Free Fatty Acids Species Research Articles

Feeding dogs a high-fat diet induces metabolic changes similar to natural aging, including dyslipidemia, hyperinsulinemia, and peripheral insulin resistance.

The goal of this study was to characterize changes induced by a high-fat diet in body composition, insulin levels and sensitivity, blood lipids, and other key biomarkers also associated with the metabolic dysfunction that occurs with natural aging. 24 male Beagle dogs, 3 to 7 years of age, of mixed castration status. Dogs were randomly assigned to continue twice daily feeding of the commercial adult maintenance diet (n = 12, including 2 intact) that they were previously fed or to a high-fat diet (12, including 2 intact) for 17 weeks between December 1, 2021, and April 28, 2022. Assessments included body composition (weight, body condition score, and adipose mass determined by deuterium enrichment), clinical chemistries, plasma fatty acid quantification, oral glucose tolerance test, and histology of subcutaneous and visceral adipose biopsy samples. The high-fat diet led to increased body weight, body condition score, fat mass and adipocyte size, hyperinsulinemia and peripheral insulin resistance, and elevations in serum lipids, including cholesterol, triglycerides, and several species of free fatty acids. Leptin levels increased in dogs fed a high-fat diet but not in control dogs. There were no significant changes in routine clinical chemistry values in either group. Feeding a high-fat diet for 17 weeks led to potentially deleterious changes in metabolism similar to those seen in natural aging in dogs, including hyperinsulinemia, insulin resistance, and dyslipidemia. A high-fat diet model may provide insights into the similar metabolic dysfunction that occurs during natural aging.

Oxygen Exposure and Tolerance Shapes the Cell Wall-Associated Lipids of the Skin Commensal Cutibacterium acnes.

Cutibacterium acnes is one of the most abundant bacteria on the skin. Being exposed to oxygen and oxic stress, the secretion of the bacterial antioxidant protein RoxP ensures an endogenous antioxidant system for the preservation of skin health. To investigate the impact of the antioxidant RoxP on oxidation of the bacteria, wildtype and an isogenic roxp mutant were cultured in anaerobic and oxic conditions. The carbonylated status of proteins were recorded, as were the most significant modifications in a relative intensity of free fatty acids (FFA) and lipids containing fatty acids (FA), such as di- (DG) and triglycerides (TG), di- (DGDG) and sulfoquinozyldiacylglycerol (SQDG) and ceramides. Concerning the fatty acid types, it was observed that the free fatty acids contained mainly C12:0-C26:0 in hydroxy and acylated forms, the DG contained mainly C29:0-C37:0, the TG contained mainly C19:0-C33:0, and the DGDG/SQDGs contained very long fatty acids (C29:0-C37:0) demonstrating the interdependence of de novo synthesis of lipids and RoxP. The area of DGDG peaks (924.52, 929.56 and 930.58) were affected by bacterial growth conditions, with the exception of m/z 910.61. Moreover, the FFA unsaturation is wider in the SQDG species (C30:0 to C36:6) than in DG, TG or free FFA species. It could be concluded that both environmental oxidative statuses, as well as the prevalence of bacterial antioxidant systems, significantly shape the lipidome of C. acnes.

Effects of perinatal stress on the metabolites and lipids in plasma of dairy goats

Dairy goats experience metabolic stress during the peripartal period, and their ability to navigate this stage of lactation is related to the occurrence and development of metabolic diseases. Unlike dairy cows, there is a lack of comprehensive analysis of changes in the plasma profiles of peripartal dairy goats, particularly using high-throughput techniques. A subset of 9 clinically-healthy dairy goats were used from a cohort of 96 primiparous Guanzhong dairy goats (BCS, 2.75 ± 0.15). Blood samples were collected at seven time points around parturition (d 21, 14, 7 before parturition, the day of kidding, and d 7, 14, 21 postpartum), were analyzed using untargeted metabolomics and targeted lipidomics. The orthogonal partial least squares discriminant analysis model revealed a total of 31 differential metabolites including p-cresol sulfate, pyruvic acid, cholic acid, and oxoglutaric acid. The pathway enrichment analysis identified phenylalanine metabolism, aminoacyl-tRNA biosynthesis, and citrate cycle as the top three significantly-altered pathways. The Limma package identified a total of 123 differentially expressed lipids. Phosphatidylserine (PS), free fatty acids (FFA), and acylcarnitines (ACs) were significantly increased on the day of kidding, while diacylglycerols (DAG) and triacylglycerols (TAG) decreased. Ceramides (Cer) and lyso-phosphatidylinositols (LPI) were significantly increased during postpartum period, while PS, FFA, and ACs decreased postpartum and gradually returned to antepartum levels. Individual species of FFA and phosphatidylcholines (PC) were segregated based on the differences in the saturation and length of the carbon chain. Overall, this work generated the largest repository of the plasma lipidome and metabolome in dairy goats across the peripartal period, which contributed to our understanding of the multifaceted adaptations of transition dairy goats.

