RESUMEN
Mutations in the human peptide:N-glycanase gene (NGLY1), which encodes a cytosolic de-N-glycosylating enzyme, cause a congenital autosomal recessive disorder. In rodents, the loss of Ngly1 results in severe developmental delay or lethality, but the underlying mechanism remains unknown. In this study, we found that deletion of Fbxo6 (also known as Fbs2), which encodes a ubiquitin ligase subunit that recognizes glycoproteins, rescued the lethality-related defects in Ngly1-KO mice. In NGLY1-KO cells, FBS2 overexpression resulted in the substantial inhibition of proteasome activity, causing cytotoxicity. Nuclear factor, erythroid 2-like 1 (NFE2L1, also known as NRF1), an endoplasmic reticulum-associated transcriptional factor involved in expression of proteasome subunits, was also abnormally ubiquitinated by SCFFBS2 in NGLY1-KO cells, resulting in its retention in the cytosol. However, the cytotoxicity caused by FBS2 was restored by the overexpression of "glycan-less" NRF1 mutants, regardless of their transcriptional activity, or by the deletion of NRF1 in NGLY1-KO cells. We conclude that the proteasome dysfunction caused by the accumulation of N-glycoproteins, primarily NRF1, ubiquitinated by SCFFBS2 accounts for the pathogenesis resulting from NGLY1 deficiency.
Asunto(s)
Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Azúcares/metabolismo , Animales , Conducta Animal , Muerte Celular , Núcleo Celular/metabolismo , Proliferación Celular , Citosol/metabolismo , Células HCT116 , Células HeLa , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Actividad Motora , Mutación/genética , Factor Nuclear 1 de Respiración/metabolismo , Polisacáridos/metabolismo , Transporte de Proteínas , UbiquitinaciónRESUMEN
N-glycanase 1 (NGLY1) deficiency is a rare inherited disorder characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, motor deficits, and other neurological symptoms. The underlying mechanisms of the NGLY1 phenotype are poorly understood, and no effective therapy is currently available. Similar to human patients, the rat model of NGLY1 deficiency, Ngly1-/-, shows developmental delay, movement disorder, somatosensory impairment, scoliosis, and learning disability. Here we show that single intracerebroventricular administration of AAV9 expressing human NGLY1 cDNA (AAV9-hNGLY1) to Ngly1-/- rats during the weaning period restored NGLY1 expression in the brain and spinal cord, concomitant with increased enzymatic activity of NGLY1 in the brain. hNGLY1 protein expressed by AAV9 was found predominantly in mature neurons, but not in glial cells, of Ngly1-/- rats. Strikingly, intracerebroventricular administration of AAV9-hNGLY1 normalized the motor phenotypes of Ngly1-/- rats assessed by the rota-rod test and gait analysis. The reversibility of motor deficits in Ngly1-/- rats by central nervous system (CNS)-restricted gene delivery suggests that the CNS is the primary therapeutic target organs for NGLY1 deficiency, and that the Ngly1-/- rat model may be useful for evaluating therapeutic treatments in pre-clinical studies.
Asunto(s)
Trastornos Congénitos de Glicosilación/fisiopatología , Actividad Motora/fisiología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Acetilglucosamina/análogos & derivados , Animales , Trastornos Congénitos de Glicosilación/enzimología , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos/administración & dosificación , Gliosis/complicaciones , Gliosis/patología , Humanos , Inflamación/patología , Inyecciones Intraventriculares , Neuronas/patología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Ratas , Ratas Transgénicas , TransgenesRESUMEN
N-Glycanase 1 (NGLY1) deficiency is a congenital disorder caused by mutations in the NGLY1 gene. Because systemic Ngly1-/- mice with a C57BL/6 (B6) background are embryonically lethal, studies on the mechanism of NGLY1 deficiency using mice have been problematic. In this study, B6-Ngly1-/+ mice were crossed with Japanese wild mice-originated Japanese fancy mouse 1 (JF1) mice to produce viable F2 Ngly1-/- mice from (JF1×B6)F1 Ngly1-/+ mice. Systemic Ngly1-/- mice with a JF1 mouse background were also embryonically lethal. Hybrid F1 Ngly1-/- (JF1/B6F1) mice, however, showed developmental delay and motor dysfunction, similar to that in human patients. JF1/B6F1 Ngly1-/- mice showed increased levels of plasma and urinary aspartylglycosamine, a potential biomarker for NGLY1 deficiency. JF1/B6F1 Ngly1-/- mice are a useful isogenic animal model for the preclinical testing of therapeutic options and understanding the precise pathogenic mechanisms responsible for NGLY1 deficiency.
