A EXPRESSÃO DA 11
β
-HIDROXISTERÓIDE
DESIDROGENASE TIPO 1 E REGULADORES CHAVE DA
ADIPOGÊNESE HUMANA NÃO ESTÃO AUMENTADOS NA
SÍNDROME DE CUSHING
Tese apresentada à Universidade Federal de São
Paulo – Escola Paulista de Medicina, para obtenção
do Título de Doutor em Ciências.
São Paulo
DANIELA ESPÍNDOLA ANTUNES
A EXPRESSÃO DA 11
β
-HIDROXISTERÓIDE
DESIDROGENASE TIPO 1 E REGULADORES CHAVE DA
ADIPOGÊNESE HUMANA NÃO ESTÃO AUMENTADOS NA
SÍNDROME DE CUSHING
Tese apresentada à Universidade Federal de São
Paulo – Escola Paulista de Medicina, para obtenção
do Título de Doutor em Ciências.
Orientador:
Prof. Dr. Claudio Elias Kater
Co-orientador:
Prof. Dr. José Antônio Silva Júnior
São Paulo
Espíndola-Antunes, Daniela
A expressão da 11β-hidroxisteróide desidrogenase tipo 1 e reguladores chave da adipogênese humana não estão aumentados na síndrome de Cushing. São Paulo, 2008.
x, 71p
Tese (Doutorado) – Programa de Pós-graduação em Endocrinologia Clínica – Universidade Federal de São Paulo. Escola Paulista de Medicina.
Orientador: Kater, Claudio Elias
Título em inglês: Expression of 11β-hydroxysteroid dehydrogenase type 1 and key regulators of human adipogenesis are not overexpressed in Cushing’s syndrome adipose depots.
iii
UNIVERSIDADE FEDERAL DE SÃO PAULO
ESCOLA PAULISTA DE MEDICINA
DEPARTAMENTO DE MEDICINA
DISCIPLINA DE ENDOCRINOLOGIA
CHEFE DO DEPARTAMENTO DE MEDICINA:
Prof. Dr. Ângelo Amato V. de Paola
COORDENADOR DO PROGRAMA DE PÓS-GRADUAÇÃO
EM ENDOCRINOLOGIA CLÍNICA:
A EXPRESSÃO DA 11
β
-HIDROXISTERÓIDE
DESIDROGENASE TIPO 1 E REGULADORES CHAVE DA
ADIPOGÊNESE HUMANA NÃO ESTÃO AUMENTADOS NA
SÍNDROME DE CUSHING
Presidente da Banca:
Prof. Dr. Claudio Elias Kater
Banca examinadora:
Prof. Dr. Ayrton Custódio Moreira
Prof. Dr. Bruno Geloneze Neto
Prof. Dr. João Bosco Pesquero
Profa. Dra. Regina Célia Mello Santiago Moisés
Suplentes:
Prof. Dr. Alfredo Halpern
v
In Harvey Cushing everyone recognized a person of brilliant
intellect and of great personal charm. His influence upon all who
came in contact with him was deep and inspiring and especially to
his students and associates he had a lasting effect upon their lives.
W.G. Mac Callum in: Biographical Memoir of Harvey Cushing (1869-1939). National Academy of Sciences of the United States of America, 1940.
A minha mãe e melhor amiga, Lenita Espíndola Antunes, que com sua
sabedoria e humildade me ensinou a viver com alegria e vontade de acertar; a
valorizar a família, a profissão e os amigos. Também foi com ela que aprendi a
ser grata por todas as graças (e foram muitas) e ensinamentos que a
Inteligência Superior e a Natureza me proporcionaram!
Ao meu pai, Osmar Antunes da Silva Dorninger, exemplo de inteligência e
honestidade, que sempre acreditou na medicina como a melhor profissão para
sua filha. Meu companheiro e grande torcedor desde a época do segundo grau e
cursinho, até os dias de hoje na pós-graduação. Papai, eu não seria tão feliz em
qualquer outra profissão!
A minha querida irmã, Denise Espíndola Antunes, minha alma gêmea, amiga
inseparável e segunda mãe. Meu exemplo de organização, de luta,
companheirismo e meiguice.
Ao meu namorado, Rafael Daher de Miranda, que mesmo longe se fez sentir
muito presente nos momentos de glória e nas dificuldades, tornando meus dias
mais leves e felizes. A você que me faz acreditar que tudo vai valer a pena!
À minha gatinha, Zinha, que torna minhas horas de estudo prazerosas. Minha
companheira ávida por novos artigos impressos a qualquer hora do dia, muito
vii
AGRADECIMENTOS
Ao meu orientador e amigo, Prof. Dr. Claudio Elias Kater, quem eu já admirava pelos
conhecimentos, inteligência, didática e polidez desde a época de acadêmica. Agradeço a
oportunidade e confiança em mim depositada nesses quatro anos e meio, nos quais pude
trabalhar com o mestre e aprender sobre Adrenal e Hipertensão. Aquele professor que
falava sobre as doenças e fisiologia das adrenais nos congressos é muito mais que um
cientista. É um ser humano extraordinário, iluminado e de valores; uma pessoa de bem
com a vida que vibra com as nossas vitórias, mas que também apóia nas dificuldades.
Agradeço pelas manhãs de sábado, feriados, sem falar nas tardes e noites, que emprestou
seu raciocínio para a discussão dos projetos em quem eu estava trabalhando. Agradeço a
paciência em conciliar seus horários com os meus depois que passei a trabalhar também
em Goiânia. Toda a minha gratidão por ter me inspirado e proporcionado iniciar a vida
acadêmica na área de Adrenal e Esteróides. Finalmente, obrigada por me ensinar que o
raciocínio flui melhor quando se interrompe as atividades para um “cafezinho” ou para um
final de semana com a família e com os amigos. Muito obrigada por tudo, Dr. Claudio!
Ao meu co-orientador, Prof. Dr. José Antônio Silva Júnior, jovem pesquisador e já
detentor de grandes conquistas, pelo exemplo a ser seguido. Agradeço também a confiança
em mim depositada, a paciência em responder as incansáveis dúvidas e o incentivo para a
concretização deste trabalho.
Aos meus preceptores de residência médica do Hospital Geral de Goiânia, Dr. Nelson
Rassi, Dr. Luciano Sanches e Dra. Eldeci Cardoso, pelos ensinamentos valiosos para o
exercício da profissão com dignidade, ética, segurança e respeito com o paciente.
