Cultured Chick Embryo Hepatocytes Research Articles

Hexanoate and octanoate inhibit transcription of the malic enzyme and fatty acid synthase genes in chick embryo hepatocytes in culture.

Hexanoate and octanoate inhibit the triiodothyronine (T3)-induced increases in the activities of malic enzyme and fatty acid synthase in chick embryo hepatocytes in culture. Butanoate was less effective as an inhibitor, and palmitate, stearate, and oleate had no effect or small stimulatory effects. Hexanoate and octanoate inhibited the lipogenic enzyme activities at a transcriptional step, and did so within 30 min of addition. Incubation for 2 h in the absence of fatty acid reversed the inhibition of transcription caused by hexanoate. The inhibitory effect of hexanoate was selective because DNA content and transcription of the glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes were not inhibited. Hexanoate-mediated inhibition of transcription rates of the lipogenic genes was not correlated with an inhibition of binding of T3 to its nuclear receptor. 2-Bromooctanoate and carnitine stimulated the T3-induced accumulation of the mRNAs for malic enzyme and fatty acid synthase. The presence of hexanoate stimulated by 2- to 3-fold the increase caused by carnitine, suggesting that hexanoate and carnitine may regulate lipogenic gene expression by a common pathway. Hexanedioate, acetoacetate, beta-hydroxybutyrate, branched chain fatty acids, and branched chain keto acids had little or no effect on abundance of the lipogenic mRNAs. We suggest that the active inhibitor is a metabolite derived from hexanoate or octanoate, possibly an intermediate derived from an acyl-CoA derivative.

The interactions of 2,3,7,8-tetrachlorodibenzo- p-dioxin and 6-methyl-1,3,8-trichlorodibenzofuran in chick embryo hepatocytes

Treatment of chick embryo hepatocytes in ovo and in culture with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) resulted in a dose-dependent induction of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities. Significant induction was observed in the hepatocytes at TCDD doses as low as 10 −11 mol/egg ( in ovo) and 10 −10 m (in culture). In contrast, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) was a relatively weak inducer of these activities and only 10–20% of the induction responses observed for TCDD were elicited by MCDF at doses of 10 −6 mol/egg or 10 −7 m in culture. Co-treatment of the chick embryo hepatocytes with TCDD (10 −10 mol/egg in ovo; 10 −10 m in culture) and different concentrations of MCDF (10 −6 to 10 −8 mol/egg in ovo and 10 −7 and 10 −8 m in culture) resulted in minimal inhibition of TCDD-induced enzyme activities in ovo and a 37 to 50% inhibition in culture. The partial antagonist activity of MCDF in the chick embryo hepatocytes in culture paralleled the interactive effects previously reported in rodent liver and transformed rodent cell lines. TCDD (10 −7 to 10 −10 m) also caused an accumulation of hepta- and octacarboxyporphyrins in chick embryo hepatocytes (10 −7 to 10 −9 m); however, MCDF (10 −6 and 10 −5 m) elicited similar responses and MCDF did not significantly decrease the TCDD-induced porphyrogenic response in these cells. These results suggest that chick embryo hepatocytes in culture will serve as a useful model for investigating TCDD-induced gene transcription and the effects and mechanism of action of antagonists.

Ethoxyresorufin-O-deethylase (EROD) and porphyriainduction in chicken embryo hepatocyte cultures — A new bioassay of PCB, PCDD, and related chemical contamination in wildlife

Ethoxyresorufin-O-deethylase (EROD) and porphyria induction potencies of extracts from polychlorinated biphenyl (PCB) and 2,3,7,8-tetrachloro dibenzo-p-dioxin (TCDD) contaminated eggs of Herring Gulls, Larus argentatus from the Great Lakes of North America, and Great Blue Herons, Ardea herodias from British Columbia were measured in primary cultures of chicken embryo hepatocytes. The results show that EROD and porphyria induction in these cells have potential for determining the toxic potencies of complex mixtures of halogenated aromatic hydrocarbons (HAHs) extracted from wildlife tissue.

