Activity and mRNA abundance of Delta-5 and Delta-6 fatty acid desaturases in two human cell lines

We analyzed fatty acid biosynthesis in Chang and ZR-75-1 cells. Both cell lines could desaturate and further elongate substrates for Delta-5 desaturase. ZR-75-1 but not Chang cells showed Delta-6 desaturation of 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3. In both cell lines, the mRNA abundance can be related to Delta-5 or Delta-6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Delta-6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.     

Delta-6-desaturase (FADS2) inhibition and omega-3 fatty acids in skeletal muscle protein turnover

Polyunsaturated fatty acids (PUFAs) are essential dietary components. They are not only used for energy, but also act as signaling molecules. The delta-6 desaturase (D6D) enzyme, encoded by the FADS2 gene, is one of two rate limiting enzymes that convert the PUFA precursors – α-linolenic (n-3) and linoleic acid (n-6) to their respective metabolites. Alterations in the D6D enzyme activity alters fatty acid profiles and are associated with metabolic and inflammatory diseases including cardiovascular disease and type 2 diabetes. Omega-3 PUFAs, specifically its constituent fatty acids DHA and EPA, are known for their anti-inflammatory ability and are also beneficial in the prevention of skeletal muscle wasting, however the mechanism for muscle preservation is not well understood. Moreover, little is known of the effects of altering the n-6/n-3 ratio in the context of a high-fat diet, which is known to downregulate protein synthesis. Twenty C57BL6 male mice were fed a high-fat lard (HFL, 45% fat (mostly lard), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 13:1) diet for 6 weeks. Mice were then divided into 4 groups (n = 5 per group): HFL– , high-fat oil– (HFO, 45% fat (mostly Menhaden oil), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 1:3), HFL+ (HFL diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day), and HFO+ (HFO diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day). After 2 weeks on their respective diets and treatments, animals were sacrificed and gastrocnemius muscle harvested. Protein turnover signaling were analyzed via Western Blot. 4-EBP1 and ribosomal protein S6 expression were measured. A two-way ANOVA revealed no significant change in the phosphorylation of both 4EBP-1 and ribosomal protein S6 with diet or inhibitor. There was a significant reduction in STAT3 phosphorylation with the inhibition of FADS2 (p = 0.03). Additionally, we measured markers of protein degradation through levels of FOXO phosphorylation, ubiquitin, and LC3B expression; there was a trend towards increased phosphorylation of FOXO (p = 0.08) and ubiquitinated proteins (p = 0.05) with FADS2 inhibition. LC3B expression, a marker of autophagy, was significantly higher in the HFL plus FADS2 inhibition group from all other comparisons. Lastly, we analyzed activation of mitochondrial biogenesis which is closely linked with protein synthesis through PGC1-α and Cytochrome-C expression, however no significant differences were associated with either marker across all groups. Collectively, these data suggest that the protective effects of muscle mass by omega-3 fatty acids are from inhibition of protein degradation. Our aim was to determine the role of PUFA metabolites, DHA and EPA, in skeletal muscle protein turnover and assess the effects of n-3s independently. We observed that by inhibiting the FADS2 enzyme, the protective effect of n-3s on protein synthesis and proliferation was lost; concomitantly, protein degradation was increased with FADS2 inhibition regardless of diet.     

The effect of dietary polyenylphosphatidylcholine on microsomal delta-6-desaturase activity, fatty acid composition, and microviscosity in rat liver under oxidative stress

Polyenylphosphatidylcholine is a choline-glycerophospholipid containing up to 80% of total fatty acids as linoleic acid and may be an important factor in ensuring normal functioning of cell membranes. We tested the effect of a polyenylphosphatidylcholine-supplemented diet and compared it with both a trilinolein-supplemented and a laboratory chow diet on the fatty acid composition, microviscosity, and delta-6-desaturase activity of liver microsomal membranes of 12-month-old rats, in the absence or presence of oxidative stress induced by adriamycin. Polyenylphosphatidylcholine- and trilinolein-supplemented diets showed a similar increase in linoleic acid content and delta-6-desaturase activity in liver microsomes, indicating that low amounts of linoleic acid are able to partially restore the enzyme activity in old rats, independent of the source of linoleic acid. After adriamycin treatment, delta-6-desaturase activity increased in polyenylphosphatidylcholine and trilinolein groups, indicating a protective mechanism against the damage induced by polyunsaturated fatty acid peroxidation. The measurement of malondialdehyde production showed a protective effect on adriamycin-induced lipid peroxidation by polyenylphosphatidylcholine supplementation only. Microsomal membrane microviscosity did not change independent of diet and adriamycin treatment, suggesting that the response of microsomes to lipid peroxidation might be the maintenance of a given membrane order. Administration of polyenylphosphatidylcholine can prevent or minimize the liver damage induced by adriamycin treatment.     

