RESUMO
Exploring mechanisms responsible for brown adipose tissue's (BAT) high metabolic activity is crucial to exploit its energy-dissipating ability for therapeutic purposes. Basigin (Bsg), a multifunctional highly glycosylated transmembrane protein, was recently proposed as one of the 98 critical markers allowing to distinguish 'white' and 'brown' adipocytes, yet its function in thermogenic brown adipocytes is unknown. Here, we report that Bsg is negatively associated with obesity in mice. By contrast, Bsg expression increased in the mature adipocyte fraction of BAT upon cold acclimation. Additionally, Bsg levels were highly induced during brown adipocyte maturation in vitro and were further increased upon ß-adrenergic stimulation in a HIF-1α-dependent manner. siRNA-mediated Bsg gene silencing in cultured brown adipocytes did not impact adipogenesis nor mitochondrial function. However, a significant decrease in mitochondrial respiration, lipolysis and Ucp1 transcription was observed in adipocytes lacking Bsg, when activated by norepinephrine. Furthermore, using gas chromatography/mass spectrometry-time-of-flight analysis to assess the composition of cellular metabolites, we demonstrate that brown adipocytes lacking Bsg have lower levels of intracellular lactate and acetoacetate. Bsg was additionally required to regulate intracellular AcAc and tricarboxylic acid cycle intermediate levels in NE-stimulated adipocytes. Our study highlights the critical role of Bsg in active brown adipocytes, possibly by controlling cellular metabolism.
Assuntos
Adipócitos Marrons , Tecido Adiposo Marrom , Camundongos , Animais , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Basigina/metabolismo , Lipólise , Obesidade/metabolismo , Termogênese/genética , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
In a randomized crossover study in humans, high fructose feeding reduced glucose uptake in brown fat without affecting the tissue's oxidative capacity. These effects were independent of alterations in the gut microbiome.
Assuntos
Frutose , Microbioma Gastrointestinal , Tecido Adiposo Marrom , Estudos Cross-Over , Glucose , HumanosRESUMO
KEY POINTS: Afadin is a ubiquitously expressed scaffold protein with a recently discovered role in insulin signalling and glucose metabolism. Insulin-stimulated phosphorylation of Afadin at S1795 occurs in insulin-responsive tissues such as adipose tissue, muscle, liver, pancreas and heart. Afadin abundance and AfadinS1795 phosphorylation are dynamically regulated in metabolic tissues during diet-induced obesity progression. Genetic silencing of AfadinS1795 phosphorylation improves glucose homeostasis in the early stages of diet-induced metabolic dysregulation. AfadinS1795 phosphorylation contributes to the early development of obesity-related complications in mice. ABSTRACT: Obesity is associated with systemic insulin resistance and numerous metabolic disorders. Yet, the mechanisms underlying impaired insulin action during obesity remain to be fully elucidated. Afadin is a multifunctional scaffold protein with the ability to modulate insulin action through its phosphorylation at S1795 in adipocytes. In the present study, we report that insulin-stimulated AfadinS1795 phosphorylation is not restricted to adipose tissues, but is a common signalling event in insulin-responsive tissues including muscle, liver, pancreas and heart. Furthermore, a dynamic regulation of Afadin abundance occurred during diet-induced obesity progression, while its phosphorylation was progressively attenuated. To investigate the role of AfadinS1795 phosphorylation in the regulation of whole-body metabolic homeostasis, we generated a phospho-defective mouse model (Afadin SA) in which the Afadin phosphorylation site was silenced (S1795A) at the whole-body level using CRISPR-Cas9-mediated gene editing. Metabolic characterization of these mice under basal physiological conditions or during a high-fat diet (HFD) challenge revealed that preventing AfadinS1795 phosphorylation improved insulin sensitivity and glucose tolerance and increased liver glycogen storage in the early stage of diet-induced metabolic dysregulation, without affecting body weight. Together, our findings reveal that AfadinS1795 phosphorylation in metabolic tissues is critical during obesity progression and contributes to promote systemic insulin resistance and glucose intolerance in the early phase of diet-induced obesity.
