The altered serum lipidome and its diagnostic potential for Non-Alcoholic Fatty Liver (NAFL)-associated hepatocellular carcinoma

Background Non-alcoholic fatty liver disease (NAFLD) is affecting more people globally. Indeed, NAFLD is a spectrum of metabolic dysfunctions that can progress to hepatocellular carcinoma (NAFLD-HCC). This development can occur in a non-cirrhotic liver and thus, often lack clinical surveillance. The aim of this study was to develop non-invasive surveillance method for NAFLD-HCC. Methods Using comprehensive ultra-high-performance liquid chromatography mass-spectrometry, we investigated 1,295 metabolites in serum from 249 patients. Area under the receiver operating characteristic curve was calculated for all detected metabolites and used to establish their diagnostic potential. Logistic regression analysis was used to establish the diagnostic score. Findings We show that NAFLD-HCC is characterised by a complete rearrangement of the serum lipidome, which distinguishes NAFLD-HCC from non-cancerous individuals and other HCC patients. We used machine learning to build a diagnostic model for NAFLD-HCC. We quantified predictive metabolites and developed the NAFLD-HCC Diagnostic Score (NHDS), presenting superior diagnostic potential compared to alpha-fetoprotein (AFP). Patients’ metabolic landscapes show a progressive depletion in unsaturated fatty acids and acylcarnitines during transformation. Upregulation of fatty acid transporters in NAFLD-HCC tumours contribute to fatty acid depletion in the serum. Interpretation NAFLD-HCC patients can be efficiently distinguished by serum metabolic alterations from the healthy population and from HCC patients related to other aetiologies (alcohol and viral hepatitis). Our model can be used for non-invasive surveillance of individuals with metabolic syndrome(s), allowing for early detection of NAFLD-HCC. Therefore, serum metabolomics may provide valuable insight to monitor patients at risk, including morbidly obese, diabetics, and NAFLD patients. Funding The funding sources for this study had no role in study design, data collection, data analyses, interpretation or writing of the report as it is presented herein

SREBP-2-driven transcriptional activation of human SND1 oncogene

Upregulation of Staphylococcal nuclease and tudor domain containing 1 (SND1) is linked to cancer progression and metastatic spread. Increasing evidence indicates that SND1 plays a role in lipid homeostasis. Recently, it has been shown that SND1-overexpressing hepatocellular carcinoma cells present an increased de novo cholesterol synthesis and cholesteryl ester accumulation. Here we reveal that SND1 oncogene is a novel target for SREBPs. Exposure of HepG2 cells to the cholesterol-lowering drug simvastatin or to a lipoprotein-deficient medium triggers SREBP-2 activation and increases SND1 promoter activity and transcript levels. Similar increases in SND1 promoter activity and mRNA are mimicked by overexpressing nuclear SREBP-2 through expression vector transfection. Conversely, SREBP-2 suppression with specific siRNA or the addition of cholesterol/25-hydroxycholesterol to cell culture medium reduces transcriptional activity of SND1 promoter and SND1 mRNA abundance. Chromatin immunoprecipitation assays and site-directed mutagenesis show that SREBP-2 binds to the SND1 proximal promoter in a region containing one SRE and one E-box motif which are critical for maximal transcriptional activity under basal conditions. SREBP-1, in contrast, binds exclusively to the SRE element. Remarkably, while ectopic expression of SREBP-1c or -1a reduces SND1 promoter activity, knocking-down of SREBP-1 enhances SND1 mRNA and protein levels but failed to affect SND1 promoter activity. These findings reveal that SREBP-2 and SREBP-1 bind to specific sites in SND1 promoter and regulate SND1 transcription in opposite ways; it is induced by SREBP-2 activating conditions and repressed by SREBP-1 overexpression. We anticipate the contribution of a SREBPs/SND1 pathway to lipid metabolism reprogramming of human hepatoma cells.This study was supported by Gobierno Vasco grants [IT971-16 and KK2016-00036] and UPV/EHU [UFI11/20 CLUMBER]. S.A., E.A. and H.N.I. were recipients of grants from UPV/EHU and Gobierno Vasco

RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease

[EN]Objective Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD. Design RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3(-/-)) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks. Results RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3(-/-) mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3(-/-) mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor. (PPAR.) was increased in Ripk3(-/-) mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPAR. in controlling fat deposition and fibrosis. Conclusion Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression.Main funding is provided by FEDER funds through the COMPETE programme and by national funds through Fundacao para a Ciencia e a Tecnologia to CMPR (grants SAICTPAC/0019/2015-LISBOA-01-0145--FEDER-016405 and PTDC/MED-FAR/29097/2017 -LISBOA-01-0145-FEDER-029097). Additional funding comes from research grant APEF (Portuguese Association for the Study of Liver)/BAYER 2020 to MBA. JG is funded by the Fondation pour la Recherche Medicale (ARF20170938613), the Institute of Cardiometabolism and Nutrition (PAP17NECJG), the Societe Francophone du Diabete (R19114DD) and the Mairie de Paris (Emergences -R18139DD). MBA, PMR, MMP and ALS were investigators or students funded by Fundacao para a Ciencia e a Tecnologia