Regulation of the malic enzyme and fatty acid synthase genes in chick embryo hepatocytes in culture: Corticosterone and carnitine regulate responsiveness to triiodothyronine

Triiodothyronine (T3) added to chick embryo hepatocytes between 20 and 68 h of culture caused a 30- to 40-fold increase in malic enzyme activity. This T3 response decreased as a function of time; after 1 week in culture, a 48-h incubation with T3 had no effect on hepatocyte malic enzyme activity. Neither corticosterone nor carnitine had a significant effect on malic enzyme activity in the absence of T3 at any time or on the response of malic enzyme to T3 during the first 68 h of culture; both stimulated responsiveness to T3 subsequent to 68 h. The effects of corticosterone and carnitine on malic enzyme activity were additive, suggesting different mechanisms. Corticosterone and carnitine regulated abundance of malic enzyme mRNA. For corticosterone, at least, this effect was due to regulation of transcription. Abundance of fatty acid synthase mRNA was also stimulated by T3 in chick embryo hepatocytes in culture, and its responsiveness to T3 decreased with time. Corticosterone and carnitine stimulated responsiveness to T3 at times subsequent to 68 h. Corticosterone had no effect on binding of T3 to nuclear receptors. Intracellular accumulation of long-chain fatty acids or long-chain acyl-CoAs probably did not cause the loss of responsiveness to T3 or the stimulation of that responsiveness by corticosterone or carnitine because adding serum albumin (0.5%) or long-chain fatty acids (0.25−0.5 m m) to the medium was without effect. Corticosterone and carnitine may control the levels of other metabolic intermediates or protein factors which, in turn, regulate the transcriptional response of the lipogenic genes to T3.

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes.

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Light-induced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protoporphyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.

Characterization of 3,5,3′-triiodothyronine receptors in primary cultures of hepatocytes and neurons from chick embryo

We have detected the presence of nuclear 3,5,3′-triiodothyronine (T 3) receptors in primary cultures of chick embryo hepatocytes and neurons. Hepatocytes were isolated from livers of embryos of 12, 16 and 19 days by treatment with 0.2% collagenase and hyaluronidase. They were plated at a density of 3–4 × 10 5 35 -mm petri dish in Ham's F-10 medium containing fetal calf serum, tryptose phosphate, and antibiotics. Cells were used for the binding assay at Day 3 of culture. Neurons from 8-day-old embryo brains were cultured in a serum-free medium at a density of 1.2 × 10 6 cells/35-mm petri dish and used for the binding assay after 7 days of culture. Biological activity of hepatocytes was determined by measuring insulin binding, inositol phosphate formation, and 5′-monodeiodinase activity. Neurons or glial cells in culture were identified by immunostaining with anti-neurofilaments and anti-glial fibrillary acidic protein antisera. Binding assay was performed with isolated nuclei and 0.4 M NaCl nuclear extracts. With the latter preparation, the Scatchard analysis showed, in both cells, a single, high-affinity, low-capacity T 3 receptor. In the hepatocytes of 12-, 16-, and 19-day-old embryos association constants ( K a ) were, respectively, 0.93 ± 0.02, 0.74 ± 0.03, and 0.56 ± 0.04 n M −1, whereas the maximal binding capacities (MBC) were 2.26 ± 0.2, 2.72 ± 0.33, and 1.83 ± 0.19 fmol/μg DNA (mean ± SE, n = 3). In neurons K a was 1.25 ± 0.53 n M −1 and MBC 0.59 ± 0.14 fmol/μg DNA ( n = 3). The receptor had a sedimentation coefficient of 3.4 S, an estimated M r of 59 kDa, and the following relative affinity for thyroid hormone analogues: TRIAC l-T 3 > l-T 4. These data indicate that cultured hepatocytes and neurons of chick embryo contained T3 receptors with properties similar to those described in intact tissues from this and other species. Only the MBC of neurons was 50% lower than that observed in whole brain of embryo, but was comparable to values observed in cultured neurons from other species.

Metallothionein mRNA stability in chicken and mouse cells

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.

Heme regulates hepatic 5-aminolevulinate synthase mRNA expression by decreasing mRNA half-life and not by altering its rate of transcription

Hepatic 5-aminolevulinate (ALA) synthase, the first and rate-limiting enzyme in the heme biosynthetic pathway, is known to be feedback repressed by the end product of the pathway, heme. We investigated whether heme regulates ALA synthase mRNA expression transcriptionally or post-transcriptionally in primary cultures of chick embryo hepatocytes. 2-Propyl-2-isopropylacetamide increased the rate of transcription of the ALA synthase gene, whereas heme or an inhibitor of heme biosynthesis, desferrioximine, had no effect on the drug-induced transcription rate. Heme decreased the half-life of ALA synthase mRNA from approximately 3.5 h to 1.2 as recently reported by Drew and Ades (1989, Biochem. Biophys. Res. Commun. 162, 102–107). We also found that the heme-mediated decrease in mRNA stability was prevented by cycloheximide treatment, suggesting that the heme effect was mediated by a labile protein. These results support a model for hepatic ALA synthase regulation in which inducing drugs directly stimulate ALA synthase gene transcription, whereas heme regulates ALA synthase expression post-transcriptionally by modulating mRNA stability as well as by blocking translocation of ALA synthase enzyme into the mitochondrion.