Lipidomics Reveals Elevated Plasmalogens in Women with Obesity Who Develop Preeclampsia.

Objective: Preeclampsia (PE) is a prevalent pregnancy disorder worldwide with limited preventative treatments available. Obesity triples the risk for PE, yet only 10% of women with obesity develop PE. The factors that distinguish PE from uncomplicated pregnancies in the context of obesity have not been fully established. Methods: We studied a cohort of women with obesity throughout pregnancy to identify lipid mediators and/or biomarkers of PE. Blood samples were collected at each trimester and analyzed by both targeted lipidomics and standard lipid panels. Individual lipid species were compared by PE status at each trimester, as well as by self-identified race (Black vs. White) and fetal sex. Results: Standard lipid panels and clinical measurements revealed few differences between PE and uncomplicated pregnancies. Targeted lipidomics, however, identified plasmalogen, phosphatidylethanolamine, and free fatty acid species that were elevated in the third trimester of women with PE. Furthermore, race and trimester of pregnancy were considerable sources of plasma lipidomic variation in women with obesity. Conclusions: First and second trimester individual plasma lipid species do not predict the development of PE in obese women. In the third trimester, PE patients have elevated levels of plasmalogens-a class of lipoprotein-associated phospholipids that have been implicated in the response to oxidative stress.

LipidSearch‐based manual comparative analysis of long‐chain free fatty acids in thermal processed tilapia muscles: workflow, thermal processing effect and comparative lipid analysis

AbstractLipidSearch is a useful tool to analyse lipid profiles in tissues. In this work, a liquid chromatography‐mass spectrometry‐LipidSearch‐based manual comparative analysis method was built to analyse the effects of thermal processing methods (steaming, boiling and roasting) on long‐chain free fatty acid species in tilapia muscles. Then, the fatty acid profiles of raw and thermal processed tilapia muscles were analysed. About 66 fatty acid species were present in the raw, steamed, boiled and roasted tilapia muscles, and only five fatty acid species had higher sensitivity in positive ionisation mode than negative ionisation mode of mass spectrometry. Thermal processing mainly had no obvious effects on the amounts of about 60% free fatty acid species. Two, two, five and twelve fatty acid species could be the best potential biomarkers for differentiation of raw, steamed, boiled and roasted, respectively, tilapia muscles in our experiments. This work could provide a workflow of LipidSearch analysis and basic information on the effect of thermal processing on the long‐chain free fatty acid profile of foods.

Free Fatty Acid Species Differentially Modulate the Inflammatory Gene Response in Primary Human Skeletal Myoblasts.

