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Plasma lipid levels are modulated by systemic infection and inflammation; it is unknown whether these changes reflect inflammatory responses or caused directly by pathogen presence. We explored the hypothesis that anti-inflammatory intervention via interleukin 6 receptor (IL-6R) blockade would influence plasma lipid levels during severe infection and evaluated the association of plasma lipid changes with clinical outcomes. Sarilumab (monoclonal antibody blocking IL-6R) efficacy was previously assessed in patients with coronavirus disease 2019 (COVID-19) (NCT04315298). This analysis determined whether strong inflammatory reduction by sarilumab in patients with COVID-19 pneumonia of increasing severity (severe, critical, multisystem organ dysfunction) affected plasma lipid changes between day 1 and day 7 of study therapy. Baseline lipid levels reflected the presence of acute systemic infection, characterized by very low HDL-C, low LDL-C, and moderately elevated triglycerides (TGs). Disease severity was associated with progressively more abnormal lipid levels. At day 7, median lipid levels increased more in the sarilumab versus placebo group (HDL-C +10.3%, LDL-C +54.7%, TG +32% vs. HDL-C +1.7%, LDL-C +15.4%, TG +8.8%, respectively). No significant association between lipid changes and clinical outcomes was observed. In conclusion, severe-to-critical COVID-19 pneumonia causes profound HDL-C depression that is only modestly responsive to strong anti-IL-6R inflammatory intervention. Conversely, LDL-C depression is strongly responsive to IL-6R blockade, with LDL-C levels likely returning to the predisease set point. These results advance our understanding of the complex relationship between serum lipids and infection/inflammation and suggest that HDL-C depression during acute contagious disease is driven by infection and not IL-6-mediated inflammation.
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Anticorpos Monoclonais Humanizados , Tratamento Farmacológico da COVID-19 , COVID-19 , Lipídeos , Receptores de Interleucina-6 , Humanos , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/sangue , Anticorpos Monoclonais Humanizados/uso terapêutico , Masculino , Feminino , Pessoa de Meia-Idade , COVID-19/sangue , COVID-19/complicações , Lipídeos/sangue , Idoso , Hospitalização , Resultado do Tratamento , SARS-CoV-2 , Adulto , Índice de Gravidade de DoençaRESUMO
Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/- mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.
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Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fígado/metabolismo , Hepatócitos/metabolismo , Inflamação/metabolismo , Modelos Animais de Doenças , Fibrose , Camundongos Endogâmicos C57BLRESUMO
Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.
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Estudo de Associação Genômica Ampla , Oximas , Animais , Humanos , Camundongos , Cirrose HepáticaRESUMO
OBJECTIVES: Treatments that prevent sepsis complications are needed. Circulating lipid and protein assemblies-lipoproteins play critical roles in clearing pathogens from the bloodstream. We investigated whether early inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) may accelerate bloodstream clearance of immunogenic bacterial lipids and improve sepsis outcomes. DESIGN: Genetic and clinical epidemiology, and experimental models. SETTING: Human genetics cohorts, secondary analysis of a phase 3 randomized clinical trial enrolling patients with cardiovascular disease (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab [ODYSSEY OUTCOMES]; NCT01663402), and experimental murine models of sepsis. PATIENTS OR SUBJECTS: Nine human cohorts with sepsis (total n = 12,514) were assessed for an association between sepsis mortality and PCSK9 loss-of-function (LOF) variants. Incident or fatal sepsis rates were evaluated among 18,884 participants in a post hoc analysis of ODYSSEY OUTCOMES. C57BI/6J mice were used in Pseudomonas aeruginosa and Staphylococcus aureus bacteremia sepsis models, and in lipopolysaccharide-induced animal models. INTERVENTIONS: Observational human cohort studies used genetic PCSK9 LOF variants as instrumental variables. ODYSSEY OUTCOMES participants were randomized to alirocumab or placebo. Mice were administered alirocumab, a PCSK9 inhibitor, at 5 mg/kg or 25 mg/kg subcutaneously, or isotype-matched control, 48 hours prior to the induction of bacterial sepsis. Mice did not receive other treatments for sepsis. MEASUREMENTS AND MAIN RESULTS: Across human cohort studies, the effect estimate for 28-day mortality after sepsis diagnosis associated with genetic PCSK9 LOF was odds ratio = 0.86 (95% CI, 0.67-1.10; p = 0.24). A significant association was present in antibiotic-treated patients. In ODYSSEY OUTCOMES, sepsis frequency and mortality were infrequent and did not significantly differ by group, although both were numerically lower with alirocumab vs. placebo (relative risk of death from sepsis for alirocumab vs. placebo, 0.62; 95% CI, 0.32-1.20; p = 0.15). Mice treated with alirocumab had lower endotoxin levels and improved survival. CONCLUSIONS: PCSK9 inhibition may improve clinical outcomes in sepsis in preventive, pretreatment settings.
