RESUMEN
Uptake of circulating succinate by brown adipose tissue (BAT) and beige fat elevates whole-body energy expenditure, counteracts obesity and antagonizes systemic tissue inflammation in mice. The plasma membrane transporters that facilitate succinate uptake in these adipocytes remain undefined. Here we elucidate a mechanism underlying succinate import into BAT via monocarboxylate transporters (MCTs). We show that succinate transport is strongly dependent on the proportion that is present in the monocarboxylate form. MCTs facilitate monocarboxylate succinate uptake, which is promoted by alkalinization of the cytosol driven by adrenoreceptor stimulation. In brown adipocytes, we show that MCT1 primarily facilitates succinate import. In male mice, we show that both acute pharmacological inhibition of MCT1 and congenital depletion of MCT1 decrease succinate uptake into BAT and consequent catabolism. In sum, we define a mechanism of succinate uptake in BAT that underlies its protective activity in mouse models of metabolic disease.
Asunto(s)
Adipocitos Marrones , Ácido Succínico , Masculino , Ratones , Animales , Adipocitos Marrones/metabolismo , Ácido Succínico/metabolismo , Tejido Adiposo Pardo/metabolismo , Transporte Biológico , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Lactate is abundant in rapidly dividing cells owing to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here we use a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we identify a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodelling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We find that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. This mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient-replete growth phase to stimulate timed opening of APC/C, cell division and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodelling and can overcome anti-mitotic pharmacology via mitotic slippage. In sum, we define a biochemical mechanism through which lactate directly regulates protein function to control the cell cycle and proliferation.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase , Proteínas de Ciclo Celular , Ciclo Celular , Ácido Láctico , Humanos , Anafase , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ácido Láctico/metabolismo , MitosisRESUMEN
Uptake of circulating succinate by brown adipose tissue (BAT) and beige fat elevates whole body energy expenditure, counteracts obesity, and antagonizes systemic tissue inflammation in mice. The plasma membrane transporters that facilitate succinate uptake in these adipocytes remain undefined. Here we elucidate a mechanism underlying succinate import into BAT via monocarboxylate transporters (MCTs). We show that succinate transport is strongly dependent on the proportion of it present in the monocarboxylate form. MCTs facilitate monocarboxylate succinate uptake, which is promoted by alkalinization of the cytosol driven by adrenoreceptor stimulation. In brown adipocytes, we show that MCT1 primarily facilitates succinate import, however other members of the MCT family can partially compensate and fulfill this role in the absence of MCT1. In mice, we show that acute pharmacological inhibition of MCT1 and 2 decreases succinate uptake into BAT. Conversely, congenital genetic depletion of MCT1 alone has little effect on BAT succinate uptake, indicative of additional transport mechanisms with high capacity in vivo . In sum, we define a mechanism of succinate uptake in BAT that underlies its protective activity in mouse models of metabolic disease.
RESUMEN
lncRAP2 is a conserved cytoplasmic lncRNA enriched in adipose tissue and required for adipogenesis. Using purification and in vivo interactome analyses, we show that lncRAP2 forms complexes with proteins that stabilize mRNAs and modulate translation, among them Igf2bp2. Surveying transcriptome-wide Igf2bp2 client mRNAs in white adipocytes reveals selective binding to mRNAs encoding adipogenic regulators and energy expenditure effectors, including adiponectin. These same target proteins are downregulated when either Igf2bp2 or lncRAP2 is downregulated, hindering adipocyte lipolysis. Proteomics and ribosome profiling show this occurs predominantly through mRNA accumulation, as lncRAP2-Igf2bp2 complex binding does not impact translation efficiency. Phenome-wide association studies reveal specific associations of genetic variants within both lncRAP2 and Igf2bp2 with body mass and type 2 diabetes, and both lncRAP2 and Igf2bp2 are suppressed in adipose depots of obese and diabetic individuals. Thus, the lncRAP2-Igf2bp2 complex potentiates adipose development and energy expenditure and is associated with susceptibility to obesity-linked diabetes.
RESUMEN
The TCA cycle metabolite succinate functions as an intra- and extracellular signal of metabolic stress. Based on the phenotype of UCP-1-deficient mice, Mills et al. (2021) now report in Nature Metabolism that accumulation of extracellular succinate due to impaired elimination in thermogenic fat drives liver inflammation and fibrosis through the succinate receptor SUCNR1.
Asunto(s)
Receptores Acoplados a Proteínas G , Ácido Succínico , Animales , Fibrosis , Ratones , Transducción de Señal , Estrés FisiológicoRESUMEN
Utilizing the thermogenic capacity of brown adipose tissue is a potential anti-obesity strategy; therefore, the mechanisms controlling expression of thermogenesis-related genes are of interest. Pyruvate kinase (PK) catalyzes the last step of glycolysis and exists as four isoenzymes: PK, liver, PK, red blood cell, PK, muscle (PKM1 and PKM2). PKM2 has both glycolytic and nuclear functions. Here, we report that PKM2 is enriched in brown adipose compared with white adipose tissue. Specific knockdown of PKM2 in mature brown adipocytes demonstrates that silencing of PKM2 does not lead to a decrease in PK activity, but causes a robust upregulation of thermogenic uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) gene expression. This increase is not mediated by any of the known mechanisms for PKM2-regulated gene expression, thus implying the existence of a novel mechanism for PKM2-dependent effects on gene expression.