Simple SummaryEpidemiological studies show that obesity increases the risk of muscle mass loss with age, a syndrome called sarcopenic obesity. Obesity leads to increased free fatty acids (FFAs) and excessive fat deposits, which impair the integrity of skeletal muscles by unknown mechanisms. This report indicates that FFAs directly affect human skeletal muscle cell replication and inflammatory gene expression. The structural characteristics of FFAs play a decisive role in triggering both processes. Thus, the characterization of abundant FFA species in the skeletal muscle of obese individuals may become a useful tool to predict the progression of sarcopenic obesity.Age-related loss of skeletal muscle is associated with obesity and inflammation. In animal models, intramuscular fat deposits compromise muscle integrity; however, the relevant fat components that mediate muscular inflammation are not known. Previously, we hypothesized that free fatty acids (FFAs) may directly induce inflammatory gene expression in skeletal muscle cells of obese rats. Here, we examined this hypothesis in primary human skeletal myoblasts (SkMs) using multiplex expression analysis of 39 inflammatory proteins in response to different FFA species. Multiplex mRNA quantification confirmed that the IL6, IL1RA, IL4, LIF, CXCL8, CXCL1, CXCL12 and CCL2 genes were differentially regulated by saturated and unsaturated C16 or C18 FFAs. Fluorescence staining revealed that only saturated C16 and C18 strongly interfere with myoblast replication independent of desmin expression, mitochondrial abundance and oxidative activity. Furthermore, we addressed the possible implications of 71 human receptor tyrosine kinases (RTKs) in FFA-mediated effects. Phosphorylated EphB6 and TNK2 were associated with impaired myoblast replication by saturated C16 and C18 FFAs. Our data suggest that abundant FFA species in human skeletal muscle tissue may play a decisive role in the progression of sarcopenic obesity by affecting inflammatory signals or myoblast replication.

Stability of lipids in plasma and serum: Effects of temperature-related storage conditions on the human lipidome

Large epidemiological studies often require sample transportation and storage, presenting unique considerations when applying advanced lipidomics techniques. The goal of this study was to acquire lipidomics data on plasma and serum samples stored at potential preanalytical conditions (e.g., thawing, extracting, evaporating), systematically monitoring lipid species for a period of one month. Split aliquots of 10 plasma samples and 10 serum samples from healthy individuals were kept in three temperature-related environments: refrigerator, laboratory benchtop, or heated incubator. Samples were analyzed at six different time points over 28 days using a Bligh & Dyer lipid extraction protocol followed by direct infusion into a lipidomics platform using differential mobility with tandem mass spectrometry. The observed concentration changes over time were evaluated relative to method and inter-individual biological variability. In addition, to evaluate the effect of lipase enzyme levels on concentration changes during storage, we compared corresponding fasting and post-prandial plasma samples collected from 5 individuals. Based on our data, a series of low abundance free fatty acid (FFA), diacylglycerol (DAG), and cholesteryl ester (CE) species were identified as potential analytical markers for degradation. These FFA and DAG species are typically produced by endogenous lipases from numerous triacylglycerols (TAGs), and certain high abundance phosphatidylcholines (PCs). The low concentration CEs, which appeared to increase several fold, were likely mass-isobars from oxidation of other high concentration CEs. Although the concentration changes of the high abundant TAG, PC, and CE precursors remained within method variability, the concentration trends of FFA, DAG, and oxidized CE products should be systematically monitored over time to inform analysts about possible pre-analytical biases due to degradation in the study sample sets.

Lipidomic profiling of plasma free fatty acids in type-1 diabetes highlights specific changes in lipid metabolism

Type-1 diabetes mellitus (T1DM) is associated with metabolic changes leading to alterations in glucose and lipid handling. While T1DM-associated effects on many major plasma lipids have been characterised, such effects on plasma free fatty acids (FFA) have not been fully examined. Using gas chromatography–mass spectrometry, we measured the plasma concentrations of FFA species in individuals with T1DM (n = 44) and age/sex-matched healthy controls (n = 44). Relationships between FFA species and various parameters were evaluated. Plasma concentrations of myristate (14:0), palmitoleate (16:1), palmitate (16:0), linoleate (18:2), oleate (18:1c9), cis-vaccenate (18:1c11), eicosapentaenoate (20:5), arachidonate (20:4) and docosahexanoate (22:6) were reduced in the T1DM group (p < 0.0001 for all, except p = 0.0020 for eicosapentaenoate and p = 0.0068 for arachidonate); α-linolenate (18:3) and dihomo-γ-linolenate (20:3) concentrations were unchanged. The saturated/unsaturated FFA ratio, n-3/n-6 ratio, de novo lipogenesis index (palmitate (main lipogenesis product)/linoleate (only found in diet)) and elongase index (oleate/palmitoleate) were increased in the T1DM group (p = 0.0166, p = 0.0089, p < 0.0001 and p = 0.0008 respectively). The stearoyl-CoA desaturase 1 (SCD1) index 1 (palmitoleate/palmitate) and index 2 (oleate/stearate) were reduced in T1DM (p < 0.0001 for both). The delta-(5)-desaturase (D5D) index (arachidonate/dihomo-γ-linolenate) was unchanged. Age and sex had no effect on plasma FFA concentrations in T1DM, while SCD1 index 1 was positively correlated (p = 0.098) and elongase index negatively correlated with age (p = 0.0363). HbA1c was negatively correlated with all plasma FFA concentrations measured except α-linolenate and dihomo-γ-linolenate. Correlations were observed between plasma FFA concentrations and cholesterol and HDL concentrations, but not LDL concentration or diabetes duration. Collectively, these results aid our understanding of T1DM and its effects on lipid metabolism.

