Conjugated linoleic acid isomers in mitochondria: evidence for an alteration of fatty acid oxidation

The beneficial effects exerted by low amounts of conjugated linoleic acids (CLA) suggest that CLA are maximally conserved and raise the question about their mitochondrial oxidizability. Cis-9,trans-11-C(18:2) (CLA1) and trans-10,cis-12-C(18:2) (CLA2) were compared to cis-9,cis-12-C(18:2) (linoleic acid; LA) and cis-9-C(16:1) (palmitoleic acid; PA), as substrates for total fatty acid (FA) oxidation and for the enzymatic steps required for the entry of FA into rat liver mitochondria. Oxygen consumption rate was lowest when CLA1 was used as a substrate with that on CLA2 being intermediate between it and the respiration on LA and PA. The order of the radiolabeled FA oxidation rate was PA > LA > CLA2 > CLA1. Transesterification to acylcarnitines of the octadecadienoic acids were similar, while uptake across inner membranes of CLA1 and, to a lesser extent, of CLA2 was greater than that of LA or PA. Prior oxidation of CLA1 or CLA2 made re-isolated mitochondria much less capable of oxidising PA or LA under carnitine-dependent conditions, but without altering the carnitine-independent oxidation of octanoic acid. Therefore, the CLA studied appeared to be both poorly oxidizable and capable of interfering with the oxidation of usual FA at a step close to the beginning of the beta-oxidative cycle.     

Conjugated linoleic acid decreases prostaglandin synthesis in bovine luteal cells in vitro

Feeding conjugated linoleic acids (CLA) improves reproductive performance in dairy cows; however, the molecular mechanisms by which CLA improves reproduction are not understood. The effect of the CLA isomers, trans‐10, cis‐12 CLA and cis‐9, trans‐11 CLA on synthesis of progesterone, PGE₂, and PGF_2α, in bovine luteal cells was determined in this study. Luteal cells from three cows were cultured in medium containing 0 or 0.1 µM of trans‐10, cis‐12 CLA and cis‐9, trans‐11 CLA in varying ratios in the presence and absence of 1 µM of forskolin. Prostaglandin and progesterone concentrations were not altered by CLA isomer and ratio. Luteal cells cultured in the presence of CLA had lower PGF_2α concentrations (62.6 ± 13.4 pg/ml vs. 55.7 ± 12.2 pg/ml; P = 0.005) and, in the absence of forskolin, lower PGE₂ concentrations (65.3 ± 15.1 pg/ml vs. 32.4 ± 14.1 pg/ml; P = 0.002) in culture media, while progesterone concentrations were not altered (P = 0.63). Relative steady‐state mRNA amounts of COX‐2 (1.7‐fold decrease; P = 0.002), PGE synthase (1.5‐fold decrease; P = 0.03) and 3β‐hydroxysteroid dehydrogenase (1.6‐fold decrease; P = 0.0003) were lower in CLA‐treated cultures, but CLA did not significantly alter mRNA amounts of PGE₂ 9‐keto‐reductase, StAR, and cytochrome P450 side chain cleavage enzyme. In conclusion, a potential mechanism exists by which trans‐10, cis‐12 CLA and cis‐9, trans‐11 CLA may improve reproductive performance in dairy cows, by suppressing PGF_2α synthesis in luteal cells via attenuation of COX‐2 gene expression. Mol. Reprod. Dev. 78:328–336, 2011. © 2011 Wiley‐Liss, Inc.     

Eicosadienoic acid differentially modulates production of pro-inflammatory modulators in murine macrophages

