visceral fat
without substantial weight loss is effective in ameliorating
obesity-related comorbidity. In this sense, the “portal
hypothesis” suggests that the proximity of visceral fat to
the liver increases the fatty acid, hormone, and cytokine
delivery from adipose tissue to the liver, exacerbating
hepatic insulin resistance and increasing glucose output [37].
An important observation in the present research was the
beneficial effects of lifestyle intervention on visceral fat
depot and inflammatory state in obese adolescents. Many
studies have demonstrated benefits from aerobic training and
diet leading to an anti-inflammatory state in obese rats and
human models [11-13,38]. Classically, aerobic exercise
training is adopted as a weight loss program, inducing an
increase in the mobilization of fatty acid from adipose tissue
and leading principally to fat oxidation by skeletal muscle
that contributes to obesity control [13,15,29,38].
Adiponectin may also have antiatherogenic and antiinflammatory properties, and high circulating levels have
been related to a lower risk of coronary heart disease [4]. A
transcriptional mechanism leading to decreased adiponectin
plasma levels in obese women has been previously
demonstrated. In addition, low levels of adiponectin have
been associated with high levels of C-reactive protein
and IL-6 [39]. However, Borges et al [40] found that a
weight loss greater than 5% improved inflammatory status
in adult women by decreasing C-reactive protein and
insulin resistance, regardless of changes in adiponectin or
TNF-α levels.
In the present study, it was verified that the theoretical
equation from the correlation between adiponectin and
the visceral to subcutaneous ratio was useful in predicting
the visceral to subcutaneous ratio from the adiponectin
serum concentration.
Although the small number of participants could be a
limitation of our study, the results contribute to the
understanding of the mechanisms linking obesity and
cytokines to the inflammatory state and the importance of
lifestyle interdisciplinary therapy intervention as clinical
practice for obesity treatment.
Although the small numbers of boys and girls made it
difficult to explore our data for sex differences, girls showed
lower levels of TNF-α and a lower visceral to subcutaneous
ratio when compared with boys (before and after experimental protocol). These results corroborate the study carried
out by Cartier et al [41], which showed that premenopausal
women had lower TNF-α levels when compared with men,
thus reinforcing the idea that visceral fat greatly contributes
to an inflammatory state in obese patients.
Borges et al [40] and Cartier et al [41], using a large
number of subjects and a long-term follow-up, investigated
the roles of other inflammatory biomarkers, such as C-reactive
protein, to better elucidate the beneficial effects of the lifestyle
interdisciplinary therapy in adolescent obese patients.
Future studies are needed to better understand pathway
contributes for high cytokines levels and relation with fat
depot. In addition, these findings should be confirmed in
other populations to prevent the development of many
obesity comorbidities at a young age.
In summary, the present study demonstrated that
decreasing the visceral fat in obese adolescents showed an
anti-inflammatory effect and that this reduction was
accompanied by a decreased proinflammatory and an
increased anti-inflammatory state.
Acknowledgment
We would like to thank the patients that participated of
the study and the following funding sources: AFIP, FAPESP
2006/00684-3, FAPESP 2008/53069-0, FAPESP (CEPID/
Sleep 9814303-3 S.T) CNPq, CAPES, CENESP, FADA,
and UNIFESP-EPM, supported by the CEPE-GEO Interdisciplinary Obesity Intervention Program.
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37
5.2 Manuscrito 2
interdisciplinary therapy. Lira FS, Rosa JC, Pimentel GD, Santos RV, Carnier J, Sanches PD, do
Nascimento CM, de Piano A, Tock L, Tufik S, de Mello MT, Oyama LM, Dâmaso AR (submetido).
38
interdisciplinary therapy
Short title: Insulin resistance in obese adolescents.
Fábio Santos Lira1; Jose Cesar Rosa1; Gustavo Duarte Pimentel1; Ronaldo Vagner Santos2; June
Carnier1; Priscila de Lima Sanches1; Aline de Piano1; Lian Tock1; Sergio Tufik3; Marco Túlio de
Mello1,3; Marília Seelaender4; Claudia Maria Oller do Nascimento1; Lila Missae Oyama1,2; Ana R.
Dâmaso1,2
Departamento de Fisiologia¹, Departamento de Biociências2, Departamento de Psicobiologia3 da
Universidade Federal de São Paulo – UNIFESP. Cancer Metabolism Research Group, Institute of
Biomedical Sciences, University of São Paulo, São Paulo, Brazil4.
Corresponding author:
Fabio S. Lira, Ana R. Damaso and Lila M. Oyama
Rua Marselhesa nº 535 – Vila Clementino- São Paulo/SP – Brazil
Postal Code: 04020-060
Phone: (5511) 5572-0177 / Fax: (5511) 55720177
E-mail:fabioslira@gmail.com, ana.damaso@unifesp.br and lmoyama@gmail.com
39
Abstract
Objective: The purpose of the present study was to assess the dietary fat intake, glucose, insulin,
Homeostasis model assessment for insulin resistance HOMA, and endotoxin level and correlate them
with adipokine serum concentrations in obese adolescents who had been admitted to long-term
interdisciplinary weight-loss therapy. Design: The present study was a longitudinal clinical
intervention of interdisciplinary therapy. Adolescents (n=18, aged 15-19 y) with a body mass index >
95th percentile were admitted and evaluated at baseline and again after 1 year of interdisciplinary
therapy. We collected blood samples, and IL-6, adiponectin, and endotoxin concentrations were
measured by ELISA. Food intake was measured using 3-day diet records. In addition, we assessed
glucose and insulin levels as well as the homeostasis model assessment for insulin resistance (HOMAIR). Results: The most important finding from the present investigation was that the long-term
interdisciplinary lifestyle therapy decreased dietary fat intake and endotoxin levels and improved
HOMA-IR. We observed positive correlations between dietary fat intake and endotoxin levels, insulin
levels, and the HOMA-IR (p<0.05). In addition, endotoxin levels showed positive correlations with
IL-6 levels, insulin levels and the HOMA-IR (p<0.05). Interestingly, we observed a negative
correlation between serum adiponectin and both dietary fat intake and endotoxin levels (p<0.05).
