Copy number variation and expression of exportin-4 associates with severity of fibrosis in metabolic associated fatty liver disease

Background Liver fibrosis risk is a heritable trait, the outcome of which is the net deposition of extracellular matrix by hepatic stellate cell-derived myofibroblasts. Whereas nucleotide sequence variations have been extensively studied in liver fibrosis, the role of copy number variations (CNV) in which genes exist in abnormal numbers of copies (mostly due to duplication or deletion) has had limited exploration. Methods The impact of the XPO4 CNV on histological liver damage was examined in a cohort comprised 646 Caucasian patients with biopsy-proven MAFLD and 170 healthy controls. XPO4 expression was modulated and function was examined in human and animal models. Findings Here we demonstrate in a cohort of 816 subjects, 646 with biopsy-proven metabolic associated liver disease (MAFLD) and 170 controls, that duplication in the exportin 4 (XPO4) CNV is associated with the severity of liver fibrosis. Functionally, this occurs via reduced expression of hepatic XPO4 that maintains sustained activation of SMAD3/SMAD4 and promotes TGF-β1-mediated HSC activation and fibrosis. This effect was mediated through termination of nuclear SMAD3 signalling. XPO4 demonstrated preferential binding to SMAD3 compared to other SMADs and led to reduced SMAD3-mediated responses as shown by attenuation of TGFβ1 induced SMAD transcriptional activity, reductions in the recruitment of SMAD3 to target gene promoters following TGF-β1, as well as attenuation of SMAD3 phosphorylation and disturbed SMAD3/SMAD4 complex formation. Interpretation We conclude that a CNV in XPO4 is a critical mediator of fibrosis severity and can be exploited as a therapeutic target for liver fibrosis

Intrahepatic Expression of Fatty Acid Translocase CD36 Is Increased in Obstructive Sleep Apnea

Nocturnal intermittent hypoxia (IH) featuring obstructive sleep apnea (OSA) dysregulates hepatic lipid metabolism and might contribute to the development of non-alcoholic fatty liver disease (NAFLD) observed in OSA patients. However, further research is required to better understanding the molecular mechanisms underlying IH-induced hepatic lipid accumulation. Therefore, the aim of the present study was to determine the effects of OSA on hepatic CD36 expression and the impact of IH by using a mouse model of OSA. Histological analysis, lipid content and CD36 expression were assessed in livers from subjects who underwent liver biopsy and polygraphic study during sleep, and in livers from mice submitted to chronic IH mimicking OSA. Among those who presented OSA features, NAFLD were significantly more frequent than in control subjects with normal respiratory function (77.8 vs. 36.4%, respectively), and showed more severe liver disease. Interestingly, CD36 expression was significantly overexpressed within the liver of OSA patients with respect to controls, and a significant positive correlation was observed between hepatic levels of CD36 and the values of two well-known respiratory parameters that characterized OSA: apnea/hypopnea index (AHI) and oxygen desaturation index (ODI). Moreover, hepatic lipid accumulation as well as induction of hepatic lipogenic genes, and CD36 mRNA and protein expression were significantly higher in livers from mice exposed to IH conditions for 8 weeks than in their corresponding littermates. This study provides novel evidence that IH featuring OSA could contribute to NAFLD setup partly by upregulating hepatic CD36 expression

Ptpn1 deletion protects oval vells against lipoapotosis by favoring lipid droplet formation and dynamics

Trabajo presentado en el The international liver congress, celebrado en Londres (Inglaterra) del 22 al 26 de junio de 2022.[Background and aims]: Activation of oval cells has been related to hepatocyte injury during chronic liver diseases including nonalcoholic fatty liver disease (NAFLD). However, oval cells plasticity can be affected by the pathological environment. We previously found a protection against hepatocyte cell death by inhibiting protein tyrosine phosphatase 1B (PTP1B). Herein, we investigated the molecular and cellular processes involved in the lipotoxic susceptibility in oval cells expressing or not PTP1B. [Method]: Palmitic acid (PA) induced apoptotic cell death in wild-type (Ptpn1+/+) oval cells in parallel to oxidative stress and impaired autophagy. This lipotoxic effect was attenuated in oval cells lacking Ptpn1 that showed up-regulated antioxidant defences, increased unfolded protein response (UPR) signaling, higher endoplasmic reticulum (ER) content and elevated stearoyl CoA desaturase (Scd1) expression and activity. [Results]: These effects in Ptpn1−/− oval cells concurred with an active autophagy, higher mitochondrial efficiency and a molecular signature of starvation, favoring lipid droplet (LD) formation and dynamics. Autophagy blockade in Ptpn1−/− oval cells reduced Scd1 expression, mitochondrial fitness, LD formation and restored lipoapoptosis, an effect also recapitulated by Scd1 silencing. Importantly, oval cells with LDs were found in livers from Ptpn1−/− mice with NAFLD. [Conclusion]: Ptpn1 deficiency restrained lipoapoptosis in oval cells through a metabolic rewiring towards a “starvation-like” fate, favoring autophagy, mitochondrial fitness and LD formation. Dynamic LD-lysosomal interations likely ensured lipid recycling and, overall, these adaptations protect against lipotoxicity. The identification of LDs in oval cells from Ptpn1−/− mice with NAFLD opens new therapeutic perspectives to ensure oval cells viability and plasticity under lipotoxic liver damage

