Comparison of linoleic acid and eicosapentaenoic acid incorporation into human breast cancer cells

To gain some insight into the mechanisms involved in the opposing effects of linoleic acid (LA) and eicosapentaenoic acid (EPA) on the growth and invasiveness of MDA-MB-435 human breast cancer cells, the dynamics of the uptake by cells and the incorporation of [¹⁴C]LA and [¹⁴C]EPA into major lipid and phospholipid pools, as well as the effects of unlabeled EPA or LA on the uptake and distribution of [¹⁴C]LA or [¹⁴C]EPA, respectively, were examined. Cells were exposed to [¹⁴C]LA (1.28 μg/mL) or [¹⁴C]EPA (1.0 μg/mL) and unlabeled EPA or LA, respectively, at 0, 1, 4 and 16 μg/mL for 24 h in serum-free media. The uptake of each fatty acid (FA) was linear over time and was not affected by the presence of the opposing FA. For both FA, 80–90% was incorporated into the phospholipid fraction with the remaining 10–20% in neutral lipids. The relative distribution profile of [¹⁴C]LA among the phospholipid classes indicated a preferential incorporation into phosphatidylcholine (65%), whereas [¹⁴C]EPA was mostly found in phosphatidylethanolamine (58%). In the presence of unlabeled EPA or LA at various concentrations, corresponding dose-dependent shifts of [¹⁴C]LA or [¹⁴C]EPA from the phospholipid to the neutral lipid pool were noted, which did not alter the relative distribution of the FA among the phospholipid classes. Exogenous exposure to EPA or LA increased its content in membrane phospholipids while concurrently decreasing LA or EPA content, respectively, in a dose-dependent manner. Arachidonic acid content of membrane phospholipids remained constant. The divergent distribution profiles of LA and EPA within the phospholipid compartment provides some insight into the mechanisms of their opposing effects on MDA-MB-435 cell growth and invasiveness. Also, the effects of LA and EPA on the uptake and distribution of their opposing FA shed some light on the mechanisms mediating their competitive effects.     

Transfer of arachidonic acid from lymphocytes to macrophages

The incorporation and oxidation of arachidonic acid (AA) by rat lymphocytes (LY), the transfer of AA from LY to rat macrophages (Mϕ) in co-culture, and the subsequent functional impact on Mϕ phagocytosis were investigated. The rate of incorporation of [1-¹⁴C]AA by untreated-LY and TG (thioglycolate treated)-LY (TG-LY) was 158±8 nmol/10¹⁰ LY per h for both untreated-LY and TG-LY. The oxidation of AA was 3.4-fold higher in TG-LY as compared with untreated cells. LY from TG-injected rats had a 2.5-fold increase in the oxidation of palmitic (PA), oleic (OA), and linoleic (LA) acids. After 6 h of incubation, [¹⁴C] from AA was distributed mainly into phospholipids. The rate of incorporation into total lipids was 1071 nmol/10¹⁰ cells in untreated-LY and 636 nmol/10¹⁰ cells in TG-LY. [¹⁴C]AA was transferred from LY to co-cultured Mϕ in substantial amounts (8.7 nmol for untreated and 15 nmol per 10¹⁰ for TG cells). Exogenously added AA, PA, OA, and LA caused a significant reduction of phagocytosis by resident cells. Mϕ co-cultured with AA-preloaded LY showed a significant reduction of the phagocytic capacity (about 40% at 35 μM). LY preloaded with PA, LA, and OA also induced a reduction in phagocytic capacity of co-cultured Mϕ. TG treatment abolished the AA-induced inhibition of phagocytosis in Mϕ co-cultured with TG-LY. Therefore, the transfer of AA between leukocytes is a modulated process and may play an important role in controlling inflammatory and immune response.     

