The Effects of Octanoate and Oleate on Isocitrate Lyase Activity during the Germination of Pinus pinea Seeds

The changes of isocitrate lyase levels with respect to the catabolism of triglycerides have been studied during the germination of Pinus pinea seeds. We studied the effects of octanoate, oleate, and inhibitors of protein synthesis on isocitrate lyase during germination. Pyruvate kinase, glucose-6-P-dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase were also assayed. Octanoate and oleate inhibited the isocitrate lyase activity, similarly to cycloheximide, chloramphenicol, and actinomycin, inhibitors of protein biosynthesis. This inhibitory effect is not specific but is strikingly evident with isocitrate lyase. This inhibition was not proportional to the concentration but was proportional to the chain length of oleate and octanoate.     

A combination of octanoate and oleate promotes in vitro differentiation of porcine intramuscular adipocytes

To understand the relationship between intramuscular adipogenesis in the pig and the supply fatty acids, we established a clonal porcine intramuscular preadipocyte (PIP) line from the marbling muscle tissue of female Duroc pig. Confluent PIP cells exhibited a fibroblastic appearance. Their adipogenic ability was investigated using confluent PIP cells after exchanging growth medium for adipogenic medium containing 50 ng/mL insulin, 0.25 microM dexamethasone, 2 mM octanoate, and 200 microM oleate. Appropriate concentrations of octanoate and oleate for the induction of adipogenesis were determined from the ability of cells to accumulate lipid and the toxicity of fatty acids. When cells were cultured in differentiation medium for 8 days, large numbers of lipid droplets were observed in differentiated PIP cells, and their cytosolic TG content increased in a time-dependent manner. While oleate only induced the expression of PPARgamma mRNA, but not that of C/EBPalpha, octanoate significantly induced the expression of both PPARgamma and C/EBPalpha mRNA. Octanoate and oleate accelerated the inducing effect of insulin and dexamethasone on the expression of aP2 mRNA. These results indicate that a combination of octanoate and oleate synergistically induced PIP adipogenesis, and that the stimulation of octanoate was essential to the trigger for the adipogenesis in PIP cells.     

Fatty acid composition of two Tunisian pine seed oils

Oils were extracted from fully ripen Pinus pinea L. and Pinus halepensis Mill seeds and fatty acid composition has been established by capillary gas chromatography. Seeds are rich in lipids, 34.63-48.12% on a dry weight basis. Qualitatively, fatty acid composition of both species is identical. For P. halepensis linoleic acid is the major fatty acid (56.06% of total fatty acids) followed by oleic (24.03%) and palmitic (5.23%) acids. For P. pinea, the same fatty acids are found with the proportions 47.28%, 36.56%, and 6.67%, respectively. Extracted fatty acids from both species are mainly unsaturated, respectively, 89.87% and 88.01%. Pinus halepensis cis-5 olefinic acids are more abundant (7.84% compared to 2.24%). Results will be important as a good indication of the potential nutraceutical value of Pinus seeds as new sources of fruit oils rich in polyunsaturated fatty acids and cis-5 olefinic acids.     

Modulation of the sympathetic nerve action on carbohydrate and ketone body metabolism by fatty acids, glucagon und insulin in perfused rat liver