Conclusions: The present results indicate that reduced dietary fat intake and endotoxin level was
highly correlated with a decreased pro-inflammatory state and an improvement in HOMA-IR. In
addition, this benefits effect may be associated with an increased adiponectin level, which suggests
that the interdisciplinary therapy was effective in decreasing inflammatory markers.
Keywords: Obesity, cytokines, endotoxin, insulin resistance, interdisciplinary therapy
40
Introduction
Saturated fatty acid intake leads to inflammation, insulin resistance and a gain in body mass.
Systemic low-level inflammation has been suggested to be both a cause and a consequence of
comorbidities associated with obesity [1]. Recently, studies have proposed that the microbial ecology
in humans could be an important factor in determining energy homeostasis (i.e., obesity, diabetes, and
fatty liver) [2-4]. Lipopolysaccharide (LPS), which is also referred to as endotoxin, has been
implicated as a potent inducer of inflammation, and LPS increases TNF-α and IL-6 and reduces
adiponectin levels. In normal circumstances, only small amounts of endotoxin cross from the intestinal
lumen into systemic circulation, and the absorbed endotoxin is rapidly removed by monocytes,
particularly resident Kupffer cells within the liver. Emerging evidence has indicated that chronic
elevation of serum endotoxin levels may play a role in insulin-resistant states and obesity [3,4].
Interestingly, Pedersen [5] described how physical inactivity leads to the accumulation of
visceral fat and the activation of a network of inflammatory pathways that promote the development of
insulin resistance, atherosclerosis, obesity, neurodegeneration, and tumor growth (i.e., the
development of diseases belonging to the “diseasome of physical inactivity”).
Recently, our group has shown that long-term interdisciplinary lifestyle therapy is effective in
controlling the psychological and physiological alterations that are commonly observed in obese
patients. Despite promising results, few studies have addressed the effects of long-term
interdisciplinary intervention on dietary fat intake and endotoxin levels and their correlation with
insulin resistance and adipokine levels.
Materials and Methods
Population
Fifty-four adolescents were invited to participate in a 1-year-long Interdisciplinary Obesity
Program at the Federal University of São Paulo- Paulista Medical School to promote changes in their
sedentary lifestyle and nutritional habits. The basic requirements for participation were motivation and
41
high attendance at the therapy sessions. Thirty-nine adolescents participated until the end of the
therapy. For the present study, we selected 18 obese adolescents who lost more than 5% fat mass (the
range was 5.4% to 22.5% fat mass).
Selected 18 obese adolescents were evaluated at baseline and after long-term (1 year) weight
loss intervention. The present study was conducted in accordance with the principles of the
Declaration of Helsinki and was formally approved by the Institutional Ethical Committee (#0135/04).
Informed consent was obtained from all subjects and/or their parents, and the agreement of the
adolescents and their families to participate was voluntary.
The ages of the 18 participants ranged from 15-19 years (16.6 ± 1.67 years), and the average
body mass index (BMI) was 37.98 ± 4.60 kg/m2 (7 boys and 11 girls). All participants met the
inclusion criteria of postpubertal Stage V, based on the Tanner stages [6], and of obesity (BMI > 95th
percentile) according to the Centers for Disease Control and Prevention (CDC) reference growth
charts. Noninclusion criteria included identifiable genetic, metabolic or endocrine disease or previous
drug utilization [7].
Study Protocol and Medical Screening
Subjects were medically screened, and we assessed their pubertal stage and recorded their
anthropometric measures (i.e., height, weight, BMI and body composition). The endocrinologist
completed a clinical interview, which included questions to determine eligibility based on inclusion
and exclusion criteria. Blood samples were collected and analyzed, and an ultrasound (US) was
performed. The procedures were scheduled for the same time of day for all subjects to remove any
influence of diurnal variations. After the initial screening, obese adolescents started the
interdisciplinary weight loss program (described in a later section).
Anthropometric measurements and Body Composition
42
Subjects were weighed on a Filizola scale while wearing light clothing and no shoes, and their
weight was recorded to the nearest 0.1 kg. Height was measured using a wall-mounted stadiometer
(Sanny, model ES 2030) to the nearest 0.5 cm. Body mass index was calculated as body weight (wt)
divided by height (h) squared (wt/ht2). Body composition was estimated by plethysmography using the
BOD POD body composition system (version 1.69, Life Measurement Instruments, Concord, CA),
which is the most advanced technique available for assessing body composition. The patented air
displacement plethysmography used by the BOD POD and PEA POD is similar in principle to
hydrostatic (or "underwater") weighing. The obvious difference between them is that air is more
convenient and comfortable than water, and air displacement plethysmography provides a much
simpler and safer testing environment, better reliability and significantly improved repeatability and
accuracy [8].
Serum analysis
Blood samples were collected in the outpatient clinic at approximately 0800 h after an
overnight fast. Adipokine (IL-6 and adiponectin) concentrations were measured using commercially
available ELISA kits from eBioscience, Inc. (San Diego, CA, USA) and R&D Systems (USA)
according to the manufacturer’s manuals.
Fasting insulin concentrations were determined using commercially available ELISA kits from
Millipore (Millipore Corporate Headquarters: 290 Concord Road, Billerica, MA 01821), and glucose
concentrations were determined by an enzymatic method (Labtest ®). Homeostasis model assessment
for insulin resistance (HOMA-IR) was calculated with assessed values of glucose and insulin.
Measurement of circulating endotoxin levels
Plasma endotoxin was assayed using a chromogenic limulus amebocyte lysate (LAL) test,
which is a quantitative test for Gram-negative bacterial endotoxin (Cambrex Corporation, 8830 Biggs
43
Ford Road,Walkersville – USA). Gram-negative bacterial endotoxin catalyzes the activation of a
proenzyme in the LAL, and the initial rate of activation is directly determined by the concentration of
endotoxin. The activated enzyme catalyzes the splitting of p-nitroaniline (pNA) from the colorless
substrate Ac-lle-Glu-Ala-Arg-pNA, and the released pNA was measured photometrically at 405–410
nm following termination of the reaction. The correlation between the absorbance and the endotoxin
concentration was linear in the range of 0.1–1.0 EU/ml. For the purposes of this study, all samples
were run in duplicate within the same plate; therefore, no interassay variability was observed in this
study.