Eicosadienoic acid (Δ11,14-20:2; EDA) is a rare, naturally occurring n-6 polyunsaturated fatty acid (PUFA) found mainly in animal tissues. EDA is elongated from linoleic acid (LA), and can also be metabolized to dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA), and sciadonic acid (Δ5,11,14-20:3; SCA). Although, the metabolism of EDA has been extensively studied, there are few reports regarding how EDA might affect inflammatory processes. The objective of this study was to determine the effect of EDA on the n-6 PUFA composition and inflammatory response of murine RAW264.7 macrophages to lipopolysaccharide (LPS). EDA was taken up rapidly by macrophages and metabolized to SCA, and the percentages of both fatty acids increased in cellular phospholipids in a dose-dependent manner. The incorporation of EDA into macrophage lipids increased the proportions of LA, DGLA, and AA as well, and reduced the proportion of total monounsaturated fatty acids. When LPS were applied to the macrophages, EDA decreased the production of nitric oxide (NO), and increased that of prostaglandin E(2) (PGE(2)) and tumor necrotic factor-α. The modulation of NO and PGE(2) was due, in part, to the modified expression of inducible nitric oxide synthase and type II cyclooxygenase. The differential effects of EDA on pro-inflammatory mediators might attribute to the negative feedback mechanism associated with prolonged inflammation. Furthermore, EDA was a weaker pro-inflammatory agent than LA, and not as anti-inflammatory as SCA. This study shows that EDA can modulate the metabolism of PUFA and alter the responsiveness of macrophages to inflammatory stimulation.     

Conjugated linoleic acid and atherosclerosis: studies in animal models

Conjugated linoleic acids (CLA) are isomeric forms of linoleic acid (LA) containing two conjugated sites of unsaturation. The most abundant dietary form of CLA is the cis-9,trans-11 (c-9,t-11) isomer that is found in the fatty tissues and milk of ruminant animals. CLA can also be acquired by ingestion of supplements, which are usually equimolar mixtures of the c-9,t-11 and t-10,c-12 CLA. For more than a decade, the potential for CLA to modify atherosclerosis in animal models has been examined. However, to date, the studies have failed to reach consensus on whether CLA can be effective in reducing the incidence or severity of atherosclerotic lesions, or whether or not plasma lipid and lipoprotein levels can be improved with CLA supplementation. This review will examine the evidence for and against a role for CLA in atherosclerosis, with a focus on the rabbit, the hamster, and the apoE-deficient mouse.     

Lactobacillus rhamnosus GG decreases TNF-alpha production in lipopolysaccharide-activated murine macrophages by a contact-independent mechanism

Animal studies and human clinical trials have shown that Lactobacillus can prevent or ameliorate inflammation in chronic colitis. However, molecular mechanisms for this effect have not been clearly elucidated. We hypothesize that lactobacilli are capable of downregulating pro-inflammatory cytokine responses induced by the enteric microbiota. We investigated whether lactobacilli diminish production of tumour necrosis factor alpha (TNF-alpha) by the murine macrophage line, RAW 264.7 gamma (NO-), and alter the TNF-alpha/interleukin-10 (IL-10) balance, in vitro. When media conditioned by Lactobacillus rhamnosus GG (LGG) are co-incubated with lipopolysaccharide (LPS) or lipoteichoic acid (LTA), TNF-alpha production is significantly inhibited compared to controls, whereas IL-10 synthesis is unaffected. Interestingly, LGG-conditioned media also decreases TNF-alpha production of Helicobacter-conditioned media-activated peritoneal macrophages. Lactobacillus species may be capable of producing soluble molecules that inhibit TNF-alpha production in activated macrophages. As overproduction of pro-inflammatory cytokines, especially TNF-alpha, is implicated in pathogenesis of chronic intestinal inflammation, enteric Lactobacillus-mediated inhibition of pro-inflammatory cytokine production and alteration of cytokine profiles may highlight an important immunomodulatory role for commensal bacteria in the gastrointestinal tract.     

Advanced glycation end products upregulate angiogenic and pro-inflammatory cytokine production in human monocyte/macrophages

Glucose can react non-enzymatically with amino groups of, for example, proteins, to yield derivatives termed advanced glycation end products (AGE), which contribute to many chronic progressive diseases associated with microvascular complications. The study aimed to determine the effect of AGE-modified albumin on THP-1 cells and human monocyte-derived macrophages. Bovine serum albumin (BSA) or human serum albumin (HSA), modified by glucose-derived AGE, was prepared by incubation with glucose for differing periods of time. Alternatively, BSA was incubated with sodium cyanoborohydride and glyoxylic acid to produce N(epsilon)-(carboxymethyl)lysine-modified BSA (CML-BSA). Stimulation for 24h of THP-1 cells with BSA, incubated for 6-8 weeks with glucose, induced significant VEGF release. Human monocyte-derived macrophages stimulated with extensively glycated HSA also showed significant VEGF release, as well as upregulation of IL-8 production, incubation for 6h with extensively glycated HSA increased release of TNFalpha and expression of tissue factor. Finally, addition of CML-BSA resulted in significant induction of TNFalpha and VEGF release. We demonstrate that a range of different methods of glycation of BSA and HSA, including CML-BSA, resulted in the induction of VEGF, TNFalpha, IL-8 and expression of tissue factor, according to length of stimulation and different glycation products used, suggesting that AGE-induced activation of macrophages may contribute to vascular complications by regulation of angiogenic, inflammatory and pro-coagulant processes.     

