Metabolic Landscape of the Mouse Liver by Quantitative 31 P Nuclear Magnetic Resonance Analysis of the Phosphorome.

BACKGROUND AND AIMS: The liver plays a central role in all metabolic processes in the body. However, precise characterization of liver metabolism is often obscured by its inherent complexity. Phosphorylated metabolites occupy a prominent position in all anabolic and catabolic pathways. Here, we develop a 31 P nuclear magnetic resonance (NMR)-based method to study the liver "phosphorome" through the simultaneous identification and quantification of multiple hydrophilic and hydrophobic phosphorylated metabolites. APPROACH AND RESULTS: We applied this technique to define the metabolic landscape in livers from a mouse model of the rare disease disorder congenital erythropoietic porphyria (CEP) as well as two well-known murine models of nonalcoholic steatohepatitis: one genetic, methionine adenosyltransferase 1A knockout mice, and the other dietary, mice fed a high-fat choline-deficient diet. We report alterations in the concentrations of phosphorylated metabolites that are readouts of the balance between glycolysis, gluconeogenesis, the pentose phosphate pathway, the tricarboxylic acid cycle, and oxidative phosphorylation and of phospholipid metabolism and apoptosis. Moreover, these changes correlate with the main histological features: steatosis, apoptosis, iron deposits, and fibrosis. Strikingly, treatment with the repurposed drug ciclopirox improves the phosphoromic profile of CEP mice, an effect that was mirrored by the normalization of liver histology. CONCLUSIONS: In conclusion, these findings indicate that NMR-based phosphoromics may be used to unravel metabolic phenotypes of liver injury and to identify the mechanism of drug action.

Magnesium accumulation upon cyclin M4 silencing activates microsomal triglyceride transfer protein improving NASH.

BACKGROUND & AIMS: Perturbations of intracellular magnesium (Mg2+) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). METHODS: Serum Mg2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine. RESULTS: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. CONCLUSIONS: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. LAY SUMMARY: Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein.

Fine-Tuning of Sirtuin 1 Expression Is Essential to Protect the Liver From Cholestatic Liver Disease.

Cholestasis comprises aetiologically heterogeneous conditions characterized by accumulation of bile acids in the liver that actively contribute to liver damage. Sirtuin 1 (SIRT1) regulates liver regeneration and bile acid metabolism by modulating farnesoid X receptor (FXR); we here investigate its role in cholestatic liver disease. We determined SIRT1 expression in livers from patients with cholestatic disease, in two experimental models of cholestasis, as well as in human and murine liver cells in response to bile acid loading. SIRT1-overexpressing (SIRToe ) and hepatocyte-specific SIRT1-KO (knockout) mice (SIRThep-/- ) were subjected to bile duct ligation (BDL) and were fed with a 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet to determine the biological relevance of SIRT1 during cholestasis. The effect of NorUDCA (24-norursodeoxycholic acid) was tested in BDL/SIRToe mice. We found that SIRT1 was highly expressed in livers from cholestatic patients, mice after BDL, and Mdr2 knockout mice (Mdr2-/- ) animals. The detrimental effects of SIRT1 during cholestasis were validated in vivo and in vitro. SIRToe mice showed exacerbated parenchymal injury whereas SIRThep-/- mice evidenced a moderate improvement after BDL and 0.1% DDC feeding. Likewise, hepatocytes isolated from SIRToe mice showed increased apoptosis in response to bile acids, whereas a significant reduction was observed in SIRThep-/- hepatocytes. Importantly, the decrease, but not complete inhibition, of SIRT1 exerted by norUDCA treatment correlated with pronounced improvement in liver parenchyma in BDL/SIRToe mice. Interestingly, both SIRT1 overexpression and hepatocyte-specific SIRT1 depletion correlated with inhibition of FXR, whereas modulation of SIRT1 by NorUDCA associated with restored FXR signaling. Conclusion: SIRT1 expression is increased during human and murine cholestasis. Fine-tuning expression of SIRT1 is essential to protect the liver from cholestatic liver damage.

