RESUMO
RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a "browning" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed "CRISPR-delivery particles," denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease.
Assuntos
Tecido Adiposo Branco/metabolismo , Metabolismo Energético , Marcação de Genes/métodos , Proteína 1 de Interação com Receptor Nuclear/genética , Adipócitos/metabolismo , Tecido Adiposo Branco/citologia , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Genes Reporter , Humanos , Camundongos Endogâmicos C57BL , Proteína 1 de Interação com Receptor Nuclear/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Obesity has become the most common metabolic disorder worldwide. Promoting brown adipose tissue (BAT) and beige adipose tissue formation, and therefore, a functional increase in energy expenditure, may counteract obesity. Mice lacking type IIß regulatory subunit of adenosine 3',5' cyclic monophosphate (cAMP)-dependent protein kinase A (PKA-RIIB) display reduced adiposity and resistance to diet-induced obesity. PKA-RIIB, encoded by the Prkar2b gene, is most abundant in BAT and white adipose tissue (WAT) and in the brain. In this study, we show that mice lacking PKA-RIIB have increased energy expenditure, limited weight gain, and improved glucose metabolism. PKA-RIIB deficiency induces brownlike adipocyte in inguinal WAT (iWAT). PKA-RIIB deficiency also increases the expression of uncoupling protein 1 and other thermogenic genes in iWAT and primary preadipocytes from iWAT through a mechanism involving increased PKA activity, which is represented by increased phosphorylation of PKA substrate, cAMP response element binding protein, and P38 mitogen-activated protein kinase. Our study provides evidence for the role of PKA-RIIB deficiency in regulating thermogenesis in WAT, which may potentially have therapeutic implications for the treatment of obesity and related metabolic disorders.
Assuntos
Adipócitos Marrons , Tecido Adiposo Branco/citologia , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/deficiência , Metabolismo Energético , Termogênese , Animais , Metabolismo dos Carboidratos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Epididimo/citologia , Glucose/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Obesidade/metabolismo , Proteína Desacopladora 1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Obesity is a major health concern that contributes to the development of diabetes, hyperlipidemia, coronary artery disease, and cancer. Id proteins are helix-loop-helix transcription factors that regulate the proliferation and differentiation of cells from multiple tissues, including adipocytes. We screened mouse tissues for the expression of Id1 and found that Id1 protein is highly expressed in brown adipose tissue (BAT) and white adipose tissue (WAT), suggesting a role for Id1 in adipogenesis and cell metabolism. Id1(-/-) mice are viable but show a significant reduction in fat mass (P<0.005) over the life of the animal that was not due to decreased number of adipocytes. Analysis of Id1(-/-) mice revealed higher energy expenditure, increased lipolysis, and fatty acid oxidation, resulting in reduced triglyceride accumulation in WAT compared to Id1(+/+) mice. Serum levels of triglycerides (193.9±32.2 vs. 86.5±33.8, P<0.0005), cholesterol (189.4±33.8 vs. 110.6±8.23, P<0.0005) and leptin (1263±835 vs. 222±260, P<0.005) were significantly lower in aged Id1(-/-) mice compared to Id1(+/+) mice. Id1-deficient mice have higher resting (P<0.005) and total (P<0.05) O(2) consumption and lower respiratory exchange ratio (P<0.005), confirming that Id1(-/-) mice use a higher proportion of lipid as an energy source for the increased energy expenditure. The expression of PGC1α and UCP1 were 2- to 3-fold up-regulated in Id1(-/-) BAT, suggesting that loss of Id1 increases thermogenesis. As a consequence of higher energy expenditure and reduced fat mass, Id1(-/-) mice displayed enhanced insulin sensitivity. Id1 deficiency protected mice against age- and high-fat-diet-induced adiposity, insulin resistance, and hepatosteatosis. Our findings suggest that Id1 plays a critical role in the regulation of energy homeostasis and could be a potential target in the treatment of insulin resistance and fatty liver disease.
