Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

A strategy to detect metabolic changes induced by exposure to chemicals from large sets of condition-specific metabolic models computed with enumeration techniques.

BACKGROUND: The growing abundance of in vitro omics data, coupled with the necessity to reduce animal testing in the safety assessment of chemical compounds and even eliminate it in the evaluation of cosmetics, highlights the need for adequate computational methodologies. Data from omics technologies allow the exploration of a wide range of biological processes, therefore providing a better understanding of mechanisms of action (MoA) related to chemical exposure in biological systems. However, the analysis of these large datasets remains difficult due to the complexity of modulations spanning multiple biological processes. RESULTS: To address this, we propose a strategy to reduce information overload by computing, based on transcriptomics data, a comprehensive metabolic sub-network reflecting the metabolic impact of a chemical. The proposed strategy integrates transcriptomic data to a genome scale metabolic network through enumeration of condition-specific metabolic models hence translating transcriptomics data into reaction activity probabilities. Based on these results, a graph algorithm is applied to retrieve user readable sub-networks reflecting the possible metabolic MoA (mMoA) of chemicals. This strategy has been implemented as a three-step workflow. The first step consists in building cell condition-specific models reflecting the metabolic impact of each exposure condition while taking into account the diversity of possible optimal solutions with a partial enumeration algorithm. In a second step, we address the challenge of analyzing thousands of enumerated condition-specific networks by computing differentially activated reactions (DARs) between the two sets of enumerated possible condition-specific models. Finally, in the third step, DARs are grouped into clusters of functionally interconnected metabolic reactions, representing possible mMoA, using the distance-based clustering and subnetwork extraction method. The first part of the workflow was exemplified on eight molecules selected for their known human hepatotoxic outcomes associated with specific MoAs well described in the literature and for which we retrieved primary human hepatocytes transcriptomic data in Open TG-GATEs. Then, we further applied this strategy to more precisely model and visualize associated mMoA for two of these eight molecules (amiodarone and valproic acid). The approach proved to go beyond gene-based analysis by identifying mMoA when few genes are significantly differentially expressed (2 differentially expressed genes (DEGs) for amiodarone), bringing additional information from the network topology, or when very large number of genes were differentially expressed (5709 DEGs for valproic acid). In both cases, the results of our strategy well fitted evidence from the literature regarding known MoA. Beyond these confirmations, the workflow highlighted potential other unexplored mMoA. CONCLUSION: The proposed strategy allows toxicology experts to decipher which part of cellular metabolism is expected to be affected by the exposition to a given chemical. The approach originality resides in the combination of different metabolic modelling approaches (constraint based and graph modelling). The application to two model molecules shows the strong potential of the approach for interpretation and visual mining of complex omics in vitro data. The presented strategy is freely available as a python module ( https://pypi.org/project/manamodeller/ ) and jupyter notebooks ( https://github.com/LouisonF/MANA ).

Weight of evidence evaluation of the metabolism disrupting effects of triphenyl phosphate using an expert knowledge elicitation approach.

Identification of Endocrine-Disrupting Chemicals (EDCs) in a regulatory context requires a high level of evidence. However, lines of evidence (e.g. human, in vivo, in vitro or in silico) are heterogeneous and incomplete for quantifying evidence of the adverse effects and mechanisms involved. To date, for the regulatory appraisal of metabolism-disrupting chemicals (MDCs), no harmonised guidance to assess the weight of evidence has been developed at the EU or international level. To explore how to develop this, we applied a formal Expert Knowledge Elicitation (EKE) approach within the European GOLIATH project. EKE captures expert judgment in a quantitative manner and provides an estimate of uncertainty of the final opinion. As a proof of principle, we selected one suspected MDC -triphenyl phosphate (TPP) - based on its related adverse endpoints (obesity/adipogenicity) relevant to metabolic disruption and a putative Molecular Initiating Event (MIE): activation of peroxisome proliferator activated receptor gamma (PPARγ). We conducted a systematic literature review and assessed the quality of the lines of evidence with two independent groups of experts within GOLIATH, with the objective of categorising the metabolic disruption properties of TPP, by applying an EKE approach. Having followed the entire process separately, both groups arrived at the same conclusion, designating TPP as a "suspected MDC" with an overall quantitative agreement exceeding 85%, indicating robust reproducibility. The EKE method provides to be an important way to bring together scientists with diverse expertise and is recommended for future work in this area.

Mitochondrial alterations in fatty liver diseases.

