Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Mercury inhibition of fatty acid synthesis in chicks.

Male chicks were fed a commercial ration and were given drinking water which contained 0, 50, 100, 150, 200 or 300 mug of mercury/ml as mercuric chloride from hatching to 3 weeks of age. In one experiment, the mercuric chloride was administered by injection into the abdominal cavity rather than in the drinking water. At 3 weeks the chicks were killed, and the livers were removed and weighed. The activity of fatty acid synthetase in the 800 X gav supernatant fractions of the liver homogenates and in vivo incorporation of [14C]acetate into liver and carcass fatty acids and respiratory 14CO2 was determined as indicated. Administration of mercury at a treatment level of 300 mug/ml of drinking water depressed growth, feed and water consumption, liver weight, hepatic fatty acid synthetase activity, and in vivo incorporation of [14C]acetate into liver and carcass fatty acids, and increased the production of respiratory 14CO2 as compared with controls. In experiments in which graded doses of mercury were administered, body weights, liver weights, and feed and water intakes of the chicks receiving 0, 50 and 100 mug of mercury/ml of drinking water were similar to each other, but these parameters were severely depressed by 200 mug of mercury/ml of drinking water. Mercury caused a dose-related decrease of fatty acid synthetase activity. Incorporation of [14C]acetate into carcass fatty acid was depressed by 50 and 200 mug of mercury/ml of drinking water; incorporation into liver fatty acids and production of respiratory 14CO2 was not affected by mercury. Intra-abdominal injection of 6 mg of mercury/100 g body weight (as mercuric chloride) into well alimented chicks depressed hepatic fatty acid synthetase activity at 1 h post-injection. The data are consistent with the hypothesis that a portion of the effects of mercury on fatty acid synthesis are direct rather than a secondary effect of inanition.     

Alteration of polyunsaturated fatty acid status and metabolism in health and disease

Essential polyunsaturated fatty acids (PUFA) cannot be synthesised in the body and must be ingested by food. A balanced intake of both n-6 and n-3 PUFA is essential for good health. PUFA are the basic constituents of phospholipid membranes and determine cellular membrane fluidity and modulate enzyme activities, carriers and membrane receptors. They are also precursors of active metabolites known collectively as eicosanoids (prostaglandins, prostacyclins, thromboxanes and leukotrienes) which regulate our cellular functions. Studies indicate that n-3 PUFA have anti-inflammatory, antithrombotic, antiarrhythmic actions and immuno-modulating properties. Erythrocyte fatty acid status is a reflection of dietary fat intake. It also explores PUFA metabolism and gives information about the integration of these fatty acids into cellular membranes. Thus, erythrocyte fatty acid analysis can detect PUFA insufficiencies and imbalances from the diet, but also metabolic abnormalities and lipid peroxidation. It can be helpful in the prevention and the control of chronic diseases in which PUFA alterations have been observed as coronary heart diseases, hypertension, cancer, diabetes, inflammatory and auto-immune disorders, atopic eczema, Alzheimer dementia, major depression, schizophrenia, multiple sclerosis, etc.     

Fatty acid oxidation and fatty acid synthesis in energy restricted rats

The importance of fat oxidation and fatty acid synthesis were examined in rats fed approximately one half their ad libitum food intake for a period of 13 days followed by 7 days of ad libitum feeding (refed rats). This study was undertaken because previous reports demonstrated that refed rats rapidly accumulated body fat. Our results confirmed this observation: refed rats accrued body fat and body weight at rates that were approximately 3 times higher than controls. Evidence for a period of increased metabolic efficiency was demonstrated by measuring the net energy requirement for maintenance over the refeeding period: refed rats had a reduced metabolic rate during the period of energy restriction (approximately 30% lower than control) and this persisted up to 2 days after the reintroduction of ad libitum feeding. The major factor responsible for the rapid fat gain was a depressed rate of fatty acid oxidation. Calculations of protein and carbohydrate intake over the refeeding period showed that the simplest explanation for the decrease in fatty acid oxidation is fat sparing. This is possible because of the large increase in dietary carbohydrate and protein intake during the refeeding period when metabolic rates are still depressed. The increased carbohydrate and protein may adequately compensate for the increasing energy requirements of the ER rats over the refeeding period affording rats the luxury of storing the excess dietary fat energy.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids

The conversion of the plant-derived omega-3 (n-3) α-linolenic acid (ALA, 18:3n-3) to the long-chain eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) can be increased by ALA sufficient diets compared to ALA deficient diets. Diets containing ALA above an optimal level result in no further increase in DHA levels in animals and humans. The present study evaluates means of maximizing plasma DHA accumulation by systematically varying both linoleic acid (LA, 18:2n-6) and ALA dietary level. Weanling rats were fed one of 54 diets for three weeks. The diets varied in the percentage of energy (en%) of LA (0.07–17.1 en%) and ALA (0.02–12.1 en%) by manipulating both the fat content and the balance of vegetable oils. The peak of plasma phospholipid DHA (>8% total fatty acids) was attained as a result of feeding a narrow dietary range of 1–3 en% ALA and 1–2 en% LA but was suppressed to basal levels (～2% total fatty acids) at dietary intakes of total polyunsaturated fatty acids (PUFA) above 3 en%. We conclude it is possible to enhance the DHA status of rats fed diets containing ALA as the only source of n-3 fatty acids but only when the level of dietary PUFA is low (<3 en%).     

Brugia malayi: microfilarial polyunsaturated fatty acid composition and synthesis

The polyunsaturated fatty acid composition of Brugia malayi microfilariae was analyzed by gas chromatography and compared to that of sera from B. malayi-infected jirds. The essential fatty acid, linoleic acid (18:2 omega 6), was the most abundant fatty acid present in both microfilarial total lipids and phospholipids as well as in jird sera. In contrast, arachidonic acid (20:4 omega 6), as well as the 18:3 omega 6, 20:2 omega 6, and 20:3 omega 6 intermediates that are formed in the enzymatic conversion of linoleic acid to arachidonic acid, were proportionally more abundant in microfilariae than in jird sera. To assess the capacity of microfilariae to transform linoleic acid into arachidonic acid, B. malayi microfilariae were incubated with [14C]linoleic acid. Microfilarial lipids were extracted and resolved by high-pressure liquid chromatography and thin-layer chromatography. A portion of [14C]linoleic acid incorporated by microfilariae was converted to [14C]arachidonic acid. Thus, microfilariae can not only incorporate exogenous arachidonic acid, as previously demonstrated, but can also synthesize arachidonic acid from exogenous linoleic acid. The capacity of microfilariae to utilize specific host polyunsaturated fatty acids raises the possibility that intravascular filarial parasites may synthesize eicosanoid metabolites of arachidonic acid that could mediate filarial-host cell interactions.     

One-pot synthesis of polyunsaturated fatty acid amides with anti-proliferative properties

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells.     

Fatty acid synthase inhibition in human breast cancer cells leads to malonyl-CoA-induced inhibition of fatty acid oxidation and cytotoxicity.

Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy.     

Elongase reactions as control points in long-chain polyunsaturated fatty acid synthesis

Background

Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA.

Methodology/Principal Findings

The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C₂₀ and C₂₂ polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3.

Conclusions

The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.     

Dietary n-6 polyunsaturated fatty acid deprivation increases docosahexaenoic acid metabolism in rat brain

Dietary n-6 polyunsaturated fatty acid (PUFA) deprivation in rodents reduces brain arachidonic acid (20:4n-6) concentration and 20:4n-6-preferring cytosolic phospholipase A(2) (cPLA(2) -IVA) and cyclooxygenase (COX)-2 expression, while increasing brain docosahexaenoic acid (DHA, 22:6n-3) concentration and DHA-selective calcium-independent phospholipase A(2) (iPLA(2) )-VIA expression. We hypothesized that these changes are accompanied by up-regulated brain DHA metabolic rates. Using a fatty acid model, brain DHA concentrations and kinetics were measured in unanesthetized male rats fed, for 15 weeks post-weaning, an n-6 PUFA 'adequate' (31.4 wt% linoleic acid) or 'deficient' (2.7 wt% linoleic acid) diet, each lacking 20:4n-6 and DHA. [1-(14) C]DHA was infused intravenously, arterial blood was sampled, and the brain was microwaved at 5 min and analyzed. Rats fed the n-6 PUFA deficient compared with adequate diet had significantly reduced n-6 PUFA concentrations in brain phospholipids but increased eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid n-3 (DPAn-3, 22:5n-3), and DHA (by 9.4%) concentrations, particularly in ethanolamine glycerophospholipid (EtnGpl). Incorporation rates of unesterified DHA from plasma, which represent DHA metabolic loss from brain, were increased 45% in brain phospholipids, as was DHA turnover. Increased DHA metabolism following dietary n-6 PUFA deprivation may increase brain concentrations of antiinflammatory DHA metabolites, which with a reduced brain n-6 PUFA content, likely promotes neuroprotection and alters neurotransmission.     

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ¹²-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.