In-depth lipidomic analysis of tri-, di-, and mono-acylglycerols released from milk fat after in vitro digestion

Milk fat is arguably one of the most complex fats found in nature and varies widely between animal species. Analysis of its digestion products is tremendously challenging, due to the complexity, diversity, and large range of concentrations of triacylglycerols (TAGs) and their digestion products (i.e. diacylglycerols (DAGs), monoacylglycerols (MAGs), and free fatty acids (FFAs)). Therefore, a method combined the solid phase extraction (SPE), high-performance liquid chromatography (HPLC) and multi-dimension mass spectrometry (MDMS) was developed to identify and semi-quantify the TAGs, DAGs and MAGs in milk fat after in vitro digestion. Up to 105, 64, 14 and 30 species of TAGs, DAGs, MAGs, and FFAs were determined with their concentrations of 0.01–22.3, 0.01–39.2, 0.01–47.8, and 0.04–191.0 mg/g fat, respectively, during the in vitro digestion of cow and sheep milk. The validation of the method shows that this method was precise and reliable.

Abstract 2235: Prostatic fatty acids correlate with the progression and racial disparity of prostate cancer

Abstract Objectives: This study is aimed to correlate the levels of prostatic fatty acids with the pathogenesis, progression and racial disparity of prostate cancer (PCa). Methods: Total fatty acids (TFA) and free fatty acids (FFA) were measured in 26 fresh frozen PCa tissues from 13 African American (AA) and 13 Caucasian American (CA) patients, and 21 benign prostatic tissues (BPT) from 12 AA and 9 CA men by gas chromatography with flame ionization detection and electrospray ionization mass spectrometry, respectively. Results: In all populations, TFA in total was significantly higher in PCa than in ABP (1.8-fold, p=0.023). Saturated TFA (STFA) was 1.6-fold higher (p=0.024), mono-unsaturated TFA (MUTFA) was 1.9-fold higher (p=0.027), and poly-unsaturated TFA (PUTFA) was 2.1-fold higher (p=0.045) in PCa than in BPT. Six out of 16 individual TFA species were significantly higher in PCa than in BPT. Three out of 24 TFA parameters were significantly higher in high than in low grade PCa; and six out of 24 TFA parameters were significantly higher in high than low stage PCa. FFA in total was significantly higher in PCa than in BPT (1.3-fold, p=0.038). Saturated FFA (SFFA) in total was 1.2-fold higher (p=0.24), mono-unsaturated FFA (MUFFA) in total was 1.7-fold higher (p=0.027), and poly-unsaturated FFA (PUFFA) in total was 1.7-fold higher (p=0.045) in PCa than in BPT. Four out of 10 FFA species were significantly higher in PCa than in BPT. In stratified populations, the difference of TFA in total was not significant between AA PCa and AA BPT (p=0.71), but it was significantly higher in CA PCa than in CA BPT (2.7-fold, p=0.003). There were only 2 out of 16 TFA species in AA PCa significantly higher than in AA BPT, whereas 11 out of 16 TFA species in CA PCa significantly higher than in CA BPT. The differences in all groups of TFAs, including STFA, MSTFA, PUTFA, n-3, n-6 and n-9 TFA were not significant between AA PCa and AA BPT, however they were all significantly higher in CA PCa than in CA BPT. Out of 24 fatty acid parameters, two in AA population but 14 in CA population were significantly higher in high than low grade PCa, and one in AA population but 15 in CA population were significantly higher in high than low stage PCa. None of 14 FFA parameters were significantly different between AA PCa and AA BPT, but 10 of those were significantly higher in CA PCa than in CA BPT. Intriguingly, TFA in total, STFA, MUTFA, PUTFA, n-3TFA, n-6 TFA, n-9 TFA and ratio of n-6 to n-3 were all higher in AA BPT than CA BPT, but all lower in AA PCa than in CA PCa. Conclusion: This study suggests that increasing in majority of prostatic fatty acids (both TFA and FFA) is risk for pathogenesis and progression of PCa. Higher levels of prostatic fatty acids in BPT are greater risk for PCa pathogenesis in AA men, whereas higher levels of prostatic fatty acids contribute more to the progression of PCa in CA men. Citation Format: Xinchun Zhou, Jinghe Mao, Hao Mei, Timera Brown, Joshua Agee, Steven Bigler, Ruth Welti. Prostatic fatty acids correlate with the progression and racial disparity of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2235.