Bacterial antigen translocation and age as BMI-independent contributing factors on systemic inflammation in NAFLD patients

Background & Aims Low‐grade systemic inflammation is a crucial landmark in NAFLD favouring disease progression and comorbidities. We evaluated the input of circulating bacterial antigens on systemic markers of inflammation in NAFLD patients. Patients & Methods Multicenter cross‐sectional study including consecutive patients with biopsy‐proven NAFLD. Demographic, metabolic and fibrosis‐related variables were collected. Circulating bacterial antigens were quantified in blood. Toll‐like receptor SNPs were genotyped. Serum cytokine levels were evaluated. Peripheral blood mononuclear cell response to bacterial antigens was evaluated in vitro. Results Three hundred and fifteen patients from five Spanish hospitals were distributed by BMI. At least, one bacterial antigenic type was found in 66 patients with BMI 30 (77.3%) (P = .014). HOMA‐IR was significantly higher in the presence of circulating antigens among patients with BMI < 30. NASH and significant fibrosis in non‐obese patients were more frequent in the presence of at least two circulating antigenic types. Allelic frequencies of TLR variants were similar to controls and did not affect clinical or laboratory parameters. Pro‐inflammatory cytokines were significantly increased in patients with bacterial antigens, regardless of BMI. TLR gene and protein expression levels were significantly increased in PBMCs from patients with bacterial antigens. Antigen concentrations independently influenced TNF‐α and IL‐6, in both BMI subgroups of patients. Age independently influenced TNF‐α and IL‐6 in non‐obese patients, and TNF‐α in obese patients. Conclusion Serum circulating bacterial antigens as well as age were BMI‐independent factors related to increased systemic inflammation in NAFLD and provides insight on the multifaceted sources of inflammation in these patients.This work has been funded by grants PI16/0967 and PI17/0535 from Instituto de Salud Carlos III, Madrid, Spain, and PROMETEO 2016/001 from Generalitat Valenciana, Valencia, Spain. IGH was recipient of a Young Investigator Grant by CIBERehd, Instituto de Salud Carlos III, Madrid, Spain

Sleep apnea-COPD overlap syndrome is associated with larger left carotid atherosclerotic plaques

BackgroundLittle is known about whether the overlap syndrome (OS) combining features of chronic obstructive pulmonary disease (COPD) and sleep apnea-hypopnea syndrome increases the risk of stroke associated with COPD itself.MethodsWe prospectively studied 74 COPD patients and 32 subjects without lung disease. Spirometry and cardiorespiratory polygraphy were used to assess the pulmonary function of the study population and ultrasound measurements of intima media thickness (IMT) as well as the volume of plaques in both carotid arteries were also evaluated.ResultsPolygraphic criteria of OS were met in 51% of COPD patients. We found that 79% of patients with OS and 50% of COPD patients without OS had atherosclerotic plaques in the left carotid artery (p = 0.0509). Interestingly, the mean volume of atherosclerotic plaques was significantly higher in the left carotid artery of COPD patients with OS (0.07 ± 0.02 ml) than in those without OS (0.04 ± 0.02 ml, p = 0.0305). However, regardless of the presence of OS, no significant differences were observed in both presence and volume of atherosclerotic plaques in the right carotid artery of COPD patients. Adjusted-multivariate linear regression revealed age, current smoking and the apnea/hypopnea index (OR = 4.54, p = 0.012) as independent predictors of left carotid atherosclerotic plaques in COPD patients.ConclusionsThis study suggests that the presence of OS in COPD patients is associated with larger left carotid atherosclerotic plaques, indicating that OS might be screened in all COPD patients to identify those with higher risk of stroke

Hypoxia-inducible factor 2α drives hepatosteatosis through the fatty acid translocase CD36