Assuntos
Envelhecimento/metabolismo , Metabolismo Energético/fisiologia , Fígado Gorduroso/metabolismo , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Resistência à Insulina/fisiologia , Adipócitos/citologia , Adipogenia/fisiologia , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Ácidos Graxos/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/prevenção & controle , Feminino , Fibroblastos/citologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Gravidez , Termogênese/fisiologiaRESUMO
BACKGROUND: Previous studies of mixed background mice have demonstrated that targeted deletion of Vgf produces a lean, hypermetabolic mouse that is resistant to diet-, lesion-, and genetically-induced obesity. To investigate potential mechanism(s) and site(s) of action of VGF, a neuronal and endocrine secreted protein and neuropeptide precursor, we further analyzed the metabolic phenotypes of two independent VGF knockout lines on C57Bl6 backgrounds. RESULTS: Unlike hyperactive VGF knockout mice on a mixed C57Bl6-129/SvJ background, homozygous mutant mice on a C57Bl6 background were hypermetabolic with similar locomotor activity levels to Vgf+/Vgf+ mice, during day and night cycles, indicating that mechanism(s) other than hyperactivity were responsible for their increased energy expenditure. In Vgf-/Vgf- knockout mice, morphological analysis of brown and white adipose tissues (BAT and WAT) indicated decreased fat storage in both tissues, and decreased adipocyte perimeter and area in WAT. Changes in gene expression measured by real-time RT-PCR were consistent with increased fatty acid oxidation and uptake in BAT, and increased lipolysis, decreased lipogenesis, and brown adipocyte differentiation in WAT, suggesting that increased sympathetic nervous system activity in Vgf-/Vgf- mice may be associated with or responsible for alterations in energy expenditure and fat storage. In addition, uncoupling protein 1 (UCP1) and UCP2 protein levels, mitochondrial number, and mitochondrial cristae density were upregulated in Vgf-/Vgf- BAT. Using immunohistochemical and histochemical techniques, we detected VGF in nerve fibers innervating BAT and Vgf promoter-driven reporter expression in cervical and thoracic spinal ganglia that project to and innervate the chest wall and tissues including BAT. Moreover, VGF peptide levels were quantified by radioimmunoassay in BAT, and were found to be down-regulated by a high fat diet. Lastly, despite being hypermetabolic, VGF knockout mice were cold intolerant. CONCLUSION: We propose that VGF and/or VGF-derived peptides modulate sympathetic outflow pathways to regulate fat storage and energy expenditure.
Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Composição Corporal/genética , Metabolismo Energético/genética , Neuropeptídeos/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/inervação , Tecido Adiposo Branco/citologia , Análise de Variância , Animais , Western Blotting , Distribuição da Gordura Corporal , Temperatura Corporal/genética , Calorimetria , Temperatura Baixa , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ingestão de Alimentos/genética , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/metabolismo , Canais Iônicos/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Atividade Motora/genética , Fatores de Crescimento Neural , Vias Neurais/metabolismo , Neurônios/metabolismo , Neuropeptídeos/genética , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radioimunoensaio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Tempo , Proteína Desacopladora 1RESUMO
Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha) is an attractive candidate gene for type 2 diabetes, as genes of the oxidative phosphorylation (OXPHOS) pathway are coordinatively downregulated by reduced expression of PGC-1 alpha in skeletal muscle and adipose tissue of patients with type 2 diabetes. Here we demonstrate that transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) had reduced white adipose tissue (WAT) mass, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and enhanced expression of the OXPHOS genes, coordinated by increased levels of PGC-1 alpha and 5'-AMP-activated protein kinase (AMPK) in WAT. As accelerated polyamine flux caused by SSAT overexpression depleted the ATP pool in adipocytes of SSAT mice and N(1),N(11)-diethylnorspermine-treated wild-type fetal fibroblasts, we propose that low ATP levels lead to the induction of AMPK, which in turn activates PGC-1 alpha in WAT of SSAT mice. Our hypothesis is supported by the finding that the phenotype of SSAT mice was reversed when the accelerated polyamine flux was reduced by the inhibition of polyamine biosynthesis in WAT. The involvement of polyamine catabolism in the regulation of energy and glucose metabolism may offer a novel target for drug development for obesity and type 2 diabetes.