Primary Culture Of Chicken Hepatocytes Research Articles

Effects of dietary sodium butyrate on hepatic biotransformation and pharmacokinetics of erythromycin in chickens

Butyrate, a commonly applied feed additive in poultry nutrition, can modify the expression of certain genes, including those encoding cytochrome P450 (CYP) enzymes. In comparative in vitro and in vivo experiments, the effect of butyrate on hepatic CYP genes was examined in primary cultures of chicken hepatocytes and in liver samples of chickens collected from animals that had been given butyrate as a feed additive. Moreover, the effect of butyrate on the biotransformation of erythromycin, a marker substance for the activity of enzymes of the CYP3A family, was investigated in vitro and in vivo. Butyrate increased the expression of the avian-specific CYP2H1 both in vitro and in vivo. In contrast, the avian CYP3A37 expression was decreased in hepatocytes following butyrate exposure, but not in the in vivo model. CYP1A was suppressed by butyrate in the in vitro experiments, and overexpressed in vivo in butyrate-fed animals. The concomitant incubation of hepatocytes with butyrate and erythromycin led to an increased CYP2H1 expression and a less pronounced inhibition of CYP3A37. In in vivo pharmacokinetic experiments, butyrate-fed animals given a single i.m. injection of erythromycin, a slower absorption phase (longer T(half-abs) and delayed T(max)) but a rapid elimination phase of this marker substrate was observed. Although these measurable differences were detected in the pharmacokinetics of erythromycin, it is unlikely that a concomitant application of sodium butyrate with erythromycin or other CYP substrates will cause clinically significant feed-drug interaction in chickens.

Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes

Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B (apoB) levels. To identify the mechanisms underlying the effect of octanoate on very low-density lipoprotein (VLDL) secretion, chicken primary hepatocytes were incubated with either fatty acid-bovine serum albumin (BSA) complexes or BSA alone. Addition of octanoate to culture medium significantly reduced VLDL-triacylglycerol (TG), VLDL-cholesterol and apoB secretion from hepatocytes compared to both control cultures with BSA only and palmitate treatments, but did not modulate intracellular TG accumulation. However, no differences in cellular microsomal triglyceride transfer protein levels were observed in the cultures with saturated fatty acid. In pulse-chase studies, octanoate treatment resulted in reduced apoB-100 synthesis, in agreement with its promotion of secretion. This characteristic effect of octanoate was confirmed by addition of a protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), to hepatocyte cultures. Analysis showed that the level of apoB mRNA was lower in cultures supplemented with octanoate than in the control cultures, but no significant changes were observed in the levels of apolipoprotein A-I, fatty acid synthase and 3-hydroxy-3-methylglutaryl-CoA reductase mRNA as a result of octanoate treatment. Time-course studies indicate that a 50% reduction in apoB mRNA levels requires 12 h of incubation with octanoate. We conclude that octanoate reduced VLDL secretion by the specific down-regulation of apoB gene expression and impairment of subsequent synthesis of apoB, not by the modulation of intracellular apoB degradation, which is known to be a major regulatory target of VLDL secretion of other fatty acids.

Octanoate inhibits very low-density lipoprotein secretion in primary cultures of chicken hepatocytes

The effects of octanoate, a medium-chain fatty acid, on very low-density lipoprotein (VLDL) secretion in primary cultures of chicken hepatocytes were compared with those of palmitate. Palmitate added to the incubation media at concentrations up to 0.36 mM increased intracellular triacylglycerol (TG) accumulation and VLDL-TG secretion in a concentration-dependent manner, whereas the addition of octanoate alone (0.21–0.6 mM) did not change these parameters. VLDL-TG secretion from hepatocytes cultured in media to which 0.6 or 1.0 mM octanoate had been added in the presence of 0.21 mM palmitate was significantly lower than that obtained under control incubation conditions (0.21 mM palmitate only). The addition of 1.0 mM octanoate to the incubation media with or without 0.21 mM palmitate decreased VLDL apolipoprotein B (apoB) secretion. These results demonstrate that the addition of octanoate to primary cultures of chicken hepatocytes reduces VLDL secretion in respect of both TG and apoB secretion. It is suggested that medium-chain fatty acids are a factor modulating VLDL secretion, which plays a key role in fat deposition in chickens.

