Free Palmitic Acid Research Articles

The presence of free palmitic acid modulates the effects of lutein on structural and dynamic properties of lipid membranes

Free fatty acids, like palmitic acid (PA), and xanthophyll pigments, like lutein (LUT) are the natural membrane compounds in plants. To study the effect of PA on LUT and their organization, a model membrane of 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) enriched with 2 mol% PA and 1 mol% LUT was formed. Molecular mechanisms underlying the interaction between these two compounds were examined with application of molecular spectroscopy techniques, e.g., visible spectroscopy, electron paramagnetic resonance and Fourier transform infrared. We determined the monomeric/dimeric organization of LUT in the membrane. We proved that the presence of PA in the lipid phase facilitated and stabilized the formation of LUT structures in the membrane. Lutein with PA did not form strong molecular aggregates like H- and J-structures. We presented the simplified model membrane that could be a suitable representation of the physiological process of de-esterification of PA from LUT appearing in natural biomembranes in humans.

Abstract 5302: Unmasking aggressive tumor profiles in lung adenocarcinomas through the molecular abundances and catalytic action of serine hydrolases

Abstract (a) Current proteomic methods quantify solely the catalytically active portion of enzymes without normalizing the active portion to the total enzyme amount, thus compromising comparison of the results. We take advantage of a SWATH/DIA-MS workflow and describe a method based on depletion-dependent ABPP (dd-ABPP) able to concurrently determine catalytically active enzymes and their total molecular abundances, as well as quantify the contextual sample proteomes. (b) We monitored high quality peptides of more than 190 SH activities and molecular abundances of around 4000 contextual tissue proteins in advanced lung adenocarcinomas (LUAD). While we found that LUAD tumors display enhanced proteolytic activities compared with control lung tissue, the most prominent characteristic of an aggressive tumor phenotype was enhanced lipolysis of metabolic SHs emphasized by IAH1, ABHD12, LYPLA2 and ABHD10. (c) Molecular signatures of the activity profiles of 23 SHs and 59 contextual tissue proteins discriminate aggressive tumors at the time of diagnosis. Enhanced detection of S-palmitoylated proteins correlating by active-enzyme capture uncovers their functional links with the metabolic SHs related to lipoprotein depalmitoylation via enzymes displaying increased de-palmitoylase activities and enhanced fatty acid metabolism in aggressive LUAD. To further validate our findings, we analysed LUAD tumors for panels of saturated and unsaturated fatty acid (C8-C22) levels. Strikingly, we detected a statistically significant increase in monounsaturated palmitoleic acid (C16:1) in the aggressive compared with less-aggressive tumors. Notably, the primary source of palmitoleic acid in the cellular environments results from an excess of free palmitic acid and its conversion by rate-limiting enzyme Stearoyl-CoA desaturase 1 (SCD) that is enhanced in LUAD cancer. (d) We detected a significant increase in palmitoleic acid levels in aggressive tumors. To a certain extent our data support previous findings on the active process of lipid desaturation in lung adenocarcinoma cells as the main source of energy for cell proliferation. Our approach also revealed that changes in the active lipase fractions between subtypes do not follow changes in enzyme protein quantities, suggesting that lipase regulations are related rather to peculiarities of the enzyme protein sequence, a distorted enzyme-inhibitor equilibrium, or a distorted molecular composition of the tissue itself. Since the inhibitors of serine lipases are promising targets in other RAS-mutant carcinomas, and the inhibitors of a rate-limiting SCD1 enzyme prevent palmitic acid conversion to a monounsaturated form, these metabolic vulnerabilities detected in aggressive LUAD will be helpful for future therapeutic strategies in lung cancer. Citation Format: Tatjana Sajic, Stephan Arni, Rudolf Aebersold, Sven Hillinger. Unmasking aggressive tumor profiles in lung adenocarcinomas through the molecular abundances and catalytic action of serine hydrolases. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5302.

Enriched dietary saturated fatty acids induce trained immunity via ceramide production that enhances severity of endotoxemia and clearance of infection.

