Effect Of Exogenous Fatty Acids Research Articles

Abstract 2581: Dietary fat influences pancreatic cancer progression by altering cell membrane lipid content to impact the PI3K/AKT pathway

Abstract Pancreatic cancer (PC) is one of the few cancers for which incidence and mortality continue to rise despite increasing knowledge of its etiology and risk factors. Amongst the latter, dietary patterns are significantly associated with PC risk. Due to its relatively slow progression, preventive strategies represent a simple means to improve outcomes. We are interested in understanding the influence of diet on PC prevention and have studied the effects of polyunsaturated fatty acids (PUFAs) in the progression of the disease both in vitro (via PC cell lines) and in vivo (via EL-KrasG12D and p48Cre/LSL-KrasG12D mouse models that recapitulate IPMN- and PanIN-like lesions, respectively). Our data demonstrate that dietary PUFAs are incorporated into plasma membrane phospholipids (PL) affecting signal transduction, particularly PI3K/AKT signaling, supporting the emergence of membrane-targeted therapies. We evaluated the effects of diets supplemented with omega-3 (ω-3) or omega-6 (ω-6) PUFAs on neoplastic lesion development. This work supports that ω-3 reduces while ω-6 accelerates lesion penetrance, tumor formation and proliferation associated with changes in pAKT. These results were also recapitulated in vitro to confirm that reduced activity of the PI3K/AKT pathway when incubated with Docosahexaenoic Acid (DHA), the primary component of the ω-3 enriched diet. This effect was abrogated by over-activation of the pathway when dosed with the main component of the ω-6 enriched diet, Linoleic Acid (LA). Since PI3K depends on its binding to PIP2 in the membrane for its activity, we next aimed to study if PUFAs were altering PL composition. By using IF and stably-transfected PC cells with a fluorescent translocation biosensor to monitor PIP2 and PIP3 lipids in the membrane, we discovered an effect of exogenous fatty acids expressed as a ratio of PIP2 to PIP3 in the plasma membrane. Enriched areas of membrane GFP staining (or PIP3) were observed in LA-treated Panc-1 cells. DHA treatment prevented PIP3 localization in the membrane of tumor cells, keeping the GFP signal diffuse in the cytoplasm. Using the PIP2 biosensor we sought to study the affinity of PI3K for PIP2 depending on the incorporation of DHA or LA in PIP2. DHA treatment reduced PI3K interaction (not total PI3K) with PIP2 resulting in lower levels of PIP3 in the membrane and reduced pAKT activation. Our results are encouraging because these PUFAs impinge on AKT, a downstream target of Kras which serves as a logical alternative to the elusive therapies for Kras and often toxic effects of PI3K/AKT pathway inhibition. Citation Format: Carolina Torres, Georgina Mancinelli, Emily Chen, Jose Cordoba-Chacon, Danielle Pins, Ronald McKinney, Sara Saeed, Karla Castellanos, Giulia Orsi, Megha Singhal, Sam Grimaldo, Poorna Chandra Rao Yalagala, Papasani Subbaiah, Cecilia Leal, Paul Grippo. Dietary fat influences pancreatic cancer progression by altering cell membrane lipid content to impact the PI3K/AKT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2581.

Up-regulated spinosad pathway coupling with the increased concentration of acetyl-CoA and malonyl-CoA contributed to the increase of spinosad in the presence of exogenous fatty acid