Expression of cFABP and PPAR in trophoblast cells: effect of PPAR ligands on linoleic acid uptake and differentiation

Throughout gestation, fetal growth depends, in part, on placental transfer of maternal essential fatty acid (EFA) and long-chain polyunsaturated fatty acid. All fatty acid (FA) can cross lipid bilayer by simple diffusion, such as those in the syncytiotrophoblasts, the multinucleated, terminally differentiated trophoblast cells. The trophoblasts differentiation process is accompanied by an increase of human chorionic gonadotropin (hCG) secretion and an inhibition of Human Achaete-Scute Homologue-2 expression (Hash-2). Furthermore, a number of FA-binding proteins (FABPs) have been identified in membrane and cytoplasm of mammalian cells, which are thought to facilitate the transfer of FA across membranes and their intracellular channeling. Thus, the aim of this study was to investigate the implication of cFABPs in linoleic acid (LA) uptake by human trophoblast cells according to differentiation. Moreover, since peroxisome proliferator-activated receptor (PPARs) regulate the expression of cFABP and play an important role in trophoblast cells differentiation, the effects of PPARs ligands are verified on cFABP expression and differentiation. Herein, we reported the increase of the expression of liver and heart FABP (L- and H-FABP) upon differentiation of trophoblast cells, an inhibition of PPAR alpha and beta, while PPAR gamma levels remains unchanged. The nonselective peroxisome-proliferating agents, bezafibrate and LA, impaired trophoblast differentiation, and reduced L- and H-FABP expression. Furthermore, cobalt, a chemical agent known to mimic hypoxia, inhibits trophoblast cells differentiation and diminishes H-, L-FABP and PPARs expression. Finally, both treatments show no influence on LA uptake by trophoblast cells. In conclusion, this study showed that there is no correlation between the expression of H- and L-FABP and LA uptake by trophoblast cells and that bezafibrate and LA greatly impaired trophoblast cells differentiation.     

Upregulation of tumor necrosis factor-alpha expression by trans10-cis12 conjugated linoleic acid enhances phagocytosis of RAW macrophages via a peroxisome proliferator-activated receptor gamma-dependent pathway

The aim of this study was to examine whether tumor necrosis factor (TNF)-alpha expression in the phagocytic activity of RAW macrophages by trans10-cis12 (10t-12c) conjugated linoleic acid (CLA) is associated with peroxisome proliferator-activated receptor gamma (PPARgamma) activation. 10t-12c CLA induced the TNF-alpha expression in RAW macrophages. Phagocytic activity of naive RAW macrophages was increased either by recombinant mouse (rm) TNF-alpha or by culture supernatant from 10t-12c CLA-treated RAW macrophages. This phagocytic activity was inhibited by addition of anti-rmTNF-alpha polyclonal antibody (pAb). 10t-12c CLA also increased the level of PPARgamma protein and mRNA in RAW macrophages. When naive RAW macrophages were incubated with the culture supernatant from RAW macrophages treated with 10t-12c CLA plus GW 9662, a PPARgamma antagonist, their phagocytic activity was significantly inhibited. In addition, GW 9662 antagonized the effect of 10t-12c CLA in stimulating TNF-alpha expression. These results suggest that 10t-12c CLA modulates the phagocytic activity of RAW macrophages by upregulating TNF-alpha expression via a PPARgamma-dependent pathway.     