Decreased production of inflammatory mediators in human osteoarthritic chondrocytes by conjugated linoleic acids

Osteoarthritic chondrocytes (OC) produce excessive prostaglandin E₂ (PGE₂) and nitric oxide (NO), which function as inflammation mediators in the pathogenesis of osteoarthritis (OA). This study examined the effect of CLA alone and in combination with other PUFA on the FA composition and the production of PGE₂ and NO in OC cultures isolated from OA patients. Human OC were grown in monolayer and treated with one of the following PUFA treatments: CLA, CLA+arachidonic acid (CLA/AA), CLA_EPA (CLA/EPA), linoleic acid (LA), LA+AA (LA/AA), LA+EPA (LA/EPA), and ethanol (as a vehicle control) at 10 and 20 μM for 6 d. Supplementation of PUFA at 10 μM for 6 d did not introduce any cytotoxic effects or morphological changes in OC, whereas 20 μM resulted in apoptosis. Cultures of OC treated with CLA, CLA/AA, and CLA/EPA had higher concentrations of CLA isomers, and these isomers were not detected in other treatments. Supplementation of CLA or LA alone to the OC led to a lower PGE₂ production compared to the control. Combination of CLA/EPA resulted in the lowest PGE₂ production in cultured OC. OC cultures treated with CLA were lower in NO production than the control, whereas the LA/AA treatment demonstrated the lowest NO production. The fact that CLA alone or in combination with other PUFA modulated PGE₂ and NO production in human OC cultures suggests that these 18∶2 isomers may have the potential to influence OA pathogenesis.     

Ketone bodies,glucose and glutamine as lipogenic precursors in human diploid fibroblasts

Incorporation of [¹⁴C] from acetoacetate, D(-)- and L(+)-3-hydroxybutyrate, glucose, glutamine, acetate and palmitate in cellular lipids were studied in cultures in human diploid fibroblasts (HDF). The results showed that acetoacetate was 2–10 times more effective as a lipogenic precursor than was either D- or L-3-hydroxybutyrate. Its extent of incorporation into lipids was 2- to 8-fold more than the other precursors examined under conditions when the overall rates of nonsaponifiable and saponi-fiable lipogenesis as measured by³H₂O incorporation were essentially unchanged. Acetoacetate supported both saponifiable and nonsaponifiable lipid syntheses with half-saturation values (K_m app.) of 185 μM and 30 μM, respectively. Glucose stimulated acetoacetate incorporation into lipids whereas, conversely, acetoacetate inhibited [¹⁴C] glucose incorporation into lipids. The presence of low density lipoproteins (LDL) cholesterol (@40 μg cholesterol/mL) inhibited the incorporation of [¹⁴C] from acetoacetate 56% into nonsaponifiable lipids; the inhibition was consistently higher (75%) when [¹⁴C] glucose or glutamine were the precrusors. The loss of 3-hydroxy-3-methyl-glutaryl CoA (HMG CoA) reductase activity upon addition of LDL-cholesterol was greater than the suppression of [¹⁴C] incorporation from acetoacetate or glucose into nonsaponifiable lipids. In the presence of glucose, [¹⁴C] acetoacetate was incorporated into 3-βOH sterols (digitonin precipitable). 7.7±1.1 times more effectively than was [¹⁴C] glucose. The results suggest that HDF would be a suitable model to investigate the effects of various precrusors of HMG CoA on the rate of cholesterol biosynthesis.     

Inhibition of cholesterol synthesis by cyclopropylamine derivatives of squalene in human hepatoblastoma cells in culture

Two squalene derivatives, trisnorsqualene cyclopropylamine and trisnorsqualeneN-methylcyclopropylamine, were synthesized and tested for inhibition of lanosterol and squalene epoxide formation from squalene in rat hepatic microsomes, and for the inhibition of cholesterol syntheses in human cultured hepatoblastoma (HepG2) cells. Trisnorsqualene cyclopropylamine inhibited [³H]-squalene conversion to [³H]squalene epoxide in microsomes (IC₅₀=5.0 μM), indicating that this derivative inhibited squalene mono-oxygenase. Trisnorsqualenen-methylcyclopropylamine inhibited [³H]squalene conversion to [³H]lanosterol (IC₅₀=12.0 μM) and caused [³H]-squalene epoxide to accumulate in microsomes, indicating that this derivative inhibited 2,3-oxidosqualene cyclase. Cholesterol biosynthesis from [¹⁴C]acetate in HepG2 cells was inhibited by both derivatives (IC₅₀=1.0 μM for trisnorsqualene cyclopropylamine; IC₅₀=0.5 μM for trisnorsqualeneN-methylcyclopropylamine). Cells incubated with trisnorsqualene cyclopropylamine accumulated [¹⁴C]squalene, while cells incubated with trisnorsqualeneN-methylcyclopropylamine accumulated [¹⁴C]squalene epoxide and [¹⁴C]squalene diepoxide. The concentration range of inhibitor which caused these intermediates to accumulate coincided with that which inhibited cholesterol synthesis. The results indicate that cyclopropylamine derivatives of squalene are effective inhibitors of cholesterol synthesis, and that substitutions at the nitrogen affect enzyme selectivity and thus the mechanism of action of the compounds.     