Asunto(s)
Trastornos Congénitos de Glicosilación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/sangre , Acetilglucosamina/genética , Animales , Trastornos Congénitos de Glicosilación/sangre , Trastornos Congénitos de Glicosilación/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/sangre , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genéticaRESUMEN
N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1-/- rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1-/- rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1-/- rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1-/- rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1-/- rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.
Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Trastornos del Movimiento/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Trastornos Congénitos de Glicosilación/metabolismo , Trastornos Congénitos de Glicosilación/patología , Modelos Animales de Enfermedad , Degradación Asociada con el Retículo Endoplásmico/genética , Enfermedades Hereditarias del Ojo , Técnicas de Inactivación de Genes , Glicosilación , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Enfermedades del Aparato Lagrimal , Trastornos del Movimiento/patología , Mutación/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Sistema Nervioso Periférico/metabolismo , Sistema Nervioso Periférico/patología , Fenotipo , Complejo de la Endopetidasa Proteasomal/genética , RatasRESUMEN
Dysferlinopathies, which are muscular diseases caused by mutations in the dysferlin gene, remain serious medical problems due to the lack of therapeutic agents. Herein, we report the design, synthesis, and structure-activity relationships of a 2,6-disubstituted 3H-imidazo[4,5-b]pyridine series, which was identified from the phenotypic screening of chemicals that increase the level of dysferlin in myocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Optimization studies with cell-based phenotypic assay led to the identification of a highly potent compound, 19, with dysferlin elevation effects at double-digit nanomolar concentrations. In addition, the molecular target of our chemical series was identified as tubulin, through a tubulin polymerization assay and a competitive binding assay using a photoaffinity labeling probe.
Asunto(s)
Imidazoles/química , Distrofia Muscular de Cinturas/tratamiento farmacológico , Piridinas/química , Moduladores de Tubulina/uso terapéutico , Sitios de Unión , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Disferlina/metabolismo , Células Hep G2 , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Simulación del Acoplamiento Molecular , Distrofia Muscular de Cinturas/patología , Proteína MioD/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Estructura Terciaria de Proteína , Piridinas/farmacología , Piridinas/uso terapéutico , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017-1029.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Células Madre Pluripotentes Inducidas/trasplante , Células Musculares/metabolismo , Distrofia Muscular de Cinturas/tratamiento farmacológico , Adulto , Femenino , Humanos , Persona de Mediana Edad , FenotipoRESUMEN
We attempted to clarify the therapeutic capability of antagonists of the farnesoid X receptor (FXR), a nuclear receptor that regulates lipid and bile acid metabolism. Herein, we report the antidyslipidemic effects of a novel synthesized FXR antagonist, compound-T1, utilizing a dyslipidemic hamster model. Compound-T1 selectively inhibited chenodeoxycholic acid-induced FXR activation (IC 50, 2.1 nmol·L-1). A hamster model of diet-induced hyperlipidemia was prepared to investigate the antidyslipidemic effects of compound-T1 through comparative studies of the nonstatin lipid-modulating agents ezetimibe, cholestyramine, and torcetrapib. In the hamster model, compound-T1 (6 mg·kg-1·day-1, p.o.) increased the level of plasma high-density lipoprotein (HDL)-cholesterol (+22.2%) and decreased the levels of plasma non-HDL-cholesterol (-43.6%) and triglycerides (-31.1%). Compound-T1 also increased hepatic cholesterol 7α-hydroxylase expression and fecal bile acid excretion, and decreased hepatic cholesterol content. Moreover, the hamster model could reflect clinical results of other nonstatin agents. Torcetrapib especially increased large HDL particles compared with compound-T1. Additionally, in the human hepatoma Huh-7 cells, compound-T1 enhanced apolipoprotein A-I secretion at a concentration close to its IC 50 value for FXR. Our results indicated the usefulness of the hamster model in evaluating FXR antagonists and nonstatin agents. Notably, compound-T1 exhibited beneficial effects on both blood non-HDL-cholesterol and HDL-cholesterol, which are thought to involve enhancement of cholesterol catabolism and apolipoprotein A-I production. These findings aid the understanding of the antidyslipidemic potential of FXR antagonists with a unique lipid and bile acid modulation.