Conjugated linoleic acid exhibits stimulatory and inhibitory effects on prostanoid production in human endothelial cells and platelets

In addition to their reported antitumorigenic properties, various conjugated linoleic acid (CLA) isomers have also been shown to decrease prostanoid synthesis as a result of inhibiting the cyclooxygenase (COX) enzyme. We have previously reported that several CLA isomers inhibited both platelet aggregation and formation of thromboxane A(2) (TXA(2)), a proaggregatory and vasoconstrictive agent. Since the interaction between platelets and vascular endothelial cells is essential to maintaining vascular homeostasis, we decided to investigate the effects of various CLA isomers on the production of endothelial prostacyclin (PGI(2)), a potent vasodilator and inhibitor of platelet function. Using interleukin 1-beta (IL1-beta)-stimulated human umbilical vein endothelial cells (HUVECs), we initially established that HUVECs of passage #2 should be used since these cells were most responsive to thrombin-induced conversion of endogenous arachidonic acid to PGI(2), as monitored by the formation of its stable, inactive metabolite, 6-ketoPGF(1alpha). In the first part of the study, the effects of CLA isomers in the free fatty acid form were tested. The 10(E), 12(Z)- and 9(Z), 11(E)-CLA isomers inhibited thrombin-induced 6-ketoPGF(1alpha) formation with I(50)'s of 2.6 and 5.5 microM, whereas the 9(Z), 11(Z)- and 9(E), 11(E)-CLA were ineffective at concentrations up to 60 microM. The inhibitory effect of the 10(E), 12(Z)-CLA was irreversible. Next, the effects of CLA incorporation into HUVECs on PGI(2) generation was determined. An average 8-fold stimulation of 6-ketoPGF(1alpha) formation was obtained with quiescent IL1-beta-exposed HUVECs pretreated for 18 h with 25 microM 9(Z), 11(Z)-CLA, whereas cells preincubated with the 10(E), 12(Z) isomer enhanced this eicosanoid 3-fold. Such IL1-beta-treated HUVECs prelabeled with 25 microM 9(Z), 11(Z)-CLA became refractory to thrombin stimulation, as measured by 6-ketoPGF(1alpha) production, whereas a small, statistically insignificant, inhibition was observed upon thrombin treatment of HUVECs prelabeled with the 10(E), 12(Z) isomer. Qualitative similar results were obtained with resting or thrombin-stimulated platelets containing these esterified CLA isomers indicating that these effects occur with cells that contain either the COX-1 or COX-2 isozymes. The results of this in vitro study indicate that the effects of CLA on cellular prostanoid formation in endothelial cells and platelets can be either inhibitory or stimulatory, and this seems to depend not only on the specific CLA isomer and whether or not the CLA is in the free fatty acid form or esterified into cellular lipids, but also whether cells are in the resting or stimulated state. These findings suggest that in vivo, CLA might have multiple, complex effects on vascular homeostasis.     

Nitric oxide production and regulation of endothelial nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: evidence for involvement of peroxisome proliferator-activated receptor (PPAR) gamma-dependent and PPARgamma-independent signaling pathways

Recently, peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been reported to increase endothelial NO, but the signaling mechanisms involved are unknown. Using troglitazone, a PPARgamma ligand known as an antidiabetic compound, we investigated the molecular mechanism of its effect on NO production in bovine aortic endothelial cells. Troglitazone increased endothelial NO production in a dose- and time-dependent manner with no alteration in endothelial nitric-oxide synthase (eNOS) expression. The maximal increase ( approximately 3.1-fold) was achieved with 20 microm troglitazone treatment for 12 h, and this increase was accompanied by increases in the expression of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, and in Akt phosphorylation. Analysis with antibodies specific for each phosphorylated site demonstrated that troglitazone (20 microm treatment for 12 h) significantly increased both the phosphorylation of Ser(1179) of eNOS (eNOS-Ser(1179)) and the dephosphorylation of eNOS-Ser(116) but did not alter eNOS-Thr(497) phosphorylation. Treatment with anti-VEGF antibody to scavenge the increased VEGF induced by troglitazone partially inhibited troglitazone-stimulated NO production. This was accompanied by the attenuation of troglitazone-stimulated increases in the phosphorylation of Akt and eNOS-Ser(1179) with no alteration in eNOS-Ser(116) dephosphorylation. We also found that bisphenol A diglycidyl ether, a PPARgamma antagonist, partially inhibited troglitazone-stimulated NO production with a concomitant reduction in VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser(1179) phosphorylation but with no alteration in eNOS-Ser(116) dephosphorylation induced by troglitazone. Taken together, our results demonstrate that prolonged treatment with troglitazone increases endothelial NO production by at least two independent signaling pathways: PPARgamma-dependent, VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser(1179) phosphorylation and PPARgamma-independent, eNOS-Ser(116) dephosphorylation.     

