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SUMMARYCell culture is generally considered to be hyperoxic. However, the importance of cellular oxygen consumption is often underappreciated, with rates of oxygen consumption often sufficient to cause hypoxia at cell monolayers. We initially focused on cultured adipocytes as a terminally differentiated cell-type with substantial oxygen consumption rates to support diverse cellular functions. Under standard conditions, cultured adipocytes are hypoxic and highly glycolytic. Increasing oxygen diverted glucose flux toward mitochondria and resulted in thousands of gene expression changes that pointed toward alleviated physiological transcriptional responses to hypoxia. Phenotypically, providing more oxygen increased adipokine secretion and rendered adipocytes more sensitive to insulin and lipolytic stimuli. The functional benefits of increasing pericellular oxygen were transferable to other cellular systems including hPSC-derived hepatocytes and cardiac organoids. Our findings suggest that oxygen is limiting in many terminally-differentiated cell culture systems, and that controlling oxygen availability can improve the quality and translatability of cell models.

Summary 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 β-adrenergic stimuli. This new protocol is scalable, enabling the study of human BAs at early stages of development., Graphical Abstract, Highlights • This is a novel, robust and scalable protocol to differentiate hPSCs into BAs • The protocol recapitulates the different stages of human BAT development • hPSC-derived BAs show transcriptomic and functional features of bona fide human BAs • It represents a useful tool for temporal analysis of human BAs differentiation, Obese patients possess low amounts of BAT, so an efficient way to expand its mass and activation is necessary to treat obesity and complications. There is limited knowledge about human BAT physiology and accessing it is challenging. To overcome this, Carobbio and colleagues developed a new protocol to differentiate human PSCs into functional brown adipocytes, recapitulating human BAT developmental path.

Increasing brown adipose tissue (BAT) mass and activation has been proposed as a potential therapeutic strategy to treat obesity and associated cardiometabolic complications. Given that obese and diabetic patients possess low amounts of BAT, an efficient way to expand their BAT mass would be necessary if BAT is to be useful. Currently, there is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Moreover, to have access to human BAT is challenging, given its low volume and being anatomically dispersed. These constrain makes detailed mechanistic studies related to BAT development and function in humans virtually impossible. To overcome these limitations, we have developed a human-relevant new protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs). Unique to this protocol is that it is chemically-defined to recapitulate a physiological step-by-step developmental path of BAT that captures transient paraxial mesoderm and BAT progenitor states, on its way to reaching the adipocyte stage finally. These hPSC-derived BAs express brown adipocyte and thermogenic markers, are insulin sensitive, and respond to β-adrenergic stimuli. This new protocol is a scalable tool to study human BAs during development.

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., Highlights • Human NASH biopsies’ transcriptomics analysis features metabolic pathway rewiring. • SCAP/SREBP/INSIG1 modulation promotes lipid/cholesterol synthesis/remodelling in NASH. • Loss of Insig1 promotes lipid remodelling, preventing hepatic lipotoxicity in NASH. • Loss of Insig1 improves liver damage and wound healing and restrains NASH progression.

[Background and Aims] Vibration-controlled transient elastography (VCTE), point shear wave elastography (pSWE), 2-dimensional shear wave elastography (2DSWE), magnetic resonance elastography (MRE), and magnetic resonance imaging (MRI) have been proposed as non-invasive tests for patients with non-alcoholic fatty liver disease (NAFLD). This study evaluated their diagnostic accuracy for liver fibrosis and non-alcoholic steatohepatitis (NASH)., [Methods] PubMED/MEDLINE, EMBASE and the Cochrane Library were searched for studies examining the diagnostic accuracy of these index tests, against histology as the reference standard, in adult patients with NAFLD. Two authors independently screened and assessed methodological quality of studies and extracted data. Summary estimates of sensitivity, specificity and area under the curve (sAUC) were calculated for fibrosis stages and NASH, using a random effects bivariate logit-normal model., [Results] We included 82 studies (14,609 patients). Meta-analysis for diagnosing fibrosis stages was possible in 53 VCTE, 11 MRE, 12 pSWE and 4 2DSWE studies, and for diagnosing NASH in 4 MRE studies. sAUC for diagnosis of significant fibrosis were: 0.83 for VCTE, 0.91 for MRE, 0.86 for pSWE and 0.75 for 2DSWE. sAUC for diagnosis of advanced fibrosis were: 0.85 for VCTE, 0.92 for MRE, 0.89 for pSWE and 0.72 for 2DSWE. sAUC for diagnosis of cirrhosis were: 0.89 for VCTE, 0.90 for MRE, 0.90 for pSWE and 0.88 for 2DSWE. MRE had sAUC of 0.83 for diagnosis of NASH. Three (4%) studies reported intention-to-diagnose analyses and 15 (18%) studies reported diagnostic accuracy against pre-specified cut-offs., [Conclusions] When elastography index tests are acquired successfully, they have acceptable diagnostic accuracy for advanced fibrosis and cirrhosis. The potential clinical impact of these index tests cannot be assessed fully as intention-to-diagnose analyses and validation of pre-specified thresholds are lacking., [Lay summary] Non-invasive tests that measure liver stiffness or use magnetic resonance imaging (MRI) have been suggested as alternatives to liver biopsy for assessing the severity of liver scarring (fibrosis) and fatty inflammation (steatohepatitis) in patients with non-alcoholic fatty liver disease (NAFLD). In this study, we summarise the results of previously published studies on how accurately these non-invasive tests can diagnose liver fibrosis and inflammation, using liver biopsy as the reference. We found that some techniques that measure liver stiffness had a good performance for the diagnosis of severe liver scarring., This work has been undertaken as part of the LITMUS (Liver Investigation: Testing Marker Utility in Steatohepatitis) project. The LITMUS project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No. 777377. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and Europen Federation of Pharmaceutical Industries and Associations (efpia.eu).