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Deficiency in the adipose-derived hormone leptin or leptin receptor signaling causes class 3 obesity in individuals with genetic loss-of-function mutations in leptin or its receptor LEPR and metabolic and liver disease in individuals with hypoleptinemia secondary to lipoatrophy such as in individuals with generalized lipodystrophy. Therapies that restore leptin-LEPR signaling may resolve these metabolic sequelae. We developed a fully human monoclonal antibody (mAb), REGN4461 (mibavademab), that activates the human LEPR in the absence or presence of leptin. In obese leptin knockout mice, REGN4461 normalized body weight, food intake, blood glucose, and insulin sensitivity. In a mouse model of generalized lipodystrophy, REGN4461 alleviated hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, and hepatic steatosis. In a phase 1, randomized, double-blind, placebo-controlled two-part study, REGN4461 was well tolerated with an acceptable safety profile. Treatment of individuals with overweight or obesity with REGN4461 decreased body weight over 12 weeks in those with low circulating leptin concentrations (<8 ng/ml) but had no effect on body weight in individuals with higher baseline leptin. Furthermore, compassionate-use treatment of a single patient with atypical partial lipodystrophy and a history of undetectable leptin concentrations associated with neutralizing antibodies to metreleptin was associated with noteable improvements in circulating triglycerides and hepatic steatosis. Collectively, these translational data unveil an agonist LEPR mAb that may provide clinical benefit in disorders associated with relatively low leptin concentrations.
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Resistência à Insulina , Lipodistrofia Generalizada Congênita , Animais , Camundongos , Humanos , Leptina/uso terapêutico , Ensaios de Uso Compassivo , Receptores para Leptina/metabolismo , Lipodistrofia Generalizada Congênita/tratamento farmacológico , Obesidade/tratamento farmacológico , Anticorpos/uso terapêutico , Peso CorporalRESUMO
Asynchronous skeletal muscle degeneration/regeneration is a hallmark feature of Duchenne muscular dystrophy (DMD); however, traditional -omics technologies that lack spatial context make it difficult to study the biological mechanisms of how asynchronous regeneration contributes to disease progression. Here, using the severely dystrophic D2-mdx mouse model, we generated a high-resolution cellular and molecular spatial atlas of dystrophic muscle by integrating spatial transcriptomics and single-cell RNAseq datasets. Unbiased clustering revealed nonuniform distribution of unique cell populations throughout D2-mdx muscle that were associated with multiple regenerative timepoints, demonstrating that this model faithfully recapitulates the asynchronous regeneration observed in human DMD muscle. By probing spatiotemporal gene expression signatures, we found that propagation of inflammatory and fibrotic signals from locally damaged areas contributes to widespread pathology and that querying expression signatures within discrete microenvironments can identify targetable pathways for DMD therapy. Overall, this spatial atlas of dystrophic muscle provides a valuable resource for studying DMD disease biology and therapeutic target discovery.
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Músculo Esquelético , Distrofia Muscular de Duchenne , Animais , Camundongos , Humanos , Músculo Esquelético/metabolismo , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/metabolismo , Progressão da Doença , Modelos Animais de DoençasRESUMO
Body fat distribution is a heritable risk factor for cardiovascular and metabolic disease. In humans, rare Inhibin beta E (INHBE, activin E) loss-of-function variants are associated with a lower waist-to-hip ratio and protection from type 2 diabetes. Hepatic fatty acid sensing promotes INHBE expression during fasting and in obese individuals, yet it is unclear how the hepatokine activin E governs body shape and energy metabolism. Here, we uncover activin E as a regulator of adipose energy storage. By suppressing ß-agonist-induced lipolysis, activin E promotes fat accumulation and adipocyte hypertrophy and contributes to adipose dysfunction in mice. Mechanistically, we demonstrate that activin E elicits its effect on adipose tissue through ACVR1C, activating SMAD2/3 signaling and suppressing PPARG target genes. Conversely, loss of activin E or ACVR1C in mice increases fat utilization, lowers adiposity, and drives PPARG-regulated gene signatures indicative of healthy adipose function. Our studies identify activin E-ACVR1C as a metabolic rheostat promoting liver-adipose cross talk to restrain excessive fat breakdown and preserve fat mass during prolonged fasting, a mechanism that is maladaptive in obese individuals.