Trained immunity is an innate immune memory response that is induced by a primary inflammatory stimulus that sensitizes monocytes and macrophages to a secondary pathogenic challenge, reprogramming the host response to infection and inflammatory disease. Dietary fatty acids can act as inflammatory stimuli, but it is unknown if they can act as the primary stimuli to induce trained immunity. Here we find mice fed a diet enriched exclusively in saturated fatty acids (ketogenic diet; KD) confer a hyper-inflammatory response to systemic lipopolysaccharide (LPS) and increased mortality, independent of diet-induced microbiome and hyperglycemia. We find KD alters the composition of the hematopoietic stem cell compartment and enhances the response of bone marrow macrophages, monocytes, and splenocytes to secondary LPS challenge. Lipidomics identified enhanced free palmitic acid (PA) and PA-associated lipids in KD-fed mice serum. We found pre-treatment with physiologically relevant concentrations of PA induces a hyper-inflammatory response to LPS in macrophages, and this was dependent on the synthesis of ceramide. In vivo, we found systemic PA confers enhanced inflammation and mortality in response to systemic LPS, and this phenotype was not reversible for up to 7 days post-PA-exposure. Conversely, we find PA exposure enhanced clearance of Candida albicans in Rag1-/- mice. Lastly, we show that oleic acid, which depletes intracellular ceramide, reverses PA-induced hyper-inflammation in macrophages and enhanced mortality in response to LPS. These implicate enriched dietary SFAs, and specifically PA, in the induction of long-lived innate immune memory and highlight the plasticity of this innate immune reprogramming by dietary constituents.

Enzymatic modification of triglycerides in conventional and surfactant-free microemulsions and in olive oil

Modification of the acylglycerol profile by interesterification of triacyglycerols (TAGs) can improve certain properties of food products. TAGs enriched in palmitic acid (PA) at the sn-2 position and in unsaturated fatty acids at the sn-1,3 position of the glycerol backbone are an important source of nutrients and energy for humans. In the present study, commercially available lipases have been used for the enzymatic interesterification of TAGs to form lipids containing PA and oleic acid (OA). Lipases A and B from Candida antarctica (CaL A and CaL B) and Thermomyces lanuginosus lipase (TLL) were tested in different environments from organic solvents to non-conventional systems such as w/o microemulsions (in the absence and presence of surfactants) and olive oil. The effects of reaction temperature, substrate molar ratio, and external energy were investigated. AOT microemulsions offered an excellent microenvironment for the interesterification reaction, while lecithin microemulsion affected TLL specificity. Surfactant-free microemulsions were also tested in terms of optimization of production process, however lower reaction yields were obtained. For the commercial immobilized CaL B, the ratio PA:triolein affected the reaction yield with the 2:1 ratio showing promising results in organic solvent environment. The main purpose of the present study was to elucidate the appropriate conditions for the enzymatic modification of olive oil. Several factors were tested such as substrates’ ratio, enzyme’s concentration, reaction temperature and external energy input. Overall, the ideal conditions for the formulation of the desired TAGs included the use of 50 mM free PA as substrate, 3 mg of immobilized CaL B/mL of olive oil while the reaction temperature was set at 40 °C and the duration was 72 h.

Effect of concentrate enriched with palmitic acid versus rapeseed oil on dairy performance, milk fatty acid composition, and mammary lipogenic gene expression in mid-lactation Holstein cows

