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1.
J Intern Med ; 276(4): 364-77, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24717051

RESUMO

Obesity and related diseases are a major cause of human morbidity and mortality and constitute a substantial economic burden for society. Effective treatment regimens are scarce, and new therapeutic targets are needed. Brown adipose tissue, an energy-expending tissue that produces heat, represents a potential therapeutic target. Its presence is associated with low body mass index, low total adipose tissue content and a lower risk of type 2 diabetes mellitus. Knowledge about the development and function of thermogenic adipocytes in brown adipose tissue has increased substantially in the last decade. Important transcriptional regulators have been identified, and hormones able to modulate the thermogenic capacity of the tissue have been recognized. Intriguingly, it is now clear that humans, like rodents, possess two types of thermogenic adipocytes: the classical brown adipocytes found in the interscapular brown adipose organ and the so-called beige adipocytes primarily found in subcutaneous white adipose tissue after adrenergic stimulation. The presence of two distinct types of energy-expending adipocytes in humans is conceptually important because these cells might be stimulated and recruited by different signals, raising the possibility that they might be separate potential targets for therapeutic intervention. In this review, we will discuss important features of the energy-expending brown adipose tissue and highlight those that may serve as potential targets for pharmacological intervention aimed at expanding the tissue and/or enhancing its function to counteract obesity.


Assuntos
Tecido Adiposo Marrom/fisiologia , Obesidade/fisiopatologia , Obesidade/terapia , Adipócitos/classificação , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo Energético , Hormônios/fisiologia , Humanos , Gordura Subcutânea/citologia , Termogênese
3.
Nat Genet ; 20(4): 374-6, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9843211

RESUMO

Fkhl0 is a member of the forkhead family of winged helix transcriptional regulators. Genes encoding forkhead proteins are instrumental during embryogenesis in mammals, in particular during development of the nervous system. Here we report that mice with a targeted disruption of the Fkh10 locus exhibit circling behaviour, poor swimming ability and abnormal reaching response-all common findings in mice with vestibular dysfunction. These animals also fail to elicit a Preyer reflex in response to a suprathreshold auditory stimulation, as seen in mice with profound hearing impairment. Histological examination of the inner ear reveals a gross structural malformation of the vestibulum as well as the cochlea. These structures have been replaced by a single irregular cavity in which neither proper semicircular ducts nor cochlea can be identified. We also show that at 9.5 days post coitum (dpc), Fkh10 is exclusively expressed in the otic vesicle. These findings implicate Fkh10 as an early regulator necessary for development of both cochlea and vestibulum and identify its human homologue FKHL10 as a previously unknown candidate deafness gene at 5q34.


Assuntos
Orelha Interna/embriologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Estimulação Acústica , Animais , Comportamento Animal , Orelha Interna/fisiopatologia , Fatores de Transcrição Forkhead , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Doenças Vestibulares/genética , Doenças Vestibulares/fisiopatologia
4.
Int J Obes (Lond) ; 34 Suppl 1: S43-6, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20935666

RESUMO

Obesity is endemic in many regions of the world and a forerunner of several serious and sometimes fatal diseases such as ischemic heart disease, stroke, kidney failure and neoplasia. Although we know its origin--it results when energy intake exceeds energy expenditure--at present, the only proven therapy is bariatric surgery. This is a major abdominal procedure that, for reasons that are largely unknown (it cannot be explained solely by a reduction in ventricular volume), significantly reduces energy intake, but because of cost and limited availability, it will most likely be reserved for only a small fraction of those who stand to gain from effective antiobesity treatment. Clearly, alternative ways to treat obesity are needed. Another way to combat excessive accumulation of white adipose tissue would be to increase energy expenditure. Rodents, hibernators and human infants all have a specialized tissue--brown adipose tissue (BAT)--with the unique capacity to regulate energy expenditure by a process called adaptive thermogenesis. This process depends on the expression of uncoupling protein-1 (UCP1), which is a unique marker for BAT. UCP1 is an inner mitochondrial membrane protein that short circuits the mitochondrial proton gradient, so that oxygen consumption is no longer coupled to adenosine triphosphate synthesis. As a consequence, heat is generated. Mice lacking ucp-1 are severely compromised in their ability to maintain normal body temperature when acutely exposed to cold and they are also prone to become obese. We have shown that, in mice, BAT protects against diet-induced obesity, insulin resistance and type 2 diabetes. This is based on prevention of excessive accumulation of triglyceride in non-adipose tissues such as muscle and liver. Ectopic triglyceride storage at these locations is associated with initiation of insulin resistance and, ultimately, development of type 2 diabetes.


