RESUMEN
We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5'-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2-/-) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2-/- mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2-/- mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2-/- versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2-/- versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.
Asunto(s)
5'-Nucleotidasa/genética , Dieta Alta en Grasa/efectos adversos , Eliminación de Gen , Resistencia a la Insulina , Aumento de Peso , Animales , Glucosa/metabolismo , Lipólisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/metabolismo , Obesidad/genética , Obesidad/prevención & controlRESUMEN
Liver X receptors limit cellular lipid uptake by stimulating the transcription of Inducible Degrader of the LDL Receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis. Unexpectedly, analysis of tissue-specific knockout mice revealed that IDOL affects energy balance, not through its actions in peripheral metabolic tissues (liver, adipose, endothelium, intestine, skeletal muscle), but by controlling lipoprotein receptor abundance in neurons. Single-cell RNA sequencing of the hypothalamus demonstrated that IDOL deletion altered gene expression linked to control of metabolism. Finally, we identify VLDLR rather than LDLR as the primary mediator of IDOL effects on energy balance. These studies identify a role for the neuronal IDOL-VLDLR pathway in metabolic homeostasis and diet-induced obesity.
Asunto(s)
Metabolismo Energético/fisiología , Neuronas/metabolismo , Receptores de LDL/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Animales , Glucemia/metabolismo , Dieta , Metabolismo Energético/genética , Hipotálamo/metabolismo , Resistencia a la Insulina , Ratones , Ratones Noqueados , Obesidad/metabolismo , Obesidad/prevención & control , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
AMP-activated protein kinase (AMPK) plays a key role in energy homeostasis and is activated in response to contraction-induced ATP depletion in skeletal muscle via a rise in intracellular AMP/ADP concentrations. AMP can be deaminated by AMP-deaminase (AMPD) to IMP, which is hydrolyzed to inosine by cytosolic 5'-nucleotidase II (NT5C2). AMP can also be hydrolyzed to adenosine by cytosolic 5'-nucleotidase 1A (NT5C1A). Previous gene silencing and overexpression studies indicated control of AMPK activation by NT5C enzymes. In the present study using gene knockout mouse models, we investigated the effects of NT5C1A and NT5C2 deletion on intracellular adenine nucleotide levels and AMPK activation in electrically stimulated skeletal muscles. Surprisingly, NT5C enzyme knockout did not lead to enhanced AMP or ADP concentrations in response to contraction, with no potentiation of increases in AMPK activity in extensor digitorum longus (EDL) and soleus mouse muscles. Moreover, dual blockade of AMP metabolism in EDL using an AMPD inhibitor combined with NT5C1A deletion did not enhance rises in AMP and ADP or increased AMPK activation by electrical stimulation. The results on muscles from the NT5C knockout mice contradict previous findings where AMP levels and AMPK activity were shown to be modulated by NT5C enzymes.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , 5'-Nucleotidasa , Animales , Activación Enzimática , Eliminación de Gen , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nucleótidos/metabolismo , SolubilidadRESUMEN
AMP-activated protein kinase (AMPK) plays a central role in regulating metabolism and energy homeostasis. It achieves its function by sensing fluctuations in the AMP:ATP ratio. AMP deaminase (AMPD) converts AMP into IMP, and the AMPD1 isoenzyme is expressed in skeletal muscles. Here, effects of pharmacological inhibition and genetic deletion of AMPD were examined in contracting skeletal muscles. Pharmacological AMPD inhibition potentiated rises in AMP, AMP:ATP ratio, AMPK Thr172, and acetyl-CoA carboxylase (ACC) Ser218 phosphorylation induced by electrical stimulation, without affecting glucose transport. In incubated extensor digitorum longus and soleus muscles from Ampd1 knockout mice, increases in AMP levels and AMP:ATP ratio by electrical stimulation were potentiated considerably compared with muscles from wild-type mice, whereas enhanced AMPK activation was moderate and only observed in soleus, suggesting control by factors other than changes in adenine nucleotides. AMPD inhibitors could be useful tools for enhancing AMPK activation in cells and tissues during ATP-depletion.