Effect of the Δ<Superscript>6</Superscript>-desaturase inhibitor SC-26196 on PUFA metabolism in human cellsinhibitor SC-26196 on PUFA metabolism in human cells

The objective of this study was to determine the effect of 2,2-diphenyl-5-(4-{[(1E)-pyridin-3-yl-methylidene]-amino}piperazin-1-yl)pentanenitrile (SC-26196), a Δ⁶-desaturase inhibitor, on PUFA metabolism in human cells. SC-26196 inhibited the desaturation of 2 μM [1-¹⁴C] 18∶2n−6 by 87–95% in cultured human skin fibroblasts, coronary artery smooth muscle cells, and astrocytes. By contrast, SC-26196 did not affect the conversion of [1-¹⁴C]20∶3n−6 to 20∶4 in the fibroblasts, demonstrating that it is selective for Δ⁶-desaturase. The IC₅₀ values for inhibition of the desaturation of 2 μM [1-¹⁴C] 18∶3n−3 and [3-¹⁴C]24∶5n−3 in the fibroblasts, 0.2–0.4 μM, were similar to those for the inhibition of [1-¹⁴C] 18∶2n−6 desaturation, and the rates of recovery of [1-¹⁴C] 18∶2n−6 and [3-¹⁴C] 24∶5n−3 desaturation after removal of SC-26196 from the culture medium also were similar. SC-26196 reduced the conversion of [3-¹⁴C] 22∶5n−3 and [3-¹⁴C] 24∶5n−3 to DHA by 75 and 84%, respectively, but it had no effect on the retroconversion of [3-¹⁴C] 24∶6n−3 to DHA. These results demonstrate that SC-26196 effectively inhibits the desaturation of 18- and 24-carbon PUFA and, therefore, decreases the synthesis of arachidonic acid, EPA, and DHA in human cells. Furthermore, they provide additional evidence that the conversion of 22∶5n−3 to DHA involves Δ⁶-desaturation.     

Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets

This study was designed to elucidate changes in rabbit platelet lipids induced by a cholesterol rich diet and to explore the possible correlation of these lipid changes with platelet abnormalities. Pronounced biochemical alterations were observed when serum cholesterol levels of 700–1000 mg% were reached. Hypercholesterolemic (HC) platelets contained 37% more neutral lipids and 16% less phospholipids than the controls. Lysolecithin, cholesterol esters and phosphatidylinositol (PI) levels were increased in HC platelets, and the levels of phosphatidylcholine (PC) were decreased. The cholesterol/phospholipid molar ratio of lipidemic platelets increased from 0.55±0.011 to 0.89±0.016 (P<0.01) in eight weeks. HC platelets had 90% more arachidonic acid (AA) in the PI than normal platelets. No significant changes in AA of PC were observed. Platelet function was monitored by the uptake and release of [¹⁴C]serotonin in platelet rich plasma (PRP), using varying concentrations of collagen as an aggregating agent. The uptake of [¹⁴C]serotonin in HC and normal platelets ranged from 78–94%. The percent of [¹⁴C]serotonin released from normal and HC platelets was proportional to the concentration of collagen. However, lipidemic platelets were hyperreactive to low concentrations of collagen. Incorporation of 50 μM acetylsalicylic acid into the aggregating medium suppressed the release of [¹⁴C]serotonin in normal PRP by more than 90%, but had only a partial effect on lipidemic PRP.     