Assuntos
Tecido Adiposo Marrom/fisiologia , Resistência à Insulina/fisiologia , Canais Iônicos/fisiologia , Proteínas Mitocondriais/fisiologia , Obesidade/fisiopatologia , Termogênese/fisiologia , Animais , Cirurgia Bariátrica , Diabetes Mellitus Tipo 2/etiologia , Ingestão de Energia/fisiologia , Humanos , Resistência à Insulina/genética , Canais Iônicos/genética , Camundongos , Proteínas Mitocondriais/genética , Obesidade/genética , Obesidade/cirurgia , Proteína Desacopladora 1
5.
J Clin Invest ; 96(6): 2914-23, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8675663

RESUMO

The brown fat-specific mitochondrial uncoupling protein (UCP) provides a mechanism for generating heat by uncoupling respiration and oxidative phosphorylation. It has been suggested that this system of thermogenesis can provide a defense against obesity. To test this idea, we created a transgenic mouse in which the fat-specific aP2 gene promoter directed Ucp expression in white fat and provided for the constitutive expression of Ucp in brown fat. Transgenic mice showed both Ucp mRNA and immunoreactive UCP in white fat at 2-10% the level normally measured in brown fat. A reduction in subcutaneous fat of aP2-Ucp C57BL/6J mice was observed at 3 mo of age. When the transgene was expressed in Avy genetically obese mice reductions in total body weight and subcutaneous fat stores were observed. Female transgenic Avy mice at 13 mo of age weighed 35 grams, a weight indistinguishable from nontransgenic C57BL/6J mice. Gonadal fat showed an increase in a novel adipocyte derivative that did not accumulate lipids and that constituted approximately 80% of the mass of the tissue in Avy transgenic. A major effect of aP2-Ucp in brown fat was to reduce endogenous gene expression by as much as 95%. The results suggest that UCP synthesized from the aP2 gene promoter is thermogenically active and capable of reducing fat stores.


Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Variação Genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteína P2 de Mielina/genética , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Obesidade/genética , Obesidade/prevenção & controle , Regiões Promotoras Genéticas , Tecido Adiposo/fisiologia , Tecido Adiposo/fisiopatologia , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Marrom/fisiopatologia , Animais , Sequência de Bases , Regulação da Temperatura Corporal , Peso Corporal , Primers do DNA , Éxons , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Feminino , Canais Iônicos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Proteínas Mitocondriais , Dados de Sequência Molecular , Tamanho do Órgão , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Caracteres Sexuais , Proteína Desacopladora 1
6.
Mol Cell Biol ; 12(10): 4622-33, 1992 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1406652

RESUMO

When preadipocytes differentiate into adipocytes, several differentiation-linked genes are activated. Lipoprotein lipase (LPL) is one of the first genes induced during this process. To investigate early events in adipocyte development, we have focused on the transcriptional activation of the LPL gene. For this purpose, we have cloned and fused different parts of intragenic and flanking sequences with a chloramphenicol acetyltransferase reporter gene. Transient transfection experiments and DNase I hypersensitivity assays indicate that several positive as well as negative elements contribute to transcriptional regulation of the LPL gene. When reporter gene constructs were stably introduced into preadipocytes, we were able to monitor and compare the activation patterns of different promoter deletion mutants at selected time points representing the process of adipocyte development. We could delimit two cis-regulatory elements important for gradual activation of the LPL gene during adipocyte development in vitro. These elements, LP-alpha (-702 to -666) and LP-beta (-468 to -430), contain a striking similarity to a consensus sequence known to bind the transcription factors HNF-3 and fork head. Results of gel mobility shift assays and DNase I and exonuclease III in vitro protection assays indicate that factors with DNA-binding properties similar to those of the HNF-3/fork head family of transcription factors are present in adipocytes and interact with LP-alpha and LP-beta. We also demonstrate that LP-alpha and LP-beta were both capable of conferring a differentiation-linked expression pattern to a heterolog promoter, thus mimicking the expression of the endogenous LPL gene during adipocyte differentiation. These findings indicate that interactions with LP-alpha and LP-beta could be a part of a differentiation switch governing induction of the LPL gene during adipocyte differentiation.