Deregulated neddylation in liver fibrosis.

Hepatic fibrosis is a global health problem currently without effective therapeutic approaches. Even though the ubiquitin-like posttranslational modification of neddylation, that conjugates Nedd8 (neural precursor cell expressed developmentally downregulated) to specific targets, is aberrant in many pathologies, its relevance in liver fibrosis (LF) remained unexplored. Our results show deregulated neddylation in clinical fibrosis and both in mouse bileductligation- and CCl4 -induced fibrosis. Importantly, neddylation inhibition, by using the pharmacological inhibitor, MLN4924, reduced liver injury, apoptosis, inflammation, and fibrosis by targeting different hepatic cell types. On one hand, increased neddylation was associated with augmented caspase 3 activity in bile-acid-induced apoptosis in mouse hepatocytes whereas neddylation inhibition ameliorated apoptosis through reduction of expression of the Cxcl1 and Ccl2 chemokines. On the other hand, chemokine receptors and cytokines, usually induced in activated macrophages, were reduced after neddylation inhibition in mouse Kupffer cells. Under these circumstances, decreased hepatocyte cell death and inflammation after neddylation inhibition could partly account for reduction of hepatic stellate cell (HSC) activation. We provide evidence that augmented neddylation characterizes activated HSCs, suggesting that neddylation inhibition could be important for resolving LF by directly targeting these fibrogenic cells. Indeed, neddylation inhibition in activated HSCs induces apoptosis in a process partly mediated by accumulation of c-Jun, whose cullin-mediated degradation is impaired under these circumstances. CONCLUSION: Neddylation inhibition reduces fibrosis, suggesting neddylation as a potential and attractive therapeutic target in liver fibrosis. (Hepatology 2017;65:694-709).

TRAIL-producing NK cells contribute to liver injury and related fibrogenesis in the context of GNMT deficiency.

Glycine-N-methyltransferase (GNMT) is essential to preserve liver homeostasis. Cirrhotic patients show low expression of GNMT that is absent in hepatocellular carcinoma (HCC) samples. Accordingly, GNMT deficiency in mice leads to steatohepatitis, fibrosis, cirrhosis, and HCC. Lack of GNMT triggers NK cell activation in GNMT(-/-) mice and depletion of TRAIL significantly attenuates acute liver injury and inflammation in these animals. Chronic inflammation leads to fibrogenesis, further contributing to the progression of chronic liver injury regardless of the etiology. The aim of our study is to elucidate the implication of TRAIL-producing NK cells in the progression of chronic liver injury and fibrogenesis. For this we generated double TRAIL(-/-)/GNMT(-/-) mice in which we found that TRAIL deficiency efficiently protected the liver against chronic liver injury and fibrogenesis in the context of GNMT deficiency. Next, to better delineate the implication of TRAIL-producing NK cells during fibrogenesis we performed bile duct ligation (BDL) to GNMT(-/-) and TRAIL(-/-)/GNMT(-/-) mice. In GNMT(-/-) mice, exacerbated fibrogenic response after BDL concurred with NK1.1(+) cell activation. Importantly, specific inhibition of TRAIL-producing NK cells efficiently protected GNMT(-/-) mice from BDL-induced liver injury and fibrogenesis. Finally, TRAIL(-/-)/GNMT(-/-) mice showed significantly less fibrosis after BDL than GNMT(-/-) mice further underlining the relevance of the TRAIL/DR5 axis in mediating liver injury and fibrogenesis in GNMT(-/-) mice. Finally, in vivo silencing of DR5 efficiently protected GNMT(-/-) mice from BDL-liver injury and fibrogenesis, overall underscoring the key role of the TRAIL/DR5 axis in promoting fibrogenesis in the context of absence of GNMT. Overall, our work demonstrates that TRAIL-producing NK cells actively contribute to liver injury and further fibrogenesis in the pathological context of GNMT deficiency, a molecular scenario characteristic of chronic human liver disease.

