RESUMEN
OBJECTIVE: Simultaneous activation of ß2- and ß3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of ß1-ARs - and thus inducing cardiovascular complications - are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel ß2-and ß3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models. METHODS: In the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective ß-AR agonist isoprenaline across various rodent ß-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5 mg/kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis. RESULTS: Exposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis. CONCLUSIONS: Our results demonstrate that ATR-127 is a highly effective, novel ß2- and ß3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies.
Asunto(s)
Tejido Adiposo Pardo , Ratones Endogámicos C57BL , Músculo Esquelético , Animales , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Masculino , Ratas , Obesidad/metabolismo , Obesidad/tratamiento farmacológico , Hígado Graso/metabolismo , Hígado Graso/tratamiento farmacológico , Termogénesis/efectos de los fármacos , Agonistas Adrenérgicos/farmacologíaRESUMEN
ß2-Adrenergic receptor (ß2AR) agonists have been reported to stimulate glucose uptake (GU) by skeletal muscle cells and are therefore highly interesting as a possible treatment for type 2 diabetes (T2D). The chirality of compounds often has a great impact on the activity of ß2AR agonists, although this has thus far not been investigated for GU. Here we report the GU for a selection of synthesized acyclic and cyclic ß-hydroxy-3-fluorophenethylamines. For the N-butyl and the N-(2-pentyl) compounds, the (R) and (R,R) (3d and 7e) stereoisomers induced the highest GU. When the compounds contained a saturated nitrogen containing 4- to 7-membered heterocycle, the (R,R,R) enantiomer of the azetidine (8a) and the pyrrolidine (9a) had the highest activity. Altogether, these results provide pivotal information for designing novel ß2AR agonist for the treatment of T2D.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2 , Diabetes Mellitus Tipo 2 , Humanos , Agonistas Adrenérgicos , Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Agonistas Adrenérgicos beta/química , Agonistas Adrenérgicos beta/farmacología , Aminas , Transporte Biológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucosa , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
Induction of thermogenesis in brown and brite adipocytes has recently emerged as a therapeutic target for novel anti obesogenic therapies necessitating the development of methods that can accurately measure heat production in these cells. Modern isothermal microcalorimetric techniques allow for the high throughput quantitative measurement of cellular heat production with limited sample material. Here, we describe the application of this technique for the measurement of thermogenesis in both floating and adherent adipocytes from various murine depots and human cell lines.
Asunto(s)
Adipocitos Beige , Adipocitos , Humanos , Animales , Ratones , Adipocitos/metabolismo , Línea Celular , Termogénesis , Adipocitos Marrones/metabolismoRESUMEN
The activation of thermogenesis in adipose tissue has emerged as an important target for the development of novel anti-obesity therapies. Using multi-well isothermal microcalorimetry, we have demonstrated that mature murine brown and brite adipocytes produce quantifiable heat upon ß3-AR stimulation, independently of any anaerobic mechanisms. Additionally, in brite adipocytes lacking UCP1 protein, ß3-AR stimulation still induces heat production, albeit to a much lower extent than in their wildtype counterparts, suggesting that UCP1 is an essential component of adrenergic induced thermogenesis in murine brite adipocytes exvivo. Similarly, we could observe an increase in heat production in human-derived adipocytes (hMADS) upon ß-AR stimulation. Collectively, these results establish the use of isothermal microcalorimetry as a sensitive and accurate technique for measuring thermogenic responses in intact mature brite adipocytes from murine and human origin.