Asunto(s)
AMP Desaminasa/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Inhibidores Enzimáticos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/metabolismo , AMP Desaminasa/antagonistas & inhibidores , AMP Desaminasa/genética , Acetil-CoA Carboxilasa/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Estimulación Eléctrica , Inhibidores Enzimáticos/química , Glucosa/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Nucleótidos de Purina/metabolismo , Ratas , Ratas WistarRESUMEN
The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a target for novel type 2 diabetes and obesity therapies based on the premise that lowering of tissue glucocorticoids will have positive effects on body weight, glycemic control, and insulin sensitivity. An 11ß-HSD1 inhibitor (compound C) inhibited liver 11ß-HSD1 by >90% but led to only small improvements in metabolic parameters in high-fat diet (HFD)-fed male C57BL/6J mice. A 4-fold higher concentration produced similar enzyme inhibition but, in addition, reduced body weight (17%), food intake (28%), and glucose (22%). We hypothesized that at the higher doses compound C might be accessing the brain. However, when we developed male brain-specific 11ß-HSD1 knockout mice and fed them the HFD, they had body weight and fat pad mass and glucose and insulin responses similar to those of HFD-fed Nestin-Cre controls. We then found that administration of compound C to male global 11ß-HSD1 knockout mice elicited improvements in metabolic parameters, suggesting "off-target" mechanisms. Based on the patent literature, we synthesized another 11ß-HSD1 inhibitor (MK-0916) from a different chemical series and showed that it too had similar off-target body weight and food intake effects at high doses. In summary, a significant component of the beneficial metabolic effects of these 11ß-HSD1 inhibitors occurs via 11ß-HSD1-independent pathways, and only limited efficacy is achievable from selective 11ß-HSD1 inhibition. These data challenge the concept that inhibition of 11ß-HSD1 is likely to produce a "step-change" treatment for diabetes and/or obesity.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Metabolismo Energético/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hipoglucemiantes/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Triazoles/farmacología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Tejido Adiposo/metabolismo , Animales , Glucemia , Peso Corporal , Encéfalo/metabolismo , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/efectos adversos , Relación Dosis-Respuesta a Droga , Femenino , Genotipo , Glucosa/metabolismo , Hipoglucemiantes/química , Insulina/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Estructura Molecular , Pirazoles/química , Pirimidinas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triazoles/químicaRESUMEN
Metabolic syndrome is growing in importance with the rising levels of obesity, type 2 diabetes, and insulin resistance. Metabolic syndrome shares many characteristics with Cushing's syndrome, which has led to investigation of the link between excess glucocorticoids and metabolic syndrome. Indeed, increased glucocorticoids from intracellular regeneration by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) drives insulin resistance and increases adiposity, but these metabolic changes are assumed to be due to increased circulating glucocorticoids. We hypothesized that increasing the substrate for 11ß-HSD1 (11-dehydrocorticosterone, 11-DHC) would adversely affect metabolic parameters. We found that chronic administration of 11-DHC to male C57BL/6J mice resulted in increased circulating glucocorticoids, and down-regulation of the hypothalamic-pituitary-adrenal axis. This elevated 11ß-HSD1-derived corticosterone led to increased body weight gain and adiposity and produced marked insulin resistance. Surprisingly liver-specific 11ß-HSD1 knockout (LKO) mice given 11-DHC did not show any of the adverse metabolic effects seen in wild-type mice. This occurred despite the 11-DHC administration resulting in elevated circulating corticosterone, presumably from adipose tissue. Mice with global deletion of 11ß-HSD1 (global knockout) were unaffected by treatment with 11-DHC, having no increase in circulating corticosterone and exhibiting no signs of metabolic impairment. Taken together, these data show that in the absence of 11ß-HSD1 in the liver, mice are protected from the metabolic effects of 11-DHC administration, even though circulating glucocorticoids are increased. This implies that liver-derived intratissue glucocorticoids, rather than circulating glucocorticoids, contribute significantly to the development of metabolic syndrome and suggest that local action within hepatic tissue mediates these effects.