Agradeço o despertar e o incentivo pela área acadêmica. Sou-lhes muito grata por todo o
suporte durante a residência em Endocrinologia e pela compreensão durante minha jornada
Universidade Federal de Goiás. Obrigada pelo despertar pela Endocrinologia e pelo
exemplo como profissional e professora.
À Prof. Dra. Ieda Verreschi, pela receptividade no laboratório de esteróides e no
ambulatório de gônadas. Obrigada pelas discussões de casos complicados e pelos
ensinamentos científicos sempre recheados de muita ética e de um “olhar” especial para o
PACIENTE.
Ao grupo da Unidade de Adrenal e Hipertensão, Dra. Martha Huayllas, Dra. Regina do
Carmo Silva e Dra. Dolores Pardini, pelos ensinamentos, incentivo e coleguismo.
Aos meus colegas de Pós-Graduação, Flávia Amanda Costa Barbosa, Viviane Chaves de
Carvalho, Maria Sílvia S. Caetano e Marcos Neres, pela ajuda na seleção de pacientes com
síndrome de Cushing, pela amizade e torcida.
Ao grupo do laboratório de esteróides, Lílian Fukusima Hayashi, Sâmia S. Cavassani,
Kelly C. de Oliveira e Ivonne F. Bianco, pelo suporte técnico e pela convivência prazerosa.
Aos cirurgiões e residentes da UNIFESP, Hospital do Servidor Público Estadual e Hospital
Brigadeiro, que auxiliaram na coleta de amostras de tecido adiposo, em especial ao Prof.
Dr. Cássio Andreoni.
À amiga Gláucia Carneiro, que me acolheu desde os primeiros dias de estágio na
UNIFESP, quando ainda éramos residentes de Endocrinologia. Obrigada pela amizade e
prontidão na ajuda e solução de problemas de qualquer ordem. Muito obrigada também
pelas calorosas discussões de estatística e qualquer assunto que dissesse respeito às nossas
ix
À minha grande amiga Monike Lourenço Dias Rodrigues, pela amizade, força e
companheirismo em todos os momentos da realização desse trabalho. Ao mesmo tempo
em que agradeço todo o suporte durante minha estada em São Paulo, desejo que essa troca
de idéias continue em Goiânia e, estou certa, que mais cedo ou mais tarde, teremos o
prazer de trabalharmos juntas e construir mais sonhos. Você é uma pessoa muito especial e
uma mente brilhante!
Às minhas tias Leide, Leni e Lênis Espíndola pelo carinho e pelas orações.
À minha avó Maria Antonieta de Amorim, pelas palavras confortantes e pelo carinho.
Às secretárias da pós-graduação Amarylis Cândida Salsano e Yeda Queiroga Confessor
pela atenção, prontidão e paciência.
Aos pacientes, sem os quais não seria possível a realização desse estudo.
SUMÁRIO
Dedicatória . . . vi
Agradecimentos . . . vii
1. INTRODUÇÃO . . . 01
2. ARTIGO 1: . . . . . .
11 -Hydroxysteroid Dehydrogenase Type 1 and Key Regulators of Human Adipogenesis Are Not Overexpressed in Cushing’s Syndrome Adipose Depots.
09
3. ARTIGO 2: . . .
Adipose Tissue 11 -Hydroxysteroid Dehydrogenase Type 1 in Obesity and in Cushing’s Syndrome.
36
5. PRINCIPAIS ACHADOS, CONCLUSÕES E NOVAS DIREÇÕES. . . 45
Pacientes com síndrome de Cushing têm adipogênese anormal e desenvolvem
obesidade de distribuição central com adelgaçamento de tecido subcutâneo, alterações
parcialmente reversíveis após tratamento ou suspensão do glicocorticóide (1,2). As
conseqüências metabólicas deletérias do hipercortisolismo crônico, como hipertensão,
dislipidemia, intolerância a glicose, obesidade visceral e osteoporose, entre outras, são bem
conhecidas. Entretanto, os mecanismos envolvidos na distribuição do tecido adiposo
mediados pelos glicocorticóides não são completamente compreendidos.
O cortisol aumenta, direta ou indiretamente, a massa total de tecido adiposo e o
redistribui da periferia para depósitos viscerais. A gordura visceral é biologicamente mais
ativa e associada às complicações da obesidade e à morte cardiovascular prematura (3,4). Os
glicocorticóides são conhecidos como hormônios catabólicos e, agudamente, ativam a
lipólise, liberando ácidos graxos livres na circulação. Entretanto, em 1997, Bujalska et al. (5-)
realizaram estudo in vitro com pré-adipócitos humanos e sugeriram que a obesidade poderia ser “uma doença de Cushing do omento”. A enzima apontada como responsável pela
superprodução local de cortisol era a 11β-hidroxiesteróide desidrogenase tipo 1 (11β-HSD1), descrita por Lakshmi e Monder em 1988 (6). Inicialmente, a 11β-HSD1 foi referida como uma versão menos potente da 11β-HSD tipo 2, que tem ação de desidrogenase, inativando o cortisol à cortisona e protegendo os receptores mineralocorticóides não seletivos (7).
Contudo, sabe-se, atualmente, que a 11β-HSD1 é dependente de NADP(H) e funciona como uma redutase na maioria das células e tecidos in vivo, convertendo a cortisona ao ativo cortisol (8).
Desde a descrição da 11β-HSD1 e, principalmente, após a associação da super-expressão dessa enzima com a obesidade visceral, houve um grande interesse da comunidade
científica na compreensão de suas funções na fisiopatologia da obesidade e da síndrome
metabólica. Apesar da expressão da 11β-HSD1 ser um assunto controverso, a maioria dos estudos apontam para uma super-expressão da enzima em tecido adiposo subcutâneo na
obesidade humana (9,10,11), sendo poucos os estudos realizados em omento (11). A
expressão aumentada da 11β-HSD1 também tem sido associada à resistência à insulina e às citocinas pró-inflamatórias (9,10,11). Além disso, inibição farmacológica da 11β-HSD1 melhora a sensibilidade insulínica em humanos (13). A 11β-HSD1 tornou-se, então, um alvo promissor para o tratamento da síndrome metabólica e do diabetes melito. Paralelamente, a
INTRODUÇÃO
3 para explicar uma questão não resolvida na síndrome de Cushing, o acúmulo de gordura
visceral. Um dos artigos que compõem essa tese faz revisão da história da 11β-HSD1 e suas correlações com obesidade, síndrome metabólica e síndrome de Cushing.