Intestinal Dysbiosis Is Associated with Altered Short-Chain Fatty Acids and Serum-Free Fatty Acids in Systemic Lupus Erythematosus.

Metabolic impairments are a frequent hallmark of systemic lupus erythematosus (SLE). Increased serum levels of free fatty acids (FFA) are commonly found in these patients, although the underlying causes remain elusive. Recently, it has been suggested that factors other than inflammation or clinical features may be involved. The gut microbiota is known to influence the host metabolism, the production of short-chain fatty acids (SCFA) playing a potential role. Taking into account that lupus patients exhibit an intestinal dysbiosis, we wondered whether altered FFA levels may be associated with the intestinal microbial composition in lupus patients. To this aim, total and specific serum FFA levels, fecal SCFA levels, and gut microbiota composition were determined in 21 SLE patients and 25 healthy individuals. The Firmicutes to Bacteroidetes (F/B) ratio was strongly associated with serum FFA levels in healthy controls (HC), even after controlling for confounders. However, this association was not found in lupus patients, where a decreased F/B ratio and increased FFA serum levels were noted. An altered production of SCFA was related to the intestinal dysbiosis in lupus, while SCFA levels paralleled those of serum FFA in HC. Although a different serum FFA profile was not found in SLE, specific FFA showed distinct patterns on a principal component analysis. Immunomodulatory omega-3 FFA were positively correlated to the F/B ratio in HC, but not in SLE. Furthermore, divergent associations were observed for pro- and anti-inflammatory FFA with endothelial activation biomarkers in lupus patients. Overall, these findings support a link between the gut microbial ecology and the host metabolism in the pathological framework of SLE. A potential link between intestinal dysbiosis and surrogate markers of endothelial activation in lupus patients is supported, FFA species having a pivotal role.

Effects of Chain Length and Saturability of Fatty Acids on Phospholipids and Proteins in Plasma Membranes of Bovine Mammary Gland.

Free fatty acids (FFAs) in plasma are essential substrates for de novo synthesis of milk fat, or directly import into mammary cells. The physico-chemical properties of mammary cells membrane composition affected by FFAs with different chain lengths and saturability are unclear yet. Employing GC, FTIR and fluorescence spectroscopy, the adsorption capacity, phospholipids content, membrane proteins conformation, lipid peroxidation product, and free sulfhydryl of plasma membranes (PMs) interacted with different FFAs were determined. The mammary cells PMs at 38 and 39.5°C showed different adsorption capacities: acetic acid (Ac)>stearic acid (SA)>β-hydroxybutyric acid (BHBA)>trans10, cis12 CLA. In the FTIR spectrum, the major adsorption peaks appeared at 2920 and 2850cm-1 for phospholipids, and at 1628 and 1560cm-1 for membrane proteins. The intensities of PMs-FFAs complexes were varied with the FFAs species and their initial concentrations. The β-sheet and turn structures of membrane proteins were transferred into random coil and α-helix after BHBA, SA and trans10, cis12 CLA treatments compared with Ac treatment. The quenching effects on the fluorescence of endogenous membrane protein, 1, 8-ANS, NBD-PE, and DHPE entrapped in PMs by LCFA were different from those of short chain FFAs. These results indicate that the adsorption of FFAs could change membrane protein conformation and polarity of head group in phospholipids. This variation of the mammary cells PMs was regulated by carbon chain length and saturability of FFAs.