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Corticosterona/análogos & derivados , Glucocorticoides/metabolismo , Hígado/enzimología , Síndrome Metabólico/etiología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Adiposidad , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Corticosterona/administración & dosificación , Corticosterona/efectos adversos , Corticosterona/sangre , Corticosterona/metabolismo , Regulación hacia Abajo , Glucocorticoides/administración & dosificación , Glucocorticoides/efectos adversos , Glucocorticoides/sangre , Hiperinsulinismo/etiología , Hiperfagia/etiología , Hiperfagia/fisiopatología , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipotálamo-Hipofisario/fisiopatología , Resistencia a la Insulina , Hígado/metabolismo , Masculino , Síndrome Metabólico/sangre , Síndrome Metabólico/metabolismo , Síndrome Metabólico/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sistema Hipófiso-Suprarrenal/metabolismo , Sistema Hipófiso-Suprarrenal/fisiopatología , Aumento de PesoRESUMEN
Platelet-derived growth factor receptor α (PDGFRα) is a receptor tyrosine kinase that promotes cell survival and is expressed in both the tumor and the stromal components of human cancers. We have developed a fully human monoclonal antibody, MEDI-575, that selectively binds to human PDGFRα with high affinity, with no observable affinity for murine PDGFRα. To more fully characterize the role of PDGFRα in the regulation of tumor stroma, we evaluated the in vivo antitumor effects of MEDI-575 in tumor-bearing severe combined immunodeficient (SCID) mice and in genetically altered SCID mice expressing human PDGFRα in place of murine PDGFRα. We used the Calu-6 non-small cell lung cancer model because it lacks an in vitro proliferative response to PDGFRα activation. Antitumor activity was observed when the study was performed in mice expressing the human receptor, but no activity was observed in the mice expressing the murine receptor. Immunohistologic analysis of the tumors from mice expressing human PDGFRα showed a highly significant reduction in stromal fibroblast content and only minor changes in tumor proliferative index in tumors exposed to MEDI-575 compared with the results seen in vehicle-treated tumors or in tumors from mice expressing murine PDGFRα. Additional in vitro studies indicated that exposure of primary cancer-associated fibroblasts to MEDI-575 can directly affect proliferation and key signaling pathways in these cells. These results highlight the potential for observing antitumor activity with MEDI-575 through modulation of the stromal component of tumors and confirm that the PDGFRα pathway can play a role in maintaining a tumor microenvironment conducive to tumor growth.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Fibroblastos/efectos de los fármacos , Neoplasias Pulmonares/patología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Ratones SCID , Células 3T3 NIH , Trasplante de Neoplasias , Fosforilación , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/patología , Trasplante HeterólogoRESUMEN
ADAMTS1, a secreted multifunctional metalloproteinase with disintegrin and thrombospondin motifs, is an early response gene of parathyroid hormone (PTH) in osteoblasts. Mice engineered to lack Adamts1 are smaller compared to wild-type (WT) mice and ADAMTS1 metalloproteinase activity has been shown to increase osteoblastic growth in collagen gels. However, there are no reports investigating the consequence of Adamts1 over-expression on bone tissue in vivo. Here, we analyze bones of female and male transgenic (TG) mice over-expressing mouse Adamts1 using peripheral quantitative computed tomography to evaluate its effect on bone shape and mineral density. Western blotting of protein extracts and immunohistochemistry of bone sections reveal increased presence of Adamts1 protein in TG bones compared to WT bones. Phenotypic analyses of femur show that female TG mice have reduced metaphyseal total density, trabecular bone mineral density and trabecular mineral content. In contrast, male TG mice which were without changes in the metaphysis showed increased total density and cortical density at the mid-diaphysis cortical site. Female TG mice showed no significant changes at the cortical site compared to WT mice. Furthermore, diaphyseal endosteal compartment was only affected in male TG mice. Along these lines, Adamts1 increased blood levels of PTH only in females whereas it reduced osteocalcin levels only in males. These results reveal that Adamts1 has an impact on bone mineral density and thus further confirm Adamts1 as a potent regulator of bone remodeling.
