RESUMO
The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.
Assuntos
Adipócitos , Diferenciação Celular , Oxigênio , Oxigênio/metabolismo , Adipócitos/metabolismo , Adipócitos/citologia , Humanos , Técnicas de Cultura de Células/métodos , Animais , Glicólise , Hepatócitos/metabolismo , Hipóxia Celular , Mitocôndrias/metabolismo , Camundongos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células Cultivadas , Glucose/metabolismo , Macrófagos/metabolismoRESUMO
The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) is increasing, and translational animal models are needed to develop novel treatments for this disease. The physiology and metabolism of pigs have a relatively high resemblance to humans, and the present study aimed to characterise choline-deficient, and high-fat diet (CDAHFD) fed Göttingen Minipigs as a novel animal model of MASLD/MASH. Göttingen Minipigs were fed CDAHFD for up to 5 months, and the phenotype was investigated by analysis of plasma parameters and repeated collection of liver biopsies. Furthermore, changes in hepatic gene expression during the experiment were explored by RNA sequencing. For a subset of the minipigs, the diet was changed from CDAHFD back to chow to investigate if the liver pathology was reversible. Göttingen Minipigs on CDAHFD gained bodyweight, and plasma levels of cholesterol, AST, ALT, ALP and GGT were increased. CDAHFD-fed minipigs developed hepatic steatosis, inflammation, and fibrosis, which in 5 of 16 animals progressed to cirrhosis. During an 11-week chow reversal period, steatosis regressed while fibrosis persisted. Regarding inflammation, the findings were less clear, depending on the type of readout. MASH Human Proximity Scoring (combined evaluation of transcriptional, phenotypic and histopathological parameters) showed that CDAHFD-fed Göttingen Minipigs resemble human MASLD/MASH better than most rodent models. In conclusion, CDAHFD-fed minipigs develop a MASH-like phenotype which in several aspects resemble the changes observed in human patients with MASLD/MASH. Furthermore, repeated collection of liver biopsies allow detailed characterisation of histopathological changes over time in individual animals.
RESUMO
Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.
Assuntos
Modelos Animais de Doenças , Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Masculino , Fígado/metabolismo , Fígado/patologia , Doenças Metabólicas/metabolismo , Doenças Metabólicas/etiologia , Dieta Ocidental/efeitos adversos , Estudos Retrospectivos , Cirrose Hepática/metabolismo , Cirrose Hepática/etiologiaRESUMO
OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a silent pandemic associated with obesity and the metabolic syndrome, and also increases cardiovascular- and cirrhosis-related morbidity and mortality. A complete understanding of adaptive compensatory metabolic programmes that modulate non-alcoholic steatohepatitis (NASH) progression is lacking. METHODS AND RESULTS: Transcriptomic analysis of liver biopsies in patients with NASH revealed that NASH progression is associated with rewiring of metabolic pathways, including upregulation of de novo lipid/cholesterol synthesis and fatty acid remodelling. The modulation of these metabolic programmes was achieved by activating sterol regulatory element-binding protein (SREBP) transcriptional networks; however, it is still debated whether, in the context of NASH, activation of SREBPs acts as a pathogenic driver of lipotoxicity, or rather promotes the biosynthesis of protective lipids that buffer excessive lipid accumulation, preventing inflammation and fibrosis. To elucidate the pathophysiological role of SCAP/SREBP in NASH and wound-healing response, we used an Insig1 deficient (with hyper-efficient SREBPs) murine model challenged with a NASH-inducing diet. Despite enhanced lipid and cholesterol biosynthesis, Insig1 KO mice had similar systemic metabolism and insulin sensitivity to Het/WT littermates. Moreover, activating SREBPs resulted in remodelling the lipidome, decreased hepatocellular damage, and improved wound-healing responses. CONCLUSIONS: Our study provides actionable knowledge about the pathways and mechanisms involved in NAFLD pathogenesis, which may prove useful for developing new therapeutic strategies. Our results also suggest that the SCAP/SREBP/INSIG1 trio governs transcriptional programmes aimed at protecting the liver from lipotoxic insults in NASH.
Assuntos
Colesterol/biossíntese , Progressão da Doença , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Biomarcadores/metabolismo , Dieta Ocidental , Feminino , Humanos , Resistência à Insulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/genética , TranscriptomaRESUMO
Increasing brown adipose tissue (BAT) mass and activation is a therapeutic strategy to treat obesity and complications. Obese and diabetic patients possess low amounts of BAT, so an efficient way to expand their mass is necessary. There is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Accessing human BAT is challenging, given its low volume and anatomical dispersion. These constraints make detailed BAT-related developmental and functional mechanistic studies in humans virtually impossible. We have developed and characterized functionally and molecularly a new chemically defined protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs) that overcomes current limitations. This protocol recapitulates step by step the physiological developmental path of human BAT. The BAs obtained express BA and thermogenic markers, are insulin sensitive, and responsive to ß-adrenergic stimuli. This new protocol is scalable, enabling the study of human BAs at early stages of development.