This study was performed to characterize the effect of a concentrate supplemented with free palmitic acid (4% on a DM basis; PA) or rapeseed oil (4% on a DM basis; RO) compared with a no-added-lipid control concentrate (CT) on the performance of dairy cows fed a corn silage-based diet over a 9-wk period. After a 3-wk pre-experimental period, 54 Holstein cows were randomly allocated to 3 experimental treatments to receive forage ad libitum with a fixed amount of CT, RO, or PA (8 kg/d for 2-yr-old primiparous; 10 kg/d for older cows). During the experiment, dry matter intake, milk yield and composition, fatty acid (FA) yields and FA profile, and feed efficiency were determined. At wk 9 of the experimental period, the mRNA levels of 10 genes involved in lipid metabolism in mammary tissue biopsy samples were measured. Compared with CT, RO and PA increased forage intake. Compared with CT, RO increased concentrate intake, the value being intermediate for PA. Compared with CT, RO increased milk yield (+2.0 kg/d) and decreased milk fat and protein content (-3.8 and -1.2 g/kg, respectively), whereas PA increased milk fat content (+4.1 g/kg). Compared with CT and RO treatments, PA increased milk fat yield (+179 g/d) and 3.5% fat-corrected milk and energy-corrected milk output (+2.8 and +2.3 kg/d, respectively), and thus improved feed efficiency (+7.3%). Compared with CT treatment, RO increased milk contents of the sum of >C16 FA, monounsaturated FA, polyunsaturated FA, trans FA, and n-3 FA, whereas PA decreased these FA contents (except n-3 FA) and also decreased n-6 FA. The variations in milk fat yield and content and FA secretion at wk 9 were not associated with modifications in mammary expression of 10 genes involved in major lipid pathways, except for the transcription factor PPARG1, which tended to be higher in PA versus RO treatment. This study demonstrated that PA improved milk fat yield and feed efficiency compared with RO and suggests that factors other than gene expression, such as substrate availability for mammary metabolism or other levels of regulation (transcriptional, posttranscriptional, translational or posttranslational), could play a key role in milk fat and FA responses to changes in diet composition in cows.

Maternal Plasma Lipids During Pregnancy, Insulin-like Growth Factor-1, and Excess Fetal Growth.

Maternal lipids during pregnancy and placental growth factors are associated with excess fetal growth. However, how these factors interact to increase the risk of delivering large-for-gestational-age (LGA) neonates remains unclear. In this study, we investigated the relationship between maternal plasma triglycerides (TGs) and free fatty acids (FFAs) during pregnancy, cord blood insulin-like growth factors (IGF), and LGA. In a cell model, we studied the effect of different FAs on placental IGF-1 secretion. This cohort study included pregnant women with term pregnancy and without diabetes or hypertensive disorders in pregnancy. Maternal fasting plasma TGs and FFAs were measured in the second trimester. Cord blood IGF-1, IGF-2, and IGF binding protein-1 and protein-3 were measured at the time of delivery. A human trophoblast cell line, 3A-sub-E, was used to evaluate the effect of different FFAs on placental IGF-1 secretion. We recruited 598 pregnant women-neonate pairs. Maternal plasma TG (180 mg/dL [152.5-185.5 mg/dL] vs 166 mg/dL [133-206 mg/dL], P = .04) and cord blood IGF-1 concentrations (72.7 ± 23.0 vs 54.1 ± 22.8 ng/mL, P < .001) were higher in the LGA group and were significantly associated with birth weight z score. Maternal plasma free palmitic acid (PA) and stearic acid (SA), but not oleic acid (OA) or linoleic acid (LA), were significantly associated with cord blood IGF-1 concentrations. In 3A-sub-E cells, treatment with PA, SA, and LA, but not OA, induced IGF-1 expression and secretion. Certain FFAs can induce placental IGF-1 secretion, which suggests a potential pathophysiology linking maternal plasma lipids and LGA.