Fatty acid uptake and metabolism in Hep G2 human-hepatoma cells

The aim of this work was to study the fatty acid metabolism of the human-hepatoma cell line Hep G2. The cultured cells were incubated with either a saturated (palmitic, stearic) or a polyunsaturated (linoleic, -linolenic, eicosatrienoic n-6) radioactive fatty acid. The fatty acids were incorporated into all the basic lipid classes as well as into the main phospholipid subclasses in the cellular membranes. All the fatty acids tested provided a source of carbon for lower members of the saturated fatty-acid family or for cholesterol through -oxidation and a new cycle ofde novo synthesis. Moreover, all radioactive fatty-acid precursors, whether saturated or unsaturated, were anabolized to higher derivatives within their own family. In the case of saturated fatty acids, palmitic and stearic, they were readily monodesaturated to their corresponding products, thus demonstrating the presence of a 9 desaturase. Linoleate and -linolenate were both desaturated and elongated to all the subsequent members of their respective n-6 and n-3 families. These latter observations provide evidence for the incidence of desaturation at the 6 and 5 positions along with the existence of an elongating capacity for fatty acids of all families and chain lengths. In addition, the cellular steady-state fatty-acid profile was seen to be significantly different from the spectrum of exogenous fatty acids available in the growth medium. We conclude that the Hep G2 human-hepatoma line represents an appropriate and relevant experimental model system for investigating the fatty-acid metabolism of adult human liverin vivo.     

Single nucleotide polymorphisms in the FADS gene cluster are associated with delta-5 and delta-6 desaturase activities estimated by serum fatty acid ratios

Genetic variability in the FADS1-FADS2 gene cluster [encoding delta-5 (D5D) and delta-6 (D6D) desaturases] has been associated with plasma long-chain PUFA (LCPUFA) and lipid levels in adults. To better understand these relationships, we further characterized the association between FADS1-FADS2 genetic variability and D5D and D6D activities in adolescents. Thirteen single nucleotide polymorphisms (SNPs) were genotyped in 1,144 European adolescents (mean ± SD age: 14.7 ± 1.4 y). Serum phospholipid fatty acid levels were analyzed using gas chromatography. D5D and D6D activities were estimated from the C20:4n-6/C20:3n-6 and C20:3n-6/C18:2n-6 ratios, respectively. Minor alleles of nine SNPs were associated with higher 18:2n-6 levels (1.9E-18 ≤ P ≤ 6.1E-5), lower C20:4n-6 levels (7.1E-69 ≤ P ≤ 1.2E-12), and lower D5D activity (7.2E-44 ≤ P ≤ 4.4E-5). All haplotypes carrying the rs174546 minor allele were associated with lower D5D activity, suggesting that this SNP is in linkage disequilibrium with a functional SNP within FADS1. In contrast, only the rs968567 minor allele was associated with higher D6D activity (P = 1.5E-6). This finding agrees with an earlier in vitro study showing that the minor allele of rs968567 is associated with a higher FADS2 promoter activity. These results suggest that rare alleles of several SNPs in the FADS gene cluster are associated with higher D6D activity and lower D5D activity in European adolescents.     

Production of arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid Delta6-desaturase, Delta6-elongase, and Delta5-desaturase genes

The liverwort Marchantia polymorpha L. synthesizes arachidonic (ARA) and eicosapentaenoic acids (EPA) from linoleic and alpha-linolenic acids respectively by a series of reactions catalyzed by Delta6-desaturase, Delta6-elongase, and Delta5-desaturase. Overexpression of the M. polymorpha genes encoding these enzymes in transgenic M. polymorpha plants resulted in 3- and 2-fold accumulation of ARA and EPA respectively, as compared to those in the wild type. When these three genes were introduced and co-expressed in tobacco plants, in which long-chain polyunsaturated fatty acids (LCPUFAs) are not native cellular components, ARA and EPA represented up to 15.5% and 4.9% respectively of the total fatty acid in the leaves. Similarly in soybean, C20-LCPUFAs represented up to 19.5% of the total fatty acids in the seeds. These results suggest that M. polymorpha can provide genes crucial to the production of C20-LCPUFAs in transgenic plants.     