Asunto(s)
Proteínas ADAM/metabolismo , Densidad Ósea/fisiología , Fémur/fisiología , Proteínas ADAM/genética , Proteína ADAMTS1 , Animales , Femenino , Fémur/diagnóstico por imagen , Fémur/enzimología , Regulación Enzimológica de la Expresión Génica , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Osteocalcina/metabolismo , Adhesión en Parafina , Hormona Paratiroidea/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Caracteres Sexuales , Tomografía Computarizada por Rayos XRESUMEN
Melanin-concentrating hormone (MCH) plays an important role in energy balance. The current studies were carried out on a new line of mice lacking the rodent MCH receptor (MCHR1(-/-) mice). These mice confirmed the previously reported lean phenotype characterized by increased energy expenditure and modestly increased caloric intake. Because MCH is expressed in the lateral hypothalamic area, which also has an important role in the regulation of the autonomic nervous system, heart rate and blood pressure were measured by a telemetric method to investigate whether the increased energy expenditure in these mice might be due to altered autonomic nervous system activity. Male MCHR1(-/-) mice demonstrated a significantly increased heart rate [24-h period: wild type 495 +/- 4 vs. MCHR1(-/-) 561 +/- 8 beats/min (P < 0.001); dark phase: wild type 506 +/- 8 vs. MCHR1(-/-) 582 +/- 9 beats/min (P < 0.001); light phase: wild type 484 +/- 13 vs. MCHR1(-/-) 539 +/- 9 beats/min (P < 0.005)] with no significant difference in mean arterial pressure [wild type 110 +/- 0.3 vs. MCHR1(-/-) 113 +/- 0.4 mmHg (P > 0.05)]. Locomotor activity and core body temperature were higher in the MCHR1(-/-) mice during the dark phase only and thus temporally dissociated from heart rate differences. On fasting, wild-type animals rapidly downregulated body temperature and heart rate. MCHR1(-/-) mice displayed a distinct delay in the onset of this downregulation. To investigate the mechanism underlying these differences, autonomic blockade experiments were carried out. Administration of the adrenergic antagonist metoprolol completely reversed the tachycardia seen in MCHR1(-/-) mice, suggesting an increased sympathetic tone.
Asunto(s)
Sistema Nervioso Autónomo/fisiología , Frecuencia Cardíaca/genética , Frecuencia Cardíaca/fisiología , Receptores de la Hormona Hipofisaria/genética , Receptores de la Hormona Hipofisaria/fisiología , Tejido Adiposo/fisiología , Animales , Sistema Nervioso Autónomo/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Presión Sanguínea/fisiología , Composición Corporal/genética , Composición Corporal/fisiología , Temperatura Corporal/genética , Temperatura Corporal/fisiología , Peso Corporal/genética , Peso Corporal/fisiología , Calorimetría Indirecta , Clonación Molecular , Ingestión de Alimentos/genética , Ingestión de Alimentos/fisiología , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Ayuno/fisiología , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Actividad Motora/fisiología , Parasimpatolíticos/farmacología , Simpaticolíticos/farmacologíaRESUMEN
It is well recognized that the hypothalamus is of central importance in the regulation of food intake and fat mass. Recent studies indicate that it also plays an important role in the regulation of bone mass. Melanin concentrating hormone (MCH) is highly expressed in the hypothalamus and has been implicated in regulation of energy homeostasis. We developed MCHR1 inactivated mice to evaluate the physiological role of this receptor. Interestingly, the MCHR1(-/-) mice have osteoporosis, caused by a reduction in the cortical bone mass, while the amount of trabecular bone is unaffected. The reduction in cortical bone mass is due to decreased cortical thickness. Serum levels of c-telopeptide, a marker of bone resorption, are increased in MCHR1(-/-) mice, indicating that the MCHR1(-/-) mice have a high bone turnover osteoporosis. In conclusion, the MCHR1(-/-) mice have osteoporosis, indicating that MCHR1-signalling is involved in a tonic stimulation of bone mass.