RESUMEN
During the 11th Stock Conference held in Montreal, Quebec, Canada, world-leading experts came together to present and discuss recent developments made in the field of brown adipose tissue biology. Owing to the vast capacity of brown adipose tissue for burning food energy in the process of thermogenesis, and due to demonstrations of its presence in adult humans, there is tremendous interest in targeting brown adipose tissue as an anti-obesity tissue in humans. However, the future of such therapeutic approaches relies on our understanding of the origin, development, recruitment, activation and regulation of brown adipose tissue in humans. As reviewed here, the 11th Stock Conference was organized around these themes to discuss the recent progress made in each aspect, to identify gaps in our current understanding and to further provide a common groundwork that could support collaborative efforts aimed at a future therapy for obesity, based on brown adipose tissue thermogenesis.
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Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/fisiología , Fármacos Antiobesidad/metabolismo , Metabolismo Energético/fisiología , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Obesidad/terapia , Termogénesis/fisiología , Tejido Adiposo Pardo/metabolismo , Fármacos Antiobesidad/uso terapéutico , Regulación de la Temperatura Corporal , Peso Corporal , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Obesidad/fisiopatología , Proteína Desacopladora 1RESUMEN
BACKGROUND: The amount of intra-thoracic fat, of which mediastinal adipose tissue comprises the major depot, is related to various cardiometabolic risk factors. Autopsy and imaging studies indicate that the mediastinal depot in adult humans could contain brown adipose tissue (BAT). To gain a better understanding of this intra-thoracic fat depot, we examined possible BAT characteristics of human mediastinal in comparison with subcutaneous adipose tissue. MATERIALS AND METHODS: Adipose tissue biopsies from thoracic subcutaneous and mediastinal depots were obtained during open-heart surgery from 33 subjects (26 male, 63.7±13.8 years, body mass index 29.3±5.1 kg m(-2)). Microarray analysis was performed on 10 patients and genes of interest confirmed by quantitative PCR (qPCR) in samples from another group of 23 patients. Adipocyte size was determined and uncoupling protein 1 (UCP1) protein expression investigated with immunohistochemistry. RESULTS: The microarray data showed that a number of BAT-specific genes had significantly higher expression in the mediastinal depot than in the subcutaneous depot. Higher expression of UCP1 (24-fold, P<0.001) and PPARGC1A (1.7-fold, P=0.0047), and lower expression of SHOX2 (0.12-fold, P<0.001) and HOXC8 (0.14-fold, P<0.001) in the mediastinal depot was confirmed by qPCR. Gene set enrichment analysis identified two gene sets related to mitochondria, which were significantly more highly expressed in the mediastinal than in the subcutaneous depot (P<0.01). No significant changes in UCP1 gene expression were observed in the subcutaneous or mediastinal depots following lowering of body temperature during surgery. UCP1 messenger RNA levels in the mediastinal depot were lower than those in murine BAT and white adipose tissue. In some mediastinal adipose tissue biopsies, a small number of multilocular adipocytes that stained positively for UCP1 were observed. Adipocytes were significantly smaller in the mediastinal than the subcutaneous depot (cross-sectional area 2400±810 versus 3260±980 µm(2), P<0.001). CONCLUSIONS: Human mediastinal adipose tissue displays some characteristics of BAT when compared with the subcutaneous depot at microscopic and molecular levels.
RESUMEN
Only with the development of the uncoupling protein 1 (UCP1)-ablated mouse has it become possible to strictly delineate the physiological significance of the thermogenic capacity of brown adipose tissue. Considering the presence of active brown adipose tissue in adult humans, these insights may have direct human implications. In addition to classical nonshivering thermogenesis, all adaptive adrenergic thermogeneses, including diet-induced thermogenesis, is fully dependent on brown adipocyte activity. Any weight-reducing effect of ß(3)-adrenergic agonists is fully dependent on UCP1 activity, as is any weight-reducing effect of leptin (in excess of its effect on reduction of food intake). Consequently, in the absence of the thermogenic activity of brown adipose tissue, obesity develops spontaneously. The ability of brown adipose tissue to contribute to glucose disposal is also mainly related to thermogenic activity. However, basal metabolic rate, cold-induced thermogenesis, acute cold tolerance, fevers, nonadaptive adrenergic thermogenesis and processes such as angiogenesis in brown adipose tissue itself are not dependent on UCP1 activity. Whereas it is likely that these conclusions are also qualitatively valid for adult humans, the quantitative significance of brown adipose tissue for human metabolism--and the metabolic consequences for a single individual possessing more or less brown adipose tissue--awaits clarification.