Asunto(s)
Adipocitos Marrones/enzimología , Adipocitos Marrones/metabolismo , Regulación hacia Abajo , Piruvato Quinasa/metabolismo , Termogénesis/genética , Animales , Línea Celular , Femenino , Factores de Crecimiento de Fibroblastos/genética , Ratones , Piruvato Quinasa/deficiencia , Piruvato Quinasa/genética , Proteína Desacopladora 1/genéticaRESUMEN
Thermogenesis by brown and beige adipose tissue, which requires activation by external stimuli, can counter metabolic disease1. Thermogenic respiration is initiated by adipocyte lipolysis through cyclic AMP-protein kinase A signalling; this pathway has been subject to longstanding clinical investigation2-4. Here we apply a comparative metabolomics approach and identify an independent metabolic pathway that controls acute activation of adipose tissue thermogenesis in vivo. We show that substantial and selective accumulation of the tricarboxylic acid cycle intermediate succinate is a metabolic signature of adipose tissue thermogenesis upon activation by exposure to cold. Succinate accumulation occurs independently of adrenergic signalling, and is sufficient to elevate thermogenic respiration in brown adipocytes. Selective accumulation of succinate may be driven by a capacity of brown adipocytes to sequester elevated circulating succinate. Furthermore, brown adipose tissue thermogenesis can be initiated by systemic administration of succinate in mice. Succinate from the extracellular milieu is rapidly metabolized by brown adipocytes, and its oxidation by succinate dehydrogenase is required for activation of thermogenesis. We identify a mechanism whereby succinate dehydrogenase-mediated oxidation of succinate initiates production of reactive oxygen species, and drives thermogenic respiration, whereas inhibition of succinate dehydrogenase supresses thermogenesis. Finally, we show that pharmacological elevation of circulating succinate drives UCP1-dependent thermogenesis by brown adipose tissue in vivo, which stimulates robust protection against diet-induced obesity and improves glucose tolerance. These findings reveal an unexpected mechanism for control of thermogenesis, using succinate as a systemically-derived thermogenic molecule.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Ácido Succínico/metabolismo , Termogénesis/fisiología , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Adipocitos/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/enzimología , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/enzimología , Tejido Adiposo Blanco/metabolismo , Animales , Femenino , Masculino , Metabolómica , Ratones , Obesidad/metabolismo , Obesidad/prevención & control , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa/metabolismo , Ácido Succínico/farmacología , Termogénesis/efectos de los fármacos , Proteína Desacopladora 1/metabolismoRESUMEN
Brown adipose tissue is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. ß-Adrenergic stimulation of brown adipocytes leads to an increase in oxygen consumption and induction of a thermogenic gene program that includes uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21). In kinase inhibitor screens, we have identified glycogen synthase kinase 3 (GSK3) as a negative regulator of basal and ß-adrenergically stimulated Fgf21 expression in cultured brown adipocytes. In addition, inhibition of GSK3 also caused increased Ucp1 expression and oxygen consumption. ß-Adrenergic stimulation triggered an inhibitory phosphorylation of GSK3 in a protein kinase A (PKA)-dependent manner. Mechanistically, inhibition of GSK3 activated the mitogen activated protein kinase (MAPK) kinase 3/6-p38 MAPK-activating transcription factor 2 signaling module. In summary, our data describe GSK3 as a novel negative regulator of ß-adrenergic signaling in brown adipocytes.
Asunto(s)
Adipocitos Marrones/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Glucógeno Sintasa Quinasa 3/genética , Termogénesis/genética , Proteína Desacopladora 1/genética , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Metabolismo Energético/genética , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Resistencia a la Insulina/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Consumo de Oxígeno/genética , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/administración & dosificación , Receptores Adrenérgicos beta/genética , Transducción de Señal/efectos de los fármacosRESUMEN
During thermogenic activation, brown adipocytes take up large amounts of glucose. In addition, cold stimulation leads to an upregulation of glycolytic enzymes. Here we have investigated the importance of glycolysis for brown adipocyte glucose consumption and thermogenesis. Using siRNA-mediated knockdown in mature adipocytes, we explored the effect of glucose transporters and glycolytic enzymes on brown adipocyte functions such as consumption of glucose and oxygen. Basal oxygen consumption in brown adipocytes was equally dependent on glucose and fatty acid oxidation, whereas isoproterenol (ISO)-stimulated respiration was fueled mainly by fatty acids, with a significant contribution from glucose oxidation. Knockdown of glucose transporters in brown adipocytes not only impaired ISO-stimulated glycolytic flux but also oxygen consumption. Diminishing glycolytic flux by knockdown of the first and final enzyme of glycolysis, i.e., hexokinase 2 (HK2) and pyruvate kinase M (PKM), respectively, decreased glucose uptake and ISO-stimulated oxygen consumption. HK2 knockdown had a more severe effect, which, in contrast to PKM knockdown, could not be rescued by supplementation with pyruvate. Hence, brown adipocytes rely on glucose consumption and glycolytic flux to achieve maximum thermogenic output, with glycolysis likely supporting thermogenesis not only by pyruvate formation but also by supplying intermediates for efferent metabolic pathways.
