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.

Alpha-1 antitrypsin deficiency: A conformational disease associated with lung and liver manifestations

Alpha-1 antitrypsin (A1AT) is a serine anti-protease produced chiefly by the liver. A1AT deficiency is a genetic disorder characterized by serum levels of less than 11 μmol/L and is associated with liver and lung manifestations. The liver disease, which occurs in up to 15% of A1AT-deficient individuals, is a result of toxic gain-of-function mutations in the A1AT gene, which cause the A1AT protein to fold aberrantly and accumulate in the endoplasmic reticulum of hepatocytes. The lung disease is associated with loss-of-function, specifically decreased anti-protease protection on the airway epithelial surface. The so-called 'Z' mutation in A1AT deficiency encodes a glutamic acid-to-lysine substitution at position 342 in A1AT and is the most common A1AT allele associated with disease. Here we review the current understanding of the molecular pathogenesis of A1AT deficiency and the best clinical management protocols. © Springer Science+Business Media B.V. 2008.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

Misonidazole binding in murine liver tissue: a marker for cellular hypoxia in vivo

The hepatic microcirculation is believed to cause variable cellular oxygenation within the organ. In this study a marker of cellular hypoxia was used to demonstrate liver oxygen tension gradients in vivo. Covalent binding of misonidazole adducts to cellular macromolecules is enhanced by hypoxia. Autoradiographs of liver from mice treated with radiolabeled misonidazole demonstrated enhanced binding of adducts within hepatocytes surrounding hepatic veins. Livers from both hypoxic and normal mice had characteristic autoradiographic grain patterns reflecting regional oxygen tension variation in vivo. Differential binding of misonidazole adducts formed in hypoxic cells could have an application in studies of liver physiology and biochemistry.

Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.

Macroautophagic cargo sequestration assays

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy.

Localization of polymorphic N-acetyltransferase (NAT2) in tissues of inbred mice

Like humans, mice exhibit polymorphism in the N-acetylation of aromatic amines, many of which are toxic and/or carcinogenic. Mice have three N-acetyltransferase (Nat) genes, Nat1, Nat2 and Nat3, and Nat2 is known to be polymorphic. There is a dramatic difference in the acetylation of NAT2 substrates by blood from fast (C57BL/6J) compared with slow acetylator (A/J) mice. However, the acetylation of these substrates by liver cytosols from the two strains is very similar. In order to determine whether the expression of the NAT2 protein corresponded with the activities measured, a polyclonal antipeptide antisera was raised against the C-terminal decapeptide of NAT2 and characterized using recombinant murine NAT2 antigen. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that the anti-NAT2 antiserum bound in a concentration-dependent fashion to recombinant NAT2. Immunochemical analysis of mouse liver cytosols from C57BL/6J or A/J livers indicated that the level of NAT2 protein expressed in the two strains was similar. Immunohistochemical staining of C57BL/6J liver with anti-NAT2 antiserum showed that NAT2 was expressed in hepatocytes throughout the liver although the intensity of staining in the perivenous (centrilobular) region was higher than that in the periportal region. NAT2 was also detected in epithelial cells in the lung, kidney, bladder, small intestine and skin as well as in erythrocytes and lymphocytes in the spleen and hair follicles and sebaceous glands in the skin. Characterization of the distribution of NAT2 will be of value in elucidating the role of polymorphic N-acetylation in protecting the organism from environmental insults as well as in endogenous metabolism.

Vitamin D and insulin resistance

Vitamin D is a steroid hormone, which in active form binds to the vitamin D receptor. Expression of the vitamin D receptor in diverse cell types (pancreatic islet cells, myocytes, hepatocytes and adipocytes) raises the suspicion that vitamin D may be involved in multiple cellular processes, including the response to insulin. Insulin resistance is a characteristic feature of type 2 DM, and its attenuation may reduce the incidence of type 2 DM and cardiovascular disease. In observational studies, low serum 25-hydroxyvitamin D (25-OHD) concentrations are associated with an increased risk of type 2 DM. It has been suggested that increasing serum 25-OHD concentrations may have beneficial effects on glucose and insulin homeostasis. However, cross-sectional and interventional studies of vitamin D supplementation provide conflicting results and demonstrate no clear beneficial effect of vitamin D on insulin resistance. These studies are complicated by inclusion of different patient cohorts, different 25-OHD assays and different doses and preparations of vitamin D. Any possible association may be confounded by alterations in PTH, 1,25-dihydroxyvitamin D or tissue vitamin D concentrations. We identified 39 studies via MEDLINE and PUBMED. We review the evidence from 10 studies (seven observational and three interventional) examining vitamin D and type 2 DM incidence, and 29 studies (one prospective observational, 12 cross-sectional and 16 interventional trials) examining vitamin D and insulin resistance. Based on this data, it is not possible to state that vitamin D supplementation has any effect on type 2 DM incidence or on insulin resistance. Data from the multiple ongoing randomized controlled trials of vitamin D supplementation due to report over the next few years should help to clarify this area.