Asunto(s)
Aclimatación/fisiología , Tejido Adiposo Pardo/fisiología , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Termogénesis/fisiología , Aclimatación/genética , Animales , Frío , Regulación de la Expresión Génica , Humanos , Canales Iónicos/genética , Ratones , Proteínas Mitocondriales/genética , Tiritona/genética , Tiritona/fisiología , Proteína Desacopladora 1RESUMEN
BACKGROUND: The regulatory gene pathways that accompany loss of adipose tissue in cancer cachexia are unknown and were explored using pangenomic transcriptome profiling. METHODS: Global gene expression profiles of abdominal subcutaneous adipose tissue were studied in gastrointestinal cancer patients with (n=13) or without (n=14) cachexia. RESULTS: Cachexia was accompanied by preferential loss of adipose tissue and decreased fat cell volume, but not number. Adipose tissue pathways regulating energy turnover were upregulated, whereas genes in pathways related to cell and tissue structure (cellular adhesion, extracellular matrix and actin cytoskeleton) were downregulated in cachectic patients. Transcriptional response elements for hepatic nuclear factor-4 (HNF4) were overrepresented in the promoters of extracellular matrix and adhesion molecule genes, and adipose HNF4 mRNA was downregulated in cachexia. CONCLUSIONS: Cancer cachexia is characterised by preferential loss of adipose tissue; muscle mass is less affected. Loss of adipose tissue is secondary to a decrease in adipocyte lipid content and associates with changes in the expression of genes that regulate energy turnover, cytoskeleton and extracellular matrix, which suggest high tissue remodelling. Changes in gene expression in cachexia are reciprocal to those observed in obesity, suggesting that regulation of fat mass at least partly corresponds to two sides of the same coin.
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Tejido Adiposo/metabolismo , Caquexia/genética , Neoplasias/genética , Transducción de Señal/genética , Pérdida de Peso/genética , Anciano , Caquexia/etiología , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Humanos , Masculino , Neoplasias/complicaciones , Neoplasias/metabolismo , Obesidad/genética , Obesidad/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Calcium-activated nonselective cation channels (NSC(Ca)) in brown adipocytes are inhibited by several nucleotides acting on the cytosolic side of the membrane. We used excised inside-out patches from rat brown adipocytes to identify important nucleotide structures for NSC-channel inhibition. We found that 100 microM 5'-AMP inhibited NSC-channel activity more than did ATP or ADP. Adenosine was a weak inhibitor, whereas adenine and ribose-5-phosphate had no effect. The channel activity was effectively blocked by 10 microM AMP, but it was unaffected by 10 microM cAMP, CMP, GMP, IMP, TMP or UMP. Dose-response studies yielded IC(50)-values of 4 microM for AMP and 32 microM for cAMP. dAMP was as effective as AMP, but all 5'-phosphate group modifications on AMP dramatically lowered the inhibitory effect. 10 microM of the AMP precursor adenylosuccinate weakly inhibited the channel activity. An increase in AMP concentration from 1 to 10 microM shifted the EC(50) for Ca(2+) activation almost 1 order of magnitude; a Schild plot analysis yielded a K(B) value of 0.3 microM for AMP. We conclude that AMP is the most efficacious endogenous nucleotide inhibitor of the brown adipocyte nonselective cation channel (NSC(Ca/AMP)) yet identified and that there is functional competition between Ca(2+) and AMP. The brown adipocyte NSC(Ca/AMP) thus appears to be functionally different from the NSC(Ca,PKA) in the exocrine pancreas and the NSC-(Ca,cAMP) in the endocrine pancreas, but similar to the NSC(Ca/AMP) in the endocrine pancreas.