Vildagliptin, a dipeptidyl peptidase-4 inhibitor, reduces betel-nut induced carcinogenesis in female mice

AimBetel-nut, a popular masticatory among Southeast Asian populations is a class I carcinogen, previously associated with dyslipidemia and aberrant lipid metabolism, and is reported to be used more frequently by females, than males. This study investigates the potential of repurposing the anti-diabetic drug, vildagliptin, a dipeptidyl peptidase-4 inhibitor, for alleviating the oncogenic condition in female Swiss Albino mice administered an aqueous extract of betel-nut (AEBN) orally (2 mg ml−1) for 24 weeks. Main methodsTissues were investigated by histopathological, immunohistochemical and apoptosis assays. Biochemical analyses of oxidative stress markers and lipid profile were performed using different tissues and sera. The expressions of different proteins involved in lipid metabolism and oncogenic pathways were evaluated by Western blotting. Key findingsAEBN induced carcinogenesis primarily in the liver by significantly impairing AMPK signaling, inducing oxidative stress, activating Akt/mTOR signaling, increasing Ki-67 immunoreactivity and cyclin D1 expression, and significantly diminishing apoptosis. Co-administration of AEBN with vildagliptin (10 mg kg−1 body weight) for 8 weeks reduced liver dysplasia, and significantly decreased free palmitic acid, increased free oleic acid, normalized lipid profile, decreased oxidative stress, cyclin D1 expression, Ki-67 immunoreactivity, and Bcl2 expression, and increased the ratio of apoptotic/non-apoptotic cells. Mechanistically, vildagliptin elicited these physiological and molecular alterations by restoring normal AMPK signaling and reducing the cellular expressions of FASN and HMGCR, restoring AMPK-dependent phosphorylation of p53 at Ser-15 and reducing Akt/mTOR signaling. SignificanceThese results indicate that vildagliptin may alleviate betel-nut induced carcinogenesis in the liver of female mice.

Enzymatic synthesis and characterization of maltoheptaose-based sugar esters

In this study, maltoheptaose (G7)-based sugar esters were synthesized from maltoheptaose and fatty acids (C10–C16) using a commercial lipase. With the exception of dimethyl sulfoxide (DMSO; 76.4%, w/v), G7 showed only limited solubility in organic solvents. Among the fatty acids, palmitic acid (PA) was the best substrate for G7-based ester formation. G7-PA ester was successfully synthesized as the monoester structure exclusively in 10% DMSO of t-butanol with a 22% conversion yield. NMR and enzymatic analyses of the purified monoester product revealed that the ester bond in the G7 was located at C-6 of the glucose at the reducing end. The G7-PA monoester showed the melting temperature at 56.3 °C that was 6.5 °C lower than that of the free PA and exhibited a different endothermic pattern from the free G7. The G7-PA monoester exhibited excellent emulsifier potential with more even droplet size distribution compared with the commercial sucrose esters for an oil-in-water emulsion system.

Myristic acid specifically stabilizes diacylglycerol kinase δ protein in C2C12 skeletal muscle cells

Decreased levels of the δ isozyme of diacylglycerol kinase (DGK) in skeletal muscle attenuate glucose uptake and, consequently, are critical for the pathogenesis of type 2 diabetes. We recently found that free myristic acid (14:0), but not free palmitic acid (16:0), increased the DGKδ protein levels and enhanced glucose uptake in C2C12 myotube cells. However, it has been unclear how myristic acid regulates the level of DGKδ2 protein. In the present study, we characterized the myristic acid-dependent increase of DGKδ protein. A cycloheximide chase assay demonstrated that myristic acid, but not palmitic acid, markedly stabilized DGKδ protein. Moreover, other DGK isozymes, DGKη and ζ, as well as glucose uptake-related proteins, such as protein kinase C (PKC) α, PKCζ, Akt and glycogen synthase kinase 3β, failed to be stabilized by myristic acid. Furthermore, DGKδ was not stabilized in cultured hepatocellular carcinoma cells, pancreas carcinoma cells or neuroblastoma cells, and only a moderate stabilizing effect was observed in embryonic kidney cells. A proteasome inhibitor and a lysosome inhibitor, MG132 and chloroquine, respectively, partly inhibited DGKδ degradation, suggesting that myristic acid prevents, at least in part, the degradation of DGKδ by the ubiquitin-proteasome system and the autophagy-lysosome pathway. Overall, these results strongly suggest that myristic acid attenuates DGKδ protein degradation in skeletal muscle cells and that this attenuation is fatty acid-, protein- and cell line-specific. These new findings provide novel insights into the molecular mechanisms of the pathogenesis of type 2 diabetes mellitus.