Rat liver was perfused in situ via the portal vein without recirculation: 1) Nerve stimulation (20 Hz, 2 ms, 20 V) increased glucose output and shifted lactate uptake to output; the alterations were diminished by oleate but not octanoate. 2) Glucagon (1nM) stimulated glucose output maximally also in the presence of the fatty acids, so that nerve stimulation could not increase it further. The hormone also enhanced lactate uptake and nerve stimulation counteracted this effect. The counteraction was diminished by oleate but not octanoate. 3) Insulin (100nM) slightly lowered glucose output and had no effect on lactate balance. It antagonized the increase of glucose output by nerve stimulation, but left the shift of lactate uptake to release unaffected. These events were not influenced by the fatty acids. 4) Nerve stimulation decreased ketone body production from oleate and octanoate. 5) Glucagon increased ketogenesis from oleate, but not octanoate. In the presence of glucagon nerve stimulation also lowered ketogenesis. This decrease was diminished in the presence of oleate. 6) Insulin lowered ketogenesis from oleate but not octanoate. In the presence of insulin nerve stimulation decreased ketogenesis; the relative change was independent of the fatty acids. The complex interactions between fatty acids, glucagon and insulin in the modulation of sympathetic nerve actions can be summarized as follows: Oleate, which enters the mitochondria via the carnitine system, but not octanoate, which enters independently from this system, as well as insulin but not glucagon effectively modulated the nerve actions on carbohydrate metabolism. Glucagon but not insulin modulated the nerve effects on ketogenesis from oleate but not octanoate. The regulatory interactions between substrates, hormones and nerves can best be explained on the basis of the model of metabolic zonation.     

Metabolism of saturated fatty acids by Paramecium tetraurelia

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.     

Effects of oleate,palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells

The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3-$\text{OHB}\text{AcAc}$) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.     

Antisense expression of 3-oxoacyl-ACP reductase affects whole plant productivity and causes collateral changes in activity of fatty acid synthase components

Brassica napus cv Westar plants were transformed with 3-oxoacyl-ACP reductase (KR) in antisense orientation, driven by either the cauliflower mosaic virus 35S promoter or a seed-specific acyl carrier protein promoter to determine the effects on plant productivity and on the activity of other fatty acid synthase (FAS) components. In plants with altered KR activity, total seed yield was reduced in all cases. In less severely affected plant lines, seeds had a normal appearance and composition but the yield of seeds was reduced by approximately 50%. In more severely affected lines, reductions in both seed fatty acid content and the number of seeds produced per plant were evident, resulting in a 90% reduction in fatty acid synthesized per plant. These phenotypes were independent of the promoter used. In severely affected lines, a large proportion of seeds showed precocious germination, and these had a reduced oleate content and increased levels of polyunsaturated 18-carbon fatty acids, compared with normal seeds of the same line. This reduction in 18:1 fatty acids was mimicked on imbibition of seeds with a normal appearance, indicating a preferential use of oleate moieties in precocious germination events. The reduction in activity of KR was mirrored for a second fatty acid synthase component, enoyl-ACP reductase, indicating a mechanism to maintain the ratio of fatty acid synthase components throughout embryogenesis.     

Effects of culture age and hexoses on fatty acid oxidation by Leishmania major

The effect of culture age on the rate of oxidation of short-, medium, and long-chain fatty acids by Leishmania major promastigotes was investigated. Promastigotes from 5-day stationary phase cultures oxidized several saturated fatty acids about 3-to-4-fold faster than cells from late log phase cultures, but [10-14C]oleate was oxidized 9-fold faster. The increase in rate of oxidation was partially reversed within 5 h and almost completely reversed within 30 h after resuspending cells from a 5-day stationary culture in fresh medium. Addition of acetate, leucine, or alanine caused moderate inhibitions of [1-14C]palmitate oxidation, while glycerol had little effect. Glucose, however, was a powerful inhibitor of the oxidation of [1-14C]palmitate and of [1-14C]octanoate. Mannose and fructose were also strong inhibitors of palmitate oxidation, but neither galactose, 2-deoxyglucose or 6-deoxyglucose caused appreciable inhibition. The extent of inhibition by acetate increased with increasing culture age, whereas inhibition by glucose decreased. In addition to demonstrating a reversible rise in beta-oxidation capacity with culture age, these data also demonstrate a hitherto unrecognized strong and culture age-dependent inhibition of fatty acid oxidation by glucose.     

Effect of long- and medium-chain fatty acids on insulin secretion from pieces of rat pancreas.