Asunto(s)
Adipocitos Beige/fisiología , Termogénesis/genética , Proteína Desacopladora 1/genética , Animales , Calorimetría , Masculino , Ratones , Proteína Desacopladora 1/metabolismoRESUMEN
AIMS/HYPOTHESIS: Chronic stimulation of ß2-adrenoceptors, opposite to acute treatment, was reported to reduce blood glucose levels, as well as to improve glucose and insulin tolerance in rodent models of diabetes by essentially unknown mechanisms. We recently described a novel pathway that mediates glucose uptake in skeletal muscle cells via stimulation of ß2-adrenoceptors. In the current study we further explored the potential therapeutic relevance of ß2-adrenoceptor stimulation to improve glucose homeostasis and the mechanisms responsible for the effect. METHODS: C57Bl/6N mice with diet-induced obesity were treated both acutely and for up to 42 days with a wide range of clenbuterol dosages and treatment durations. Glucose homeostasis was assessed by glucose tolerance test. We also measured in vivo glucose uptake in skeletal muscle, insulin sensitivity by insulin tolerance test, plasma insulin levels, hepatic lipids and glycogen. RESULTS: Consistent with previous findings, acute clenbuterol administration increased blood glucose and insulin levels. However, already after 4 days of treatment, beneficial effects of clenbuterol were manifested in glucose homeostasis (32% improvement of glucose tolerance after 4 days of treatment, p < 0.01) and these effects persisted up to 42 days of treatment. These favourable metabolic effects could be achieved with doses as low as 0.025 mg kg-1 day-1 (40 times lower than previously studied). Mechanistically, these effects were not due to increased insulin levels, but clenbuterol enhanced glucose uptake in skeletal muscle in vivo both acutely in lean mice (by 64%, p < 0.001) as well as during chronic treatment in diet-induced obese mice (by 74%, p < 0.001). Notably, prolonged treatment with low-dose clenbuterol improved whole-body insulin sensitivity (glucose disposal rate after insulin injection increased up to 1.38 ± 0.31%/min in comparison with 0.15 ± 0.36%/min in control mice, p < 0.05) and drastically reduced hepatic steatosis (by 40%, p < 0.01) and glycogen (by 23%, p < 0.05). CONCLUSIONS/INTERPRETATION: Clenbuterol improved glucose tolerance after 4 days of treatment and these effects were maintained for up to 42 days. Effects were achieved with doses in a clinically relevant microgram range. Mechanistically, prolonged treatment with a low dose of clenbuterol improved glucose homeostasis in insulin resistant mice, most likely by stimulating glucose uptake in skeletal muscle and improving whole-body insulin sensitivity as well as by reducing hepatic lipids and glycogen. We conclude that selective ß2-adrenergic agonists might be an attractive potential treatment for type 2 diabetes. This remains to be confirmed in humans. Graphical abstract.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Clenbuterol/uso terapéutico , Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Glucosa/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Animales , Homeostasis/efectos de los fármacos , Resistencia a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/metabolismoRESUMEN
Surveying microglia, the resident macrophage-like cells in the central nervous system, continuously screen their surroundings to sense imbalance in tissue homeostasis. Their activity is tightly regulated in both a pro- and anti-inflammatory manner. We have previously shown that the lipoglycoproteins WNT-3A and WNT-5A drive pro-inflammatory transformation in primary mouse microglia cells, arguing that WNTs have a role in the modulation of the central nervous system immune response. In this study, we address the effects of recombinant WNT-3A and WNT-5A on lipopolysaccharide (LPS)-activated mouse primary microglia to investigate the putative anti-inflammatory modulation of microglia by WNTs. While both WNT-3A and WNT-5A alone induce an up-regulation of cyclooxygenase 2 (COX2), a generic pro-inflammatory microglia marker, LPS exceeds these effects dramatically. However, combination of LPS and WNTs results in a dose-dependent decrease in LPS-induced cyclooxygenase 2 protein and mRNA expression. In conclusion, our data suggest that WNTs have a dual and context-dependent effect on microglia acting in a homeostatic pro- and anti-inflammatory manner.
Asunto(s)
Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Proteínas Wnt/metabolismo , Proteína Wnt3A/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Microglía/inmunología , Microglía/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas/farmacología , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Wnt/farmacología , Proteína Wnt-5a , Proteína Wnt3A/farmacologíaRESUMEN
WNT-3A is a secreted lipoglycoprotein that engages Class Frizzled receptors and LDL receptor related protein 5/6 (LRP5/6) for cellular communication. Generally, WNT-3A mediates WNT/ß-catenin signaling to regulate TCF/LEF-dependent gene expression. We have previously shown that ß-catenin levels are elevated in proinflammatory microglia of Alzheimer's disease patients and that WNT-3A can evoke a strong proinflammatory response in primary microglia. In order to investigate the underlying mechanisms, we focus here on the pharmacological dissection of WNT-3A-induced signaling to ß-catenin and to the extracellular signal-regulated kinases 1/2 (ERK1/2) in mouse primary microglia. Both pathways are induced by WNT-3A with slightly different kinetics, suggesting that they might be pharmacologically separable. Inhibition of heterotrimeric Gαi/o proteins by pertussis toxin blocks WNT-3A-induced LRP6 phosphorylation, disheveled shift, ß-catenin stabilization and phosphorylation of ERK1/2. On the other hand LRP6 blockade by Dickkopf 1 treatment abrogated the WNT/ß-catenin pathway without affecting WNT/ERK1/2 signaling. In the opposite way, inhibition of ßγ subunits, phospholipase C (PLC), intracellular calcium and MEK1/2, the upstream kinase of ERK1/2, blocked ERK1/2 phosphorylation but not ß-catenin stabilization. In summary, the data suggest a central role of Gαi/o for both ß-catenin-dependent and -independent pathways. WNT-3A-induced ERK1/2 phosphorylation is mediated by ßγ subunits, PLC, intracellular calcium and MEK1/2. Furthermore, we show that cyclooxygenase 2 (COX2), a generic proinflammatory marker of microglia, is induced by WNT-3A through ERK1/2-dependent pathways arguing that ß-catenin-independent signaling downstream of WNT-3A is of physiological importance for the proinflammatory regulation of microglia.