Multivariate Statistical Analysis Applied to an IL6 Signal Transduction Model in Hepatocytes

This paper introduces the application of linear multivariate statistical techniques, including partial least squares (PLS), canonical correlation analysis (CCA) and reduced rank regression (RRR), into the area of Systems Biology. This new approach aims to extract the important proteins embedded in complex signal transduction pathway models.The analysis is performed on a model of intracellular signalling along the janus-associated kinases/signal transducers and transcription factors (JAK/STAT) and mitogen activated protein kinases (MAPK) signal transduction pathways in interleukin-6 (IL6) stimulated hepatocytes, which produce signal transducer and activator of transcription factor 3 (STAT3).A region of redundancy within the MAPK pathway that does not affect the STAT3 transcription was identified using CCA. This is the core finding of this analysis and cannot be obtained by inspecting the model by eye. In addition, RRR was found to isolate terms that do not significantly contribute to changes in protein concentrations, while the application of PLS does not provide such a detailed picture by virtue of its construction.This analysis has a similar objective to conventional model reduction techniques with the advantage of maintaining the meaning of the states prior to and after the reduction process. A significant model reduction is performed, with a marginal loss in accuracy, offering a more concise model while maintaining the main influencing factors on the STAT3 transcription.The findings offer a deeper understanding of the reaction terms involved, confirm the relevance of several proteins to the production of Acute Phase Proteins and complement existing findings regarding cross-talk between the two signalling pathways.

AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo

A major problem in gene therapy and tissue replacement is accessibility of tissue-specific stem cells. One solution is to isolate tissue-specific stem cells from differentiating embryonic stem (ES) cells. Here, we show that liver progenitor cells can be purified from differentiated ES cells using alpha-fetoprotein (AFP) as a marker. By knocking the green fluorescent protein (GFP) gene into the AFP locus of ES cells and differentiating the modified ES cells in vitro, a subpopulation of GFP(+) and AFP-expressing cells was generated. When transplanted into partially hepatectomized lacZ-positive ROSA26 mice, GFP(+) cells engrafted and differentiated into lacZ-negative and albumin-positive hepatocytes. Differentiation into hepatocytes also occurred after transplantation of GFP(+) cells in apolipoprotein-E- (ApoE) or haptoglobin-deficient mice as demonstrated by the presence of ApoE-positive hepatocytes and ApoE mRNA in the liver of ApoE-deficient mice or by haptoglobin in the serum and haptoglobin mRNA in the liver of haptoglobin-deficient mice. This study describes the first isolation of ES-cell-derived liver progenitor cells that are viable mediators of liver-specific functions in vivo.

Regulation of lipid metabolism in adipocytes and hepatocytes by hexarelin through scavenger receptor CD36

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides.

Targeted Stimuli-Responsive Dextran Conjugates for Doxorubicin Delivery to Hepatocytes

A targeted, stimuli-responsive, polymeric drug delivery vehicle is being developed in our lab to help alleviate severe side-effects caused by narrow therapeutic window drugs. Targeting specific cell types or organs via proteins, specifically, lectin-mediated targeting holds potential due to the high specificity and affinity of receptor-ligand interactions, rapid internalization, and relative ease of processing. Dextran, a commercially available, biodegradable polymer has been conjugated to doxorubicin and galactosamine to target hepatocytes in a three-step, one-pot synthesis. The loading of doxorubicin and galactose on the conjugates was determined by absorbance at 485 nm and elemental analysis, respectively. Conjugation efficiency based on the amount loaded of each reactant varies from 20% to 50% for doxorubicin and from 2% to 20% for galactosamine. Doxorubicin has also been attached to dextran through an acid-labile hydrazide bond. Doxorubicin acts by intercalating with DNA in the nuclei of cells. The fluorescence of doxorubicin is quenched when it binds to DNA. This allows a fluorescence-based cell-free assay to evaluate the efficacy of the polymer conjugates where we measure the fluorescence of doxorubicin and the conjugates in increasing concentrations of calf thymus DNA. Fluorescence quenching indicates that our conjugates can bind to DNA. The degree of binding increases with polymer molecular weight and substitution of doxorubicin. In cell culture experiments with hepatocytes, the relative uptake of polymer conjugates was evaluated using flow cytometry, and the killing efficiency was determined using the MTT cell proliferation assay. We have found that conjugate uptake is much lower than that of free doxorubicin. Lower uptake of conjugates may increase the maximum dose of drug tolerated by the body. Also, non-galactosylated conjugate uptake is lower than that of the galactosylated conjugate. Microscopy indicates that doxorubicin localizes almost exclusively at the nucleus, whereas the conjugates are present throughout the cell. Doxorubicin linked to dextran through a hydrazide bond was used to achieve improved killing efficiency. Following uptake, the doxorubicin dissociates from the polymer in an endosomal compartment and diffuses to the nucleus. The LC₅₀ of covalently linked doxorubicin is 7.4 μg/mL, whereas that of hydrazide linked doxorubicin is 4.4 μg/mL.