Fatty liver diseases can result from common metabolic diseases, as well as from xenobiotic exposure and excessive alcohol use, all of which have been shown to exert toxic effects on hepatic mitochondrial functionality and dynamics. Invasive or complex methodology limits large-scale investigations of mitochondria in human livers. Nevertheless, abnormal mitochondrial function, such as impaired fatty acid oxidation and oxidative phosphorylation, drives oxidative stress and has been identified as an important feature of human steatohepatitis. On the other hand, hepatic mitochondria can be flexible and adapt to the ambient metabolic condition to prevent triglyceride and lipotoxin accumulation in obesity. Experience from studies on xenobiotics has provided important insights into the regulation of hepatic mitochondria. Increasing awareness of the joint presence of metabolic disease-related (lipotoxic) and alcohol-related liver diseases further highlights the need to better understand their mutual interaction and potentiation in disease progression. Recent clinical studies have assessed the effects of diets or bariatric surgery on hepatic mitochondria, which are also evolving as an interesting therapeutic target in non-alcoholic fatty liver disease. This review summarises the current knowledge on hepatic mitochondria with a focus on fatty liver diseases linked to obesity, type 2 diabetes and xenobiotics.

Transcriptomic analysis in zebrafish larvae identifies iron-dependent mitochondrial dysfunction as a possible key event of NAFLD progression induced by benzo[a]pyrene/ethanol co-exposure.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide epidemic for which environmental contaminants are increasingly recognized as important etiological factors. Among them, the combination of benzo[a]pyrene (B[a]P), a potent environmental carcinogen, with ethanol, was shown to induce the transition of steatosis toward steatohepatitis. However, the underlying mechanisms involved remain to be deciphered. In this context, we used high-fat diet fed zebrafish model, in which we previously observed progression of steatosis to a steatohepatitis-like state following a 7-day-co-exposure to 43 mM ethanol and 25 nM B[a]P. Transcriptomic analysis highlighted the potent role of mitochondrial dysfunction, alterations in heme and iron homeostasis, involvement of aryl hydrocarbon receptor (AhR) signaling, and oxidative stress. Most of these mRNA dysregulations were validated by RT-qPCR. Moreover, similar changes were observed using a human in vitro hepatocyte model, HepaRG cells. The mitochondria structural and functional alterations were confirmed by transmission electronic microscopy and Seahorse technology, respectively. Involvement of AhR signaling was evidenced by using in vivo an AhR antagonist, CH223191, and in vitro in AhR-knock-out HepaRG cells. Furthermore, as co-exposure was found to increase the levels of both heme and hemin, we investigated if mitochondrial iron could induce oxidative stress. We found that mitochondrial labile iron content was raised in toxicant-exposed larvae. This increase was prevented by the iron chelator, deferoxamine, which also inhibited liver co-exposure toxicity. Overall, these results suggest that the increase in mitochondrial iron content induced by B[a]P/ethanol co-exposure causes mitochondrial dysfunction that contributes to the pathological progression of NAFLD.

Xenobiotic-Induced Aggravation of Metabolic-Associated Fatty Liver Disease.

Metabolic-associated fatty liver disease (MAFLD), which is often linked to obesity, encompasses a large spectrum of hepatic lesions, including simple fatty liver, steatohepatitis, cirrhosis and hepatocellular carcinoma. Besides nutritional and genetic factors, different xenobiotics such as pharmaceuticals and environmental toxicants are suspected to aggravate MAFLD in obese individuals. More specifically, pre-existing fatty liver or steatohepatitis may worsen, or fatty liver may progress faster to steatohepatitis in treated patients, or exposed individuals. The mechanisms whereby xenobiotics can aggravate MAFLD are still poorly understood and are currently under deep investigations. Nevertheless, previous studies pointed to the role of different metabolic pathways and cellular events such as activation of de novo lipogenesis and mitochondrial dysfunction, mostly associated with reactive oxygen species overproduction. This review presents the available data gathered with some prototypic compounds with a focus on corticosteroids and rosiglitazone for pharmaceuticals as well as bisphenol A and perfluorooctanoic acid for endocrine disruptors. Although not typically considered as a xenobiotic, ethanol is also discussed because its abuse has dire consequences on obese liver.

Advanced preclinical models for evaluation of drug-induced liver injury - consensus statement by the European Drug-Induced Liver Injury Network [PRO-EURO-DILI-NET].