Growth, stool consistency and bone mineral content in healthy term infants fed sn-2-palmitate-enriched starter infant formula: A randomized, double-blind, multicentre clinical trial

Palmitate in breast milk is predominantly located in the triacylglycerol sn-2 position, while infant formulae contain palmitate predominantly in the sn-1 and sn-3 positions. During digestion, palmitate in the sn-1 and sn-3 positions is hydrolyzed to free palmitic acid that can subsequently complex with calcium to form insoluble soaps; this may partially explain why formula-fed infants have harder stools than breast-fed infants. This large (n=488) randomized, double-blind, multicentre trial investigated whether increasing the sn-2 palmitate content of infant formula improves stool consistency and bone mineral content (measured by dual-energy x-ray absorptiometry), without affecting growth or health. From ∼1 week to 4 months of age, infants were exclusively fed one of three formulae: i) control formula (CF; 16% of total palmitate at sn-2; n=162), (ii) experimental formula 1 (EF1; 43% of total palmitate at sn-2; n=166) or (iii) experimental formula 2 (EF2; 51% of total palmitate at sn-2; n=160). Intention-to-treat analysis showed softer stools in both EF groups (vs. CF) at ages 2 weeks and 1 and 2 months (p≤0.01), but not 3 and 4 months. At 4 months, all groups had similar growth outcomes while bone mineral content was significantly higher in EF1 (p=0.0012) and EF2 (p=0.0002) compared with CF. Comparison of reported adverse events up to 12 months revealed no differences among groups. All 3 infant formulae exhibited equally good digestive tolerance. Formulae enriched in sn-2 palmitate fed in early infancy are safe, improve stool consistency (from 2 weeks to 2 months) and increase bone mineral content (at 4 months).

Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar–endothelial co-cultures

Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle-mediated cardiovascular health effects. We hypothesized that the presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. We investigated the interaction between direct exposure to MWCNTs and PA on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar–endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect measured in the lower chamber on HUVECs and THP-1 cells. The exposure to MWCNTs, including a short (NM400) and long (NM402) type of entangled fibers, was associated with elevated levels of reactive oxygen species as well as a decrease in the intracellular glutathione concentration in HUVEC and A549 monocultures. Both effects were found to be independent of the presence of PA. MWCNT exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did not promote THP-1 adhesion to HUVECs in the lower chamber. When THP-1 macrophages were present on the cell culture inserts, there was a modest increase in the adhesion and an increase in interleukin-6 and interleukin-8 levels in the lower chamber whereas no tumor necrosis factor was detected. Overall, this study showed that direct exposure of HUVECs to MWCNTs was associated with oxidative stress and monocyte adhesion and the presence of PA increased the adhesion when exposed to NM400.

Impact of caffeine on β cell proliferation and apoptosis under the influence of palmitic acid

We examined the influence of caffeine on the proliferation and apoptosis of β cells cultured in vitro in the presence of the free fatty acid palmitic acid (PA). Different concentrations of caffeine (1-100 mM) and free fatty PA were added to cultured β cells. The MTT assay was used to analyze cell proliferative activity; flow cytometry was used to measure apoptosis and calculate the apoptosis rate. Compared with the blank control group, cells cultured with 500 mM PA for 24, 48, 72, and 96 h showed inhibition of pancreatic β cell proliferative activity. In the 10 and 25 mM caffeine groups cultured for 48, 72, and 96 h, β cell proliferative activity was much higher than that in the 500 mM PA group. The apoptosis rate in the 500 mM PA group was 40.55 ± 20.33%, which was higher than that in the blank control group. The apoptosis rates in the 10 and 25 mM caffeine group and the PA group were 19.12 ± 10.56 and 20.97 ± 9.75%, respectively, which was lower than that in the 500 mM PA group. At some concentrations, caffeine can improve free fatty PA levels and guide pancreatic β cell proliferation inhibition and cell apoptosis.