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Diabetes Mellitus Tipo 2 , Lipólise , Humanos , Camundongos , Animais , Ativinas/metabolismo , Adiposidade/genética , Diabetes Mellitus Tipo 2/metabolismo , PPAR gama/metabolismo , Obesidade/metabolismo , Tecido Adiposo/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismoRESUMO
Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.
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Fígado Gorduroso , Interleucina-6 , Humanos , Camundongos , Animais , Interleucina-6/metabolismo , Receptor gp130 de Citocina/metabolismo , Gotículas Lipídicas/metabolismo , Hepatócitos/metabolismo , Glicoproteínas , LipídeosRESUMO
The interleukin-6 (IL-6) family cytokines signal through gp130 receptor homodimerization or heterodimerization with a second signaling receptor and play crucial roles in various cellular processes. We determined cryo-electron microscopy structures of five signaling complexes of this family, containing full receptor ectodomains bound to their respective ligands ciliary neurotrophic factor, cardiotrophin-like cytokine factor 1 (CLCF1), leukemia inhibitory factor, IL-27, and IL-6. Our structures collectively reveal similarities and differences in the assembly of these complexes. The acute bends at both signaling receptors in all complexes bring the membrane-proximal domains to a ~30 angstrom range but with distinct distances and orientations. We also reveal how CLCF1 engages its secretion chaperone cytokine receptor-like factor 1. Our data provide valuable insights for therapeutically targeting gp130-mediated signaling.
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Antígenos CD , Interleucina-6 , Receptor gp130 de Citocina/metabolismo , Interleucina-6/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Microscopia Crioeletrônica , Antígenos CD/metabolismo , Glicoproteínas de Membrana/metabolismo , Citocinas/metabolismoRESUMO
A highly effective 2-step system for site-specific antibody modification and conjugation of the monoclonal antibody Herceptin (commercially available under Trastuzumab) in a cysteine-independent manner was used to generate labelled antibodies for in vivo imaging. The first step contains redox-activated chemical tagging (ReACT) of thioethers via engineered methionine residues to introduce specific alkyne moieties, thereby offering a novel easy way to fundamentally change the process of antibody bioconjugation. The second step involves modification of the introduced alkyne via azide-alkyne cycloaddition 'click' conjugation. The versatility of this 2-step approach is demonstrated here by the selective incorporation of a fluorescent dye but can also be applied to a wide variety of different conjugation partners depending on the desired application in a facile manner. Methionine-modified antibodies were characterised in vitro, and the diagnostic potential of the most promising variant was further analysed in an in vivo xenograft animal model using a fluorescence imaging modality. This study demonstrates how methionine-mediated antibody conjugation offers an orthogonal and versatile route to the generation of tailored antibody conjugates with in vivo applicability.
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Metionina , Neoplasias , Animais , Humanos , Trastuzumab , Anticorpos Monoclonais/química , Racemetionina , Alcinos/química , Azidas/químicaRESUMO
BACKGROUND: Sarcopenia is defined as age-related low muscle mass and function, and can also describe the loss of muscle mass in certain medical conditions, such as sarcopenic obesity. Sarcopenic obesity describes loss of muscle and function in obese individuals; however, as sarcopenia is an age-related condition and obesity can occur in any age group, a more accurate term is obesity with low lean muscle mass (OLLMM). Given limited data on OLLMM (particularly in those aged < 65 years), the purpose of this study was to estimate the prevalence of OLLMM in adults aged ≥ 20 years in the USA. METHODS: Data from the National Health and Nutrition Examination Survey (NHANES) 2017-2018 and 1999-2006 were used. OLLMM was defined as an appendicular lean mass, adjusted for body mass index (BMI), cut-off point < 0.789 for males and < 0.512 for females, measured by dual-energy X-ray absorptiometry (DXA). DXA was only measured in individuals 20-59 years old in NHANES 2017-2018; we therefore utilized logistic regression models to predict OLLMM from NHANES 1999-2006 for those aged ≥ 60 years. The prevalence of OLLMM was estimated overall, and by sex, age, race/ethnicity, and clinical subgroup (high BMI, prediabetes, type 2 diabetes mellitus [T2DM], non-alcoholic fatty liver disease [NAFLD] with fibrosis, or post-bariatric surgery). Prevalence estimates were extrapolated to the USA population using NHANES sampling weights. RESULTS: We estimated that, during 2017-2018, 28.7 million or 15.9% of the USA population had OLLMM. The prevalence of OLLMM was greater in older individuals (8.1%, aged 20-59 years vs 28.3%, aged ≥ 60 years), highest (66.6%) in Mexican-American females aged ≥ 60 years, and lowest (2.6%) in non-Hispanic Black males aged 20-59 years. There was a higher prevalence of OLLMM in adults with prediabetes (19.7%), T2DM (34.5%), NAFLD with fibrosis (25.4%), or post-bariatric surgery (21.8%), compared with those without each condition. CONCLUSIONS: Overall, the burden of OLLMM in the USA is substantial, affecting almost 30 million adults. The prevalence of OLLMM increased with age, and among those with prediabetes, T2DM, NAFLD with fibrosis, or post-bariatric surgery. A unified definition of OLLMM will aid diagnosis and treatment strategies.
