RESUMO
CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Edição de Genes/normas , Genoma/genética , Mutação , Especificidade por Substrato/genética , Animais , Proteínas Associadas a CRISPR/genética , Feminino , Humanos , Mutação INDEL , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/genética , Transgenes/genéticaRESUMO
Aging, obesity, and insulin resistance are associated with low levels of PGC1α and PGC1ß coactivators and defective mitochondrial function. We studied mice deficient for PGC1α and PGC1ß [double heterozygous (DH)] to investigate their combined pathogenic contribution. Contrary to our hypothesis, DH mice were leaner, had increased energy dissipation, a pro-thermogenic profile in BAT and WAT, and improved carbohydrate metabolism compared to wild types. WAT showed upregulation of mitochondriogenesis/oxphos machinery upon allelic compensation of PGC1α4 from the remaining allele. However, DH mice had decreased mitochondrial OXPHOS and biogenesis transcriptomes in mitochondria-rich organs. Despite being metabolically healthy, mitochondrial defects in DH mice impaired muscle fiber remodeling and caused qualitative changes in the hepatic lipidome. Our data evidence first the existence of organ-specific compensatory allostatic mechanisms are robust enough to drive an unexpected phenotype. Second, optimization of adipose tissue bioenergetics is sufficient to maintain a healthy metabolic phenotype despite a broad severe mitochondrial dysfunction in other relevant metabolic organs. Third, the decrease in PGC1s in adipose tissue of obese and diabetic patients is in contrast with the robustness of the compensatory upregulation in the adipose of the DH mice.
Assuntos
Tecido Adiposo/metabolismo , Mitocôndrias/genética , Proteínas Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fatores de Transcrição/genética , Envelhecimento/genética , Animais , Modelos Animais de Doenças , Metabolismo Energético/genética , Heterozigoto , Resistência à Insulina/genética , Masculino , Camundongos , Obesidade/genética , Termogênese/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. RESULTS: To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. CONCLUSIONS: Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.
Assuntos
Colesterol/sangue , Hipercolesterolemia/genética , Fígado/metabolismo , Pró-Proteína Convertase 9/genética , Animais , Modelos Animais de Doenças , Edição de Genes , Genoma , Humanos , Hipercolesterolemia/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Oxytocin (Oxt) and its receptor (Oxtr) gene system has been implicated in cardiomyogenesis and cardioprotection; however, effects of chronic activation of Oxtr are not known. We generated and investigated transgenic (TG) mice that overexpress Oxtr specifically in the heart. METHODS AND RESULTS: Cardiac-specific overexpression of Oxtr was obtained by having the α-major histocompatibility complex promoter drive the mouse Oxtr gene (α-Mhc-Oxtr). Left ventricular (LV) function and remodeling were assessed by magnetic resonance imaging and echocardiography. In α-Mhc-Oxtr TG mice, LV ejection fraction was severely compromised at 14 weeks of age compared with wild-type (WT) littermates (25 ± 6% vs 63 ± 3%; P < .001). LV end-diastolic volume was larger in the TG mice (103 ± 6 µL vs 67 ± 5 µL; P < .001). α-Mhc-Oxtr TG animals displayed cardiac fibrosis, atrial thrombus, and increased expression of pro-fibrogenic genes. Mortality of α-Mhc-Oxtr TG animals was 45% compared with 0% (P < .0001) of WT littermates by 20 weeks of age. Most cardiomyocytes of α-Mhc-Oxtr TG animals but not WT littermates (68.0 ± 12.1% vs 5.6 ± 2.4%; P = .008) were positive in staining for nuclear factor of activated T cells (NFAT). To study if thrombin inhibitor prevents thrombus formation, a cohort of 7-week-old α-Mhc-Oxtr TG mice were treated for 12 weeks with AZD0837, a potent thrombin inhibitor. Treatment with AZD0837 reduced thrombus formation (P < .05) and tended to attenuate fibrosis and increase survival. CONCLUSIONS: Cardiac-specific overexpression of Oxtr had negative consequences on LV function and survival in mice. The present findings necessitate further studies to investigate potential adverse effects of chronic Oxt administration. We provide a possible mechanism of Oxtr overexpression leading to heart failure by nuclear factor of activated T cell signaling. The recapitulation of human heart failure and the beneficial effects of the antithrombin inhibitor render the α-Mhc-Oxtr TG mice a promising tool in drug discovery for heart failure.