To assess recovery of endotoxin within the assay, known concentrations of recombinant
endotoxin (0.25 and 1.00 EU/ml) were added to diluted plasma to determine whether the expected
concentration correlated with the actual observed value and whether there were any variations due to
reaction with plasma contents. Lyophilized endotoxin (E. coli origin) was used to generate a standard
curve with the chromogenic LAL test kit in accordance with the manufacturer's instructions.
Visceral and Subcutaneous Adiposity Measurements
All abdominal ultrasonographic procedures and measurements of visceral and subcutaneous
fat tissue were performed by the same physician who was blinded to the subjects’ assignment group.
This physician was a specialist in imaging diagnostics using a 3.5-MHz multifrequency transducer
(broad band), which reduces the risk of misclassification. The intra-examination coefficient of
variation for US was 0.8%.
We took US measurements of intra-abdominal (¨visceral¨) and subcutaneous fat. Ultrasounddetermined subcutaneous fat was defined as the distance between the skin and external face of the
recto abdominis muscle, and visceral fat was defined as the distance between the internal face of the
same muscle and the anterior wall of the aorta. Cutoff points to define visceral obesity by
ultrasonographic parameters were based on previous methodological descriptions by Ribeiro-Filho et
al. [10].
44
Clinical Intervention
Dietary Program
Energy intake was set at the levels recommended by the dietary reference intake for subjects
with low levels of physical activity of the same age and gender following a balanced diet [13]. No
drugs or antioxidants were recommended. Once a week, adolescents had a dietetics lesson, which
provided information on the food pyramid, diet record assessment, weight loss diets and miracle diets,
food labels, dietetics, fat-free and low-calorie foods, fats (kinds, sources and substitute foods), fast
food calories and nutritional composition, good nutritional choices in special occasions, healthy
sandwiches, shakes and products to promote weight loss, functional foods and decisions on food
choices. All patients received individual nutritional consultation during the intervention program. In
addition, a dietitian encouraged the parents to call if they needed extra information.
Exercise program
During the one-year interdisciplinary intervention period, adolescents followed a personalized
aerobic training program that included a 60-minute session completed three times each week (180minute/week) under the supervision of a sports therapist. Each program was developed according to
the results of an initial oxygen uptake test for aerobic exercises (cycle-ergometer and treadmill). The
intensity was set at a work load corresponding to a ventilatory threshold of 1 (50% to 70% of oxygen
uptake test). Adolescents were under heart-rate monitoring during the aerobic sessions. The exercise
program was based on the 2009 recommendations of the American College of Sports Medicine [11].
Psychological intervention
Diagnoses of common psychological problems associated with obesity, such as depression,
disturbances of body image, anxiety and decreased self-esteem, were established by validated
questionnaires. During the interdisciplinary intervention, the adolescents had weekly psychological
support group sessions. During these sessions, the adolescents discussed body image; alimentary
45
disorders, including bulimia, anorexia nervosa and binge eating; the signs, symptoms and health
consequences of these disorders; the relationship between feelings and food; and family problems,
such as alcoholism. Individual psychological therapy was recommended if individuals were found to
have nutritional or behavioral problems [12].
Statistical analysis
The data distribution was checked by Bartlett's test for equal variances, and the data are reported as
the mean ± SD. Statistical outliers within each treatment group were identified using Grubbs’ test
(GraphPad Software) and subsequently removed. All remaining data were analyzed by GraphPad Prism
(version 5.00). The differences between groups for all parameters were assessed by a paired Student’s t
test. The Pearson correlation coefficient was calculated to assess the relationship between variables,
and all analyses were carried out with the significance level set at p<0.05.
Results
Long-term therapy was effective in reducing body weight (-15%, p<0.001), BMI (- 15%,
p<0.001), percent fat (- 24%, p<0.001), and fat mass (-33%, p<0.001). These results are shown in
Table 1.
Characteristics of the food intake in obese adolescents are shown in Table 2. Energy intake
was reduced 38% (p<0.00001), carbohydrate intake was reduced 28% (p<0.002), protein intake was
reduced 43% (p<0.00004), and fat intake was reduced 47% (p<0.00002).
Glucose, insulin, and endotoxin levels as well as the homeostasis model assessment for insulin
resistance (HOMA-IR) are shown in Table 3. Glucose (the range before therapy was 4.38 – 5.66
µU/mL, and the range after therapy was 4.32 – 4.77 µU/mL, p<0.02), insulin (the range before therapy
was 7.10 – 23.3 µU/mL, and the range after therapy was 5.00 – 18.9 µU/mL, p<0.001), HOMA (the
range before therapy was 1.45 – 5.23 µU/mL, and the range after therapy was 1.01 – 3.87 µU/mL,
46
p<0.002), and endotoxin (the range before therapy was 0.112 – 0.394 in Log EU/mL, and the range
after therapy was 0.095 – 0.309 in Log EU/mL, p<0.0003) were reduced after therapy.
Pearson correlation analyses showed positive correlations between dietary fat intake and
endotoxin levels (r=0.36, p<0.01), dietary fat diet intake and insulin levels (r=0.38, p<0.05), and
dietary fat intake and HOMA (r=0.41, p<0.04). In addition, there was a negative correlation between
dietary fat intake and adiponectin (r= -0.42, p<0.01) (Figure 1A-D).
We also observed positive correlations between endotoxin and IL-6 levels (r=0.35, p<0.03),
endotoxin and insulin levels (r=0.36, p<0.05), and endotoxin levels and HOMA (r=0.35, p<0.05).
Interestingly, we observed a negative correlation between endotoxin and adiponectin levels (r=-0.28,
p<0.06) (Figure 2A-D).
Discussion
The present study showed that long-term therapy was effective in reducing dietary fat intake
and endotoxin levels. In addition, these data were positively correlated with improvements in insulin
resistance in obese adolescents.