Background & Aims: Molecular mechanisms by which hypoxia might contribute to hepatosteatosis, the earliest stage in non-alcoholic fatty liver disease (NAFLD) pathogenesis, remain still to be elucidated. We aimed to assess the impact of hypoxia-inducible factor 2α (HIF2α) on the fatty acid translocase CD36 expression and function in vivo and in vitro. Methods: CD36 expression and intracellular lipid content were determined in hypoxic hepatocytes, and in hypoxic CD36- or HIF2α -silenced human liver cells. Histological analysis, and HIF2α and CD36 expression were evaluated in livers from animals in which von Hippel-Lindau (Vhl) gene is inactivated (Vhl -deficient mice), or both Vhl and Hif2a are simultaneously inactivated (Vhl Hif2α -deficient mice), and from 33 biopsy-proven NAFLD patients and 18 subjects with histologically normal liver. Results: In hypoxic hepatocytes, CD36 expression and intracellular lipid content were augmented. Noteworthy, CD36 knockdown significantly reduced lipid accumulation, and HIF2A gene silencing markedly reverted both hypoxia-induced events in hypoxic liver cells. Moreover livers from Vhl -deficient mice showed histologic characteristics of non-alcoholic steatohepatitis (NASH) and increased CD36 mRNA and protein amounts, whereas both significantly decreased and NASH features markedly ameliorated in Vhl Hif2α -deficient mice. In addition, both HIF2α and CD36 were significantly overexpressed within the liver of NAFLD patients and, interestingly, a significant positive correlation between hepatic transcript levels of CD36 and erythropoietin (EPO), a HIF2α -dependent gene target, was observed in NAFLD patients. Conclusions: This study provides evidence that HIF2α drives lipid accumulation in human hepatocytes by upregulating CD36 expression and function, and could contribute to hepatosteatosis setup. f/f f/f /f f/f f/f f/fThis work was supported by PI13/01299, PI17/00535 and CIBEREHD from Instituto de Salud Carlos III (ISCIII/FEDER, Spain) to CGM; CP14/00181, PI16/00823 and PI19/00123 (ISCIII/FEDER, Spain), and Beca Eduardo Gallego 2016 (Fundación Francisco Cobos, Spain) to AGR; SAF2016-76815 (Ministerio de Economía y Competitividad/FEDER, Spain), 534/C/2016 (TV3 Marató, Spain) and CIBERCV (ISCIII/FEDER, Spain) to JA

Liver injury in non-alcoholic fatty liver disease is associated with urea cycle enzyme dysregulation

The main aim was to evaluate changes in urea cycle enzymes in NAFLD patients and in two preclinical animal models mimicking this entity. Seventeen liver specimens from NAFLD patients were included for immunohistochemistry and gene expression analyses. Three-hundred-and-eighty-two biopsy-proven NAFLD patients were genotyped for rs1047891, a functional variant located in carbamoyl phosphate synthetase-1 (CPS1) gene. Two preclinical models were employed to analyse CPS1 by immunohistochemistry, a choline deficient high-fat diet model (CDA-HFD) and a high fat diet LDLr knockout model (LDLr −/−). A significant downregulation in mRNA was observed in CPS1 and ornithine transcarbamylase (OTC1) in simple steatosis and NASH-fibrosis patients versus controls. Further, age, obesity (BMI > 30 kg/m), diabetes mellitus and ALT werefound to be risk factors whereas A-allele from CPS1 was a protective factor from liver fibrosis. CPS1 hepatic expression was diminished in parallel with the increase of fibrosis, and its levels reverted up to normality after changing diet in CDA-HFD mice. In conclusion, liver fibrosis and steatosis were associated with a reduction in both gene and protein expression patterns of mitochondrial urea cycle enzymes. A-allele from a variant on CPS1 may protect from fibrosis development. CPS1 expression is restored in a preclinical model when the main trigger of the liver damage disappears.The research leading to these results has received funding from the Consejería de Salud de la Junta de Andalucía under grant agreement PC-0148-2016-0148 and PE-0451-2018 and Instituto de Salud Carlos III under grant agreements CD21/00095, PI16/01842, PI19/01404, PI19/00589, IFI18/00041, FI20/00201, CD18/00126 and EHD18PI04/2021. Rocío Gallego-Durán has received the Andrew K Burroughs Fellowship from European Association for the Study of the Liver (EASL), Aprendizaje de Nuevas Tecnologías fellowship from Asociación Española para el Estudio del Hígado (AEEH) and CIBERehd Grant to support researcher’s mobility

