Liver gene expression in relation to hepatic steatosis and lipid secretion in two duck species.

The susceptibility to development of hepatic steatosis is known to differ between Muscovy and Pekin ducks. Although some experiments were conducted to decipher these differences, few data have been produced to analyse the role of specific genes in this process. For this purpose, expression levels of genes involved in lipid (ATP citrate lyase, malic enzyme 1, fatty acid synthase, stearoyl-CoA desaturase 1, diacylglycerol O-acyl transferase 2, microsomal triglyceride transfer protein, apolipoprotein A1, apolipoprotein B, sterol regulatory element binding factor 1, hepatocyte nuclear factor 4, choline/ethanolamine phosphotransferase 1, carnitine palmitoyl transferase 1A, peroxisome proliferator-activated receptor alpha and sterol O-acyltransferase) and carbohydrate (activating transcription factor 4 or cAMP-response element binding protein, mitochondrial malate dehydrogenase 2 and carbohydrate responsive element binding protein) metabolism and in other functions were analysed in the liver of Pekin and Muscovy ducks fed ad libitum or overfed. A specific positive effect of feeding was observed on the expression of genes involved mainly in fatty acids and TG synthesis and glycolysis, and negative effect on genes involved in beta-oxidation. Interestingly, a strong species effect was also observed on stearoyl-CoA desaturase 1 and to a lesser extent on diacylglycerol O-acyl transferase 2 expression, leading to large differences in expression levels between Pekin and Muscovy overfed ducks, which could explain the difference in lipid metabolism and steatosis ability observed between the two duck species. These results should shed light on gene expression that might underlie susceptibility to hepatic steatosis in humans.

Quantitative real-time PCR primer design, cDNA amplification and sequence analysis from 22 genes mainly associated with lipid metabolism in Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) ducks.

Few genomic tools are available in ducks. To produce some new resources, we have designed Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) duck-specific primers for 22 genes involved mainly in lipid metabolism, and to a lesser extent in carbohydrate metabolism and other functions. Primers were designed according to duck sequences when available and otherwise from the corresponding conserved regions in chicken and human sequences. These primers allowed quantitative RT-PCR amplification of RNA from Pekin and Muscovy ducks. Amplified cDNA products from both species were sequenced and were found to be very similar to chicken sequences (about 94%). This work provides additional genomic resources and polymorphism information for some genes in duck species and represents a first step towards gene expression analyses in Pekin and Muscovy ducks.

Adipogenic genes expression in relation to hepatic steatosis in the liver of two duck species.

Some studies have shown that expression of peroxisome proliferator-activated receptor gamma (PPARG), a key regulator of adipogenesis, and of some adipocyte-specific genes or adipokines are expressed in hepatic steatosis, leading to the concept of 'adipogenic hepatic steatosis' or 'hepatic adiposis.' Most of these studies were conducted in genetic obese mouse models or after manipulation of gene expression. The relevance of this concept to other species and more physiological models was here addressed in ducks which are able to develop hepatic steatosis after overfeeding. The expression of PPARG and other adipocyte-specific genes was thus analyzed in the liver of ducks fed ad libitum or overfed and compared with those observed in adipose tissues. Pekin (Anas platyrhynchos) and Muscovy ducks (Cairina moschata) were analyzed, as metabolic responses to overfeeding differ according to these two species, Muscovy ducks having a greater ability to synthesize and store lipids in the liver than Pekin ducks. Our results indicate that adipocyte-specific genes are expressed in the liver of ducks, PPARG and fatty acid-binding protein 4 being upregulated and adiponectin and leptin receptor downregulated by overfeeding. However, these expression levels are much lower than those observed in adipose tissue suggesting that fatty liver cells are not transformed to adipocytes, although some hepato-specific functions are decreased in fatty liver when compared with normal liver.

A new hypotensive polyunsaturated fatty acid dietary combination regulates oleic acid accumulation by suppression of stearoyl CoA desaturase 1 gene expression in the SHR model of genetic hypertension.