A ação biológica dos glicocorticóides é mediada pela sua interação com receptores de
glicocorticóide (GR), cuja expressão correlaciona-se com a expressão de 11β-HSD1. Fígado e pulmão de ratos, no período embrionário imediatamente anterior ao nascimento, período no
qual o nível sérico de corticosterona diminui, mostram maior expressão de 11β-HSD1 para compensar sua diminuição e aumentar a densidade de GR (14). Os GR pertencem à
superfamília dos receptores de esteróide, tiróide e retinóides. Duas isoformas de GR foram
descritas em humanos: GRα e GRβ, que se originam do mesmo gene por variantes de processamento de RNA. GRα é a isoforma predominante e mostra atividade de ligação com esteróide. Em condições fisiológicas, o splice alternativo leva à produção de GRα (15). Estudos têm reportado expressão de GRβ em tipos celulares específicos, maioria inflamatória, com super-expressão em estados de resistência aos glicocorticóides, como
asma, colite ulcerativa, leucemia linfocítica e polipose nasal (16). GRβ é inativo e incapaz de se ligar a todos os agonistas e antagonistas já testados.
A ação dos corticosteróides é, então, em parte regulada pela 11β-HSD1 antes da sua ligação ao receptor. Estudos prévios mostraram que a ação de redutase da 11β-HSD1 leva à diferenciação de pré-adipócitos em adipócitos maduros (17) e, provavelmente, à maior
expressão de GR. Pedersen et al. (18), mostraram que o tecido adiposo visceral contem
quatro vezes mais GR que o depósito subcutâneo. Recentemente, foi investigada a relação
entre obesidade e diferenciação de pré-adipócitos in vitro (19) e, ao contrário do que se esperava, os resultados indicaram potencial limitado de diferenciação de pré-adipócitos em
indivíduos com obesidade central, concluindo que o baixo potencial de diferenciação deve
ser, pelo menos em parte, conseqüente a menor expressão de GR. Por outro lado,
administração de metilprednisolona por uma semana diminuiu os níveis de GR e da proteína
de GR em tecido adiposo subcutâneo de indivíduos saudáveis (20).
A expressão da 11β-HSD1 é regulada por diversos fatores, dentre eles o receptor gama ativado pelo proliferador do peroxissomo (PPARγ). Os PPARγ estão entre os fatores de transcrição mais importantes no processo de diferenciação dos adipócitos. São o terceiro
aminoácidos adicionais na porção amino-terminal (21). Os PPARγ2 são expressos exclusivamente em tecido adiposo, enquanto os PPARγ1 são mais amplamente expressos, apesar de mais abundantes no tecido adiposo (22).
Os PPARγ são induzidos precocemente no processo de diferenciação dos pré-adipócitos. A exposição de pré-adipócitos humanos a agonistas dos PPARγ, tiazolidinedionas (TZD), induz diferenciação dos mesmos (23). Por outro lado, a transdução prévia destas
células com adenovírus expressando um mutante negativo e dominante do PPARγ bloqueia esse processo (21). Vários estudos têm mostrado que o aumento da gordura corporal
associada ao tratamento com TZD é mediado principalmente pelo acúmulo de gordura
subcutânea, enquanto que o volume de gordura visceral é reduzido ou não se altera (22,24).
Ligantes do PPARγ estão envolvidos na regulação do metabolismo lipídico e glicídico, sendo utilizados no tratamento do diabetes melito tipo 2 como drogas
sensibilizadoras da insulina. É provável que o PPARγ no tecido adiposo seja o alvo principal das TZD que aumentam a sensibilidade à insulina no tecido hepático e muscular, sugerindo
que os PPARγ controlem a expressão de genes envolvidos na sinalização do tecido adiposo para outros tecidos. Além disso, ligantes dos PPARγ regulam outros genes adipocitários que devem contribuir para a sensibilidade à insulina, como a adiponectina e a 11β-HSD1. Ligantes do PPARγ, TZD e não-TZD, diminuem a expressão desta enzima em adipócitos 3T3-L1 (25) e em tecido subcutâneo humano (26). Há sugestão de que a redução da
expressão de 11β-HSD1 nos adipócitos deva promover a sensibilidade insulínica, seja pela redução da expressão de genes induzidos pelos glicocorticóides nos adipócitos, seja pela
redução da secreção dos glicocorticóides (27).
Adicionalmente, as TZD induzem mudanças fenotípicas em adipócitos de ratos,
reduzindo o tamanho de adipócitos viscerais e aumentando seu potencial de estoque de
lipídeos (28). A ocorrência natural de mutantes do PPARγ também atesta o papel crucial desse promotor na adipogênese e na distribuição de gordura. Mutações com perda de função
no domínio de ligação PPARγ humano causam lipodistrofia, com perda de gordura subcutânea de membros e região glútea e relativa preservação de depósitos subcutâneos e
viscerais (22). Contudo, estudos a respeito da expressão dos PPARγ1 e PPARγ2 em tecido adiposo humano são conflitantes, dado o pequeno número de pacientes estudados e a
variabilidade de parâmetros considerados.
INTRODUÇÃO
5 por hipercortisolismo crônico e distribuição anormal de depósitos de gordura, cujos
mecanismos permanecem não solucionados. Assim, os objetivos do presente estudo foram:
(i) quantificar a expressão gênica da 11β-HSD1, GRα, PPARγ1 e PPARγ2 em tecido adiposo subcutâneo e visceral de pacientes do sexo feminino com síndrome de Cushing e controles
obesas e não obesas, (ii) avaliar os efeitos da exposição ao hipercortisolismo crônico na
expressão dos referidos genes, e (iii) correlacionar os achados moleculares com dados
clínicos
Abreviações:
Circunferência abdominal: CA
11β-Hidroxisteróide desidrogenase Tipo 1: 11β-HSD1 Índice de massa corporal: IMC
Receptor glicocorticóide isoforma alfa: GRα
Receptor gama ativado pelo proliferador do peroxissomo: PPARγ Tecido adiposo subcutâneo: SAT
Tecido adiposo visceral: VAT
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INTRODUÇÃO
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Diederic S. Rosiglitazone decreases 11β-hydroxysteroid dehydrogenase type 1 in subcutaneous adipose tissue. Clin Endocrinol 2007;67:419-425.
27.Rangwala SM, Lazar MA. Peroxisome proliferator-activated receptor γ in diabetes and metabolism. Trends Pharmacol Sciences 2004; 25:331-336.
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2. Artigo 1
11β-Hydroxysteroid Dehydrogenase Type 1 and Key Regulators of Human
Adipogenesis Are Not Overexpressed in Cushing’s Syndrome Adipose Depots.
Espíndola-Antunes D, Goto EM, Guimarães AO, Pesquero JB, Silva JA Jr, Kater CE.