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Diabetes Mellitus Tipo 2 , Hepatopatia Gordurosa não Alcoólica , Estado Pré-Diabético , Sarcopenia , Masculino , Adulto , Feminino , Humanos , Idoso , Adulto Jovem , Pessoa de Meia-Idade , Sarcopenia/epidemiologia , Inquéritos Nutricionais , Hepatopatia Gordurosa não Alcoólica/complicações , Diabetes Mellitus Tipo 2/complicações , Prevalência , Estado Pré-Diabético/complicações , Obesidade/complicações , Obesidade/epidemiologia , Fibrose , Músculos , Composição CorporalRESUMO
Body fat distribution is a major, heritable risk factor for cardiometabolic disease, independent of overall adiposity. Using exome-sequencing in 618,375 individuals (including 160,058 non-Europeans) from the UK, Sweden and Mexico, we identify 16 genes associated with fat distribution at exome-wide significance. We show 6-fold larger effect for fat-distribution associated rare coding variants compared with fine-mapped common alleles, enrichment for genes expressed in adipose tissue and causal genes for partial lipodystrophies, and evidence of sex-dimorphism. We describe an association with favorable fat distribution (p = 1.8 × 10-09), favorable metabolic profile and protection from type 2 diabetes (~28% lower odds; p = 0.004) for heterozygous protein-truncating mutations in INHBE, which encodes a circulating growth factor of the activin family, highly and specifically expressed in hepatocytes. Our results suggest that inhibin ßE is a liver-expressed negative regulator of adipose storage whose blockade may be beneficial in fat distribution-associated metabolic disease.
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Diabetes Mellitus Tipo 2 , Subunidades beta de Inibinas/genética , Tecido Adiposo , Adiposidade/genética , Diabetes Mellitus Tipo 2/genética , Exoma/genética , Humanos , MutaçãoRESUMO
BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).
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Proteínas Reguladoras de Apoptose , Mutação em Linhagem Germinativa , Hepatopatias , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Predisposição Genética para Doença/genética , Predisposição Genética para Doença/prevenção & controle , Humanos , Fígado/metabolismo , Hepatopatias/genética , Hepatopatias/metabolismo , Hepatopatias/prevenção & controle , Transaminases/genética , Sequenciamento do ExomaRESUMO
Amino acids, the building blocks of proteins in the cells and tissues, are of fundamental importance for cell survival, maintenance, and proliferation. The liver plays a critical role in amino acid metabolism and detoxication of byproducts such as ammonia. Urea cycle disorders with hyperammonemia remain difficult to treat and eventually necessitate liver transplantation. In this study, ornithine transcarbamylase deficient (Otcspf-ash ) mouse model was used to test whether knockdown of a key glutamine metabolism enzyme glutaminase 2 (GLS2, gene name: Gls2) or glutamate dehydrogenase 1 (GLUD1, gene name: Glud1) could rescue the hyperammonemia and associated lethality induced by a high protein diet. We found that reduced hepatic expression of Gls2 but not Glud1 by AAV8-mediated delivery of a short hairpin RNA in Otcspf-ash mice diminished hyperammonemia and reduced lethality. Knockdown of Gls2 but not Glud1 in Otcspf-ash mice exhibited reduced body weight loss and increased plasma glutamine concentration. These data suggest that Gls2 hepatic knockdown could potentially help alleviate risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle.