Elongation predominates over desaturation in the metabolism of 18∶3n−3 and 20∶5n−3 in turbot (Scophthalmus maximus) brain astroglial cells in primary culture

The origin of docosahexaenoic acid (DHA, 22∶6n−3) that accumulates in turbot brain during development was investigated by studying the incorporation and metabolismvia the desaturase/elongase pathways of [1-¹⁴C]-labelled polyunsaturated fatty acids (PUFA) in primary cultures of brain astrocytic glial cells. There was little specificity evident in the total incorporation of PUFAs into the turbot astrocytes. However, specificity was apparent in the distribution of the various PUFAs among the individual lipid classes. In particular, there was very specific incorporation of [¹⁴C]arachidonic acid (AA, 20∶4n−6) into phosphatidylinositol balanced by a lower incorporation of this acid into total diradyl glycerophosphocholines. [¹⁴C]-Linolenic acid (LNA, 18∶3n−3) and [¹⁴C]eicosapentaenoic acid (EPA, 20∶5n−3) were metabolizedvia the desaturase/elongase pathways to a significantly greater extent than [¹⁴C]linoleic acid (18∶2n−6) and [¹⁴C]AA. The turbot astrocytes expressed very little Δ5 desaturase activity and only low levels of Δ4 desaturation activity. Although the percentages were small, approximately 4–5 times as much labelled DHA was produced from [¹⁴C]EPA compared with [¹⁴C]LNA. However, it was concluded that very little DHA in the turbot brain could result from the metabolism of LNA and EPA in astrocytic glial cells.     

Plasma clerance and hepatic utilization of stearic,myristic and linoleic acids introducedvia chylomicrons in rats

The primary objective of the present study was to compare the rates of plasma clearance and hepatic utilization of stearic (18∶0), myristic (14∶0) and linoleic (18∶2) acids, as introducedvia chylomicrons. Lymph chylomicrons were specifically labeledin vivo with [¹⁴C]stearic and (SA), [¹⁴C]myristic acid (MA), or [¹⁴C]linoleic acid (LA) by infusing donor rats intraduodenally with the labeled fatty acids in a lipid emulsion. Following intravenous injection of recipient rats with the labeled chylomicrons, the rates of plasma clearance and incorporation of the label in triglycerides (TG), phospholipids (PL) and other lipids in the liver were compared at 5, 15 and 30 min. [¹⁴C]SA was cleared at a slightly faster rate (t_1/2=7.0 min) than [¹⁴C]MA (t_1/2=8.1 min) and [¹⁴C]LA (t_1/2=8.0 min) (P<0.05). [¹⁴C]SA was accumulated in the liver at a significantly faster rate than [¹⁴C]MA and [¹⁴C]LA. At the peak (15 min) of hepatic uptake, 30.3% of [¹⁴C]SA, 26.2% of [¹⁴C]LA and 21.9% of [¹⁴C]MA were recovered in the liver. At 30 min, 33.5% of [¹⁴C]SA was taken up by the liver, whereas 27.8% of [¹⁴C]LA and only 15.2% of [¹⁴C]MA were removed. In the liver, the percentage of [¹⁴C]SA incorporated into PL steadily increased with time, whereas the percent-age incorporated into TG decreased. [¹⁴C]SA was preferentially incorporated into PL at all time intervals, as compared with [¹⁴C]MA and [¹⁴C]LA. At 30 min, 38.6% of [¹⁴C]SA was found in PL, and only 5.2% of [¹⁴C]MA and 12.0% of [¹⁴C]LA were present in PL. A large proportion of hepatic [¹⁴C]MA remained unesterified (free fatty acid) throughout the 30-min period, with a small proportion incorporated into PL and TG. Of the total liver¹⁴C radioactivity recovered at 30 min, 63.8% of [¹⁴C]MA, 48.8% of [¹⁴C]LA and 25.5% of [¹⁴C]SA were found unesterified. During 30 min, a significantly greater amount of [¹⁴C]MA (76.9%) was oxidized in both the liver and the peripheral tissue combined, compared with [¹⁴C]LA (64.7%) and [¹⁴C]SA (61.2%). A higher proportion of [¹⁴C]LA was incorporated into TG than into PL at all time intervals. No differences were noted in the relative distribution of¹⁴C in cholesterol and other lipids among the three fatty acids. Using labeled fatty acids incorporatedin vivo into chylomicrons, the present study demonstrated that SA, MA and LA are distinctly different in their metabolic behavior. During the initial 30 min after their entry into the blood, 92–95% of the fatty acids were cleared. During this early phase of metabolism, [¹⁴C]SA was preferentially utilized for liver PL synthesis, whereas [¹⁴C]LA was better incorporated into TG. [¹⁴C]MA was poorly incorporated into hepatic lipids, but was preferentially oxidized in the liver or utilized by the peripheral tissue.     

