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[Background]: The gut microbiome and iron status are known to play a role in the pathophysiology of non-alcoholic fatty liver disease (NAFLD), although their complex interaction remains unclear., [Results]: Here, we applied an integrative systems medicine approach (faecal metagenomics, plasma and urine metabolomics, hepatic transcriptomics) in 2 well-characterised human cohorts of subjects with obesity (discovery n = 49 and validation n = 628) and an independent cohort formed by both individuals with and without obesity (n = 130), combined with in vitro and animal models. Serum ferritin levels, as a markers of liver iron stores, were positively associated with liver fat accumulation in parallel with lower gut microbial gene richness, composition and functionality. Specifically, ferritin had strong negative associations with the Pasteurellaceae, Leuconostocaceae and Micrococcaea families. It also had consistent negative associations with several Veillonella, Bifidobacterium and Lactobacillus species, but positive associations with Bacteroides and Prevotella spp. Notably, the ferritin-associated bacterial families had a strong correlation with iron-related liver genes. In addition, several bacterial functions related to iron metabolism (transport, chelation, heme and siderophore biosynthesis) and NAFLD (fatty acid and glutathione biosynthesis) were also associated with the host serum ferritin levels. This iron-related microbiome signature was linked to a transcriptomic and metabolomic signature associated to the degree of liver fat accumulation through hepatic glucose metabolism. In particular, we found a consistent association among serum ferritin, Pasteurellaceae and Micrococcacea families, bacterial functions involved in histidine transport, the host circulating histidine levels and the liver expression of GYS2 and SEC24B. Serum ferritin was also related to bacterial glycine transporters, the host glycine serum levels and the liver expression of glycine transporters. The transcriptomic findings were replicated in human primary hepatocytes, where iron supplementation also led to triglycerides accumulation and induced the expression of lipid and iron metabolism genes in synergy with palmitic acid. We further explored the direct impact of the microbiome on iron metabolism and liver fact accumulation through transplantation of faecal microbiota into recipient’s mice. In line with the results in humans, transplantation from ‘high ferritin donors’ resulted in alterations in several genes related to iron metabolism and fatty acid accumulation in recipient’s mice., [Conclusions]: Altogether, a significant interplay among the gut microbiome, iron status and liver fat accumulation is revealed, with potential significance for target therapies., This work was supported by EU-FP7 FLORINASH (Health-F2-2009-241913) to R.B., M.F., J.M.F.R., E.H. and J.K.N. Infrastructure support was provided by the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre (BRC). L.H. was in receipt of an MRC Intermediate Research Fellowship in Data Science (grant number MR/L01632X/1, UK Med-Bio). This work was also partly supported by funding to M.-E.D. (EU METACARDIS under agreement HEALTH-F4-2012-305312, Neuron II under agreement 291840 and the MRC MR/M501797/1) and by grants from the French National Research Agency (ANR-10-LABX-46 [European Genomics Institute for Diabetes]), from the National Center for Precision Diabetic Medicine – PreciDIAB, which is jointly supported by the French National Agency for Research (ANR-18-IBHU-0001), by the European Union (FEDER), by the Hauts-de-France Regional Council (Agreement 20001891/NP0025517) and by the European Metropolis of Lille (MEL, Agreement 2019_ESR_11) and by Isite ULNE (R-002-20-TALENT-DUMAS), also jointly funded by ANR (ANR-16-IDEX-0004-ULNE) the Hauts-de-France Regional Council (Agreement 20002045) and by the European Metropolis of Lille (MEL). J.M.-P. is funded by the Miguel Servet Program from the Instituto de Salud Carlos III (ISCIII CP18/00009), co-funded by the European Social Fund ‘Investing in your future’. María Arnoriaga Rodríguez is funded by a predoctoral Río Hortega contract (CM19/00190, co-funded by European Social Fund ‘Investing in your future’) from the Instituto de Salud Carlos III, Spain. This work was supported by grants to AM from the Spanish Ministry of Science and Innovation (PID2019-105969GB-I00) and Generalitat Valenciana (project Prometeo/2018/133).