Assuntos
Resistência à Insulina , Animais , Dieta Hiperlipídica , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas dos Microfilamentos , FosforilaçãoRESUMO
Objective: The ß-site APP-cleaving enzyme 1 (BACE1) is a rate-limiting step in ß-amyloid (Aß) production in Alzheimer's disease (AD) brains, but recent evidence suggests that BACE1 is also involved in metabolic regulation. Here, we aimed to assess the effects of highfat diet (HFD) on metabolic and cognitive phenotypes in the diabetic BACE1 knock-in mice (PLB4) and WT controls; we additionally examined whether these phenotypes can be normalized with a synthetic retinoid (Fenretinide, Fen) targeting weight loss.Methods: Five-month old male WT and PLB4 mice were fed either (1) control chow diet, (2) 45%-saturated fat diet (HFD), (3) HFD with 0.04% Fen (HFD + Fen) or (4) control chow diet with 0.04% Fen (Fen) for 10 weeks. We assessed basic metabolic parameters, circadian rhythmicity, spatial habituation (Phenotyper) and working memory (Y-maze). Hypothalami, forebrain and liver tissues were assessed using Western blots, qPCR and ELISAs.Results: HFD feeding drastically worsened metabolism and induced early mortality (-40%) in otherwise viable PLB4 mice. This was ameliorated by Fen, despite no effects on glucose intolerance. In HFD-fed WT mice, Fen reduced weight gain, glucose intolerance and hepatic steatosis. The physiological changes induced in WT and PLB4 mice by HFD (+/-Fen) were accompanied by enhanced cerebral astrogliosis, elevated PTP1B, phopsho-eIF2α and altered hypothalamic transcription of Bace1, Pomc and Mc4r. Behaviourally, HFD feeding exacerbated spatial memory deficits in PLB4 mice, which was prevented by Fen and linked with increased full-length APP, normalized brain Aß*56 oligomerization and astrogliosis.Conclusions: HFD induces early mortality and worsened cognition in the Alzheimer's-like BACE1 mice- partial prevention was achieved with Fenretinide, without improvements in glucose homeostasis.
Assuntos
Doença de Alzheimer , Fenretinida , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Cognição , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus regulates food intake and whole-body metabolism. NAD+ regulates multiple cellular processes controlling energy metabolism. Yet, its role in hypothalamic AgRP neurons to control food intake is poorly understood. Here, we aimed to assess whether genetic deletion of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in NAD+ production, affects AgRP neuronal function to impact whole-body metabolism and food intake. Metabolic parameters during fed and fasted states, and upon systemic ghrelin and leptin administration were studied in AgRP-specific Nampt knockout (ARNKO) mice. We monitored neuropeptide expression levels and density of AgRP neurons in ARNKO mice from embryonic to adult age. NPY cells were used to determine effects of NAMPT inhibition on neuronal viability, energy status, and oxidative stress in vitro. In these cells, NAD+ depletion reduced ATP levels, increased oxidative stress, and promoted cell death. Agrp expression in the hypothalamus of ARNKO mice gradually decreased after weaning due to progressive AgRP neuron degeneration. Adult ARNKO mice had normal glucose and insulin tolerance, but exhibited an elevated respiratory exchange ratio (RER) when fasted. Remarkably, fasting-induced food intake was unaffected in ARNKO mice when evaluated in metabolic cages, but fasting- and ghrelin-induced feeding and body weight gain decreased in ARNKO mice when evaluated outside metabolic cages. Collectively, deletion of Nampt in AgRP neurons causes progressive neurodegeneration and impairs fasting and ghrelin responses in a context-dependent manner. Our data highlight an essential role of Nampt in AgRP neuron function and viability.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Citocinas/fisiologia , Ingestão de Alimentos , Jejum , Grelina/farmacologia , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Nicotinamida Fosforribosiltransferase/fisiologia , Proteína Relacionada com Agouti/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismoRESUMO
OBJECTIVE: Increasing adaptive thermogenesis by stimulating browning in white adipose tissue is a promising method of improving metabolic health. However, the molecular mechanisms underlying this transition remain elusive. Our study examined the molecular determinants driving the differentiation of precursor cells into thermogenic adipocytes. METHODS: In this study, we conducted temporal high-resolution proteomic analysis of subcutaneous white adipose tissue (scWAT) after cold exposure in mice. This was followed by loss- and gain-of-function experiments using siRNA-mediated knockdown and CRISPRa-mediated induction of gene expression, respectively, to evaluate the function of the transcriptional regulator Y box-binding protein 1 (YBX1) during adipogenesis of brown pre-adipocytes and mesenchymal stem cells. Transcriptomic analysis of mesenchymal stem cells following induction of endogenous Ybx1 expression was conducted to elucidate transcriptomic events controlled by YBX1 during adipogenesis. RESULTS: Our proteomics analysis uncovered 509 proteins differentially regulated by cold in a time-dependent manner. Overall, 44 transcriptional regulators were acutely upregulated following cold exposure, among which included the cold-shock domain containing protein YBX1, peaking after 24 h. Cold-induced upregulation of YBX1 also occurred in brown adipose tissue, but not in visceral white adipose tissue, suggesting a role of YBX1 in thermogenesis. This role was confirmed by Ybx1 knockdown in brown and brite preadipocytes, which significantly impaired their thermogenic potential. Conversely, inducing Ybx1 expression in mesenchymal stem cells during adipogenesis promoted browning concurrent with an increased expression of thermogenic markers and enhanced mitochondrial respiration. At a molecular level, our transcriptomic analysis showed that YBX1 regulates a subset of genes, including the histone H3K9 demethylase Jmjd1c, to promote thermogenic adipocyte differentiation. CONCLUSION: Our study mapped the dynamic proteomic changes of murine scWAT during browning and identified YBX1 as a novel factor coordinating the genomic mechanisms by which preadipocytes commit to brite/beige lineage.