Triiodothyronine-induced accumulations of malic enzyme, fatty acid synthase, acetyl-coenzyme A carboxylase, and their mRNAs are blocked by protein kinase inhibitors. Transcription is the affected step.

Addition of triiodothyronine (T3) to chick-embryo hepatocytes in culture causes increased accumulations of malic enzyme, fatty acid synthase, acetyl-CoA carboxylase and their mRNAs. H-8 and other protein kinase inhibitors inhibited the T3-induced accumulations of these lipogenic enzymes and their mRNAs but had no effect on the activities of 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, enzymes not induced by T3 in chick-embryo hepatocytes. H-8 also had no effect on the activities of malic enzyme, fatty acid synthase, and acetyl-CoA carboxylase in hepatocytes not treated with T3. Synthesis of soluble protein, levels of mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, and induction of metallothionein mRNA by Zn2+ were unaffected by H-8 at concentrations that inhibited the T3-induced accumulation of lipogenic enzymes and their mRNAs. H-8 inhibited T3-induced transcription of the genes for both malic enzyme and fatty acid synthase but had little effect on transcription of the beta-actin or glyceraldehyde-3-phosphate dehydrogenase genes or on total RNA synthesis in isolated nuclei. H-8 also had no effect on binding of T3 to its nuclear receptor. In isolated nuclei, H-8 inhibited phosphorylation of total protein by 15-20%. Phosphorylation of only one major protein was consistently and substantially inhibited, indicating that the effect of H-8 was selective. These results suggest that on-going protein phosphorylation is required specifically for stimulation of transcription of the lipogenic genes by T3.

Elevation of δ-aminolevulinic acid synthase and cytochrome PB 1 P450 messenger RNA levels by dihydropyridines, dihydroquinolines, sydnones, and N-ethylprotoporphyrin IX

A series of compounds that increase the activity of δ-aminolevulinic acid synthase (ALAS) in chick embryo hepatocyte cultures were studied for their effects on steady-state levels of mRNA for ALAS and phenobarbital-inducible cytochrome PB, P450. N-Ethylprotoporphyrin IX (N-EtPP), which is believed to lower heme levels by inhibition of ferrochelatase (FC), had little effect on steady-state ALAS mRNA levels. 3,5-Diethoxycarbonyl-l,4-dihydro-2,6-dimethyl-4-isobutylpyridine (4-isobutyl DDC), which is believed to lower heme levels by repetitive destruction of the heme moiety of cytochrome P450, increased steady-state levels of ALAS mRNA by approximately 2-fold. 3,5-Diethoxycarbonyl-1, 4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC) which inhibits FC activity and destroys the heme moiety of cytochrome P450, increased ALAS mRNA levels approximately 4-fold. A combination of N-EtPP and 4-isobutyl DDC produced a synergistic increase in ALAS mRNA levels to approximately 6-fold over control levels. The synergistic increase in ALAS activity observed previously with this combination can be explained, at least in part, by a synergistic increase in ALAS mRNA levels. Other porphyrinogenic agents, which function as mechanism-based inactivators of cytochrome P450 and elevate ALAS activity, were found to elevate ALAS mRNA. These compounds included 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), 2,4-diethyl-2-methyl-1,2-dihydroquinoline (DMDQ), and 2,2,4-trimethyl-1,2,dihydroquinoline (TMDQ). The elevation of ALAS mRNA by these porphyrinogenic agents is probably due to their lowering of cellular heme levels by a combination of ferrochelatase inhibition and repetitive destruction of the heme moiety of cytochrome P450. The lowering of heme levels should result in an enhancement of ALAS mRNA half-life as it has been demonstrated by others that heme shortens the half-life of ALAS mRNA. It was of interest that some of these drug treatments also caused an elevation in steady-state levels of cytochrome PB 1 P450 mRNA; the exception was TTMS, which along with its analogue 3-(2-phenylethyl)-4-methylsydnone (PEMS), did not alter cytochrome PB 1 P450 mRNA levels. Increases in steady-state levels of cytochrome pb 1 P450 mRNA subsequent to increases in steady-state levels of ALAS mRNA were observed with 4-ethyl DDC, 4-isobutyl DDC, DMDQ, and TMDQ. The data obtained with N-EtPP and a combination of N-EtPP and 4-isobutyl DDC on cytochrome PB 1 P450 mRNA levels do not support the contention that heme functions as a positive regulator of cytochrome P450 gene expression.