Liver osteopontin is required to prevent the progression of age-related nonalcoholic fatty liver disease

[EN] Osteopontin (OPN), a senescence-associated secretory phenotype factor, is increased in patients with nonalcoholic fatty liver disease (NAFLD). Cellular senescence has been associated with age-dependent hepatosteatosis. Thus, we investigated the role of OPN in the age-related hepatosteatosis. For this, human serum samples, animal models of aging, and cell lines in which senescence was induced were used. Metabolic fluxes, lipid, and protein concentration were determined. Among individuals with a normal liver, we observed a positive correlation between serum OPN levels and increasing age. This correlation with age, however, was absent in patients with NAFLD. In wild-type (WT) mice, serum and liver OPN were increased at 10 months old (m) along with liver p53 levels and remained elevated at 20m. Markers of liver senescence increased in association with synthesis and concentration of triglycerides (TG) in 10m OPN-deficient (KO) hepatocytes when compared to WT hepatocytes. These changes in senescence and lipid metabolism in 10m OPN-KO mice liver were associated with the decrease of 78 kDa glucose-regulated protein (GRP78), induction of ER stress, and the increase in fatty acid synthase and CD36 levels. OPN deficiency in senescent cells also diminished GRP78, the accumulation of intracellular TG, and the increase in CD36 levels. In 20m mice, OPN loss led to increased liver fibrosis. Finally, we showed that OPN expression in vitro and in vivo was regulated by p53. In conclusion, OPN deficiency leads to earlier cellular senescence, ER stress, and TG accumulation during aging. The p53-OPN axis is required to inhibit the onset of agerelated hepatosteatosis.This work was supported by Ayudas para apoyar grupos de investigación del sistema Universitario Vasco (IT971‐16 to P.A.), MINECO‐FEDER (SAF2017‐87301‐R to M.L.M‐Ch) MCIU/AEI/FEDER, UE (RTI2018‐095134‐B‐100 to P.A. and RTI2018‐099413‐B‐I00 to RN, Asociación Española contra el Cáncer, Canceres raros (M.L.M‐Ch), La Caixa Foundation (to M.L.M‐Ch), Ayudas Fundación BBVA a equipos de Investigación Científica 2018 (to M.L.M‐Ch), Xunta de Galicia (RN: 2015‐CP080 and 2016‐ PG057), Fundación BBVA (RN), and European Foundation for the Study of Diabetes (RN). ISCIII‐FEDER PI17/00535 (to C.G‐M.), ISCIII‐FEDER CP14/00181 and PI16/00823 (to A.G‐ R.), and Francisco Cobos Foundation (to A.G‐R.). CiC bioGUNE thanks MINECO for the Severo Ochoa Excellence Accreditation (SEV‐2016‐ 0644

Development and Validation of Hepamet Fibrosis Scoring System A Simple, Noninvasive Test to Identify Patients With Nonalcoholic Fatty Liver Disease With Advanced Fibrosis

Background & Aims Fibrosis affects prognoses for patients with nonalcoholic fatty liver disease (NAFLD). Several non-invasive scoring systems have aimed to identify patients at risk for advanced fibrosis, but inconclusive results and variations in features of patients (diabetes, obesity and older age) reduce their diagnostic accuracy. We sought to develop a scoring system based on serum markers to identify patients with NAFLD at risk for advanced fibrosis. Methods We collected data from 2452 patients with NAFLD at medical centers in Italy, France, Cuba, and China. We developed the Hepamet fibrosis scoring system using demographic, anthropometric, and laboratory test data, collected at time of liver biopsy, from a training cohort of patients from Spain (n = 768) and validated the system using patients from Cuba (n = 344), Italy (n = 288), France (n = 830), and China (n = 232). Hepamet fibrosis score (HFS) were compared with those of previously developed fibrosis scoring systems (the NAFLD fibrosis score [NFS] and FIB-4). The diagnostic accuracy of the Hepamet fibrosis scoring system was assessed based on area under the receiver operating characteristic (AUROC) curve, sensitivity, specificity, diagnostic odds ratio, and positive and negative predictive values and likelihood ratios. Results Variables used to determine HFS were patient sex, age, homeostatic model assessment score, presence of diabetes, levels of aspartate aminotransferase, and albumin, and platelet counts; these were independently associated with advanced fibrosis. HFS discriminated between patients with and without advanced fibrosis with an AUROC curve value of 0.85 whereas NFS or FIB-4 did so with AUROC values of 0.80 (P = .0001). In the validation set, cut-off HFS of 0.12 and 0.47 identified patients with and without advanced fibrosis with 97.2% specificity, 74% sensitivity, a 92% negative predictive value, a 76.3% positive predictive value, a 13.22 positive likelihood ratio, and a 0.31 negative likelihood ratio. HFS were not affected by patient age, body mass index, hypertransaminasemia, or diabetes. The Hepamet fibrosis scoring system had the greatest net benefit in identifying patients who should undergo liver biopsy analysis and led to significant improvements in reclassification, reducing the number of patients with undetermined results to 20% from 30% for the FIB-4 and NFS systems (P < .05). Conclusions Using clinical and laboratory data from patients with NAFLD, we developed and validated the Hepamet fibrosis scoring system, which identified patients with advanced fibrosis with greater accuracy than the FIB-4 and NFS systems. the Hepamet system provides a greater net benefit for the decision-making process to identify patients who should undergo liver biopsy analysis