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Adenosina Monofosfato/metabolismo , Tejido Adiposo Pardo/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/metabolismo , Adenosina Monofosfato/administración & dosificación , Adenosina Monofosfato/análogos & derivados , Tejido Adiposo Pardo/efectos de los fármacos , Animales , AMP Cíclico/administración & dosificación , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Mice devoid of the original uncoupling protein UCP1 have provided opportunities to delineate UCP1 function in a series of biochemical and physiological contexts. The isolated brown-fat mitochondria from such mice are fully coupled (without the addition of GDP), but still exhibit a depressed capacity for ATP synthesis. However, they only show a 2-fold decrease in sensitivity to the de-energizing effect of free fatty acids, compared with UCP1-containing mitochondria, whereas they possess a (UCP1-independent) 50-fold higher sensitivity than liver mitochondria; the fatty acid sensitivities in wild-type and UCP1-deficient mitochondria may, however, be of different natures. Despite the fact that brown-fat cells from UCP1-ablated mice cannot produce heat when stimulated by noradrenaline ('norepinephrine') or fatty acids, UCP1-ablated mice can be induced to tolerate extended cold exposure, but the heat then fully results from shivering thermogenesis. Recruitable or adaptive (by cold acclimation or adaptation to a cafeteria diet) adrenergically-stimulated thermogenesis does not exist in the UCP1-ablated animals, demonstrating the unique ability of UCP1 to mediate recruitable non-shivering thermogenesis. In addition to information on the function of UCP1, the UCP1-ablated mice can be used to gain information concerning the function of the UCP1 homologues. Thus whereas an uncoupling function of the UCP1 homologues cannot be excluded, UCP1-ablated animals clearly lack any ability to recruit any UCP1 homologue to functionally replace the loss of thermogenesis resulting from UCP1. UCP1 (thermogenin) thus remains the only protein the activity of which can be recruited for the purpose of facultative thermogenesis.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Mitocondrias/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Frío , Relación Dosis-Respuesta a Droga , Guanosina Difosfato/farmacología , Canales Iónicos , Potenciales de la Membrana , Ratones , Ratones Transgénicos , Proteínas Mitocondriales , Temperatura , Proteína Desacopladora 1RESUMEN
Adaptive nonshivering thermogenesis may have profound effects on energy balance and is therefore therefore is a potential mechanism for counteracting the development of obesity. The molecular basis for adaptive nonshivering thermogenesis has remained a challenge that sparked acute interest with the identification of proteins (UCP2, UCP3, etc.) with high-sequence similarity to the original uncoupling protein-1 (UCP1), which is localized only in brown adipose tissue. Using UCP1-ablated mice, we examined whether any adaptive nonshivering thermogenesis could be recruited by acclimation to cold. Remarkably, by successive acclimation, the UCP1-ablated mice could be made to subsist for several weeks at 4C during which they had to constantly produce heat at four times their resting levels. Despite these extreme requirements for adaptive nonshivering thermogenesis, however, no substitution of shivering by any adaptive nonshivering thermogenic process occurred. Thus, although the existence of, for example, muscular mechanisms for adaptive nonshivering thermogenesis has recurrently been implied, we did not find any indication of such thermogenesis. Not even during prolonged and enhanced demand for extra heat production was any endogenous hormone or neurotransmitter able to recruit any UCP1-independent adaptive nonshivering thermogenic process in muscle or in any other organ, and no proteins other than UCP1-not even UCP2 or UCP3-therefore have the ability to mediate adaptive nonshivering thermogenesis in the cold.
Asunto(s)
Regulación de la Temperatura Corporal/fisiología , Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Desacopladores , Adaptación Fisiológica , Animales , Temperatura Corporal , Frío , Canales Iónicos , Ratones , Proteínas Mitocondriales , Tiritona , Termogénesis , Proteína Desacopladora 1RESUMEN
Arotinolol, a clinically used alpha/beta-adrenergic blocker, has been demonstrated to be an anti-obesity agent. The anti-obesity effect of arotinolol was suggested to be the result of direct activation of thermogenesis in brown-fat cells. We tested the ability of arotinolol to stimulate thermogenesis (oxygen consumption) in isolated brown-fat cells and in intact animals. Arotinolol stimulated thermogenesis in brown-fat cells isolated from mouse and hamster. A relatively low sensitivity to the beta-adrenergic antagonist propranolol (pK(B) approximately 6) indicated that arotinolol interacted with the beta3-adrenergic receptor. On the beta3-receptor, arotinolol was a very weak (EC50 approximately 20 microM) and only partial (approximately 50%) agonist, but arotinolol also demonstrated the properties of being a beta3-receptor antagonist with a pK(B) of 5.7. In intact animals, only the antagonistic action of arotinolol could be observed. Because arotinolol is only a very weak and partial agonist on the beta3-receptors, direct stimulation of thermogenesis in brown adipose tissue is unlikely to be sufficient to cause significant weight loss. It may be necessary to invoke additional pathways to explain the anti-obesity effects of chronic treatment with arotinolol.
