AIM： To investigate and compare the hepatogenic transdifferentiation of adipose tissue-derived stem cells （ADSC） and bone marrow-derived mesenchymal stem cells （BMSC） in vitro. Transdifferentiation ...of BMSC into hepatic cells in vivo has been described. Adipose tissue represents an accessible source of ADSC, with similar characteristics to BMSC. METHODS： BMSCs were obtained from patients undergoing total hip arthroplasty and ADSC from human adipose tissue obtained from lipectomy. Cells were grown in medium containing 15% human serum. Cultures were serum deprived for 2 d before cultivating under similar pro-hepatogenic conditions to those of liver development using a 2-step protocol with sequential addition of growth factors, cytokines and hormones. Hepatic differentiation was RT-PCR-assessed and liver-marker genes were immunohistochemically analysed.RESULTS： BMSC and ADSC exhibited a fibroblastic morphology that changed to a polygonal shape when cells differentiated. Expression of stem cell marker Thyl decreased in differentiated ADSC and BMSC. However, the expression of the hepatic markers, albumin and CYPs increased to a similar extent in differentiated BMSC and ADSC. Hepatic gene activation could be attributed to increased liver-enriched transcription factors （C/EBPβ and HNF4α）, as demonstrated by adenoviral expression vectors.CONCLUSION： Mesenchymal stem cells can be induced to hepatogenic transdifferentiation in vitro. ADSCs have a similar hepatogenic differentiation potential to BMSC, but a longer culture period and higher proliferation capacity. Therefore, adipose tissue may be an ideal source of large amounts of autologous stem cells, and may become an alternative for hepatocyte regeneration, liver cell transplantation or preclinical drug testing.
The present study was designed to define an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the metabolic and cytotoxic/apoptotic effects could be ...separated. This was accomplished by defining the experimental conditions of lipid exposure that lead to significant intracellular fat accumulation in the absence of overt cytotoxicity, therefore allowing to differentiate between cytotoxic and apoptotic effects. Palmitic (C16:0) and oleic (C18:1) acids are the most abundant fatty acids (FFAs) in liver triglycerides in both normal subjects and patients with nonalcoholic fatty liver disease (NAFLD). Therefore, human hepatocytes and HepG2 cells were incubated with a mixture of different proportions of saturated (palmitate) and unsaturated (oleate) FFAs to induce fat-overloading. Similar intracellular levels of lipid accumulation as in the human steatotic liver were achieved. Individual FFAs have a distinct inherent toxic potential. Fat accumulation, cytotoxicity and apoptosis in cells exposed to the FFA mixtures were investigated. The FFA mixture containing a low proportion of palmitic acid (oleate/palmitate, 2:1 ratio) is associated with minor toxic and apoptotic effects, thus representing a cellular model of steatosis that mimics benign chronic steatosis. On the other hand, a high proportion of palmitic acid (oleate/palmitate, 0:3 ratio) might represent a cellular model of steatosis in which saturated FFAs promote an acute harmful effect of fat overaccumulation in the liver. These hepatic cellular models are apparently suitable to experimentally investigate the impact of fat overaccumulation in the liver excluding other factors that could influence hepatocyte behaviour.
Triglyceride accumulation in nonalcoholic fatty liver (NAFL) results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged ...helix/forkhead box (Fox) transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB). Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.
Scope
Gut microbiota contributes to non‐alcoholic fatty liver disease (NAFLD) pathogenesis by multiple mechanisms not yet completely understood. Novel differential features between germ‐free mice ...(GFm) transplanted with protective or non‐protective cecal microbiota against NAFLD are investigated.
Methods and results
Gut microbiota composition, plasma, and fecal bile acids (BAs) and liver mRNAs are quantified in GFm recipients from four donor mice differing in NAFLD severity (control diet, high‐fat diet HFD‐responder, HFD‐non‐responder, and quercetin‐supplemented HFD). Transplanted GFm are on control or HFD for 16‐weeks. Multivariate analysis shows that GFm colonized with microbiota from HFD‐non‐responder and quercetin supplemented‐HFD donors (protected against NAFLD) clusters together, whereas GFm colonized with microbiota from control and HFD‐responder mice (non‐protected against NAFLD) establishes another cluster. Protected phenotype is associated with increased gut Desulfovibrio and Oscillospira, reduced gut Bacteroides and Oribacterium, lower primary and higher secondary BAs in plasma and feces, induction of hepatic BA transporters, and repression of hepatic lipogenic and BA synthesis genes.
Conclusion
Protective gut microbiota associates with increased specific secondary BAs, which likely inhibit lipogenic pathways and enhance bile flow in the liver. This novel cross‐talk between gut and liver, via plasma BAs, that promotes protection against NAFLD may have clinical and nutritional relevance.
A microbiota‐derived protected phenotype against non‐alcoholic fatty liver disease (NAFLD) in mice is reported. This phenotype associates with increased gut Desulfovibrio and Oscillospira, reduced gut Bacteroides and Oribacterium, lower primary and higher secondary bile acids (Bas) in plasma and feces, induction of hepatic BA transporters, and repression of hepatic lipogenic and BA synthesis genes.