Assuntos
Tecido Adiposo/citologia , Regulação Enzimológica da Expressão Gênica , Lipase Lipoproteica/genética , Regiões Promotoras Genéticas , Células 3T3 , Tecido Adiposo/metabolismo , Animais , Sequência de Bases , Southern Blotting , Diferenciação Celular/genética , Clonagem Molecular , DNA , Humanos , Lipase Lipoproteica/metabolismo , Camundongos , Dados de Sequência Molecular , Mapeamento por Restrição , Fatores de Transcrição/metabolismo
7.
Mol Cell Biol ; 14(1): 59-67, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8264627

RESUMO

Previous studies on the regulation of a Ucp minigene in transgenic mice demonstrated that the sequences necessary for brown-fat-specific expression and inducibility by norepinephrine were located in the 5' flanking region between 1 and 2.8 kb from the transcriptional start site. We have investigated this region in more detail in cultured mouse brown adipocyte tumor cells. Deletion analysis of two types of chloramphenicol acetyltransferase reporter gene constructs under control of either the Ucp promoter or a heterologous herpes simplex virus-tk promoter defined an enhancer in a 220-bp HindIII-XbaI fragment which was essential for both brown fat specificity and norepinephrine inducibility. Site-directed mutagenesis of the reporter gene constructs established that independent mutations to a cyclic AMP-responsive element (CRE-2) or one of two TTCC motifs (BRE [brown fat regulatory element]), all within 17 bp, eliminated transient expression. Competitive DNA mobility shift assays with probes of the CRE and BRE motifs indicate that nuclear proteins interact with these motifs in a cooperative, synergistic manner. While these CRE-BRE probes do not show changes in binding which is dependent on norepinephrine treatment, a probe containing a third TTCC motif located 130 bp downstream of BRE-1 does show this dependency. The results indicate that a complex interaction of the CRE and BRE motifs, which cannot be functionally separated, control Ucp expression.


Assuntos
Tecido Adiposo Marrom/metabolismo , Proteínas de Transporte/genética , Elementos Facilitadores Genéticos , Proteínas de Membrana/genética , Animais , Sequência de Bases , Células Cultivadas , Mapeamento Cromossômico , DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reguladores , Canais Iônicos , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Norepinefrina/farmacologia , Deleção de Sequência , Transfecção , Desacopladores/metabolismo , Proteína Desacopladora 1
8.
Mol Cell Biol ; 9(11): 4587-95, 1989 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2601690

RESUMO

Adipocytes constitute a major part of the bone marrow stroma in vivo and may play an active role in lymphohematopoiesis. Earlier studies had shown that the bone marrow stromal cell clone BMS2 was capable of adipocyte differentiation in vitro, in addition to its well-defined ability to support B lymphopoiesis. We now demonstrate that the process of adipogenesis in this functional bone marrow stromal cell clone can be inhibited by the cytokines interleukin-1 alpha, tumor necrosis factor, and transforming growth factor beta. Exposure of preadipocyte BMS2 cells to these agents blocked the induction of adipocyte differentiation as assessed by morphologic criteria and analysis of the neutral lipid content. Both interleukin-1 alpha and tumor necrosis factor elicited a rapid transient elevation in the steady-state mRNA levels of c-fos, c-jun, and JE. When added to differentiated adipocytes, the three cytokines continued to act as adipogenic antagonists. This was indicated by concentration- and time-dependent decreases in the activity of an adipocyte-specific enzyme, lipoprotein lipase. These changes in enzyme activity correlated directly with a decrease in steady-state levels of lipoprotein lipase mRNA. Another RNA marker of adipocyte differentiation (adipsin) was less influenced by the adipogenic antagonists. This may reflect the longer half-life of this mRNA transcript compared with those of lipoprotein lipase. Our results dramatically demonstrate that the differentiation state of bone marrow stromal cells can be modulated by exogenous factors in vitro. It is also the first report that transformation growth factor beta regulates the activity of lipoprotein lipase. These data suggest potential physiologic actions for these cytokines in vivo within the overall context of lymphohematopoiesis.


Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea , Interleucina-1/farmacologia , Fatores de Crescimento Transformadores/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Tecido Adiposo/metabolismo , Northern Blotting , Medula Óssea/metabolismo , Diferenciação Celular , Linhagem Celular , Fator D do Complemento , Relação Dose-Resposta a Droga , Indução Enzimática , Lipídeos/análise , Lipase Lipoproteica/biossíntese , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Proto-Oncogenes , RNA Mensageiro/análise , Serina Endopeptidases/genética , Fatores de Tempo
9.
Lymphology ; 39(2): 84-94, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16910099

RESUMO

FOXC2 mutations cause the lymphatic/ocular disorder Lymphedema-Distichiasis (LD), and Foxc2 haploinsufficient mice mimic this disorder. To determine if FOXC2 overexpression might also cause lymphatic and/or ocular abnormalities, we performed dynamic lymphatic imaging (Evans blue dye), ocular tissue examination, and metabolic profiles in mice: transgenic for FOXC2 with an adipocyte (aP2) promoter (aP2-FOXC2 Tg), heterozygous for targeted disruption of Foxc2 (Foxc2+/-), or compound heterozygous and transgenic (Foxc2+/-, Tg) compared to wild-type controls (WT). Foxc2+/-; aP2-FOXC2 Tg; and Foxc2+/-, Tg, exhibited LD's distinctive hyperplastic lymphatic phenotype characterized by increased number of lymphatic channels and lymph nodes as well as retrograde lymph reflux. Foxc2+/-, and Foxc2+/-, Tg but not aP2-FOXC2 Tg or WT showed an abnormal ocular phenotype. Previously described alterations in brown/ white fat distribution and lean phenotype in aP2-FOXC2 transgenics were confirmed. AP2-FOXC2 Tg immunohistochemistry disclosed aberrant FOXC2 expression in ectopic sites, especially embryonic heart. Lymphatic system links with fat metabolism are discussed.


Assuntos
Modelos Animais de Doenças , Pestanas/anormalidades , Fatores de Transcrição Forkhead/fisiologia , Linfedema/genética , Adipócitos/química , Animais , Proteínas de Ligação a Ácido Graxo/genética , Ácidos Graxos/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Glucose/metabolismo , Heterozigoto , Humanos , Técnicas Imunoenzimáticas , Insulina/metabolismo , Anormalidades Linfáticas/genética , Anormalidades Linfáticas/patologia , Linfedema/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos
10.
Biochim Biophys Acta ; 1535(2): 174-85, 2001 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-11342006

RESUMO

UNLABELLED: It is well-established that high levels of cAMP or glucose can produce insulin resistance. The aim of this study was to characterize the interaction between these agents and insulin with respect to adipose tissue/muscle glucose transporter isoform (glucose transporter 4, GLUT4) gene regulation in cultured 3T3-F442A adipocytes and to further elucidate the GLUT4-related mechanisms in insulin resistance. Insulin (10(4) microU/ml) treatment for 16 h clearly increased GLUT4 mRNA level in cells cultured in medium containing 5.6 mM glucose but not in cells cultured in medium with high glucose (25 mM). 8-Bromo-cAMP (1 or 4 mM) or N(6)-monobutyryl cAMP, a hydrolyzable and a non-hydrolyzable cAMP analog, respectively, markedly decreased the GLUT4 mRNA level irrespective of glucose concentrations. In addition, these cAMP analogs also inhibited the upregulating effect of insulin on GLUT4 mRNA level. Interestingly, the tyrosine phosphatase inhibitor vanadate (1-50 microM) clearly increased GLUT4 mRNA level in a time- and concentration-dependent manner. Furthermore, cAMP-induced inhibition of the insulin effect was also prevented by vanadate. In parallel to the effects on GLUT4 gene expression, both insulin, vanadate and cAMP produced similar changes in cellular GLUT4 protein content and cAMP impaired the effect of insulin to stimulate (14)C-deoxyglucose uptake. In contrast, insulin, vanadate or cAMP did not alter insulin receptor (IR) mRNA or the cellular content of IR protein. IN CONCLUSION: (1) Both insulin and vanadate elicit a stimulating effect on GLUT4 gene expression in 3T3-F442A cells, but a prerequisite is that the surrounding glucose concentration is low. (2) Cyclic AMP impairs the insulin effect on GLUT4 gene expression, but this is prevented by vanadate, probably by enhancing the tyrosine phosphorylation of signalling peptides and/or transcription factors. (3) IR gene and protein expression is not altered by insulin, vanadate or cAMP in this cell type. (4) The changes in GLUT4 gene expression produced by cAMP or vanadate are accompanied by similar alterations in GLUT4 protein expression and glucose uptake, suggesting a role of GLUT4 gene expression for the long-term regulation of cellular insulin action on glucose transport.


