RESUMEN
Regenerating pancreatic ß-cells is a potential curative approach for diabetes. We previously identified the small molecule CID661578 as a potent inducer of ß-cell regeneration, but its target and mechanism of action have remained unknown. We now screened 257 million yeast clones and determined that CID661578 targets MAP kinase-interacting serine/threonine kinase 2 (MNK2), an interaction we genetically validated in vivo. CID661578 increased ß-cell neogenesis from ductal cells in zebrafish, neonatal pig islet aggregates and human pancreatic ductal organoids. Mechanistically, we found that CID661578 boosts protein synthesis and regeneration by blocking MNK2 from binding eIF4G in the translation initiation complex at the mRNA cap. Unexpectedly, this blocking activity augmented eIF4E phosphorylation depending on MNK1 and bolstered the interaction between eIF4E and eIF4G, which is necessary for both hypertranslation and ß-cell regeneration. Taken together, our findings demonstrate a targetable role of MNK2-controlled translation in ß-cell regeneration, a role that warrants further investigation in diabetes.
Asunto(s)
Factor 4E Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Animales , Línea Celular , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Recién Nacido , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Pez Cebra/metabolismoRESUMEN
Cidea is a gene highly expressed in thermogenesis-competent (UCP1-containing) adipose cells, both brown and brite/beige. Here, we initially demonstrate a remarkable adipose-depot specific regulation of Cidea expression. In classical brown fat, Cidea mRNA is expressed continuously and invariably, irrespective of tissue recruitment. However, Cidea protein levels are regulated posttranscriptionally, being conspicuously induced in the thermogenically recruited state. In contrast, in brite fat, Cidea protein levels are regulated at the transcriptional level, and Cidea mRNA and protein levels are proportional to tissue "briteness." Although routinely followed as a thermogenic molecular marker, Cidea function is not clarified. Here, we employed a gain-of-function approach to examine a possible role of Cidea in the regulation of thermogenesis. We utilized transgenic aP2-hCidea mice that overexpress human Cidea in all adipose tissues. We demonstrate that UCP1 activity is markedly suppressed in brown-fat mitochondria isolated from aP2-hCidea mice. However, mitochondrial UCP1 protein levels were identical in wild-type and transgenic mice. This implies a regulatory effect of Cidea on UCP1 activity, but as we demonstrate that Cidea itself is not localized to mitochondria, we propose an indirect inhibitory effect. The Cidea-induced inhibition of UCP1 activity (observed in isolated mitochondria) is physiologically relevant since the mice, through an appropriate homeostatic compensatory mechanism, increased the total amount of UCP1 in the tissue to exactly match the diminished thermogenic capacity of the UCP1 protein and retain unaltered nonshivering thermogenic capacity. Thus, we verified Cidea as being a marker of thermogenesis-competent adipose tissues, but we conclude that Cidea, unexpectedly, functions molecularly as an indirect inhibitor of thermogenesis.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Mitocondrias/metabolismo , ARN Mensajero/metabolismo , Termogénesis/genética , Proteína Desacopladora 1/genética , Tejido Adiposo Pardo/patología , Tejido Adiposo Blanco/patología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Calorimetría Indirecta , Frío , Humanos , Ratones , Ratones Transgénicos , Consumo de Oxígeno , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Desacopladora 1/metabolismoRESUMEN
Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α1-adrenoceptor coupled via Gq), clonidine (α2 via Gi) or CL316243 (ß3 via Gs) or via ß1-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration-response relationship (IC50 0.3µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades.
