RESUMO
OBJECTIVE: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. METHODS: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. RESULTS: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. CONCLUSIONS: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Ansiedade/metabolismo , Sequência de Bases , Encéfalo/metabolismo , Mapeamento Cromossômico , Bases de Dados Genéticas , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox , Proteínas de Homeodomínio/fisiologia , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Proteínas do Tecido Nervoso/fisiologia , Sistemas Neurossecretores/metabolismo , Obesidade/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: Although reduced glutathione (rGSH) is decreased in obese mice and humans, block of GSH synthesis by buthionine sulfoximine (BSO) results in a lean, insulin-sensitive phenotype. Data is lacking about the effect of BSO on GSH precursors, cysteine and glutamate. Plasma total cysteine (tCys) is positively associated with stearoyl-coenzyme A desaturase (SCD) activity and adiposity in humans and animal models. OBJECTIVE: To explore the phenotype, amino acid and fatty acid profiles in BSO-treated mice. DESIGN: Male C3H/HeH mice aged 11 weeks were fed a high-fat diet with or without BSO in drinking water (30 mmol/L) for 8 weeks. Amino acid and fatty acid changes were assessed, as well as food consumption, energy expenditure, locomotor activity, body composition and liver vacuolation (steatosis). RESULTS: Despite higher food intake, BSO decreased particularly fat mass but also lean mass (both P<0.001), and prevented fatty liver vacuolation. Physical activity increased during the dark phase. BSO decreased plasma free fatty acids and enhanced insulin sensitivity. BSO did not alter liver rGSH, but decreased plasma total GSH (tGSH) and rGSH (by ~70%), and liver tGSH (by 82%). Glutamate accumulated in plasma and liver. Urine excretion of cysteine and its precursors was increased by BSO. tCys, rCys and cystine decreased in plasma (by 23-45%, P<0.001 for all), but were maintained in liver, at the expense of decreased taurine. Free and total plasma concentrations of the SCD products, oleic and palmitoleic acids were decreased (by 27-38%, P <0.001 for all). CONCLUSION: Counterintuitively, block of GSH synthesis decreases circulating tCys, raising the question of whether the BSO-induced obesity-resistance is linked to cysteine depletion. Cysteine-supplementation of BSO-treated mice is warranted to dissect the effects of cysteine and GSH depletion on energy metabolism.
Assuntos
Aminoácidos/metabolismo , Peso Corporal , Ácidos Graxos/metabolismo , Glutationa/deficiência , Fenótipo , Compostos de Sulfidrila/metabolismo , Tecido Adiposo/citologia , Aminoácidos/sangue , Animais , Composição Corporal , Butionina Sulfoximina/metabolismo , Ingestão de Alimentos , Metabolismo Energético , Ácidos Graxos/sangue , Glutationa/urina , Insulina/metabolismo , Fígado/citologia , Fígado/metabolismo , Locomoção , Masculino , Camundongos , Compostos de Sulfidrila/sangueRESUMO
AIMS/HYPOTHESIS: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2ß and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2ß increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice. METHODS: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a). RESULTS: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age. CONCLUSIONS/INTERPRETATION: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis. ACCESS TO RESEARCH MATERIALS: All reagents are available upon request.
Assuntos
Resistência à Insulina/fisiologia , Leptina/metabolismo , Obesidade/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Adipócitos/metabolismo , Animais , Western Blotting , Linhagem Celular , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Glucose/metabolismo , Homeostase/genética , Homeostase/fisiologia , Hipotálamo/metabolismo , Insulina/metabolismo , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function in vitro and in vivo as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate in vitro. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO's demethylase activity could be therapeutically useful for the treatment of obesity.
Assuntos
Fármacos Antiobesidade/farmacologia , Glicina/análogos & derivados , Isoquinolinas/farmacologia , Oxigenases de Função Mista/antagonistas & inibidores , Obesidade/tratamento farmacológico , Oxo-Ácido-Liases/antagonistas & inibidores , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Glicina/farmacologia , Concentração Inibidora 50 , Masculino , Camundongos Endogâmicos C57BL , Oxigenases de Função Mista/metabolismo , Obesidade/metabolismo , Oxo-Ácido-Liases/metabolismoRESUMO
Certain human diseases affecting the biliary tree can be modeled in rats by ingestion of the hepatobiliary toxin alpha-naphthylisothiocyanate (ANIT). Phosphorus magnetic resonance spectroscopy (MRS) allows the noninvasive monitoring of cell dynamics through detection of phosphodiesters (PDE) and phosphomonoesters (PME). Hepatic (31)P MRS techniques were therefore used to study the toxic effects of low-dose chronic ANIT ingestion, with a view toward providing biomarkers sensitive to hepatobiliary dysfunction and cholestatic liver injury. Rats were fed an ANIT supplemented diet at three doses (ANIT_0.05%, ANIT_0.04%, and ANIT_0.025%) for 2 weeks. Data from in vivo MRS were compared with results from pair-fed controls (PFCs). Blood and tissue samples were collected at 2 weeks for clinical chemistry, histology, and (1)H magic angle spinning MRS. Increases in PDE, relative to total phosphorus (tPh), were detected in both the ANIT_0.05% and ANIT_0.04% groups (0.07 ± 0.01 and 0.08 ± 0.01, respectively) relative to PFC groups (0.03 ± 0.01 and 0.05 ± 0.01, respectively). An increase in PME/tPh was observed in the ANIT_0.05% group only (0.17 ± 0.02) relative to PFC_0.05% (0.12 ± 0.01). Ex vivo (1)H MRS findings supported this, wherein measured phosphocholines (PCs) were increased in ANIT_0.05% and ANIT_0.04% groups. Increases in relative total choline (tCho) distinguished the ANIT_0.05% group from the ANIT_0.04% group. Markers of hepatotoxicity such as raised total bilirubin and alkaline phosphatase were found at all ANIT doses. Histological findings included a dose-related increase in both severity of biliary hyperplasia and focal hepatocellular necrosis. Here, we found that ANIT-induced moderate hepatobiliary dysfunction was associated with a relative increase in phosphodiesters in vivo and PCs ex vivo. Raised PME/tPh in vivo and tCho ex vivo were also present at high doses corresponding to a higher incidence of marked biliary hyperplasia and moderate hepatocellular necrosis.