Meibum Color and Free Fatty Acid Composition in Patients With Meibomian Gland Dysfunction.

We measured the components of meibum in patients with meibomian gland dysfunction (MGD) and control subjects and then examined the relation between meibum composition and clinical parameters. Thirty-eight patients with MGD (13 men and 25 women; mean age ± SD, 66.9 ± 15.0 years) and 20 control subjects (8 men and 12 women; 64.5 ± 6.7 years) were enrolled. Ocular symptom score, keratoconjunctival staining score, tear film breakup time, and Schirmer's test value were determined. Lid margin abnormalities and meibomian gland morphology were assessed for upper and lower eyelids, and meibum properties were evaluated at temporal, central, and nasal sites of each lid. Free fatty acid (FFA) composition of meibum was analyzed by liquid chromatography-Fourier transform mass spectrometry. Upper meibum color score was significantly correlated with epiphora and sticky sensation in MGD patients. Meibum grade, color, or viscosity did not differ significantly among the sites evaluated. A total of 103 species of FFA--including very long chain (such as C36 and C37) and odd-numbered chain (such as C17, C19, and C21) FFAs--were detected in meibum. Free fatty acid composition differed between clear and colored (cloudy or yellow) meibum, with unsaturated FFAs tending to be more abundant in colored meibum. Free fatty acid composition of human meibum correlates with meibum color as determined with a slit-lamp microscope. This finding may provide insight into the pathogenesis of MGD.

The Association of Lipoprotein Lipase and Phosphatidylinositol 3‐Kinase with Macrophage Lipid Accumulation

Lipoprotein lipase (LPL) is an extracellular enzyme that hydrolyzes triglycerides and phospholipids within lipoproteins. LPL is expressed in several tissues, including macrophages within atherosclerotic lesions, where it has been shown to promote foam cell formation. Our laboratory previously showed that the hydrolysis products generated by LPL from total lipoproteins, and specifically the free fatty acids (FFA), increased the phosphorylation of the signaling molecule protein kinase B (or Akt). Thus, we hypothesized that the FFA liberated from total lipoproteins by LPL promote lipid accumulation in macrophages in part through a mechanism associated with the phosphatidylinositol 3-kinase (PI3K) signaling pathway. To test this hypothesis, THP-1 macrophages were incubated with a mixture of purified FFA (which matched the concentrations of the FFA species liberated by LPL lipoprotein hydrolysis) for 18 hours in the absence or presence of the PI3K inhibitor LY294002. As expected, Oil Red O staining showed an increased cellular accumulation of lipids following the FFA mixture treatment. THP-1 macrophages in the presence of both the FFA mixture and LY294002 exhibited a 20% decrease of Oil Red O staining (n=9, p<0.0001). However, no differences in Oil Red O staining were observed between THP-1 macrophages incubated with individual species of FFA in the absence or presence of LY294002, thus suggesting that multiple FFA species together are necessary to modulate the PI3K signaling pathway. Overall, our data show that the FFA mixture indeed promotes macrophage lipid accumulation partially through a PI3K pathway. Funded by the Natural Sciences and Engineering Research Council of Canada (grant #402185/2011).

The functional Q84R polymorphism of TRIB3 gene is associated with diabetic nephropathy in Chinese type 2 diabetic patients

Increased oxidative stress and circulating free fatty acids (FFA) has been suggested to involve in the pathogenesis of diabetic nephropathy. TRIB3 can inhibit FFA and reactive oxygen species (ROS) stimulated podocyte production of MCP-1. Smoking increases the production of reactive oxygen species, which accelerates oxidative stress under hyperglycemia. To determine whether the Q84R polymorphism (rs2295490), alone or in combination with smoking, contributes to the development of diabetic nephropathy, a case–control study was performed in 812 Chinese patients with type 2 diabetes. Among patients, 214 had diabetic nephropathy with microalbuminuria (n=156) or overt albuminuria (n=58), and 598 did not show either of these symptoms but had diabetes for ≥10years and were not undergoing antihypertension treatment. After adjustment for confounders, TRIB3 single-nucleotide polymorphism rs2295490 was associated with DN (OR 1.318, 95% CI 1.075, 1.653, p=0.017); smoking was also an independent risk factor for diabetic nephropathy (1.42 [1.25–2.04], p<0.001). In addition, we identified possible synergistic effects; i.e., the high-risk group (smokers with the AG+GG genotype) showed 2.13 times higher risk (1.51–3.96, p<0.001) of diabetic nephropathy than the low-risk group (nonsmokers with the AA genotype) in a multiple logistic regression analysis controlled for the confounders, but no departure from additivity was found. Our results indicate that smoking and the TRIB3 G-allele is associated with an increased risk of diabetic nephropathy, which supports the hypothesis that oxidative stress contributes to the development of diabetic nephropathy.

Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL.

Regulation of Hypothalamic-Pituitary-Adrenal Axis by Circulating Free Fatty Acids in Male Wistar Rats: Role of Individual Free Fatty Acids

We previously showed that a fall in the plasma free fatty acid (FFA) level increases plasma corticosterone levels in rats by activating the hypothalamic-pituitary-adrenal axis. In the present study, we tested whether this regulation is mediated by specific or all species of FFAs. Nicotinic acid (NA) (30 μmol/h) was infused in rats to decrease plasma FFAs and increase plasma ACTH and corticosterone. The NA infusion was combined with an infusion of lipids with different FFA compositions to selectively prevent falls in individual FFA levels; coconut, olive, and safflower oils (n = 7 for each), which are predominantly (>70%) composed of saturated, monounsaturated (oleic acid), and polyunsaturated (linoleic acid) FFAs, respectively, were used. At an infusion rate (0.1 g/h) that only partially prevented a fall in the total FFA level, coconut oil, but not olive or safflower oil, completely prevented NA-induced increases in plasma ACTH or corticosterone, suggesting that these responses are mainly mediated by saturated FFAs. In addition, quantification of individual FFA species in the blood using FFA-specific fluorescent probes revealed that plasma corticosterone and ACTH correlated significantly with plasma palmitate but not with other FFAs, such as oleate, linoleate, or arachidonate. Taken together, our data suggest that the regulation of the hypothalamic-pituitary-adrenal axis by FFAs is mainly mediated by the saturated fatty acid palmitate, but not by unsaturated fatty acids, such as oleate and linoleate.

Mechanisms of Fuel Surfeit Detoxification in Pancreatic β-cells

Elevated glucose and free fatty acids (FFA) cause pancreatic β-cell dysfunction, depletion of insulin stores and apoptosis (glucolipotoxicity). If much knowledge has been gained on the biochemical basis of glucolipotoxicity, little is known as to how the β-cell detoxifies fuel excess. We tested the hypothesis that β-cells averts metabolic stress caused by fuel surfeit through glucose metabolism via the glycerolipid/FFA cycle and the subsequent release of lipolysis-derived glycerol and long-chain FFA. Isolated rat islets exposed for 1 h to elevated glucose concentrations showed enhanced glycerolipid/FFA cycling and converted significant amount of glucose carbon to FFA and exported a large fraction of them out of the cell. The stearic and palmitic acids were the predominant FFA species released into the medium. Glycerol release, an index of lipolysis, and glucose utilization did not show saturation even at 16 mmol/L glucose, unlike glucose oxidation, ATP production and insulin secretion. Incorporation of glucose carbons to "inert" triglycerides and cholesterol esters were also elevated with increasing glucose concentration. The results suggest that in islets external glucose concentration is increased, the pathways involved in signalling for secretion and energy production reach saturation earlier than detoxification processes and that at high glucose islets convert a significant portion of the sugar into glycerol and FFA, which are exported outside the β-cell. The identification of fuel surfeit detoxification mechanisms by a targeted metabolomic approach should reveal additional pathways of β-cell fuel detoxification. Targeting fuel detoxification processes may provide a novel approach to preserve β-cell mass and function.