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue for which there is a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause of ALF because of its extensive worldwide use as an analgesic. Despite intensive efforts utilising current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful discoveries by detailing the requirements for more realistic human-based systems to assess hepatotoxicity and guide future drug safety testing. We present novel insights and discuss major players in APAP pathophysiology, and describe emerging in vitro and in vivo pre-clinical models, as well as advanced imaging and in silico technologies, which may improve prediction of clinical outcomes of DILI.

Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanism-based toxicity.

Steatosis is a liver lesion reported with numerous pharmaceuticals. Prior studies showed that severe impairment of mitochondrial fatty acid oxidation (mtFAO) constantly leads to lipid accretion in liver. However, much less is known about the mechanism(s) of drug-induced steatosis in the absence of severe mitochondrial dysfunction, although previous studies suggested the involvement of mild-to-moderate inhibition of mtFAO, increased de novo lipogenesis (DNL), and impairment of very low-density lipoprotein (VLDL) secretion. The objective of our study, mainly carried out in human hepatoma HepaRG cells, was to investigate these 3 mechanisms with 12 drugs able to induce steatosis in human: amiodarone (AMIO, used as positive control), allopurinol (ALLO), D-penicillamine (DPEN), 5-fluorouracil (5FU), indinavir (INDI), indomethacin (INDO), methimazole (METHI), methotrexate (METHO), nifedipine (NIF), rifampicin (RIF), sulindac (SUL), and troglitazone (TRO). Hepatic cells were exposed to drugs for 4 days with concentrations decreasing ATP level by less than 30% as compared to control and not exceeding 100 × Cmax. Among the 12 drugs, AMIO, ALLO, 5FU, INDI, INDO, METHO, RIF, SUL, and TRO induced steatosis in HepaRG cells. AMIO, INDO, and RIF decreased mtFAO. AMIO, INDO, and SUL enhanced DNL. ALLO, 5FU, INDI, INDO, SUL, RIF, and TRO impaired VLDL secretion. These seven drugs reduced the mRNA level of genes playing a major role in VLDL assembly and also induced endoplasmic reticulum (ER) stress. Thus, in the absence of severe mitochondrial dysfunction, drug-induced steatosis can be triggered by different mechanisms, although impairment of VLDL secretion seems more frequently involved, possibly as a consequence of ER stress.

Molecular Networking for Drug Toxicities Studies: The Case of Hydroxychloroquine in COVID-19 Patients.

Using drugs to treat COVID-19 symptoms may induce adverse effects and modify patient outcomes. These adverse events may be further aggravated in obese patients, who often present different illnesses such as metabolic-associated fatty liver disease. In Rennes University Hospital, several drug such as hydroxychloroquine (HCQ) have been used in the clinical trial HARMONICOV to treat COVID-19 patients, including obese patients. The aim of this study is to determine whether HCQ metabolism and hepatotoxicity are worsened in obese patients using an in vivo/in vitro approach. Liquid chromatography high resolution mass spectrometry in combination with untargeted screening and molecular networking were employed to study drug metabolism in vivo (patient's plasma) and in vitro (HepaRG cells and RPTEC cells). In addition, HepaRG cells model were used to reproduce pathophysiological features of obese patient metabolism, i.e., in the condition of hepatic steatosis. The metabolic signature of HCQ was modified in HepaRG cells cultured under a steatosis condition and a new metabolite was detected (carboxychloroquine). The RPTEC model was found to produce only one metabolite. A higher cytotoxicity of HCQ was observed in HepaRG cells exposed to exogenous fatty acids, while neutral lipid accumulation (steatosis) was further enhanced in these cells. These in vitro data were compared with the biological parameters of 17 COVID-19 patients treated with HCQ included in the HARMONICOV cohort. Overall, our data suggest that steatosis may be a risk factor for altered drug metabolism and possibly toxicity of HCQ.

Moderate chronic ethanol consumption exerts beneficial effects on nonalcoholic fatty liver in mice fed a high-fat diet: possible role of higher formation of triglycerides enriched in monounsaturated fatty acids.