Asunto(s)
Benzoatos/uso terapéutico , Dislipidemias/tratamiento farmacológico , Hipolipemiantes/uso terapéutico , Metabolismo de los Lípidos/efectos de los fármacos , Piperidinas/uso terapéutico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Animales , Apolipoproteína A-I/metabolismo , Benzoatos/administración & dosificación , Ácidos y Sales Biliares/análisis , Línea Celular Tumoral , Colestenonas/sangre , Colesterol/sangre , Cricetinae , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Dislipidemias/sangre , Dislipidemias/metabolismo , Heces/química , Humanos , Hipolipemiantes/administración & dosificación , Masculino , Mesocricetus , Piperidinas/administración & dosificación , Triglicéridos/sangreRESUMEN
BACKGROUND: The effects of farnesoid X receptor (FXR) antagonists on plasma lipid profile in mice have not been investigated thus far. The aim of this study was to investigate the antidyslipidemic effects of an FXR antagonist in dyslipidemic mice, and to clarify the mechanisms underlying the lipid modulatory effect. METHODS: Compound-T0 (1-100â¯mg/kg) was orally administered to C57BL/6J mice fed a Western-type diet or low-density lipoprotein receptor knockout (LDLR-/-) mice fed a Western-type diet for a week, and plasma lipid levels were investigated. Effects on lipid clearance, hepatic triglyceride secretion after Triton WR-1339 challenge, and intestinal lipid absorption were investigated after multiple dosing. RESULTS: Compound-T0 significantly increased plasma level of non-high-density lipoprotein cholesterol in both C57BL/6 and LDLR-/- mice; in addition, it significantly increased plasma triglyceride level in LDLR-/- mice. Compound-T0 failed to enhance the clearance of 3,3'-dioctadecylindocarbocyanine (DiI)-labeled LDL in C57BL/6J mice. Although compound-T0 did not affect triglyceride clearance and hepatic triglyceride secretion, it significantly increased intestinal [3H]cholesterol absorption in LDLR-/- mice. CONCLUSIONS: It was found that the FXR antagonist, compound-T0 exacerbated dyslipidemia in mice because it enhanced intestinal lipid absorption via acceleration of bile acid excretion.
Asunto(s)
Benzoatos/farmacología , Dislipidemias/inducido químicamente , Lípidos/sangre , Hígado/efectos de los fármacos , Piperidinas/farmacología , Pirazoles/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Animales , Ácidos y Sales Biliares/metabolismo , Biomarcadores/sangre , Colesterol/sangre , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Dislipidemias/sangre , Dislipidemias/genética , Predisposición Genética a la Enfermedad , Absorción Intestinal/efectos de los fármacos , Eliminación Intestinal/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Triglicéridos/sangreRESUMEN
Neuromedin U (NMU), a neuropeptide originally isolated from porcine spinal cord, has multiple physiological functions and is involved in obesity and inflammation. Excessive fat accumulation in the liver leads to non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), which is closely associated with obesity. NAFLD and NASH develop and progress via complex pathophysiological processes, and it remains unclear to what extend the NMU system contributes to the risk of obesity-related disorders such as NAFLD and NASH. Here, we demonstrate that the NMU system plays a role in NAFLD/NASH pathogenesis. In the normal mouse liver, NMU mRNA was not detectable, and expression of the mRNA encoding neuromedin U receptor 1 (NMUR1), the peripheral receptor of NMU, was low. However, the expression of both was significantly increased in the livers of NASH mice. Furthermore, overproduction of NMU induced the mouse liver by hydrodynamic injection, exacerbated NASH pathogenesis. These data indicate a novel role for the peripheral NMU system, providing new insights into the pathogenesis of NAFLD/NASH.