1 CUMULUS CELLS PROTECT OOCYTES AGAINST POTENTIAL LIPOTOXICITY FROM ELEVATED FREE FATTY ACID CONCENTRATIONS IN FOLLICULAR FLUID

Metabolic conditions characterized by elevated free fatty acid levels in the blood are often associated with reduced fertility performance. Increased concentrations of saturated free fatty acids can induce lipotoxicity in cumulus–oocyte-complexes in vitro, while unsaturated fatty acids like oleic acid are mostly harmless and able to counteract the impact of saturated fatty acids (Aardema et al. 2011 Biol. Reprod. 85, 62–69). This study investigates the impact of elevated free fatty acids in the blood on the follicular fluid and the lipid of cumulus and oocytes derived from these follicles. Furthermore, in vitro maturing oocytes were exposed to free fatty acid concentrations measured in follicles of control and metabolically stressed animals from this study to determine the impact on oocyte developmental competence. Cyclic heifers (n = 12) were synchronized (7 days CIDR®) and superstimulated from Day 10 of the synchronized cycle [4 days of Folltropin-V® (Bioniche Animal Health Inc., Belleville, ON, Canada) in decreasing doses; in total 200 mg]. Heifers received ad libitum grass silage, apart from the experimental group (n = 6), which was metabolically stressed during the period of superstimulation. Ovaries were collected by ovariectomy at final maturation, 22 h after the induced LH peak. Follicular fluids and cumulus–oocyte complexes (COC) were collected from follicles of ≥8 mm. To determine the free fatty acid and lipid composition, blood, follicular fluid, cumulus cells, and oocytes were analyzed with mass spectrometry. The COC (4 runs, 400 per group) derived from slaughterhouse ovaries were in vitro matured in a standard medium without or with the dominating free fatty acids, saturated palmitic and stearic and unsaturated oleic acid, in concentrations measured in follicular fluid of control (80, 70, and 100 µM) and experimental heifers (150, 100, and 200 µM) and fertilized and cultured until the blastocyst stage. Culture data were analyzed by one-way ANOVA and lipid data by two-sample t-test (P ≤ 0.05 considered significant). Procedures were approved by the Institutional Animal Care and Use Committee. Metabolic stress resulted in elevated free fatty acid levels in blood (from 430 ± 70 to 1048 ± 190 µM) and follicular fluid (from 357 ± 72 to 670 ± 133 µM), with relatively high oleic acid concentrations in follicular fluid (+10%). The increased levels of free fatty acids in follicular fluid resulted in a massive increase of fatty acids in the cumulus cells, but oocytes did only show marginal changes. In line with this, maturation in the presence of elevated palmitic, stearic, and oleic acid did not impair oocyte developmental competence and resulted in comparable blastocyst rates for the standard medium and the free fatty acid control and metabolic stress medium (31 ± 8.7, 34 ± 7.8, and 28 ± 1.7%). Thus, cumulus cells appear to protect oocytes against potential lipotoxicity from elevated free fatty acid concentrations by the accumulation of these fatty acids. This work was funded by Pfizer Animal Health.

Thermochemolysis using TMAAc and TMAH reagents as means to differentiate between free acids and esters

The reactivity of aromatic and aliphatic esters in on-line thermochemolysis in the presence of methylation reagents was studied using model compounds. Guaiacyl and 2-nonyl palmitates were synthesised, representing aromatic and aliphatic ester bonds, respectively. These model compounds were analysed by on-line thermochemolysis using tetramethylammonium acetate (TMAAc) and tetramethylammonium hydroxide (TMAH) in order to differentiate free acids from esterified acids. The released palmitic acid with TMAH for both model compounds was close to the theoretical palmitic acid content in the model compounds, even if part of the aliphatic ester had not reacted with TMAH. The free palmitic acid content by TMAAc was one third of the theoretical value for aromatic ester and only a few percentages for aliphatic ester. The result indicated that the less basic reagent is able to hydrolyse the aromatic ester linkage to some extent, whereas the aliphatic ester remains intact. Thus, differentiation of free acids by TMAAc from the esterified acids cannot be reliably performed from matrices containing aromatic esters. It was found that transesterification due to the use of methanol as a solvent with TMAAc is an insignificant reaction in on-line thermochemolysis.

