Nonalcoholic Fatty Liver Disease: Focus on Lipoprotein and Lipid Deregulation

Obesity with associated comorbidities is currently a worldwide epidemic and among the most challenging health conditions in the 21st century. A major metabolic consequence of obesity is insulin resistance which underlies the pathogenesis of the metabolic syndrome. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. It comprises a disease spectrum ranging from simple steatosis (fatty liver), through nonalcoholic steatohepatitis (NASH) to fibrosis, and ultimately liver cirrhosis. Abnormality in lipid and lipoprotein metabolism accompanied by chronic inflammation is the central pathway for the development of metabolic syndrome-related diseases, such as atherosclerosis, cardiovascular disease (CVD), and NAFLD. This paper focuses on pathogenic aspect of lipid and lipoprotein metabolism in NAFLD and the relevant mouse models of this complex multifactorial disease

Determination of reference genes for circadian studies in different tissues and mouse strains

<p>Abstract</p> <p>Background</p> <p>Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration.</p> <p>Results</p> <p>In the present study the expression of ten candidate reference genes (<it>Actb</it>, <it>Eif2a</it>, <it>Gapdh</it>, <it>Hmbs</it>, <it>Hprt1</it>, <it>Ppib</it>, <it>Rn18s</it>, <it>Rplp0</it>, <it>Tbcc </it>and <it>Utp6c</it>) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and <it>Crem </it>KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (<it>Actb</it>) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons.</p> <p>Conclusions</p> <p>Genetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.</p

Effect of CAR activation on selected metabolic pathways in normal and hyperlipidemic mouse livers

<p>Abstract</p> <p>Background</p> <p>Detoxification in the liver involves activation of nuclear receptors, such as the constitutive androstane receptor (CAR), which regulate downstream genes of xenobiotic metabolism. Frequently, the metabolism of endobiotics is also modulated, resulting in potentially harmful effects. We therefore used 1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) to study the effect of CAR activation on mouse hepatic transcriptome and lipid metabolome under conditions of diet-induced hyperlipidemia.</p> <p>Results</p> <p>Using gene expression profiling with a dedicated microarray, we show that xenobiotic metabolism, PPARα and adipocytokine signaling, and steroid synthesis are the pathways most affected by TCPOBOP in normal and hyperlipidemic mice. TCPOBOP-induced CAR activation prevented the increased hepatic and serum cholesterol caused by feeding mice a diet containing 1% cholesterol. We show that this is due to increased bile acid metabolism and up-regulated removal of LDL, even though TCPOBOP increased cholesterol synthesis under conditions of hyperlipidemia. Up-regulation of cholesterol synthesis was not accompanied by an increase in mature SREBP2 protein. As determined by studies in CAR -/- mice, up-regulation of cholesterol synthesis is however CAR-dependent; and no obvious CAR binding sites were detected in promoters of cholesterogenic genes. TCPOBOP also affected serum glucose and triglyceride levels and other metabolic processes in the liver, irrespective of the diet.</p> <p>Conclusion</p> <p>Our data show that CAR activation modulates hepatic metabolism by lowering cholesterol and glucose levels, through effects on PPARα and adiponectin signaling pathways, and by compromising liver adaptations to hyperlipidemia.</p

Novel Insights into the Downstream Pathways and Targets Controlled by Transcription Factors CREM in the Testis

The essential role of the Crem gene in normal sperm development is widely accepted and is confirmed by azoospermia in male mice lacking the Crem gene. The exact number of genes affected by Crem absence is not known, however a large difference has been observed recently between the estimated number of differentially expressed genes found in Crem knock-out (KO) mice compared to the number of gene loci bound by CREM. We therefore re-examined global gene expression in male mice lacking the Crem gene using whole genome transcriptome analysis with Affymetrix microarrays and compared the lists of differentially expressed genes from Crem−/− mice to a dataset of genes where binding of CREM was determined by Chip-seq. We determined the global effect of CREM on spermatogenesis as well as distinguished between primary and secondary effects of the CREM absence. We demonstrated that the absence of Crem deregulates over 4700 genes in KO testis. Among them are 101 genes associated with spermatogenesis 41 of which are bound by CREM and are deregulated in Crem KO testis. Absence of several of these genes in mouse models has proven their importance for normal spermatogenesis and male fertility. Our study showed that the absence of Crem plays a more important role on different aspects of spermatogenesis as estimated previously, with its impact ranging from apoptosis induction to deregulation of major circadian clock genes, steroidogenesis and the cell-cell junction dynamics. Several new genes important for normal spermatogenesis and fertility are down-regulated in KO testis and are therefore possible novel targets of CREM