Selective incorporation of n-3 and n-6 fatty acids in essential fatty acid deficient rats in response to short-term oil feeding

Essential fatty acid deficient male Sprague Dawley rats were fed for 7 days a fat-free semi-synthetic diet supplemented with 10% by weight of different oil supplements. The oil supplement was a mixture of olive, safflower and linseed oils prepared at different proportions so the dietary n-9/n-6/n-3 ratios were approximate 2/1/1, 1/2/1, 1/1/2, and 1/1/1. The fatty acid compositions of plasma and liver lipids were then examined. Our results show polyunsaturated n-6 and n-3 fatty acids were selectively incorporated into plasma and liver phospholipids, and also into plasma cholesteryl esters. A preferential incorporation of n-6 over n-3 fatty acids into plasma cholesteryl esters and phospholipids was also observed.     

Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.     

Synthesis and processing of human serum apolipoprotein AII in vitro and in Hep G2 cells

The synthesis and structure of the primary translation product of apo AII in a human liver poly(A+) mRNA primed cell-free system and its cotranslational modification was studied parallel to studies in vivo with Hep G2 cells, a human hepatoma cell line. The primary translation product is a preproprotein containing 100 amino acid residues, which is cleaved by the signal peptidase of endoplasmic reticulum to pro-apo AII with the loss of the N-terminal pre-sequence consisting of 18 amino acid residues. Hep G2 cells contain about equal amounts of the proform of apolipoprotein AII and of mature apo AII. Approximately in the same ratio pro- and mature apo AII are secreted into the medium. Determination of the partial amino-acid sequence by automated Edman degradation of the labelled prepro- and proforms of apo AII led to the segmentation of the N-terminus of the primary translation product, consisting of 23 amino acid residues, into the pre-sequence (18 residues) and the pro-sequence (5 residues) with terminal Arg-Arg-residues at the cleavage site to apo AII. We must therefore correct our previously postulated 17 and 6 residues containing segmentation. So far no information has been obtained in which compartment and at what stage of posttranslational events the dimerization occurs by formation of the single disulfide bond at position Cys6 in the mature apo AII structure, leading to the symmetrical molecule.     

Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor

Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acysyst-P^® (Endotronic) with a total fiber surface area of 7.2 m² (6×1.2 m²) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics-and serum-free IMDM medium, supplemented with 50g/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20–40 g protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.     

Uptake and metabolic conversion of saturated and unsaturated fatty acids in Hep2 human larynx tumor cells

Research on fatty acid metabolism in cultured human larynx tumor cells Hep2 was carried out.The cells were incubated with either a saturated (palmitic) or a polyunsaturated (linoleic, alpha-linolenic and eicosatrienoic (n-6)) radioactive fatty acid (0.66 pM, 24 h). The best incorporation capacity was observed in the linoleic acid followed by alpha-linolenic, palmitic and eicosatrienoic acids. All fatty acids tested were anabolized to higher derivatives within their own family. Palmitic acid was primarily monodesaturated rather than elongated, proving to have a very active A9 desaturase activity.With respect to polyunsaturated acid metabolism, the conversion of alpha-linolenic acid to higher homologs, although better than linoleic acid, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells. This impairment in higher polyunsaturated fatty acid biosynthesis, reflected in the low levels of arachidonic acid in the fatty acid composition, would not reside in the A5 desaturation step since Hep2 cells can readily convert eicosatrienoic acid into arachidonic acid. Considering the potential regulatory role of specific polyunsaturated fatty acids in the cell proliferative control, the knowledge of the metabolism of fatty acids in this human tumor cell would be important for designing future experiments in order to clarify the mechanism involved in balance, proliferation and cell death.     

5-Azacytidine increases the total cellular copper content and basal level metallothionein mRNA accumulation of human Hep G2 cells.

In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells with hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper-inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed.     