Differential potency of atropisomers of polychlorinated biphenyls on cytochrome P450 induction and uroporphyrin accumulation in the chick embryo hepatocyte culture

The atropisomers of 2,2',3,4,6-pentachlorobiphenyl (PeCB), 2,2',3,4,4',6-hexachlorobiphenyl (HeCB), and 2,2',3,3',4,4',6,6'-octachlorobiphenyl (OCB) were studied in the chick embryo hepatocyte culture to determine if chirality plays a role in the recognition events associated with the induction of cytochromes P450 and the accumulation of uroporphyrin (URO). Concentration-related induction of cytochrome P450 content, ethoxyresorufin- O-deethylase (EROD) and benzphetamine Ndemethylase (BPDM) activities were measured. The rank order of potency for total cytochrome P450 induction was HeCB > OCB ⩾ PeCB. The (+)- and (-)-enantiomers of PeCB and OCB were of equal potencies as inducers of cytochromes P450, whereas the (+)-HeCB was greater than the (-)-HeCB. HeCB was a much more potent inducer of EROD activity than was either PeCB or OCB. EROD activity was induced to a much greater extent by the (+)-enantiomers of all compounds, with the (-)enantiomers of PeCB and OCB being inactive. BPDM activity was induced by all three compounds in the order of OCB ⩾ HeCB > PeCB. The (-)-enantiomers were more potent inducers of BPDM activities than were the (+)-enantiomers, except for HeCB, in which the (+)- was more potent than the (-)enantiomer. Analysis of porphyrin accumulation in cultures treated with δ-aminolevulinic acid revealed that (+)-HeCB caused the greatest percent URO accumulation, which also correlated with the greatest increase in EROD activity. All other enantiomers caused up to 47% URO accumulation, which did not correlate with an increase in EROD activity.

Triiodothyronine stimulates and cyclic AMP inhibits transcription of the gene for malic enzyme in chick embryo hepatocytes in culture

In chick embryo hepatocytes in culture, insulin and triiodothyronine (T3) increase malic enzyme activity and the abundance of malic enzyme mRNA by at least 50-fold, and glucagon or cAMP blocks this effect. Steps regulated by these hormones were defined by measuring transcriptional activity with the nuclear run-on assay and multiple fragments of the malic enzyme gene as probes. T3 alone caused a significant increase in transcription within 1 h, with a maximal increase of 30-40-fold occurring by 24 h. When T3 was added with insulin, 80% of the maximum rate was reached in 1 h. Insulin alone had no effect on transcription of the malic enzyme gene; it amplified the response to T3 in the first few hours after adding T3 but did not alter T3's maximal effect. Cyclic AMP for 1 h completely inhibited the increase in transcription caused by T3. The size and speed of the responses of the malic enzyme gene to T3 and cAMP suggest regulation of transcription initiation. T3-stimulated transcription of the malic enzyme gene did not require ongoing protein synthesis despite the fact that inhibitors of protein synthesis inhibited the T3-stimulated accumulation of its mRNA. T3 may directly activate transcription of this gene via its receptor. The pattern of DNase I hypersensitivity of the malic enzyme gene in chick embryo hepatocytes was the same as that in fed chick liver. Insulin, T3, and cAMP had no effect on that pattern. In chick embryo hepatocytes in culture, factors involved in regulation of transcription by insulin, T3, and cAMP may be bound to DNA independently of hormonal treatment.

Isolation and partial characterization of the gene for goose fatty acid synthase.