Decreased endotoxin levels have been found with consumption of a low-fat diet compared
with a high-fat diet [13]. In addition, the type of fatty acid in the diet could have important effects on
endotoxinemia. Recently, several studies have shown that omega-3 ( -3) fatty acids, particularly
eicosapentaenoic acid (EPA), reduces endotoxin and pro-inflammatory cytokine concentrations
[14,15]. Moreover, Oz et al. [16] demonstrated that a diet rich in EPA, docosahexaenoic acid (DHA),
and prebiotic fructooligosaccharides (FOS) protects against LPS-induced systemic inflammatory
responses. In contrast to the present study, Al-Attas et al. [3] showed that a diet-controlled program in
diabetic individuals did not significantly decrease endotoxin levels compared with individuals who
only received insulin. Interestingly, herbs used in food dishes reduce the production of LPS and proinflammatory cytokines [17]. In the present study, we observed that interdisciplinary therapy was able
to decrease the fat intake, which was sufficient to reduce endotoxin concentrations and insulin
resistance.
47
The present study found a positive correlation between endotoxin and both pro-inflammatory
cytokines (especially, IL-6) and insulin resistance. After interdisciplinary therapy, endotoxinemia, proinflammatory status and insulin resistance were decreased. These results showed the importance of
making lifestyle changes (i.e., nutritional modification) to reduce the pro-inflammatory state in obese
individuals. We have previously shown that long-term therapy is effective in reducing body fat
(especially visceral fat), TNF-α and IL-6 and increasing IL-10 and adiponectin. In addition, we
observed a positive correlation between pro-inflammatory cytokines (IL-6 and TNF-α levels) and
visceral fat [9].
Creely et al. [2] found that circulating serum endotoxin was higher in type 2 diabetes mellitus
(T2DM) patients than in lean healthy subjects, and endotoxin can activate the innate immune pathway
in isolated abdominal adipocytes to stimulate secretion of pro-inflammatory cytokines. Creely et al.
suggested that the subclinical inflammation seen in type 2 diabetes patients was related to the increase
in endotoxin. Mehta et al. [18] observed that endotoxemia (3 ng/kg intravenous bolus in healthy
adults) induced an elevation in TNF-α and systemic insulin resistance in humans. Furthermore, insulin
resistance measured at 24 h post-LPS was preceded by specific modulation of adipose inflammatory
and insulin signaling pathways. Leuwer et al. [19] have shown that endotoxinemia leads to major
increases in inflammatory adipokine (TNF-α, IL-6, and MCP-1) gene expression in white adipose
tissue in mice. In addition, previous studies in human adipose tissue have shown that obesity and
T2DM induce upregulation of inflammatory genes [2]. These results are in agreement with the present
study in which endotoxin showed a close correlation with IL-6, which was reduced after 1 year of
interdisciplinary therapy.
Although we did not directly analyze the effect of exercise, we cannot exclude that the
exercise protocol used in the present study contributed to the beneficial effects of the interdisciplinary
therapy in obese adolescents. Many studies [9,20,21] have actually demonstrated the benefits of
exercise training, which induces an anti-inflammatory state in obese rat and human models. Bradley et
al. suggested that voluntary exercise in diet-induced obese mice reduced adiposity despite continued
consumption of a high-fat diet. In addition, exercise normalized insulin sensitivity and decreased
adipose tissue inflammation (reduced IKK-β gene expression) in these obese mice [22] . These data
48
demonstrated the positive role of exercise training in preventing the development of several diseases,
including obesity, diabetes, and fatty liver.
Starkie et al. [23] demonstrated that an intravenous infusion of endotoxin induced a two to
threefold increase in the plasma TNF-α level. When human subjects adopted an acute exercise
protocol (75% VO2max), however, the production of TNF-α elicited by low-grade endotoxemia was
inhibited. Similarly, Chen et al. [24] found that chronically exercised rats exhibited minor pathological
changes in the heart, liver, and lung after endotoxemia. In addition, Lira et al. [25] observed that a
lifestyle associated with high-intensity, high-volume exercise induced favorable changes in chronic
low-grade inflammatory markers and may reduce the risk for obesity, diabetes and cardiovascular
diseases.
Although the small number of participants could be a limitation of the present study, the
results contribute to the understanding of the mechanisms linking insulin resistance in obesity and
endotoxinemia to the inflammatory state. In addition, the present study highlights the importance of
lifestyle interdisciplinary therapy intervention as clinical practice for obesity treatment.
In summary, the present study demonstrated that reduced dietary fat intake and endotoxin
level was correlated with a decreased pro-inflammatory state and an improvement in insulin
resistance, which may be associated with an increased adiponectin level. Taken together, these results
suggest that interdisciplinary therapy is effective in decreasing inflammatory markers related to
obesity.
Conflicts of interest
We declare that there are no conflicts of interest (including financial and other relationships)
for each author.
49
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Figure 1A-1D. Correlation Pearson between of fat diet intake and endotoxin, insulin,
adiponectin and HOMA level in obese adolescents (n = 18).
Figure 2A-2D. Correlation Pearson between of endotoxin and IL-6, insulin, adiponectin and
HOMA level in obese adolescents (n = 18).
Table 1. Characteristics of the obese adolescents (n = 18).
Table 2. Characteristics of the food intake in obese adolescents (n = 18).
Table 3. Glucose, insulin, IL-6, adiponectin, HOMA-IR, and endotoxin levels in obese
adolescents (n = 18).
53
Results.
54
55
56
5.3 Manuscrito 3
3T3-L1 adipocytes. Lira FS; Rosa JC; Pimentel GD; Seelaender M; Damaso AR, Oyama LM and
Oller do Nascimento C. (submetido).
57
3T3-L1 adipocytes.
Fábio Santos Lira1; José Cesar Rosa1; Gustavo Duarte Pimentel1; Marília Seelaender2; Ana Raimunda
Damaso1, Lila Missae Oyama1 and Claudia Oller do Nascimento1
1
Departamento de Fisiologia, Universidade Federal de São Paulo, São Paulo, Brasil.
2
Cancer and Metabolism Group, Institute of Biomedical Sciences, University of São Paulo, São Paulo,
Brazil.
Running head: Actions anti-inflammatory the Adiponectin and IL-10.
Correspondence to:
Claudia Maria Oller do Nascimento
Departamento de Fisiologia, Universidade Federal de São Paulo, São Paulo, Brasil.
Rua Botucatu, 862, 2º andar, São Paulo, SP, Brazil. CEP: 04023-060.
e-mail: claudia.oller@unifesp.com
58
Abstract
Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by
differentiated adipocytes and are involved in energy homeostasis, insulin sensibility and the antiinflammatory response. These two adipokines are reduced in obese subjects, which favors increased
activation of nuclear factor kappa B (NF-κB) and leads to elevation of pro-inflammatory adipokines.
