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1.
Am J Physiol Regul Integr Comp Physiol ; 324(1): R109-R119, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36409022

RESUMO

The fundamental body functions that determine maximal O2 uptake (V̇o2max) have not been studied in Aqp5-/- mice (aquaporin 5, AQP5). We measured V̇o2max to globally assess these functions and then investigated why it was found altered in Aqp5-/- mice. V̇o2max was measured by the Helox technique, which elicits maximal metabolic rate by intense cold exposure of the animals. We found V̇o2max reduced in Aqp5-/- mice by 20%-30% compared with wild-type (WT) mice. As AQP5 has been implicated to act as a membrane channel for respiratory gases, we studied whether this is caused by the known lack of AQP5 in the alveolar epithelial membranes of Aqp5-/- mice. Lung function parameters as well as arterial O2 saturation were normal and identical between Aqp5-/- and WT mice, indicating that AQP5 does not contribute to pulmonary O2 exchange. The cause for the decreased V̇o2max thus might be found in decreased O2 consumption of an intensely O2-consuming peripheral organ such as activated brown adipose tissue (BAT). We found indeed that absence of AQP5 greatly reduces the amount of interscapular BAT formed in response to 4 wk of cold exposure, from 63% in WT to 25% in Aqp5-/- animals. We conclude that lack of AQP5 does not affect pulmonary O2 exchange, but greatly inhibits transformation of white to brown adipose tissue. As under cold exposure, BAT is a major source of the animals' heat production, reduction of BAT likely causes the decrease in V̇o2max under this condition.


Assuntos
Tecido Adiposo Marrom , Troca Gasosa Pulmonar , Animais , Camundongos , Tecido Adiposo Marrom/metabolismo , Termogênese/fisiologia , Pulmão , Consumo de Oxigênio , Temperatura Baixa
2.
Mamm Genome ; 33(1): 120-122, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34328547

RESUMO

Improving reproducibility and replicability in preclinical research is a widely discussed and pertinent topic, especially regarding ethical responsibility in animal research. INFRAFRONTIER, the European Research Infrastructure for the generation, phenotyping, archiving, and distribution of model mammalian genomes, is addressing this issue by developing internal quality principles for its different service areas, that provides a quality framework for its operational activities. This article introduces the INFRAFRONTIER Quality Principles in Systemic Phenotyping of genetically altered mouse models. A total of 11 key principles are included, ranging from general requirements for compliance with guidelines on animal testing, to the need for well-trained personnel and more specific standards such as the exchange of reference lines. Recently established requirements such as the provision of FAIR (Findable, Accessible, Interoperable, Reusable) data are also addressed. For each quality principle, we have outlined the specific context, requirements, further recommendations, and key references.


Assuntos
Genoma , Mamíferos , Animais , Modelos Animais de Doenças , Camundongos , Reprodutibilidade dos Testes
3.
Handb Exp Pharmacol ; 257: 399-423, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31541322

RESUMO

Using standardized guidelines in preclinical research has received increased interest in light of recent concerns about transparency in data reporting and apparent variation in data quality, as evidenced by irreproducibility of results. Although the costs associated with supporting quality through a quality management system are often obvious line items in laboratory budgets, the treatment of the costs associated with quality failure is often overlooked and difficult to quantify. Thus, general estimations of quality costs can be misleading and inaccurate, effectively undervaluing costs recovered by reducing quality defects. Here, we provide examples of quality costs in preclinical research and describe how we have addressed misconceptions of quality management implementation as only marginally beneficial and/or unduly burdensome. We provide two examples of implementing a quality management system (QMS) in preclinical experimental (animal) research environments - one in Europe, the German Mouse Clinic, having established ISO 9001 and the other in the United States, the University of Kentucky (UK), having established Good Laboratory Practice-compliant infrastructure. We present a summary of benefits to having an effective QMS, as may be useful in guiding discussions with funders or administrators to promote interest and investment in a QMS, which ultimately supports shared, mutually beneficial outcomes.