Polyketides are important compounds with a staggering range of biological and medicinal activities. Previous studies have demonstrated that the addition of fatty acids can increase polyketides production. However, a detailed metabolic explanation of this phenomenon has not been established. The aim of this study was to explain the positive effect of exogenous fatty acids on polyketides production. Spinosyns are polyketide-derived macrolides. In our study, spinosyns were used, as an example, to study the positive effect of exogenous fatty acids on their production. In the presence of exogenous fatty acids, gene expression assays indicated that the transcription of de novo fatty acid biosynthesis was significantly decreased and the transcriptions of β-oxidation and spinosad biosynthesis were up-regulated. The decreased de novo fatty acid synthesis transcription and the increased β-oxidation transcription resulted in the increase of acetyl-CoA and malonyl-CoA. It is the up-regulated spinosad pathway coupling with the enhanced concentration of acetyl-CoA and malonyl-CoA that contributed to the increase of spinosad. Taken together, a metabolic link among de novo fatty acid synthesis, β-oxidation, and spinosad biosynthesis at the presence of exogenous fatty acids was established. The results presented here enable researchers to better understand why added fatty acids can increase polyketides production.

Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei

Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C12-C18 fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the zeta-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.

Inhibitory effects of halothane on the thermogenic pathway in brown adipocytes: localization to adenylyl cyclase and mitochondrial fatty acid oxidation

Volatile anesthetics such as halothane efficiently inhibit nonshivering thermogenesis as well as the cellular manifestation of that phenomenon: norepinephrine-induced respiration in brown adipocytes. To identify the molecular site(s) of action of such anesthetics, we have examined the effect of halothane on the sequential intracellular steps from the interaction of norepinephrine with isolated brown adipocytes to the stimulation of mitochondrial respiration (=thermogenesis). We did not identify an inhibition at the level of the adrenergic receptors, but a first site of inhibition was identified as the generation of cAMP by adenylyl cyclase; this led to inhibition of norepinephrine-induced expression of the uncoupling protein-1 (UCP1) gene and reduced norepinephrine-induced lipolysis as secondary effects. Although an inhibition of lipolysis in itself would inhibit thermogenesis, circumvention of this inhibition revealed that a second, postlipolytic, site of inhibition existed: halothane also inhibited the stimulatory effect of exogenous fatty acids on cellular respiration. This inhibition was independent of the presence of UCP1 in the mitochondria of the cells and was thus not directly on the thermogenic uncoupling mechanism. Since not only fatty acid oxidation but also pyruvate oxidation were inhibited by halothane in isolated mitochondria, whereas glycerol-3-phosphate oxidation was not, the second site of action of halothane, evident when cyclase/lipolytic inhibition was circumvented, was located to the respiratory chain, complex I. The results thus explain the inhibition of nonshivering thermogenesis by identifying two sites of action of halothane in brown adipocytes. In addition, the results may open for new formulations of the molecular background to anesthesia.

Effect of Exogenous Fatty Acids on in vitro Development of Rat Embryos

Effect of Exogenous Fatty Acids on in vitro Development of Rat Embryos

Effect of exogenous fatty acids with greater than 22 carbon atoms (very long chain fatty acids) on superoxide production by human neutrophils.

The effects of exogenous long chain fatty acids (LCFA) and very long chain fatty acids (VLCFA) on superoxide production by human neutrophils were compared. Superoxide production was greater and more rapid in response to arachidonic (20:4 (n-6)), eicosapentanoic (20:5 (n-3)), and docosahexanoic (22:6 (n-3)) acids than for triacontatetranoic (30:4 (n-6)), dotriacontatetranoic (32:4 (n-6)), and tetratriacontahexanoic (34:6 (n-3)) acids, although all of these fatty acids gave responses larger than FMLP. A similar decline in activity with increasing carbon chain length was observed for the monoenoic VLCFA (22:1 (n-9) to 34:1 (n-9)). 32:4 (n-6) did not affect responses to a maximally stimulatory concentration of 20:4 (n-6). However, the simultaneous addition of 20:4 (n-6) and 30:4 (n-6) gave additive responses if suboptimal dosages of 20:4 (n-6) were used. This suggests that the LCFA and VLCFA may use the same signal transduction systems. In addition, 30:4 (n-6) was only 10% as effective as was 20:4 (n-6) at gaining access to the organic solvent extractable cellular fraction. This figure correlated with the relative biologic potency of 20:4 (n-6) and 30:4 (n-6), suggesting that the extent of association with the cell may regulate the biologic activity of the fatty acids. The saturates, arachidic (20:0) and cerotic (26:0) acids, were either inactive or poor activators in all assay systems examined. The failure of 20:0 to induce superoxide production and the lower responses to 30:4 (n-6) and 34:6 (n-3) were not because of extracellular Ca2+, because the biologic potency of these fatty acids was not greatly enhanced by removing Ca2+ from the extracellular medium. In contrast, 20:4 (n-6)- and 22:6 (n-3)-induced superoxide production was markedly increased under Ca(2+)-free conditions.