S-Adenosylmethionine increases circulating very-low density lipoprotein clearance in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Very-low-density lipoproteins (VLDLs) export lipids from the liver to peripheral tissues and are the precursors of low-density-lipoproteins. Low levels of hepatic S-adenosylmethionine (SAMe) decrease triglyceride (TG) secretion in VLDLs, contributing to hepatosteatosis in methionine adenosyltransferase 1A knockout mice but nothing is known about the effect of SAMe on the circulating VLDL metabolism. We wanted to investigate whether excess SAMe could disrupt VLDL plasma metabolism and unravel the mechanisms involved. METHODS: Glycine N-methyltransferase (GNMT) knockout (KO) mice, GNMT and perilipin-2 (PLIN2) double KO (GNMT-PLIN2-KO) and their respective wild type (WT) controls were used. A high fat diet (HFD) or a methionine deficient diet (MDD) was administrated to exacerbate or recover VLDL metabolism, respectively. Finally, 33 patients with non-alcoholic fatty-liver disease (NAFLD); 11 with hypertriglyceridemia and 22 with normal lipidemia were used in this study. RESULTS: We found that excess SAMe increases the turnover of hepatic TG stores for secretion in VLDL in GNMT-KO mice, a model of NAFLD with high SAMe levels. The disrupted VLDL assembly resulted in the secretion of enlarged, phosphatidylethanolamine-poor, TG- and apoE-enriched VLDL-particles; special features that lead to increased VLDL clearance and decreased serum TG levels. Re-establishing normal SAMe levels restored VLDL secretion, features and metabolism. In NAFLD patients, serum TG levels were lower when hepatic GNMT-protein expression was decreased. CONCLUSIONS: Excess hepatic SAMe levels disrupt VLDL assembly and features and increase circulating VLDL clearance, which will cause increased VLDL-lipid supply to tissues and might contribute to the extrahepatic complications of NAFLD.

SIRT1 controls liver regeneration by regulating bile acid metabolism through farnesoid X receptor and mammalian target of rapamycin signaling.

UNLABELLED: Sirtuin1 (SIRT1) regulates central metabolic functions such as lipogenesis, protein synthesis, gluconeogenesis, and bile acid (BA) homeostasis through deacetylation. Here we describe that SIRT1 tightly controls the regenerative response of the liver. We performed partial hepatectomy (PH) to transgenic mice that overexpress SIRT1 (SIRT). SIRT mice showed increased mortality, impaired hepatocyte proliferation, BA accumulation, and profuse liver injury after surgery. The damaging phenotype in SIRT mice correlated with impaired farnesoid X receptor (FXR) activity due to persistent deacetylation and lower protein expression that led to decreased FXR-target gene expression; small heterodimer partner (SHP), bile salt export pump (BSEP), and increased Cyp7A1. Next, we show that 24-norUrsodeoxycholic acid (NorUDCA) attenuates SIRT protein expression, increases the acetylation of FXR and neighboring histones, restores trimethylation of H3K4 and H3K9, and increases miR34a expression, thus reestablishing BA homeostasis. Consequently, NorUDCA restored liver regeneration in SIRT mice, which showed increased survival and hepatocyte proliferation. Furthermore, a leucine-enriched diet restored mammalian target of rapamycin (mTOR) activation, acetylation of FXR and histones, leading to an overall lower BA production through SHP-inhibition of Cyp7A1 and higher transport (BSEP) and detoxification (Sult2a1) leading to an improved liver regeneration. Finally, we found that human hepatocellular carcinoma (HCC) samples have increased presence of SIRT1, which correlated with the absence of FXR, suggesting its oncogenic potential. CONCLUSION: We define SIRT1 as a key regulator of the regenerative response in the liver through posttranscriptional modifications that regulate the activity of FXR, histones, and mTOR. Moreover, our data suggest that SIRT1 contributes to liver tumorigenesis through dysregulation of BA homeostasis by persistent FXR deacetylation.