Assuntos
Cardiomiopatias/genética , Regulação da Expressão Gênica , Miocárdio/metabolismo , RNA/genética , Receptores de Ocitocina/genética , Animais , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Imagem Cinética por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/patologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Ocitocina/biossínteseRESUMO
Free fatty acid receptor 2 (Ffar2), also known as GPR43, is activated by short-chain fatty acids (SCFA) and expressed in intestine, adipocytes, and immune cells, suggesting involvement in lipid and immune regulation. In the present study, Ffar2-deficient mice (Ffar2-KO) were given a high-fat diet (HFD) or chow diet and studied with respect to lipid and energy metabolism. On a HFD, Ffar2-KO mice had lower body fat mass and increased lean body mass. The changed body composition was accompanied by improved glucose control and lower HOMA index, indicating improved insulin sensitivity in Ffar2-KO mice. Moreover, the Ffar2-KO mice had higher energy expenditure accompanied by higher core body temperature and increased food intake. The liver weight and content of triglycerides as well as plasma levels of cholesterol were lower in the Ffar2-KO mice fed a HFD. A histological examination unveiled decreased lipid interspersed in brown adipose tissue of the Ffar2-KO mice. Interestingly, no significant differences in white adipose tissue (WAT) cell size were observed, but significantly lower macrophage content was detected in WAT from HFD-fed Ffar2-KO compared with wild-type mice. In conclusion, Ffar2 deficiency protects from HFD-induced obesity and dyslipidemia at least partly via increased energy expenditure.
Assuntos
Gorduras na Dieta/administração & dosagem , Hiperglicemia/prevenção & controle , Hipoglicemia/prevenção & controle , Obesidade/prevenção & controle , Receptores Acoplados a Proteínas G/fisiologia , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/patologia , Animais , Composição Corporal , Regulação da Temperatura Corporal , Dieta , Dislipidemias/sangue , Dislipidemias/prevenção & controle , Metabolismo Energético , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Homeostase , Resistência à Insulina , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , Obesidade/patologia , Receptores Acoplados a Proteínas G/genéticaRESUMO
We investigated whether ghrelin action at the level of the ventral tegmental area (VTA), a key node in the mesolimbic reward system, is important for the rewarding and motivational aspects of the consumption of rewarding/palatable food. Mice with a disrupted gene encoding the ghrelin receptor (GHS-R1A) and rats treated peripherally with a GHS-R1A antagonist both show suppressed intake of rewarding food in a free choice (chow/rewarding food) paradigm. Moreover, accumbal dopamine release induced by rewarding food was absent in GHS-R1A knockout mice. Acute bilateral intra-VTA administration of ghrelin increased 1-hour consumption of rewarding food but not standard chow. In comparison with sham rats, VTA-lesioned rats had normal intracerebroventricular ghrelin-induced chow intake, although both intake of and time spent exploring rewarding food was decreased. Finally, the ability of rewarding food to condition a place preference was suppressed by the GHS-R1A antagonist in rats. Our data support the hypothesis that central ghrelin signaling at the level of the VTA is important for the incentive value of rewarding food.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Grelina/farmacologia , Sistema Límbico/efeitos dos fármacos , Mesencéfalo/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Recompensa , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Condicionamento Clássico/efeitos dos fármacos , Dopamina/metabolismo , Injeções , Masculino , Camundongos , Camundongos Knockout , Núcleo Accumbens/efeitos dos fármacos , Receptores de Grelina/efeitos dos fármacos , Receptores de Grelina/genética , Transdução de Sinais/efeitos dos fármacos , Paladar/efeitos dos fármacosRESUMO
BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD) characterised by liver fat accumulation, inflammation and progressive fibrosis. Emerging data indicate that genetic susceptibility increases risks of NAFLD, NASH and NASH-related cirrhosis. AIMS: To review NASH genetics and discuss the potential for precision medicine approaches to treatment. METHOD: PubMed search and inclusion of relevant literature. RESULTS: Single-nucleotide polymorphisms in PNPLA3, TM6SF2, GCKR, MBOAT7 and HSD17B13 are clearly associated with NASH development or progression. These genetic variants are common and have moderate-to-large effect sizes for development of NAFLD, NASH and hepatocellular carcinoma (HCC). The genes play roles in lipid remodelling in lipid droplets, hepatic very low-density lipoprotein (VLDL) secretion and de novo lipogenesis. The PNPLA3 I148M variant (rs738409) has large effects, with approximately twofold increased odds of NAFLD and threefold increased odds of NASH and HCC per allele. Obesity interacts with PNPLA3 I148M to elevate liver fat content and increase rates of NASH. Although the isoleucine-to-methionine substitution at amino acid position 148 of the PNPLA3 enzyme inactivates its lipid remodelling activity, the effect of PNPLA3 I148M results from trans-repression of another lipase (ATGL/PNPLA2) by sequestration of a shared cofactor (CGI-58/ABHD5), leading to decreased hepatic lipolysis and VLDL secretion. In homozygous Pnpla3 I148M knock-in rodent models of NAFLD, targeted PNPLA3 mRNA knockdown reduces hepatic steatosis, inflammation and fibrosis. CONCLUSION: The emerging genetic and molecular understanding of NASH paves the way for novel interventions, including precision medicines that can modulate the activity of specific genes associated with NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/terapia , Medicina de Precisão/tendências , Alelos , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Progressão da Doença , Predisposição Genética para Doença , Testes Genéticos/métodos , Testes Genéticos/tendências , Humanos , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Hepatopatia Gordurosa não Alcoólica/patologia , Polimorfismo de Nucleotídeo Único , Medicina de Precisão/métodos , PrognósticoRESUMO
To investigate the role of the central neuromedin U (NMU) signaling system in body weight and energy balance regulation, we examined the effects of long-term intracerebroventricular (icv) infusion of NMU in C57Bl/6 mice and in mice lacking the gene encoding NMU receptor 2. In diet-induced obese male and female C57BL/6 mice, icv infusion of NMU (8 microg x day(-1) x mouse(-1)) for 7 days decreased body weight and total energy intake compared with vehicle treatment. However, these parameters were unaffected by NMU treatment in lean male and female C57BL/6 mice fed a standard diet. In addition, female (but not male) NMUR2-null mice had increased body weight and body fat mass when fed a high-fat diet but lacked a clear body weight phenotype when fed a standard diet compared with wild-type littermates. Furthermore, female (but not male) NMUR2-null mice fed a high-fat diet were protected from central NMU-induced body weight loss compared with littermate wild-type mice. Thus, we provide the first evidence that long-term central NMU treatment reduces body weight, food intake, and adiposity and that central NMUR2 signaling is required for these effects in female but not male mice.
Assuntos
Peso Corporal/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Neuropeptídeos/farmacologia , Receptores de Neurotransmissores/genética , Tecido Adiposo/anatomia & histologia , Tecido Adiposo/efeitos dos fármacos , Animais , Composição Corporal/efeitos dos fármacos , Composição Corporal/genética , Peso Corporal/genética , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiologia , Ingestão de Energia/efeitos dos fármacos , Ingestão de Energia/genética , Metabolismo Energético/genética , Feminino , Deleção de Genes , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/administração & dosagem , Receptores de Neurotransmissores/metabolismo , Receptores de Neurotransmissores/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de TempoRESUMO
The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta) has been implicated in important metabolic processes. A mouse lacking PGC-1beta (PGC1betaKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1betaKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1beta ablation was partially compensated by up-regulation of PGC-1alpha in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1betaKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1beta was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1betaKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1betaKO mice have impaired mitochondrial function. Lack of PGC-1beta also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1beta plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.