Possible role of estrogen metabolism and aldo-keto reductase activity in chemoresistance of ovarian cancer

High-grade serous ovarian cancer (HGSOC) is the most aggressive and chemoresistant form of epithelial ovarian cancer (OC) and is responsible for ~80% of OC-related deaths. OC is associated with disturbed estrogen action. In postmenopausal patients, estrogens are formed locally from steroid precursors. Enzymes of the AKR1C subfamily are associated with resistance to chemotherapeutic agents and are involved in the biosynthesis and metabolism of steroid hormones, thus may contribute to the growth of hormone-dependent tumors. To date, the interplay of estrogen synthesis and aldo-keto reductase activity in HGSOC chemoresistance remains unclear. The aim of this study was to investigate the differences in targeted transcriptomics of HGSOC cell lines with different sensitivity to carboplatin: OVSAHO, OVCAR-3, Kuramochi, OVCAR-4, Caov- 3, and COV362, and to evaluate the differences in correlation patterns between targeted gene expression profiles in platinum-sensitive and -resistant patients using publicly available data (PAD) (cBioPortal). We first determined the expression of genes involved in estrogen biosynthesis/metabolism (STS, SULT1E1, HSD17B1, HSD17B2, HSD17B14, PAPSS1, PAPSS2), steroid transport (SLCO1A2, SLCO1B3, SLCO2B1, SLCO4A1, SLCO4C1, ABCC1, ABCC4, ABCC11, ABCG2, SLC51A, SLC51B), estrogen action (ESR1, ESR2, GPER) and oxidative metabolism (CYP1A1, CYP1A2, CYP1B1, SULT1A1, SULT2B1, SULT1E1, UGTB7, COMT, NOQ1, NOQ2, GSTP1), NFE2L2 and AKR1C1-3 by qPCR. Next, by using PAD we conducted a correlation analysis using the Pearson correlation coefficient for gene expression data of targeted genes in OC patients. The patients were classified into two groups based on their response to platinum treatment: sensitive and resistant. The correlation matrix was computed independently for each group. Expression analysis revealed that the estrogen receptor ESR2, the efflux transporter ABCG2 and aldo-keto reductase AKR1C1 were highly expressed in the most resistant cell lines COV362 and Caov-3. The mRNA levels of estrogen biosynthesis and oxidative metabolism genes STS, HSD17B14, NOQ1, and GSTP1 increased with carboplatin resistance in the HGSOC cell lines. These results indicate the potential of ESR2, STS, HSD17B14, NOQ1, GSTP1, and ABCG2 as predictive markers for HGSOC chemoresistance. Furthermore, analysis of PAD revealed different correlation profiles between genes in sensitive and resistant patients. In chemoresistant were found a moderately to strong positive correlations (p<0.001) between gene pairs including AKR1C1– AKR1C3, AKR1C1 – NFE2L2, AKR1C1 – SULT1E1, NOQ1 – HSD17B14, COMT – SULT1A1, ABCG2 – SLC515. In chemosensitive patients was found a strong positive correlation (p<0.001) between gene pair CYP1B1 – SULT1E1. The correlation differences between sensitive and resistant OC patients suggest possible gene regulatory networks or molecular interactions contributing to the heterogeneity of response to platinum in OC. Further studies are ongoing to elucidate the mechanism of the interplay between local estrogen metabolism and aldo-keto reductase activity in HGSOC chemoresistanceBook of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