Cytostatic activity of in vitro generated macrophages: evidence for a prostaglandin-independent reversible cytostatic mechanism

Culture of spleen cells for 5 days has previously been shown to result in the generation of strongly adherent cells from nonadherent precursors. In the current report it is shown that the majority (85-95%) of adherent cells are Mac-1+, FcR+, Thy 1.2- macrophages. Expression of effector activity by these macrophages requires exposure to activating signals. Coculture of the macrophages with Con A-stimulated spleen cells results in the expression of cytostatic activity against lymphocytic and monocytic tumor cell lines. Significant cytostatic activity is apparent within 6 hr after addition of the activating cells. Culture supernates of Con A-stimulated spleen cells (CAS-CM) are not effective in inducing cytostatic activity in the adherent macrophage population either alone or in the presence of additional Con A. However, stimulation of the culture generated macrophages with LPS in the presence of CAS-CM does induce cytostatic activity. The effector cell must be metabolically active in order to effect cytostasis insofar as heat fixation of the culture generated macrophage population eliminates effector activity. Proliferation of the tumor cells is significantly reduced after a 4-hr incubation period with the activated macrophages and is reduced two- to threefold after an 8- to 12-hr incubation period. The cytostatic effect is rapidly reversible. Proliferative activity of the tumor cells returned to control level within 12-24 hr after removal from activated macrophages. Cell cycle analysis indicated that the target cells were not arrested in a single stage of cell cycle, although an increase in frequency of cells in G1-phase was observed. Fluorescence analysis of bromodeoxyuridine (BrdU) incorporation rate demonstrated that the rate of DNA synthesis was reduced in all of the cells in the target population and that the mean rate of BrdU incorporation of the inhibited cells was three- to fivefold lower than control cells. RNA and protein synthesis were not affected to the same degree as DNA synthesis. The cytostatic effect was not mediated by prostaglandins or thymidine insofar as addition of indomethacin and 2-deoxycytidine did not prevent the cytostatic activity of the macrophages. The supernates of activated macrophages contained little inhibitory activity especially when indomethacin was included in the culture medium (19% inhibition of tumor cell proliferation by 1:1 dilution of supernate). The activity that was present could be eliminated by dialysis against fresh culture medium using Spectropor membranes with a 1000-Da molecular cutoff.(ABSTRACT TRUNCATED AT 400 WORDS)     

Magnesium decreases inflammatory cytokine production: a novel innate immunomodulatory mechanism

MgSO(4) exposure before preterm birth is neuroprotective, reducing the risk of cerebral palsy and major motor dysfunction. Neonatal inflammatory cytokine levels correlate with neurologic outcome, leading us to assess the effect of MgSO(4) on cytokine production in humans. We found reduced maternal TNF-α and IL-6 production following in vivo MgSO(4) treatment. Short-term exposure to a clinically effective MgSO(4) concentration in vitro substantially reduced the frequency of neonatal monocytes producing TNF-α and IL-6 under constitutive and TLR-stimulated conditions, decreasing cytokine gene and protein expression, without influencing cell viability or phagocytic function. In summary, MgSO(4) reduced cytokine production in intrapartum women, term and preterm neonates, demonstrating effectiveness in those at risk for inflammation-associated adverse perinatal outcomes. By probing the mechanism of decreased cytokine production, we found that the immunomodulatory effect was mediated by magnesium and not the sulfate moiety, and it was reversible. Cellular magnesium content increased rapidly upon MgSO(4) exposure, and reduced cytokine production occurred following stimulation with different TLR ligands as well as when magnesium was added after TLR stimulation, strongly suggesting that magnesium acts intracellularly. Magnesium increased basal I?Bα levels, and upon TLR stimulation was associated with reduced NF-κB activation and nuclear localization. These findings establish a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis.     