Primary culture of chicken hepatocytes as an in vitro model for determining the influence of dioxin.

An easy method for primary culture of chicken hepatocytes was developed to study the influence of dioxin on birds. Chicken hepatocytes could maintain gene expression and protein secretion of albumin for a long period in serum-free medium with free atmosphere exchange at 37 degrees C. Moreover, the cells showed a sensitive response to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) by monitoring the expression of P450 1A, theta GST (theta-GST) and albumin genes.

Ascorbic acid supplementation to primary culture of chicken hepatocytes with non-serum medium

Chicken liver is lack of ascorbic acid biosynthesis system, different from mammals and highly evoluted birds. Chicken hepatocytes cultured without ascorbate was expected to have lower ascorbate amounts than physiological levels. Intracellular was decreased as compared with intact liver by cell preparation performed with in situ collagenase perfusion. We added ascorbate to a primary culture of chicken hepatocytes in order to restore the amount of ascorbate. Serum-free Leivobitz's L-15 medium which do not contain ascorbate was used for control medium. Cells were cultured with several concentrations of ascorbate for 24 or 48 h. After ascorbate supplementation for 24 to 48 h, cellular ascorbate concentration increased depending on the dose of medium ascorbate. Medium lactate dehydrogenase activity derived from hepatocytes, an index of cell injury, decreased upon 5–100 mg/l of ascorbate supplementation for 48 h. Tyrosine aminotransferase activity, an index of liver function, increased following culture with 50 and 100 mg/l ascorbate for 48 h. The activities, however, decreased by supplementation with 1000 mg/l of ascorbate. In conclusion hepatocytes lost intracellular ascorbate during preparation by in situ collagenase perfusion. Supplementation of ascorbate restored cellular ascorbate concentration, lowered cell injury and raised tyrosine aminotransferase activitv in primary cultured chicken hepatocytes. Ascorbate treatment for 48 h at 50 mg/l was the best combination in this study for primary culture of chicken hepatpcyte with non-serum L-15 medium

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.

A Possible Procedure to Prepare Mono Fatty Acid-Enriched Plasma Membrane in Chicken Hepatocytes

In a viewpoint to explore an experimental model to study the regulatory mechanism of hepatic lipogenesis by n-3 fatty acids on hepatic lipogenesis, three experiments were undertaken to prepare mono fatty acid-enriched plasma membrane phospholipid in chicken hepatocytes. Incubation with 500μM α-linolenic acid for 24h in the primary culture of chicken hepatocytes significantly increased α-linolenic acid content and decreased oleic acid content in the plasma membrane phospholipid. In a same fashion, addition of linoleic acid or oleic acid to the media resulted in the higher proportion of the corresponding fatty acid in the plasma membrane. In the subsequent incubation in a fatty acid-free media of α-linolenic acid-enriched cells, α-linolenic acid content decreased gradually with a half life of 24h. Our results suggest that fatty acid supplementation to the culture medium modifies the fatty acid composition of plasma membrane phospholipid of chicken hepatocytes. The present chicken primary culture system may be applicable for investigation of regulatory mechanism of lipid metabolism.

Octanoate Metabolism in Primary Culture of Chicken Hepatocytes: Evidence for High-Capacity Octanoate Esterification

[14C]Octanoate metabolism was measured in chicken primary hepatocytes, as a function of time in culture. [14C]Octanoate was essentially converted to oxidative products (CO2 and ketone bodies) in freshly isolated cells while [14C]palmitate was exclusively esterified in triacylglycerols (TG). In contrast, in cultured cells, [14C] octanoate was exclusively recovered in TG as radiolabeled octanoate (33%), but also as newly synthetised fatty acids mainly palmitate (37%), stearate (18%), oleate (4.4%), decanoate (2.4%), and myristate (2.2%). Therefore, it may be suggested that the enhanced lipogenic conditions induced by culture enhance octanoate flux toward TG synthesis in this model. It is further suggested that chicken hepatocytes in primary culture could be a useful model to study regulatory mechanisms of several lipogenic enzymes.