Analyzing 12,361 all-cause cirrhosis cases and 790,095 controls from eight cohorts, we identify a common missense variant in the Mitochondrial Amidoxime Reducing Component 1 gene (MARC1 p.A165T) that associates with protection from all-cause cirrhosis (OR 0.91, p = 2.3*10−11). This same variant also associates with lower levels of hepatic fat on computed tomographic imaging and lower odds of physician-diagnosed fatty liver as well as lower blood levels of alanine transaminase (-0.025 SD, 3.7*10−43), alkaline phosphatase (-0.025 SD, 1.2*10−37), total cholesterol (-0.030 SD, p = 1.9*10−36) and LDL cholesterol (-0.027 SD, p = 5.1*10−30) levels. We identified a series of additional MARC1 alleles (low-frequency missense p.M187K and rare protein-truncating p.R200Ter) that also associated with lower cholesterol levels, liver enzyme levels and reduced risk of cirrhosis (0 cirrhosis cases for 238 R200Ter carriers versus 17,046 cases of cirrhosis among 759,027 non-carriers, p = 0.04) suggesting that deficiency of the MARC1 enzyme may lower blood cholesterol levels and protect against cirrhosis., Author summary Cirrhosis is a leading cause of death worldwide. However, the genetic underpinnings of cirrhosis remain poorly understood. In this study, we analyze twelve thousand individuals with cirrhosis and identify a common missense variant in a gene called MARC1 that protects against cirrhosis. Carriers of this missense variant also have lower blood cholesterol levels, lower liver enzyme levels and reduced liver fat. We identify an additional two low-frequency coding variants in MARC1 that are also associated with lower cholesterol levels, lower liver enzyme levels and protection from cirrhosis. Finally, we identify an individual homozygous for a predicted loss-of-function variant in MARC1 who exhibits very low blood LDL cholesterol levels. These genetic findings suggest that MARC1 deficiency may lower blood cholesterol levels and protect against cirrhosis, pointing to MARC1 as a potential therapeutic target for liver disease.

OBJECTIVES: We evaluated the ability of the Fatty Liver Index (FLI), a surrogate marker of hepatic steatosis, to predict the development of type 2 diabetes (T2D) at 3 years follow-up in a Spanish cohort with prediabetes from a prospective observational study in primary care (PREDAPS). METHODS: FLI was calculated at baseline for 1,142 adult subjects with prediabetes attending primary care centers, and classified into three categories: FLI 60 and FLI ≥60, respectively. The most significant variables increasing the risk of developing T2D were metabolic syndrome (hazard ratio [HR] = 3.02; 95% confidence interval [CI] = 2.14-4.26) and FLI ≥60 (HR = 4.52; 95%CI = 2.10-9.72). Moreover, FLI ≥60 was independently associated with T2D incidence: the HR was 4.97 (95% CI: 2.28-10.80) in the base regression model adjusted by sex, age and educational level, and 3.21 (95%CI: 1.45-7.09) in the fully adjusted model. CONCLUSIONS: FLI may be considered an easy and valuable early indicator of high risk of incident T2D in patients with prediabetes attended in primary care, which could allow the adoption of effective measures needed to prevent and reduce the progression of the disease. OBJECTIVES: We evaluated the ability of the Fatty Liver Index (FLI), a surrogate marker of hepatic steatosis, to predict the development of type 2 diabetes (T2D) at 3 years follow-up in a Spanish cohort with prediabetes from a prospective observational study in primary care (PREDAPS). METHODS: FLI was calculated at baseline for 1,142 adult subjects with prediabetes attending primary care centers, and classified into three categories: FLI 60 and FLI ≥60, respectively. The most significant variables increasing the risk of developing T2D were metabolic syndrome (hazard ratio [HR] = 3.02; 95% confidence interval [CI] = 2.14-4.26) and FLI ≥60 (HR = 4.52; 95%CI = 2.10-9.72). Moreover, FLI ≥60 was independently associated with T2D incidence: the HR was 4.97 (95% CI: 2.28-10.80) in the base regression model adjusted by sex, age and educational level, and 3.21 (95%CI: 1.45-7.09) in the fully adjusted model. CONCLUSIONS: FLI may be considered an easy and valuable early indicator of high risk of incident T2D in patients with prediabetes attended in primary care, which could allow the adoption of effective measures needed to prevent and reduce the progression of the disease. OBJECTIVES: We evaluated the ability of the Fatty Liver Index (FLI), a surrogate marker of hepatic steatosis, to predict the development of type 2 diabetes (T2D) at 3 years follow-up in a Spanish cohort with prediabetes from a prospective observational study in primary care (PREDAPS). METHODS: FLI was calculated at baseline for 1,142 adult subjects with prediabetes attending primary care centers, and classified into three categories: FLI 60 and FLI ≥60, respectively. The most significant variables increasing the risk of developing T2D were metabolic syndrome (hazard ratio [HR] = 3.02; 95% confidence interval [CI] = 2.14-4.26) and FLI ≥60 (HR = 4.52; 95%CI = 2.10-9.72). Moreover, FLI ≥60 was independently associated with T2D incidence: the HR was 4.97 (95% CI: 2.28-10.80) in the base regression model adjusted by sex, age and educational level, and 3.21 (95%CI: 1.45-7.09) in the fully adjusted model. CONCLUSIONS: FLI may be considered an easy and valuable early indicator of high risk of incident T2D in patients with prediabetes attended in primary care, which could allow the adoption of effective measures needed to prevent and reduce the progression of the disease. OBJECTIVES: We evaluated the ability of the Fatty Liver Index (FLI), a surrogate marker of hepatic steatosis, to predict the development of type 2 diabetes (T2D) at 3 years follow-up in a Spanish cohort with prediabetes from a prospective observational study in primary care (PREDAPS). METHODS: FLI was calculated at baseline for 1,142 adult subjects with prediabetes attending primary care centers, and classified into three categories: FLI 60 and FLI ≥60, respectively. The most significant variables increasing the risk of developing T2D were metabolic syndrome (hazard ratio [HR] = 3.02; 95% confidence interval [CI] = 2.14-4.26) and FLI ≥60 (HR = 4.52; 95%CI = 2.10-9.72). Moreover, FLI ≥60 was independently associated with T2D incidence: the HR was 4.97 (95% CI: 2.28-10.80) in the base regression model adjusted by sex, age and educational level, and 3.21 (95%CI: 1.45-7.09) in the fully adjusted model. CONCLUSIONS: FLI may be considered an easy and valuable early indicator of high risk of incident T2D in patients with prediabetes attended in primary care, which could allow the adoption of effective measures needed to prevent and reduce the progression of the disease.

Summary Background & aims To assess the possible association between a validated Dietary Inflammatory Index (DII) and specific dietary components with suitable non-invasive markers of liver status in overweight and obese subjects within the PREDIMED study. Methods A cross-sectional study encompassing 794 randomized overweight and obese participants (mean ± SD age: 67.0 ± 5.0 y, 55% females) from the PREDIMED (PREvencion con DIeta MEDiterranea) trial was conducted. DII is a validated tool evaluating the effect of diet on six inflammatory biomarkers (IL-1b, IL-4, IL-6, IL-10, TNF-α and C-reactive protein). Furthermore, a validated 137-item food-frequency-questionnaire was used to obtain the information about the food intake. In addition, anthropometric measurements and several non-invasive markers of liver status were assessed and the Fatty Liver Index (FLI) score was calculated. Results A higher DII and lower adherence to Mediterranean diet (MeDiet) were associated with a higher degree of liver damage (FLI > 60) in obese as compared to overweight participants. Furthermore, the DII score was positively associated with relevant non-invasive liver markers (ALT, AST, GGT and FLI) and directly affected FLI values. Interestingly, a positive correlation was observed between liver damage (>50th percentile FLI) and nutrients and foods linked to a pro-inflammatory dietary pattern. Conclusions This study reinforced the concept that obesity is associated with liver damage and revealed that the consumption of a pro-inflammatory dietary pattern might contribute to obesity and fatty liver disease features. These data suggest that a well-designed precision diet including putative anti-inflammatory components could specifically prevent and ameliorate non-alcoholic fatty liver manifestations in addition to obesity.

Introduction & Aims Cryopreservation of serum samples is a standard procedure for biomedical research in tertiary centers. However, studies evaluating the long-term biological stability of direct liver fibrosis markers using cryopreserved samples are scarce. Methods We compared the stability of hyaluronic acid (HA), tissue inhibitor of metalloproteinases (TIMP-1) and amino-terminal propeptide of type III procollagen (PIIINP) in 225 frozen serum samples of HCV-infected patients with a paired liver biopsy for up to 25 years (1990–2014). Moreover, we assessed the diagnostic accuracy (AUROC) of the Enhanced Liver Fibrosis (ELF®) score to identify significant fibrosis (F2-4) and its predictive capacity to identify clinical events during follow-up. Results Seventy-six patients (39,8%) had mild fibrosis (F0-1) and 115 (60,2%) significant fibrosis (F2-4). HA, PIIINP and TIMP-1 values remained stable during the period from 1995 to 2014 while those of 1990–94 were slightly higher. We did not find significant differences in the median ELF® values during the 20-year period from 1995–2014 in patients with mild (from 8,4 to 8,7) and significant fibrosis (from 9,9 to 10,9) (p = ns between periods and fibrosis stages). The AUROCs of ELF® to identify significant fibrosis were high in all the periods (from 0,85 to 0,91). The ELF® score showed a good predictive capability to identify clinical events during follow-up. Conclusions The biological stability of direct serum markers (HA, PIIINP and TIMP-1) using HCV-infected samples cryopreserved for 20 years is good. Therefore, the diagnostic accuracy of the ELF® score to identify significant fibrosis and clinical events during follow-up is very high.