The altered serum lipidome and its diagnostic potential for Non-Alcoholic Fatty Liver (NAFL)-associated hepatocellular carcinoma

[Background] Non-alcoholic fatty liver disease (NAFLD) is affecting more people globally. Indeed, NAFLD is a spectrum of metabolic dysfunctions that can progress to hepatocellular carcinoma (NAFLD-HCC). This development can occur in a non-cirrhotic liver and thus, often lack clinical surveillance. The aim of this study was to develop non-invasive surveillance method for NAFLD-HCC.[Methods] Using comprehensive ultra-high-performance liquid chromatography mass-spectrometry, we investigated 1,295 metabolites in serum from 249 patients. Area under the receiver operating characteristic curve was calculated for all detected metabolites and used to establish their diagnostic potential. Logistic regression analysis was used to establish the diagnostic score.[Findings] We show that NAFLD-HCC is characterised by a complete rearrangement of the serum lipidome, which distinguishes NAFLD-HCC from non-cancerous individuals and other HCC patients. We used machine learning to build a diagnostic model for NAFLD-HCC. We quantified predictive metabolites and developed the NAFLD-HCC Diagnostic Score (NHDS), presenting superior diagnostic potential compared to alpha-fetoprotein (AFP). Patients’ metabolic landscapes show a progressive depletion in unsaturated fatty acids and acylcarnitines during transformation. Upregulation of fatty acid transporters in NAFLD-HCC tumours contribute to fatty acid depletion in the serum.[Interpretation] NAFLD-HCC patients can be efficiently distinguished by serum metabolic alterations from the healthy population and from HCC patients related to other aetiologies (alcohol and viral hepatitis). Our model can be used for non-invasive surveillance of individuals with metabolic syndrome(s), allowing for early detection of NAFLD-HCC. Therefore, serum metabolomics may provide valuable insight to monitor patients at risk, including morbidly obese, diabetics, and NAFLD patients.We thank all funding sources: The laboratory of JBA is supported by the Novo Nordisk Foundation (14040, 0058419), Danish Cancer Society (R98-A6446, R167-A10784, R278-A16638), and the Danish Medical Research Council (4183-00118A, 1030-00070B). Data used for validation in this study provided by JMB was funded by the Spanish Ministry of Economy and Competitiveness and ’Instituto de Salud Carlos III’ grants (PI18/01075, Miguel Servet Programme CON14/00129 and CPII19/00008) co-financed by ’Fondo Europeo de Desarrollo Regional’ (FEDER); CIBERehd, Spain; IKERBASQUE, Basque foundation for Science, Spain; BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD); Department of Health of the Basque Country (2017111010), Euskadi RIS3 (2019222054, 2020333010); Department of Industry of the Basque Country (Elkartek: KK-2020/00008), AECC Scientific Foundation and European Commission Horizon 2020 program (ESCALON project no.: 825510). Similarly, MAJ was funded by grants from the Fondo Nacional De Ciencia y Tecnología de Chile (FONDECYT #1191145 to M.A.) and the Comisión Nacional de Investigación, Ciencia y Tecnología (CONICYT, AFB170005, CARE Chile UC).Peer reviewe

Adiponectin, leptin, and IGF-1 are useful diagnostic and stratification biomarkers of NAFLD

[EN] Background: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease where liver biopsy remains the gold standard for diagnosis. Here we aimed to evaluate the role of circulating adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) levels as non-invasive NAFLD biomarkers and assess their correlation with the metabolome. Materials and Methods: Leptin, adiponectin, and IGF-1 serum levels were measured by ELISA in two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls (discovery: 38 NAFLD, 13 controls; validation: 194 NAFLD, 31 controls) and correlated with clinical data, histology, genetic parameters, and serum metabolomics. Results: In both cohorts, leptin increased in NAFLD vs. controls (discovery: AUROC 0.88; validation: AUROC 0.83; p < 0.0001). The leptin levels were similar between obese and non-obese healthy controls, suggesting that obesity is not a confounding factor. In the discovery cohort, adiponectin was lower in non-alcoholic steatohepatitis (NASH) vs. non-alcoholic fatty liver (AUROC 0.87; p < 0.0001). For the validation cohort, significance was attained for homozygous for PNPLA3 allele c.444C (AUROC 0.63; p < 0.05). Combining adiponectin with specific serum lipids improved the assay performance (AUROC 0.80; p < 0.0001). For the validation cohort, IGF-1 was lower with advanced fibrosis (AUROC 0.67, p<0.05), but combination with international normalized ratio (INR) and ferritin increased the assay performance (AUROC 0.81; p < 0.01). Conclusion: Serum leptin discriminates NAFLD, and adiponectin combined with specific lipids stratifies NASH. IGF-1, INR, and ferritin distinguish advanced fibrosis.CR was funded by FEDER through the COMPETE program and by national funds through Fundação para a Ciência e a Tecnologia (PTDC/MED-FAR/29097/2017—LISBOA-01- 0145-FEDER-029097) and by European Horizon 2020 (H2020- MSCA-RISE-2016-734719). This work was also supported by Fundação para a Ciência e Tecnologia (PD/BD/135467/2017) and Portuguese Association for the Study of Liver/MSD 2017. JB was funded by Spanish Carlos III Health Institute (ISCIII) (PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129 and CPII19/00008), co-financed by Fondo Europeo de Desarrollo Regional (FEDER), Instituto de Salud Carlos III (CIBERehd, Spain), La Caixa Scientific Foundation (HR17-00601), Fundación Científica de la Asociación Española Contra el Cáncer, and European Horizon 2020 (ESCALON project: H2020-SC1-BHC-2018-2020)

Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles

Background and Aims We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors. Approach and Results We analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL-apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6, and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A. Conclusions Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification.National Institutes of Health (R01DK123763, R01DK119437, HL151328, P30DK52574, P30DK56341, and UL1TR002345); Ministerio de Economía y Competitividad de España (SAF2017-88041-R); Ministerio de Economía y Competitividad de España for the Severo Ochoa Excellence Accreditation (SEV-2016-0644); CIBERehd (Biomedical Research Center in Hepatic and Digestive Diseases) and Netherlands Organization for Applied Scientific Research Program (PMC13 and PMC15); Spanish Carlos III Health Institute (PI15/01132 and PI18/01075); Miguel Servet Program (CON14/00129 and CPII19/00008); Fondo Europeo de Desarrollo Regional, CIBERehd, Department of Industry of the Basque Country (Elkartek: KK-2020/00008); La Caixa Scientific Foundation (HR17-00601); Liver Investigation: Testing Marker Utility in Steatohepatitis consortium funded by the Innovative Medicines Initiative Program of the European Union (777377), which receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA; Newcastle NIHR Biomedical Research Center; Czech Ministry of Health (RVO-VFN64165/2020); Fondo Nacional De Ciencia y Tecnología de Chile (1191145); and the Comisión Nacional de Investigación, Ciencia y Tecnología (AFB170005, CARE Chile UC); Agencia Nacional de Investigación y Desarrollo (ANID ACE 210009); European Union's Horizon 2020 Research and Innovation Program (825510)

Adiponectin, Leptin, and IGF-1 Are Useful Diagnostic and Stratification Biomarkers of NAFLD

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease where liver biopsy remains the gold standard for diagnosis. Here we aimed to evaluate the role of circulating adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) levels as non-invasive NAFLD biomarkers and assess their correlation with the metabolome.Materials and Methods: Leptin, adiponectin, and IGF-1 serum levels were measured by ELISA in two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls (discovery: 38 NAFLD, 13 controls; validation: 194 NAFLD, 31 controls) and correlated with clinical data, histology, genetic parameters, and serum metabolomics.Results: In both cohorts, leptin increased in NAFLD vs. controls (discovery: AUROC 0.88; validation: AUROC 0.83; p &lt; 0.0001). The leptin levels were similar between obese and non-obese healthy controls, suggesting that obesity is not a confounding factor. In the discovery cohort, adiponectin was lower in non-alcoholic steatohepatitis (NASH) vs. non-alcoholic fatty liver (AUROC 0.87; p &lt; 0.0001). For the validation cohort, significance was attained for homozygous for PNPLA3 allele c.444C (AUROC 0.63; p &lt; 0.05). Combining adiponectin with specific serum lipids improved the assay performance (AUROC 0.80; p &lt; 0.0001). For the validation cohort, IGF-1 was lower with advanced fibrosis (AUROC 0.67, p &lt; 0.05), but combination with international normalized ratio (INR) and ferritin increased the assay performance (AUROC 0.81; p &lt; 0.01).Conclusion: Serum leptin discriminates NAFLD, and adiponectin combined with specific lipids stratifies NASH. IGF-1, INR, and ferritin distinguish advanced fibrosis

Metabolomics and lipidomics in NAFLD: biomarkers and non-invasive diagnostic tests.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide and is often associated with aspects of metabolic syndrome. Despite its prevalence and the importance of early diagnosis, there is a lack of robustly validated biomarkers for diagnosis, prognosis and monitoring of disease progression in response to a given treatment. In this Review, we provide an overview of the contribution of metabolomics and lipidomics in clinical studies to identify biomarkers associated with NAFLD and nonalcoholic steatohepatitis (NASH). In addition, we highlight the key metabolic pathways in NAFLD and NASH that have been identified by metabolomics and lipidomics approaches and could potentially be used as biomarkers for non-invasive diagnostic tests. Overall, the studies demonstrated alterations in amino acid metabolism and several aspects of lipid metabolism including circulating fatty acids, triglycerides, phospholipids and bile acids. Although we report several studies that identified potential biomarkers, few have been validated

RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease

International audienceObjective Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD.Design RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3 −/−) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks.Results RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3 −/− mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3 −/− mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor γ (PPARγ) was increased in Ripk3 −/− mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPARγ in controlling fat deposition and fibrosis.Conclusion Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression