Soluble CD163 is not increased in visceral fat and steatotic liver and is even suppressed by free fatty acids in vitro

Visceral fat differs from subcutaneous fat by higher local inflammation and increased release of IL-6 and free fatty acids (FFA) which contribute to hepatic steatosis. IL-6 has been shown to upregulate the monocyte/macrophage specific receptor CD163 whose soluble form, sCD163, is increased in inflammatory diseases. Here, it was analyzed whether CD163 and sCD163 are differentially expressed in the human fat depots and fatty liver. CD163 mRNA and protein were similarly expressed in paired samples of human visceral and subcutaneous fat, and comparable levels in portal venous and systemic venous blood of liver-healthy controls indicate that release of sCD163 from visceral adipose tissue was not increased. CD163 was also similarly expressed in steatotic liver when compared to non-steatotic tissues and sCD163 was almost equal in the respective sera. Concentrations of sCD163 were not affected when passing the liver excluding substantial hepatic removal/release of this protein. A high concentration of IL-6 upregulated CD163 protein while physiological doses had no effect. However, sCD163 was not increased by any of the IL-6 doses tested. FFA even modestly decreased CD163 and sCD163. The anti-inflammatory mediators fenofibrate, pioglitazone, and eicosapentaenoic acid (EPA) did not influence sCD163 levels while CD163 was reduced by EPA. These data suggest that in humans neither visceral fat nor fatty liver are major sources of sCD163.

Low- vs. high-glycemic load diets: effects on postprandial plasma free fatty acids

Thesis (Master's)--University of Washington, 2016-06

Weight-related differences in glucose metabolism and free fatty acid production in two South African population groups

OBJECTIVE The effects of free fatty acids (FFA), leptin, tumour necrosis factor (TNF) alpha and body fat distribution on in vivo oxidation of a glucose load were studied in two South African ethnic groups. DESIGN AND MEASUREMENTS Anthropometric and various metabolic indices were measured at fasting and during a 7h oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis and subcutaneous and visceral fat mass was assessed using a five- and two-level CT-scan respectively. Glucose oxidation was evaluated by measuring the ratio of (13)CO(2) to (12)CO(2) in breath following ingestion of 1-(13)C-labelled glucose. SUBJECTS Ten lean black women (LBW), ten obese black women (OBW), nine lean white women (LWW) and nine obese white women (OWW) were investigated after an overnight fast. RESULTS Visceral fat levels were significantly higher (P < 0.01) in obese white than black women, despite similar body mass indexes (BMIs). There were no ethnic differences in glucose oxidation however; in the lean subjects of both ethnic groups the area under the curve (AUC) was higher than in obese subjects (P < 0.05 for both) and was found to correlate negatively with weight (r = -0.69, P < 0.01) after correcting for age. Basal TNF alpha concentrations were similar in all groups. Percentage suppression of FFAs at 30 min of the OCTT was 24 +/- 12% in OWW and - 38 +/- 23% (P < 0.05) in OBW, ie the 30 min FFA level was higher than the fasting level in the latter group. AUC for FFAs during the late postprandial period (120 - 420 min) was significantly higher in OWW than OBW (P < 0.01) and LWW (P < 0.01) and correlated positively with visceral fat mass independent of age (r = 0.78, P < 0.05) in the OWW only. Leptin levels were higher (P < 0.01) both at fasting and during the course of the OCTT in obese women from both ethnic groups compared to the lean women. CONCLUSIONS Glucose oxidation is reduced in obese subjects of both ethnic groups; inter- and intra-ethnic differences were observed in visceral fat mass and FFA production and it is possible that such differences may play a role in the differing prevalences of obesity-related disorders that have been reported in these two populations.

Influence of cold storage time on the protein changes and fatty acids deterioration of (Rutilus frisi kutum) during frozen storage

The main aim of this research was to identify fatty acids composition of Caspian sea of White fish Rutilus frisi kutum tissue and their changes during one year cold storage (-18Ċ).The secondary aim was to determine the changes of moisture, ash, protein, fat, and to investigate the effects of storage time on peroxide, TBAi, FFA, and extractability of myofibrillar proteins of the fish tissue during one year cold storage (-18 Ċ). 10 samples of (Rutilus frisi kutum) were randomly collected from Anzali landings. The samples were frozen at -30 Ċ and kept in cold storage at -18Ċ for one year. According to time table, the samples were examined. The results showed that 27 fatty acids were identified. The unsaturated fatty acids (UFA) and saturated fatty acids (SFA) were 74/09 and 21/63 %, respectively, in fresh tissue. So that DHA (C22:6) oleic acid (C18:1c) had high amounts (15/07 ,20/57 ) among the UFA and palmitic acid (C16:0) was the most (13/09 %) among the SFA. The effects of freezing and cold storage on fish tissue showed that UFA and SFA contents have reached to 58/79 and 22/17 %, respectively, at the end of cold storage. It indicated that these compound change to each other during frozen storage. Also ω-3 and ω-6 series of fatty acids was 24/22 and 15/56% in fresh tissue, but their contents decreased to 8/68 and 5/11% at the end of period. Among the fatty acids C22:6, C18:1c and C16:0 had the most changes. The changes of fatty acids were significantly at 95% level expected for C18:0. Results showed that moisture, ash, protein, and fat contents were 75/9±0/03, 1/28±0/012, 21/8±0/2, and 4/1±0/01 % respectively, in fresh tissue. The moisture, ash, protein, and fat contents were 72/3±0/04, 1/83±0/05, 1/91±0/01 and 19/9±0/01 % respectively, at the end of storage period. Lipid damage was measured on the basis of free fatty acids (FFA), peroxide value (PV), and Thiobarbituric acid index (TBA-i). PV, TBARS and FFA concentration of frozen Caspian Sea white fish stored at -18 Ċ the temporal variation of these three variables were statistically significant (p<0.001). Results of White fish myofibrillar proteins showed aggregation of bound reduced for stored at 12 months. SDS-PAGE analysis revealed that, the intensity of the myosin heavy chain and actin bound was reduced with increasing storage time. SDS-PAGE patterns showed that myosin heavy chain was much more susceptible to hydrolysis than actin. Key words: Rutilus frisi kutum, frozen storage, ω-3, ω-6, protein myofibrillar

Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency, such that acetate (C2) has been described as FFA2 selective while propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations marked variation in ligand-independent, constitutive activity was identified. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity while the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the 2nd extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity, and in most cases also yielded corresponding changes in SCFA potency.

The acute effects of differential dietary fatty acids on PDHa activity in human skeletal muscle at the onset of exercise

Pyruvate dehydrogenase (PDH) is an important regulator of carbohydrate oxidation during exercise and its activity can be down-regulated by an increase in dietary fat. The purpose of this study was to determine the acute metabolic effects of differential dietary fatty acids on the activation of PDH in its active form (PDHa) at rest and at the onset of moderate-intensity exercise. University-aged male subjects (n=7) underwent 2 fat loading trials spaced at least 2 weeks apart. Subjects consumed saturated (SFA) or polyunsaturated (PUFA) fat over the course of 5 hours. Following this, participants cycled at 65% VO2 max for 15 min. Muscle biopsies were taken prior to and following fat loading and at 1 min exercise. Plasma free fatty acids increased from 0.15 ± 0.07 to 0.54 ± 0.19 mM over 5 hours with SFA and from 0.1 1 ± 0.04 to 0.35 ±0.13 mM with PUFA. PDHa activity was unchanged following fat loading, but increased at the onset of exercise in the SFA trial, from 1 .4 ± 0.4 to 2.2 ± 0.4 /xmol/min/kg wet wt. This effect was negated in the PUFA trial (1 .2 ± 0.3 to 1 .3 ± 0.3 pimol/min/kg wet wt.). PDH kinase (PDK) was unchanged in both trials, suggesting that the attenuation of PDHa activity with PUFA was a result of changes in the concentrations of intramitochondrial effectors, more specifically intramitochondrial NADH or Ca^*. Our findings suggest that attenuated PDHa activity participates in the preferential oxidation of PUFA during moderateintensity exercise.

Frying of potato chips in a blend of canola oil and palm olein: changes in levels of individual fatty acids and tocols

The changes occurring in the levels of nutritionally relevant oil components were assessed during repeated frying of potato chips in a blend of palm olein and canola oil (1:1 w/w). The blend suffered minimal reductions in omega-3 and omega-6 polyunsaturated fatty acids. There was no significant difference between the fatty acid composition of the oil extracted from the product and that of the frying medium, in all three cases. The blend also contained a significant amount of tocols which add a nutritional value to the oil. The concentration of the tocols was satisfactorily retained over the period of oil usage, in contrast to the significant loses observed in the case of the individual oils. The blend also performed well when assessed by changes in total polar compounds, free fatty acids, p-anisidine value. When fried in used oil, the product oil content increased progressively with oil usage time. This study shows that blended frying oils can combine good stability and nutritional quality

Intracellular pH in adipocytes: effects of free fatty acid diffusion across the plasma membrane, lipolytic agonists, and insulin.

The main function of white adipose tissue is to store nutrient energy in the form of triglycerides. The mechanism by which free fatty acids (FFA) move into and out of the adipocyte has not been resolved. We show here that changes in intracellular pH (pH1) in adipocytes correlate with the movement of FFA across cellular membranes as predicted by the Kamp and Hamilton model of passive diffusion of FFA. Exposure of fat cells to lipolytic agents or external FFA results is a rapid intracellular acidification that is reversed by metabolism of the FFA or its removal by albumin. In contrast, insulin causes an alkalinization of the cell, consistent with its main function to promote esterification. Inhibition of Na+/H+ exchange in adipocytes does not prevent the changes in pHi caused by FFA, lipolytic agents, or insulin. A fatty acid dimer, which diffuses into the cell but is not metabolized, causes an irreversible acidification. Taken together, the data suggest that changes in pHi occur in adipocytes in response to the passive diffusion of un-ionized FFA (flip-flop) into and out of the cell and in response to their metabolism and production within the cell. These changes in pHi may, in turn, modulate hormonal signaling and metabolism with significant impact on cell function.

Fatty acids, monocytes and ageing

Elevated free fatty acids (FFA) are a feature of ageing and a risk factor for metabolic disorders such as cardiovascular disease (CVD) and type-2 diabetes (T2D). Elevated FFA contribute to insulin resistance, production of inflammatory cytokines and expression of adhesion molecules on immune cells and endothelial cells, risk factors for CVD and T2D. Molecular mechanisms of FFA effects on monocyte function and how FFA phenotype is affected by healthy ageing remain poorly understood. This thesis evaluated the effects of the two major FFA in plasma, oleate and palmitate on monocyte viability, cell surface antigen expression, and inflammatory activation in THP-1 monocytes. Palmitate but not oleate increased cell surface expression of CD11b and CD36 after 24h, independent of mitochondrial superoxide, but dependent on de novo synthesis of ceramides. LPS-mediated cytokine production in THP-1 monocytes was enhanced and decreased following incubation with palmitate and oleate respectively. In a model of monocyte-macrophage differentiation, palmitate induced a pro-inflammatory macrophage phenotype which required de novo ceramide synthesis, whilst oleate reduced cytokine secretion, producing a macrophage with enhanced clearance apoptotic cells. Plasma fatty acid analysis in young and mid-life populations revealed age-related increases in both the SFA and MUFA classes, especially the medium and very long chain C14 and C24 fatty acids, which were accompanied by increases in the estimated activities of desaturase enzymes. Changes were independently correlated with increased PBMC CD11b, plasma TNF-a and insulin resistance. In conclusion, the pro-atherogenic phenotype, enhanced LPS responses in monocytes, and pro-inflammatory macrophage in the presence of palmitate but not oleate is reliant upon de novo ceramide synthesis. Age-related increases in inflammation, cell surface integrin expression are related to increases in both the MUFA and SFA fatty acids, which in part may be explained by altered de novo fatty acid synthesis.

Ageing, adipose tissue, fatty acids and inflammation

A common feature of ageing is the alteration in tissue distribution and composition, with a shift in fat away from lower body and subcutaneous depots to visceral and ectopic sites. Redistribution of adipose tissue towards an ectopic site can have dramatic effects on metabolic function. In skeletal muscle, increased ectopic adiposity is linked to insulin resistance through lipid mediators such as ceramide or DAG, inhibiting the insulin receptor signalling pathway. Additionally, the risk of developing cardiovascular disease is increased with elevated visceral adipose distribution. In ageing, adipose tissue becomes dysfunctional, with the pathway of differentiation of preadipocytes to mature adipocytes becoming impaired; this results in dysfunctional adipocytes less able to store fat and subsequent fat redistribution to ectopic sites. Low grade systemic inflammation is commonly observed in ageing, and may drive the adipose tissue dysfunction, as proinflammatory cytokines are capable of inhibiting adipocyte differentiation. Beyond increased ectopic adiposity, the effect of impaired adipose tissue function is an elevation in systemic free fatty acids (FFA), a common feature of many metabolic disorders. Saturated fatty acids can be regarded as the most detrimental of FFA, being capable of inducing insulin resistance and inflammation through lipid mediators such as ceramide, which can increase risk of developing atherosclerosis. Elevated FFA, in particular saturated fatty acids, maybe a driving factor for both the increased insulin resistance, cardiovascular disease risk and inflammation in older adults.

Macrophage polarisation by fatty acids is PPARgamma-dependent

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. We investigated the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Palmitate increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300µM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300µM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis. We have also explored whether specific dietary fatty acids drive monocyte to macrophage polarisation via metabolic pathways. Here we show that monocytes pre-incubated with the saturated fatty acid palmitate increase production of inflammatory cytokines such as TNFa and IL-6 in response to a phorbol myristate differentiation trigger. This increases mitochondrial superoxide production, reduces dependency on oxidative phosphorylation through ceramide-dependent inhibition of PPARgamma activity and increases TNFa production, again via a mechanism that requires ceramide production.