Transcriptional Control of the Stearoyl-CoA Desaturase-1 Gene by Polyunsaturated Fatty Acids 1

The effect of exogenous fatty acids on expression of the stearoyl-CoA destaurase-1 (SCD1) gene was assessed both in vivo and ex vivo. Mice fed a fat-free diet or a diet containing a largely monounsaturated (18:1) fat, i.e.,olive oil, expressed high levels of hepatic SCD1 mRNA. In contrast, in mice fed diets containing primarily polyunsaturated (18:2 and 18:3) fats, expression of the hepatic SCD1 message was markedly suppressed. Similar experiments with pure fatty acid esters showed that arachidonate (20:4) and linoleate (18:2) were far more potent in down-regulating expression of the hepatic SCD1 message than oleate (18:1), an end-product (as its CoA thioester) of the SCD1-catalyzed reaction. The reduction of hepatic SCD1 mRNA appears to be primarily due to inhibition of SCD1 gene transcription since polyunsaturated fatty acids caused a decrease in run-on transcription of the gene comparable to the decrease in message level. Consistent with the effects observed in vivo, unsaturated fatty acids suppressed the expression of SCD1 mRNA by rat hepatocytes cultured in serum-free medium. Suppression increased with degree of unsaturation with arachidonic (20:4) and eicosapentaenoic (20:5) acids,causing a ≥90% reduction in the level of SCD1 message. Thus, the SCD1 gene, like the fatty acid synthase and S14 genes, undergoes coordinate transcriptional down-regulation in response to unsaturated fatty acids.

Effect of exogenous fatty acids on reperfusion arrhythmias in isolated working perfused hearts.

Exogenous fatty acids may promote arrhythmias during ischemia and reperfusion, perhaps by increasing myocardial concentrations of long-chain acylcarnitines. We therefore studied the effects of high concentrations of fatty acids on reperfusion arrhythmias and acylcarnitine accumulation in isolated working rat hearts subjected to regional ischemia and reperfusion. Hearts were perfused with buffer containing 3% albumin, 5.9 mM K+, and either 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate. After 15 min of aerobic work, the left anterior descending artery was reversibly ligated for 10 min and released, and the hearts were subsequently reperfused for 3 min. Although ischemic zone acylcarnitine accumulation after reperfusion was significantly greater in glucose plus palmitate-perfused hearts (247 +/- 149 vs. 717 +/- 176 nmol/g dry wt in glucose- vs. palmitate-perfused hearts, respectively), no significant differences in the incidence (67 vs. 44%) or duration (95 +/- 17 vs. 56 +/- 17 s) of ventricular fibrillation (VF) were seen in glucose or glucose plus palmitate hearts, respectively. Because low K+ concentration ([K+]) has been reported to increase reperfusion arrhythmias in similar models, we reduced perfusate [K+] to 4.0 mM. This significantly increased the incidence and duration of VF in hearts perfused with glucose alone but had no effect in palmitate-perfused hearts. We conclude that acylcarnitine accumulation is not arrhythmogenic in this model and that fatty acids may actually have antiarrhythmic effects if exogenous [K+] is low.