PURPOSE: Several clinical studies suggested that light-to-moderate alcohol intake could alleviate nonalcoholic fatty liver disease (NAFLD), but the underlying mechanism is still poorly understood. METHODS: Mice fed a high-fat diet (HFD) were submitted or not to moderate ethanol intake for 3 months (ca. 10 g/kg/day) via drinking water. Biochemical, analytical and transcriptomic analyses were performed in serum and liver. RESULTS: Serum ethanol concentrations in ethanol-treated HFD mice comprised between 0.5 and 0.7 g/l throughout the experiment. NAFLD improvement was observed in ethanol-treated HFD mice as assessed by reduced serum transaminase activity. This was associated with less microvesicular and more macrovacuolar steatosis, the absence of apoptotic hepatocytes and a trend towards less fibrosis. Liver lipid analysis showed increased amounts of fatty acids incorporated in triglycerides and phospholipids, reduced proportion of palmitic acid in total lipids and higher desaturation index, thus suggesting enhanced stearoyl-coenzyme A desaturase activity. mRNA expression of several glycolytic and lipogenic enzymes was upregulated. Genome-wide expression profiling and gene set enrichment analysis revealed an overall downregulation of the expression of genes involved in collagen fibril organization and leukocyte chemotaxis and an overall upregulation of the expression of genes involved in oxidative phosphorylation and mitochondrial respiratory chain complex assembly. In addition, mRNA expression of several proteasome subunits was upregulated in ethanol-treated HFD mice. CONCLUSIONS: Moderate chronic ethanol consumption may alleviate NAFLD by several mechanisms including the generation of non-toxic lipid species, reduced expression of profibrotic and proinflammatory genes, restoration of mitochondrial function and possible stimulation of proteasome activity.

The GOLIATH Project: Towards an Internationally Harmonised Approach for Testing Metabolism Disrupting Compounds.

The purpose of this project report is to introduce the European "GOLIATH" project, a new research project which addresses one of the most urgent regulatory needs in the testing of endocrine-disrupting chemicals (EDCs), namely the lack of methods for testing EDCs that disrupt metabolism and metabolic functions. These chemicals collectively referred to as "metabolism disrupting compounds" (MDCs) are natural and anthropogenic chemicals that can promote metabolic changes that can ultimately result in obesity, diabetes, and/or fatty liver in humans. This project report introduces the main approaches of the project and provides a focused review of the evidence of metabolic disruption for selected EDCs. GOLIATH will generate the world's first integrated approach to testing and assessment (IATA) specifically tailored to MDCs. GOLIATH will focus on the main cellular targets of metabolic disruption-hepatocytes, pancreatic endocrine cells, myocytes and adipocytes-and using an adverse outcome pathway (AOP) framework will provide key information on MDC-related mode of action by incorporating multi-omic analyses and translating results from in silico, in vitro, and in vivo models and assays to adverse metabolic health outcomes in humans at real-life exposures. Given the importance of international acceptance of the developed test methods for regulatory use, GOLIATH will link with ongoing initiatives of the Organisation for Economic Development (OECD) for test method (pre-)validation, IATA, and AOP development.

Large-Scale Modeling Approach Reveals Functional Metabolic Shifts during Hepatic Differentiation.

Being able to explore the metabolism of broad metabolizing cells is of critical importance in many research fields. This article presents an original modeling solution combining metabolic network and omics data to identify modulated metabolic pathways and changes in metabolic functions occurring during differentiation of a human hepatic cell line (HepaRG). Our results confirm the activation of hepato-specific functionalities and newly evidence modulation of other metabolic pathways, which could not be evidenced from transcriptomic data alone. Our method takes advantage of the network structure to detect changes in metabolic pathways that do not have gene annotations and exploits flux analyses techniques to identify activated metabolic functions. Compared to the usual cell-specific metabolic network reconstruction approaches, it limits false predictions by considering several possible network configurations to represent one phenotype rather than one arbitrarily selected network. Our approach significantly enhances the comprehensive and functional assessment of cell metabolism, opening further perspectives to investigate metabolic shifts occurring within various biological contexts.

Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells.

Although mitochondriotoxicity plays a major role in drug-induced hepatotoxicity, alteration of mitochondrial DNA (mtDNA) homeostasis has been described only with a few drugs. Because it requires long drug exposure, this mechanism of toxicity cannot be detected with investigations performed in isolated liver mitochondria or cultured cells exposed to drugs for several hours or a few days. Thus, a first aim of this study was to determine whether a 2-week treatment with nine hepatotoxic drugs could affect mtDNA homeostasis in HepaRG cells. Previous investigations with these drugs showed rapid toxicity on oxidative phosphorylation but did not address the possibility of delayed toxicity secondary to mtDNA homeostasis impairment. The maximal concentration used for each drug induced about 10% cytotoxicity. Two other drugs, zalcitabine and linezolid, were used as positive controls for their respective effects on mtDNA replication and translation. Another goal was to determine whether drug-induced mitochondriotoxicity could be modulated by lipid overload mimicking nonalcoholic fatty liver. Among the nine drugs, imipramine and ritonavir induced mitochondrial effects suggesting alteration of mtDNA translation. Ritonavir toxicity was stronger in nonsteatotic cells. None of the nine drugs decreased mtDNA levels. However, increased mtDNA was observed with five drugs, especially in nonsteatotic cells. The mtDNA levels could not be correlated with the expression of key factors involved in mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), PGC1ß, and AMP-activated protein kinase α-subunit. Hence, drug-induced impairment of mtDNA translation might not be rare, and increased mtDNA levels could be a frequent adaptive response to slight energy shortage. Nevertheless, this adaptation could be impaired by lipid overload.