11
β
-Hydroxysteroid Dehydrogenase Type 1 and Key Regulators of
Human Adipogenesis Are Not Overexpressed in Cushing’s Syndrome
Adipose Depots
Daniela Espíndola-Antunes1, Eduardo M. Goto2, Alessander O. Guimarães3,
João B. Pesquero3, José A. Silva Junior2,4, Claudio E. Kater1
1
Division of Endocrinology and Metabolism, Department of Medicine; 2Department of
Pathology; and 3Department of Biophysics, Federal University of Sao Paulo (UNIFESP),
and 4Nove de Julho University (UNINOVE), Sao Paulo, SP, Brazil
Address for correspondence:
Claudio E. Kater, Associate Professor of Medicine
Division of Endocrinology, Department of Medicine
Universidade Federal de São Paulo
Rua Pedro de Toledo, 781 – 13o. andar
04039-032 São Paulo, SP - Brasil
Email: kater@unifesp.br
Running head:11 -HSD1, GRα and PPARγ in Cushing’s syndrome
Abbreviations: 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; GRα,
glucocorticoid receptor alpha; PPARγ, peroxisome proliferator-activated receptor gamma;
AC, abdominal circumference; BMI, Body mass index; CS, Cushing’s syndrome; SF,
ARTIGO 1
11 ABSTRACT
Objective: To quantify and evaluate the effects of chronic exposure to
hypercortisolism on the expression of 11β-hydroxysteroid dehydrogenase type 1
(11-βHSD1), glucocorticoid receptor-α (GRα) and peroxisome proliferator-activated
receptor-γ (PPARγ) in adipose depots of patients with Cushing’s syndrome.
Design and methods: Samples of visceral (VAT) and subcutaneous adipose tissue
(SAT) were obtained during elective abdominal surgery from female patients with
Cushing’s syndrome (n=10), and obese (n=15) and nonobese controls (n=10), in whom
body mass index (BMI), abdominal circumference (AC), and salivary cortisol (SF) were
previously determined. 11β-HSD1, GRα, PPARγ1 and PPARγ2 mRNA expressions were
quantified by real-time PCR.
Results: 11β-HSD1 gene expressions in SAT and VAT of Cushing’s patients were not
different from nonobese and were upregulated in obese (P<0.0001 and P=0.019,
respectively). Additionally, GRα mRNA expression was downregulated in SAT of obese
and Cushing’s patients (P<0.0001 for both). PPARγ2 mRNA expression was higher in
VAT of obese than in Cushing’s patients (P<0.05) and lower in SAT than nonobese. In the
whole group, 11β-HSD1, PPARγ1 and PPARγ2 levels showed no correlations with SF.
Moreover, 11β-HSD1 mRNA expression correlated positively with GRα in VAT (r=0.6,
P<0.0001); in SAT, it correlated positively with PPARγ1 (r= 0.77; P<0.0001) and
negatively with PPARγ2 (r=-0.54, P=0.01). Finally, the best predictor of BMI and AC was
PPARγ2 mRNA in SAT (and not in VAT).
Conclusion: Chronic hypercortisolism, as seen in Cushing’s syndrome, do not result in
upregulation of 11β-HSD1 and PPARγ gene expressions in adipose depots, in contrast to
INTRODUCTION
Glucocorticoids (GC) have a major role in determining adipose tissue distribution and
metabolism. Subjects with endogenous or exogenous hypercortisolism develop a central
obesity pattern that is reversible upon treatment or GC withdrawal (1). The mechanisms
involved in GC-mediated adipose tissue distribution are not completely understood.
Part of GC action is regulated at a pre-receptor level by 11β-hydroxysteroid
dehydrogenase type 1 (11β-HSD1), an NADPH-dependent enzyme highly expressed in the
liver and adipose tissue, where it is co-localized with the GC receptor-α (GRα). In most
intact cells and tissues, 11β-HSD1 functions as a reductase, converting inactive cortisone
to active cortisol (2). GC regulate multiple processes in the adipose tissue: (a) they
influence fat cell size, so that enlarged abdominal fat cells are seen in Cushing’s syndrome
(3); (b) promote differentiation of human pre-adipocytes into mature adipocytes, increasing
fat cell number (4-5); and (c) activate lipolysis, releasing free fatty acids into circulation.
However, chronic exposure to cortisol, as seen in Cushing’s syndrome, is associated with a
2-3 fold increase in lipoprotein lipase activity, resulting in lower lipolytic capacity (3).
The nuclear receptor, peroxisome proliferator-activated receptor-γ (PPARγ), has a
central role in the entire adipogenesis program, promoting not only the conversion of
fibroblasts into adipocytes, but also the transdifferentiation of myoblasts into adipocytes
(6-7). Diverse promoters coupled with alternate splicing of the PPARγ gene gives rise to
three mRNA isoforms: PPARγ1 and PPARγ3, which encodes the same protein product,
and PPARγ2 which is identical to PPARγ1, except for an additional 28 amino-acids at its
N-terminal (8). PPARγ2 is virtually adipose tissue-specific, whereas PPARγ1 is widely
expressed (9), albeit more abundantly in the adipose tissue. Exposure of primary human
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13 sensitizing drugs, induces adipocyte differentiation (10), whereas in vitro loss-of-function experiments block this process (11). PPARγ activation is extensively manifested in the
mature fat cell phenotype, including morphological changes, lipid accumulation, and the
acquisition of insulin sensitivity (12). It has been hypothesized that tissue-specific
deregulation of cortisol metabolism may be involved in the complex pathophysiology of
the metabolic syndrome and obesity. Transgenic mice overexpressing 11β-HSD1 in
adipose tissue develop obesity with all features of the metabolic syndrome (13), whereas
11β-HSD1-knockout mice are protected from both (14). The bulk of evidences points to an
overexpression and increased activity of 11β-HSD1 also in human adipose tissue (15-18),
although there are contrasting data (19). Serum cortisol levels are not elevated in obesity
(20,21); instead, it may be locally increased in the adipose tissue due to a greater activity of
11β-HSD1.
Furthermore, several studies in humans have demonstrated that treatment with TZD
leads to selective accumulation of subcutaneous adipose tissue (SAT), with concomitant
lack of change or reduced adiposity of visceral depots (22). PPARγ ligands regulate genes
that may contribute to insulin sensitivity, such as 11β-HSD1. TZD and non-TZD PPARγ
agonists markedly reduce 11β-HSD1 gene expression in 3T3-L1 adipocytes (23), visceral
depots in rats (24) and subcutaneous depots in the human (25). However, PPARγ
expression in human adipose tissue is still a matter of debate (26-28).