The vitamin D receptor (VDR) must be relevant to liver lipid metabolism because
deficient mice are protected from hepatosteatosis. Therefore, our objective was to define the role of VDR on the ...overall lipid metabolism in human hepatocytes. We developed an adenoviral vector for human VDR and performed transcriptomic and metabolomic analyses of cultured human hepatocytes upon VDR activation by vitamin D (VitD). Twenty percent of the VDR responsive genes were related to lipid metabolism, including
,
,
, and
(glycerolipid metabolism);
,
, and
(phospholipid metabolism); and
,
, and
(uptake of fatty acids, betaine, and glycerol, respectively). They were rapidly induced (4-6 h) upon VDR activation by 10 nM VitD or 100 µM lithocholic acid (LCA). Most of these genes were also upregulated by VDR/VitD in mouse livers in vivo. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) metabolomics demonstrated intracellular accumulation of triglycerides, with concomitant decreases in diglycerides and phosphatidates, at 8 and 24 h upon VDR activation. Significant alterations in phosphatidylcholines, increases in lyso-phosphatidylcholines and decreases in phosphatidylethanolamines and phosphatidylethanolamine plasmalogens were also observed. In conclusion, active VitD/VDR signaling in hepatocytes triggers an unanticipated coordinated gene response leading to triglyceride synthesis and to important perturbations in glycerolipids and phospholipids.
REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) is a global strategy and regulation policy of the EU that aims to improve the protection of human health and the ...environment through the better and earlier identification of the intrinsic properties of chemical substances. It entered into force on 1st June 2007 (EC 1907/2006). REACH and EU policies plead for the use of robust high-throughput "omic" techniques for the in vitro investigation of the toxicity of chemicals that can provide an estimation of their hazards as well as information regarding the underlying mechanisms of toxicity. In agreement with the 3R's principles, cultured cells are nowadays widely used for this purpose, where metabolomics can provide a real-time picture of the metabolic effects caused by exposure of cells to xenobiotics, enabling the estimations about their toxicological hazards. High quality and robust metabolomics data sets are essential for precise and accurate hazard predictions. Currently, the acquisition of consistent and representative metabolomic data is hampered by experimental drawbacks that hinder reproducibility and difficult robust hazard interpretation. Using the differentiated human liver HepG2 cells as model system, and incubating with hepatotoxic (acetaminophen and valproic acid) and non-hepatotoxic compounds (citric acid), we evaluated in-depth the impact of several key experimental factors (namely, cell passage, processing day and storage time, and compound treatment) and instrumental factors (batch effect) on the outcome of an UPLC-MS metabolomic analysis data set. Results showed that processing day and storage time had a significant impact on the retrieved cell's metabolome, while the effect of cell passage was minor. Meta-analysis of results from pathway analysis showed that batch effect corrections and quality control (QC) measures are critical to enable consistent and meaningful estimations of the effects caused by compounds on cells. The quantitative analysis of the changes in metabolic pathways upon bioactive compound treatment remained consistent despite the concurrent causes of metabolomic data variation. Thus, upon appropriate data retrieval and correction and by an innovative metabolic pathway analysis, the metabolic alteration predictions remained conclusive despite the acknowledged sources of variability.
It is estimated that only a few marketed drugs are able to directly induce liver steatosis. However, many other drugs may exacerbate or precipitate fatty liver in the presence of other risk factors ...or in patients prone to non-alcoholic fatty liver disease. On the other hand, current in vitro tests for drug-induced steatosis in preclinical research are scarce and not very sensitive or reproducible. In the present study, we have investigated the effect of well-characterized steatotic drugs on the expression profile of 47 transcription factors (TFs) in human hepatoma HepG2 cells and found that these drugs are able to up- and down-regulate a substantial number of these factors. Multivariate data analysis revealed a common TF signature for steatotic drugs, which consistently and significantly repressed FOXA1, HEX and SREBP1C in cultured cells. This signature was also observed in the livers of rats and in cultured human hepatocytes. Therefore, we selected these three TFs as predictive biomarkers for iatrogenic steatosis. With these biomarkers, a logistic regression analysis yielded a predictive model, which was able to correctly classify 92 % of drugs. The developed algorithm also predicted that ibuprofen, nifedipine and irinotecan are potential steatotic drugs, whereas troglitazone is not. In summary, this is a sensitive, specific and simple RT-PCR test that can be easily implemented in preclinical drug development to predict drug-induced steatosis. Our results also indicate that steatotic drugs affect expression of both common and specific subsets of TF and lipid metabolism genes, thus generating complex transcriptomic responses that cause or contribute to steatosis in hepatocytes.
MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite ...measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (−) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids. Graphical abstract A novel analytical workflow for the LC-MS-based metabolomic analysis of adherent cultured hepatic cells was developed. Three key steps were evaluated: a) cell harvesting, which includes cell detachment and metabolism quenching; b) metabolite extraction; and c) LC-MS analysis, assessing the use of RP and HILIC chromatographies. The final protocol allowed us to extend the metabolome coverage and enabled the detection of a wide range of metabolites from highly polar compounds to a wide range of lipids
The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug–drug interactions. Drug metabolism is a major ...determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human
in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed
in vitro actually reflect that of the liver
in vivo? By comparing the metabolism of a model compound both
in vitro and
in vivo in the same individual, a good correlation between the
in vitro and
in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.
This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation (HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New ...sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that: (1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver; (2) Organs with steatosis (≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained; (3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT; (4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and (5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a very useful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used.
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