However, the effects of adiponectin and IL-10 on NF-κB DNA binding activity (NF-κBp50 and NFκBp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4), such as MYD88 and
TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of
lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-κB
pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased
the nuclear activity NF-κB (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited elevated IL6 levels and activated NF-κB (p50 and p65) DNA binding. Taken together, the present results provide
evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response. In
addition, inhibition of NF-κB signaling pathways may be an excellent strategy for the treatment of
inflammation in obese individuals.
Keywords: adiponectin, interleukin 10, 3T3-L1, lipopolysaccharide, NF-κB pathway
59
Introduction
White adipose tissue plays a role in energy storage and insulation from environmental
temperature and trauma. Recent advances in adipose biology have provided convincing evidence that
adipocytes also secrete multiple proteins (i.e., adipokines) that influence metabolism in peripheral
tissues (Oller do Nascimento et al, 2009; Trayhurn and Wood, 2005). Obesity has been shown to
cause an increase in plasma concentrations of a number of pro-inflammatory (e.g., IL-6, TNF-α)
markers that are expressed and released by adipocytes (Cani et al. 2007). In addition,, the proinflammatory status in obesity promotes a decrease in anti-inflammatory adipokines, such as
adiponectin and IL-10 plasma concentrations (Jung et al. 2008; Lira et al. 2011a).
Previous studies (Ajuwon and Spurlock 2005; Zoico et al. 2009) have shown that nuclear
factor κB (NF-κB) transcription factor is a key mediator of inflammation in adipose tissue. New data
have shown a close relationship between toll-like receptor 4 (TLR-4) and activation of the NF-κB
pathway, which leads to an elevation of pro-inflammatory adipokine gene and protein expression in
adipose tissue (Tsukumo et al. 2007; Rosa Neto et al. 2011).
Cani et al. (2007) showed that increased endotoxin levels in obesity may be a key factor for
the initiation of inflammation in adipose tissue, and the prototypical endotoxin, lipopolysaccharide
(LPS), acts on TLR-4. Toll-like receptors are transmembrane proteins that play an important role in
recognizing microbial pathogens and mediating whole body inflammation (Gleeson et al. 2006). They
are highly expressed in cells of the innate immune system (Muzio and Mantovani 2000). In addition,
TLR-2 and TLR-4 are also found in various other cell types, including adipocytes, hepatocytes, and
myocytes (Lin et al. 2000; Lang et al. 2003).
Studies have shown that adiponectin and interleukin 10 (IL-10), two adipocyte-derived
cytokines, act as potent inhibitors of inflammatory responses (Yamaguchi et al. 2005; Ajuwon and
Spurlock 2005; Zoico et al. 2009; Lira et al. 2009).
Zoico et al. (2009) demonstrated that globular adiponectin and full-length adiponectin
decreased NF-κB activity in 3T3-L1 adipocytes by 50 and 40%, respectively, compared with the NF-
60
κB activation induced by LPS alone. This result demonstrated the important anti-inflammatory role of
adiponectin to combat obesity-mediated inflammation.
Interestingly, Turner et al. (2010) explored the anti-inflammatory effects of IL-10 in primary
human cultures of differentiated adipocytes and found that IL-10 was ineffective against TLR-4induced cytokine secretion. Human adipocytes, however, do not express the IL-10 receptor, which has
been shown to respond to IL-10 in the murine 3T3-L1 adipocyte model.
The effects of adiponectin and IL-10 on NF-κB DNA binding activity (NF-κBp50 and NFκBp65) and the expression of proteins involved in the signaling of the toll-like receptor (TLR-2 and
TLR-4), such as MYD88 and TRAF6, in lipopolysaccharide-treated 3T3-L1 adipocytes are not clear.
In the present study, we analyzed the effects of adiponectin, IL-10, and the combination of
adiponectin and IL-10 on TLR-2, TLR-4 and the NF-κB pathway in 3T3-L1 adipocytes in the
presence of LPS.
61
Materials and Methods
Cell culture
3T3-L1 cells were obtained from American Type Culture Collection and cultured at 37°C in
5% CO2 / 95% humidified air. The cells were maintained in Dulbecco´s Eagle modified medium
(DMEM) with 25 mM glucose, 1.0 mM pyruvate, 4.02 mM L-alanine-glutamine and 10% fetal bovine
serum (Gibco, New York, USA). Cell differentiation began 24 h after confluence and took 4 days in a
medium containing 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 5 μg/mL
insulin (Sigma). After differentiation, the cells were cultured for 8 days in growth medium containing
5 μg/ml insulin.
Treatment
In the ten days after differentiation, the cells were pretreated for a 24-h period with a medium
containing 0.5% serum fetal bovine. The cells were harvested 24 h later. Cells were treated with
adiponectin (50 ng/mL), IL-10 (5 ng/mL) or a combination of adiponectin (50 ng/mL) and IL-10 (5
ng/mL) for 24 hours in the presence of LPS (100 ng/mL), and the cells were harvested 24 h later. In
the control plates (C), the medium was changed, but no treatment was added. The cultured medium
and adipocytes were collected in tubes and stored at -80°C.
Preliminary experiments were performed to determine the concentrations of adiponectin and
IL-10 (data not shown). In addition, we previously ran a time-course experiment (3, 6, 12 and 24 hours
of treatment).
Determination of the IL-6 level in the adipocyte culture medium
Quantitative assessment of the IL-6 level in the culture medium was performed using an
enzyme linked immunosorbent assay (ELISA) (DuoSet ELISA, R&D Systems, Minneapolis, MN).
The IL-6 (DY506) assay sensitivity was found to be 5.0 pg/ml, and the range was 31.2 – 2,000 pg/ml.
The intra- and inter-assay variability of the IL-6 kit was 2.7–5.2%. All samples were run in triplicate,
and the mean value was used for analysis. The protein concentration of 3T3-L1 adipocytes was
62
determined by the Bradford assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA)
as a standard. The results are expressed in pg/μg protein.