Mathematical model for low density lipoprotein (LDL) endocytosis by hepatocytes

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL-LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed.

Mathematical modelling of competitive LDL/VLDL binding and uptake by hepatocytes

Elevated levels of low-density-lipoprotein cholesterol (LDL-C) in the plasma are a well-established risk factor for the development of coronary heart disease. Plasma LDL-C levels are in part determined by the rate at which LDL particles are removed from the bloodstream by hepatic uptake. The uptake of LDL by mammalian liver cells occurs mainly via receptor-mediated endocytosis, a process which entails the binding of these particles to specific receptors in specialised areas of the cell surface, the subsequent internalization of the receptor-lipoprotein complex, and ultimately the degradation and release of the ingested lipoproteins' constituent parts. We formulate a mathematical model to study the binding and internalization (endocytosis) of LDL and VLDL particles by hepatocytes in culture. The system of ordinary differential equations, which includes a cholesterol-dependent pit production term representing feedback regulation of surface receptors in response to intracellular cholesterol levels, is analysed using numerical simulations and steady-state analysis. Our numerical results show good agreement with in vitro experimental data describing LDL uptake by cultured hepatocytes following delivery of a single bolus of lipoprotein. Our model is adapted in order to reflect the in vivo situation, in which lipoproteins are continuously delivered to the hepatocyte. In this case, our model suggests that the competition between the LDL and VLDL particles for binding to the pits on the cell surface affects the intracellular cholesterol concentration. In particular, we predict that when there is continuous delivery of low levels of lipoproteins to the cell surface, more VLDL than LDL occupies the pit, since VLDL are better competitors for receptor binding. VLDL have a cholesterol content comparable to LDL particles; however, due to the larger size of VLDL, one pit-bound VLDL particle blocks binding of several LDLs, and there is a resultant drop in the intracellular cholesterol level. When there is continuous delivery of lipoprotein at high levels to the hepatocytes, VLDL particles still out-compete LDL particles for receptor binding, and consequently more VLDL than LDL particles occupy the pit. Although the maximum intracellular cholesterol level is similar for high and low levels of lipoprotein delivery, the maximum is reached more rapidly when the lipoprotein delivery rates are high. The implications of these results for the design of in vitro experiments is discussed.

The effect of sixteen medicinal plants used in the Brazilian pharmacopoeia on the expression and activity of glutathione S-transferase in hepatocytes and leukemia cells

Glutathione S-transferase (GST) is a family of enzymes involved in the detoxification of electrophilic compounds. Different classes of GST are expressed in various organs, such as liver, lungs, stomach and others. Expression of GST can be modulated by diet components and plant-derived compounds. The importance of controlling GST expression is twofold: increasing levels of GST are beneficial to prevent deleterious effects of toxic and carcinogenic compounds, while inhibition of GST in tumor cells may help overcoming tumor resistance to chemotherapy. A screening of 16 plants used in the Brazilian pharmacopoeia tested their effects on GST expression in hepatocytes and Jurkat (leukemia) T-cells. The methanol extracts of five plants inhibited GST expression in hepatocytes. Three plants significantly inhibited and four others induced GST expression in Jurkat cells. Among these, the extracts of Bauhinia forficata Link. (Leguminosae) and Cecropia pachystachya Trec. (Urticaceae) inhibited GST expression at relatively low concentrations. With the exception of B. forficata, all plants were cytotoxic when administered to Jurkat cells at high doses (1 mg/mL) and some extracts were considerably cytotoxic even at lower concentrations.

Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics. (C) 2009 Elsevier Inc. All rights reserved.

The small molecule, genistein, increases hepcidin expression in human hepatocytes

Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin-6 (IL-6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28-52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole-mount in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction. Genistein's stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3-binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein-treated hepatocytes indicated that genistein up-regulated 68% of the transcripts that were up-regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up-regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. Conclusion: Genistein is the first small-molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.