Fatty acid synthase is regulated by diet and hormones, with regulation being primarily transcriptional. In chick embryo hepatocytes in culture, triiodothyronine stimulates accumulation of enzyme and transcription of the gene. Since the 5'-flanking region of this gene is likely involved in hormonal regulation of its expression, we have isolated and partially characterized an avian fatty acid synthase gene. A genomic DNA library was constructed in a cosmid vector and screened with cDNA clones that contained sequence complementary to the 3' end of goose fatty acid synthase mRNA. A genomic clone (approximately 35 kilobase pairs (kb] was isolated, and a 6.5-kb EcoRI fragment thereof contained DNA complementary to the 3' noncoding region of fatty acid synthase mRNA. Additional cosmid libraries were screened with 5' fragments of previously isolated genomic clones, resulting in the isolation of five overlapping cosmid DNAs. The entire region of cloned DNA spans approximately 105 kb. Exon-containing fragments were identified by hybridization with end-labeled poly(A)+ RNA and by hybridization of labeled exon-containing genomic DNA fragments to fatty acid synthase mRNA. A new set of cDNA clones spanning approximately 3.2 kb was isolated from a lambda-ZAP goose liver cDNA library using the 5'-most exon-containing fragment of the 5'-most genomic DNA clone. This region of mRNA contains a 5'-untranslated sequence and a continuous open reading frame which includes a region that codes for the essential cysteine of the beta-ketoacyl synthase domain. The entire fatty acid synthase gene spans about 50 kb. The 5' 15 kb of the gene contain 7 exons. S1 nuclease and primer extension analyses were used to identify a single site for initiation of transcription, 174 nucleotides upstream from the putative translation initiation codon. Putative "TATA" and "CCAAT" boxes are located 28 and 60 base pairs (bp), respectively, upstream of the site of initiation of transcription. The 5'-flanking 597 bp of DNA contains G/C-rich sequences including several "GC" boxes corresponding to binding sites for the nuclear transcription factor Sp1. Putative sites for AP-2, C/EBP, and the triiodothyronine and glucocorticoid receptors also were found in this region. A chimeric DNA, containing approximately 1.6 kb of 5'-flanking sequence and 139 bp of untranslated sequence of the goose fatty acid synthase gene ligated to the bacterial chloramphenicol acetyl-transferase (CAT) gene, was transfected into chick embryo hepatocytes in culture. Cells treated with triiodothyronine contained increased chloramphenicol acetyltransferase and fatty acid synthase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Triiodothyronine stimulates transcription of the fatty acid synthase gene in chick embryo hepatocytes in culture. Insulin and insulin-like growth factor amplify that effect.

Hepatic fatty acid synthase is regulated by nutritional state. Starvation decreases and refeeding increases the activity of avian fatty acid synthase, principally by regulating transcription of the gene (Back, B. W., Goldman, M. J., Fisch, J.E., Ochs, R.A., and Goodridge, A.G. (1986) J. Biol. Chem. 261, 4190-4197). In chick embryo hepatocytes in culture, the stimulatory effect of feeding on fatty acid synthase activity is mimicked by adding triiodothyronine and insulin; the inhibitory effect of starvation is mimicked by adding glucagon or cyclic AMP. We now show that triiodothyronine alone stimulates transcription of fatty acid synthase by 4- to 6-fold, about the same as the increase in fatty acid synthase mRNA. When added alone, insulin has little or no effect on transcription, mRNA level, or enzyme activity. In combination with triiodothyronine, however, insulin amplifies the response to triiodothyronine by about 2-fold, leading to an overall increase of about 10-fold. Insulin-like growth factor 1 (IGF-1) has the same effect as insulin, no effect by itself, and amplification of the stimulation by triiodothyronine. A maximally effective dose of insulin has no effect in the presence of a maximally effective dose of IGF-1, suggesting regulation by a common pathway. It takes much less IGF-1 than insulin to achieve a given effect, suggesting that both insulin and IGF-1 may act through IGF-1 receptors. Plasma levels of IGF-1 are decreased by starvation and increased by feeding (reviewed by Froesch, E.R., and Zapf, J. (1985) Diabetologia 28, 485-493). Thus, IGF-1 may play a physiological role in the regulation of hepatic fatty acid synthase during transitions between the starved and fed states, roles previously assigned primarily to insulin and glucagon. IGF-1 regulates transcription of the fatty acid synthase gene. Insulin and IGF-1 also have similar effects on activity, mRNA abundance, and transcription of the malic enzyme gene. Glucagon or dibutyryl cyclic AMP inhibit fatty acid synthase activity and mRNA level in hepatocytes in culture by 70-80% and 60%, respectively, but have no effect on transcription of the fatty acid synthase gene, suggesting a post-transcriptional mode of regulation for cyclic AMP.