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Glutaminase/metabolismo , Hiperamonemia , Doença da Deficiência de Ornitina Carbomoiltransferase , Distúrbios Congênitos do Ciclo da Ureia , Amônia , Animais , Modelos Animais de Doenças , Glutaminase/genética , Glutamina/metabolismo , Humanos , Hiperamonemia/metabolismo , Fígado/metabolismo , Camundongos , Ornitina Carbamoiltransferase/genética , Doença da Deficiência de Ornitina Carbomoiltransferase/metabolismo , Ureia/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/genética , Distúrbios Congênitos do Ciclo da Ureia/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is a global health-care problem with limited therapeutic options. To obtain a cellular resolution of pathogenesis, 82,168 single-cell transcriptomes (scRNA-seq) across different NAFLD stages were profiled, identifying hepatocytes and 12 other non-parenchymal cell (NPC) types. scRNA-seq revealed insights into the cellular and molecular mechanisms of the disease. We discovered a dual role for hepatic stellate cells in gene expression regulation and in the potential to trans-differentiate into myofibroblasts. We uncovered distinct expression profiles of Kupffer cells versus monocyte-derived macrophages during NAFLD progression. Kupffer cells showed stronger immune responses, while monocyte-derived macrophages demonstrated a capability for differentiation. Three chimeric NPCs were identified including endothelial-chimeric stellate cells, hepatocyte-chimeric endothelial cells, and endothelial-chimeric Kupffer cells. Our work identified unanticipated aspects of mouse with NAFLD at the single-cell level and advanced the understanding of cellular heterogeneity in NAFLD livers.
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NASH is a chronic liver disease that affects 3%-6% of individuals and requires urgent therapeutic developments. Isolating the key cell types in the liver is a necessary step towards understanding their function and roles in disease pathogenesis. However, traditional isolation methods through gradient centrifugation can only collect one or a few cell types simultaneously and pose technical difficulties when applied to NASH livers. Taking advantage of identified cell surface markers from liver single-cell RNAseq, here we established the combination of gradient centrifugation and antibody-based cell sorting techniques to isolate five key liver cell types (hepatocytes, endothelial cells, stellate cells, macrophages and other immune cells) from a single mouse liver. This method yielded high purity of each cell type from healthy and NASH livers. Our five-in-one protocol simultaneously isolates key liver cell types with high purity under normal and NASH conditions, enabling for systematic and accurate exploratory experiments such as RNA sequencing.
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Separação Celular , Fígado/citologia , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Biomarcadores , Separação Celular/métodos , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Imunofluorescência , Hepatócitos/metabolismo , Imunofenotipagem , Macrófagos/metabolismo , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/etiologiaRESUMO
Energy balance is controlled by interconnected brain regions in the hypothalamus, brainstem, cortex, and limbic system. Gene expression signatures of these regions can help elucidate the pathophysiology underlying obesity. RNA sequencing was conducted on P56 C57BL/6NTac male mice and E14.5 C57BL/6NTac embryo punch biopsies in 16 obesity-relevant brain regions. The expression of 190 known obesity-associated genes (monogenic, rare, and low-frequency coding variants; GWAS; syndromic) was analyzed in each anatomical region. Genes associated with these genetic categories of obesity had localized expression patterns across brain regions. Known monogenic obesity causal genes were highly enriched in the arcuate nucleus of the hypothalamus and developing hypothalamus. The obesity-associated genes clustered into distinct "modules" of similar expression profile, and these were distinct from expression modules formed by similar analysis with genes known to be associated with other disease phenotypes (type 1 and type 2 diabetes, autism, breast cancer) in the same energy balance-relevant brain regions.