Inhibiting Δ9-Desaturase Activity Impairs Triacylglycerol Secretion in Cultured Chicken Hepatocytes

The relationship between endogenous oleic acid produced by hepatic Δ9-desaturase and the secretion of VLDL-triglycerides was investigated in a primary culture of chicken hepatocytes. When the fatty acid compositions of the secreted and intracellular triglycerides (TG) (or triacylglycerols) were compared, an imbalance between monoenes and saturated fatty acids was observed, with the secreted TG being significantly more unsaturated than the intracellular TG. The addition of a mixture of cyclopropenic fatty acids (specific inhibitors of fatty acid desaturation) to the culture medium of cells 24 h before measurement of their Δ9-desaturase activity and TG secretion rate caused a significant impairment of both desaturase activity and TG secretion, without affecting total TG synthesis. However, the addition of oleic acid to the culture medium of cells treated with cyclopropenic fatty acids restored the TG secretion rate. Palmitic acid did not restore the TG secretion rate and linoleic acid partly restored the TG secretion rate. Finally, even in the presence of oleic acid in the culture medium of secreting cells, those which had been treated with cyclopropenic fatty acids had a significantly lower TG secretion rate than nontreated cells. Taken together, these results show that TG secretion is highly dependent on the Δ9-desaturase activity and suggest that oversecretion of VLDL-TG in chickens and subsequent fattening could originate in a high hepatic Δ9-desaturation of saturated fatty acids.

Primary culture of chicken hepatocytes in serum-free medium (pH 7.8) secreted albumin and transferrin for a long period in free gas exchange with the atmosphere

To study the liver functions of chicken, we examined the primary culture of chicken hepatocytes, and found an easy method of long-term culture with free atmosphere exchange. Chicken hepatocytes were obtained by collagenase perfusion and cultured at 37°C as a monolayer without substratum in serum-free L-15 medium (pH 7.8) with free atmosphere exchange. The amounts of albumin and transferrin in medium were assayed by ELISA. The culture of chicken hepatocytes was maintained in the serum-free L-15 medium (pH 7.8) at 37°C with free atmosphere exchange for 20 days. The amount of albumin secreted in the medium decreased to low levels early in culture; however, this was followed by marked increase from day 9 to day 17 of culture. The amount of transferrin was constant until day 6, then it too increased considerably with further culture. We reported an easy method for the simple monolayer culture of chicken hepatocytes in serum-free L-15 medium (pH 7.8) with free atmosphere exchange over an extended period. Expression of liver-specific functions, viz. albumin and transferrin synthesis, was observed after 1 week of culture.

Stearoyl-CoA desaturase activity in primary culture of chicken hepatocytes. influence of insulin, glucocorticoid, fatty acids and cordycepin

1. 1. Stearoyl-CoA desaturase (Δ9-desaturase) activity was measured in chicken primary hepatocytes, as a function of time in culture. 2. 2. When using fasted donor animals, the desaturase activity was low at the beginning of culture and then increased steadily to a maximum value between 30 and 70 hr of culture. When hepatocyte cultures were prepared from fed animals, enzyme activity was high at the beginning of culture and maintained thereafter at similar values to those obtained in cultured hepatocytes from fasted animals after 30 hr of culture. 3. 3. Insulin significantly enhanced enzyme activity when added to the culture medium at a 10 −9M concentration, and a small stimulating effect was also observed with 10 −6M dexamethasone. 4. 4. Linoleic acid (0.5 mM) added to the culture medium as albuminic complex partly inhibited Δ9-desaturase activity. 5. 5. Cordycepin (3' deoxyadenosine) decreased enzyme activity when present at a 3 μg/ml concentration in the culture medium. 6. 6. Taken together, the induction of enzyme activity in culture, its impairment by cordycepin and response to insulin and linoleic acid strongly suggest that synthesis and translation of the Δ9-desaturase mRNA occur in chicken hepatocytes in primary culture, and that this cellular model may be a useful tool for further studies on Δ9-desaturase regulatory mechanisms.