Protein analysis by western blotting
3T3-L1 adipocyte cells were homogenized in 1.0 mL of solubilization buffer at 4 C [1%
Triton X-100, 100 mM Tris-HCl (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride,
10 mM EDTA, 10 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1
mg aprotinin/mL]. Insoluble material was removed by centrifugation for 30 min at 9,000 x g in a 70 Ti
rotor (Beckman, Fullerton, CA, USA) at 4 C. The protein concentration of the supernatants was
determined with a BCA assay (Bio-Rad, Hercules, CA, USA). Proteins were denatured by boiling (5
min) in Laemmli sample buffer (Laemmli 1970) containing 100 mM DTT and subjected to 10% SDSPAGE in a Bio-Rad miniature slab gel apparatus.
Electrotransfer of proteins from the gel to nitrocellulose membranes was performed for
~1.30 h/4 gels at 15 V (constant) in a Bio-Rad semi-dry transfer apparatus. Nonspecific protein
binding to the nitrocellulose was reduced by preincubation for 2 h at 22°C in blocking buffer (5%
nonfat dry milk, 10 mM Tris, 150 mM NaCl and 0.02% Tween 20). The nitrocellulose membranes
were incubated overnight at 4°C with antibodies against IL-6R, TLR2, TLR4, MYD88, TRAF6, and
alpha-tubulin (Santa Cruz Biotechnology, CA, USA), which were all diluted 1:1,000 in blocking
buffer with 1% BSA. After incubation, the membranes were washed for 30 min in blocking buffer
without BSA. The blots were subsequently incubated with peroxidase-conjugated secondary antibody
for 1 h at 22 C. For the evaluation of protein loading, membranes were stripped and reblotted with
anti-alpha-tubulin antibody as appropriate. Specific bands were detected by chemiluminescence, and
visualization/capture was performed by exposure of the membranes to RX films. Band intensities were
quantified by optical densitometry of developed autoradiographs (Scion Image software, Scion
Corporation, Frederick, MD, USA).
63
Nuclear extraction
3T3-L1 adipocyte cells were rapidly removed and homogenized in accordance with the
manufacturer’s instructions for the Panomics Nuclear Extraction Kit (AY2002). The nuclear fraction
was stored at -80°C.
NF-κBp50 and NF-κBp65 DNA binding assay
Nuclear-localized NF-ΚB was quantified using a Transcription Factor ELISA Kit to detect the
DNA binding of the p50 and p65 subunits of NF-ΚB (Panomics, Fremont, CA; EK 1110 and 1120,
respectively). All reagents required for preparing nuclear extracts and performing ELISA assays were
included, and all reagents were used as described by the manufacturer.
Statistical analysis
The results are expressed as the mean ± S.E.M. We used Student’s t test to compare the
treatment effects (control vs. LPS; LPS vs. LPS plus adiponectin; LPS vs. LPS plus IL-10; and LPS
vs. LPS plus adiponectin and IL-10). Values of p<0.05 were considered to be statistically significant.
Results
Time course
Figure 1A-D shows that the IL-6 level in the culture medium after 3, 6, 12 and 24 h was
higher in the LPS, LPS plus adiponectin, LPS plus IL-10, and LPS plus adiponectin and IL-10 groups
compared with the control group (p<0.05).
Interestingly, the IL-6 level was lower in LPS group after 3 hours compared with the other
treatments (p<0.05).
After 6 hours, IL-10 and LPS reduced the IL-6 level compared with LPS alone. Conversely,
LPS plus adiponectin increased the IL-6 level compared with LPS alone, LPS plus IL-10 and LPS plus
IL-10 and adiponectin (p<0.05).
In addition, adipokines incubated with LPS for 24 h had an approximately 25% decrease in the
IL-6 level in the culture medium.
64
We observed similar results for NF-κBp50 nuclear activity in adipocytes after 3 and 6 hours of
treatment among groups (Figure 2A and B); however, LPS treatment for 12 h and 24 h caused an
increase in NF-κBp50 nuclear activity in adipocytes compared with the others groups (p<0.01) (Figure
2C and D).
The NF-κBp65 nuclear activity in adipocytes was increased by LPS treatment at all examined
time points compared with control and adipokine-treated groups. The addition of adiponectin, IL-10 or
adiponectin plus IL-10 decreased the effect of LPS (Figure 3A-D) on NF-κBp65 nuclear activity in
adipocytes. After 6, 12 and 24 h of adipokine treatment, this parameter was similar to the control
group (Figure 3B-D).
After we verified the best time response for our treatments with adipokines, we chose to assess
the 24-h time point, and we divided our study into three distinct experiments. We assessed the effects
of adiponectin, IL-10, and adiponectin with IL-10 on TLR-2, TLR-4 and the NF-κB pathway in
adipocytes in the presence the LPS.
Previous studies have shown that LPS induces NF-κB activation and IL-6 production in
adipocytes (do Nascimento et al, 2004; Ajuwon and Spurlock, 2005; Song et al, 2006; Creely et al,
2007; Zoico et al, 2009). In addition, we observed that LPS increased TLR-4, MYD88 and TRAF6
expression. These data demonstrated a classic LPS-mediated pro-inflammatory response in adipocytes.
These results are shown in Figure 4A-D and Figure 5A-D.
Figures 6A-D and 7A-D show the effects of adiponectin on the LPS-induced inflammatory
response in adipocytes. Compared with LPS alone, adiponectin reduced the IL-6 level and both NFκBp50 and NF-κBp65 nuclear activity in adipocytes (p<0.05). In addition, adiponectin increased TLR2, MYD88 and TRAF6 protein expression compared with LPS alone (p<0.05).
Figures 8A-D and 9A-D show the effects of IL-10 on the LPS-induced inflammatory response
in adipocytes. Compared with LPS alone, IL-10 reduced the IL-6 level and both NF-κBp50 and NFκBp65 nuclear activity in adipocytes (p<0.05). We did not observe any effects of IL-10 on TLR-2,
MYD88, or TRAF6 protein expression.
Figures 10A-D and 11A-D show the effects of adiponectin combined with IL-10 on the LPSinduced inflammatory response in adipocytes. Compared with LPS alone, the combination of
65
adiponectin and IL-10 reduced the IL-6 level, MYD88 protein expression and both NF-κBp50 and
NF-κBp65 nuclear activity in adipocytes (p<0.01).