Assuntos
Adipócitos/efeitos dos fármacos , AMP Cíclico/farmacologia , Glucose/farmacologia , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Células 3T3 , Adipócitos/metabolismo , Animais , Western Blotting , Meios de Cultura , Desoxiglucose/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/análise , Transportador de Glucose Tipo 4 , Resistência à Insulina/genética , Camundongos , Proteínas de Transporte de Monossacarídeos/análise , Proteínas de Transporte de Monossacarídeos/genética , Proteínas Tirosina Fosfatases/antagonistas & inibidores , RNA Mensageiro/análise , Vanadatos/farmacologia
11.
J Mol Biol ; 296(2): 351-9, 2000 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-10669593

RESUMO

Transcription factors of the forkhead type share a highly conserved DNA-binding domain of about 100 amino acid residues. FREAC-11, expressed in adipocytes, belongs to this class. Here, we report on NMR studies that established the three-dimensional structure of the FREAC-11, DNA-binding domain. Although apparent similarities to the structures of other members within the forkhead family are observed, the structure also reveals some remarkable differences. Along with the complementary dynamics, the data provide insight into the fundamentals of sequence specificity within a highly conserved motif.


Assuntos
Tecido Adiposo/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , DNA/genética , DNA/metabolismo , Fatores de Transcrição Forkhead , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Soluções , Especificidade por Substrato
12.
Mol Endocrinol ; 7(8): 972-8, 1993 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8232317

RESUMO

Buffalo rat liver cells were stably transfected with an expression vector containing rat GH (rGH) receptor cDNA. Transfected cells expressed rGH receptor mRNA and specifically bound GH with high affinity. When transfected cells were stimulated with GH, levels of lipoprotein lipase (LPL) mRNA were increased in a time- and dose-dependent fashion, while glyceraldehyde-3-phosphate-dehydrogenase mRNA levels were unaffected. No GH binding or LPL mRNA could be detected in untransfected cells. Treatment of transfected cells with actinomycin D inhibited the GH-stimulated increase in LPL mRNA, indicating that GH acts at a transcriptional level. When protein synthesis was inhibited using cycloheximide, basal levels of LPL mRNA were increased, and there was no GH stimulation. This suggests that LPL gene expression is constantly repressed by a labile protein. Chloramphenicol acetyltransferase constructs containing the human LPL promoter could be regulated by GH. In conclusion, stimulation of the rGH receptor in stably transfected Buffalo rat liver cells results in specific induction of LPL gene expression. This provides a novel model to study the mechanism of GH action, particularly in relation to gene regulation.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Hormônio do Crescimento/farmacologia , Lipase Lipoproteica/biossíntese , Receptores da Somatotropina/fisiologia , Transdução de Sinais , Animais , Sequência de Bases , Linhagem Celular , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Indução Enzimática , Gliceraldeído-3-Fosfato Desidrogenases/genética , Lipase Lipoproteica/genética , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos BUF , Proteínas Recombinantes de Fusão/metabolismo
13.
Int J Obes Suppl ; 5(Suppl 1): S23-7, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27152171