Application of GPCR Structures for Modelling of Free Fatty Acid Receptors

Five G protein-coupled receptors (GPCRs) have been identified to be activated by free fatty acids (FFA). Among them, FFA1 (GPR40) and FFA4 (GPR120) bind long-chain fatty acids, FFA2 (GPR43) and FFA3 (GPR41) bind short-chain fatty acids and GPR84 binds medium-chain fatty acids. Free fatty acid receptors have now emerged as potential targets for the treatment of diabetes, obesity and immune diseases. The recent progress in crystallography of GPCRs has now enabled the elucidation of the structure of FFA1 and provided reliable templates for homology modelling of other FFA receptors. Analysis of the crystal structure and improved homology models, along with mutagenesis data and structure activity, highlighted an unusual arginine charge pairing interaction in FFA1-3 for receptor modulation, distinct structural features for ligand binding to FFA1 and FFA4 and an arginine of the second extracellular loop as a possible anchoring point for FFA at GPR84. Structural data will be helpful for searching novel small molecule modulators at the FFA receptors.

EFFECT OF INTERMITTENT FRYING ON FATTY ACIDS, VITAMIN E, LIPID OXIDATION AND ACRYLAMIDE IN OILS AND PLANTAIN CHIPS COLLECTED FROM SMALL- SCALE PRODUCERS IN CAMEROON

Deep-fat frying is susceptible to induce the formation of undesirable products as lipid oxidation products and acrylamide in fried foods. Plantain chips produced by small-scale producers are sold to consumers without any control. The objective of this study was to evaluate the quality of plantain chips from local producers in relation to production process parameters and oils, and to identify the limiting factors for the production of acrylamide in plantain chips. Samples of frying oils and plantain chips prepared with either palm olein or soybean oil were collected from 10 producers in Yaoundé. Quality parameters determined in this study were: fatty acid composition of the oils, determined by gas chromatography (GC) of free acid methyl ester; trans fatty acids, determined by Fourier transform infra-red spectroscopy; Tocopherols and tocotrienols as markers of nutritional quality were analyzed by High Performance Liquid Chromatography in isocratic mode. Free fatty acids and acylglycerols as markers of lipid hydrolysis were analyzed by GC of trimethylsilyl derivatives of glycerides. Conjugated dienes, Anisidine value and viscosity as markers of lipid oxidation and thermal decomposition of the oils; acrylamide which is formed through Maillard reaction and identified as a toxic compound in various fried products. Asparagine content of the raw fresh plantain powder was also determined. Fatty acid composition of palm oleins was stable within a day of intermittent frying. In soybean oils, about 57% and 62.5% of linoleic and linolenic acids were lost but trans fatty acids were not detected. Soybean oils were partly hydrolysed leading to the formation of free fatty acids, monoacylglycerols and diacylglycerols. In both oils, tocopherols and tocotrienols contents decreased significantly by about 50%. Anisidine value (AV) and polymers contents increased slightly in fried palm oleins while conjugated hydroperoxides, AV and polymers greatly increased in soybean oils. Acrylamide was not detected in the chips. This is explained by the absence of asparagine in the raw plantains, the other acrylamide precursors being present. This study shows that the plantain chips prepared at the small-scale level in Yaounde with palm olein are of good quality regarding oxidation and hydrolysis parameters and the absence of acrylamide. In contrast, oxidation developed with soybean oil whose usage for frying should be questioned. Considering that asparagine is the limiting factor for the formation of acrylamide in plantain chips, its content depending on several factors such as production parameters and maturity stage should be explored.

Interaction of glucose and long chain fatty acids (C18) on antioxidant defences and free radical damage in porcine vascular smooth muscle cells in vitro.

Aims/hypothesis: Abnormalities of glucose and fatty acid metabolism in diabetes are believed to contribute to the development of oxidative stress and the long term vascular complications of the disease therefore the interactions of glucose and long chain fatty acids on free radical damage and endogenous antioxidant defences were investigated in vascular smooth muscle cells. Methods: Porcine vascular smooth muscle cells were cultured in 5 mmol/l or 25 mmol/l glucose for ten days. Fatty acids, stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and gamma-linolenic acid (18:3) were added with defatted bovine serum albumin as a carrier for the final three days. Results. Glucose (25 mmol/l) alone caused oxidative stress in the cells as evidenced by free radical-mediated damage to DNA, lipids, and proteins. The addition of fatty acids (0.2 mmol/l) altered the profile of free radical damage; the response was J-shaped with respect to the degree of unsaturation of each acid, and oleic acid was associated with least damage. The more physiological concentration (0.01 mmol/l) of gamma-linolenic acids was markedly different in that, when added to 25 mmol/l glucose it resulted in a decrease in free radical damage to DNA, lipids and proteins. This was due to a marked increase in levels of the antioxidant, glutathione, and increased gene expression of the rate-limiting enzyme in glutathione synthesis, gamma-glutamylcysteine synthetase. Conclusion/Interpretation: The results clearly show that glucose and fatty acids interact in the production of oxidative stress in vascular smooth muscle cells.