Uptake and oxidation of malonaldehyde by cultured mammalian cells

Primary cultures of rat skin fibroblasts were used as a model system to investigate the cellular uptake and oxidation of malonaldehyde (MA). The cells were grown in a medium containing 10⁻⁵ M, 10⁻⁴ M or 10⁻³ M concentrations of [1,3-¹⁴C]MA. There was a limited, concentration-dependent uptake of MA by 24 hr (∼4% at all concentrations). The uptake of [1,2-¹⁴C]acetate by 24 hr was ∼24%; 83–89% of the¹⁴C in the MA taken up was oxidized to¹⁴CO₂ by 24 hr and ∼5% was recovered in the major lipids. Despite its low uptake and rapid oxidation to CO₂, pretreatment of the cells with 10⁻³ M MA for 24 hr produced a latent inhibition of [¹⁴C]glucose oxidation. Limited cellular uptake of MA may explain the tolerance of cells grown in culture to relatively high MA concentrations.     

Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3-L1 preadipocytes

Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt. 1), cells were cultured in media containing a crude mixture of CLA isomers or pure LA at 0, 10, 50, or 200 μM for 4 d. Preadipocyte proliferation (cell number, ³H-thymidine incorporation into DNA) decreased as the level of CLA increased in the cultures. In contrast, LA had no impact on DNA synthesis. In Experiment 2a, postconfluent cultures were grown in media containing a crude mixture of CLA isomers or LA at 0, 10, 50, or 200 μM for the next 6 d. Postconfluent cultures supplemented with 50–200 μM CLA had less triglyceride (TG) and were smaller in size than cultures supplemented with similar amounts of LA. In Experiment 2b, postconfluent cultures supplemented with 200 μM of a crude mixture of CLA isomers or LA were harvested on days 1, 3, 6, or 9. Differences in TG content of cultures supplemented with 200 μM CLA compared to control and LA-supplemented cultures became apparent after 3 d of culture. Experiments 3a and 3b examined whether the fatty acid vehicle (BSA vs. ethanol) or the vitamin E status (±0.2 mM α-tocopherol) of the cultures altered CLA’s impact on preadipocyte TG content. In Experiment 3a, ethanol-treated cultures had more TG than non-ethanol-treated cultures regardless of the fatty acid treatment. In Experiment 3b, cultures treated with 100 μM of either a crude mixture of CLA or the trans-10, cis-12 CLA isomer without supplemental vitamin E for 6 d had less TG than CLA-treated cultures containing vitamin E. In Experiment 4, postconfluent cultures were grown in media containing 100 μM LA or either a crude mixture of CLA isomers or the trans-10, cis-12 CLA isomer for 24–96 h to assess CLA’s influence on the cell cycle and indices of apoptosis. Cultures treated with 100 μM CLA for 24–96 h had more apoptotic cells than BSA- or LA-treated cultures. Furthermore, cultures treated for 48 h with CLA had fewer cells in the S-phase than control cultures. The effects of the trans-10,cis-12 CLA isomer were more pronounced than those of the crude mixture of CLA isomers. These data suggest that CLA may exert its antiobesity effects by inhibiting proliferation, attenuating TG content, and/or inducing apoptosis in (pre)adipocytes.     

Pharmacological modulation of fatty acid desaturation and of cholesterol biosynthesis in THP-1 cells