Effect of exogenous fatty acids on lipid synthesis, marker‐enzymes, and development of glial cells maintained in serum‐free culture

Glial cells were isolated from the cerebra of 7-day-old rats and maintained in culture in a chemically defined medium that favours the development of oligodendrocytes. Acetate, butyrate, or albumin-bound hexanoate, octanoate, decanoate, laurate, myristate, palmitate, oleate, linoleate, or arachidonate was added to the culture medium. The incorporation of [3H]acetate into fatty acids and cholesterol and [35S]sulphate into sulphatide, and the activities of the oligodendrocyte marker enzymes 2',3'-cyclic-nucleotide 3'-phosphodiesterase and glycerol 3-phosphate dehydrogenase were measured. The composition of the glial cell population (the number of astrocytes and oligodendrocytes) in these cultures was studied by immunocytochemistry. Results show that 1) long-chain fatty acids depress the synthesis of fatty acids, cholesterol, and sulphatide; and 2) the presence of long-chain, in contrast to short-chain, fatty acids in the culture medium lowers the activities of 2',3'-cyclic-nucleotide 3'-phosphodiesterase and glycerol 3-phosphate dehydrogenase and decreases the number of oligodendrocytes. Our results suggest that long-chain fatty acids exert a negative influence on the development of oligodendrocytes in the culture system used.

Arachidonic and oleic acids stimulate zygote formation in the presence of mating pheromone in the yeast, Saccharomyces cerevisiae

Effect of exogenous fatty acids on zygote formation in Saccharomyces cerevisiae was studied. Arachidonic and oleic acids considerably stimulated zygote formation, but other fatty acids tested, linoleic, linolenic, stearic and palmitic acids, did not. Pretreatment experiments with arachidonic acid showed that the stimulation of zygote formation by the fatty acid required the presence of mating pheromone.

Inhibitory effect of exogenous arachidonic acid on alveolar macrophage 5-lipoxygenase metabolism. Role of ATP depletion.

Although a variety of agonists have the capacity to stimulate the release of endogenous arachidonic acid (AA) from macrophage lipids and its subsequent metabolism to both cyclooxygenase and 5-lipoxygenase eicosanoids, including leukotrienes (LT), previous work suggests that exogenously supplied arachidonate is metabolized preferentially to cyclooxygenase products and mono-hydroxyeicosatetranenoic acids rather than LT. We have compared the metabolism of endogenous and exogenous AA in cultured rat alveolar macrophages and have further examined the effect of exogenous fatty acids on eicosanoid synthesis stimulated by the calcium ionophore A23187 and the particulate agonist zymosan. As reported by others, exogenous AA was metabolized to large amounts of cyclooxygenase products, 12-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid, but far lesser amounts of all 5-lipoxygenase products. However, whereas exogenous AA augmented the synthesis of cyclooxygenase metabolites in both A23187- and zymosan-stimulated cells, it dose-dependently inhibited the synthesis of LT. Inasmuch as unsaturated fatty acids, including AA, uncouple oxidative phosphorylation, and because 5-lipoxygenase activity depends on ATP, we examined the possibility that ATP depletion was responsible for the inhibition of LT synthesis by exogenous AA. Arachidonate depleted cellular ATP in a dose-dependent fashion similar to its inhibition of LT synthesis. In addition, the other unsaturated fatty acids, linolenic and eicosatrienoic, but not the saturated fatty acids, palmitic or stearic, both depleted ATP and inhibited A23187-induced LT synthesis. These data suggest that the relative lack of synthesis of LT from exogenous AA is related to the ability of this unsaturated fatty acid to function as an inhibitor, as well as a substrate, of 5-lipoxygenase. Our results further suggest that this inhibition of 5-lipoxygenase may be mediated by ATP depletion.