Asunto(s)
Regulación de la Expresión Génica , Hígado/metabolismo , Neuropéptidos/biosíntesis , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Animales , Hígado/patología , Masculino , Ratones , Ratones Endogámicos ICR , Neuropéptidos/química , Neuropéptidos/farmacología , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/patología , Receptores de Neurotransmisores/metabolismo , PorcinosRESUMEN
Sarcopenia and cachexia present characteristic features of a decrease in skeletal muscle mass and strength, anorexia, and lack of motivation. Treatments for these diseases have not yet been established, although selective androgen receptor modulators (SARMs) are considered as therapeutic targets. We previously reported that a novel SARM compound, SARM-2f, exhibits anabolic effect on muscles, with less stimulatory effect on prostate weight compared with testosterone, in rat Hershberger assays and cancer cachexia models. In this study, we studied the mechanism of action for SARM-2f selectivity and also assessed whether the muscle increase by this compound might lead to improvement of muscle function and physical activity. First, we examined the tissue distribution of SARM-2f. Tissue concentration was 1.2-, 1.6-, and 1.9-fold as high as the plasma concentration in the levator ani muscle, brain, and prostate, respectively. This result showed that the tissue-selective pharmacological effect did not depend on SARM-2f concentration in the tissues. The ability of SARM-2f to influence androgen receptor (AR)-mediated transcriptional activation was examined by reporter assays using human normal prostate epithelial cells (PrEC) and skeletal muscle cells (SKMC). SARM-2f exerted higher activity against AR in SKMC than in PrEC. Mammalian two hybrid assays showed different co-factor recruitment patterns between SARM-2f and dihydrotestosterone. Next, we studied the effect of SARM-2f on motivation and physical functions such as sexual behavior and motor activities in castrated rat or mouse models. SARM-2f restored the sexual behavior that was lost by castration in male rats. SARM-2f also increased voluntary running distance and locomotor activities. These results suggest that tissue-specific AR regulation by SARM-2f, but not tissue distribution, might account for its tissue specific androgenic effect, and that the muscle mass increase by SARM-2f leads to improvement of physical function. Together, these findings suggest that SARM-2f might represent an effective treatment for sarcopenia and cachexia.
Asunto(s)
Actividad Motora , Orquiectomía , Pirrolidinonas/farmacología , Receptores Androgénicos/efectos de los fármacos , Conducta Sexual Animal , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Pirrolidinonas/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores Androgénicos/fisiología , Distribución Tisular , Transcripción GenéticaRESUMEN
Although chronic kidney disease (CKD) is strongly associated with onsets of cardiovascular disease (CVD), the pathogenic mechanism between these diseases has not been fully understood. To develop and validate new therapeutic strategies for this complication, appropriate experimental models that reflect the complexity of the underlying pathophysiology are needed. The Osborne-Mendel (OM) rat was identified as an atherosclerosis-prone and a premature-death rat strain among 16 inbred rat strains when fed high-cholesterol containing diet. When fed high-cholesterol diet, OM rats showed simultaneous occurrence of aortic aneurysm, aortic dissection, peripheral artery occlusion, and left atrial thrombosis. OM rats had significantly lower max dP/dt and higher min dP/dt than F344 rats did, indicating impaired left ventricle contractility and relaxation. OM rats developed renal dysfunction, showing increased urinary albumin excretion. OM rats also showed mild hypertension, decreased endothelial function, and enhanced coagulation and platelet aggregation, compared with F344 rats. We now report that OM rat would be a novel spontaneous animal model which simultaneously demonstrates cardiac and renal dysfunction, and CVD events. This model could be a useful model for the pre-clinical testing of pharmacological therapies and could provide new insight into potential targets and pathways for the treatment of CKD and CVD.
