Altered fatty acid metabolism and composition in cultured astrocytes under hyperammonemic conditions

In vitro ¹H- and ¹³C-NMR spectroscopy was used to investigate the effect of ammonia on fatty acid synthesis and composition in cultured astrocytes. Cells were incubated 3 and 24 h with 5 mM ammonia in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. An increase of de novo synthesized fatty acids and the glycerol subunit of lipids was observed after 3 h treatment with ammonia (35% and 40% over control, respectively), the initial time point examined. Both parameters further increased significantly to 85% and 60% over control after 24 h ammonia treatment. Three hours incubation with ammonia increased the synthesis of diacylglycerides, while formation of triacylglycerides was decreased (40% over and 15% under control, respectively). The degradation of fatty acids was not affected by ammonia treatment. Furthermore, ammonia caused alterations in the composition of fatty acids, e.g. increased mono- and decreased polyunsaturated fatty acids (85% over and 15% under control concentrations, respectively). The decrease of polyunsaturated fatty acids was even more pronounced in isolated astrocytic mitochondria (39% lower than controls). Our results suggest ammonia-induced abnormalities in astrocytic membranes, which may be related to astrocytic mitochondrial dysfunction in hyperammonemic states. Most of the observed effects of ammonia on fatty acid synthesis and composition were ameliorated when glutamine synthetase was inhibited by methionine sulfoximine, supporting a pathological role of glutamine in ammonia toxicity. This study further emphasizes the importance of investigating the relative contribution of exogenous ammonia, effects of glutamine and of glutamine-derived ammonia on astrocytes and astrocytic mitochondria.     

The impact of overexpression and deficiency of fatty acid translocase (FAT)/CD36

Fatty acid translocase (FAT)/CD36 has been associated with diverse normal and pathologic processes. These include scavenger receptor functions (uptake of apoptotic cells and modified lipid), lipid metabolism and fatty acid transport, adhesion, angiogenesis, modulation of inflammation, transforming growth factor- activation, atherosclerosis, diabetes and cardiomyopathy. Although CD36 was identified more than 25 years ago, it is only with the advent of recent genetic technology that in vivo evidence has emerged for its physiologic and pathologic relevance. As these in vivo studies are expanded, we will gain further insight into the mechanism(s) by which CD36 transmits a cellular signal, and this will allow the design of specific therapeutics that impact on a particular function of CD36.     

CD36 deficiency in mice impairs lipoprotein lipase-mediated triglyceride clearance

CD36 is involved in high-affinity peripheral FFA uptake. CD36-deficient (cd36(-)(/)(-)) mice exhibit increased plasma FFA and triglyceride (TG) levels. The aim of the present study was to elucidate the cause of the increased plasma TG levels in cd36(-)(/)(-) mice. cd36(-)(/)(-) mice showed no differences in hepatic VLDL-TG production or intestinal [(3)H]TG uptake compared with wild-type littermates. cd36(-)(/)(-) mice showed a 2-fold enhanced postprandial TG response upon an intragastric fat load (P < 0.05), with a concomitant 2.5-fold increased FFA response (P < 0.05), suggesting that the increased FFA in cd36(-/-) mice may impair LPL-mediated TG hydrolysis. Postheparin LPL levels were not affected. However, the in vitro LPL-mediated TG hydrolysis rate as induced by postheparin plasma of cd36(-)(/)(-) mice in the absence of excess FFA-free BSA was reduced 2-fold compared with wild-type plasma (P < 0.05). This inhibition was relieved upon the addition of excess FFA-free BSA. Likewise, increasing plasma FFA in wild-type mice to the levels observed in cd36(-)(/)(-) mice by infusion prolonged the plasma half-life of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles by 2.5-fold (P < 0.05). We conclude that the increased plasma TG levels observed in cd36(-)(/)(-) mice are caused by decreased LPL-mediated hydrolysis of TG-rich lipoproteins resulting from FFA-induced product inhibition of LPL.     

Cardiac triglyceride accumulation following acute lipid excess occurs through activation of a FoxO1-iNOS-CD36 pathway

Obesity due to nutrient excess leads to chronic pathologies including type 2 diabetes and cardiovascular disease. Related to nutrient excess, FoxO1 has a role in regulating fatty acid uptake and oxidation and triglyceride (TG) storage by mechanisms that are largely unresolved. We examined the mechanism behind palmitate (PA)-induced TG accumulation in cardiomyocytes. To mimic lipid excess, rat ventricular myocytes were incubated with albumin-bound PA (1 mM) or rats were administered Intralipid (20%). PA-treated cardiomyocytes showed a substantial increase in TG accumulation, accompanied by amplification of nuclear migration of phospho-p38 and FoxO1, iNOS induction, and translocation of CD36 to the plasma membrane. PA also increased Cdc42 protein and its tyrosine nitration, thereby rearranging the cytoskeleton and facilitating CD36 translocation. These effects were duplicated by TNF-α and reversed by the iNOS inhibitor 1400 W. PA increased the nuclear interaction between FoxO1 and NF-κB, reduced the nuclear presence of PGC-1α, and downregulated expression of oxidative phosphorylation proteins. In vivo a robust increase in cardiac TGs after Intralipid administration was also associated with augmentation of nuclear FoxO1 and iNOS expression. Impeding this FoxO1-iNOS-CD36 pathway could decrease cardiac lipid accumulation and oxidative/nitrosative stress and help ameliorate the cardiovascular complications associated with obesity and diabetes.     

The biochemistry of headgroup exchange during triacylglycerol synthesis in canola

Many pathways of primary metabolism are substantially conserved within and across plant families. However, significant differences in organization and fluxes through a reaction network may occur, even between plants in closely related genera. Assessing and understanding these differences is key to appreciating metabolic diversity, and to attempts to engineer plant metabolism for higher crop yields and desired product profiles. To better understand lipid metabolism and seed oil synthesis in canola (Brassica napus), we have characterized four canola homologues of the Arabidopsis (Arabidopsis thaliana) ROD1 gene. AtROD1 encodes phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), the enzyme that catalyzes a major flux of polyunsaturated fatty acids (PUFAs) in oil synthesis. Assays in yeast indicated that only two of the canola genes, BnROD1.A3 and BnROD1.C3, encode active isozymes of PDCT, and these genes are strongly expressed during the period of seed oil synthesis. Loss of expression of BnROD1.A3 and BnROD1.C3 in a double mutant, or by RNA interference, reduced the PUFA content of the oil to 26.6% compared with 32.5% in the wild type. These results indicate that ROD1 isozymes in canola are responsible for less than 20% of the PUFAs that accumulate in the seed oil compared with 40% in Arabidopsis. Our results demonstrate the care needed when translating results from a model species to crop plants.     

Obesity alters the gustatory perception of lipids in the mouse: plausible involvement of lingual CD36

A relationship between orosensory detection of dietary lipids, regulation of fat intake, and body mass index was recently suggested. However, involved mechanisms are poorly understood. Moreover, whether obesity can directly modulate preference for fatty foods remains unknown. To address this question, exploration of the oral lipid sensing system was undertaken in diet-induced obese (DIO) mice. By using a combination of biochemical, physiological, and behavioral approaches, we found that i) the attraction for lipids is decreased in obese mice, ii) this behavioral change has an orosensory origin, iii) it is reversed in calorie-restricted DIO mice, revealing an inverse correlation between fat preference and adipose tissue size, iv) obesity suppresses the lipid-mediated downregulation of the lipid-sensor CD36 in circumvallate papillae, usually found during the refeeding of lean mice, and v) the CD36-dependent signaling cascade controlling the intracellular calcium levels ([Ca²⁺]_i) in taste bud cells is decreased in obese mice. Therefore, obesity alters the lipid-sensing system responsible for the oral perception of dietary lipids. This phenomenon seems to take place through a CD36-mediated mechanism, leading to changes in eating behavior.     

In vivo 19F NMR chemical-shift imaging of Ancistrocladus species

Summary ¹⁹F nuclear magnetic resonance (NMR) imaging and¹⁹F NMR chemical-shift imaging (¹⁹F CSI) have been used to localize fluorinated compounds administered to stems ofAncistrocladus heyneanus andA. abbreviatus for the elucidation of biosynthetic pathways in living plants. This first application of¹⁹F CSI on plants proved CSI to be a valuable technique for mapping fluorinated molecules in plants. Exemplarily using trifluoroacetate as a model compound allowed to select appropriate feeding methods and to optimize both concentration and duration of the application to the plant. The time course of the uptake and distribution of trifluoroacetate was monitored by both¹⁹F imaging and¹⁹F CSI. Fluorinated metabolites formed by uptake of 3-fluoro-3-deoxy-D-glucose were detected with¹⁹F CSI.Abbreviations 3-FDG 3-fluoro-3-deoxy-D-glucose - CSI chemicalshift imaging - NMR nuclear magnetic resonance - SNR signal-to-noise ratio - TFA trifluoroacetate Dedicated to Professor Manfred Christi on the occasion of his 60th birthday     

Chylomicron remnants are increased in the postprandial state in CD36 deficiency

The clustering of risk factors including dyslipidemia, hyperglycemia, and hypertension is highly atherogenic along with the excess of remnants from triglyceride (TG)-rich lipoproteins. CD36 is involved in the uptake of long-chain fatty acids (LCFAs) in muscles and small intestines. Patients with CD36 deficiency (CD36-D) have postprandial hypertriglyceridemia, insulin resistance, and hypertension. To investigate the underlying mechanism of postprandial hypertriglyceridemia in CD36-D, we analyzed lipoprotein profiles of CD36-D patients and CD36-knockout (CD36-KO) mice after oral fat loading (OFL). In CD36-D patients, plasma triglycerides, apolipoprotein B-48 (apoB-48), free fatty acids (FFAs), and free glycerol levels were much higher after OFL than those of controls, along with increases in chylomicron (CM) remnants and small dense low-density lipoprotein (sdLDL) particles. In CD36-KO mice, lipoproteins smaller than CM in size in plasma and intestinal lymph were markedly increased after OFL and mRNA levels of genes involved in FFA biosynthesis, such as fatty acid binding protein (FABP)-1 and FAS, were significantly increased. These results suggest that CD36-D might increase atherosclerotic risk by enhancing plasma level of CM remnants due to the increased synthesis of lipoproteins smaller than CM in size in the intestine.     