RESUMO

Brown adipose tissue (BAT) is capable of transforming chemically stored energy, in the form of triglycerides, into heat. Recent studies have shown that metabolically active BAT is present in a large proportion of adult humans, where its activity correlates with a favorable metabolic status. Hence, the tissue is now regarded as an interesting target for therapies against obesity and associated diseases such as type 2 diabetes, the hypothesis being that an induction of BAT would be beneficial for these disease states. Apart from the association between BAT activity and a healthier metabolic status, later studies have also shown a positive correlation between BAT volume and both bone cross-sectional area and bone mineral density, suggesting that BAT might stimulate bone anabolism. The aim of this review is to give the reader a brief overview of the BAT research field and to summarize and discuss recent findings regarding BAT being a potential player in bone metabolism.

14.
Endocrinology ; 133(5): 2327-33, 1993 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8404685

RESUMO

Follicular development involves both proliferation and differentiation of thecal and granulosa cells. The process is regulated by gonadotropins and paracrine and autocrine factors, including steroid hormones, presumably by the induction of different genes at specific time points. In the present study, the expression and distribution of the CCAAT enhancer-binding protein-alpha (C/EBP alpha) were studied in immature ovaries and in ovaries in which follicular growth and development were initiated with PMSG, whereas ovulation and luteal formation were induced by the injection of hCG. Ovaries were collected before and at different time points after PMSG (0, 6, 24, and 48 h) and hCG (0.25, 1, 3, 10, and 24 h) treatment for analyses of the contents of C/EBP alpha mRNA and protein and the cell-specific immunohistochemical localization of the protein. C/EBP alpha mRNA increased to maximal levels 24 h after PMSG treatment. The effect was specific for the ovary, as C/EBP alpha mRNA in the uterus did not change. C/EBP alpha mRNA decreased 10 h after hCG treatment and increased again in newly formed corpora lutea. Immunohistochemistry and immunoblotting demonstrated a similar increase in C/EBP alpha during follicular development. To examine the involvement of specific hormones in the regulation of C/EBP alpha, hypophysectomized immature rats were injected sequentially with estradiol and FSH. This treatment resulted in a substantial increase in C/EBP alpha mRNA and protein. These results demonstrate that C/EBP alpha is hormonally regulated in the ovary and suggest a role for C/EBP alpha during differentiation of ovarian cells and follicular development.


Assuntos
Gonadotropina Coriônica/farmacologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas Equinas/farmacologia , Proteínas Nucleares/genética , Folículo Ovariano/fisiologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular , Corpo Lúteo/fisiologia , Feminino , Imunofluorescência , Hibridização de Ácido Nucleico , Folículo Ovariano/citologia , Ovário/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Útero/metabolismo
15.
Endocrinology ; 140(2): 835-43, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9927313

RESUMO

The C/EBP (CCAAT/enhancer-binding protein) family of transcription factors is important for differentiation, lipid biosynthesis, and metabolism. Here, we demonstrate for the first time the presence of C/EBP alpha, beta, delta, and zeta messenger RNA (mRNA) and protein in Sertoli cell primary cultures. Treatment with FSH or 8-CPTcAMP strongly induced C/EBP beta mRNA above basal levels with rapid and transient kinetics in Sertoli cell primary cultures as well as in whole testes from hypophysectomized rats. Whereas C/EBP beta mRNA was induced approximately 50-fold, C/EBP delta mRNA was induced 5- to 8-fold by cAMP in Sertoli cells. Messenger RNA for C/EBP beta and delta were induced by inhibition of protein synthesis with cycloheximide and cycloheximide acted synergistically with cAMP. Immunoblots with C/EBP antibodies demonstrated a strong induction of C/EBP beta, delta, and zeta by cAMP. Electrophoretic mobility shift analysis of nuclear proteins from cAMP-treated Sertoli cells using a C/EBP consensus oligonucleotide and antibodies revealed specific binding of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP beta antibody. Transfections of Sertoli cells with a C/EBP reporter construct showed approximately 3-fold induction of reporter gene activity by cAMP. In contrast, the reporter gene vector with a mutated form of the C/EBP binding site, was almost unresponsive to cAMP in transfections of Sertoli cells. Furthermore, C/EBP beta expression increased the activities of two promoters known to be cAMP-responsive in Sertoli cells. Thus, the early induction of C/EBP isoforms by cAMP may play a role in FSH-dependent regulation of late response genes in Sertoli cells.