Asunto(s)
Adipocitos Marrones/metabolismo , Receptores ErbB/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptores Adrenérgicos/metabolismo , Adipocitos Marrones/efectos de los fármacos , Antagonistas Adrenérgicos/farmacología , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Agonistas de Receptores Adrenérgicos beta 3/farmacología , Animales , Clonidina/farmacología , Dioxoles/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Norepinefrina/farmacología , Fosforilación , Quinazolinas/farmacología , Tirfostinos/farmacologíaRESUMEN
Analogs of the incretin hormones Gip and Glp-1 are used to treat type 2 diabetes and obesity. Findings in experimental models suggest that manipulating several hormones simultaneously may be more effective. To identify small molecules that increase the number of incretin-expressing cells, we established a high-throughput in vivo chemical screen by using the gip promoter to drive the expression of luciferase in zebrafish. All hits increased the numbers of neurogenin 3-expressing enteroendocrine progenitors, Gip-expressing K-cells, and Glp-1-expressing L-cells. One of the hits, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor, additionally decreased glucose levels in both larval and juvenile fish. Knock-down experiments indicated that nfatc4, a downstream mediator of DYRKs, regulates incretin+ cell number in zebrafish, and that Dyrk1b regulates Glp-1 expression in an enteroendocrine cell line. DYRK inhibition also increased the number of incretin-expressing cells in diabetic mice, suggesting a conserved reinforcement of the enteroendocrine system, with possible implications for diabetes.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Animales , Descubrimiento de Drogas , Polipéptido Inhibidor Gástrico/metabolismo , Polipéptido Inhibidor Gástrico/uso terapéutico , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/uso terapéutico , Glucosa/metabolismo , Incretinas/metabolismo , Incretinas/uso terapéutico , Ratones , Tirosina , Pez Cebra/metabolismoRESUMEN
Caveolae and caveolin have been implicated as being involved in the signal transduction of many receptors, including the EGF, PDGF, LPA and beta3-adrenergic receptors. To investigate the role of caveolin-1 (Cav1) in these signaling pathways in brown adipose tissue, primary brown adipocyte cultures from Cav1-ablated mice and wild-type mice were investigated. In pre-adipocytes, Cav1-ablation affected neither the G-protein coupled LPA receptor signaling to Erk1/2, nor the receptor tyrosine kinases PDGF- or EGF-receptor signaling to Erk1/2. Mature primary Cav1-/- brown adipocytes accumulated lipids and expressed aP2 to the same extent as did wild-type cells. However, the cAMP levels induced by the beta3-adrenergic receptor agonist CL316,243 were lower in the Cav1-/- cultures, with an unchanged EC50 for CL316,243. Also the response to the direct adenylyl cyclase agonist forskolin was reduced. Thus, in brown adipocytes, Cav1 is apparently required for an intact response to adenylyl cyclase-linked agonists/activators, whereas other signaling pathways examined function without Cav1.
Asunto(s)
Adipocitos Marrones/fisiología , Caveolina 1/metabolismo , Receptores ErbB/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Adipocitos Marrones/citología , Agonistas de Receptores Adrenérgicos beta 3 , Agonistas Adrenérgicos beta/metabolismo , Animales , Caveolina 1/genética , Colforsina/metabolismo , AMP Cíclico/metabolismo , Dioxoles/metabolismo , Receptores ErbB/genética , Lisofosfolípidos/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Adrenérgicos beta 3/genética , Receptores del Ácido Lisofosfatídico/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
With the finding that brown adipose tissue is present and negatively correlated to obesity in adult man, finding the mechanism(s) of how to activate brown adipose tissue in humans could be important in combating obesity, type 2 diabetes, and their complications. In mice, the main regulator of nonshivering thermogenesis in brown adipose tissue is norepinephrine acting predominantly via ß(3)-adrenergic receptors. However, vast majorities of ß(3)-adrenergic agonists have so far not been able to stimulate human ß(3)-adrenergic receptors or brown adipose tissue activity, and it was postulated that human brown adipose tissue could be regulated instead by ß(1)-adrenergic receptors. Therefore, we have investigated the signaling pathways, specifically pathways to nonshivering thermogenesis, in mice lacking ß(3)-adrenergic receptors. Wild-type and ß(3)-knockout mice were either exposed to acute cold (up to 12 h) or acclimated for 7 wk to cold, and parameters related to metabolism and brown adipose tissue function were investigated. ß(3)-knockout mice were able to survive both acute and prolonged cold exposure due to activation of ß(1)-adrenergic receptors. Thus, in the absence of ß(3)-adrenergic receptors, ß(1)-adrenergic receptors are effectively able to signal via cAMP to elicit cAMP-mediated responses and to recruit and activate brown adipose tissue. In addition, we found that in human multipotent adipose-derived stem cells differentiated into functional brown adipocytes, activation of either ß(1)-adrenergic receptors or ß(3)-adrenergic receptors was able to increase UCP1 mRNA and protein levels. Thus, in humans, ß(1)-adrenergic receptors could play an important role in regulating nonshivering thermogenesis.