Regulation of Voltage-Gated Calcium Channel Activity by Palmitoylation: A Fatty Acid Tale

The phospholipid phosphatidylinositol 4,5-bisphospate (PIP2) interacts with voltage-gated Ca2+ channels to facilitate their opening. Conversely, inhibition of channel activity following activation of Gq-protein coupled receptors is associated with PIP2 breakdown. PIP2's freed fatty acid tails appear to remain associated with channels, stabilizing closed conformations. My lab is testing a model where an accessory β-subunit CaVβ2a acts as a phospholipid mimic resistant to metabolism; its two palmitoyl groups bind to the channel at a site normally occupied by PIP2's two fatty acid tails. To test this model, we have concentrated on examining N-type Ca2+ channel modulation in a recombinant system where CaV2.2 is coexpressed with α2δ-1 and one of four CaVβ subunits. M1 muscarinic or neurokinin-1 receptor stimulation inhibits N-current from CaVβ1b-, CaVβ3-, or CaVβ4-containing channels, but enhances N-current from CaVβ2a-containing channels. Exogenously applied arachidonic acid produces the same pattern of modulation. Further studies with mutated, chimeric CaVβ subunits and free palmitic acid revealed palmitoylation of CaVβ2a is essential for loss of inhibition. Loss of inhibition appears independent of kinetic changes that occur with different channel complexes. In contrast, channel mutations that reorient CaVβ2a's relative position to CaV2.2 rescue inhibition suggesting that in these experiments, the palmitoyl groups become sufficiently displaced that endogenously released arachidonic acid can once again bind to the inhibitory site. These findings suggest a new dynamic function for palmitoylation and predict that other doubly palmitoylated proteins reach up into the membrane with their lipid fingers to interact with and change the functioning of transmembrane proteins.(Funded by the NIH, AHA and the UMass Medical School).

Regularities of endogenous lipid metabolites formation in phorbol 12-miristate 13-acetate-stimulated peripheral blood lymphocytes at leukemia

Regularities of biologically active lipid metabolites formation in dynamics (5, 10, 30, 60 s) by phorbol 12-miristate 13-acetate stimulation in [14C]palmitic acid have been investigated in normal and leukemia peripheral blood lymphocytes prelabeled with [14C]palmitate. In normal cells there was two-phase formation of 1,2-diacylglycerol (5, 30 s), lysophosphatidylcholine (10, 60 s), as well as free palmitic acid at 10 s of stimulation. Under the identical experimental conditions there was inhibition of investigated lipid release processes at early (5 and 10 s) stages of stimulation of leukemic lymphocytes. At later (30, 60 s) terms of these lymphocytes the activation, basically, similar to norm changes in the formation of palmitic acid-containing metabolites except free palmitic acid (the level of which raised only at 60 second of the post-stimulation) was found. Various protein kinases C are involved in the regulation of investigated lipid levels at certain stages of signal transduction both in norm, and in blast cells. Short-term (5, 10 s) activations of healthy donors lymphocytes are coupled to functioning of Ca2+-independent isoforms of protein kinase C. The inhibition of this protein kinase C in leukemic cells leads to normalization of the investigated lipid release. The data obtained suggests disorders of early membrane-bound reactions in agonist - and a protein kinase C-mediated processes of formation palmitic acid-containing lipid metabolites in the leukemic cells in comparison with the norm.

Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects

Background Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry. Objective To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals. Methods Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor. Results Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E 2 was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D 2 products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B 4 has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes–eoxins were detectable and metabolized to eoxin E 4. Two main vascular prostanoids: prostacyclin and thromboxane B 2 were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF 2α isoprostane was increased in smokers. Conclusion Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment.