Assuntos
AMP Cíclico/fisiologia , Receptores de Peptídeos de Invertebrados/metabolismo , Células de Sertoli/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Eletroforese , Hormônio Foliculoestimulante/farmacologia , Hipofisectomia , Isomerismo , Masculino , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Peptídeos de Invertebrados/genética , Especificidade por Substrato , Testículo/metabolismo
16.
J Clin Endocrinol Metab ; 80(12): 3483-8, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8530587

RESUMO

Recent clinical studies indicate that GH is important for bone remodeling. Patients with GH deficiency exhibit decreased bone density, and GH substitution increases bone density in these patients. The aim of the present study was to investigate the presence of GH receptors and the effects of GH on cultured human osteoblast-like cells. Primary cultures of human osteoblast-like cells were established from trabecular bone. Northern blot analysis, using a probe recognizing exon 10 of the human GH receptor, revealed a 4.7-kilobase transcript corresponding to the human GH receptor. Cultured osteoblast-like cells expressed, as determined by RNase protection assay, approximately one fourth of the GH receptor messenger RNA levels found in liver. Binding studies using 125I-labeled GH revealed a single class of receptors with approximately 2000 binding sites per cell and an association constant (Ka) of 2.6 x 10(9) M-1. GH stimulation of the cultured cells resulted in increased [3H]thymidine incorporation, suggesting that the GH receptors are functional. In summary, the present study shows that cultured human osteoblast-like cells express functional GH receptors.


Assuntos
Osteoblastos/metabolismo , Receptores da Somatotropina/metabolismo , Sequência de Bases , Células Cultivadas , Hormônio do Crescimento/metabolismo , Humanos , Sondas Moleculares/genética , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Receptores da Somatotropina/genética , Timidina/metabolismo
17.
J Clin Endocrinol Metab ; 79(3): 820-5, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8077367

RESUMO

The influence of cortisol, in the presence of insulin, on the regulation of lipoprotein lipase (LPL) activity was studied in human adipose tissue, using a tissue incubation technique. Tissue pieces were preincubated for 3 days in a control medium containing insulin (7175 pmol/L), then incubated for 2 additional days in the control medium with and without cortisol (1000 nmol/L). After the 5 days of incubation, the levels of LPL messenger ribonucleic acid (mRNA), relative LPL synthesis, and LPL activity (total and heparin releasable) were studied. Cortisol exposure for 2 days increased all of the variables related to LPL. The average increase was 2.5-fold for LPL mRNA, 3.0-fold for relative LPL synthesis, 5.2-fold for total LPL activity, and 9.4-fold for heparin-releasable LPL activity compared to that in controls without cortisol. The results confirm previous findings that cortisol, in the presence of insulin, has a marked stimulatory effect on LPL activity in human adipose tissue in vitro. New data have been presented on the mechanisms of cortisol regulation of LPL activity. They involve both an increased level of LPL mRNA, leading to increased relative LPL synthesis, and additional posttranslational regulation.


Assuntos
Tecido Adiposo/enzimologia , Hidrocortisona/farmacologia , Lipase Lipoproteica/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Adulto , Idoso , Relação Dose-Resposta a Droga , Feminino , Humanos , Hidrocortisona/administração & dosagem , Insulina/farmacologia , Lipase Lipoproteica/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo
18.
J Clin Endocrinol Metab ; 80(3): 936-41, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7883853