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Tecido Adiposo Branco/metabolismo , Termogênese/genética , Termogênese/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adipócitos Marrons/metabolismo , Adipogenia , Tecido Adiposo Marrom/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Proteômica , Gordura Subcutânea/metabolismo , Transcriptoma , Regulação para CimaRESUMO
Activation of thermogenic adipose tissue is linked to improved metabolic outcomes in mice and humans. Dissipation of energy as heat during thermogenesis relies on sufficient innervation of fat by sympathetic nerve fibers, a process recently proposed to be regulated by the adipose-specific calsyntenin3ß (Clstn3ß)-S100b axis. Here we aimed 1) to assess enrichment patterns of CLSTN3ß, S100b as well as the previously annotated neuronal CLSTN3α in perirenal brown and subcutaneous white human fat specimens, and 2) to investigate if the novel Clstn3ß is dynamically regulated by changes in environmental temperatures and nutritional stress in thermogenic adipose tissues in mice. We provide evidence for CLSTN3ß enrichment in multilocular perirenal fat located anatomically in the proximity to both the adrenal gland and sympathetic nerve bundles innervating the kidney in humans. Moreover, transcript levels of CLSTN3ß, but not S100b or CLSTN3α, positively correlate with uncoupling protein 1 (UCP1) expression in human adipose tissue. Our results further show that Clsnt3ß is preferentially expressed in brown adipocytes and is highly responsive to changes in environmental temperature and obesity state in mice. Collectively, this brief communication highlights CLSTN3ß as a hallmark of thermogenic adipose depots in mice and humans.
Assuntos
Tecido Adiposo Marrom/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Dieta Hiperlipídica/efeitos adversos , Resposta ao Choque Térmico , Proteínas de Membrana/metabolismo , Obesidade/fisiopatologia , Termogênese , Tecido Adiposo Marrom/metabolismo , Adulto , Idoso , Animais , Proteínas de Ligação ao Cálcio/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Gordura SubcutâneaRESUMO
AIM: Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signalling in these neurons promotes development of obesity. The role of the rate-limiting enzyme in the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied. METHODS: We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet-induced obesity, by fasting and re-feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin-induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)-mediated FK866 administration. RESULTS: NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet-induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD+ , increased levels of ROS, and affected the expression of Agrp, Pomc and genes related to mitochondrial function. Finally, ICV-induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting- and ghrelin-induced increases in food intake. CONCLUSION: Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting- and ghrelin-induced food intake.
Assuntos
Jejum/metabolismo , Grelina/metabolismo , Hipotálamo/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Acrilamidas/administração & dosagem , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , Linhagem Celular , Ingestão de Alimentos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Piperidinas/administração & dosagem , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismoRESUMO
Insulin orchestrates metabolic homeostasis through a complex signaling network for which the precise mechanisms controlling its fine-tuning are not completely understood. Here, we report that Afadin, a scaffold protein, is phosphorylated on S1795 (S1718 in humans) in response to insulin in adipocytes, and this phosphorylation is impaired with obesity and insulin resistance. In turn, loss of Afadin enhances the response to insulin in adipose tissues via upregulation of the insulin receptor protein levels. This happens in a cell-autonomous and phosphorylation-dependent manner. Insulin-stimulated Afadin-S1795 phosphorylation modulates Afadin binding with interaction partners in adipocytes, among which HDAC6 preferentially interacts with phosphorylated Afadin and acts as a key intermediate to suppress insulin receptor protein levels. Adipose tissue-specific Afadin depletion protects against insulin resistance and improves glucose homeostasis in diet-induced obese mice, independently of adiposity. Altogether, we uncover a novel insulin-induced cellular feedback mechanism governed by the interaction of Afadin with HDAC6 to negatively control insulin action in adipocytes, which may offer new strategies to alleviate insulin resistance.