In THP-1 cells, simvastatin decreases, in a concentration-dependent manner, cholesterol synthesis and increases linoleic acid (LA) conversion to its long-chain derivatives, in particular to arachidonic acid, activating Δ6 and Δ5 fatty acid (FA) desaturases. The intermediates in cholesterol synthesis, mevalonate and geranylgeraniol, partially reverse the effects of simvastatin on the LA conversion. The aims of this work were to evaluate: (i) the correlation between cholesterol synthesis and desaturase activity and (ii) the possible involvement of protein isoprenylation in desaturase activity, assessed through pharmacological treatments. THP-1 cells were incubated with [1-¹⁴C]LA or with [1-¹⁴C]di-homo-γ-linolenic acid (DHGLA) and treated with simvastatin or with curcumin and nicardipine, inhibitors of desaturases. Curcumin was more active than nicardipine in inhibiting LA and DHGLA conversion: 20 μM curcumin, alone or with simvastatin, totally inhibited Δ6 and Δ5 desaturation steps; 10 μM nicardipine only partially inhibited the enzymes, being more active on Δ5 desaturase. Simvastatin treatment decreased the incorporation of acetate in cholesterol (−93.8%) and cholesterol esters (−70.2%), as expected. Curcumin and nicardipine also decreased cholesterol synthesis and potentiated simvastatin. Finally, the isoprenylation inhibitors (perillic acid and GGTI-286) neither affected the conversion of LA nor inhibited the Δ5 desaturase activity. In conclusion, our results indicate that there is no direct relationship between cholesterol synthesis and desaturase activity. In fact, simvastatin decreased cholesterol synthesis and enhanced LA conversion (mainly Δ5 desaturation), whereas curcumin and nicardipin decreased Δ5 desaturation, with a limited effect on cholesterol synthesis.     

Effect of ethanol on transport from rat intestine during high and low rates of oleate absorption

Long-chain fatty acids (LCFA) are transported predominantly in the intestinal lymph when rates of LCFA absorption are high, and oral ethanol has been shown to enhance this lymphatic transport. A greater proportion of absorbed LCFA is transported via portal blood when rates of LCFA absorption are low. We tested the hypothesis in unanesthetized lymph-fistula rats that ethanol might also enhance the mucosal absorption and lymphatic transport of oleic acid when oleate absorption rates were low. The results did not support this hypothesis. Ethanol enhanced oleate absorption and transport from the intestine when 360 μmol, but not when 8 μmol of [¹⁴C] oleate was infused intraduodenally over 4 hr. There were major differences in intestinal mucosal metabolism of high and low loads of oleic acid. After the high load, the proportion of intestinal [¹⁴C] phospholipid to [¹⁴C] neutral lipid was 8∶92. This ratio changed to 37∶63, and the percentage of neutral¹⁴C as triglyceride decreased from 87 to 68% when the low load of oleate was infused. We suggest that a portion of absorbed LCFA is incorporated into phospholipid and transported as high-density lipoproteins in portal blood. This portal pathway for LCFA was uninfluenced by ethanol in the present experiments.     

Differential alterations of ethanolamine and choline phosphoglyceride metabolism by clofibrate and retinoic acid in human fibroblasts are not mediated by phorbol ester-sensitive protein kinase C

Peroxisomal proliferators and retinoids have been reported to interact to regulate lipid metabolism, particularly β-oxidation of fatty acids. Based on postulated interactions of these agents at the levels of receptors and response elements, we examined whether interactions exist between the peroxisomal proliferator, clofibrate (CLF), and retinoic acid (RA) in modulation of phospholipid turnover in cultured human skin fibroblasts. Treatment of cultured cells with either 25 μM CLF or 1 μM RA alone decreased [¹⁴C]ethanolamine incorporation into ethanolamine phosphoglycerides (EPG) by 20–30%, and simultaneous exposure to both agents resulted in additive inhibition. By contrast, [³H]choline incorporation into phospholipid was stimulated 5–30% by incubation with either agent; when CLF and RA were administered together, the stimulatory effects were additive. Different types of pulse-chase studies examining effects on uptake, biosynthesis, and degradation of labelled phospholipids indicated stimulation of EPG degradation and inhibition of phosphatidylcholine degradation by CLF; no effect on catabolism of either phospholipid was observed with RA. Combinations of modifiers of protein kinase activity [4β-12-O-tetradecanoylphorbol-13-acetate (β-TPA), 1-(5-isoquino-linesulfonyl)-2-methylpiperazine dihydrochloride,N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride,bis-indolylmaleimide, staurosporine] indicated that β-TPA-responsive protein kinases were not involved. Accordingly, CLF and RA regulate biosynthesis and degradation of ethanolamine and choline phosphoglycerides in cultured skin fibroblasts by different mechanisms that do not involve classical protein kinase C (PKC) isoforms, even though turnover of phospholipids generating lipid activators of PKC occurs.     