The microstructure of the stratum corneum lipid barrier: Mid-infrared spectroscopic studies of hydrated ceramide:palmitic acid:cholesterol model systems

The current mid-infrared spectroscopic study is a systematic investigation of hydrated stratum corneum lipid barrier model systems composed of an equimolar mixture of a ceramide, free palmitic acid and cholesterol. Four different ceramide molecules (CER NS, CER NP, CER NP-18:1, CER AS) were investigated with regard to their microstructure arrangement in a stratum corneum lipid barrier model system. Ceramide molecules were chosen from the sphingosine and phytosphingosine groups. The main differences in the used ceramide molecules result from their polar head group architecture as well as hydrocarbon chain properties. The mixing properties with cholesterol and palmitic acid are considered. This is feasible by using perdeuterated palmitic acid and proteated ceramides. Both molecules can be monitored separately, within the same experiment, using mid-infrared spectroscopy; no external label is necessary.At physiological relevant temperatures, between 30 and 35°C, orthorhombic as well as hexagonal chain packing of the ceramide molecules is observed. The formation of these chain packings are extremely dependent on lipid hydration, with a decrease in ceramide hydration favouring the formation of orthorhombic hydrocarbon chain packing, as well as temperature. The presented data suggest in specific cases phase segregation in ceramide and palmitic acid rich phases. However, other ceramides like CER NP-18:1 show a rather high miscibility with palmitic acid and cholesterol. For all investigated ternary systems, more or less mixing of palmitic acid with cholesterol is observed. The investigated stratum corneum mixtures exhibit a rich polymorphism from crystalline domains with heterogeneous lipid composition to a “fluid” homogeneous phase. Thus, a single gel phase is not evident for the presented stratum corneum model systems.The study shows, that under skin physiological conditions (pH 5.5, hydrated, 30–35°C) ternary systems composed of an equimolar ratio of ceramides, free palmitic acid and cholesterol may form gel-like domains delimitated by a liquid-crystalline phase boundary. The presented results support the microstructural arrangement of the stratum corneum lipids as suggested by the domain mosaic model.

The Ca2+ channel β subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current

In superior cervical ganglion (SCG) neurons, stimulation of M1 receptors (M1Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M1Rs. In addition, techniques that diminish AA's concentration during M1R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M1R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the β subunit (CaVβ) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M1Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by CaV2.2 and coexpressed with CaVβ1b, CaVβ3, or CaVβ4 but enhanced activity of N channels containing CaVβ2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of CaVβ2a, CaVβ3, and CaVβ4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of CaVβ with CaV2.2. Further experiments with mutated, chimeric CaVβ subunits and free palmitic acid revealed that palmitoylation of CaVβ2a is essential for loss of inhibition. The data presented here fit a model in which CaVβ2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated CaVβ2a toggles M1R- or NK-1R–mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M1Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated CaVβ2a.

Production of hydroxy-fatty acid derivatives from waste oil by Escherichia coli cells producing fungal cytochrome P450foxy

Cytochrome P450foxy (P450foxy) is a fatty acid (FA) monooxygenase that is characterized by self-sufficient catalysis and high turnover numbers due to the fused structure of cytochrome P450 and its reductase. Here we found that resting recombinant Escherichia coli cells producing P450foxy converted saturated FA with a chain length of 7-16 carbon atoms to their omega-1 to omega-3 hydroxy derivatives. Most products were recovered from the culture supernatant. Decanoic acid was most efficiently converted to omega-1 to omega-3 hydroxy decanoic acids in the order of omega-1>omega-2>omega-3, with a total product yield of 47%. We also found that P450foxy was more active against physiological fatty acyl esters such as monopalmitoyl glycerol, monopalmitoyl phospholipid, and palmitoyl CoA than free palmitic acid. The bacteria producing P450foxy were applicable as biocatalysts in the production of omega-1 hydroxy palmitic acid from lard, vegetable, and soy sauce oil wastes from the food industry.