Human antigen R contributes to hepatic stellate cell activation and liver fibrosis.

UNLABELLED: RNA-binding proteins (RBPs) play a major role in the control of messenger RNA (mRNA) turnover and translation rates. We examined the role of the RBP, human antigen R (HuR), during cholestatic liver injury and hepatic stellate cell (HSC) activation. HuR silencing attenuated fibrosis development in vivo after BDL, reducing liver damage, oxidative stress, inflammation, and collagen and alpha smooth muscle actin (α-SMA) expression. HuR expression increased in activated HSCs from bile duct ligation mice and during HSC activation in vitro, and HuR silencing markedly reduced HSC activation. HuR regulated platelet-derived growth factor (PDGF)-induced proliferation and migration and controlled the expression of several mRNAs involved in these processes (e.g., Actin, matrix metalloproteinase 9, and cyclin D1 and B1). These functions of HuR were linked to its abundance and cytoplasmic localization, controlled by PDGF, by extracellular signal-regulated kinases (ERK) and phosphatidylinositol 3-kinase activation as well as ERK/LKB1 (liver kinase B1) activation, respectively. More important, we identified the tumor suppressor, LKB1, as a novel downstream target of PDGF-induced ERK activation in HSCs. HuR also controlled transforming growth factor beta (TGF-ß)-induced profibrogenic actions by regulating the expression of TGF-ß, α-SMA, and p21. This was likely the result of an increased cytoplasmic localization of HuR, controlled by TGF-ß-induced p38 mitogen-activated protein kinase activation. Finally, we found that HuR and LKB1 (Ser428) levels were highly expressed in activated HSCs in human cirrhotic samples. CONCLUSION: Our results show that HuR is important for the pathogenesis of liver fibrosis development in the cholestatic injury model, for HSC activation, and for the response of activated HSC to PDGF and TGF-ß.

Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation.

UNLABELLED: Hu antigen R (HuR) is a central RNA-binding protein regulating cell dedifferentiation, proliferation, and survival, which are well-established hallmarks of cancer. HuR is frequently overexpressed in tumors correlating with tumor malignancy, which is in line with a role for HuR in tumorigenesis. However, the precise mechanism leading to changes in HuR expression remains unclear. In the liver, HuR plays a crucial role in hepatocyte proliferation, differentiation, and transformation. Here, we unraveled a novel mean of regulation of HuR expression in hepatocellular carcinoma (HCC) and colon cancer. HuR levels correlate with the abundance of the oncogene, murine double minute 2 (Mdm2), in human HCC and colon cancer metastases. HuR is stabilized by Mdm2-mediated NEDDylation in at least three lysine residues, ensuring its nuclear localization and protection from degradation. CONCLUSION: This novel Mdm2/NEDD8/HuR regulatory framework is essential for the malignant transformation of tumor cells, which, in turn, unveils a novel signaling paradigm that is pharmacologically amenable for cancer therapy.

Non-invasive detection of drug toxicity in rats by solid-phase extraction and MALDI-TOF analysis of urine samples.

An access to fast and non-invasive techniques to infer or predict the drug-induced injury caused by newly developed drugs and to monitor therapeutic efficacy of established drugs during treatment are of the outmost importance in pharmaceutical industry and clinical diagnosis. Peptidome and low molecular weight proteome profiling is an emerging technique that allows the recognition of distinctive patterns and differentiation among diverse physiopathological conditions. In this article, we evaluated the utility of peptide/small protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with WCX magnetic bead-based solid-phase extraction as a screening tool for drug toxicity assessment in urine samples. Given that drug-induced injury is primarily reflected in liver, three different, well-described hepatotoxic drugs were chosen for this work. These were: carbon tetrachloride (CCl(4)) which induces liver fibrosis, D(+)-galactosamine as a model for acute liver injury, and Escherichia coli-derived lipopolysaccharide to study the damage caused by endotoxins. The profiles obtained with a correct clustering analysis show that this methodology can be used as a non-invasive and straightforward approach to test for potential drug toxicity. Pharmaceutical research and drug development studies could benefit from this methodology as liver injury inducer compounds could be easily detected in vivo by non-invasive means, accelerating the launch of safer drugs to the market.