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Encéfalo/metabolismo , Metabolismo Energético/genética , Obesidade/metabolismo , Animais , Embrião de Mamíferos , Regulação da Expressão Gênica , Predisposição Genética para Doença , Masculino , Camundongos , Obesidade/genética , RNA-SeqRESUMO
PROBLEM: In 2009 the National Confidential Enquiry into Patient Outcome and Death suggested only 50% of patients with acute kidney injury (AKI) receive good standards of care. In response National Health Service (NHS) England mandated the use of electronic AKI alerts within secondary care. However, we recognised AKI is not just a secondary care problem, where primary care has a crucial role to play in prevention, early detection and management as well as post-AKI care. METHODS: AKI alerts were implemented in primary and secondary care services for a population of 480 000. Comparisons were made in AKI incidence, peak creatinine following AKI and renal recovery in the years before and after using Byar's approximation (95% CI). INTERVENTION: A complex quality improvement initiative was implemented based on the design and integration of an AKI alerting system within laboratory information management systems for primary and secondary care, with an affixed URL for clinicians to access a care bundle of AKI guidelines on safe prescribing, patient advice and early contact with nephrology. RESULTS: The intervention was associated with an 8% increase in creatinine testing (n=32 563). Hospital acquired AKI detection increased by 6%, while community acquired AKI detection increased by 3% and AKI stage 3 detected in primary care fell by 14%. The intervention overall had no effect on AKI severity but did improve follow-up testing and renal recovery. Importantly hospital AKI 3 recoveries improved by 22%. In a small number of AKI cases, the algorithm did not produce an alert resulting in a reduction in follow-up testing compared with preintervention levels. CONCLUSION: The introduction of AKI alerts in primary and secondary care, in conjunction with access to an AKI care bundle, was associated with higher rates of repeat blood sampling, AKI detection and renal recovery. Validating accuracy of alerts is required to avoid patient harm.
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Injúria Renal Aguda , Atenção Secundária à Saúde , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/epidemiologia , Injúria Renal Aguda/terapia , Diagnóstico Precoce , Eletrônica , Humanos , Medicina EstatalRESUMO
Angiopoietin-like protein (ANGPTL)3 regulates plasma lipids by inhibiting LPL and endothelial lipase (EL). ANGPTL3 inactivation lowers LDL-C independently of the classical LDLR-mediated pathway and represents a promising therapeutic approach for individuals with homozygous familial hypercholesterolemia due to LDLR mutations. Yet, how ANGPTL3 regulates LDL-C levels is unknown. Here, we demonstrate in hyperlipidemic humans and mice that ANGPTL3 controls VLDL catabolism upstream of LDL. Using kinetic, lipidomic, and biophysical studies, we show that ANGPTL3 inhibition reduces VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation. This suggests that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production. Mechanistically, we discovered that EL is a key mediator of ANGPTL3's novel pathway. Our experiments revealed that, although dispensable in the presence of LDLR, EL-mediated processing of VLDL becomes critical for LDLR-independent particle clearance. In the absence of EL and LDLR, ANGPTL3 inhibition perturbed VLDL catabolism, promoted accumulation of atypical remnants, and failed to reduce LDL-C. Taken together, we uncover ANGPTL3 at the helm of a novel EL-dependent pathway that lowers LDL-C in the absence of LDLR.
Assuntos
Proteínas Semelhantes a Angiopoietina/metabolismo , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Proteína 3 Semelhante a Angiopoietina , Animais , Endotélio/metabolismo , Humanos , Camundongos , Receptores de LDL/metabolismoRESUMO
OBJECTIVE: Reelin (RELN) is a large glycoprotein involved in synapse maturation and neuronal organization throughout development. Deficits in RELN signaling contribute to multiple psychological disorders, such as autism spectrum disorder, schizophrenia, and bipolar disorder. Nutritional stress alters RELN expression in brain regions associated with these disorders; however, the involvement of RELN in the neural circuits involved in energy metabolism is unknown. The RELN receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR) are involved in lipid metabolism and expressed in the hypothalamus. Here we explored the involvement of RELN in hypothalamic signaling and the impact of diet-induced obesity (DIO) on this system. METHODS: Adult male mice were fed a chow diet or maintained on a high-fat diet (HFD) for 12-16 weeks. HFD-fed DIO mice exhibited decreased ApoER2 and VLDLR expression and increased RELN protein in the hypothalamus. Electrophysiology was used to determine the mechanism by which the central fragment of RELN (CF-RELN) acts on arcuate nucleus (ARH) satiety-promoting proopiomelanocortin (POMC) neurons and the impact of DIO on this circuitry. RESULTS: CF-RELN exhibited heterogeneous presynaptic actions on inhibitory inputs onto ARH-POMC-EGFP neurons and consistent postsynaptic actions. Additionally, central administration of CF-RELN caused a significant increase in ARH c-Fos expression and an acute decrease in food intake and body weight. CONCLUSIONS: We conclude that RELN signaling is modulated by diet, that RELN is involved in synaptic signaling onto ARH-POMC neurons, and that altering central CF-RELN levels can impact food intake and body weight.