Discussion
The present study showed that treatment with anti-inflammatory adipokines was effective in
reducing activation of inflammatory pathways, especially the NF-κB pathway. In addition, antiinflammatory adipokines decreased IL-6 levels.
In agreement with previous studies, the LPS administration utilized in the present study caused
an increase in IL-6, IL-6R, TLR-4, MYD88, and TRAF6 protein expression and the nuclear activity of
NF-κB (p50 and p65) DNA binding (Ajuwon and Spurlock, 2005; Zoico et al. 2010; Lira et al.
2011b).
One of the questions addressed in the present study was how adiponectin would affect the
inflammatory response in the presence of LPS. We observed that the addition of adiponectin reduced
NF-κB (both p50 and p65) activation.
Ajuwon and Spurlock (2005) showed that adiponectin may be a local regulator of
inflammation in the adipocyte and adipose tissue via its regulation of the NF-κB and PPARγ2
transcription factors. They used primary adipocytes from pig subcutaneous adipose tissue with or
without LPS and adiponectin. Although LPS induced an increase in NF-κB activation, adiponectin
suppressed both NF-κB activation and the induction of IL-6 expression by LPS. Similar results were
obtained in 3T3-L1 adipocytes. In addition, adiponectin antagonized the LPS-induced increase in
TNF-α mRNA expression and tended to diminish its accumulation in the culture media in 3T3-L1
adipocytes. Adiponectin also induced an upregulation of PPARγ2 mRNA.
Similar results were found in a study by Zoico et al. (2010), which showed that adiponectin
(two isoforms of adiponectin: globular and full length) significantly suppressed LPS-induced
expression of IL-6 mRNA in adipocytes and reduced the concentration of IL-6 in culture media.
Adiponectin pretreatment significantly reduced the increase in monocyte chemotactic protein 1 (MCP1) mRNA in adipocytes exposed to LPS. In culture media, the increase in MCP-1 detected after LPS
stimulation was significantly attenuated after pretreatment with adiponectin. In 3T3-L1, adiponectin
66
reduced NF-κB activity by 50% compared with the NF-κB activation induced by LPS alone.
Moreover, adiponectin significantly attenuated IkappaB-alpha and IKK gene expression.
Using macrophages, Park et al. (2007) demonstrated the mechanism by which adiponectin
suppresses the inflammatory pathway. They showed that adiponectin initially increases TNF-α
production by macrophages via ERK1/2, Egr-1 and NF-κB-dependent mechanisms, which leads to
increased expression of IL-10 and an eventual dampening of LPS-mediated cytokine production.
Traditionally, LPS is specific for TLR-4, and TLR-2 is a receptor for bacterial lipoproteins
(Tsan and Gao, 2004). Lin et al. (2000) reported that acute LPS induced TLR-2 expression, which was
consistent with the notion that TLR-4, but not TLR-2, is constitutively present on the cell surface of
3T3-L1 adipocytes. In addition, TLR-4 activation results in induction of TLR-2, and this newly
synthesized TLR-2 translocates to the cell surface where it can contribute to increased signaling.
Unexpectedly, adiponectin addition in the culture medium of 3T3-L1 adipocytes increased
protein expression of TLR-2, MYD88 and TRAF6 compared with the levels induced by LPS alone,
which demonstrated that the reduced NF-κB (both p50 and p65) activation caused by adiponectin was
not related to an effect on the TLR-4 pathway; however, this could be associated with a decrease in
IL-6.
One possible interpretation is that TLR-4 recruits TLR-2 into a complex. Alternatively, TLR-4
activation could result in the activation of intracellular effectors that could associate with TLR-2. This
interaction may also be involved in other intracellular signaling pathways because both IL-6 and NFқB are reduced. In addition, TLR-2, MYD88 and TRAF6 were increased, and they could participate in
other noninflammatory pathways.
The present study also investigated the effects of IL-10 on the LPS-induced inflammatory
response. We observed that IL-10 reduced the IL-6 level and NF-κB (both p50 and p65) DNA binding.
In contrast with adiponectin, fewer studies have been conducted to investigate the effects of IL-10 in
adipocytes and the inflammatory response.
Interleukin 10 inhibits the production of several cytokines, such as TNF-α, IL-1β and IL-6, in
a variety of cell types. Several studies have shown that the production of IL-10 is increased in
inflammatory processes and predominantly plays an immune modulating role in these conditions (Lira
67
et al. 2009; Lira et al. 2011b). In obesity, adiponectin and IL-10 serum levels are decreased, which
leads to a pro-inflammatory status (Jung et al. 2008).
Compared with control cells, Bradley et al. (2008) showed that 3T3-L1 adipocytes incubated
with palmitic acid for 24 h exhibited a 70% increase in TNF-α production and up to a 75% decrease in
IL-10 production. Furthermore, NF-κB DNA binding activity increased fourfold in response to
palmitic acid.
Turner et al. (2010) examined the anti-inflammatory effects of IL-10 in primary human
adipocytes and showed that IL-10 did not inhibit TLR-4-induced cytokine secretion. Interestingly, a
different result was observed in the 3T3-L1 adipocyte model. The authors suggested that the receptor
for IL-10 was absent in human adipocytes.
Cintra et al. (2008) administered an endogenous IL-10 inhibitor for 5 days in male Swiss mice
and demonstrated an increase in hepatic expression of inflammatory markers, such as TNF-α, IL-6, IL1β and F4/80. This increase in inflammatory markers was accompanied by a significant negative
modulation
of
insulin
signal
transduction
through
the
insulin
receptor/IRS1-IRS2/PI3-
kinase/Akt/FOXO1 pathway and through an increase in hepatic signaling proteins involved in
gluconeogenesis and lipid synthesis.
Strategies such as energy restriction and exercise training have been utilized to promote
increases in the IL-10 serum level and adipose tissue production, which would reduce the
inflammatory status (Jung et al. 2008; Lira et al. 2009, Lira et al. 2011a; Yamashita et al. 2010). The
pathway that mediates this IL-10 effect in 3T3-L1 adipocytes, however, is unknown. We demonstrated
that the addition of IL-10 to the culture medium decreased NF-κB DNA binding activity in 3T3-L1
adipocytes independent of the TLR pathway.