Assuntos
Coração/fisiologia , Fígado/fisiologia , Mitocôndrias/fisiologia , Termogênese/fisiologia , Transativadores/genética , Transativadores/fisiologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/citologia , Adrenérgicos/farmacologia , Animais , Distribuição da Gordura Corporal , Peso Corporal , Temperatura Baixa , Dieta Aterogênica , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Feminino , Regulação da Expressão Gênica , Frequência Cardíaca/efeitos dos fármacos , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas , Músculo Esquelético/fisiologia , Norepinefrina/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Termogênese/genética , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is becoming a leading cause of advanced chronic liver disease. The progression of NAFLD, including nonalcoholic steatohepatitis (NASH), has a strong genetic component, and the most robust contributor is the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 encoding the 148M protein sequence variant. We hypothesized that suppressing the expression of the PNPLA3 148M mutant protein would exert a beneficial effect on the entire spectrum of NAFLD. METHODS: We examined the effects of liver-targeted GalNAc3-conjugated antisense oligonucleotide (ASO)-mediated silencing of Pnpla3 in a knock-in mouse model in which we introduced the human PNPLA3 I148M mutation. RESULTS: ASO-mediated silencing of Pnpla3 reduced liver steatosis (p = 0.038) in homozygous Pnpla3 148M/M knock-in mutant mice but not in wild-type littermates fed a steatogenic high-sucrose diet. In mice fed a NASH-inducing diet, ASO-mediated silencing of Pnpla3 reduced liver steatosis score and NAFLD activity score independent of the Pnpla3 genotype, while reductions in liver inflammation score (p = 0.018) and fibrosis stage (p = 0.031) were observed only in the Pnpla3 knock-in 148M/M mutant mice. These responses were accompanied by reduced liver levels of Mcp1 (p = 0.026) and Timp2 (p = 0.007) specifically in the mutant knock-in mice. This may reduce levels of chemokine attracting inflammatory cells and increase the collagenolytic activity during tissue regeneration. CONCLUSION: This study provides the first evidence that a Pnpla3 ASO therapy can improve all features of NAFLD, including liver fibrosis, and suppress the expression of a strong innate genetic risk factor, Pnpla3 148M, which may open up a precision medicine approach in NASH.
Assuntos
Lipase/genética , Cirrose Hepática/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Oligonucleotídeos Antissenso/genética , Fosfolipases A2 Independentes de Cálcio/genética , Animais , Feminino , Inativação Gênica , Humanos , Lipase/metabolismo , Cirrose Hepática/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Fosfolipases A2 Independentes de Cálcio/metabolismoRESUMO
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.
Assuntos
Metabolismo Energético/fisiologia , Neurônios/metabolismo , Receptores de LDL/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Glicemia/metabolismo , Dieta , Metabolismo Energético/genética , Hipotálamo/metabolismo , Resistência à Insulina , Camundongos , Camundongos Knockout , Obesidade/metabolismo , Obesidade/prevenção & controle , Ubiquitina-Proteína Ligases/genéticaRESUMO
The adipocyte-derived hormone adiponectin regulates glucose and lipid metabolism and influences the risk for developing obesity, type 2 diabetes, and cardiovascular disease. Adiponectin binds to two different seven-transmembrane domain receptors termed AdipoR1 and AdipoR2. To study the physiological importance of these receptors, AdipoR1 gene knockout mice (AdipoR1(-/-)) and AdipoR2 gene knockout mice (AdipoR2(-/-)) were generated. AdipoR1(-/-) mice showed increased adiposity associated with decreased glucose tolerance, spontaneous locomotor activity, and energy expenditure. However, AdipoR2(-/-) mice were lean and resistant to high-fat diet-induced obesity associated with improved glucose tolerance and higher spontaneous locomotor activity and energy expenditure and reduced plasma cholesterol levels. Thus, AdipoR1 and AdipoR2 are clearly involved in energy metabolism but have opposing effects.