Chronic and low exposure to a pharmaceutical cocktail induces mitochondrial dysfunction in liver and hyperglycemia: Differential responses between lean and obese mice.

Pharmaceuticals are found in the environment but the impact of this contamination on human and animal health is poorly known. The liver could be particularly targeted since a significant number of these drugs are hepatotoxic, in particular via oxidative stress and mitochondrial dysfunction. Notably, the latter events can also be observed in liver diseases linked to obesity, so that the obese liver might be more sensitive to drug toxicity. In this study, we determined the effects of a chronic exposure to low doses of pharmaceuticals in wild-type and obese mice, with a particular focus on mitochondrial function. To this end, wild-type and ob/ob mice were exposed for 4 months to a cocktail of 11 pharmaceuticals provided in drinking water containing 0.01, 0.1, or 1 mg/L of each drug. At the end of the treatment, liver mitochondria were isolated and different parameters were measured. Chronic exposure to the pharmaceuticals reduced mitochondrial respiration driven by succinate and palmitoyl-l-carnitine in wild-type mice and increased antimycin-induced ROS production in ob/ob mice. Hyperglycemia and hepatic histological abnormalities were also observed in treated ob/ob mice. Investigations were also carried out in isolated liver mitochondria incubated with the mixture, or with each individual drug. The mitochondrial effects of the mixture were different from those observed in treated mice and could not be predicted from the results obtained with each drug. Because some of the 11 drugs included in our cocktail can be found in water at relatively high concentrations, our data could be relevant in environmental toxicology. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1375-1389, 2017.

Bisphenol a induces steatosis in HepaRG cells using a model of perinatal exposure.

Human exposure to bisphenol A (BPA) could favor obesity and related metabolic disorders such as hepatic steatosis. Investigations in rodents have shown that these deleterious effects are observed not only when BPA is administered during the adult life but also with different protocols of perinatal exposure. Whether perinatal BPA exposure could pose a risk in human is currently unknown, and thus appropriate in vitro models could be important to tackle this major issue. Accordingly, we determined whether long-term BPA treatment could induce steatosis in human HepaRG cells by using a protocol mimicking perinatal exposure. To this end, the kinetics of expression of seven proteins differentially expressed during liver development was determined during a 4-week period of cell culture required for proliferation and differentiation. By analogy with data reported in rodents and humans, our results indicated that the period of cell culture around day 15 and day 18 after seeding could be considered as the "natal" period. Consequently, HepaRG cells were treated for 3 weeks with BPA (from 0.2 to 2000 nM), with a treatment starting during the proliferating period. BPA was able to induce steatosis with a nonmonotonic dose response profile, with significant effects on neutral lipids and triglycerides observed for the 2 nM concentration. However, the expression of many enzymes involved in lipid and carbohydrate homeostasis was unchanged in exposed HepaRG cells. The expression of other potential BPA targets and enzymes involved in BPA biotransformation was also determined, giving answers as well as new questions regarding the mechanisms of action of BPA. Hence, HepaRG cells provide a valuable model that can prove useful for the toxicological assessment of endocrine disruptors on hepatic metabolisms, in particular in the developing liver. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1024-1036, 2017.

A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen.

Obesity and nonalcoholic fatty liver disease (NAFLD) can increase susceptibility to hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are poorly understood. For acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during NAFLD. The first aim of our study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, human HepaRG cells were incubated for one week with stearic acid or oleic acid, in the presence of different concentrations of insulin. Although cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids, CYP2E1 activity was significantly increased only by stearic acid. CYP2E1 activity was reduced by insulin and this effect was reproduced in cultured primary human hepatocytes. Next, APAP cytotoxicity was assessed in HepaRG cells with or without lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations showed that the loss of ATP and glutathione was almost always greater in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole, recovery of ATP was significantly higher in the presence of stearate with low (2.5mM) or high (20mM) concentrations of APAP. Levels of APAP-glucuronide were significantly enhanced by insulin. Hence, HepaRG cells can be used as a valuable model of NAFLD to unveil important metabolic and hormonal factors which can increase susceptibility to drug-induced hepatotoxicity.