There are striking similarities between Cushing’s and the metabolic syndrome, namely
the combination of visceral obesity, systemic arterial hypertension, dyslipidemia and
glucose intolerance. Besides, omental adipose stromal cells cultured with cortisol showed
increased activity of 11β-HSD1 (4,29). All these observations led to the speculation that
11β-HSD1 gene expression is upregulated in the visceral adipose tissue (VAT) of subjects
Relying on the evidences that 11β-HSD1 mRNA expression is closely related to 11β
-HSD1 activity (16), we examined the hypothesis that 11β-HSD1, GRα and PPARγ are
distinctively expressed in subcutaneous and visceral compartments of Cushing’s syndrome
and obese patients.
Research design and methods:
Subjects/Procedures
The study, previously approved by the local Ethics Committee, encompassed 10 female
patients with adrenal Cushing’s syndrome and 25 female control subjects who gave their
full, informed, written consent.
Body mass index (BMI), abdominal (AC) and hip circumferences were determined.
The control group was stratified according to BMI, into two subgroups: the nonobese
(BMI<30 Kg/m2) with 10 subjects, and the obese with 15, including 7 class I/II (BMI≥30
and <40 Kg/m2) and 8 class III (BMI≥ 40 Kg/m2). Exclusion criteria included the presence
of any inflammatory and/or malignant condition, diabetes, fasting impaired glucose (>5.6
mmol/liter) or current use of medications known to interfere with 11 -HSD1 expression or
function.
The diagnosis of adrenal Cushing’s syndrome was established by elevated 23:00h
salivary cortisol (SF), lack of cortisol suppression after overnight 1mg oral dexamethasone,
increased 24h urinary free cortisol excretion and undetectable plasma ACTH levels, and
confirmed in all on pathology grounds.
Approximately 2g subcutaneous and visceral adipose tissue samples were obtained
from the patients with Cushing’s syndrome during videolaparoscopic adrenalectomy for a
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15 surgery (colecistectomy in 13 and bariatric surgery in 10; ovarian cystectomy in one and
tubal sterilization in one). Samples were immediately frozen in dry ice and stored at -70°C
until RNA extraction.
The night before surgery, all patients remained fast after 22:00h and were instructed to
collect saliva at 23:00h in a specific collector (Salivette®, Sarstedt, Germany) after oral
hygiene with filtered water; the material was kept under refrigeration until the next
morning.
Measurement of salivary cortisol
Saliva samples were centrifuged at 2,000 rpm and kept frozen until assay. Salivary
cortisol was measured in 25µl saliva aliquots by an in-house radioimmunoassay (RIA)
without previous extraction or chromatography, as previously described (30). In brief, the
intra- and inter-assay coefficients of variation were 4.4% and 5.1%, respectively, with a
detection limit of 10 ng/dL.
Tissue preparation and reverse transcriptase (RT)
Total RNA was isolated from SAT and VAT by TRIzol reagent (Gibco BRL,
Gaithersburg, MD, USA), according to the manufacturer’s protocol. RNA was subjected to
DNase I digestion, and quantified using spectrophotometric analyses (ND-1000,
NanoDrop, Wilmington, DE, USA). RNA integrity was assessed by electrophoresis on 1%
agarose gel. A standard curve for each pair of primers was generated by serial dilution of
Real-Time PCR
PCR was performed in a 7000 Sequence Detection System (ABI Prism, Applied
Biosystems, Foster City, CA, USA) using the SYBRGreen core reaction kit (Applied
Biosystems). Primers used for 11β-HSD1, GRα, PPARγ1, and PPARγ2 mRNA
quantifications were as follows:
11β-HSD1, 5´-GCAGCCTCAGCACACTACATTG-3´ (forward),
5´-GGTGATGTGGTTGAGAATGAGC-3´ (reverse)
(GenBankTM accession number J00691);
GRα, 5´-CCCCAGGTAAAGAGACGAATG-3´ (forward),
5´-CGGTAAAATGAGAGGCTTGCA-3´ (reverse)
(GenBankTM accession number NM_030851);
PPARγ1, 5´-TGAACCACCCTGAGTCCTCACA-3´ (forward),
5´-CGTGTTCCGTGACAATCTGTCT-3´ (reverse)
(GenBankTM accession number NM_138712.3);
PPARγ2, 5´-GGCAATTGAATGTCGTGTCTGT-3´ (forward),
5´-TGCAAGGCATTTCTGAAACC-3´ (reverse)
(GenBankTM accession number NM_015869.4).
All reactions were multiplexed with the housekeeping human 18S gene with the
following sequence: 5´-GTAACCCGTTGAACCCCATT-3´ (forward),
5´-CCATCCAATCGGTAGTAGCG-3´ (reverse).
All results were confirmed using the housekeeping ARPO gene (data not shown).
Each sample was run in duplicate and the mean of duplicate was used to calculate
transcript level. Quantitative values for 11β-HSD1, GRα, PPARγ1, PPARγ2, and 18S
mRNA transcription were obtained from the threshold cycle (Ct) number, where the
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17 detected. Melting curves were generated at the end of every run to ensure product
uniformity. The relative target gene expression level was normalized on the basis of 18S
expression as endogenous RNA control.
ΔCt values of the samples were determined by subtracting the average Ct value of 11β
-HSD1, GRα, PPARγ1, and PPARγ2 mRNA from the average Ct value of the internal
control 18S gene.Reactions were performed as follows: 50°C for 2 min, 95°C for 10 min,
and then 50 cycles of 95°C for 15 sec and 60°C for 30s, and a dissociation cycle.
Statistical analysis
Statistical analysis was performed using the SPSS software 16.0.1 version for
Windows (SPSS Inc. Chicago, IL, USA). Comparisons between groups were performed by
ANOVA for variables with a normal distribution and Kruskal-Wallis for non-parametric
variables, whereas Pearson and Spearman tests were used to verify correlation between
variables. Multiple regression analyses were employed to adjust for the influence of BMI
and AC. The data are expressed as mean±SD, unless otherwise stated. Differences were
considered statistically significant when P was less than 5%.
RESULTS
Subjects characteristics
Clinical and biochemical characteristics of patients with CS, and nonobese and obese
subjects are shown in table 1. BMI of Cushing’s patients were similar to nonobese
(28.5±4.0 vs 25.5±2.2 Kg/m2), and significantly lower than obese class I/II and class III (33.5±3.7 Kg/m2; P=0.043 and 46.3±4.3 Kg/m2; P<0.0001, respectively). Abdominal and
(125±15.2 vs 97.8±17.7 cm; P<0.001 and 136.7±17.6 vs 103.9±11.8 cm; P<0.0001, respectively), nonobese (P<0.0001 for both) and obese I/II (P=0.031 and P=0.001,
respectively). Similar to BMI, abdominal and hip circumferences in Cushing’s syndrome
were closer to those in nonobese.