The effect of fatty acids on insulin secretion in vitro was investigated. Pieces of pancreas from fed rats were incubated for 15 or 90 min at low (0.6 mg/ml) and high (3.0 mg/ml) glucose concentrations with and without either sodium octanoate or sodium oleate. A highly significant difference in insulin secretion between low and high glucose concentrations indicated viability and responsiveness of the incubated tissue. No statistically significant effect of octanoate or oleate on insulin secretion was found at either low or high glucose concentration. Thus, no support was found for the concept that medium- and long-chain free fatty acids are insulin secretagogues.     

Effect of glutamate on arachidonic acid production from Mortierella alpina

AIMS: To evaluate the effect of glutamate on arachidonic acid production from Mortierella alpina. METHODS AND RESULTS: Cell growth, arachidonic acid production, proportions of poly-unsaturated fatty acids (PuFAs) in fatty acids and glucose-6-phosphate dehydrogenase (G6PDH) activity were analysed when glutamate concentration was 0.8 g l(-1). Biomass and arachidonic acid production were higher in the culture containing glutamate than those in the control culture, and both reached their maximum of 25 g l(-1) and 1.4 g l(-1) after 7 d, respectively. The proportions of some PuFAs, oleic acid, gamma-linolenic acid and dihomo-gamma-linolenic acid were decreased while linoleic acid and arachidonic acid were enhanced by glutamate addition. Glutamate addition enhanced G6PDH activity compared with the control during the whole culture process. CONCLUSIONS: Addition of 0.8 g l(-1) glutamate was beneficial to enhance arachidonic acid production from Mortierella alpina, which was a result of activating the pentose phosphate pathway (PPP). SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the addition of glutamate and regulation of PPP had a positive influence on arachidonic acid synthesis in Mortierella species.     

Gravitational stress on germinating Pinus pinea seeds

In the germination of lipid-rich seeds, the glyoxylate cycle plays a control role in that, bypassing the two decarboxylative steps of the Krebs cycle; it allows the net synthesis of carbohydrates from lipids. The activity of isocitrate lyase, the key enzyme of the glyoxylate cycle, is an indicator of the state of seed germination: stage of germination, growth of embryo, activation and progress of protein synthesis, depletion of lipidic supplies. In order to investigate the effects of gravity on seed germination, we carried out a study on the time pattern of germination of Pinus pinea seeds that were subjected to a hypergravitational stress (1000 g for 64 h at 4 degrees C), either in a dry or in a wet environment, before to be placed in germination plates. During the whole time of germination, we monitored the state of embryo growth and the most representative enzymes of the main metabolic pathways. In treated wet seeds, we observed an average germination of only 20% with a slowdown of the enzyme activities assayed and a noticeable degradation of lipidic reserves with respect to the controls. These differences in germination are not found for dry seeds.     

Occurrence of cyclic AMP and related enzymes during germination of Pinus pinea seeds

The occurrence of cAMP, adenylate cyclase and cAMP phosphodiesterase has been tested in Pinus pinea seed during germination. The study has been carried out on dormant and imbibed seeds, seedlings, endospermic residues, roots and cotyledons. cAMP has been detected by the protein binding method and its occurrence has been verified by HPLC detections. cAMP phosphodiesterase shows a very high activity at acidic pH, while being completely inactive at pH 7.4. At this pH value, well detectable levels of adenylate cyclase have been observed. Therefore, the classical pathway of synthesis and breakdown of cAMP, already accepted for animal and bacterial cells, seems to be operating in Pinus pinea plant too.     