Lack of excision of introns from primary transcripts of certain chicken vitellogenin II minigenes.

Two truncated versions of the chicken vitellogenin II gene VTGII were designed and constructed to include all known essential regulatory elements of the complete gene. Both pCB123 and pCB123/4 contain 945 base pairs (bp) of the 5'-flanking sequence, introns and exons 1-3, and a subset of the remaining 32 exons of VTGII, inserted into a pBluescript SK (+/-) vector. pCB123/4 contains 752 bp of legitimate VTGII 3'-flanking sequences, while the 3' end of pCB123 terminates at the VTGII cDNA end, followed by AT-tailing and vector sequences carried over during cloning. Expression of these plasmids was tested following their lipofection into primary cultures of chicken hepatocytes established from day 14 embryos. Poly(A)+ RNA derived from pCB123 was detected by Northern blotting and reverse transcription-polyacrylamide chain reaction. No evidence was observed for appropriate hormonal control of expression, despite the presence of 17 beta-estradiol or colipofection with the estrogen receptor clone pHEO. VTGII sequences at the 3' end of pCB123/4 led to an apparent destabilization of the RNA transcript. Unexpectedly, unprocessed pCB123 transcripts of varying lengths accumulated in the cells. These experiments constitute the first reported attempts to express authentic VTGII coding sequences in cultured cells and highlight the dilemma of which introns to include in a minigene. Despite reports that some minigenes are expressed more efficiently if one or two introns are included, other minigenes may be expressed more effectively in the absence of introns. In the case of a complex gene with many introns, such as VTGII, there may be a preferential order in which introns are removed from the primary construct. The truncation of complex genes to give functional minigenes for transgenic studies may require considerable experimentation.

Lipogenic enzyme and apoprotein messenger RNAs in long-term primary culture of chicken hepatocytes

Hepatocytes isolated from 9-week-old chickens were cultured in a serum-free, hormonally defined medium. Relative amounts of mRNAs coding for lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, delta 9 desaturase, malic enzyme) and apoproteins (apoprotein A1 and apoprotein B) were determined until the 12th day. beta-actin and albumin mRNA, as well as albumin secretion, were also assessed. Cellular metabolic activity appeared to be very low for the first days of culture, but increased after the 7th day. All the mRNAs studied, except for that of malic enzyme, were present from this time throughout the culture lifespan. The biological significance of the observed results and the relevance of this chicken hepatocyte culture system for long-term metabolic and genetic studies are discussed.

Stimulation of insulin-like growth factor I production in primary cultures of chicken hepatocytes by chicken growth hormone

Extracts of conditioned media from primary cultures of chicken hepatocytes stimulated [ 3H]thymidine incorporation into chick embryo fibroblasts demonstrating that the cells released mitogen(s) into the medium. There was also a simultaneous release of insulin-like growth factor I (IGF-I) irnmunoreactivity into the medium. Acid chromatography of freeze-dried extracts of conditioned media by high performance gel permeation chromatography demonstrated that IGF-I immunoreactivity eluted in a major peak with a molecular weight of 7500 Da and a minor peak with a molecular weight of 55,000 Da. The release of IGF-I immunoreactivity was increased by pituitary-derived chicken growth hormone (cGH) in a dose-dependent manner with half-maximum stimulation occurring at a cGH concentration of 40 ng/ml and maximum stimulation at cGH concentrations greater than 800 ng/ml. These results demonstrate that cultured chicken hepatocytes produce IGF-I and that this can be stimulated by cGH in vitro.