Asunto(s)
Adipocitos Marrones/efectos de los fármacos , Adipocitos Marrones/metabolismo , Agonistas Adrenérgicos beta/farmacología , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Ácidos Grasos/metabolismo , Isoproterenol/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Oxidación-Reducción/efectos de los fármacos , Termogénesis/efectos de los fármacosRESUMEN
Brown adipose tissue (BAT) metabolism influences glucose homeostasis and metabolic health in mice and humans. Sympathetic stimulation of ß-adrenergic receptors in response to cold induces proliferation, differentiation, and UCP1 expression in pre-adipocytes and mature brown adipocytes. Here we show that spleen tyrosine kinase (SYK) is upregulated during brown adipocyte differentiation and activated by ß-adrenergic stimulation. Deletion or inhibition of SYK, a kinase known for its essential roles in the immune system, blocks brown and white pre-adipocyte proliferation and differentiation in vitro, and results in diminished expression of Ucp1 and other genes regulating brown adipocyte function in response to ß-adrenergic stimulation. Adipocyte-specific SYK deletion in mice reduces BAT mass and BAT that developed consisted of SYK-expressing brown adipocytes that had escaped homozygous Syk deletion. SYK inhibition in vivo represses ß-agonist-induced thermogenesis and oxygen consumption. These results establish SYK as an essential mediator of brown fat formation and function.
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Adipocitos Marrones/enzimología , Tejido Adiposo Pardo/metabolismo , Diferenciación Celular , Quinasa Syk/metabolismo , Adipocitos Marrones/citología , Animales , Proliferación Celular , Células Cultivadas , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Quinasa Syk/genética , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
Brown adipose tissue takes up large amounts of glucose during cold exposure in mice and humans. Here we report an induction of glucose transporter 1 expression and increased expression of several glycolytic enzymes in brown adipose tissue from cold-exposed mice. Accordingly, these genes were also induced after ß-adrenergic activation of cultured brown adipocytes, concomitant with accumulation of hypoxia inducible factor-1α (HIF-1α) protein levels. HIF-1α accumulation was dependent on uncoupling protein 1 and generation of mitochondrial reactive oxygen species. Expression of key glycolytic enzymes was reduced after knockdown of HIF-1α in mature brown adipocytes. Glucose consumption, lactate export and glycolytic capacity were reduced in brown adipocytes depleted of Hif-1α. Finally, we observed a decreased ß-adrenergically induced oxygen consumption in Hif-1α knockdown adipocytes cultured in medium with glucose as the only exogenously added fuel. These data suggest that HIF-1α-dependent regulation of glycolysis is necessary for maximum glucose metabolism in brown adipocytes.
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Adipocitos Marrones/metabolismo , Glucosa/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Adipocitos Marrones/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Células Cultivadas , Frío , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Glucólisis/genética , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Termogénesis/efectos de los fármacos , Termogénesis/genéticaRESUMEN
Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to ß-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.
RESUMEN
The tumor suppressor p53 (TRP53 in mice) is known for its involvement in carcinogenesis, but work during recent years has underscored the importance of p53 in the regulation of whole body metabolism. A general notion is that p53 is necessary for efficient oxidative metabolism. The importance of UCP1-dependent uncoupled respiration and increased oxidation of glucose and fatty acids in brown or brown-like adipocytes, termed brite or beige, in relation to energy balance and homeostasis has been highlighted recently. UCP1-dependent uncoupled respiration in classic interscapular brown adipose tissue is central to cold-induced thermogenesis, whereas brite/beige adipocytes are of special importance in relation to diet-induced thermogenesis, where the importance of UCP1 is only clearly manifested in mice kept at thermoneutrality. We challenged wild-type and TRP53-deficient mice by high-fat feeding under thermoneutral conditions. Interestingly, mice lacking TRP53 gained less weight compared with their wild-type counterparts. This was related to an increased expression of Ucp1 and other PPARGC1a and PPARGC1b target genes but not Ppargc1a or Ppargc1b in inguinal white adipose tissue of mice lacking TRP53. We show that TRP53, independently of its ability to bind DNA, inhibits the activity of PPARGC1a and PPARGC1b. Collectively, our data show that TRP53 has the ability to regulate the thermogenic capacity of adipocytes through modulation of PPARGC1 activity.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Termogénesis/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adipocitos/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Células Cultivadas , Dieta Alta en Grasa , Femenino , Regulación de la Expresión Génica , Canales Iónicos/genética , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteína Desacopladora 1 , Aumento de Peso/fisiologíaRESUMEN
BACKGROUND: Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. RESULTS: The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α. CONCLUSIONS: UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.