Conjugated linoleic acid and hepatic lipogenesis in mouse: role of the mitochondrial citrate carrier

Conjugated linoleic acid (CLA) is able to reduce adiposity by affecting lipid metabolism. In particular, CLA administration to mice reduces body fat mass with a concomitant lipid accumulation in the liver. We investigated the effects of CLA on the activity of the mitochondrial citrate carrier (CIC), which is implicated in hepatic lipogenesis. The transport activity of the CIC, measured both in intact mitochondria and in the proteoliposomes, progressively increased with the duration of CLA feeding. An increase in the CIC activity of approximately 1.7-fold was found in 16 week CLA-treated mice with respect to control animals. A kinetic analysis showed a 1.6-fold increase in the V(max) of citrate transport but no change in the K(m) value. Western blot experiments revealed an increase of approximately 1.7-fold in the expression of CIC after CLA treatment. A strict correlation between the increase in CIC activity and the stimulation of the cytosolic lipogenic enzymes was also found. These data indicate that the CIC may play a role in the onset of hepatic steatosis in CLA-fed mice by supplying the carbon source for de novo fatty acid synthesis.     

Microbial production of a novel trihydroxy unsaturated fatty acid from linoleic acid

A bacterium isolated from a dry soil sample collected from McCalla, AL, USA, converted linoleic acid to a novel compound, 12,13,17-trihydroxy-9 (Z)-octadecenoic acid (THOA). The organism is a Gram-positive, non-motile rod (0.5 μ m × 2 μ m). It was identified as a species of Clavibacter ALA2. The product was purified by high pressure liquid chromatography, and its structure was determined by ¹H and ¹³C nuclear magnetic resonance and Fourier transform infrared spectroscopies, and by mass spectrometer. Maximum production of THOA with 25% conversion of the substrate was reached after 5–6 days of reaction. THOA was not further metabolized by strain ALA2. This is the first report of a 12,13,17-trihydroxy unsaturated fatty acid and its production by microbial transformation. Some dihydroxy intermediates were also detected. THOA has a structure similar to those of known plant self-defense substances. Received 13 January 1997/ Accepted in revised form 05 May 1997     

Conjugated linoleic acid prevents the development of essential hypertension in spontaneously hypertensive rats

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid found in beef, lamb, and dairy products. CLA has attracted considerable attention over the past several decades because of its potentially beneficial biologic effects, including protective effects against several cancers, atherosclerosis, and obesity. In previous studies, we provided evidence that dietary CLA could prevent the development of obesity-related hypertension in obese animals. Here, we show that CLA suppresses the development of non-obese essential hypertension in spontaneously hypertensive rats (SHRs). After 4 weeks of feeding with CLA, the increase of systolic blood pressure was significantly suppressed compared with rats fed linoleic acid. Abdominal adipose tissue weight was also significantly lowered in CLA-fed SHRs. Content of arachidonic acid, the substrate of eicosanoid production, was not changed, but accumulation of oleic acid, the lipogenesis end-product, was markedly decreased in the membrane phospholipids of CLA-fed SHRs. In addition, we found increased level of plasma adiponectin, suggested as a regulatory factor of hypertension, through the enhancement of mRNA expression in CLA-fed SHRs. We speculate that the antihypertensive effect of dietary CLA may be due to the increase of plasma adiponectin level and associated with the alleviation of membrane abnormality in SHRs.     

Mechanisms of action of conjugated linoleic acid: evidence and speculation

Conjugated linoleic acid (CLA) has been shown to inhibit carcinogenesis and atherosclerosis, enhance immunologic function while protecting against the catabolic effects of immune stimulation, affect body composition change (reducing body fat gain while enhancing lean body mass gain), and stimulate the growth of young rats. We discuss possible biochemical mechanisms that underlie these physiological effects. We emphasize the importance of considering the effects, both individually and combined, of the two CLA isomers (cis-9, trans-11 CLA and trans-10, cis-12 CLA) that have been shown to exhibit biological activity and which appear to exert their effects via different biochemical mechanisms.