Choline-phosphate cytidyltransferase activity and phosphatidylcholine synthesis in rat granular pneumocytes are increased with exogenous fatty acids

We investigated the effect of exogenous fatty acids on phosphatidylcholine (PC) and disaturated phosphati-dylcholine (DSPC) synthesis by rat granular pneumocytes in primary culture. Synthesis of PC and DSPC from [ 3- methyl]choline, as evaluated by increasing specific activity (pmol choline incorporated /μ g phosphorus), was linear for 3 h. Exogenous palmitic, oleic, linoleic, or linolenic acid (100 μM each) increased the synthesis of PC by approx. 50% during incubation for 3 h. In contrast, synthesis of DSPC was increased only by palmitic acid. The increase in DSPC synthesis was approx. 150% after 3 h. Conversion of choline phosphate to PC was increased in the presence of palmitic or oleic acid as indicated by pulse-chase studies with [ 3H- methylcholine in the intact cells. Cells incubated for 3 h with either oleic or palmitic acid showed increased choline-phosphate cytidyltransferase activity in the cells and the microsomal fraction. In addition, oleic acid increased the activity of this enzyme in the cytosolic fraction. The distribution of this enzyme in cytosolic and microsomal fraction was 24 and 76% in the cells incubated with palmitic acid and 32 and 68% in control cells. These results suggest that exogenous fatty acids stimulate the de novo pathway of PC synthesis in granular pneumocytes by increasing the microsomal choline-phosphate cytidyltransferase activity.

Physiological effect of exogenous fatty acids on lipid metabolism and prostaglandin precursors

Physiological effect of exogenous fatty acids on lipid metabolism and prostaglandin precursors

A biphasic effect of exogenous oleate on the rate of cholesterol biosynthesis by rat hepatocytes

Short-term (0–1 h) incubations of rat hepatocytes with oleate (2 mM) resulted in a decrease in the rate of cholesterol synthesis compared to controls incubated in the absence of fatty acid. However, during this period the activity of hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase was higher in the oleate-incubated cells. After longer incubation periods in the presence of oleate there was a higher rate of cholesterol synthesis than in the corresponding non-oleate controls and HMG-CoA reductase activity remained elevated. This biphasic effect provides an explanation for previous contradictory reports concerning the effect of exogenous fatty acids on the rate of cholesterol synthesis in liver. The present studies also suggest that in some physiological situations, the rate of cholesterol synthesis is determined by substrate supply rather than by HMG-CoA reductase activity.

Effect of exogenous fatty acids on growth, membrane fluidity, and phospholipid fatty acid composition in yeast.

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-delta 11-eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 15:0, and 16:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.

Lipid compositional manipulation in Acholeplasma laidlawii B. Effect of exogenous fatty acids on fatty acid composition and cell growth when endogenous fatty acid production is inhibited.

A variety of potential inhibitors of de novo fatty acid biosynthesis have been tested for activity in Acholeplasma laidlawii B. Two compounds, avidin and N,N-dimethyl-4-oxo-2trans-dodecenamide (CM-55), an antimicrobial fatty amide, strongly inhibit de novo biosynthesis without nonspecific toxic effects at moderate dosages. Avidin is the more potent inhibitor, abolishing de novo fatty acid synthesis and greatly reducing the chain elongation of exogenous fatty acids at level of 25 U/l. CM-55 gives complete inhibition of de novo biosynthesis only at low temperatures and inhibits exogenous fatty acid elongation to a variable extent. However, CM-55 is still a more potent antilipogenic agent in this organism than is the fungal antibiotic cerulenin. Cells cultured with avidin grow only when one or more exogenous medium- or long-chain fatty acids are added to the growth medium. The extent of cell growth under these conditions depends primarily on the physical properties of the exogenous fatty acid(s). In general, fatty acids giving diacylglycerolipids of very high or very low fluidity are unsuitable growth substrates, while those whose diacylglycerol derivatives are of intermediate fluidity support fair to good cell growth.