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Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/fisiología , Agonistas de Receptores Adrenérgicos beta 3 , Antagonistas Adrenérgicos beta/farmacología , Propanolaminas/farmacología , Antagonistas de Receptores Adrenérgicos beta 3 , Agonistas Adrenérgicos beta/farmacología , Animales , Cricetinae , Relación Dosis-Respuesta a Droga , Femenino , Calor , Masculino , Mesocricetus , Ratones , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta 3/fisiologíaRESUMEN
Although it has generally been assumed that protein kinase A (PKA) is essential for brown adipose tissue function, this has not as yet been clearly demonstrated. H89, an inhibitor of PKA, was used here to inhibit PKA activity. In cell extracts, it was confirmed that norepinephrine stimulated PKA activity, which was abolished by H89 treatment. In isolated brown adipocytes, H89 inhibited adrenergically induced thermogenesis (with an IC(50) of approx. 40 microM), and in cultured cells, adrenergically stimulated expression of the uncoupling protein-1 (UCP1) gene was abolished by H89 (full inhibition with 50 microM). However, H89 has been reported to be an adrenergic antagonist on beta(1)/beta(2)-adrenoceptors (AR). Although adrenergic stimulation of thermogenesis and UCP1 gene expression are mediated via beta(3)-ARs, it was deemed necessary to investigate whether H89 also had antagonistic potency on beta(3)-ARs. It was found that EC(50) values for beta(3)-AR-selective stimulation of cAMP production (with BRL-37344) in brown adipose tissue membrane fractions and in intact cells were not affected by H89. Similarly, the EC(50) of adrenergically stimulated oxygen consumption was not affected by H89. As H89 also abolished forskolin-induced UCP1 gene expression, and potentiated selective beta(3)-AR-induced cAMP production, H89 must be active downstream of cAMP. Thus, no antagonism of H89 on beta(3)-ARs could be detected. We conclude that H89 can be used as a pharmacological tool for elucidation of the involvement of PKA in cellular signalling processes regulated via beta(3)-ARs, and that the results are concordant with adrenergic stimulation of thermogenesis and UCP1 gene expression in brown adipocytes being mediated via a PKA-dependent pathway.
Asunto(s)
Adipocitos/efectos de los fármacos , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Isoquinolinas/farmacología , Proteínas de la Membrana/metabolismo , Sulfonamidas , Adipocitos/metabolismo , Animales , Proteínas Portadoras/química , Células Cultivadas , Colforsina/antagonistas & inhibidores , AMP Cíclico/metabolismo , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Canales Iónicos , Proteínas de la Membrana/química , Ratones , Proteínas Mitocondriales , Norepinefrina/antagonistas & inhibidores , Consumo de Oxígeno/efectos de los fármacos , Receptores Adrenérgicos beta 3/metabolismo , Transducción de Señal , Proteína Desacopladora 1RESUMEN
The uniqueness of UCP1 (as compared to UCP2/UCP3) is evident from expression analysis and ablation studies. UCP1 expression is positively correlated with metabolic inefficiency, being increased by cold acclimation (in adults or perinatally) and overfeeding, and reduced in fasting and genetic obesity. Such a simple relationship is not observable for UCP2/UCP3. Studies with UCP1-ablated animals substantiate the unique role of UCP1: the phenomenon of adaptive adrenergic non-shivering thermogenesis in the intact animal is fully dependent on the presence of UCP1, and so is any kind of cold acclimation-recruited non-shivering thermogenesis; thus UCP2/UCP3 (or any other proteins or metabolic processes) cannot substitute for UCP1 physiologically, irrespective of their demonstrated ability to show uncoupling in reconstituted systems or when ectopically expressed. Norepinephrine-induced thermogenesis in brown-fat cells is absolutely dependent on UCP1, as is the uncoupled state and the recoupling by purine nucleotides in isolated brown-fat mitochondria. Although very high UCP2/UCP3 mRNA levels are observed in brown adipose tissue of UCP1-ablated mice, there is no indication that the isolated brown-fat mitochondria are uncoupled; thus, high expression of UCP2/UCP3 does not necessarily confer to the mitochondria of a tissue a propensity for being innately uncoupled. Whereas the thermogenic effect of fatty acids in brown-fat cells is fully UCP1-dependent, this is not the case in brown-fat mitochondria; this adds complexity to the issues concerning the mechanisms of UCP1 function and the pathway from beta(3)-adrenoceptor stimulation to UCP1 activation and thermogenesis. In addition to amino acid sequences conserved in all UCPs as part of the tripartite structure, all UCPs contain certain residues associated with nucleotide binding. However, conserved amongst all UCP1s so far sequenced, and without parallel in all UCP2/UCP3, are two sequences: 144SHLHGIKP and the C-terminal sequence RQTVDC(A/T)T; these sequences may therefore be essential for the unique thermogenic function of UCP1. The level of UCP1 in the organism is basically regulated at the transcriptional level (physiologically probably mainly through the beta(3)-adrenoceptor/CREB pathway), with influences from UCP1 mRNA stability and from the delay caused by translation. It is concluded that UCP1 is unique amongst the uncoupling proteins and is the only protein able to mediate adaptive non-shivering thermogenesis and the ensuing metabolic inefficiency.