Asunto(s)
Aneurisma de la Aorta/fisiopatología , Arteriopatías Oclusivas/fisiopatología , Cardiopatías/fisiopatología , Enfermedades Renales/fisiopatología , Enfermedad Arterial Periférica/fisiopatología , Trombosis/fisiopatología , Animales , Aneurisma de la Aorta/etiología , Arteriopatías Oclusivas/etiología , Presión Sanguínea/efectos de los fármacos , Colesterol en la Dieta/administración & dosificación , Colesterol en la Dieta/toxicidad , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Atrios Cardíacos , Cardiopatías/etiología , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Enfermedades Renales/etiología , Masculino , Enfermedad Arterial Periférica/etiología , Ratas Endogámicas F344 , Ratas Endogámicas , Especificidad de la Especie , Análisis de Supervivencia , Trombosis/etiología , Factores de TiempoRESUMEN
AIM: Experimental models of non-alcoholic steatohepatitis (NASH) are still required for understanding the pathophysiology of this disease. This study aimed to examine whether disease progression is accelerated by combining dyslipidemic genetic modification and dietary challenges and develop NASH-associated hepatic fibrosis, cirrhosis, and carcinoma in a short period. METHODS: Low-density lipoprotein receptor knockout mice were fed a modified choline-deficient amino acid-defined diet, including 1 w/w% cholesterol and 41 kcal% fat, and was comprehensively profiled over 1 year. RESULTS: Microvesicular and macrovesicular steatosis in the liver was observed from the first week after starting the modified choline-deficient amino acid-defined diet. Macrovesicular steatosis was exacerbated with time and was observed in almost all hepatocytes at week 8, but slightly decreased at week 16. Infiltration of macrophages and neutrophils, and upregulation of hepatic inflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß were also observed from week 1. Plasma hepatic transaminase activities were increased at week 1, reached a peak at week 4, and gradually decreased thereafter. In parallel with increases in hepatic gene expression of collagen-I, the hepatic fibrosis area expanded after week 4 and massively spread all over the liver by week 8. Hepatocellular hyperplasia was observed from week 24. Hepatocellular adenoma and carcinoma were observed from week 31 and 39, respectively. CONCLUSION: These results suggest that, in a rodent NASH model with the combination of genetic modification and dietary challenges, hepatic steatosis, inflammatory cell infiltration and hepatic injury, hepatic fibrosis, hepatocellular hyperplasia, adenoma, and carcinoma can be developed in a relatively short period.
RESUMEN
AIM: Low-density lipoprotein receptor knockout (LDLR-KO) mice fed a modified choline-deficient and amino acid-defined (mCDAA) diet show non-alcoholic steatohepatitis (NASH)-like pathophysiology. In order to pharmacologically benchmark this model, effects of pioglitazone (a thiazolidinedione) and candesartan cilexetil (an angiotensin II type 1 receptor blocker) on steatosis and liver fibrosis were examined. METHODS: Pioglitazone (10 mg/kg) and candesartan cilexetil (3 mg/kg) were given orally once daily to LDLR-KO mice under mCDAA diet for 7 weeks. Blood biochemistry and hepatic histology were assessed, and hepatic gene expression levels and triglyceride content were measured. RESULTS: Pioglitazone suppressed hepatic COL1A1 gene expression by 43% and attenuated hepatic fibrosis areas by 49%. Pioglitazone also decreased plasma alanine aminotransferase levels, liver weight, hepatic triglyceride content, and hepatic expression of other fibrosis-related genes such as TGFB1, SPP1, TIMP1, and IL6. Candesartan cilexetil suppressed hepatic COL1A1 gene expression by 33%, whereas the other end-points including hepatic fibrosis areas were not affected. CONCLUSIONS: Pioglitazone showed anti-fibrotic effects accompanied by improving hepatic transaminase activity and hepatic lipid accumulation, but the effect of candesartan cilexetil was only limited, unlike previous reports for angiotensin II type 1 receptor blockers. As the pharmacological effects of pioglitazone in the current animal model are similar to those reported in patients with NASH, this model may represent some aspects of the pathophysiology of NASH. Further profiling using other agents or mechanisms that have been tested in the clinic will better clarify the utility of the animal model.
RESUMEN
Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3(-/-) mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3(-/-) mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan.