Salivary cortisol at 23:00h was noticeably higher in Cushing’s (1,114±805 ng/dL) than
in nonobese (182±105 ng/dL; P<0.0001), obese class I/II (126.6±70 ng/dL; P<0.0001) and
class III (108.5±40 ng/dL; P<0.001), but did not differ among the latter 3 groups.
When no differences were observed in a specific variable between class I/II and class III
obese, all of these patients were grouped together. Means (±SD) of age, BMI, AC and SF
in the whole obese group were: 49.6±15.4 years; 40.3±7.6 Kg/m2; 115.6±16.4 cm and
118±57 ng/dL, respectively.
Table 1. Clinical and biochemical characteristics of the female patients studied.
Cushing’s syndrome (n=10)
Nonobese (n=10)
Obese class I/II (n=7)
Obese class III (n=8) Age (years) 42.1±17.7
[21 – 81]
41.5 ±17 [20 – 80]
58.6±14.5 [42 – 79]
41.9±12 [27 – 57]
BMI (Kg/m2) 28.5±4.0**,*** [22.6 – 35.3]
25.5±2.2 [21.3 - 28.3]
33.5±3.7† [30 – 39]
46.3±4.3††,& [27 – 57]
Abdominal
circumference (cm)
97.8±17.7*** [96 – 120]
87.9±7.3 [78 – 100]
104.8±10.3 [90-120]
125±15.2††,& [105-153]
Hip circumference (cm)
103.9±11.8*** [84 – 130]
96.5±4.3 [91 – 103]
110.5±7.8 [102 – 120]
136.7±17.6††,& [112 – 158]
23:00h Salivary cortisol (ng/dL)
1,114±805*,**,*** [293 – 2,680]
182±105 [57 – 347]
126.6±70 [22 – 235]
108.5±40 [53 – 152]
All data are mean±SD, followed by range in brackets.
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19 Comparison of 11β-HSD1 and GRα mRNA levels in subcutaneous and visceral
adipose tissue
In Cushing’s syndrome, 11β-HSD1 expressions in SAT and VAT were not statistically
different from those in nonobese (0.35±0.1 vs 0.17±0.06 and 1.3±0.56 vs 0.95±0.36, respectively) (Figure 1), whereas they were significantly lower than in obese only in SAT
(0.35±0.1 vs 0.64±0.3; P=0.01). In obese, 11β-HSD1 mRNA levels were higher than in nonobese patients both in SAT and VAT (0.64±0.3 vs 0.17±0.06; P<0.0001 and 1.6±0.7 vs 0.95±0.36; P=0.019, respectively).
GRα mRNA expressions in SAT of Cushing’s and obese subjects were significantly
lower than in nonobese (0.14±0.08 and 0.17± 0.09 vs 0.36±0.1; P<0.0001 for both) (Figure 1). However, in VAT, GRα mRNA expression was higher in obese class I/II than in
Cushing’s patients (2.81±1.6 vs 1.07±0.26; P<0.05).
Cushing’s
11β-HSD1/18S mRNA
VAT SAT
Obese Nonobese
0 1.0
0.5 2.0
1.5 0 1.0
0.5 2.0
1.5
GRα/18S mRNA
*
* * * * *
* *
* *
11β-HSD1: * P<0.01 (vs obese), ** P<0.001 (vs nonobese), *** P<0.02 (vs nonobese); GRα: * P<0.0001 (vs nonbese), ** P<0.05 (class I/II vs CS).
A significant correlation between visceral, but not subcutaneous, 11β-HSD1 and GRα
mRNA levels was observed when the analyses was performed either with the whole group
(nonobese, obese and Cushing’s, r=0.6; P<0.0001) or with Cushing’s syndrome and obese
subjects individually (r=0.87; P=0.001 and r=0.74; P=0.002, respectively). (Figure 2)
R=0.6 P<0.0001 0 1 2 3 4 5 6
0 0,5 1 1,5 2 2,5 3 3,5
GR α m RNA /1 8 S
11βHSD-1 mRNA/18S
0 1 2 3 4 5 6
0 0,5 1 1,5 2 2,5 3 3,5
11βHSD-1 mRNA/18S
GR α m R NA /18S
11βHSD-1 mRNA/18S
GR α m RNA /1 8 S 0 1 2 3 4 5 6
0 0,5 1 1,5 2 2,5
R=0.87 P=0.001
R=0.74
P=0.002 Whole group
Cushing’s syndrome Obese subjects
r=0.87 P=0.001 r=0.74 P=0.002 r=0.6 P<0.0001
Figure 2. Correlations between 11β-HSD1 and GRα mRNA expressions in visceral adipose tissue of the whole group of patients (nonobese, obese and Cushing’s) and in Cushing’s and obese patients separately.
Comparison of PPARγ and 11β-HSD1 levelsin subcutaneous and visceral adipose
tissue
PPARγ1 mRNA expression in SAT of obese class III was significantly higher than in
nonobese patients (1.84±0.8 vs 0.79±0.2; P<0.05), whereas in VAT it was significantly lower (1.27± 0.6 vs 3.1±1.6; P=0.013). (Figure 3)
PPARγ2 mRNA expression was significantly reduced in SAT of Cushing’s and obese
class I/II and class III patients as compared to nonobese (0.05±0.03, 0.090±0.1, and
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21 significantly greater expression of PPARγ2 mRNA in VAT than obese class III and
Cushing’s patients (1.2±0.8 vs 0.14±0.9 and 0.2±0.13; P<0.05, respectively).
Cushing’s
PPARγ1/18S mRNA
VAT SAT
Obese I I I Nonobese
0 0.8 1.2
PPARγ2/18S mRNA
0.4 0 2.0 3.0
1.0
Obese I / I I
*
*
*
* *
* * *
PPARγ1: * P<0.05 (vs nonbese), ** P<0.013 (vs nonobese); PPARγ2: * P<0.05 (vs nonbese), ** P<0.05 (vs Cushing’s).
Figure 3. PPARγ1 and PPARγ2 mRNA expressions in subcutaneous
(SAT) and visceral adipose tissues (VAT) of nonobese, obese (classes I/II and III) and Cushing’s patients.