We also examined the effects of adiponectin combined with IL-10 on the LPS-induced
inflammatory response. Interestingly, we observed that the combination of adiponectin and IL-10
reduced the IL-6 level, the protein expression of MYD88, and NF-κB (both p50 and p65) DNA
binding.
Almost all TLRs have a common signaling pathway in which MYD88 adaptor molecules form
a molecular complex with TLR-initiated signaling events. MYD88 also interacts with downstream IL-
68
1R-associated kinase (IRAKs) (Arancibia et al., 2007), and TRAF6 regulates distinct processes of
innate and adaptive immunity mediated by IkB kinases (IKK) that regulate NF-κB (Dadgostar and
Cheng, 1998; Bradley and Pober, 2001). The combination of adiponectin and IL-10 altered the
MYD88-dependent pathway, which led to a decrease in NF-κB (both p50 and p65) DNA binding.
Tsan and Gao (2004) reported that TLR1/2, TLR2/6, and TLR4 (but not other TLRs) induced NF-κB
activation through a MYD88-dependent pathway.
More studies are needed to fully elucidate the comprehensive pathway involved with antiinflammatory responses in adipocytes.
In summary, we demonstrated that adiponectin, IL-10 and the combination of adiponectin and
IL-10 all reduced NF-κB (both p50 and p65) DNA binding in 3T3-L1 adipocytes exposed to LPS,
which may have resulted from a reduction in IL-6 production rather than an inhibition of the TLR-4
pathway. The present results suggest that anti-inflammatory adipokines may be utilized as strategies to
reduce the pro-inflammatory state exhibited in obese people.
Acknowledgements
This work was supported by FAPESP (08/54733-0).
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Legends Figure
Figure 1. Time course of IL-6 release in the culture medium of 3T3-L1 adipocytes treated with LPS,
LPS+IL-10, LPS+adiponectin. . n=6 for all groups.* Values are means ± SE. * p<0.05 in relation to
control, # p<0.05 in relation to LPS.
Figure 2 - Time course of DNA-binding activity of NF-kBp50 in 3T3-L1 adipocytes treated with LPS,
LPS+IL-10, LPS+adiponectin. n=6 for all groups.* Values are means ± SE. * p<0.05 in relation to
Figure 2 - Time course of DNA-binding activity of NF-kBp65 in 3T3-L1 adipocytes treated with LPS,
LPS+IL-10, LPS+adiponectin. n=6 for all groups. Values are means ± SE. * p<0.05 in relation to
Figure 4 – Effect of LPS for 24h on the IL-6 level in culture medium and on the 3T3-L1 protein
expression of IL-6R, TLR-2 and TLR-4. n=6 for all groups. Values are means ± SE. * p<0.05 and ***
p<0,001 in relation to control.
Figure 5 – Effect of LPS for 24h on the 3T3-L1 protein expression of MYD88, TRAF6, NF-kBp50,
NF-kBp65. n=6 for all groups. Values are means ± SE. * p<0.05 and *** p<0,001 in relation to
control.
Figure 6 – Effect of adiponectin treatment for 24h on IL-6 level in the culture medium and on protein
expression of IL-6R, TLR-2 and TLR-4 in 3T3-L1 adipocyte treated with LPS. n=6 for all groups.
Values are means ± SE. * p<0.05 in relation to LPS.
Figure 7 - Effect of adiponectin treatment for 24h on protein expression of MYD88, TRAF6, NFkBp50, NF-kBp65 in 3T3-L1 adipocyte treated with LPS. n=6 for all groups. Values are means ± SE.
* p<0.05 in relation to LPS.
Figure 8 – Effect of IL-10 treatment for 24h on IL-6 level in the culture medium and on protein
expression of IL-6R, TLR-2 and TLR-4 in 3T3-L1 adipocyte treated with LPS. n=6 for all groups.
Values are means ± SE. * p<0.05 in relation to LPS.
Figure 9 - Effect of IL-10 treatment for 24h on protein expression of MYD88, TRAF6, NF-kBp50,
NF-kBp65 in 3T3-L1 adipocyte treated with LPS. n=6 for all groups. Values are means ± SE. *
p<0.05 in relation to LPS.
Figure 10 – Effect of IL-10 plus adiponectin treatment for 24h on IL-6 level in the culture medium and
on protein expression of IL-6R, TLR-2 and TLR-4 in 3T3-L1 adipocyte treated with LPS. n=6 for all
groups. Values are means ± SE. * p<0.05 in relation to LPS.
Figure 11 - Effect of IL-10 plus adiponectin treatment for 24h on protein expression of MYD88,
TRAF6, NF-kBp50, NF-kBp65 in 3T3-L1 adipocyte treated with LPS. n=6 for all groups. Values are
means ± SE. * p<0.05 in relation to LPS.
72
Results
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74
75
76
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78
6. Considerações Finais
Frente às questões levenatadas em nossa tese, podemos afirmar que:
correlacionados com a elevação das adipocinas anti-inflamatórias (adiponectina e IL10);
correlacionados com a redução das citocinas pró-inflamatórias (TNF-α e IL-6),
endotoxina e dos depósitos de gordura corporal;
Os efeitos anti-inflamatórios da adiponectina e da IL-10 em adipócitos 3T3-L1 são
dissociados;
A ação anti-inflamatória, individual ou conjunta, da adiponectina e IL-10 em adipócitos
3T3-L1, não é mediada por alterações na via de sinalização do TLR-4, mas sim do NFκB;
A terapia interdicisplinar de longo prazo foi capaz de elevar as adipocinas anti-inflamatória
(adiponectina e IL-10), e concomitantemente, promoveu redução do depósito de gordura visceral, IL6, TNF-α e endotoxina sérica. Esses eventos direcionaram para melhora do quadro da resistência à
ação da insulina nos adolescentes obesos. Adicionalmente, observamos que a concentração sérica de
adiponectina pode ser um bom preditor da relação entre gordura visceral e subcutânea.
O estudo in vitro em células adiposas 3T3-L1 confirmou que tais adipocinas anti-inflamatórias
tem papel importante na redução da produção de IL-6 e na inibição da ligação do NF-κB com DNA,
favorecendo desta maneira redução da via inflamatória. No entanto, tais eventos parece não ser
dependetendes dos TLRs, sendo necessários mais estudos para elucidar as vias envolvidas neste
processo.
79
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