RESUMEN
Adipocyte fatty acid-binding protein, aP2, is a member of the intracellular fatty acid binding protein family. Previously, studies have shown increased insulin sensitivity in aP2-deficient mice with dietary obesity. Here, we asked whether aP2-related alterations in lipolytic response and insulin production are features of obesity-induced insulin resistance and investigated the effects of aP2-deficiency on glucose homeostasis and lipid metabolism in ob/ob mice, a model of extreme obesity. ob/ob mice homozygous for the aP2 null allele (ob/ ob-aP2-/-) became more obese than ob/ob mice as indicated by significantly increased body weight and fat pad size but unaltered body length. However, despite their extreme adiposity, ob/ob-aP2-/- animals were more insulin-sensitive compared with ob/ob controls, as demonstrated by significantly lower plasma glucose and insulin levels and better performance in both insulin and glucose tolerance tests. These animals also showed improvements in dyslipidemia and had lower plasma triglyceride and cholesterol levels. Lipolytic response to beta-adrenergic stimulation and lipolysis-associated insulin secretion was significantly reduced in ob/ob-aP2-/- mice. Interestingly, glucose-stimulated insulin secretion, while virtually abolished in ob/ob controls, was significantly improved in ob/ob-aP2-/- animals. There were no apparent morphological differences in the structure or size of the pancreatic islets between genotypes. Taken together, the data indicate that in obesity, aP2-deficiency not only improves peripheral insulin resistance but also preserves pancreatic beta cell function and has beneficial effects on lipid metabolism.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Glucosa/metabolismo , Metabolismo de los Lípidos , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Obesidad/metabolismo , Tejido Adiposo/fisiología , Envejecimiento/fisiología , Animales , Peso Corporal/fisiología , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/metabolismo , Femenino , Homeostasis/fisiología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Leptina/metabolismo , Lipólisis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/genética , Páncreas/metabolismo , Caracteres SexualesRESUMEN
Severe quantitative and qualitative brown adipocyte defects are common in obesity. To investigate whether aberrant expression of tumor necrosis factor alpha (TNF-alpha) in obesity is involved in functional brown fat atrophy, we have studied genetically obese (ob/ob) mice with targeted null mutations in the genes encoding the two TNF receptors. The absence of both TNF receptors or p55 receptor alone resulted in a significant reduction in brown adipocyte apoptosis and an increase in beta(3)-adrenoreceptor and uncoupling protein-1 expression in obese mice. Increased numbers of multilocular functionally active brown adipocytes, and improved thermoregulation was also observed in obese animals lacking TNF-alpha function. These results indicate that TNF-alpha plays an important role in multiple aspects of brown adipose tissue biology and mediates the abnormalities that occur at this site in obesity.
Asunto(s)
Adipocitos/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Apoptosis , Obesidad/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adaptación Fisiológica , Adipocitos/citología , Tejido Adiposo Pardo/citología , Animales , Antígenos CD/genética , Temperatura Corporal , Proteínas Portadoras/metabolismo , Frío , AMP Cíclico/biosíntesis , Etiquetado Corte-Fin in Situ , Canales Iónicos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Proteínas Mitocondriales , Mutación , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 3 , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Desacopladores/metabolismo , Proteína Desacopladora 1RESUMEN
Tumour necrosis factor-alpha (TNFalpha) is a multifunctional cytokine that exerts a myriad of biological actions in numerous different tissues including adipocytes through its two distinct cell surface receptors. To address the role of each TNF receptor in the biological actions of TNFalpha in adipocytes, we have developed four new preadipocyte cell lines. These were established from wild type controls (TNFR1(+/+)R2(+/+)) and from mice lacking TNFR1 (TNFR1(-/-)), TNFR2 (TNFR2(-/-)) or both (TNFR1(-/-)R2(-/-)). All four new cell lines can fully differentiate to form mature adipocytes, under appropriate culture conditions, as judged by cell morphology, expression of multiple adipogenic markers and the ability to mediate agonist-stimulated lipolysis and insulin-stimulated glucose transport. In wild type (TNFR1(+/+)R2(+/+)) and TNFR2(-/-) adipocytes, TNFalpha stimulated lipolysis and inhibited insulin-stimulated glucose transport as well as insulin receptor autophosphorylation. In contrast, these activities were completely lost in the TNFR1(-/-)R2(-/-) and TNFR1(-/-) cells. Taken together, these studies demonstrate that TNFalpha-induced lipolysis, as well as inhibition of insulin-stimulated glucose transport are predominantly mediated by TNFR1 and that the presence of TNFR2 is not necessary for these functions. This new experimental system promises to be useful in dissecting the molecular pathways activated by each TNF receptor in mediating the biological functions of TNFalpha in differentiated adipocytes.