RESUMO

The in vitro effects of GH on human adipose tissue lipoprotein lipase (LPL) activity and messenger ribonucleic acid (mRNA) levels were studied using a tissue incubation technique. After preincubation for 3 days, abdominal sc adipose tissue pieces were exposed to cortisol (1000 nmol/L) for 3 days to induce LPL activity. Addition of GH (50 micrograms/L) to the cortisol-containing medium during the last 24 h (day 6) caused a decrease by 84 +/- 4% (P < 0.01) in heparin-releasable LPL activity and by 65 +/- 4% (P < 0.01) in total LPL activity. Moreover, the heparin-releasable fraction was reduced from 42% of the total LPL activity with cortisol alone to 17% when both GH and cortisol were present in the incubation medium during the last 24 h (P < 0.01). The reduction in LPL activity in response to GH was not accompanied by a decrease in the level of LPL mRNA measured by a solution hybridization ribonuclease protection assay. In adipose tissue incubated in the control medium for 6 days, the addition of GH alone during the last 24 h caused an insignificant decrease in heparin-releasable LPL activity. Low control activities limited the scope for further decrease. It is concluded that GH counteracts the potent stimulatory effect of glucocorticoids on LPL activity without affecting LPL mRNA levels. Therefore, the inhibition of LPL activity by GH probably occurs during translation and/or posttranslational processing of the enzyme, and the mechanism may involve a decreased channeling of the lipase to the cell surface.


Assuntos
Tecido Adiposo/enzimologia , Hormônio do Crescimento/farmacologia , Lipase Lipoproteica/antagonistas & inibidores , Adulto , Idoso , Feminino , Humanos , Hidrocortisona/farmacologia , Fator de Crescimento Insulin-Like I/fisiologia , Lipase Lipoproteica/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise
19.
Gene ; 84(2): 391-7, 1989 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-2612912

RESUMO

The coding sequence of guinea pig lipoprotein lipase (LPL) is organized into nine exons and spans a region of approximately 14 kb of the guinea pig genome. A non-conforming 5'-splice site is located on the first intron, which exhibits a 12-nucleotide perfect match with the 5'-end of the second exon. A previously described tryptic cleavage site is located on exon V, close to the 3' end of this exon. A similarity to vitellogenin resides on exons IV and V, and a putative active site is found on exon IV. A novel similarity to a fatty-acid-binding protein is noted on exon VI, adjacent to the postulated heparin-binding region. We suggest that free fatty acids (FFA) and heparin to some extent share the same site of interaction on the LPL molecule; and that a high local concentration of FFA can displace LPL from its site of action--the vascular endothelium--by competing for binding to heparan sulfate.


Assuntos
Éxons , Genes , Lipase Lipoproteica/genética , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Splicing de RNA , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA Recombinante , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Cobaias , Heparina/metabolismo , Humanos , Íntrons , Lipase Lipoproteica/metabolismo , Camundongos , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Suínos , alfa-Amilases/genética
20.
Gene ; 64(1): 97-106, 1988 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-3396878

RESUMO

Levels of mRNA for lipoprotein lipase (LPL) in guinea pig epididymal adipose tissue, heart and liver were determined by dot blot analysis of total RNA using a cDNA probe complementary to the coding region, and compared to the LPL activity. For adipose tissue we also measured the incorporation of radioactivity into immunoprecipitable LPL after pulse-labeling with [35S]methionine. LPL activity was 93%, LPL mRNA 82% and LPL synthesis 85% lower in epididymal fat pads from animals fasted for 48 h compared to rigorously fed animals. In contrast, neither LPL activity nor LPL mRNA levels differed in heart. A single dose of tumor necrosis factor (TNF) decreased LPL activity and LPL mRNA in fat pads with no effects in heart. In the liver, TNF caused a marked increase in LPL mRNA levels, which are normally very low. Northern-blot analysis confirmed a previous observation that the patterns of mRNA species differ between heart, in which a 3.8-kb mRNA dominates, and adipose tissue, in which the LPL mRNAs of 3.3 and 2.1 kb occur in similar abundance as the 3.8-kb species.


Assuntos
Tecido Adiposo/enzimologia , Regulação da Expressão Gênica , Genes/efeitos dos fármacos , Lipase Lipoproteica/genética , Fígado/enzimologia , Miocárdio/enzimologia , Fenômenos Fisiológicos da Nutrição , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Cobaias , Lipase Lipoproteica/biossíntese , Masculino , Especificidade de Órgãos , RNA Mensageiro/efeitos dos fármacos , Valores de Referência
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