Mid-infrared fibre evanescent wave spectroscopy of serum allows fingerprinting of the hepatic metabolic status in mice.

Non-alcoholic fatty liver disease is associated with obesity, diabetes, and metabolic syndrome. The detection of systemic metabolic changes associated with alterations in the liver status during non-alcoholic fatty liver disease could improve patient follow-up. The aim of the present study was to evaluate the potential of mid-infrared fibre evanescent wave spectroscopy as a minimum-invasive method for evaluating the liver status during non-alcoholic fatty liver disease. Seventy-five mice were subjected to a control, high-fat or high-fat-high carbohydrate diets. We analysed the serum biochemical parameters and mRNA levels of hepatic genes by quantitative RT-PCR. Steatosis was quantified by image analysis. The mid-infrared spectra were acquired from serum, and then analysed to develop a predictive model of the steatosis level. Animals subjected to enriched diets were obese. Hepatic steatosis was found in all animals. The relationship between the spectroscopy-predicted and observed levels of steatosis, expressed as percentages of the liver biopsy area, was not linear. A transition around 10% steatosis was observed, leading us to consider two distinct predictive models (<10% and >10%) based on two different sets of discriminative spectral variables. The model performance was evaluated using random cross-validation (10%). The hypothesis that additional metabolic changes occur beyond this transition was supported by the fact that it was associated with increased serum ALT levels, and Col1α1 chain mRNA levels. Our data suggest that mid-infrared spectroscopy combined with statistical analysis allows identifying serum mid-infrared signatures that reflect the liver status during non-alcoholic fatty liver disease.

The effect of water suppression on the hepatic lipid quantification, as assessed by the LCModel, in a preclinical and clinical scenario.

OBJECTIVE: To investigate the effect of water suppression on the hepatic lipid quantification, using the LCModel. MATERIALS AND METHODS: MR spectra with and without water suppression were acquired in the liver of mice at 4.7 T and patients at 3 T, and processed with the LCModel. The Cramér-Rao Lower Bound (CRLB) values of the seven lipid resonances were determined to assess the impact of water suppression on hepatic lipid quantification. A paired t test was used for comparison between the CRLBs obtained with and without water suppression. RESULTS: For the preclinical data, in the high (low) fat fraction subset an overall impairment in hepatic lipid quantification, i.e. an increase of CRLBs (no significant change of CRLBs) was observed in spectra acquired with water suppression. For the clinical data, there were no substantial changes in the CRLB with water suppression. Because (1) the water suppression does not overall improve the quantification of the lipid resonances and (2) the MR spectrum without water suppression is always acquired for fat fraction calculation, the optimal data-acquisition strategy for liver MRS is to acquire only the MR spectrum without water suppression. CONCLUSION: For quantification of hepatic lipid resonances, it is advantageous to perform MR spectroscopy without water suppression in a clinical and preclinical scenario (at moderate fields).

Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease.

The worldwide epidemic of obesity and insulin resistance favors nonalcoholic fatty liver disease (NAFLD). Insulin resistance (IR) in the adipose tissue increases lipolysis and the entry of nonesterified fatty acids (NEFAs) in the liver, whereas IR-associated hyperinsulinemia promotes hepatic de novo lipogenesis. However, several hormonal and metabolic adaptations are set up in order to restrain hepatic fat accumulation, such as increased mitochondrial fatty acid oxidation (mtFAO). Unfortunately, these adaptations are usually not sufficient to reduce fat accumulation in liver. Furthermore, enhanced mtFAO without concomitant up-regulation of the mitochondrial respiratory chain (MRC) activity induces reactive oxygen species (ROS) overproduction within different MRC components upstream of cytochrome c oxidase. This event seems to play a significant role in the initiation of oxidative stress and subsequent development of nonalcoholic steatohepatitis (NASH) in some individuals. Experimental investigations also pointed to a progressive reduction of MRC activity during NAFLD, which could impair energy output and aggravate ROS overproduction by the damaged MRC. Hence, developing drugs that further increase mtFAO and restore MRC activity in a coordinated manner could ameliorate steatosis, but also necroinflammation and fibrosis by reducing oxidative stress. In contrast, physicians should be aware that numerous drugs in the current pharmacopoeia are able to induce mitochondrial dysfunction, which could aggravate NAFLD in some patients.