RESUMEN
Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, ß-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.
Asunto(s)
Dieta , Fructoquinasas/deficiencia , Errores Innatos del Metabolismo de la Fructosa/genética , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/genética , Dieta Alta en Grasa , Ingestión de Alimentos/genética , Fructoquinasas/genética , Fructoquinasas/metabolismo , Errores Innatos del Metabolismo de la Fructosa/metabolismo , Fructosafosfatos/sangre , Intolerancia a la Glucosa/genética , Resistencia a la Insulina , Metabolismo de los Lípidos/genética , Ratones NoqueadosRESUMEN
Mutations of the AMP-activated kinase gamma 2 subunit (AMPKγ2), N488I (AMPKγ2NI) and R531G (AMPKγ2RG), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiac-specific transgenic overexpression of human AMPKγ2NI or AMPKγ2RG leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, non-physiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2NI or AMPKγ2RG mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2NI and AMPKγ2RG, respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2NI or AMPKγ2RG in the heart, both lines developed WPW syndrome and cardiac hypertrophy; however, these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2WT mice, AMPKγ2NI and AMPKγ2RG mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high fat diet (HFD). Surprisingly, AMPKγ2RG but not AMPKγ2NI mice fed with an HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2NI and AMPKγ2RG mutations at physiological levels can induce beneficial metabolic effects but that this is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2RG in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of an HFD.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Mutación , Insuficiencia Renal/genética , Síndrome de Wolff-Parkinson-White/genética , Animales , Apoptosis , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Inflamación/genética , Inflamación/patología , Riñón/metabolismo , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Insuficiencia Renal/patología , Síndrome de Wolff-Parkinson-White/patologíaRESUMEN
OBJECTIVE: To phenotypically characterize ADAMTS-4- and ADAMTS-5-double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice. METHODS: Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5-double-knockout animals. Twelve-week-old and 1-year-old male and female ADAMTS-4/5-double-knockout mice were compared with age- and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5-double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5-double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation. RESULTS: There were no genotype-related phenotype differences between ADAMTS-4/5-double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5-double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5-double-knockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5-double-knockout mice was ablated, to a level comparable with that in ADAMTS-5-knockout mice. CONCLUSION: Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5-double-knockout mice were comparable with those observed with deletion of ADAMTS-5 alone.
Asunto(s)
Proteínas ADAM/genética , Osteoartritis de la Cadera/fisiopatología , Osteoartritis de la Rodilla/fisiopatología , Procolágeno N-Endopeptidasa/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Agrecanos/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Genotipo , Articulación de la Cadera/enzimología , Articulación de la Cadera/patología , Inestabilidad de la Articulación/patología , Inestabilidad de la Articulación/fisiopatología , Articulación de la Rodilla/enzimología , Articulación de la Rodilla/patología , Masculino , Ratones , Ratones Noqueados , Osteoartritis de la Cadera/patología , Osteoartritis de la Rodilla/patología , Fenotipo , Procolágeno N-Endopeptidasa/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Gob-5 is a member of the calcium-activated chloride channel family and has been associated with allergic response in mouse models of pulmonary inflammation. Gene expression of Gob-5 has been shown to be induced in allergic airways and has been strongly associated with mucin gene regulation and goblet cell hyperplasia. We investigated the physiologic role of Gob-5 in murine models of pulmonary inflammation using mice deficient in Gob-5. After sensitization and aerosol challenge with ovalbumin (OVA), Gob-5 knockout mice exhibit significantly increased bronchoalveolar lavage (BAL) inflammation as compared with wild-type controls. The augmented inflammation in BAL consisted predominantly of neutrophils. Examination of perivascular inflammation revealed that tissue inflammation was decreased in OVA-challenged Gob-5-/- mice. OVA-challenged Gob-5 knockout mice also had decreased goblet cell hyperplasia as well as decreased mucus production. These mice also had decreased airway hypersensitivity after cholinergic provocation with methacholine. Gob-5 knockout mice were also challenged via intranasal LPS, a TLR-4 agonist. Gob-5-/- mice responded with increased neutrophilic BAL inflammation and decreased perivascular tissue inflammation as compared with wild-type controls. There was little effect on goblet cell hyperplasia and mucus production after LPS challenge. These observations reinforce findings that associate Gob-5 with goblet cell hyperplasia and mucus production in the allergic immune response, but also implicate Gob-5 in the regulation of tissue inflammation in the innate immune response.
Asunto(s)
Canales de Cloruro/fisiología , Células Caliciformes/patología , Mucoproteínas/fisiología , Neumonía/genética , Neumonía/patología , Resistencia de las Vías Respiratorias , Animales , Antígenos/toxicidad , Líquido del Lavado Bronquioalveolar/citología , Quimiocinas/metabolismo , Canales de Cloruro/genética , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Caliciformes/efectos de los fármacos , Células Caliciformes/metabolismo , Hiperplasia/genética , Hiperplasia/patología , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Mucoproteínas/genética , Moco/metabolismo , Ovalbúmina/toxicidad , Neumonía/inducido químicamenteRESUMEN
Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.
Asunto(s)
Cartílago Articular/metabolismo , Modelos Animales de Enfermedad , Eliminación de Gen , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Osteoartritis/metabolismo , Proteínas ADAM , Proteína ADAMTS5 , Animales , Dominio Catalítico , Endopeptidasas/química , Endopeptidasas/deficiencia , Endopeptidasas/genética , Endopeptidasas/metabolismo , Exones/genética , Cabeza Femoral , Placa de Crecimiento/metabolismo , Articulaciones/patología , Articulaciones/fisiopatología , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Ratones , Ratones Noqueados , Osteoartritis/genética , Osteoartritis/patología , Osteoartritis/fisiopatología , Proteoglicanos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
OBJECTIVE: To determine the importance of the enzymatic activity of ADAMTS-4 in normal growth and development and to evaluate the role of ADAMTS-4 in the progression of osteoarthritis (OA). METHODS: We generated catalytic domain-deleted ADAMTS-4-transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS-4 in the progression of the disease. The response of wild-type (WT) and ADAMTS-4-knockout (ADAMTS-4-KO) articular cartilage to interleukin-1 and retinoic acid challenge in vitro was also evaluated. RESULTS: ADAMTS-4-KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS-4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS-4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE(373-374)ARGS site was clearly evident after exposure of articular cartilage from ADAMTS-4-KO mice to inflammatory cytokines. CONCLUSION: Although expression of the ADAMTS-4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS-4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS-4 is not the primary enzyme responsible for aggrecan degradation at the TEGE(373-374)ARGS site. The elucidation of the relative importance of ADAMTS-4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.