In the whole group, there was a strong positive correlation between 11β-HSD1 and
PPARγ1 mRNA expressions in SAT (r= 0.79; P<0.001), but not in VAT (Figure 4); in
contrast, a strong positive correlation between both was observed in VAT of Cushing’s
patients alone (r=0.88; P=0.001).
In addition, when the whole group was analyzed, a significant and inverse correlation
between 11β-HSD1 and PPARγ2 mRNA levels was observed in SAT (r=-0.54; P=0.01),
but not in VAT. Instead, a strong positive correlation was observed between both in obese
0 1 2 3 4 5 6 7
0 0,5 1 1,5
Obese class I/II 0 0,2 0,4 0,6 0,8 1
0 0,5 1 1,5
R=0.77
P<0.001 R=-0.54P=0.01
11β-HSD1 mRNA/18S
0 0,5 1 1,5 2 2,5
0 1 2 3
R=0.83 P=0.01 PPA R γ 2 m R N A /1 8S
11β-HSD1 mRNA/18S 0
1 2 3 4
0 0,5 1 1,5 2 2,5
R=0.87 P=0.001
11β-HSD1 mRNA/18S
PPA R γ 1 m R N A /18S
B) PPARγ2 in the whole group
11β-HSD1 mRNA/18S
PPA R γ 2 m R NA/18S VAT PPA R γ 1 m R N A /18S PPA R γ 2 m R N A /18S SAT Whole group r=-0.54 P=0.01 SAT Cushing’s VAT r=0.88 P=0.001 r=0.79 P<0.001 r=0.83 P=0.01
Figure 4. Correlations between 11β-HSD1 and PPARγ1 and PPARγ2 expressions in subcutaneous adipose tissue (SAT) of the whole group of patients (upper 2 panels) and in visceral adipose tissue (VAT) in Cushing’s and obese class I/II patients separately (lower 2 panels).
Association of 11β-HSD1, GRα, and PPARγ mRNA with anthropometric and
biochemical parameters
In the whole group, 11β-HSD1 expressions in SAT and VAT were positively and
significantly correlated with BMI (r=0.43; P=0.01 and r=0.35; P=0.041, respectively) and
with AC (r=0.36; P=0.033 and r=0.4; P=0.008, respectively) (Table 2). Individually, 11β
-HSD1 expression correlated with BMI in SAT of nonobese (r=0.63; P=0.049) and with AC
in VAT of Cushing’s patients (r=0.66; P=0.038). In contrast, GRα mRNA levels in SAT,
but not in VAT, were negatively correlated with BMI and AC in the whole group (r=-0.37;
P=0.028 and r=-0.45; P=0.007, respectively), but did not correlate with either in individual
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23 In the whole group, a positive and significant correlation was observed in SAT between
PPARγ1 and both BMI and AC (r=0.50; P=0.002 and r=0.35; P=0.034, respectively),
whereas an inverse correlation was observed between PPARγ2 and AC (r=-0.59;
P<0.0001). In contrast, PPARγ1 and PPARγ2 gene expressions correlated negatively with
BMI in VAT (significant only for PPARγ1: r= -0.4; P= 0.017) (Table 3), whereas no
correlations were seen for AC, except for PPARγ1 in Cushing’s alone (r=0.68; P=0.028).
Table 2. Correlation of 11β-HSD1, GRα, PPARγ1, and PPARγ2 mRNA levels with anthropometric parameters in the whole group (nonobese, obese and Cushing’s patients).
11β-HSD1 GRα PPARγ1 PPARγ2
SAT VAT SAT VAT SAT VAT SAT VAT
BMI (Kg/m2 )
r= +0.43 P= 0.01
r= +0.35 P= 0.04
r= -0.37 P= 0.028
NS r= +0.5 P= 0.002
r= -0.4 P= 0.017
NS NS
AC (cm) r= +0.36 P= 0.03
r= +0.40 P= 0.008
r= -0.45 P= 0.007
NS r= +0.35 P= 0.034
NS r= -0.59
P<0.001 NS
BMI: Body mass index; AC: Abdominal circumference
Salivary cortisol had a strong positive correlation with PPARγ1 expression (r= 0.9;
P=0.037) in VAT but not in SAT of obese class III patients, and did not correlate with 11β
-HSD1, GRα, or PPARγ2 in any group or altogether in VAT or SAT.
When 11β-HSD1, GRα, PPARγ1, and PPARγ2 were analyzed altogether in the whole
group (by stepwise multiple regression), PPARγ2 in SAT was found to be the best
predictor of both AC and BMI; however, when only 11β-HSD1 and GRα genes are
considered, the former was the best predictor of BMI and the latter the best predictor of
DISCUSSION
In our study, there were no significant differences between 11β-HSD1 mRNA
expressions in SAT and VAT from Cushing’s patients as compared to nonobese controls.
Furthermore, 11β-HSD1 expression in VAT was greater in obese than in Cushing’s
patients, a finding confirmed in the only published study in Cushing’s syndrome (34) that
reported no differences in 11β-HSD1 mRNA expression in omental biopsies as compared
to normal weight controls. Of interest, both ours and Mariniello’s study (34) are not in
accordance with previous in vitro experiments, which demonstrated increased activity and expression of 11β-HSD1 in human omental adipose stromal cells cultured with cortisol,
suggesting that obesity could be “Cushing’s disease of the omentum” (5), a finding
recently confirmed by Lee et al (29). Thus, it is anticipated that both systemic
hypercortisolism and cortisol generated from 11β-HSD1 in an autocrine manner, could
promote adipocyte differentiation (4) and proliferation (35), as seen in stromal cells in
vitro. However, chronic exposure to cortisol in vivo, as observed in CS in our study, seems instead to downregulate 11β-HSD1 expression in both subcutaneous and visceral
compartments and 11β-HSD1 expression is remarkably correlated with its activity (16).
Also, we demonstrate that 11β-HSD1 mRNA expression is upregulated in both SAT and
VAT of obese subjects. These findings are in agreement with preliminary studies
performed in SAT (15-18). Although VAT appears biologically more active than SAT and
responsible for obesity-related complications and increased mortality (31), there is only a
few controversial studies in VAT in obesity: one observed increased 11β-HSD1 expression
(32) and others did not (19,33). Despite similarities between Cushing’s syndrome and the
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25 The expression of PPARγ isoforms in human adipose depots and their correlation with
obesity parameters are vastly controversial in the literature. Previous studies have shown:
(a) increased PPARγ2 levels in obesity and no correlations of PPARγ1 with BMI (26); (b)
increased PPARγ2 levels in obesity and inverse correlation of PPARγ1 with BMI (28) and
inverse correlation of PPARγ2 with BMI (27); and (c) increased levels of PPARγ1 in
overweight patients (36) and no correlations with BMI (37). The dispute could be partially
explained by the considerable variability of the parameters studied, such as the adipose
compartment (whole versus freshly isolated tissue), gender, degree of obesity, transcriptional factors involved in fat storage control (27) and in short-term regulators (37),
including degree of intra-operatory stress.