Assuntos
Controle de Qualidade , Animais , Guias como Assunto , Camundongos , Estados Unidos
4.
Pflugers Arch ; 468(2): 213-27, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26490457

RESUMO

Glutamine, the most abundant amino acid in mammals, is critical for cell and organ functions. Its metabolism depends on the ability of cells to take up or release glutamine by transporters located in the plasma membrane. Several solute carrier (SLC) families transport glutamine, but the SLC38 family has been thought to be mostly responsible for glutamine transport. We demonstrate that despite the large number of glutamine transporters, the loss of Snat3/Slc38a3 glutamine transporter has a major impact on the function of organs expressing it. Snat3 mutant mice were generated by N-ethyl-N-nitrosurea (ENU) mutagenesis and showed stunted growth, altered amino acid levels, hypoglycemia, and died around 20 days after birth. Hepatic concentrations of glutamine, glutamate, leucine, phenylalanine, and tryptophan were highly reduced paralleled by downregulation of the mTOR pathway possibly linking reduced amino acid availability to impaired growth and glucose homeostasis. Snat3-deficient mice had altered urea levels paralleled by dysregulation of the urea cycle, gluconeogenesis, and glutamine synthesis. Mice were ataxic with higher glutamine but reduced glutamate and gamma-aminobutyric acid (GABA) levels in brain consistent with a major role of Snat3 in the glutamine-glutamate cycle. Renal ammonium excretion was lower, and the expression of enzymes and amino acid transporters involved in ammoniagenesis were altered. Thus, SNAT3 is a glutamine transporter required for amino acid homeostasis and determines critical functions in various organs. Despite the large number of glutamine transporters, loss of Snat3 cannot be compensated, suggesting that this transporter is a major route of glutamine transport in the liver, brain, and kidney.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Encéfalo/metabolismo , Rim/metabolismo , Fígado/metabolismo , Mutação , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Gluconeogênese , Glucose/metabolismo , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Serina-Treonina Quinases TOR/metabolismo
5.
Mamm Genome ; 27(11-12): 587-598, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27671791

RESUMO

Animal models resembling human mutations are valuable tools to research the features of complex human craniofacial syndromes. This is the first report on a viable dominant mouse model carrying a non-synonymous sequence variation within the endothelin receptor type A gene (Ednra c.386A>T, p.Tyr129Phe) derived by an ENU mutagenesis program. The identical amino acid substitution was reported recently as disease causing in three individuals with the mandibulofacial dysostosis with alopecia (MFDA, OMIM 616367) syndrome. We performed standardized phenotyping of wild-type, heterozygous, and homozygous Ednra Y129F mice within the German Mouse Clinic. Mutant mice mimic the craniofacial phenotypes of jaw dysplasia, micrognathia, dysplastic temporomandibular joints, auricular dysmorphism, and missing of the squamosal zygomatic process as described for MFDA-affected individuals. As observed in MFDA-affected individuals, mutant Ednra Y129F mice exhibit hearing impairment in line with strong abnormalities of the ossicles and further, reduction of some lung volumetric parameters. In general, heterozygous and homozygous mice demonstrated inter-individual diversity of expression of the craniofacial phenotypes as observed in MFDA patients but without showing any cleft palates, eyelid defects, or alopecia. Mutant Ednra Y129F mice represent a valuable viable model for complex human syndromes of the first and second pharyngeal arches and for further studies and analysis of impaired endothelin 1 (EDN1)-endothelin receptor type A (EDNRA) signaling. Above all, Ednra Y129F mice model the recently published human MFDA syndrome and may be helpful for further disease understanding and development of therapeutic interventions.


Assuntos
Alopecia/genética , Disostose Mandibulofacial/genética , Receptor de Endotelina A/genética , Alopecia/fisiopatologia , Animais , Genótipo , Humanos , Disostose Mandibulofacial/fisiopatologia , Camundongos , Mutação , Fenótipo , Transdução de Sinais
6.
Biochim Biophys Acta Mol Basis Dis ; 1869(7): 166760, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37230398

RESUMO

The alternative oxidase, AOX, provides a by-pass of the cytochrome segment of the mitochondrial respiratory chain when the chain is unavailable. AOX is absent from mammals, but AOX from Ciona intestinalis is benign when expressed in mice. Although non-protonmotive, so does not contribute directly to ATP production, it has been shown to modify and in some cases rescue phenotypes of respiratory-chain disease models. Here we studied the effect of C. intestinalis AOX on mice engineered to express a disease-equivalent mutant of Uqcrh, encoding the hinge subunit of mitochondrial respiratory complex III, which results in a complex metabolic phenotype beginning at 4-5 weeks, rapidly progressing to lethality within a further 6-7 weeks. AOX expression delayed the onset of this phenotype by several weeks, but provided no long-term benefit. We discuss the significance of this finding in light of the known and hypothesized effects of AOX on metabolism, redox homeostasis, oxidative stress and cell signaling. Although not a panacea, the ability of AOX to mitigate disease onset and progression means it could be useful in treatment.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons , Mitocôndrias , Animais , Camundongos , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Membranas Mitocondriais/metabolismo , Fenótipo , Fatores de Transcrição/metabolismo , Mamíferos/metabolismo
7.
J Physiol ; 590(24): 6413-24, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23045339