The differential effect of eicosapentaenoic acid and oleic acid on lipid synthesis and VLDL secretion in rabbit hepatocytes

The suppression of plasma very low density lipoprotein (VLDL) triglyceride levels by dietary fish oils rich in polyunsaturated n−3 fatty acids has been attributed to decreased hepatic VLDL secretion. To investigate the effect of n−3 fatty acids on lipid metabolism and VLDL secretion in a tissue culture system, we incubated rabbit hepatocytes with oleic acid and eicosapentaenoic acid (EPA) and examined [³H]glycerol and [¹⁴C]fatty acid incorporation into hepatocyte triglyceride and phospholipid and into media VLDL. Glycerol incorporation studies showed that EPA failed to stimulate VLDL triglyceride secretion from hepatocytes as occurred with oleic acid (P<0.05). Oleic acid preferentially enhanced hepatocyte triglyceride synthesis while EPA stimulated significantly phospholipid synthesis (P<0.01). Varying the relative concentrations of oleic acid and EPA at a constant total fatty acid concentration corroborated preferential triglyceride synthesis from oleic acid. Synthesis shifted predominantly to phospholipids with increasing concentrations of EPA and lower levels of oleic acid. Incorporation of the [¹⁴C]fatty acids (800 μM) followed similar patterns: 87% of [¹⁴C]oleic acid was incorporated into hepatocyte triglyceride and 44% of [¹⁴C]EPA was assimilated in hepatocyte phospholipid (p<0.001). Fatty acids at trace concentrations (53 nM) showed a more divergent pattern of lipid incorporation: 60% of [¹⁴C]oleic acid was incorporated into triglyceride while 91% of [¹⁴CEPA was incorporated into phospholipid (p<0.001). We conclude that in primary rabbit hepatocyte culture, which appears to be a useful model to study lipid metabolism and VLDL secretion, EPA is avidly incorporated into phospholipid while oleic acid predominantly becomes esterified in triglyceride. In addition, EPA, unlike oleic acid, fails to stimulate hepatocyte VLDL secretion. These divergent effects on hepatocyte lipid metabolism are, at least in part, likely to be responsible for fish oil induced suppression of plasma triglycerides.     

Thioglycolate-elicited rat macrophages exhibit alterations in incroporation and oxidation of fatty acids

Incorporation and oxidation of fatty acids (FA) were investigated in resident and thioglycolate-clicited (TG-elicited) rat macrophages (Mϕ). Both cell types presented a time-dependent incorporation of [¹⁴C]-labeled palmitic acid (PA), oleic acid (OA), linoleic acid (LA), and arachidonic acid (AA) up to 6h. The total amount of [¹⁴C]-FA incorporated by resident Mϕ after 6 h was: AA>PA=LA>OA. TG-elicited cells presented a 50% reduction in the incorporation of LA, PA, and AA, whereas that of OA remained unchanged as compared to resident Mϕ. The FA were oxidized by resident Mϕ as follows: LA>OA>PA>AA. TG elicitation promoted a reduction of 42% in LA oxidation and a marked increase in AA oxidation (280%). The increased oxidation of AA in TG-elicited cells may account for the lower production of prostaglandins in Mϕ under these conditions. The full significance of these findings for Mϕ function, however, remains to be examined.     

Effects of phosphatidylcholines on de novo synthesis and excretion of sterol by L-929 fibroblasts

The effects on [¹⁴C] sterol synthesis and excretion by exposure of L-929 cells to several phosphatidylcholines (PC) has been examined. No significant effects were noted on either parameter during a 6-hr period if exposure of cells to the phospholipid preceded the addition of [1-¹⁴C] acetate by just 30 min. However, if cultures were grown in media containing delipidized serum and 2×10⁻⁵ M PC through 2 or more subculturings prior to adding [1-¹⁴C] acetate, the amount of [¹⁴C] sterol increased in both cells and medium by 70–200% when saturated or monounsaturated PC were used. Dilinoleylphosphatidylcholine (18∶2 PC) at the same concentration did not stimulate synthesis or excretion of newly synthesized sterol. Total cellular sterol was determined by gas chromatography, and was only marginally affected by long-term exposure to dipalmitylphosphatidylcholine (16∶0 PC), whereas the total sterol of the medium increased by 4-fold over a 19-hr period. Cultures which had been exposed to 16∶0 PC through 3 subculturings continued to display enhanced de novo sterol synthesis, but not excretion, for up to 5 hr after replacement with fresh medium lacking 16∶0 PC. The disparity in response to 2×10⁻⁵ M levels of 16∶0 PC and 18∶2 PC may relate to differences in metabolism of cellular fatty acids, whereas relatively small changes in the cellular fatty acid composition were noted with 16∶0 PC-treated cells. The results indicate that extracellular PC can promote sterol synthesis and excretion by L-929 cells, and that the magnitude of this response is influenced by the time of exposure to the phospholipid and by its fatty acid composition.     