Effect of exogenous fatty acids on the retention of phospholipid acyl groups by mouse L fibroblasts

Exogenous oleic or linoleic acid, given at a high but nontoxic level (1 mg fatty acid/day for 20 · 10 6 cells in 50 ml medium), caused substantial redistribution of the otherwise permanently retained phospholipid acyls in mouse L fibroblasts. 18–40% of the preformed phospholipid acyls were shifted to triglycerides but most returned to phospholipids when the supply of exogenous fatty acid was removed. The phospholipid acyls could be reshuttled back to triglycerides again whenever an adequate amount of exogenous fatty acid was provided. Daily changes of medium containing oleic acid bound to bovine serum albumin caused a still greater total loss of phospholipid acyls into the medium. The removal of the prelabeled phospholipid acyls also occurred with phospholipid acyls which had been synthesized from [1- 14C]acetate 3 days earlier. The results demonstrate the fact that the apparent permanently retained phospholipid acyl groups found in L-cells could in fact be displaced through experimental manipulations.

Effect of exogenous fatty acids on biotin deprived death of Saccharomyces cerevisiae.

The effect of exogenous fatty acids on cell growth and death of the biotin-requiring yeast Saccharomyces cerevisiae BA-1 was examined with respect to the mechanism of synthetic pathway of fatty acid under biotin starvation. At a growth temperature of 30°C, exogenous unsaturated fatty acids, such as palmitoleic, oleic, linoleic, and linolenic acids which promote the cell growth and suppress death effectively, were incorporated intactly into the cellular fatty acids, whereas the saturated fatty acid, palmitic acid, which supports growth but some what inhibits death, was once incorporated, and about 60% of incorporated palmitic acid was found to be desaturated. However, at an elevated temperature of 36°C, even palmitic acid showed similar effects to unsaturated fatty acids in cell growth and death; followed by an increased desaturation of palmitic acid. Thus the data indicate that palmitic acid, as well as unsaturated fatty acids directly compensate for the deficiency of endogenously synthesized fatty acids ...

Membrane lipid biosynthesis in Acholeplasma laidlawii B : Investigations into the in vivo regulation of the quantity and hydrocarbon chain lengths of de novo biosynthesized fatty acids in response to exogenously supplied fatty acids

The regulation of the nature and quantity of the fatty acids produced in vivo by Acholeplasma laidlawii B in the presence of various exogenous fatty acids has been investigated. In the presence of exogenous medium- or long-chain fatty acids, the organism appears to reduce the amounts of de novo biosynthesized fatty acids in its cellular lipid pool by two distinct mechanisms: an excretion of biosynthesized fatty acids to the growth medium as free fatty acids, and a reduction in total de novo biosynthetic output. These two mechanisms do not suffice to maintain constant total membrane lipid levels, but they do appear to significantly moderate the effect of exogenous fatty acids on the level of membrane lipid. In the presence of short-chain fatty acids, total membrane lipid levels are not elevated. Exogenous fatty acids can cause shifts in the average chain length of de novo biosynthesized fatty acids; the magnitudes and directions of these shifts can be correlated with the specificity of the exogenous species for esterification to the 1- or the 2-position of the glycerol moiety of membrane glycerolipids. As the various endogenously synthesized fatty acids differ in their positional specificity for glycerolipid esterification, we propose that the competition of an exogenous species with significant specificity for a particular position with the endogenously derived fatty acids specific for that position can selectively depress the synthesis of such endogenously derived species, thereby altering the overall product spectrum of de novo fatty acid biosynthesis in vivo.

Intestinal microflora in rats: isolation and characterization of strictly anaerobic bacteria requiring long-chain fatty acids.

Three strains of strictly anaerobic bacteria, isolated from the cecal contents of rats, have strict requirements for long-chain fatty acids. The effect of exogenous fatty acids on the growth and fatty acid composition of the bacteria was examined. Biohydrogenation of linoleic acid into octadecenoic acid was observed. These observations suggest that long-chain fatty acids in the intestine are factors in controlling the localization and the population levels of indigenous bacteria in vivo in rats.