Assuntos
Metabolismo Energético/fisiologia , Receptores de Superfície Celular/metabolismo , Proteínas Quinases Ativadas por AMP , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Adiposidade/genética , Adiposidade/fisiologia , Animais , Peso Corporal/fisiologia , Encéfalo/patologia , Metabolismo Energético/genética , Comportamento Alimentar , Feminino , Glucose/metabolismo , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/genética , Atividade Motora/fisiologia , Complexos Multienzimáticos/metabolismo , PPAR alfa/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Adiponectina , Receptores de Superfície Celular/genética , Transdução de Sinais , Testículo/citologia , Fatores de TempoRESUMO
α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene edited PiZ mice. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Fenótipo , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/genética , Adenoviridae/genética , Animais , Proliferação de Células , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Transgênicos , Transdução Genética , Transgenes , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/patologia , Deficiência de alfa 1-Antitripsina/terapiaRESUMO
The hypothalamic peptide melanin-concentrating hormone (MCH) plays important roles in energy homeostasis. Animals overexpressing MCH develop hyperphagia, obesity, and insulin resistance. In this study, mice lacking both the MCH receptor-1 (MCHr1 knockout) and leptin (ob/ob) double-null mice (MCHr1 knockout ob/ob) were generated to investigate whether the obesity and/or the insulin resistance linked to the obese phenotype of ob/ob mice was attenuated by ablation of the MCHr1 gene. In MCHr1 knockout ob/ob mice an oral glucose load resulted in a lower blood glucose response and markedly lower insulin levels compared with the ob/ob mice despite no differences in body weight, food intake, or energy expenditure. In addition, MCHr1 knockout ob/ob mice had higher locomotor activity and lean body mass, lower body fat mass, and altered body temperature regulation compared with ob/ob mice. In conclusion, MCHr1 is important for insulin sensitivity and/or secretion via a mechanism not dependent on decreased body weight.
Assuntos
Peso Corporal , Resistência à Insulina , Leptina/deficiência , Obesidade/metabolismo , Receptores de Somatostatina/fisiologia , Animais , Composição Corporal , Regulação da Temperatura Corporal , Hormônio Liberador da Corticotropina/análise , Ingestão de Alimentos , Glucose/metabolismo , Camundongos , Camundongos Obesos , Atividade Motora , RNA Mensageiro/análise , Receptores para Leptina , Receptores de Somatostatina/deficiência , Estearoil-CoA Dessaturase/genéticaRESUMO
It is well known that peripherally administered growth hormone (GH) results in decreased body fat mass. However, GH-deficient patients increase their food intake when substituted with GH, suggesting that GH also has an appetite stimulating effect. Transgenic mice with an overexpression of bovine GH in the central nervous system (CNS) were created to investigate the role of GH in CNS. This study shows that overexpression of GH in the CNS differentiates the effect of GH on body fat mass from that on appetite. The transgenic mice were not GH-deficient but were obese and showed increased food intake as well as increased hypothalamic expression of agouti-related protein and neuropeptide Y. GH also had an acute effect on food intake following intracerebroventricular injection of C57BL/6 mice. The transgenic mice were severely hyperinsulinemic and showed a marked hyperplasia of the islets of Langerhans. In addition, the transgenic mice displayed alterations in serum lipid and lipoprotein levels and hepatic gene expression. In conclusion, GH overexpression in the CNS results in hyperphagia-induced obesity indicating a dual effect of GH with a central stimulation of appetite and a peripheral lipolytic effect.
Assuntos
Hormônio do Crescimento/genética , Hiperlipidemias/genética , Hiperfagia/genética , Resistência à Insulina/genética , Obesidade/etiologia , Tecido Adiposo/anatomia & histologia , Animais , Sequência de Bases , Glicemia/metabolismo , Peso Corporal , Calorimetria Indireta , Bovinos , Sondas de DNA , Ingestão de Energia/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Genoma , Hormônio do Crescimento/administração & dosagem , Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/fisiologia , Hiperinsulinismo/induzido quimicamente , Hiperfagia/sangue , Hiperfagia/fisiopatologia , Hipotálamo/fisiologia , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/sangueRESUMO
The G-protein coupled receptor 55 (GPR55) is activated by cannabinoids and non-cannabinoid molecules and has been speculated to play a modulatory role in a large variety of physiological and pathological processes, including in metabolically perturbed states. We therefore generated male mice deficient in the gene coding for the cannabinoid/lysophosphatidylinositol (LPI) receptor Gpr55 and characterized them under normal dietary conditions as well as during high energy dense diet feeding followed by challenge with the CB1 receptor antagonist/GPR55 agonist rimonabant. Gpr55 deficient male mice (Gpr55 KO) were phenotypically indistinguishable from their wild type (WT) siblings for the most part. However, Gpr55 KO animals displayed an intriguing nocturnal pattern of motor activity and energy expenditure (EE). During the initial 6 hours of the night, motor activity was significantly elevated without any significant effect observed in EE. Interestingly, during the last 6 hours of the night motor activity was similar but EE was significantly decreased in the Gpr55 KO mice. No significant difference in motor activity was detected during daytime, but EE was lower in the Gpr55 KO compared to WT mice. The aforementioned patterns were not associated with alterations in energy intake, daytime core body temperature, body weight (BW) or composition, although a non-significant tendency to increased adiposity was seen in Gpr55 KO compared to WT mice. Detailed analyses of daytime activity in the Open Field paradigm unveiled lower horizontal activity and rearing time for the Gpr55 KO mice. Moreover, the Gpr55 KO mice displayed significantly faster reaction time in the tail flick test, indicative of thermal hyperalgesia. The BW-decreasing effect of rimonabant in mice on long-term cafeteria diet did not differ between Gpr55 KO and WT mice. In conclusion, Gpr55 deficiency is associated with subtle effects on diurnal/nocturnal EE and motor activity behaviours but does not appear per se critically required for overall metabolism or behaviours.
Assuntos
Metabolismo Energético , Dor/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Comportamento Animal , Temperatura Corporal , Calorimetria , Antagonistas de Receptores de Canabinoides/metabolismo , Dieta Hiperlipídica , Metabolismo Energético/genética , Deleção de Genes , Estudos Longitudinais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Dor/genética , Piperidinas/metabolismo , Pirazóis/metabolismo , Receptores de Canabinoides/deficiência , Receptores de Canabinoides/genética , Rimonabanto , Sensação Térmica/genéticaRESUMO
BACKGROUND: The gut microbiota plays an important role in the development of obesity and obesity-associated impairments such as low-grade inflammation. Lingonberries have been shown to prevent diet-induced obesity and low-grade inflammation. However, it is not known whether the effect of lingonberry supplementation is related to modifications of the gut microbiota. The aim of the present study was to describe whether consumption of different batches of lingonberries alters the composition of the gut microbiota, which could be relevant for the protective effect against high fat (HF)-induced metabolic alterations. METHODS: Three groups of C57BL/6J mice were fed HF diet with or without a supplement of 20% lingonberries from two different batches (Lingon1 and Lingon2) during 11 weeks. The composition and functionality of the cecal microbiota were assessed by 16S rRNA sequencing and PICRUSt. In addition, parameters related to obesity, insulin sensitivity, hepatic steatosis, inflammation and gut barrier function were examined. RESULTS: HF-induced obesity was only prevented by the Lingon1 diet, whereas both batches of lingonberries reduced plasma levels of markers of inflammation and endotoxemia (SAA and LBP) as well as modified the composition and functionality of the gut microbiota, compared to the HF control group. The relative abundance of Akkermansia and Faecalibacterium, genera associated with healthy gut mucosa and anti-inflammation, was found to increase in response to lingonberry intake. CONCLUSIONS: Our results show that supplementation with lingonberries to an HF diet prevents low-grade inflammation and is associated with significant changes of the microbiota composition. Notably, the anti-inflammatory properties of lingonberries seem to be independent of effects on body weight gain.