Hyperphosphorylation of hepatic proteome characterizes nonalcoholic fatty liver disease in S-adenosylmethionine deficiency.

Methionine adenosyltransferase 1a (MAT1A) is responsible for hepatic S-adenosyl-L-methionine (SAMe) biosynthesis. Mat1a -/- mice have hepatic SAMe depletion, develop nonalcoholic steatohepatitis (NASH) which is reversed with SAMe administration. We examined temporal alterations in the proteome/phosphoproteome in pre-disease and NASH Mat1a -/- mice, effects of SAMe administration, and compared to human nonalcoholic fatty liver disease (NAFLD). Mitochondrial and peroxisomal lipid metabolism proteins were altered in pre-disease mice and persisted in NASH Mat1a -/- mice, which exhibited more progressive alterations in cytoplasmic ribosomes, ER, and nuclear proteins. A common mechanism found in both pre-disease and NASH livers was a hyperphosphorylation signature consistent with casein kinase 2α (CK2α) and AKT1 activation, which was normalized by SAMe administration. This was mimicked in human NAFLD with a metabolomic signature (M-subtype) resembling Mat1a -/- mice. In conclusion, we have identified a common proteome/phosphoproteome signature between Mat1a -/- mice and human NAFLD M-subtype that may have pathophysiological and therapeutic implications.

Mitochondrial dysfunction-associated microbiota establishes a transmissible refractory response to anti-TNF therapy during ulcerative colitis.

Anti-TNF therapy can induce and maintain a remission status during intestinal bowel disease. However, up to 30% of patients do not respond to this therapy by mechanisms that are unknown. Here, we show that the absence of MCJ, a natural inhibitor of the respiratory chain Complex I, induces gut microbiota changes that are critical determinants of the lack of response in a murine model of DSS-induced inflammation. First, we found that MCJ expression is restricted to macrophages in human colonic tissue. Therefore, we demonstrate by transcriptomic analysis of colon macrophages from DSS-induced mice that MCJ-deficiency is linked to the expression of genes belonging to the FcγR signaling pathway and contains an anti-TNF refractory gene signature identified in ulcerative colitis patients. The gut microbial composition changes observed upon DSS treatment in the MCJ-deficient mice revealed the increased presence of specific colitogenic members, including Ruminococcus gnavus and Oscillospira, which could be associated with the non-response to TNF inhibitors. Further, we show that the presence of a microbiota associated resistance to treatment is dominant and transmissible to responsive individuals. Collectively, our findings underscore the critical role played by macrophage mitochondrial function in the gut ecological niche that can substantially affect not only the severity of inflammation but also the ability to successfully respond to current therapies.

Hepatic levels of S-adenosylmethionine regulate the adaptive response to fasting.

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, ß-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive ß-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.

Methionine adenosyltransferase 1a antisense oligonucleotides activate the liver-brown adipose tissue axis preventing obesity and associated hepatosteatosis.

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2-mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis.

Neddylation inhibition ameliorates steatosis in NAFLD by boosting hepatic fatty acid oxidation via the DEPTOR-mTOR axis.

OBJECTIVE: Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD. METHODS: Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8. RESULTS: Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models CONCLUSIONS: Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury.

BACKGROUND & AIMS: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. METHODS: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. RESULTS: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. CONCLUSIONS: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. LAY SUMMARY: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

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

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 age-related hepatosteatosis.

Targeting Hepatic Glutaminase 1 Ameliorates Non-alcoholic Steatohepatitis by Restoring Very-Low-Density Lipoprotein Triglyceride Assembly.