In vitro mimicry of essential fatty acid deficiency in human endothelial cells by TNFalpha impact of omega-3 versus omega-6 fatty acids

Severe endothelial abnormalities are a prominent feature in sepsis with cytokines such as tumor necrosis factor (TNF)alpha being implicated in the pathogenesis. As mimic to inflammation, human umbilical vascular endothelial cells (HUVEC) were incubated with TNFalpha for 22 h, in the absence or presence of the omega-6 fatty acid (FA), arachidonic acid (AA), or the alternative omega-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). TNFalpha caused marked alterations in the PUFA profile and long chain PUFA content of total phospholipids (PL) decreased. In contrast, there was a compensatory increase in mead acid [MA, 20:3(omega-9)], the hallmark acid of the essential fatty acid deficiency (EFAD) syndrome. Corresponding changes were noted in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but not in the sphingomyelin fraction. Supplementation with AA, EPA, or DHA markedly increased the respective FA contents in the PL pools, suppressed the increase in MA, and resulted in a shift either toward further predominance of omega-6 or predominance of omega-3 FA. We conclude that short-term TNFalpha incubation of HUVEC causes an EFAD state hitherto only described for long-term malnutrition, and that endothelial cells are susceptible to differential influence by omega-3 versus omega-6 FA supplementation under these conditions.     

2- and 4-Aminobiphenyls induce oxidative DNA damage in human hepatoma (Hep G2) cells via different mechanisms

4-Aminobiphenyl (4-ABP) and its analogue, 2-aminobiphenyl (2-ABP), were examined for their ability to induce oxidative DNA damage in Hep G2 cells. Using the alkaline comet assay, we showed that 2-ABP and 4-ABP (25-200 microM) were able to induce the DNA damage in Hep G2 cells. With both compounds, formation of intracellular reactive oxygen species (ROS) was detected using flow cytometry analysis. Post-treatment of 2-ABP and 4-ABP-treated cells by endonuclease III (Endo III) or formamidopyrimidine-DNA glycosylase (Fpg) to determine the formation of oxidized pyrimidines or oxidized purines showed a significant increase of the extent of DNA migration. This indicated that oxidative DNA damage occurs in Hep G2 cells after exposure to 2-ABP and 4-ABP. This assumption was further substantiated by the fact that the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN), decreased DNA damage significantly. Furthermore, addition of the catalase (100 U/ml) caused a decrease in the DNA damage induced by 2-ABP or 4-ABP, indicating that H(2)O(2) is involved in ABP-induced DNA damage. Pre-incubation of the cells with the iron chelator desferrioxamine (DFO) (1mM) and with the copper chelator neocupronine (NC) (100 microM) also decreased DNA damage in cells treated with 200 microM 2-ABP or 200 microM 4-ABP, while the calcium chelator {1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester}(BAPTA/AM) (10 microM) decreased only DNA strand breaks in cells exposed to 4-ABP. This suggested that ions are involved in the formation of DNA strand breaks. Using RT-PCR and Western blotting, lower inhibition of the expression of the OGG1 gene and of the OGG1 protein was observed in cells treated with 4-ABP, and 2-ABP-treated cells showed a marked reduction in the expression of OGG1 gene and OGG1 protein. Taken together, our finding indicated the mechanisms of induced oxidative DNA damage in Hep G2 cell by 2-ABP and 4-ABP are different, although both tested compounds are isomers.     

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells

Physalis angulata and P. peruviana are herbs widely used in folk medicine. In this study, the aqueous and ethanol extracts prepared from the whole plant of these species were evaluated for their antihepatoma activity. Using XTT assay, three human hepatoma cells, namely Hep G2, Hep 3B and PLC/PRF/5 were tested. The results showed that ethanol extract of P. peruviana (EEPP) possessed the lowest IC50 value against the Hep G2 cells. Interestingly, all extracts showed no cytotoxic effect on normal mouse liver cells. Treatment with carbonyl cyanide m-chlorophenyl hydrazone, a protonophore, caused a reduction of membrane potential (Deltapsim) by mitochondrial membrane depolarization. At high concentrations, EEPP was shown to induce cell cycle arrest and apoptosis through mitochondrial dysfunction as demonstrated by the following observations: (i) EEPP induced the collapse of Deltapsim and the depletion of glutathione content in a dose dependent manner; (ii) pretreatment with the antioxidant (1.0 microg/ml vitamin E) protected cells from EEPP-induced release of ROS; and (iii) at concentrations 10 to 50 microg/ml, EEPP displayed a dose-dependent accumulation of the Sub-G1 peak (hypoploid) and caused G0/G1-phase arrest. Apoptosis was elicited when the cells were treated with 50 microg/ml EEPP as characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane. The results conclude that EEPP possesses potent antihepatoma activity and its effect on apoptosis is associated with mitochondrial dysfunction.