Polyunsaturated fatty acids (PUFA) are known to repress SCD-1 gene expression, key enzyme of monounsaturated fatty acid biosynthesis. Alterations of the monounsaturated/saturated fatty acids ratio have been implicated in various diseases related to the metabolic syndrome, including hypertension. We previously evidenced that lipogenesis end-products accumulated in spontaneously hypertensive rats (SHR), and that a dietary combination of n-6/n-3 PUFA had hypotensive effects. Our present objective was to test the hypothesis that these SHR liver lipid disorders might be modulated, in response to this hypotensive combination, by changes in SCD-1 expression and activity. So we studied, in hepatocytes, SCD-1 transcription by Northern blotting, as well as plasma and liver fatty acid composition by gas-liquid chromatography. Liver SCD-1 gene expression was suppressed by 50%, and in different lipid classes, relative abundance of stearic and oleic acids decreased. Consequently, the Delta9 desaturation index, calculated from the ratio of oleic vs. stearic acids, decreased. In addition, the level of circulating saturated fatty acids decreased when one of oleic acids increased. These data provided evidence that the tested hypotensive PUFA combination reverses the high monounsaturated/saturated fatty acids ratio associated to hypertension in SHR, via a regulation monounsaturated fatty acid relative abundance by repression of SCD-1 gene.

Triglyceride synthesis and secretion and lipogenesis implicated gene expression in the chicken hepatocarcinoma cell line LMH.

Avian lipogenesis was studied in the chicken hepatocarcinoma LMH cell line. The differentiated and lipogenic status of these cells was evidenced by the presence of the albumin mRNA as well as of some mRNA coding for enzymes involved in lipogenesis (acetyl-CoA carboxylase, fatty acid synthase, delta 9 desaturase) and for apoproteins (apoprotein B and A1). These results were further confirmed by the analysis of triglyceride synthesis and secretion rates in growing cells. A time course analysis showed that triglyceride metabolism was affected by cell density. Hormone responsiveness of triglyceride production was also analyzed. Insulin, triiodothyronine and glucagon to a lesser extent were shown to regulate lipogenesis of LMH cells. The results were compared with those obtained in primary cultures of chicken hepatocytes.

Characterization of a cDNA sequence encoding the peroxisome proliferator activated receptor alpha in the chicken.

Peroxisome proliferator-activated receptors (PPAR) belong to the superfamily of the nuclear hormone receptors that play an important role in lipid homeostasis. A partial complementary deoxyribonucleic acid clone encoding a PPAR alpha from chicken liver was isolated and sequenced. Comparison with human, mouse, rat, and Xenopus PPAR alpha cDNA indicates a high degree of homology, especially at the level of the inferred peptide sequence (greater than 90%). The tissue pattern of expression indicates that PPAR alpha expression in the chicken is similar to that reported in other species, i.e., high expression levels in the liver, heart, and kidney, but also occurs in the lipogenic uropygial gland specific to birds.

Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2 (SREBP-2), chromosomal localization, and tissue expression of chicken SREBP-1 and -2 genes.

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.

Effect of ibuprofen on bilirubin-albumin binding in vitro at concentrations observed during treatment of patent ductus arteriosus.

BACKGROUND: In vitro studies have shown that ibuprofen (IBU) may interfere with bilirubin-albumin binding at concentrations of 100 microg/mL and above. OBJECTIVES: The present study evaluates the in vitro bilirubin displacement over the range of IBU plasma concentrations observed in vivo during curative treatment of patent ductus arteriosus in preterm infants. METHODS: Considering that individual plasma concentrations obtained during the clinical development of IBU in preterm infants were ranging between 10 and 70 microg/mL and exceptionally above 100 microg/mL, we used the modified peroxidase method to determine total and unbound bilirubin concentrations without IBU and with IBU over this specific concentration range. RESULTS: Total bilirubin and albumin concentrations were respectively 6.6 mg/dL and 2.87 g/dL in pooled newborn plasma. No displacement of bilirubin from its albumin binding sites by IBU was observed over a range of concentrations from 10 to 100 microg/mL. Only a concentration of 200 microg/mL significantly increased the unbound bilirubin by 1.5-fold (p=0.0008). CONCLUSIONS: This in vitro study confirms displacement of bilirubin by a high IBU concentration of 200 microg/mL, however it retrieves no significant displacement over a range of concentrations up to and including 100 microg/mL, i.e. within the range of in vivo concentrations at the recommended dose regimen.

Replication of hepatitis B virus in differentiated adult rat hepatocytes transfected with cloned viral DNA.