Inhibition of ATG3 ameliorates liver steatosis by increasing mitochondrial function

Non-alcoholic fatty liver disease (NAFLD) is a major health threat in both developed and developing countries and is a precursor of the more advanced liver diseases, including non-alcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. Currently, understanding the multiple and complex molecular pathways implicated in NAFLD onset and progression is a major priority. The transcription factor p63, which belongs to a family comprising p53, p63, and p73,1 is one of many factors that contributes to the development of liver steatosis. The role of p63 as a tumor suppressor and in cell maintenance and renewal is well studied, but we have recently reported that it is also relevant in the control of lipid metabolism.2 p63 encodes multiple isoforms that can be grouped into 2 categories; isoforms with an acidic transactivation domain (TA) and those without this domain (domain negative). The TAp63α isoform is elevated in the liver of animal models of NAFLD as well as in liver biopsies from obese patients with NAFLD. Furthermore, downregulation of p63α in the liver attenuates liver steatosis in diet-induced obese (DIO) mice, while the activation of TAp63α increases hepatic fat content, mediated by the activation of IKKβ and endoplasmic reticulum stress.2 A specialized form of autophagy that degrades lipid droplets, termed “lipophagy”, is a major pathway of lipid mobilization in hepatocytes. Lipophagy is elevated in hepatoma cells upon exposure to free fatty acids,3 and reduces the fatty acid load in mouse hepatocytes.4 Its impairment has been associated with the development of fatty liver and insulin resistance3,5; in contrast, the autophagic flux is increased during the activation of hepatic stellate cells.6 In the present study, we used an unbiased proteomics approach to gain insight into novel proteins modulating lipid metabolism in the liver of mice with genetic knockdown or overexpression of TAp63α. We found that autophagy-related gene 3 (ATG3) was upregulated by TAp63α activation and downregulated after p63α inhibition. ATG3 is elevated in several animal models of NAFLD and in the liver of patients with NAFLD. Genetic overexpression of ATG3 increased the lipid load in hepatocytes, while its repression alleviated TAp63α- and diet-induced steatosis. ATG3 exerted its role in lipid metabolism by regulating SIRT1 and mitochondrial function. Collectively, these findings identify ATG3 as a novel factor implicated in the development of steatosisThis work has been supported by grants from FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (PA: RTI2018-095134-B-100; DS and LH: SAF2017-83813-C3-1-R; MLMC: RTC2019-007125-1; CD: BFU2017-87721; ML: RTI2018–101840-B-I00; GS; PID2019-104399RB-I00; RN: RTI2018-099413-B-I00 and RED2018-102379-T; MLMC: SAF2017-87301-R; TCD: RTI2018-096759-A-100), FEDER/Instituto de Salud Carlos III (AGR: PI19/00123), Xunta de Galicia (ML: 2016-PG068; RN: 2015-CP080 and 2016-PG057), Fundación BBVA (RN, GS and MLM), Proyectos Investigación en Salud (MLMC: DTS20/00138), Sistema Universitario Vasco (PA: IT971-16); Fundación Atresmedia (ML and RN), Fundación La Caixa (M.L., R.N. and M.C.), Gilead Sciences International Research Scholars Program in Liver Disease (MVR), Marató TV3 Foundation (DS: 201627), Government of Catalonia (DS: 2017SGR278) and European Foundation for the Study of Diabetes (RN and GS). This research also received funding from the European Community’s H2020 Framework Programme (ERC Synergy Grant-2019-WATCH- 810331, to RN, VP and MS). Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) and CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERdem). CIBERobn, CIBERehd and CIBERdem are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644)S