RESUMO
BACKGROUND AND PURPOSE: Melanin-concentrating hormone (MCH) is an orexigen, and while rodents express one MCH receptor (MCH1 receptor), humans, non-human primates and dogs express two MCH receptors (MCH1 and MCH2 ). MCH1 receptor antagonists have been developed for the treatment of obesity and lower body weight in rodents. However, the mechanisms for the body weight loss and whether MCH1 receptor antagonism can lower body weight in species expressing both MCH receptors are not fully understood. EXPERIMENTAL APPROACH: A novel recently identified potent MCH1 receptor antagonist, AZD1979, was studied in wild type and Mchr1 knockout (KO) mice and by using pair-feeding and indirect calorimetry in diet-induced obese (DIO) mice. The effect of AZD1979 on body weight was also studied in beagle dogs. KEY RESULTS: AZD1979 bound to MCH1 receptors in the CNS and dose-dependently reduced body weight in DIO mice leading to improved homeostasis model assessment-index of insulin sensitivity. AZD1979 did not affect food intake or body weight in Mchr1 KO mice demonstrating specificity for the MCH1 receptor mechanism. In DIO mice, initial AZD1979-mediated body weight loss was driven by decreased food intake, but an additional component of preserved energy expenditure was apparent in pair-feeding and indirect calorimetry studies. AZD1979 also dose-dependently reduced body weight in dogs. CONCLUSION AND IMPLICATIONS: AZD1979 is a novel potent MCH1 receptor antagonist that affects both food intake and energy expenditure. That AZD1979 also lowers body weight in a species expressing both MCH receptors holds promise for the use of MCH1 receptor antagonists for the treatment of human obesity.
Assuntos
Azetidinas/farmacologia , Peso Corporal/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Oxidiazóis/farmacologia , Receptores de Somatostatina/antagonistas & inibidores , Animais , Azetidinas/administração & dosagem , Azetidinas/química , Cães , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Oxidiazóis/administração & dosagem , Oxidiazóis/química , Receptores de Somatostatina/deficiência , Relação Estrutura-AtividadeRESUMO
RORγ is a nuclear hormone receptor which controls polarization of naive CD4+ T-cells into proinflammatory Th17 cells. Pharmacological antagonism of RORγ has therapeutic potential for autoimmune diseases; however, this mechanism may potentially carry target-related safety risks, as mice deficient in Rorc, the gene encoding RORγ, develop T-cell lymphoma with 50% frequency. Due to the requirement of RORγ during development, the Rorc knockout (KO) animals lack secondary lymphoid organs and have a dysregulation in the generation of CD4+ and CD8+ T cells. We wanted to extend the evaluation of RORγ deficiency to address the question whether lymphomas, similar to those observed in the Rorc KO, would develop in an animal with an otherwise intact adult immune system. Accordingly, we designed a conditional RORγ knockout mouse (Rorc CKO) where the Rorc locus could be deleted in adult animals. Based on these studies we can confirm that these animals also develop lymphoma in a similar time frame as embryonic Rorc knockouts. This study also suggests that in animals where the gene deletion is incomplete, the thymus undergoes a rapid selection process replacing Rorc deficient cells with remnant thymocytes carrying a functional Rorc locus and that subsequently, these animals do not develop lymphoblastic lymphoma.
Assuntos
Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Doenças Autoimunes/terapia , Masculino , Camundongos , Camundongos Knockout , Células Th17/imunologiaRESUMO
Inhibition of AMP deaminase (AMPD) holds the potential to elevate intracellular adenosine and AMP levels and, therefore, to augment adenosine signaling and activation of AMP-activated protein kinase (AMPK). To test the latter hypothesis, novel AMPD pan inhibitors were synthesized and explored using a panel of in vitro, ex vivo, and in vivo models focusing on confirming AMPD inhibitory potency and the potential of AMPD inhibition to improve glucose control in vivo. Repeated dosing of selected inhibitors did not improve glucose control in insulin-resistant or diabetic rodent disease models. Mice with genetic deletion of the muscle-specific isoform Ampd1 did not showany favorable metabolic phenotype despite being challenged with high-fat diet feeding. Therefore, these results do not support the development of AMPD inhibitors for the treatment of type 2 diabetes.