Non-alcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatic fat in an inflammatory/fibrotic background. Herein, we show that the hepatic high-activity glutaminase 1 isoform (GLS1) is overexpressed in NASH. Importantly, GLS1 inhibition reduces lipid content in choline and/or methionine deprivation-induced steatotic mouse primary hepatocytes, in human hepatocyte cell lines, and in NASH mouse livers. We suggest that under these circumstances, defective glutamine fueling of anaplerotic mitochondrial metabolism and concomitant reduction of oxidative stress promotes a reprogramming of serine metabolism, wherein serine is shifted from the generation of the antioxidant glutathione and channeled to provide one-carbon units to regenerate the methionine cycle. The restored methionine cycle can induce phosphatidylcholine synthesis from the phosphatidylethanolamine N-methyltransferase-mediated and CDP-choline pathways as well as by base-exchange reactions between phospholipids, thereby restoring hepatic phosphatidylcholine content and very-low-density lipoprotein export. Overall, we provide evidence that hepatic GLS1 targeting is a valuable therapeutic approach in NASH.

miR-873-5p targets mitochondrial GNMT-Complex II interface contributing to non-alcoholic fatty liver disease.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. METHODS: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. RESULTS: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid ß-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. CONCLUSION: GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment.

SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer.

BACKGROUND: Even though liver kinase B1 (LKB1) is usually described as a tumor suppressor in a wide variety of tissues, it has been shown that LKB1 aberrant expression is associated with bad prognosis in Hepatocellular Carcinoma (HCC). METHODS: Herein we have overexpressed LKB1 in human hepatoma cells and by using histidine pull-down assay we have investigated the role of the hypoxia-related post-translational modification of Small Ubiquitin-related Modifier (SUMO)ylation in the regulation of LKB1 oncogenic role. Molecular modelling between LKB1 and its interactors, involved in regulation of LKB1 nucleocytoplasmic shuttling and LKB1 activity, was performed. Finally, high affinity SUMO binding entities-based technology were used to validate our findings in a pre-clinical mouse model and in clinical HCC. FINDINGS: We found that in human hepatoma cells under hypoxic stress, LKB1 overexpression increases cell viability and aggressiveness in association with changes in LKB1 cellular localization. Moreover, by using site-directed mutagenesis, we have shown that LKB1 is SUMOylated by SUMO-2 at Lys178 hampering LKB1 nucleocytoplasmic shuttling and fueling hepatoma cell growth. Molecular modelling of SUMO modified LKB1 further confirmed steric impedance between SUMOylated LKB1 and the STe20-Related ADaptor cofactor (STRADα), involved in LKB1 export from the nucleus. Finally, we provide evidence that endogenous LKB1 is modified by SUMO in pre-clinical mouse models of HCC and clinical HCC, where LKB1 SUMOylation is higher in fast growing tumors. INTERPRETATION: Overall, SUMO-2 modification of LKB1 at Lys178 mediates LKB1 cellular localization and its oncogenic role in liver cancer. FUND: This work was supported by grants from NIH (US Department of Health and Human services)-R01AR001576-11A1 (J.M.M and M.L.M-C.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), MINECO: SAF2017-87301-R and SAF2014-52097-R integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M., respectively), BFU2015-71017/BMC MINECO/FEDER, EU (to A.D.Q. and I.D.M.), BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovacion 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M), Asociación Española contra el Cáncer (T.C.D, P·F-T and M.L.M-C), Daniel Alagille award from EASL (to T.C.D), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M and M.A), La Caixa Foundation Program (to M.L.M), Programma di Ricerca Regione-Università 2007-2009 and 2011-2012, Regione Emilia-Romagna (to E.V.), Ramón Areces Foundation and the Andalusian Government (BIO-198) (A.D.Q. and I.D.M.), ayudas para apoyar grupos de investigación del sistema Universitario Vasco IT971-16 (P.A.), MINECO:SAF2015-64352-R (P.A.), Institut National du Cancer, FRANCE, INCa grant PLBIO16-251 (M.S.R.), MINECO - BFU2016-76872-R to (E.B.). Work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (M.V-R). Finally, Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644). Funding sources had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.