Asunto(s)
Tejido Adiposo Pardo/fisiología , Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Mitocondrias/fisiología , Termogénesis , Aclimatación , Adipocitos/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Humanos , Canales Iónicos , Potenciales de la Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mitocondrias/efectos de los fármacos , Mitocondrias Hepáticas/fisiología , Proteínas Mitocondriales , Modelos Químicos , Norepinefrina , Obesidad/genética , Consumo de Oxígeno , ARN Mensajero/análisis , Desacopladores/metabolismo , Proteína Desacopladora 1RESUMEN
The CREM (cAMP-response-element modulator) gene product ICER (induced cAMP early repressor) has been proposed to function as a tumour (cell proliferation) suppressor. To investigate the generality of this concept, the expression pattern of ICER in brown adipocytes was followed; this was critical because brown adipocytes are one of few cell types in which cAMP is associated positively with cell proliferation but negatively with apoptosis. In response to the physiological stimulus of cold (which induces cell proliferation), ICER mRNA levels were increased in brown adipose tissue in vivo. In brown adipocytes in primary culture, ICER gene expression was induced by noradrenaline (norepinephrine) not only in the mature state (where noradrenaline potentiates differentiation), but also in the proliferative state of the cell cultures (where noradrenaline enhances cell proliferation). The induction was mediated via beta-receptors and the cAMP/protein kinase A pathway. The induced ICER appeared to repress its own expression and that of the beta2-adrenoceptor. It is thus evident that also in cell types in which cAMP induces proliferation, and even when these cells are in the proliferative state, ICER expression is induced by the same agents that stimulate proliferation. This can either mean that ICER is not a general tumour suppressor, or that brown adipocytes temporally or spatially avoid this role of ICER.
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Tejido Adiposo Pardo/metabolismo , División Celular/fisiología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Genes Supresores de Tumor , Norepinefrina/fisiología , Tejido Adiposo Pardo/citología , Animales , Secuencia de Bases , Células Cultivadas , Modulador del Elemento de Respuesta al AMP Cíclico , Cartilla de ADN , Proteínas de Unión al ADN/fisiología , Cinética , Masculino , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/fisiologíaRESUMEN
The nature of the sustained norepinephrine-induced depolarization in brown fat cells was examined by patch-clamp techniques. Norepinephrine (NE) stimulation led to a whole cell current response consisting of two phases: a first inward current, lasting for only 1 min, and a sustained inward current, lasting as long as the adrenergic stimulation was maintained. The nature of the sustained current was here investigated. It could be induced by the alpha(1)-agonist cirazoline but not by the beta(3)-agonist CGP-12177A. Reduction of extracellular Cl(-) concentration had no effect, but omission of extracellular Ca(2+) or Na(+) totally eliminated it. When unstimulated cells were studied in the cell-attached mode, some activity of approximately 30 pS nonselective cation channels was observed. NE perfusion led to a 10-fold increase in their open probability (from approximately 0.002 to approximately 0.017), which persisted as long as the perfusion was maintained. The activation was much stronger with the alpha(1)-agonist phenylephrine than with the beta(3)-agonist CGP-12177A, and with the Ca(2+) ionophore A-23187 than with the adenylyl cyclase activator forskolin. We conclude that the sustained inward current was due to activation of approximately 30 pS nonselective cation channels via alpha(1)-adrenergic receptors and that the effect may be mediated via an increase in intracellular free Ca(2+) concentration.