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Microglía/efectos de los fármacos , Toxina del Pertussis/toxicidad , Transducción de Señal/efectos de los fármacos , Proteína Wnt3A/farmacología , Aminoacetonitrilo/análogos & derivados , Aminoacetonitrilo/farmacología , Animales , Calcio/metabolismo , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Cinética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Fosfolipasas de Tipo C/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt3A/aislamiento & purificación , beta Catenina/metabolismoRESUMEN
BACKGROUND: WNT-5A signaling in the central nervous system is important for morphogenesis, neurogenesis and establishment of functional connectivity; the source of WNT-5A and its importance for cellular communication in the adult brain, however, are mainly unknown. We have previously investigated the inflammatory effects of WNT/ß-catenin signaling in microglia in Alzheimer's disease. WNT-5A, however, generally recruits ß-catenin-independent signaling. Thus, we aim here to characterize the role of WNT-5A and downstream signaling pathways for the inflammatory transformation of the brain's macrophages, the microglia. METHODS: Mouse brain sections were used for immunohistochemistry. Primary isolated microglia and astrocytes were employed to characterize the WNT-induced inflammatory transformation and underlying intracellular signaling pathways by immunoblotting, quantitative mRNA analysis, proliferation and invasion assays. Further, measurements of G protein activation by [γ-(35)S]GTP binding, examination of calcium fluxes and cyclic AMP production were used to define intracellular signaling pathways. RESULTS: Astrocytes in the adult mouse brain express high levels of WNT-5A, which could serve as a novel astroglia-microglia communication pathway. The WNT-5A-induced proinflammatory microglia response is characterized by increased expression of inducible nitric oxide synthase, cyclooxygenase-2, cytokines, chemokines, enhanced invasive capacity and proliferation. Mapping of intracellular transduction pathways reveals that WNT-5A activates heterotrimeric G(i/o) proteins to reduce cyclic AMP levels and to activate a G(i/o) protein/phospholipase C/calcium-dependent protein kinase/extracellular signal-regulated kinase 1/2 (ERK1/2) axis. We show further that WNT-5A-induced ERK1/2 signaling is responsible for distinct aspects of the proinflammatory transformation, such as matrix metalloprotease 9/13 expression, invasion and proliferation. CONCLUSIONS: Thus, WNT-5A-induced and G protein-dependent signaling to ERK1/2 is important for the regulation of proinflammatory responses in mouse primary microglia cells. We show for the first time that WNT-5A/G protein signaling mediates physiologically important processes in primary mammalian cells with natural receptor and G protein stochiometry. Consequently, WNT-5A emerges as an important means of astrocyte-microglia communication and we, therefore, suggest WNT-5A as a new player in neuroinflammatory conditions, such as neurodegenerative disease, hypoxia, stroke, injury and infection.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/fisiología , Mediadores de Inflamación/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Microglía/patología , Proteínas Wnt/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Microglía/fisiología , Proteína Wnt-5aRESUMEN
Microglia activation is central to the neuroinflammation associated with neurological and neurodegenerative diseases, particularly because activated microglia are often a source of proinflammatory cytokines. Despite decade-long research, the molecular cascade of proinflammatory transformation of microglia in vivo remains largely elusive. Here, we report increased ß-catenin expression, a central intracellular component of WNT signaling, in microglia undergoing a proinflammatory morphogenic transformation under pathogenic conditions associated with neuroinflammation such as Alzheimer's disease. We substantiate disease-associated ß-catenin signaling in microglia in vivo by showing age-dependent ß-catenin accumulation in mice with Alzheimer's-like pathology (APdE9). In cultured mouse microglia expressing the WNT receptors Frizzled FZD(4,5,7,8) and LDL receptor-related protein 5/6 (LRP5/6), we find that WNT-3A can stabilize ß-catenin. WNT-3A dose dependently induces LRP6 phosphorylation with downstream activation of disheveled, ß-catenin stabilization, and nuclear import. Gene-expression profiling reveals that WNT-3A stimulation specifically increases the expression of proinflammatory immune response genes in microglia and exacerbates the release of de novo IL-6, IL-12, and tumor necrosis factor α. In summary, our data suggest that the WNT family of lipoglycoproteins can instruct proinflammatory microglia transformation and emphasize the pathogenic significance of ß-catenin-signaling networks in this cell type.