Asunto(s)
Adipocitos/metabolismo , Receptores del Factor de Necrosis Tumoral/deficiencia , Factor de Necrosis Tumoral alfa/metabolismo , Adipocitos/efectos de los fármacos , Animales , Diferenciación Celular , Línea Celular , Regulación de la Expresión Génica , Glucosa/metabolismo , Insulina/farmacología , Lipólisis/efectos de los fármacos , Ratones , Ratones Noqueados , Fosforilación , Receptor de Insulina/metabolismoRESUMEN
Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-alpha in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-alpha has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-alpha.
Asunto(s)
Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Obesidad/genética , Proteínas/genética , Tejido Adiposo/química , Tejido Adiposo/metabolismo , Animales , Alimentos , Privación de Alimentos , Hiperfagia/fisiopatología , Canales Iónicos , Leptina/farmacología , Metabolismo de los Lípidos , Lípidos/análisis , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , ARN Mensajero/análisis , Factor de Necrosis Tumoral alfa/farmacología , Proteína Desacopladora 2 , Regulación hacia ArribaRESUMEN
Recent studies have shown that genetic deficiency of the adipocyte fatty acid-binding protein (aP2) results in minor alterations of plasma lipids and adipocyte development but provides significant protection from dietary obesity-induced hyperinsulinemia and insulin resistance. To identify potential mechanisms responsible for this phenotype, we examined lipolysis and insulin secretion in aP2-/- mice. Beta-adrenergic stimulation resulted in a blunted rise of blood glycerol levels in aP2-/- compared with aP2+/+ mice, suggesting diminished lipolysis in aP2-/- adipocytes. Confirming this, primary adipocytes isolated from aP2-/- mice showed attenuated glycerol and free fatty acid (FFA) release in response to dibutyryl cAMP. The decreased lipolytic response seen in the aP2-/- mice was not associated with altered expression levels of hormone-sensitive lipase or perilipin. The acute insulin secretory response to beta-adrenergic stimulation was also profoundly suppressed in aP2-/- mice despite comparable total concentrations and only minor changes in the composition of systemic FFAs. To address whether levels of specific fatty acids are different in aP2-/- mice, the plasma FFA profile after beta-adrenergic stimulation was determined. Significant reduction in both stearic and cis-11-eicoseneic acids and an increase in palmitoleic acid were observed. The response of aP2-/- mice to other insulin secretagogues such as arginine and glyburide was similar to that of aP2+/+ mice, arguing against generally impaired function of pancreatic beta-cells. Finally, no aP2 expression was detected in isolated pancreatic islet cells. These results provide support for the existence of an adipo-pancreatic axis, the proper action of which relies on the presence of aP2. Consequently, aP2's role in the pathogenesis of type 2 diabetes might involve regulation of both hyperinsulinemia and insulin resistance through its impact on both lipolysis and insulin secretion.
Asunto(s)
Proteínas Portadoras/fisiología , Ácidos Grasos/metabolismo , Insulina/metabolismo , Lipólisis , Proteína P2 de Mielina/fisiología , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Regulación de la Expresión Génica , Secreción de Insulina , Ratones , Ratones Endogámicos C57BL , Proteína P2 de Mielina/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 3RESUMEN
Obesity is associated with a cluster of abnormalities, including hypertension, insulin resistance, hyperinsulinemia, and elevated levels of both plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor beta (TGF-beta). Although these changes may increase the risk for accelerated atherosclerosis and fatal myocardial infarction, the underlying molecular mechanisms remain to be defined. Although tumor necrosis factor alpha (TNF-alpha) has been implicated in the insulin resistance associated with obesity, its role in other disorders of obesity is largely unknown. In this report, we show that in obese (ob/ob) mice, neutralization of TNF-alpha or deletion of both TNF receptors (TNFRs) results in significantly reduced levels of plasma PAI-1 antigen, plasma insulin, and adipose tissue PAI-1 and TGF-beta mRNAs. Studies in which exogenous TNF-alpha was infused into lean mice lacking individual TNFRs indicate that TNF-alpha signaling of PAI-1 in adipose tissue can be mediated by either the p55 or the p75 TNFR. However, TNF-alpha signaling of TGF-beta mRNA expression in adipose tissue is mediated exclusively via the p55 TNFR. Our results suggest that TNF-alpha is a common link between the insulin resistance and elevated PAI-1 and TGF-beta in obesity. The chronic elevation of TNF-alpha in obesity thus may directly promote the development of the complex cardiovascular risk profile associated with this condition.
Asunto(s)
Obesidad/sangre , Inhibidor 1 de Activador Plasminogénico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Regulación de la Expresión Génica , Resistencia a la Insulina , Ratones , Ratones Obesos , Obesidad/genética , Inhibidor 1 de Activador Plasminogénico/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Factores de Riesgo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Although obesity has become the most common metabolic disorder in the developed world and is highly associated with insulin resistance and noninsulin-dependent diabetes mellitus, the molecular mechanisms underlying these disorders are not clearly understood. Tumor necrosis factor-alpha (TNF-alpha) is overexpressed in obesity and is a candidate mediator of obesity-induced insulin resistance. Complete lack of TNF-alpha function through targeted mutations in TNF-alpha gene or both of its receptors results in significant improvement of insulin sensitivity in dietary, chemical, or genetic models of rodent obesity. In this study, we have analyzed the in vivo role of TNF signaling from p55 [TNF receptor (TNFR) 1] and p75 (TNFR 2) TNFR in the development of insulin resistance by generating genetically obese mice (ob/ob) lacking p55 or p75 TNFRs. In the ob/ob mice, the absence of p55 caused a significant improvement in insulin sensitivity. p75 deficiency alone did not affect insulin sensitivity but might potentiate the effects of p55 deficiency in animals lacking both TNFRs. These results indicate that TNF-alpha is a component of insulin resistance in the ob/ob model of murine obesity and p55 TNFR is the predominant receptor mediating its actions.
Asunto(s)
Resistencia a la Insulina/fisiología , Obesidad/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Animales , Composición Corporal/fisiología , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Homeostasis/fisiología , Lípidos/sangre , Ratones/genética , Ratones/crecimiento & desarrollo , Mutación/fisiología , Obesidad/metabolismo , Obesidad/patología , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genéticaRESUMEN
OBJECTIVES: A nuclear matrix protein (PC-1) was previously identified and reported to be present only in human prostate cancer but absent in tissue from the same prostate containing either benign prostatic hyperplasia (BPH) or normal prostate tissue. The PC-1 protein was identified by high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and exhibited a molecular mass of 56 kDa and an isoelectric point of 6.58. This work investigates the immunohistochemical characterization of PRO:4-216, a monoclonal antibody to PC-1. METHODS: Areas of the 2D-PAGE gels containing the human prostate cancer nuclear matrix proteins near PC-1 were isolated, eluted, and injected into mice to develop monoclonal antibodies. Antibodies were screened by immunofluorescence for nuclear reactivity to a human prostate cancer cell line (LnCaP) and by 1D and 2D Western blots for reactivity with prostate cancer nuclear matrix proteins. Monoclonal antibodies from the selected clones were affinity purified. The monoclonal antibody PRO:4-216 was used to analyze frozen tissue from 20 cancerous, 22 BPH, and 22 normal regions from fresh human prostate specimens. Tissue sections were analyzed for their immunohistochemical (IHC) (horseradish peroxidase) staining. RESULTS: Using a reference value for positive staining at an IHC score of greater than 50, 85% (17 of 20) of the cancerous, 5% (1 of 22) of the BPH, and 9% (2 of 22) of the normal prostate tissues stained positive. The one BPH and two normal tissues that stained positive were taken from prostates in which the adjacent cancerous tissue also demonstrated high IHC scores (greater than 225). CONCLUSIONS: These data demonstrate nuclear reactivity on fresh frozen human prostate cancer tissue for the monoclonal antibody PRO:4-216. PRO:4-216 may aid in distinguishing normal prostate and BPH from cancerous tissue.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Autoanticuerpos/inmunología , Proteínas Nucleares/inmunología , Neoplasias de la Próstata/inmunología , Animales , Antígenos Nucleares , Técnica del Anticuerpo Fluorescente Directa , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Hiperplasia Prostática/inmunología , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patologíaRESUMEN
Obesity is highly associated with insulin resistance and is the biggest risk factor for non-insulin-dependent diabetes mellitus. The molecular basis of this common syndrome, however, is poorly understood. It has been suggested that tumour necrosis factor (TNF)-alpha is a candidate mediator of insulin resistance in obesity, as it is overexpressed in the adipose tissues of rodents and humans and it blocks the action of insulin in cultured cells and whole animals. To investigate the role of TNF-alpha in obesity and insulin resistance, we have generated obese mice with a targeted null mutation in the gene encoding TNF-alpha and those encoding the two receptors for TNF-alpha. The absence of TNF-alpha resulted in significantly improved insulin sensitivity in both diet-induced obesity and that resulting for the ob/ob model of obesity. The TNFalpha-deficient obese mice had lower levels of circulating free fatty acids, and were protected from the obesity-related reduction in the insulin receptor signalling in muscle and fat tissues. These results indicate that TNF-alpha is an important mediator of insulin resistance in obesity through its effects on several important sites of insulin action.
Asunto(s)
Resistencia a la Insulina , Proteínas Musculares , Obesidad/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Tejido Adiposo/metabolismo , Animales , Ácidos Grasos/sangre , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4 , Homeostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Proteínas de Transporte de Monosacáridos/metabolismo , Mutación , Fosforilación , Receptor de Insulina/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Triglicéridos/sangre , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cytokines, in particular tumor necrosis factor-alpha (TNF-alpha), have significant effects on energy metabolism and appetite although their mechanisms of action are largely unknown. Here, we examined whether TNF-alpha modulates the production of leptin, the recently identified fat-specific energy balance hormone, in cultured adipocytes and in mice. TNF-alpha treatment of 3T3-L1 adipocytes resulted in rapid stimulation of leptin accumulation in the media, with a maximum effect at 6 h. This stimulation was insensitive to cycloheximide, a protein synthesis inhibitor, but was completely inhibited by the secretion inhibitor brefeldin A, indicating a posttranslational effect. Treatment of mice with TNF-alpha also caused a similar increase in plasma leptin levels. Finally, in obese TNF-alpha-deficient mice, circulating leptin levels were significantly lower, whereas adipose tissue leptin was higher compared with obese wild-type animals. These data provide evidence that TNF-alpha can act directly on adipocytes to regulate the release of a preformed pool of leptin. Furthermore, they suggest that the elevated adipose tissue expression of TNF-alpha that occurs in obesity may contribute to obesity-related hyperleptinemia.