DOI: 10.1371/journal.pone.0154601 Background: Non-alcoholic fatty liver disease (NAFLD) causes a wide spectrum of liver damage, ranging from simple steatosis to cirrhosis. However, simple steatosis (SS) and steatohepatitis (NASH) cannot yet be distinguished by clinical or laboratory features. The aim of this study was to assess the relationship between alpha-ketoglutarate and the degrees of NAFLD in morbidly obese patients. Materials and Methods: We used a gas chromatography-quadruple time-of-flight-mass spectrometry analysis to quantify alpha-ketoglutarate in serum from normal-weight subjects (n = 30) and morbidly obese women (n = 97) with or without NAFLD. Results: We found that serum levels of alpha-ketoglutarate were significantly higher in morbidly obese women than in normal-weight women. We showed that circulating levels of alpha-ketoglutarate were lower in lean controls and morbidly obese patients without NAFLD. We also found that alpha-ketoglutarate serum levels were higher in both SS and NASH than in normal liver of morbidly obese patients. However, there was no difference between SS and NASH. Moreover, we observed that circulating levels of alpha-ketoglutarate were associated with glucose metabolism parameters, lipid profile, hepatic enzymes and steatosis degree. In addition, diagnostic performance of alpha-ketoglutarate has been analyzed in NAFLD patients. The AUROC curves from patients with liver steatosis exhibited an acceptable clinical utility. Finally, we showed that the combination of biomarkers (AST, ALT and alpha-ketoglutarate) had the highest accuracy in diagnosing liver steatosis. Conclusion: These findings suggest that alpha-ketoglutarate can determine the presence of non-alcoholic fatty liver in morbidly obese patients but it is not valid a biomark

BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF) regulates angiogenesis, yet therapeutic strategies to disrupt VEGF signaling can interfere with physiologic angiogenesis. In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal production or functions, we investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of portal hypertension and liver disease. METHODS: We obtained transjugular liver biopsies from patients with hepatitis C virus-associated cirrhosis or liver tissues removed during transplantation; healthy human liver tissue was obtained from a commercial source (control). We also performed experiments with male Sprague-Dawley rats and CPEB-deficient mice (C57BL6 or mixed C57BL6/129 background) and their wild-type littermates. Secondary biliary cirrhosis was induced in rats by bile duct ligation, and portal hypertension was induced by partial portal vein ligation. Liver and mesenteric tissues were collected and analyzed in angiogenesis, reverse transcription polymerase chain reaction, polyA tail, 3' rapid amplification of complementary DNA ends, Southern blot, immunoblot, histologic, immunohistochemical, immunofluorescence, and confocal microscopy assays. CPEB was knocked down with small interfering RNAs in H5V endothelial cells, and translation of luciferase reporters constructs was assessed. RESULTS: Activation of CPEB1 promoted alternative nuclear processing within noncoding 3'-untranslated regions of VEGF and CPEB4 messenger RNAs in H5V cells, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promoted cytoplasmic polyadenylation of VEGF messenger RNA, increasing its translation; the high levels of VEGF produced by these cells led to their formation of tubular structures in Matrigel assays. We observed increased levels of CPEB1 and CPEB4 in cirrhotic liver tissues from patients, compared with control tissue, as well as in livers and mesenteries of rats and mice with cirrhosis or/and portal hypertension. Mice with knockdown of CPEB1 or CPEB4 did not overexpress VEGF or have signs of mesenteric neovascularization, and developed less-severe forms of portal hypertension after portal vein ligation. CONCLUSIONS: We identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and nonredundant functions of CPEB1 and CPEB4. Regulation of CPEB4 by CPEB1 and the CPEB4 autoamplification loop induces pathologic angiogenesis. Strategies to block the activities of CPEBs might be developed to treat chronic liver and other angiogenesis-dependent diseases. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2011-29491 and SAF2014-55473-R to MF; BFU2011-30121, BFU2014-54122-P and Consolider RNAREG CSD2009-00080 to RM; PI13/00341 to JB; PI11/01562 and PI14/00125 to PN), Generalitat de Catalunya (SGR1436 to RM; SGR1108 to JB), Fundación Botín by Banco Santander through its Santander Universities Global Division (to RM), AECC Scientific Foundation (to RM and MF), Worldwide Cancer Research (to RM and MF) and RETIC Cancer RD12/0036/0051/FEDER to PN. GFM is funded by a Juan de la Cierva contract from MINECO. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). CIBERehd is an initiative from the Instituto de Salud Carlos III. We also thank Dr Francisco X Real for his contribution to knockout mice generation.