Effect of a sustained reduction in plasma free fatty acid concentration on insulin signalling and inflammation in skeletal muscle from human subjects

Free fatty acids (FFAs) have been implicated in the pathogenesis of insulin resistance. Reducing plasma FFA concentration in obese and type 2 diabetic (T2DM) subjects improves insulin sensitivity. However, the molecular mechanism by which FFA reduction improves insulin sensitivity in human subjects is not fully understood. In the present study, we tested the hypothesis that pharmacological FFA reduction enhances insulin action by reducing local (muscle) inflammation, leading to improved insulin signalling. Insulin-stimulated total glucose disposal (TGD), plasma FFA species, muscle insulin signalling, IBα protein, c-Jun phosphorylation, inflammatory gene (toll-like receptor 4 and monocyte chemotactic protein 1) expression, and ceramide and diacylglycerol (DAG) content were measured in muscle from a group of obese and T2DM subjects before and after administration of the antilipolytic drug acipimox for 7 days, and the results were compared to lean individuals. We found that obese and T2DM subjects had elevated saturated and unsaturated FFAs in plasma, and acipimox reduced all FFA species. Acipimox-induced reductions in plasma FFAs improved TGD and insulin signalling in obese and T2DM subjects. Acipimox increased IBα protein (an indication of decreased IB kinase-nuclear factor B signalling) in both obese and T2DM subjects, but did not affect c-Jun phosphorylation in any group. Acipimox also decreased inflammatory gene expression, although this reduction only occurred in T2DM subjects. Ceramide and DAG content did not change. To summarize, pharmacological FFA reduction improves insulin signalling in muscle from insulin-resistant subjects. This beneficial effect on insulin action could be related to a decrease in local inflammation. Notably, the improvements in insulin action were more pronounced in T2DM, indicating that these subjects are more susceptible to the toxic effect of FFAs.

229 RAPID, UNTARGETED LIPID DETERMINATION IN INDIVIDUAL BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS BY HIGH-RESOLUTION DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY

Lipid structural analysis in individual pre-implantation mammalian embryos is hampered by the small amount of biological material, such that most studies use staining methods or gas chromatography analysis generate information only on the fatty acyl residues. Recent developments in high-resolution desorption electrospray ionization mass spectrometry (DESI-MS) allow the analysis of free fatty acids (FA) and glycerophospholipids (PL) in individual bovine embryos. Here, we report on the use of DESI-MS for the sensitive analysis of triacylglycerol (TAG) species, profiles of FA and PL in individual bovine oocytes and embryos. Bovine oocytes (n = 40) and blastocysts (n = 42) were frozen in a minimal volume of PBS (2 to 5 µL). Samples were directly deposited on glass slides after thawing. After drying, a volume of 500 µL of methanol : water (1 : 1, vol/vol) was carefully deposited on the surface of the glass slide and removed by orienting the glass slide vertically to eliminate PBS salts. An Orbitrap mass spectrometer was used for the experiments. Parameters for the positive ion mode were as follows: acetonitrile (ACN) supplemented with 3 µL mL–1 of AgNO3 at a 5 µL min–1 flow rate, injection time of 1000 ms, and a mass-to-charge range of m/z 400 to 1500. For the negative ion mode, the solvent combination used was acetonitrile + dimethylformamide (1 : 1, vol/vol) at a 1.0 µL min–1 flow rate, a maximum injection time of 1000 ms, and a mass-to-charge range of m/z 150 to 1000. Positive ion mode data for the detection of TAG species were acquired first, followed by acquisition of FA and PL in the negative ion mode. Detection of TAG by DESI, which is extremely useful for bovine embryo cryopreservation and metabolism research, has been performed by adding AgNO3 in the DESI spray to obtain silver adducts, which are easily recognised by the characteristic 1 : 1 abundance ratio of the 107 : 109 Ag isotopes. The most abundant fatty acyl residues present in TAG species were palmitic (P), linoleic (L), oleic (O), and stearic (S) acids, such as TAG of m/z 937, PPL (50 : 2); m/z 965, POO (52 : 1); m/z 967, POS (52 : 2); m/z 989, OOL/LLS (54 : 4); and m/z 991, OOO, SOL (54 : 3). Free FA and PL profiles collected from the same samples in the negative ion mode were similar to those in our recent report (2012 J. Mass Spectrom. 47, 29–33). Lipid attribution has been performed based on high-resolution mass analysis. Multivariate statistics from this data set will allow visualisation of differences observed in the lipid profiles among samples. In conclusion, we report the use of DESI-MS for the sensitive analysis of TAG in individual bovine oocytes and embryos and the creation of profiles of FA, PL, and TAG species in the same sample by DESI-MS.