The possibility of obtaining expression of human hepatitis B virus (HBV) genes and production of virus particles in normal liver cells from heterologous species like normal adult rat hepatocytes, by transfecting the complete HBV genome, was investigated. Various techniques for hepatocyte transfection were assayed including the usual calcium-phosphate coprecipitation technique, the Pasco and Fagan modified calcium-phosphate procedure, and the lipofection technique. Transfection efficiency was determined by measuring the production of HBV surface antigen under various culture conditions. Transfection was the most efficient when assayed 1 or 2 days after hepatocyte plating at low density. Few variations in the efficiency were observed between the different transfection procedures. We show that under these culture conditions, replication of HBV can be achieved in differentiated adult rat hepatocytes. Synthesis of relaxed circular and single-stranded DNA forms and of viral transcripts including pregenome RNA occurred in the cells whereas viral antigens and mature and immature viral particles were released into the culture medium. The production of viral proteins was always higher in hepatocytes cocultivated with rat liver epithelial cells and maintained at a low density. In contrast, viral replication was not obtained by transfecting undifferentiated rat liver epithelial cells. These results demonstrate that replication of HBV can occur in hepatocytes from mammalian species non-closely related to primates and strongly support the idea that attachment of the virus and its penetration into the cells are critical steps in the host-specificity of the infection process and that hepatic-specific regulating factors could be essential for viral replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation by dimethylsulfoxide, insulin, and corticosteroids of hepatitis B virus replication in a transfected human hepatoma cell line.

A human hepatoblastoma cell clone E4 was obtained by transfection of HepG2 cells with a plasmid DNA containing four tandem copies of hepatitis B virus (HBV) genome. Analysis of both intracellular and extracellular viral DNA revealed that this clone exhibited the main steps of the replication process previously found in normal hepatocyte primary cultures experimentally infected in vitro. Indeed, relaxed-circular, covalently closed circular, and single-stranded forms of viral DNA were identified in the cells together with complete virions and immature cores in the medium. Furthermore, the ability of these secreted particles to infect normal human hepatocyte cultures was established. These E4 cells were used to evaluate the effect of various soluble factors on HBV replication. Corticosteroids and, to a greater extent, dimethylsulfoxide (DMSO) increased intracellular viral DNA, whereas insulin reduced it dramatically. Parallel changes in the amounts of viral DNA secreted in the medium were observed. Measurement of the albumin secretion rate indicated that cellular and viral activities could be regulated, at least in part, in a coordinated manner.

Hormonal regulation of stearoyl coenzyme-A desaturase 1 activity and gene expression in primary cultures of chicken hepatocytes.

Previous studies have provided evidence for the important role of liver stearoyl-CoA desaturase (SCD) in excessive adiposity in the chicken and suggest that the difference in SCD activity between fat and lean chickens could be explained by a difference in SCD1 gene expression. In the present study, the regulation of SCD1 gene expression was analyzed as the result of insulin and glucagon action, using primary cultures of 6-week-old chicken hepatocytes. Insulin increased SCD1 activity and mRNA levels, whereas glucagon decreased dramatically both the enzyme activity and the mRNA levels. Nuclear run-on transcription assays and mRNA stability investigations demonstrated that insulin and glucagon effects on SCD1 gene expression was primarily transcriptional. Furthermore, the results indicated that the glucagon-mediated inhibition of SCD1 gene transcription was more potent than just counteracting the insulin-mediated effect. These data represent the first demonstration that the glucagon effect on the SCD1 gene expression is primarily transcriptional. Moreover, among hepatic genes involved in lipid metabolism in chicken, SCD1 is the first gene shown to be regulated at the transcriptional level by insulin, in the absence of triiodothyronine. These data point out the potency of the growing chicken hepatocyte culture model in contrast with the embryonic cell culture model as regards the investigations of the insulin effect on gene expression.

Reproducible high level infection of cultured adult human hepatocytes by hepatitis B virus: effect of polyethylene glycol on adsorption and penetration.

We have previously succeeded in infecting normal human hepatocyte primary cultures with hepatitis B virus (HBV). However, infection was subject to individual variations even in the presence of dimethyl sulfoxide (DMSO), which appeared to increase the amounts of viral DNA associated with the cells. In this study, we have defined conditions which enhance hepatitis B virus penetration into the cells, and we show that, under these conditions, infection of hepatocytes is always possible, regardless of their individual origin. We have found that addition of polyethylene glycol (PEG) to the cultures maintained in the presence of 2% DMSO at the time of infection markedly increased the infection process and made it highly reproducible. Moreover, both the tissue and species specificity were preserved. This increased HBV infection was correlated to increased amount of internalized HBV DNA and to enhanced attachment of the virions. From these results it may be assumed that PEG could favor a better interaction between virions and cells, resulting in an activated internalization of bound viral particles. Data also show that adult human hepatocyte primary cultures, which are not equally susceptible to HBV infection, are consistently capable of viral replication when the viral genome has entered the cells. This suggests that the main limitation of the in vitro HBV infection lies in the ability of human hepatocytes to specifically bind the viral particles.

Effects of polyunsaturated fatty acids and clofibrate on chicken stearoyl-coA desaturase 1 gene expression.

In chicken, adiposity is influenced by hepatic stearoyl-CoA desaturase (SCD) 1. This gene is up-regulated by low-fat high-carbohydrate diet and down-regulated by addition of polyunsaturated fatty acids (PUFA). In this study, we present evidence for an inhibition of chicken SCD1 expression by PUFA using reporter gene constructs in transient transfection assays. This inhibition does not involve the peroxisome proliferator-activated receptor pathway, in contrast with what has been observed in rodents. We were able to localise a PUFA as well as an insulin response element within the -372/+125 bp region of the promoter. Sequence analyses of this region allowed identification of several cis-regulatory elements: A sterol regulatory element (SRE) and a juxtaposed NF-Y element which have been shown to be involved in the regulation of mouse SCD genes by PUFA. In addition, we identified an overlapping Sp1/USF motif, which was described to play a role in insulin/glucose and PUFA regulation of fatty synthase, ATP-citrate-lyase, and leptin genes. These data provide the first characterisation of the chicken SCD1 promoter and putative cis-sequences involved in the regulation of this gene by PUFA and insulin.

Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide.

We investigated the possibility of infecting normal adult human hepatocytes maintained in pure cultures or in cocultures with hepatitis B virus (HBV). Several assays with different infectious sera and hepatocyte populations from various donors identified only limited HBV replication, with significant variations from one cell preparation to another. The addition of 1.5% dimethyl sulfoxide to the culture medium markedly enhanced the infection process. Indeed, hepatitis B e antigen secretion, the appearance of both HBV DNA replicative forms and major HBV transcripts, and the release of complete HBV particles into the medium were demonstrated. It is possible that the significant increase in intracellular HBV DNA in dimethyl sulfoxide-treated cells was related to enhanced adsorption of the virus. When viral particles produced by a transfected HepG2 cell line were used to infect normal hepatocytes, the same results were obtained. In addition, comparative assays with hepatocytes from three different donors showed that although high amounts of intracellular viral DNA were found in all cases, viral replicative intermediates were visualized in only one case. These findings suggest that this HBV-producing cell line could serve as a reproducible source of infectious virus and that primary culturing of human hepatocytes represents a unique tool for analyzing intracellular regulating factors which, in addition to the penetration step, modulate HBV replication.

In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region.

Hepatitis B virus (HBV) variants with a nonsense mutation in the distal pre-C region have been detected in patients positive for anti-HBe, and the complete nucleotide sequence of one cloned pre-C variant has been determined. Transfection of this HBV variant clone into the human hepatoma cell line HepG2 resulted in the appearance of major HBV transcripts, replicative forms of viral DNA evidenced by both molecular hybridization and endogenous DNA polymerase assay, as well as the expression and secretion of HBsAg and HBcAg particles. Western blotting revealed only the 21-kDa HBcAg but not the 17-kDa HBeAg. These results demonstrate the replication capacity of the HBV variant with a nonfunctional pre-C region despite its inability to express HBeAg.

Characterization of the chicken fatty acid synthase gene 5' part and promoter region.

Fatty acid synthase activity has been shown to be regulated mainly at the transcriptional level under both dietary and hormonal influences. As a first step towards elucidating the factors involved, we isolated and characterized chicken genomic clones encompassing the 5' part of the chicken fatty acid synthase gene and its flanking region. The entire region of the cloned DNA spans 30 kb, and the first three exons of the gene were mapped to a 6.3-kb genomic fragment. The transcription initiation site was determined after subcloning the cDNA which encodes the 5' end of the mRNA. The first exon, which was 129 bp long, was located approximately 5.3 kb upstream of the second exon, which contained the start codon. In the 5' flanking region, putative TATA and CAAT boxes were located 30 and 92 bp, respectively, upstream of the transcription initiation site. The 5' flanking region contained numerous sequences corresponding to consensus binding sites for transcription factors. Various lengths of flanking sequences extending up to 1028 bp upstream of the transcription initiation site and containing 100 bp of the first exon were linked to the bacterial chloramphenicol acetyltransferase gene; in this study, these constructs were analyzed in transient transfection assays in human hepatoma cells. The proximal 125-bp sequence upstream of the transcription start site was shown to be a basal promoter. The cloning and characterization of the chicken fatty-acid synthase gene provides some further insight into the regulation of fatty acid synthesis in birds as compared to mammals.

trans-Acting factors, detoxication enzymes and hepatitis B virus replication in a novel set of human hepatoma cell lines.