Asunto(s)
Aclimatación/genética , Adipocitos Marrones/metabolismo , Canales Iónicos/genética , Proteínas Mitocondriales/genética , Receptores Adrenérgicos beta 1/fisiología , Receptores Adrenérgicos beta 3/genética , Termogénesis/genética , Aclimatación/fisiología , Adipocitos Marrones/citología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Frío , Regulación hacia Abajo/genética , Epistasis Genética/fisiología , Femenino , Humanos , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas Mitocondriales/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Tiritona/genética , Tiritona/fisiología , Termogénesis/fisiología , Proteína Desacopladora 1RESUMEN
In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G(i)-protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.
Asunto(s)
Adipocitos Marrones/efectos de los fármacos , Adipocitos Marrones/metabolismo , Lisofosfolípidos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Adipocitos Marrones/citología , Animales , AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Immunoblotting , Masculino , Ratones , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Activación TranscripcionalRESUMEN
Caveolin-1 (Cav1)-ablated mice display impaired lipolysis in white adipose tissue. They also seem to have an impairment in brown adipose tissue function, implying that Cav1-ablated mice could encounter problems in surviving longer periods in cold temperatures. To investigate this, Cav1-ablated mice and wild-type mice were transferred to cold temperatures for extended periods of time, and parameters related to metabolism and thermogenesis were investigated. Unexpectedly, the Cav1-ablated mice survived in the cold. There were no differences between Cav1-ablated and wild-type mice with regard to food intake, in behavior related to shivering, or in body temperature. The Cav1-ablated mice had a halved total fat content independently of acclimation temperature. There was no difference in brown adipose tissue uncoupling protein-1 (UCP1) protein amount, and isolated brown fat mitochondria were thermogenically competent but displayed 30% higher thermogenic capacity. However, the beta(3)-adrenergic receptor amount was reduced by about one-third in the Cav1-ablated mice at all acclimation temperatures. Principally in accordance with this, a higher than standard dose of norepinephrine was needed to obtain full norepinephrine-induced thermogenesis in the Cav1-ablated mice; the higher dose was also needed for the Cav1-ablated mice to be able to utilize fat as a substrate for thermogenesis. In conclusion, the ablation of Cav1 impairs brown adipose tissue function by a desensitization of the adrenergic response; however, the desensitization is not evident in the animal as it is overcome physiologically, and Cav1-ablated mice can therefore survive in prolonged cold by nonshivering thermogenesis.
Asunto(s)
Tejido Adiposo Pardo/fisiología , Caveolina 1/fisiología , Receptores Adrenérgicos beta/fisiología , Termogénesis/fisiología , Animales , Metabolismo Basal , Composición Corporal , Temperatura Corporal/fisiología , Peso Corporal/fisiología , Calorimetría Indirecta , Frío , Ingestión de Alimentos/fisiología , Femenino , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/fisiología , Norepinefrina/farmacologíaRESUMEN
Stimulation by both adrenergic and non-adrenergic pathways can induce proliferation of brown pre-adipocytes. To understand the signalling pathways involved in non-adrenergic stimulation of cell proliferation, we examined Erk1/2 activation. In primary cultures of mouse brown pre-adipocytes, both EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) induced Erk1/2 activation. EGF-stimulated Erk1/2 activation involved Src tyrosine kinases, but not PKC or PI3K, whereas in PDGF-induced Erk1/2 activation, PI3K, PKC (probably the atypical zeta isoform) and Src were involved sequentially. Both EGF and PDGF induced PI3K-dependent Akt activation that was not involved in Erk1/2 activation. By comparing effects of signalling inhibitors (wortmannin, SH-6, TPA, Gö6983, PP2, PD98059) on EGF- and PDGF-induced Erk1/2 activation and cell proliferation (3H-thymidine incorporation), we conclude that while the signal transduction pathways initiated by these growth factors are clearly markedly different, their effects on cell proliferation can be fully explained through their stimulation of Erk1/2 activation; thus Erk1/2 is a common, essential step for stimulation of proliferation in these cells.