Asunto(s)
Tejido Adiposo Pardo/efectos de los fármacos , Conductividad Eléctrica , Canales Iónicos/fisiología , Norepinefrina/farmacología , Receptores Adrenérgicos alfa 1/fisiología , Adenilil Ciclasas/metabolismo , Tejido Adiposo Pardo/citología , Agonistas alfa-Adrenérgicos/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Calcimicina/farmacología , Calcio/fisiología , Cationes , Cloruros/metabolismo , Colforsina/farmacología , Activación Enzimática/efectos de los fármacos , Espacio Extracelular/metabolismo , Imidazoles/farmacología , Ionóforos/farmacología , Masculino , Técnicas de Placa-Clamp , Fenilefrina/farmacología , Propanolaminas/farmacología , Ratas , Ratas Sprague-Dawley , Sodio/fisiologíaRESUMEN
To examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated. Ucp1(-/-) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust ( approximately 10-fold increase in thermogenesis); Ucp1(-/-) cells only responded approximately 3% of this. Ucp1(-/-) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect in Ucp1(-/-) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells.
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Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Agonistas Adrenérgicos/metabolismo , Proteínas Portadoras/fisiología , Ácidos Grasos/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas/fisiología , Temperatura , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Proteínas Portadoras/genética , Células Cultivadas , AMP Cíclico/metabolismo , Exones , Glicerol/metabolismo , Canales Iónicos , Lauratos/farmacología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Modelos Biológicos , Norepinefrina/metabolismo , Ácido Oléico/farmacología , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/genética , Proteínas/genética , Recombinación Genética , Tretinoina/farmacología , Desacopladores/farmacología , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
To identify the signaling pathway that mediates the adrenergic stimulation of the expression of the gene for vascular endothelial growth factor (VEGF) during physiologically induced angiogenesis, we examined mouse brown adipocytes in primary culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced VEGF expression 3-fold, in a dose- and time-dependent manner (EC(50) approximately 90 nm). Also, the hypoxia-mimicking agent cobalt, as well as serum and phorbol ester, induced VEGF expression, but the effect of NE was additive to each of these factors, implying that a separate signaling mechanism for the NE-mediated induction was activated. The NE effect was abolished by propranolol and mimicked by isoprenaline or BRL-37344 and was thus mediated via beta-adrenoreceptors. The NE-induced VEGF expression was fully cAMP mediated, an effect which was inhibited by H-89 and thus was dependent on protein kinase A activity. Involvement of other adrenergic signaling pathways (alpha(1)-adrenoreceptors, Ca(2+), protein kinase C, alpha(2)-adrenoreceptors, and pertussis toxin-sensitive G(i)-proteins) was excluded. The specific inhibitor of Src tyrosine kinases, PP2, markedly reduced the stimulation by NE, which demonstrates that a cAMP-dependent Src-mediated pathway is positively connected to VEGF expression. However, inhibition of Erk1/2 MAP kinases by PD98059 was without effect. NE did not prolong VEGF mRNA half-life and its effect was thus transcriptional, and was independent of protein synthesis. These results demonstrate that adrenergic stimulation, through beta-adrenoreceptor/cAMP/protein kinase A signaling, recruits a pathway that branches off from the NE-activated Src-Erk1/2 cascade to enhance transcription of the VEGF gene.
Asunto(s)
Tejido Adiposo Pardo/fisiología , Factores de Crecimiento Endotelial/genética , Regulación de la Expresión Génica/efectos de los fármacos , Linfocinas/genética , Norepinefrina/farmacología , Receptores Adrenérgicos beta/fisiología , Vasoconstrictores/farmacología , Animales , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Factores de Crecimiento Endotelial/biosíntesis , Linfocinas/biosíntesis , Ratones , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Familia-src Quinasas/fisiologíaRESUMEN
A novel signaling pathway for mediation of beta(3)-adrenergic activation of the mitogen-activated protein kinases Erk1/2 (associated with proliferation, differentiation, and apoptosis) has recently been proposed, which implies mediation via constitutively coupled G(i)-proteins and Gbetagamma-subunits, distinct from the classical cAMP pathway of beta-adrenergic stimulation. To verify the significance of this pathway in cells in primary cultures that entopically express beta(3)-adrenoreceptors, we examined the functionality of this pathway in cultured brown adipocytes. Norepinephrine activated Erk1/2 via both beta(3) receptors and alpha(1) receptors but not via alpha(2) receptors. Forskolin induced Erk1/2 activation similarly to beta(3) activation, indicating cAMP-mediation; this induction could be inhibited with H89, implying protein kinase A mediation. The G(i)-pathway was functional in these cells, as pertussis toxin increased agonist-induced cAMP accumulation. However, pertussis toxin was unable to affect adrenergically induced Erk1/2 activation. Also, wortmannin was without effect, implying that Gbetagamma activation of the phosphatidylinositol 3-kinase pathway was not involved. PP1/2, which inhibits Src, abolished both beta(3)- and alpha(1)-induced Erk1/2 activation. Thus, the proposed novel G(i) pathway for beta(3) mediation is not universal, because it is not functional in the untransformed primary cell culture system with entopically expressed beta(3) receptors examined here. Here, the beta(3) signal is mediated classically via cAMP/protein kinase A. beta(3) and alpha(1) signals converge at Src, which thus mediates Erk1/2 activation in both pathways.