Asunto(s)
Metabolismo Energético/fisiología , Longevidad/fisiología , Fosfotransferasas (Aceptor del Grupo Fosfato)/fisiología , Animales , Composición Corporal , Peso Corporal , Línea Celular , Dexametasona/farmacología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético/genética , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Islotes Pancreáticos/enzimología , Longevidad/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Fibras Musculares Esqueléticas/enzimología , Músculo Esquelético/enzimología , Miocardio/metabolismo , Especificidad de Órganos , Fosforilación , Fosfotransferasas (Aceptor del Grupo Fosfato)/deficiencia , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Procesamiento Proteico-Postraduccional , Ratas , Proteína S6 Ribosómica/metabolismoRESUMEN
Nephropathy frequently co-occurs with metabolic syndrome in humans. Metabolic syndrome is a cluster of metabolic diseases including obesity, diabetes, hypertension, and dyslipidemia, and some previous studies revealed that dyslipidemia contributes to the progression of kidney dysfunction. To establish a new nephropathy model with metabolic syndrome, we produced human apolipoprotein B (apoB) transgenic (Tg.) SHR/NDmcr-cp (SHR-cp/cp) rats, in which dyslipidemia is exacerbated more than in an established metabolic syndrome model, SHR-cp/cp rats. Human apoB Tg. SHR-cp/cp rats showed obesity, hyperinsulinemia, hypertension, and severe hyperlipidemia. They also exhibited exacerbated early-onset proteinuria, accompanied by increased kidney injury and increased oxidative and inflammatory markers. Histological analyses revealed the characteristic features of human apoB Tg. SHR-cp/cp rats including prominent glomerulosclerosis with lipid accumulation. Our newly established human apoB Tg. SHR-cp/cp rat could be a useful model for the nephropathy in metabolic syndrome and for understanding the interaction between dyslipidemia and renal dysfunction in metabolic syndrome.
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Apolipoproteína B-100/genética , Modelos Animales de Enfermedad , Enfermedades Renales/genética , Animales , Progresión de la Enfermedad , Humanos , Hiperinsulinismo/genética , Hiperlipidemias/etiología , Hipertensión/genética , Inflamación , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/patología , Metabolismo de los Lípidos , Masculino , Síndrome Metabólico/genética , Obesidad/genética , Estrés Oxidativo , Proteinuria/genética , Ratas Transgénicas , Ratas Wistar , EsclerosisRESUMEN
Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.
Asunto(s)
Colesterol/sangre , Farnesil Difosfato Farnesil Transferasa/genética , Hígado/enzimología , Animales , Vías Biosintéticas , Colesterol/biosíntesis , Farnesil Difosfato Farnesil Transferasa/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hígado/fisiopatología , Masculino , Ratones TransgénicosRESUMEN
AIMS: We investigated antidyslipidemic effects of a farnesoid X receptor antagonist compound-T3 in non-human primates as a novel treatment approach for dyslipidemia. MAIN METHODS: Cynomolgus monkeys were fed a high-fat diet over 3 weeks. Drugs were administered to the monkeys for a week, and their plasma and fecal lipid parameters were measured. KEY FINDINGS: Compound-T3 dose-dependently decreased the plasma non-high-density lipoprotein (non-HDL) cholesterol and apolipoprotein B levels in high-fat diet-fed cynomolgus monkeys. The plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, and total fecal bile acid levels increased, suggesting that the hypocholesterolemic effects would be dependent on the activation of cholesterol catabolism in the liver. Compound-T3 significantly increased the plasma levels of HDL cholesterol and apolipoprotein A-I. In this condition, the cholesterol absorption inhibitor ezetimibe significantly decreased the plasma non-HDL cholesterol levels and increased the fecal cholesterol levels without affecting plasma HDL cholesterol and triglyceride levels. Bile acid sequestrant cholestyramine tended to decrease plasma non-HDL cholesterol and increase fecal bile acid levels. The cholesteryl ester transfer protein inhibitor torcetrapib significantly increased plasma HDL cholesterol levels without affecting plasma non-HDL cholesterol and fecal cholesterol levels. SIGNIFICANCE: The results of ezetimibe, cholestyramine, and torcetrapib treatments indicate that our high-fat diet fed monkey model would be a preferred animal model for studying non-statin type antidyslipidemic drugs. Compound-T3 significantly decreased non-HDL cholesterol levels and increased HDL cholesterol levels in the monkey model, suggesting that a farnesoid X receptor antagonist could be a therapeutic option in human dyslipidemia.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Dislipidemias/sangre , Dislipidemias/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Animales , Anticolesterolemiantes/química , Anticolesterolemiantes/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Macaca fascicularis , Masculino , Primates , Receptores Citoplasmáticos y Nucleares/metabolismo , Resultado del TratamientoRESUMEN