In our study, PPARγ1 expression was augmented in SAT and reduced in VAT of obese
subjects, whereas PPARγ2 was reduced in SAT of obese and Cushing’s and augmented in
VAT of only class I/II obese patients. In addition, PPARγ1 was positively correlated with
BMI and AC in SAT and inversely correlated in VAT (only BMI). Nevertheless, PPARγ
expression in CS is difficult to predict, as to date there is only one report in SAT of
untreated Cushing’s patients that showed a decreased PPARγ2/PPARγ1ratio, in agreement
with our data (38).
Although 11β-HSD1 mRNA expression was positively correlated with BMI and AC
both in SAT and VAT of the whole group, as shown in other studies (16-18,34,39), it did
not correlate with systemic cortisol. Salivary cortisol levels were increased in Cushing’s
syndrome, but were not associated with either 11β-HSD1 or GRα expressions. The lack of
association between 11β-HSD1 and SF was also observed in the other groups individually
or altogether, suggesting that its expression in whole adipose tissue is not regulated by
We also observed a lack of correlation between SF and PPARγ mRNA expression
(except for PPARγ1 in obese class III). However, 11β-HSD1 was positively correlated
with PPARγ1 in the whole group in SAT and in Cushing’s patients in VAT, and inversely
correlated with PPARγ2 in the whole group in SAT. Although there are no reports to date
on correlations between these genes and Cushing’s syndrome, indirect evidences point to
regulation of PPARγ expression by tissue levels of cortisol generated in an autocrine
manner. Treatment with the PPARγ agonist rosiglitazone during 8 weeks reduced 11β
-HSD1 expression and activity in subcutaneous fat of male volunteers with impaired
glucose tolerance (25). This was also observed in patients with type 2 diabetes after a
12-week treatment period, but not on a short-term period (5 12-weeks) in healthy men (40).
Moreover, in this same study, Wake DJ et al. found no reduction in adipose 11β-HSD1
activity with glucocorticoid blockade with RU486 and metyrapone alone, but a significant
reduction when rosiglitazone was added (results were limited to SAT). In addition,
experiments in rats showed that metabolic response to rosiglitazone and reduction in 11β
-HSD1 expression in white adipose tissue is not influenced by adrenalectomy (41).
Therefore, taken altogether, it is tempting to assume that it is the intra-adipocyte
glucocorticoid concentration that regulates PPARγ expression, not its serum levels.
GRα mRNA expression in VAT also showed a strong and positive correlation with
11β-HSD1 in the whole group and in obese and Cushing’s groups separately, strengthen
the theory that co-expression of these two genes may amplify glucocorticoid action locally
(2). Additionally, the inverse correlation of GRα mRNA expression with BMI and AC in
SAT is in agreement with Zoi et al (32). However, they observed an inverse correlation of
GRα mRNA expression in omental depots with BMI and visceral adiposity (32). Besides,
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27 compensatory downregulation to increased 11β-HSD1. On the other hand, GRα mRNA
expression in VAT of Cushing’s and obese subjects did not undergo downregulation,
despite the 11β-HSD1 increment in obese. Our results with the obese patients are in
keeping with a previous study (42) that also demonstrated a significant decrease in GRα
mRNA levels in SAT, whereas this phenomenon was not observed in the visceral adipose
compartment. Although 11β-HSD1 expression was not evaluated, the work of
Boullu-Ciocca (42) suggests that the downregulation of GRα gene in subcutaneous compartments
of obese patients is a consequence of local hypercortisolism due to 11β-HSD1
overexpression, as observed in our study. The absence of this protective mechanism in
VAT could contribute to obesity related metabolic complications. As for Cushing’s
syndrome, this is the first report to evaluate GRα expression in adipose tissue. Chronic
hypercortisolism in vivo seems to result in downregulation of GRα gene expression in both SAT and VAT. Indeed, health volunteers treated for one week with prednisolone had a
50% decrease both in GR protein and mRNA levels in subcutaneous abdominal biopsies
(43).
It has been recognized that large abdominal adipose depots are closely linked to
cardiovascular complications. However, some evidences suggest that 11β-HSD1 may not
hold a good relationship with body composition in VAT (19,33). Indeed, we found that the
best predictors of BMI and AC in SAT, but not in VAT, were respectively 11β-HSD1 and
GRα when only these two genes are considered, and PPARγ2 when all four genes are
analyzed. Also, biopsying subcutaneous depots is a much easier procedure, so that these
observations will facilitate methodology of further studies.
11β-HSD1 is now emerging as a key component in homeostatic adaptation, rather than
the cause of visceral obesity or metabolic syndrome. Recent studies suggest that the
whole-body rates of cortisol generated by 11β-HSD1 (44). Moreover, 11β-HSD1 undergoes
downregulation in the adipose tissue of high-fat fed mice (45). Accordingly, the lack of
11β-HSD1 increase in Cushing’s syndrome may suggest a protective mechanism against
the metabolic complications. Indeed, when the opposite occurs, e.g., weight loss in simple
obesity, 11β-HSD1 undergoes upregulation (46), although this is not a universal finding
(18). Thus, there are several evidences suggesting that 11β-HSD1 adjusts local cortisol
concentration independently of its circulating levels.
In summary, 11β-HSD1 gene expression is downregulated in Cushing’s syndrome and
is up-regulated in obesity. In addition, GRα and PPARγ2 mRNA levels are downregulated,
respectively in SAT and in SAT and VAT of Cushing’s patients. The expected
upregulation of 11β-HSD1 and PPARγ gene expressions in VAT of Cushing’s patients was
not observed. Finally, based on the evidences that 11β-HSD1 mRNA expression is closely
coupled to its activity, autocrine cortisol production, but not its serum levels, seems to play
ARTIGO 1
29 ACKNOWLEDGEMENTS
We are grateful to our colleagues, Viviane Chaves, Martha Huayllas, Flávia Amanda
Barbosa and Marcos Neres for referring patients with Cushing´s syndrome. We thank
Lilian Hayashi, Sâmia Cavassani and Kelly de Oliveira for measurement of salivary
cortisol and for technical support.
This study was supported in part by a grant from Fundação de Amparo a Pesquisa do
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