Control of glycolysis in cultured chick embryo hepatocytes. Fructose 2,6-bisphosphate content and phosphofructokinase-1 activity are stimulated by insulin and epidermal growth factor

Chick embryo hepatocytes were maintained in monolayer culture in a serum-free chemically defined medium for periods of up to 2 days. Over this time period, insulin provoked selective increases (up to 5-fold) in factors relevant to the control of glycolysis: the activities of phosphofructokinase-1 (PFK-1), phosphofructokinase-2 (PFK-2) and hexokinase isoenzymes and the content of fructose 2,6-bisphosphate (F26BP). Half-maximal effects of insulin on pFK-1 activity were in the physiological range (0.1 nM). Changes in enzyme activities and F26BP content in response to insulin were correlated with stimulation of glycolytic flux as estimated by radioisotopic flux. These data are discussed in relation to known changes which occur in hepatic glycolytic activity and PFK-1 activity in the intact chick around hatching. The effects of insulin on F26BP content, PFK-1 activity and glycolytic flux were mimicked by epidermal growth factor (EGF). In contrast, phorbol esters produced minimal actions on any of the above parameters. Our data indicate that protein kinase C is not involved in the actions of insulin or EGF in control of F26BP content or PFK-1 activity. This work indicates that the related tyrosyl kinase receptors of insulin and EGF may provoke identical responses within hepatocytes, but through the utilization of different transduction systems which merge to common control points.

Utilization of cultured chick embryo hepatocytes as in vitro bioassays for polychlorinated biphenyls (PCBs): Quantitative structure-induction relationships

The concentration-dependent induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activities by several PCB congeners was determined in primary chick embryo hepatocytes in culture. The most active inducers were the coplanar PCBs, 3,3′,4,4′-tetra- and 3,3′,4,4′5-pentachlorobiphenyl (pentaCB) with EC 50 values of 1.2 × 10 −9 and 1.5 × 10 −9 M (AHH induction) and 2.3 × 10 −9 and 1.2 × 10 −9 M (EROD induction) respectively. The corresponding EC 50 values for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were 2.4 × 10 −11 and 7.2 × 10 −11 M for AHH and EROD induction respectively. Three monoortho coplanar PCB analogs, 2,3,3′,4,4′-pentaCB, 2,3′,4,4′,5-pentaCB and 2,3,3′,4,4′,5-hexaCB also exhibited induction activity but their corresponding EC 50 values were much higher than observed for the coplanar PCBs. Several di- and triortho-substituted PCB congeners were weakly active as inducers of AHH and EROD activity. The structure-induction relationships for the coplanar and monoortho coplanar PCBs in the chick embryo hepatocytes were comparable to those previously reported in rat hepatoma H-4-II E cells and in in vivo studies.

Induction of Cytochrome P450 by Barbiturates in Chick Embryo Hepatocytes: A Quantitative Structure‐Activity Analysis

AbstractIn cultured chick embryo hepatocytes, induction of cytochrome P450 by nine 5,5‐substituted barbiturates was analyzed for a quantitative structure‐activity relationship. The data led to the following equation: Log 1/C = 1.02 (±0.16) log P + 2.75 (±0.28), r = 0.984 where C is the concentration that caused a 50% increase in cytochrome P450, P is the partition coefficient between octanol and water, and r is the correlation coefficient. The results suggest that the potency of the barbiturates was directly related to their hydrophobicity.

Effects of cigarette smoke condensate and 12-0-tetradecanoylphorbol-13-acetage on gap junction structure and function in cultured cells

The effects of cigarette smoke condensate (CSC) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on gap junction structure, quantity and function were investigated. Gap junction morphology was studied in rotary-shadowed freeze-fracture replicas of primary chick embryo hepatocytes. CSC (24 micrograms/ml) induced a strong decrease of gap junction areas; within 6 h the areas were reduced by greater than 60%. In the first 3 h of exposure, TPA (100 ng/ml) also reduced gap junction areas, but in the next 3 h a partial recovery was observed. Protoplasmic fracture face centre-to-centre particle spacings were used as a measure for gap junction coupling. CSC had a slow (although not significant) reducing effect on particle spacings, while TPA induced a reduction from 10.6 nm (control) to 10.0 nm within 3 h, indicating a reduction of coupling. Gap junctions were quantified in thin sections of cultured chick embryo hepatocytes, V79 fibroblasts, and co-cultivated hepatocytes and V79 cells. CSC did not influence gap junction numbers in any of these cultures, while TPA treatment caused a disappearance of gap junctions between hepatocytes and between hepatocytes and V79 cells in the first 12 h of cultivation. In the following 36 h a slow recovery could be observed. Gap junctions between V79 cells had already disappeared within 30 min. Metabolic co-operation between hepatocytes and hypoxanthine-guanine phosphoribosyltransferase-deficient V79 cells was quickly and continuously blocked by CSC over 27 h, whereas the phorbol ester induced a transient block. The dissimilar effects of these compounds on both gap junction structure and function indicate that they act via different mechanisms. The finding that CSC did not inhibit phorbol ester protein kinase C binding and did not activate this protein kinase in vitro supports this hypothesis.