The Sterolgene v0 cDNA microarray: a systemic approach to studies of cholesterol homeostasis and drug metabolism

<p>Abstract</p> <p>Background</p> <p>Cholesterol homeostasis and xenobiotic metabolism are complex biological processes, which are difficult to study with traditional methods. Deciphering complex regulation and response of these two processes to different factors is crucial also for understanding of disease development. Systems biology tools as are microarrays can importantly contribute to this knowledge and can also discover novel interactions between the two processes.</p> <p>Results</p> <p>We have developed a low density Sterolgene v0 cDNA microarray dedicated to studies of cholesterol homeostasis and drug metabolism in the mouse. To illustrate its performance, we have analyzed mouse liver samples from studies focused on regulation of cholesterol homeostasis and drug metabolism by diet, drugs and inflammation. We observed down-regulation of cholesterol biosynthesis during fasting and high-cholesterol diet and subsequent up-regulation by inflammation. Drug metabolism was down-regulated by fasting and inflammation, but up-regulated by phenobarbital treatment and high-cholesterol diet. Additionally, the performance of the Sterolgene v0 was compared to the two commercial high density microarray platforms: the Agilent cDNA (G4104A) and the Affymetrix MOE430A GeneChip. We hybridized identical RNA samples to the commercial microarrays and showed that the performance of Sterolgene is comparable to commercial arrays in terms of detection of changes in cholesterol homeostasis and drug metabolism.</p> <p>Conclusion</p> <p>Using the Sterolgene v0 microarray we were able to detect important changes in cholesterol homeostasis and drug metabolism caused by diet, drugs and inflammation. Together with its next generations the Sterolgene microarrays represent original and dedicated tools enabling focused and cost effective studies of cholesterol homeostasis and drug metabolism. These microarrays have the potential of being further developed into screening or diagnostic tools.</p

The role of bile acids in carcinogenesis

Bile acids are soluble derivatives of cholesterol produced in the liver that subsequently undergo bacterial transformation yielding a diverse array of metabolites. The bulk of bile acid synthesis takes place in the liver yielding primary bile acids; however, other tissues have also the capacity to generate bile acids (e.g. ovaries). Hepatic bile acids are then transported to bile and are subsequently released into the intestines. In the large intestine, a fraction of primary bile acids is converted to secondary bile acids by gut bacteria. The majority of the intestinal bile acids undergo reuptake and return to the liver. A small fraction of secondary and primary bile acids remains in the circulation and exert receptor-mediated and pure chemical effects (e.g. acidic bile in oesophageal cancer) on cancer cells. In this review, we assess how changes to bile acid biosynthesis, bile acid flux and local bile acid concentration modulate the behavior of different cancers. Here, we present in-depth the involvement of bile acids in oesophageal, gastric, hepatocellular, pancreatic, colorectal, breast, prostate, ovarian cancer. Previous studies often used bile acids in supraphysiological concentration, sometimes in concentrations 1000 times higher than the highest reported tissue or serum concentrations likely eliciting unspecific effects, a practice that we advocate against in this review. Furthermore, we show that, although bile acids were classically considered as pro-carcinogenic agents (e.g. oesophageal cancer), the dogma that switch, as lower concentrations of bile acids that correspond to their serum or tissue reference concentration possess anticancer activity in a subset of cancers. Differences in the response of cancers to bile acids lie in the differential expression of bile acid receptors between cancers (e.g. FXR vs. TGR5). UDCA, a bile acid that is sold as a generic medication against cholestasis or biliary surge, and its conjugates were identified with almost purely anticancer features suggesting a possibility for drug repurposing. Taken together, bile acids were considered as tumor inducers or tumor promoter molecules; nevertheless, in certain cancers, like breast cancer, bile acids in their reference concentrations may act as tumor suppressors suggesting a Janus-faced nature of bile acids in carcinogenesis

Detecting gene–gene interactions from GWAS using diffusion kernel principal components

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