AIMS: We previously reported anti-dyslipidemic effects of a farnesoid X receptor antagonist in monkeys. In this study, we compared the cholesterol-lowering effects of single and combined administration of a farnesoid X receptor antagonist, compound-T8, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor atorvastatin in a guinea pig model. MAIN METHODS: Plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, were measured after a single administration of compound-T8. The effects of compound-T8 or atorvastatin on plasma cholesterol levels and low-density lipoprotein (LDL) clearance were investigated after 14 or 16 days of repeated dosing, respectively. Fractional catabolic rate of plasma LDL was estimated by intravenous injection of DiI-labeled human LDL. The cholesterol-lowering effects of combination therapy were investigated after 7 days of repeated treatment. KEY FINDINGS: Compound-T8 (10 and 30 mg/kg) increased plasma 7α-hydroxy-4-cholesten-3-one levels in a dose-dependent manner. Single administration of compound-T8 (30 mg/kg) and atorvastatin (30 mg/kg) reduced plasma non-high-density lipoprotein (non-HDL) cholesterol levels by 48% and 46%, respectively, and increased clearance of plasma DiI-labeled LDL by 29% and 35%, respectively. Compound-T8 (10mg/kg) or atorvastatin (10mg/kg) reduced non-HDL cholesterol levels by 19% and 25%, respectively, and combination therapy showed an additive effect and lowered cholesterol levels by 48%. SIGNIFICANCE: Similar to atorvastatin, compound-T8 reduced plasma non-HDL cholesterol levels accompanied with accelerated LDL clearance in guinea pigs. Combination therapy additively decreased plasma non-HDL cholesterol levels. Therefore, monotherapy with a farnesoid X receptor antagonist and combination therapy of a farnesoid X receptor antagonist with atorvastatin would be attractive dyslipidemia treatment options.
Asunto(s)
Benzoatos/farmacología , LDL-Colesterol/sangre , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Pirazoles/farmacología , Pirroles/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Animales , Anticolesterolemiantes/administración & dosificación , Anticolesterolemiantes/farmacología , Atorvastatina , Benzoatos/administración & dosificación , Colestenonas/sangre , Colesterol/sangre , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Cobayas , Ácidos Heptanoicos/administración & dosificación , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Lipoproteínas HDL/sangre , Masculino , Pirazoles/administración & dosificación , Pirroles/administración & dosificación , Factores de TiempoRESUMEN
Age-related decrease of testosterone levels in blood and brain is believed to be associated with neurodegenerative diseases such as Alzheimer's disease. However, the effect of testosterone on brain function is not well understood. Therefore, we investigated the impact of testosterone deprivation on mitochondrial gene expression in the brain of male gonadectomized (GDX) rats. We found that peripheral castration led to testosterone deficiency in the brain and caused a significant reduction in protein and mRNA expression of genes encoded by mitochondrial DNA, namely NADPH dehydrogenase subunit 1, subunit 4, cytochrome b, and cytochrome c oxidase subunit 1 and subunit 3 in the hippocampus. In addition, gene expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which is a master regulator of mitochondrial biogenesis, and its downstream transcriptional factors, nuclear respiratory factors 1 and 2 and mitochondrial transcription factors A and B2, were also decreased in the hippocampus of GDX rats. These reductions in the expression of mitochondrial gene and transcriptional coactivators and factors were recovered by androgen replacement. These findings indicate that androgen plays an important role in mitochondrial gene expression in the hippocampus.