Asunto(s)
Adipocitos Marrones/enzimología , Factor de Crecimiento Epidérmico/farmacología , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Adipocitos Marrones/citología , Adipocitos Marrones/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Flavonoides/farmacología , Masculino , Ratones , Ratones Endogámicos , Proteína Quinasa C/metabolismo , Células Madre/efectos de los fármacos , Células Madre/enzimología , Familia-src Quinasas/metabolismoRESUMEN
Inhibition of notch signaling is known to induce differentiation of endocrine cells in zebrafish and mouse. After performing an unbiased in vivo screen of â¼2,200 small molecules in zebrafish, we identified an inhibitor of Cdk5 (roscovitine), which potentiated the formation of ß-cells along the intrapancreatic duct during concurrent inhibition of notch signaling. We confirmed and characterized the effect with a more selective Cdk5 inhibitor, (R)-DRF053, which specifically increased the number of duct-derived ß-cells without affecting their proliferation. By duct-specific overexpression of the endogenous Cdk5 inhibitors Cdk5rap1 or Cdkal1 (which previously have been linked to diabetes in genome-wide association studies), as well as deleting cdk5, we validated the role of chemical Cdk5 inhibition in ß-cell differentiation by genetic means. Moreover, the cdk5 mutant zebrafish displayed an increased number of ß-cells independently of inhibition of notch signaling, in both the basal state and during ß-cell regeneration. Importantly, the effect of Cdk5 inhibition to promote ß-cell formation was conserved in mouse embryonic pancreatic explants, adult mice with pancreatic ductal ligation injury, and human induced pluripotent stem (iPS) cells. Thus, we have revealed a previously unknown role of Cdk5 as an endogenous suppressor of ß-cell differentiation and thereby further highlighted its importance in diabetes.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Conductos Pancreáticos/citología , Células Madre/citología , Células Madre/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Quinasa 5 Dependiente de la Ciclina/genética , Estudio de Asociación del Genoma Completo , Genotipo , Larva/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
OBJECTIVE: Pharmacological activation of adenosine signaling has been shown to increase ß-cell proliferation and thereby ß-cell regeneration in zebrafish and rodent models of diabetes. However, whether adenosine has an endogenous role in regulating ß-cell proliferation is unknown. The objective of this study was to determine whether endogenous adenosine regulates ß-cell proliferation-either in the basal state or states of increased demand for insulin-and to delineate the mechanisms involved. METHODS: We analyzed the effect of pharmacological adenosine agonists on ß-cell proliferation in in vitro cultures of mouse islets and in zebrafish models with ß- or δ-cell ablation. In addition, we performed physiological and histological characterization of wild-type mice and mutant mice with pancreas- or ß-cell-specific deficiency in Adora2a (the gene encoding adenosine receptor A2a). The mutant mice were used for in vivo studies on the role of adenosine in the basal state and during pregnancy (a state of increased demand for insulin), as well as for in vitro studies of cultured islets. RESULTS: Pharmacological adenosine signaling in zebrafish had a stronger effect on ß-cell proliferation during ß-cell regeneration than in the basal state, an effect that was independent of the apoptotic microenvironment of the regeneration model. In mice, deficiency in Adora2a impaired glucose control and diminished compensatory ß-cell proliferation during pregnancy but did not have any overt phenotype in the basal state. Islets isolated from Adora2a-deficient mice had a reduced baseline level of ß-cell proliferation in vitro, consistent with our finding that UK432097, an A2a-specific agonist, promotes the proliferation of mouse ß-cells in vitro. CONCLUSIONS: This is the first study linking endogenously produced adenosine to ß-cell proliferation. Moreover, we show that adenosine signaling via the A2a receptor has an important role in compensatory ß-cell proliferation, a feature that could be harnessed pharmacologically for ß-cell expansion and future therapeutic development for diabetes.
Asunto(s)
Proliferación Celular , Células Secretoras de Insulina/fisiología , Receptor de Adenosina A2A/fisiología , Adenosina , Animales , Femenino , Insulina , Ratones , Embarazo , Receptor de Adenosina A2A/genéticaRESUMEN
The environmental pollutants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) cause a dramatic reduction in the size of the major adipose tissue depots and a general body weight decrease when they are added to the food of mice. We demonstrate here that this is mainly due to a reduction in food intake; this reduction was not due to food aversion. Remarkably and unexpectedly, a large part of the effect of PFOA/PFOS on food intake was dependent on the presence of the uncoupling protein 1 (UCP1) in the mice. Correspondingly, PFOA/PFOS treatment induced recruitment of brown adipose tissue mitochondria: increased oxidative capacity and increased UCP1-mediated oxygen consumption (thermogenesis). In mice pair-fed to the food intake during PFOA/PFOS treatment in wildtype mice, brown-fat mitochondrial recruitment was also induced. We conclude that we have uncovered the existence of a regulatory component of food intake that is dependent upon brown adipose tissue thermogenic activity. The possible environmental consequences of this novel PFOA/PFOS effect (a possible decreased fitness) are noted, as well as the perspectives of this finding on the general understanding of control of food intake control and its possible extension to combatting obesity.