Fatty acids from seeds of Pinus pinea L.: composition and population profiling

Pinus pinea L. is widely disseminated all over the Mediterranean Basin. Qualitatively, P. pinea fatty acid seed composition is identical and typical of the genus Pinus. This composition is made of unsaturated oil with several unusual polymethylene-interrupted unsaturated fatty acids. Linoleic acid is the major fatty acid followed by oleic, palmitic and stearic acids. Quantitatively, for all Mediterranean populations, total amounts of fatty acids seem to be fairly constant and independent from their origin. When applying principal component analysis, it seems that there is not a distinct geographical variability. Tunisian populations appear to be integral part of the Mediterranean populations without any particular structuring. Taking into account this research and the data reported in the literature, we can confirm that P. pinea expresses no significant variability. This low genetic diversity revealed by fatty acid composition can be explained by anthropogenetic diffusion of genetically homogeneous reproductive material as early as the first explorations.     

Ketogenesis in isolated rat liver mitochondria. II. Factors affecting the rate of β-oxidation

1. During fatty acid oxidation by rat liver mitochondria, the rate of β-oxidation is dependent on the relative amounts of substrate and mitochondrial protein, on the energy state of the mitochondria, on the chain length and the number of double bonds of the fatty acid and on the concentration of various compounds in the reaction medium (l-carnitine, CoASH, hexokinase, albumin).2. The rate of β-oxidation of long-chain fatty acids decreases when the ratio of albumin over fatty acid is increased. This effect is most marked in the absence of added carnitine.3. Addition of excess hexokinase decreases the rate of β-oxidation in the presence of added carnitine.4. Maximal rates of β-oxidation are observed with octanoate and decanoate (40–60 nmoles acetyl-CoA/min per mg mitochondrial protein at 25 °C).5. Odd-numbered fatty acids are oxidized at a much lower rate than the even-numbered homologues. In a low-energy state propionyl-CoA accumulates; in a high-energy state in the presence of bicarbonate, Krebs-cycle intermediates accumulate.6. l-Carnitine enhances the rate of β-oxidation of all fatty acids except butyrate. The stimulatory effect is most pronounced with odd-numbered and with long-chain fatty acids.7. In the absence of added carnitine the rate of β-oxidation of long-chain fatty acids decreases with the chain length and increases with the number of double bonds. It is suggested that the solubility of the long-chain fatty acids in the aqueous medium is the rate-limiting factor under these conditions.8. In the presence of carnitine and albumin, palmitate, oleate, linoleate and linolenate are all oxidized at about the same rate (25–30 nmoles/min per mg protein at 25 °C).9. Propionyl-CoA is not formed as an intermediate during oxidation of unsaturated fatty acids.     

Inhibition of adenosine 3':k'-monophosphate accumulation white fat acids, lactate, and beta-hydroxybutyrate.

The large increase in cyclic AMP accumulation by rat white fat cells seen in the presence of lipolytic agents plus methylxanthines and adenosine deaminase was markedly inhibited by lactate. However, lipolysis was unaffected by lactate. Octanoate, hexanoate, heptanoate, and beta-hydroxybutyrate inhibited both cyclic AMP accumulation and lipolysis by rat fat cells. The mechanism by which these acids inhibit lipolysis differs from that for long chain fatty acids such as oleate. Oleate directly inhibited triglyceride lipase activity of homogenized rat adipose tissue. In contrast, octanoate, beta-hydroxybutyrate, and lacatate had no effect on triglyceride lipase activity. Hormone-stimulated adenylate cyclase activity of rat fat cell ghosts was inhibited by oleate and 4mM octanoate but not by 1.6 mM octanoate, heptanoate, hexanoate, beta-hydroxybutyrate or lactate. None of the acids affected the soluble protein kinase activity of rat adipose tissue. There was no stimulation by lactate, butyrate, beta-hydroxybutyrate, or octanoate of the soluble or particulate cyclic AMP antilipolytic action of a short chain acid such as octanoate or hexanoate was not accompanied by any drop in total fat cell ATP. The mechanism by which lactate lowers cyclic AMP but not lipolysis remains to be established.     