Assuntos
Tecido Adiposo/metabolismo , Antígenos CD/genética , Desacetilase 6 de Histona/genética , Insulina/genética , Proteínas dos Microfilamentos/genética , Obesidade/genética , Processamento de Proteína Pós-Traducional , Receptor de Insulina/genética , Células 3T3-L1 , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/patologia , Animais , Antígenos CD/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Desacetilase 6 de Histona/metabolismo , Homeostase/genética , Humanos , Insulina/metabolismo , Insulina/farmacologia , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Fosforilação , Cultura Primária de Células , Receptor de Insulina/metabolismoRESUMO
We recently reported that brain-specific human ß-secretase 1 (BACE1) knock-in (PLB4), a mouse model of sporadic Alzheimer's disease (AD), also develops a severe diabetic phenotype characterised by impaired glucose homeostasis, decreased insulin sensitivity and a fatty liver phenotype. Hence, we here aimed to assess if targeted anti-diabetic therapies (Liraglutide and Fenretinide) would attenuate the diabetic and behavioural phenotype of these mice. PLB4 mice and wild-type (WT) controls were administered Liraglutide or Fenretinide for ten consecutive weeks alongside vehicle-treated mice. Physiological (body weight and mass composition, glucose tolerance, serum hormone concentration), behavioural (locomotor activity) and molecular assessments were performed in mice pre- and post-treatment. Liraglutide and Fenretinide treatments inhibited adiposity gain and decreased circulating serum triglyceride (with Liraglutide) and leptin (with Fenretinide) levels in PLB4 mice. We also found that PLB4 mice exhibited increased levels of serum dipeptidyl peptidase 4 (DPP4), together with up-regulated hepatic expression of Dpp4, retinol binding protein 4 (Rbp4) and sterol regulatory element-binding 1c (Srebp1c), which was normalised by both treatments. Interestingly, Liraglutide treatment slowed down habituation to a novel environment and increased secondary night activity peak in WT mice, suggesting an impact on circadian activity regulation. However, neither treatment improved glucose homeostasis in PLB4 mice, implying that impaired glucose metabolism in this genotype may not be associated with glucagon like peptide 1 (GLP-1) and/or RBP4-mediated pathways. In summary, this study provides new insights into molecular mechanisms underlying neuronal BACE1-mediated metabolic regulation and implicates BACE1 as a putative regulator of systemic DPP4 levels.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Fenretinida/uso terapêutico , Hipoglicemiantes/uso terapêutico , Liraglutida/uso terapêutico , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Diabetes Mellitus/genética , Fenretinida/farmacologia , Técnicas de Introdução de Genes , Humanos , Hipoglicemiantes/farmacologia , Liraglutida/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Resultado do TratamentoRESUMO
Gene mutations within amyloid precursor protein (APP or AßPP) and/or presenilin 1 (PS1) genes are determinants of familial Alzheimer's disease (fAD) and remain fundamental for experimental models. Here, we generated a neuronal knock-in mouse (PLB2APP) with mutated human APPSwe/Lon and investigated histopathology and behavioral phenotypes. Additionally, PLB2APP mice were cross-bred with a presenilin (PS1A246E) line to assess the impact of this gene combination. Immunohistochemistry determined amyloid-ß (Aß) pathology, astrogliosis (via GFAP labelling), and neuronal densities in hippocampal and cortical brain regions. One-year old PLB2APP mice showed higher levels of intracellular Aß in CA1, dentate gyrus, and cortical regions compared to PLBWT controls. Co-expression of PS1 reduced hippocampal but elevated cortical Aß build-up. Amyloid plaques were sparse in aged PLB2APP mice, and co-expression of PS1 promoted plaque formation. Heightened GFAP expression followed the region-specific pattern of Aß in PLB2APP and PLB2APP/PS1 mice. Behaviorally, habituation to a novel environment was delayed in 6-month-old PLB2APP mice, and overall home-cage activity was reduced in both lines at 6 and 12 months, particularly during the dark phase. Spatial learning in the water maze was impaired in PLB2APP mice independent of PS1 expression and associated with reduced spatial navigation strategies. Memory retrieval was compromised in PLB2APP mice only. Our data demonstrate that low expression of APP is sufficient to drive histopathological and cognitive changes in mice without overexpression or excessive plaque deposition. AD-like phenotypes were altered by co-expression of PS1, including a shift from hippocampal to cortical Aß pathology, alongside reduced deficits in spatial learning.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/genética , Mutação/genética , Fenótipo , Adaptação Fisiológica/genética , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Locomoção/genética , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroglia/patologia , Neurônios/patologia , Presenilina-1/genética , Fatores de Tempo , Proteínas tau/genéticaRESUMO