RESUMO

The uniporter TAT1 (Slc16a10) mediates the facilitated diffusion of aromatic amino acids (AAAs) across basolateral membranes of kidney, small intestine and liver epithelial cells, and across the plasma membrane of non-epithelial cells like skeletal myocytes. Its role for body AA homeostasis has now been investigated using newly generated TAT1 (Slc16a10) defective mice (tat1(-/-)). These mice grow and reproduce normally, show no gross phenotype and no obvious neurological defect. Histological analysis did not reveal abnormalities and there is no compensatory change in any tested AA transporter mRNA. TAT1 null mice, however, display increased plasma, muscle and kidney AAA concentration under both normal and high protein diet, although this concentration remains normal in the liver. A major aromatic aminoaciduria and a smaller urinary loss of all substrates additionally transported by l-type AA antiporter Lat2-4F2hc (Slc7a8) were revealed under a high protein diet. This suggests an epithelial transport defect as also shown by the accumulation of intravenously injected (123)I-2-I-l-Phe in kidney and l-[(3)H]Phe in ex vivo everted gut sac enterocytes. Taken together, these data indicate that the uniporter TAT1 is required to equilibrate the concentration of AAAs across specific membranes. For instance, it enables hepatocytes to function as a sink that controls the extracellular AAAs concentration. Additionally, it facilitates the release of AAAs across the basolateral membrane of small intestine and proximal kidney tubule epithelial cells, thereby allowing the efflux of other neutral AAs presumably via Lat2-4F2hc.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos Aromáticos/metabolismo , Proteínas Alimentares/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/deficiência , Sistemas de Transporte de Aminoácidos Neutros/genética , Aminoácidos Aromáticos/sangue , Animais , Proteínas Alimentares/sangue , Células Epiteliais/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Genótipo , Homeostase , Intestino Delgado/metabolismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Fenótipo , RNA Mensageiro/metabolismo
8.
Nat Commun ; 13(1): 6830, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36369285

RESUMO

Current concepts regarding the biology of aging are primarily based on studies aimed at identifying factors regulating lifespan. However, lifespan as a sole proxy measure for aging can be of limited value because it may be restricted by specific pathologies. Here, we employ large-scale phenotyping to analyze hundreds of markers in aging male C57BL/6J mice. For each phenotype, we establish lifetime profiles to determine when age-dependent change is first detectable relative to the young adult baseline. We examine key lifespan regulators (putative anti-aging interventions; PAAIs) for a possible countering of aging. Importantly, unlike most previous studies, we include in our study design young treated groups of animals, subjected to PAAIs prior to the onset of detectable age-dependent phenotypic change. Many PAAI effects influence phenotypes long before the onset of detectable age-dependent change, but, importantly, do not alter the rate of phenotypic change. Hence, these PAAIs have limited effects on aging.


Assuntos
Envelhecimento , Longevidade , Camundongos , Animais , Masculino , Longevidade/genética , Camundongos Endogâmicos C57BL , Envelhecimento/fisiologia , Fenótipo
9.
Nat Commun ; 12(1): 4957, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400653

RESUMO

Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring.


Assuntos
Infecções Bacterianas/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/virologia , Parto , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Hematopoese , Humanos , Influenza Humana/imunologia , Pulmão/imunologia , Macrófagos Alveolares , Camundongos , Camundongos Endogâmicos C57BL , Mães , Poli I-C , Gravidez
10.
EMBO Mol Med ; 13(12): e14397, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34750991

RESUMO

Mitochondrial disorders are clinically and genetically diverse, with isolated complex III (CIII) deficiency being relatively rare. Here, we describe two affected cousins, presenting with recurrent episodes of severe lactic acidosis, hyperammonaemia, hypoglycaemia and encephalopathy. Genetic investigations in both cases identified a homozygous deletion of exons 2 and 3 of UQCRH, which encodes a structural complex III (CIII) subunit. We generated a mouse model with the equivalent homozygous Uqcrh deletion (Uqcrh-/- ), which also presented with lactic acidosis and hyperammonaemia, but had a more severe, non-episodic phenotype, resulting in failure to thrive and early death. The biochemical phenotypes observed in patient and Uqcrh-/- mouse tissues were remarkably similar, displaying impaired CIII activity, decreased molecular weight of fully assembled holoenzyme and an increase of an unexpected large supercomplex (SXL ), comprising mostly of one complex I (CI) dimer and one CIII dimer. This phenotypic similarity along with lentiviral rescue experiments in patient fibroblasts verifies the pathogenicity of the shared genetic defect, demonstrating that the Uqcrh-/- mouse is a valuable model for future studies of human CIII deficiency.