Asunto(s)
Tejido Adiposo Pardo/enzimología , Proteínas de Unión al GTP/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Oncogénica pp60(v-src)/fisiología , Receptores Adrenérgicos alfa 1/fisiología , Receptores Adrenérgicos beta/fisiología , Adipocitos/enzimología , Tejido Adiposo Pardo/citología , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Humanos , Masculino , Ratones , Norepinefrina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Receptores Adrenérgicos beta 3RESUMEN
In brown adipocytes, fundamental cellular processes (cell proliferation, differentiation and apoptosis) are regulated by adrenergic stimulation, notably through beta-adrenergic receptors. The presence of all three beta-receptor subtypes has been demonstrated in brown adipose tissue. Due to the significance of the action of these receptors and indications that the subtypes govern different processes, the adrenergic regulation of the expression of the beta(1)-(,) beta(2)- and beta(3)-adrenoceptor genes was examined in murine brown-fat primary cell cultures. Moderate levels of beta(1)-receptor mRNA, absence of beta(2)-receptor mRNA and high levels of beta(3)-receptor mRNA were observed in mature brown adipocytes (day 6 in culture). Noradrenaline (norepinephrine) addition led to diametrically opposite effects on beta(1)- (markedly enhanced expression) and beta(3)-gene expression (full cessation of expression, as previously shown). beta(2)-Gene expression was induced by noradrenaline, but only transiently (<1 h). The apparent affinities (EC(50)) of noradrenaline were clearly different (7 nM for the beta(1)-gene and
Asunto(s)
Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Adrenérgicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Receptores Adrenérgicos beta/genética , Tejido Adiposo Pardo/citología , Agonistas de Receptores Adrenérgicos beta 1 , Antagonistas de Receptores Adrenérgicos beta 1 , Agonistas de Receptores Adrenérgicos beta 2 , Antagonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Células Cultivadas , Frío , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Semivida , Masculino , Ratones , Norepinefrina/agonistas , Norepinefrina/antagonistas & inhibidores , Norepinefrina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteína Quinasa C/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 3 , Transcripción Genética/efectos de los fármacosRESUMEN
The mechanism behind the distinctive non-Michaelis-Menten, bell-shaped kinetics of cAMP accumulation in brown adipocytes (which underlies the similar kinetics of UCP1 and beta(1)-adrenoreceptor gene expression) was investigated. A theoretical dual component analysis indicated that the observed dose-response curves could be constructed as the resultant of a stimulatory and an inhibitory component. Experimentally, inhibition of the alpha(1)-component of the norepinephrine response revealed the underlying existence of a much larger stimulatory beta(3)-component which displayed monophasic Michaelis-Menten kinetics. The inhibitory alpha(1)-component (which was also monophasic but had a 2-fold higher EC(50)) was mediated via an increase in [Ca(2+)](i); the protein kinase C pathway was not involved. The [Ca(2+)](i) increase which resulted in massive inhibition of cAMP accumulation was very low: <100 nM. The [Ca(2+)](i) signal stimulated a calmodulin-controlled phosphodiesterase, possibly PDE-1. The acquirement of this specific interaction pattern between beta- and alpha(1)-adrenergic stimulation was thus part of the differentiation program of the brown adipocytes. It was concluded that an array of synergistic or inhibitory alpha(1)/beta interactions occur in the adrenergic regulation of this cell type which is unique in its dependence upon adrenergic stimulation for cellular proliferation, differentiation, and metabolic function.