Effect of endurance exercise training on liver gene expression in male and female mice.

Chronic endurance exercise is a therapeutic strategy in the treatment of many chronic diseases in humans, including the prevention and treatment of metabolic diseases such as diabetes mellitus. Metabolic, cardiorespiratory, and endocrine pathways targeted by chronic endurance exercise have been identified. In the liver, however, the cellular and molecular pathways that are modified by exercise and have preventive or therapeutic relevance to metabolic disease need to be elucidated. The mouse model used in the current study allows for the quantification of a human-relevant exercise "dosage". In this study we show hepatic gene expression differences between sedentary female and sedentary male mice and that chronic exercise modifies the transcription of hepatic genes related to metabolic disease and steatosis in both male and female mice. Chronic exercise induces molecular pathways involved in glucose tolerance, glycolysis, and gluconeogenesis while producing a decrease in pathways related to insulin resistance, steatosis, fibrosis, and inflammation. Given these findings, this mouse exercise model has potential to dissect the cellular and molecular hepatic changes following chronic exercise with application to understanding the role that chronic exercise plays in preventing human diseases. Novelty: Exercise modifies the hepatic gene expression and hepatic pathways related to metabolic disease in male and female mice. Sex differences were seen in hepatic gene expression between sedentary and exercised mice. The mouse exercise model used in this study allows for application and evaluation of exercise effects in human disease.

Evaluation of impedance-based label-free technology as a tool for pharmacology and toxicology investigations.

The use of label-free technologies based on electrical impedance is becoming more and more popular in drug discovery. Indeed, such a methodology allows the continuous monitoring of diverse cellular processes, including proliferation, migration, cytotoxicity and receptor-mediated signaling. The objective of the present study was to further assess the usefulness of the real-time cell analyzer (RTCA) and, in particular, the xCELLigence platform, in the context of early drug development for pharmacology and toxicology investigations. In the present manuscript, four cellular models were exposed to 50 compounds to compare the cell index generated by RTCA and cell viability measured with a traditional viability assay. The data revealed an acceptable correlation (ca. 80%) for both cell lines (i.e., HepG2 and HepaRG), but a lack of correlation (ca. 55%) for the primary human and rat hepatocytes. In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models. In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not. Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52). Overall, despite some limitations, the xCELLigence platform is a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies.

The use of real-time cell analyzer technology in drug discovery: defining optimal cell culture conditions and assay reproducibility with different adherent cellular models.

The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p < 0.0001). The data also show that a cell index decrease is not always associated with cytotoxicity effects and that there are some confounding factors that can affect the analysis. Finally, another drawback is that the correlation analysis between cellular impedance measurements and classical toxicity endpoints has been performed on a limited number of compounds. Overall, despite some limitations, the RTCA technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.

Determination of phospholipidosis potential based on gene expression analysis in HepG2 cells.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.

Possible role for insulin-like growth factor-I in the pathogenesis of cystic endometrial hyperplasia pyometra complex in the bitch.

Cystic endometrial hyperplasia (CEH) is an important pathologic condition in the canine uterus and recognized as a common cause of illness and death in this species. The underlying cause and pathogenic mechanism responsible for this condition remains incompletely understood. Aberrant sex steroid hormone receptor expression in the uterus of dogs with CEH has been documented but not explained. In the dog there is an exceptionally high, progestin induced production of insulin-like growth factor-I (IGF-I) which is now generally accepted to be one of the most important growth factors with a high mitogenic effect on the uterus. Therefore, in this study the immunohistochemical staining intensity for IGF-I was compared among the uteri of 25 adult female dogs that had developed CEH and 14 healthy dogs in comparable stages of the estrus cycle. Specific staining for IGF-I was found in the cytoplasm epithelial cells and in smooth muscle cells of endometrium and myometrium. A marked increase in specific staining intensity for IGF-I was found in the surface epithelium, glandular epithelium and in the stroma of the uteri of dogs with CEH. The increase in IGF-I specific staining intensity was most prominent in the superficial endometrial stroma. Based on the known role of IGF-I in endometrial proliferation, it was concluded from the present study that high concentrations of IGF-I located in and around the epithelial cells of the endometrium in dogs with CEH, could play an important role in the development of CEH.