Assuntos
Doenças Mitocondriais , Animais , Complexo III da Cadeia de Transporte de Elétrons , Éxons , Homozigoto , Humanos , Camundongos , Doenças Mitocondriais/genética , Fenótipo , Deleção de Sequência
11.
G3 (Bethesda) ; 6(12): 4035-4046, 2016 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-27815347

RESUMO

The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.


Assuntos
Estudos de Associação Genética , Glicoproteínas/genética , Mutação , Fenótipo , Animais , Osso e Ossos/metabolismo , Proteínas de Ligação ao Cálcio , Mapeamento Cromossômico , Modelos Animais de Doenças , Metabolismo Energético/genética , Exoma , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Rim/metabolismo , Rim/fisiopatologia , Testes de Função Renal , Masculino , Camundongos , Camundongos Knockout , Osteíte Deformante/genética , Osteíte Deformante/metabolismo , Osteíte Deformante/patologia , Esqueleto/anormalidades
12.
Mol Aspects Med ; 34(2-3): 638-45, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23268354

RESUMO

The SLC43 family is composed of only three genes coding for the plasma membrane facilitator system l amino acid transporters LAT3 (SLC43A1; TC 2.A.1.44.1) and LAT4 (SLC43A2; TC 2.A.1.44.2), and the orphan protein EEG1 (SLC43A3; TC 2.A.1.44.3). Besides the known mechanism of transport of LAT3 and LAT4, their physiological roles still remain quite obscure. Morphants suggested a role of LAT3 in renal podocyte development in zebrafish. Expression in liver and skeletal muscle, and up-regulation by starvation suggest a role of LAT3 in the flux of branched-chain amino acids (BCAAs) from liver and skeletal muscle to the bloodstream. Finally, LAT3 is up-regulated in androgen-dependent cancers, suggesting a role in mTORC1 signaling in this type of tumors. In addition, LAT4 might contribute to the transfer of BCAAs from mother to fetus. Unfortunately, the EEG1 mouse model (EEG1(Y221∗)) described here has not yet offered a clue to the physiological role of this orphan protein.


Assuntos
Sistema y+L de Transporte de Aminoácidos/genética , Sistema y+L de Transporte de Aminoácidos/fisiologia , Aminoácidos/metabolismo , Modelos Moleculares , Família Multigênica/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Sistema y+L de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Northern Blotting , Humanos , Fígado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Neoplasias/metabolismo , Podócitos/metabolismo , Serina-Treonina Quinases TOR/metabolismo
13.
J Comp Physiol B ; 177(2): 183-92, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17009045

RESUMO

We sought to identify associations of basal metabolic rate (BMR) with morphological traits in laboratory mice. In order to expand the body mass (BM) range at the intra-strain level, and to minimize relevant genetic variation, we used male and female wild type mice (C3HeB/FeJ) and previously unpublished ENU-induced dwarf mutant littermates (David mice), covering a body mass range from 13.5 g through 32.3 g. BMR was measured at 30 degrees C, mice were killed by means of CO(2 )overdose, and body composition (fat mass and lean mass) was subsequently analyzed by dual X-ray absorptiometry (DEXA), after which mice were dissected into 12 (males) and 10 (females) components, respectively. Across the 44 individuals, 43% of the variation in the basal rates of metabolism was associated with BM. The latter explained 47% to 98% of the variability in morphology of the different tissues. Our results demonstrate that sex is a major determinant of body composition and BMR in mice: when adjusted for BM, females contained many larger organs, more fat mass, and less lean mass compared to males. This could be associated with a higher mass adjusted BMR in females. Once the dominant effects of sex and BM on BMR and tissue mass were removed, and after accounting for multiple comparisons, no further significant association between individual variation in BMR and tissue mass emerged.


Assuntos
Metabolismo Basal/fisiologia , Composição Corporal/fisiologia , Índice de Massa Corporal , Nanismo/fisiopatologia , Camundongos Mutantes/fisiologia , Animais , Metabolismo Basal/genética , Composição Corporal/genética , Tamanho Corporal/genética , Tamanho Corporal/fisiologia , Nanismo/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C3H , Tamanho do Órgão/genética , Tamanho do Órgão/fisiologia , Fenótipo
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