Effects of polychlorinated biphenyls on cytochrome P450 induction in the chick embryo hepatocyte culture

The effects of pure synthetic polychlorinated biphenyl (PCB) congeners on the induction of cytochrome P450 and associated activities were examined in cultured chick embryo hepatocytes. Dose-response effects for the induction of total cytochrome P450, ethoxyresorufin- O-deethylase (EROD) activity, and benzphetamine demethylase (BPDM) activity were studied using 10 selected tetra- to hexachlorinated PCB congeners. These studies revealed that PCBs caused effects in the chick hepatocyte culture different from previously observed effects in rat liver. Based on their effects in chick hepatocytes, the PCBs could be categorized into two groups. The first group (consisting of 3,3′,4,4′-PCB, 3,3′,4,4′,5-PCB, 3,3′,4,4′,5,5′-PCB, 2′,3,3′,4,5-PCB, 2,3,3′,4,4′,5′-PCB, and 2,3,4,4′,5-PCB) induced total cytochrome P450 2.4- to 2.9-fold and EROD activity from 1–2 pmol/min/mg protein to 162–247. There was marked variation in potency, but all these congeners had a maximal inducing dose above which cytochrome P450 concentrations and EROD activities declined. BPDM activities were increased only slightly (1.2- to 1.6-fold) at the maximal cytochrome P450 inducing dose. The second group of congeners (consisting of 2,2′,4,5,5′-PCB, 2,2′,4,4′,5,5′-PCB, and 2,2′,3,4,4′,6-PCB) induced total cytochrome P450 concentrations 4.0-fold and BPDM activities 2.2- to 2.6-fold with greatest activity occurring at the highest doses which could be added (10–50 μ m). However, EROD activities were also increased by these congeners to 60–112 pmol/min/mg protein with declining activities seen at the highest PCB doses (i.e., resembling EROD induction patterns of the first group). The EROD induction patterns with these latter PCB congeners are noteworthy since these PCBs do not induce EROD activity in the rat. For both groups of PCB congeners, EROD induction was associated with increased accumulation of uroporphyrin in cultures exposed to exogenous 5-aminolevulinate. Studies investigating the reason for the depression of cytochrome P450 concentrations and/or EROD activities by high doses of the PCBs revealed that with the first group there was slightly decreased total protein synthesis, decreased total cell heme concentrations, and decreased accumulation of radiolabeled heme synthesized from 5-[ 14C]aminolevulinate. These changes might represent nonspecific toxic effects of the first group of PCBs. However, since these changes were not seen with the second group of PCBs, it is unlikely that either inhibition of heme synthesis or toxicity cause the depression of EROD activity With high PCB doses.

Regulation of the stability of chicken embryo liver δ-aminolevulinate synthase mRNA by hemin

The effect of hemin on the stability of δ-aminolevulinate (ALA) synthase mRNA was investigated in primary cultures of chicken embryo hepatocytes. Hepatocytes were first incubated with allylisopropylacetamide (AIA) to increase the starting level of ALA synthase mRNA, and then the cells were incubated with α-amanitin (1 μg/ml) to block further transcription of ALA synthase RNA. The rates of depletion of the enzyme's mRNA were then determined by liquid hybridization analyses in cells incubated with or without hemin (10 μM) in the culture medium. The analyses indicated that hemin increased the rate of ALA synthase mRNA degradation. The half-life of the messenger in hepatocytes incubated with hemin was 80 min compared with 220 min in cells incubated without additional hemin in the culture medium. The results thus indicated a novel mechanism by which hemin modulated the biogenesis of ALA synthase in liver.