Adenosine triphosphate (ATP) and other nucleotides stimulate the hydrolysis of phosphatidylethanolamine in intact fibroblasts

Addition of adenosine triphosphate (ATP) to [¹⁴C]ethanolamine-prelabeled NIH 3T3 fibroblasts resulted in rapid formation of [¹⁴C]ethanolamine from the prelabeled cellular phosphatidylethanolamine (PtdEtn) pool. After 2-min exposure, 10 μM ATP had near maximal effects on PtdEtn hydrolysis. Several other nucleotides, including UTP, ITP, and the stable ATP analog adenosine 5′-O-(3-thiotriphosphate) (ATPγS), also had stimulatory effects on PtdEtn hydrolysis with a potency comparable to that observed with ATP. The same nucleotides which acted on PtdEtn hydrolysis also had similar stimulatory effects on the hydrolysis of phosphatidylcholine (PtdCho) in [¹⁴C]choline-labeled cells. In isolated membranes, Mg²⁺ greatly enhanced the stimulatory effects of ATP and ATPγS, but not of other nucleotides, on the hydrolysis of PtdEtn and PtdCho. Results indicate that in isolated membranes, both ATP and ATPγS stimulate phospholipid hydrolysis by two different mechanisms, but in intact cells only one of these mechanisms appears to be responsive to externally added nucleotides.     

Eicosapentaenoic and arachidonic acid: Comparison of metabolism and activity in murine epidermal cells

The biological activity, including metabolism and modulation of ornithine decarboxylase activity and DNA synthesis, of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were compared in epidermal cells from SENCAR mice. Radiolabelled AA and EPA were found to be similarly incorporated into and released from membrane phospholipids of unstimulated cultures. However, when cells were stimulated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA), the release of AA was significantly higher than the release of EPA. The extent of metabolism of AA and EPA to prostaglandins was determined in both freeze-thawed cell preparations and in viable cultured cells. In the freeze-thawed preparations, use of AA as a substrate resulted in significantly more PGF than when EPA was used as the substrate. However, more PGE₃ was formed than PGE₂. PGD levels were the same for either fatty acid precursor. Prostaglandin production was also determined in viable cultured cells since other influences such as phospholipase A₂ activity can modify prostaglandin production. Control cultures prelabelled with either AA or EPA produced similar amounts of the respective PGF, PGE, and PGD. However, TPA-stimulated cultures produced significantly higher amounts of each prostaglandin in cultures prelabelled with AA compared to cells prelabelled with EPA. HETE or HEPE production was the same both for cultured cells prelabelled with AA or EPA and for homogenates from uncultured cells incubated directly with the radiolabelled fatty acids. TPA-induced ornithine decarboxylase (ODC) was significantly higher in AA-treated cultures compared to EPA-treated cultures. AA supports DNA synthesis to a greater extent than EPA, either alone or in the presence of TPA. These findings suggest that AA and EPA do not have equivalent biological activity in mouse epidermal cells.     

Incorporation of linoleic acid and its conversion to λ-linolenic acid in fungi

The incorporation of [1-¹⁴C]linoleic acid (LA) into lipids ofMortierella ramanniana var.angulispora was studied to determine which lipid classes participated in the δ6-desaturation of [1-¹⁴C]LA. [1-¹⁴C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1-¹⁴C]LA incorporation between fungal cells at the exponential growth phase and the stationary growth phase showed that [1-¹⁴C]LA incorporation into most lipids—except for triacylglycerol (TG) and phosphatidylcholine (PC)—were greatly reduced at the stationary growth phase. Desaturation of [1-¹⁴C]LA into λ-linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse-chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly turned over, while that in TG and diacylglycerol (DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1-¹⁴C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest that LA, which has been esterified into phospholipids such as PC, PE and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids may be reflected in the GLA content in the individual lipids.