Activation of energy expenditure in thermogenic fat is a promising strategy to improve metabolic health, yet the dynamic processes that evoke this response are poorly understood. Here we show that synthesis of the mitochondrial phospholipid cardiolipin is indispensable for stimulating and sustaining thermogenic fat function. Cardiolipin biosynthesis is robustly induced in brown and beige adipose upon cold exposure. Mimicking this response through overexpression of cardiolipin synthase (Crls1) enhances energy consumption in mouse and human adipocytes. Crls1 deficiency in thermogenic adipocytes diminishes inducible mitochondrial uncoupling and elicits a nuclear transcriptional response through endoplasmic reticulum stress-mediated retrograde communication. Cardiolipin depletion in brown and beige fat abolishes adipose thermogenesis and glucose uptake, which renders animals insulin resistant. We further identify a rare human CRLS1 variant associated with insulin resistance and show that adipose CRLS1 levels positively correlate with insulin sensitivity. Thus, adipose cardiolipin has a powerful impact on organismal energy homeostasis through thermogenic fat bioenergetics.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Bege/metabolismo , Tecido Adiposo Marrom/metabolismo , Cardiolipinas/biossíntese , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Células Cultivadas , Metabolismo Energético , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Termogênese , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
This editorial highlights an article by McKee and colleagues in the current issue of Journal of Neurochemistry, in which the authors report epigenetic changes linked to one-carbon metabolism in prefrontal cortex (PFC) of murine offspring from dams fed high-fat diet to mimic maternal obesity. The group found that high-fat diet feeding in utero increases weight gain in offspring and dynamically alters DNA methylation in the PFC of male but not female brains. These epigenetic marks were associated with a shift in brain one-carbon metabolism (folate and methionine) intermediates and were normalized by early-life methyl-donor supplementation in a sex-specific manner.
Assuntos
Metilação de DNA , Dieta Hiperlipídica , Animais , Encéfalo , Carbono , Feminino , Humanos , Masculino , Camundongos , Obesidade , GravidezRESUMO
OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations. METHODS: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes. RESULTS: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations. CONCLUSIONS: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.
Assuntos
Adipogenia/genética , Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Adipócitos/citologia , Adipócitos/metabolismo , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Termogênese , Proteína Desacopladora 1/metabolismoRESUMO
AIMS: ß-Secretase 1 (BACE1) is a key enzyme in Alzheimer's disease pathogenesis that catalyses the amyloidogenic cleavage of amyloid precursor protein (APP). Recently, global Bace1 deletion was shown to protect against diet-induced obesity and diabetes, suggesting that BACE1 is a potential regulator of glucose homeostasis. Here, we investigated whether increased neuronal BACE1 is sufficient to alter systemic glucose metabolism, using a neuron-specific human BACE1 knockin mouse model (PLB4). METHODS: Glucose homeostasis and adiposity were determined by glucose tolerance tests and EchoMRI, lipid species were measured by quantitative lipidomics, and biochemical and molecular alterations were assessed by western blotting, quantitative PCR and ELISAs. Glucose uptake in the brain and upper body was measured via (18)FDG-PET imaging. RESULTS: Physiological and molecular analyses demonstrated that centrally expressed human BACE1 induced systemic glucose intolerance in mice from 4 months of age onward, alongside a fatty liver phenotype and impaired hepatic glycogen storage. This diabetic phenotype was associated with hypothalamic pathology, i.e. deregulation of the melanocortin system, and advanced endoplasmic reticulum (ER) stress indicated by elevated central C/EBP homologous protein (CHOP) signalling and hyperphosphorylation of its regulator eukaryotic translation initiation factor 2α (eIF2α). In vivo (18)FDG-PET imaging further confirmed brain glucose hypometabolism in these mice; this corresponded with altered neuronal insulin-related signalling, enhanced protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4) levels, along with upregulation of the ribosomal protein and lipid translation machinery. Increased forebrain and plasma lipid accumulation (i.e. ceramides, triacylglycerols, phospholipids) was identified via lipidomics analysis. CONCLUSIONS/INTERPRETATION: Our data reveal that neuronal BACE1 is a key regulator of metabolic homeostasis and provide a potential mechanism for the high prevalence of metabolic disturbance in Alzheimer's disease.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Intolerância à Glucose/fisiopatologia , Homeostase , Humanos , Camundongos , Obesidade/genética , Obesidade/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismoRESUMO
Models of Tau pathology related to frontotemporal dementia (FTD) are essential to determine underlying neurodegenerative pathologies and resulting tauopathy relevant behavioural changes. However, existing models are often limited in their translational value due to Tau overexpression, and the frequent occurrence of motor deficits which prevent comprehensive behavioural assessments. In order to address these limitations, a forebrain-specific (CaMKIIα promoter), human mutated Tau (hTauP301L+R406W) knock-in mouse was generated out of the previously characterised PLB1Triple mouse, and named PLB2Tau. After confirmation of an additional hTau species (~60kDa) in forebrain samples, we identified age-dependent progressive Tau phosphorylation which coincided with the emergence of FTD relevant behavioural traits. In line with the non-cognitive symptomatology of FTD, PLB2Tau mice demonstrated early emerging (~6months) phenotypes of heightened anxiety in the elevated plus maze, depressive/apathetic behaviour in a sucrose preference test and generally reduced exploratory activity in the absence of motor impairments. Investigations of cognitive performance indicated prominent dysfunctions in semantic memory, as assessed by social transmission of food preference, and in behavioural flexibility during spatial reversal learning in a home cage corner-learning task. Spatial learning was only mildly affected and task-specific, with impairments at 12months of age in the corner learning but not in the water maze task. Electroencephalographic (EEG) investigations indicated a vigilance-stage specific loss of alpha power during wakefulness at both parietal and prefrontal recording sites, and site-specific EEG changes during non-rapid eye movement sleep (prefrontal) and rapid eye movement sleep (parietal). Further investigation of hippocampal electrophysiology conducted in slice preparations indicated a modest reduction in efficacy of synaptic transmission in the absence of altered synaptic plasticity. Together, our data demonstrate that the transgenic PLB2Tau mouse model presents with a striking behavioural and physiological face validity relevant for FTD, driven by the low level expression of mutant FTD hTau.
Assuntos
Comportamento Animal/fisiologia , Demência Frontotemporal/patologia , Potenciação de Longa Duração/genética , Memória/fisiologia , Proteínas tau/genética , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Animais , Modelos Animais de Doenças , Demência Frontotemporal/fisiopatologia , Técnicas de Introdução de Genes/métodos , Camundongos Transgênicos , Emaranhados Neurofibrilares/metabolismo , Plasticidade Neuronal/genética , Transmissão Sináptica/genética , Tauopatias/patologiaRESUMO
Key neuropathological hallmarks of Alzheimer's disease (AD) are elevated levels of amyloid ß-peptide (Aß) species generated via amyloid precursor protein (APP) endoproteolysis and cleavage by the rate-limiting ß-site enzyme 1 (BACE1). Because rodents do not develop amyloid pathologies, we here investigated whether AD-like endophenotypes can be created in mice by expression of human bace1. To avoid pitfalls of existing models, we introduced hbace1 via knock-in under the control of the CaMKII α promoter into the safe HPRT locus. We report amyloidogenic processing of murine APP in the hBACE1 mice (termed PLB4), resulting in the formation of toxic APP metabolites that accumulate intra- and extraneuronally in hippocampus and cortex. Pronounced accumulation of Aß*56 and Aß hexamers in the absence of plaque deposition was detected in brain tissue from symptomatic PLB4 mice. Heightened levels of inflammation (gliosis) also appeared in several AD-related brain regions (dentate gyrus, hippocampal area CA1, piriform and parietal cortices) at 6 and 12 months of age. Behaviorally, deficits in habituation to a novel environment and semantic-like memory (social transmission of food preference) were detected from 3 to 4 months of age. Impairments in spatial learning strategies in long-term reference (water maze) and working memory (Y-maze) tasks presented at 6 months, and were distinct from reductions in locomotor activity and anxiety. Overall, our data indicate for the first time that targeted, subtle forebrain-specific expression through single gene knock-in of hBACE1 is sufficient to generate AD-relevant cognitive impairments amid corresponding histopathologies, confirming human BACE as the key parameter in amyloid pathogenesis.