Competition between fatty acids and carbohydrate or ketone bodies as metabolic fuels for the isolated perfused heart

The ability of carbohydrate fuels (lactate, pyruvate, glucose) and the ketone bodies (acetoacetate, beta-hydroxybutyrate) to compete with fatty acids as fuels of respiration in the isolated Langendorf-perfused heart was studied. Oleate and octanoate were used as fatty acid fuels since oleate requires carnitine for entry into mitochondria, whereas octanoate does not. The two ketone bodies inhibited the oxidation of both oleate and octanoate implying an intramitochondrial site of action. Pyruvate, lactate, and lactate plus glucose inhibited oleate oxidation but not octanoate oxidation, indicating a mechanism of inhibition that involves the carnitine system. Pyruvate was a more potent inhibitor than lactate at equal concentrations, but the effect of lactate could be greatly increased by dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase. The physiological and mechanistic implications of these observations are discussed.     

Changes in the albumin and globulin fractions in seeds of Pinus pinea during artificial aging

The alterations on the electrophoretic patterns of albumin and globulin fractions in both embryo and endosperms of Pinus pinea seeds during artificial ageing have been investigated. The results show that the electrophoretic patterns of aged seeds are drastically different from those of seeds with high germination capacity. These alterations were observed even when the quantitative analysis did not show any significant changes.     

Rates of ketone-body formation in the perfused rat liver

1. The rates of formation of acetoacetate and β-hydroxybutyrate by the isolated perfused rat liver were measured under various conditions. 2. The rates found after addition of butyrate, octanoate, oleate and linoleate were about 100μmoles/hr./g. wet wt. in the liver of starved rats. These rates are much higher than those found with rat liver slices. 3. The differences between the rates given by slices and by the perfused organ were much higher with the long-chain than with short-chain fatty acids. The increments caused by oleate and linoleate were 12 and 16 times as large in the perfused organ as in the slices, whereas the increments caused by butyrate and octanoate were about four times as large. 4. The rates of ketogenesis in the unsupplemented perfused liver of well-fed rats, and the increments caused by the addition of fatty acids, were about half of those in the liver from starved rats. 5. The value of the [β-hydroxybutyrate]/[acetoacetate] ratio of the medium was raised by octanoate, oleate and linoleate. 6. Carnitine did not significantly accelerate ketogenesis from fatty acids. 7. Oleate formed up to 82% of the expected yield of ketone bodies. 8. In the liver of alloxan-diabetic rats the endogenous rates of ketogenesis were raised, in some cases as high as in the liver from starved rats, after addition of oleate. 9. On addition of either β-hydroxybutyrate or acetoacetate to the perfusion medium the liver gradually adjusted the [β-hydroxybutyrate]/[acetoacetate] ratio towards the normal range. 10. The [β-hydroxybutyrate]/[acetoacetate] ratio of the medium was about 0·4 when slices were incubated, but near the physiological value of 2 when the liver was perfused. 11. The experiments demonstrate that for the study of ketogenesis slices are in many ways grossly inferior to the perfused liver.     

Fatty Acyl-CoAs as feedback regulators of hexose monophosphate shunt in rat adipocytes

Summary The high basal glucose utilization through hexose monophosphate shunt found in our experimental conditions were almost completely inhibited by oleate, octanoate and caproate. However, the inhibition of glucose oxidation due to butyrate was about 50% whereas ketone bodies and acetate did not inhibit. The rate of triacylglycerol formation was not significantly modified with the above organic acids except oleate that presented a 5-fold increase on labeling incorporation into lipids. Oleate inhibition of glucose oxidation was completely prevented by the NADPH oxidant menadione. There was no inhibition by octanoate, caproate, butyrate or ketone bodies of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or malic enzyme in adipose tissue homogenates. In contrast, specifically glucose-6-phosphate dehydrogenase was inhibited by oleoyl-CoA. The oleoyl-CoA inhibition was prevented by enzyme preincubation with low NADP concentration. The data lend further support for the hypothesis that fatty acids and NADP fulfill an important role in the modulation of the hexose monophosphate shunt activity.