A panel of four novel human hepatoma cell lines was isolated from a single tumor from a male individual. BC1, B16 and B16A2 lines were well differentiated, while cells of the B9 line were only poorly differentiated, being essentially negative for the functions analyzed. These cell lines have been surveyed for expression of a large set of plasma proteins, accumulation of liver-specific mRNAs and DNA-binding activity of ubiquitous and liver-enriched transcription factors. BC1 cells expressed the highest levels of albumin mRNA, whereas B16 and B16A2 cells accumulated the largest amounts of haptoglobin mRNA. In addition, B16 and B16A2 cells were unique in that they expressed CYP2E1 mRNA, a species absent from the available human liver cells, including HepG2 hepatoma cells, and 3-methylcholanthrene-inducible CYP1A2 mRNA. The activities of genes encoding transcription factors were evidenced in all four cell lines which expressed mRNAs for nuclear factor interleukin 6 and hepatocyte nuclear factor 1 (HNF) together with the DNA-binding activity of NFY and AP1 nuclear proteins. Strikingly, HNF-1 and HNF-4-like DNA-binding activities were restricted to BC1, B16 and B16A2 cells, supporting the idea of the potential role of these (or closely related) factors in the maintenance and/or in the establishment of the differentiated phenotype. B9 cells contained variant HNF1-like DNA-binding activity, similar to dedifferentiated rat hepatoma cells of the H5 line. CCAAT/enhancer-binding protein and HNF-3-like activities were found in all cell lines, although at a lower level and/or activity in B9 cells. Finally, transfection experiments of plasmids containing the whole hepatitis-B virus genome demonstrated that B16 cells, but not B9 cells, were able to support hepatitis-B virus replication and virion production, in agreement with the notion that HNF-1 activity is necessary for viral replication. We believe that the specific complement of transcription factors expressed in the differentiated BC1, B16 and B16A2 cells, and in the poorly differentiated B9 cells, will allow studies on the regulation of hepatic gene expression in these human lines, and will also aid the analysis of xenobiotic metabolism and the biology of hepatitis-B virus replication.

Cloning and characterization of the 5' end and promoter region of the chicken acetyl-CoA carboxylase gene.

Acetyl-CoA carboxylase is a rate-limiting enzyme in the biogenesis of long-chain fatty acids. In the present study, the 5' end and flanking region of the acetyl-CoA carboxylase (ACC) gene was analysed in the chicken. A genomic clone was isolated containing the first three exons, the third one containing the ATG codon. Using nuclease-mapping experiments and primer-extension analyses, the transcription-initiation site was located 153 nucleotides upstream of the ATG codon. In contrast with rat ACC gene expression, reverse transcriptase PCR analysis performed on chicken liver mRNA did not reveal alternative splicing in the 5'-untranslated region of these messengers. The promoter region is very G+C rich, and contains no TATA or CAAT boxes. Analysis by transient transfection in a human hepatoma cell line (HepG2) demonstrates that the promoter activity requires the presence of symmetrical sequences located upstream of the GC boxes. Transcription of this gene is found to be controlled by tri-iodothyronine in HepG2 cells, but the sequence responsible for the tri-iodothyronine response is not the consensus tri-iodothyronine-responsive element localized in the promoter. These results bring new insights to the regulation of the chicken ACC gene which differs from that of the rat.

Stearoyl-CoA desaturase 1 coding sequences and antisense RNA affect lipid secretion in transfected chicken LMH hepatoma cells.

Hepatic stearoyl CoA desaturase (SCD) activity in chickens from a fat line is higher than that of chickens from a lean line and correlates with plasma triacylglycerol concentrations. Furthermore, in these lines, the hepatic SCD1 mRNA level is positively correlated with the adipose tissue weight. To analyze the contribution of the SCD1 gene in the regulation of adiposity in the early stages of triacylglycerol secretion, SCD1 coding sequence and antisense RNA expression vectors were transfected in LMH cells. After selection, these cells were analyzed with regard to SCD1 expression and lipid secretion. The amounts of secreted triacylglycerols and phospholipids were shown to be higher in LMH cells transfected with the SCD1 gene, but reduced in those transfected with the SCD1 antisense sequences when compared to cells transfected with the vector alone (without SCD1 sequences). These results provide direct evidence that the expression of the SCD1 gene plays a major role in the triacylglycerol and phospholipid secretion process.