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-¹³C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field ¹³C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C . Brocadia fulgida synthesizes C_16:0 and iso C_16:0 fatty acids according to the known pathway of type II fatty acid biosynthesis. The ¹³C-labelling pattern of the C₈ alkyl chain of the C₂₀ [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.     

Characterization of cerebral glucose dynamics in vivo with a four-state conformational model of transport at the blood-brain barrier

Determination of brain glucose transport kinetics in vivo at steady-state typically does not allow distinguishing apparent maximum transport rate (T(max)) from cerebral consumption rate. Using a four-state conformational model of glucose transport, we show that simultaneous dynamic measurement of brain and plasma glucose concentrations provide enough information for independent and reliable determination of the two rates. In addition, although dynamic glucose homeostasis can be described with a reversible Michaelis-Menten model, which is implicit to the large iso-inhibition constant (K(ii)) relative to physiological brain glucose content, we found that the apparent affinity constant (K(t)) was better determined with the four-state conformational model of glucose transport than with any of the other models tested. Furthermore, we confirmed the utility of the present method to determine glucose transport and consumption by analysing the modulation of both glucose transport and consumption by anaesthesia conditions that modify cerebral activity. In particular, deep thiopental anaesthesia caused a significant reduction of both T(max) and cerebral metabolic rate for glucose consumption. In conclusion, dynamic measurement of brain glucose in vivo in function of plasma glucose allows robust determination of both glucose uptake and consumption kinetics.     

Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo

In this study we tested the hypothesis that the 65-kDa isoform of glutamate decarboxylase (GAD(65)) mediates activity-dependent GABA synthesis as invoked by seizures in anesthetized rats. GABA synthesis was measured following acute GABA-transaminase inhibition by gabaculine using spatially localized (1)H NMR spectroscopy before and after bicuculline-induced seizures. Experiments were conducted with animals pre-treated with vigabatrin 24 h earlier in order to reduce GAD(67) protein and also with non-treated controls. GAD isoform content was quantified by immunoblotting. GABA was higher in vigabatrin-treated rats compared to non-treated controls. In vigabatrin-treated animals, GABA synthesis was 28% lower compared to controls [p < 0.05; vigabatrin-treated, 0.043 +/- 0.011 micromol/(g min); non-treated, 0.060 +/- 0.014 micromol/(g min)] and GAD(67) was 60% lower. No difference between groups was observed for GAD(65). Seizures increased GABA synthesis in both control [174%; control, 0.060 +/- 0.014 micromol/(g min) vs. seizures, 0.105 +/- 0.043 micromol/(g min)] and vigabatrin-treated rats [214%; control, 0.043 +/- 0.011 micromol/(g min); seizures, 0.092 +/- 0.018 micromol/(g min)]. GAD(67) could account for at least half of basal GABA synthesis but only 20% of the two-fold increase observed in vigabatrin-treated rats during seizures. The seizure-induced activation of GAD(65) in control cortex occurs concomitantly with a 2.3-fold increase in inorganic phosphate, known to be a potent activator of apoGAD(65)in vitro. Our results are consistent with a major role for GAD(65) in activity-dependent GABA synthesis.     

Influence of rearing temperature on triacylglycerol storage in the pea aphid,Acyrthosiphon pisum

Total fatty acids in the pea aphid reared at low temperatures increased significantly compared to that at high rearing temperatures. This change is reflected in a large increase of myristic acid, which occurs exclusively in triacylglycerols. When aphids were moved from 25°C to a lower rearing temperature at 10°C, saturated fatty acids accumulated over time, reaching a maximum at 16th day. When aphids were moved to 4°C, a temperature below the developmental threshold, those aphids did not accumulate saturated fatty acids. Similar results were observed when aphids were exposed to sequential decrease in rearing temperature. However, both total fatty acids and myristic acid in the aphids from the treatments of sequential decreasing rearing temperature were significantly higher compared to those in the aphids from the treatments of sudden decreasing rearing temperature. This result, therefore, supports the hypothesis that cold‐adapted aphids can survive under threshold temperature for a longer period of time than noncold‐adapted aphids. Acetyl‐CoA carboxylase activity in the aphids at 25°C was twofold higher than that in the aphids at 10°C, whereas fatty acid synthase activities in the aphids reared at 25 and 10°C are similar. Aphids reared at 10°C showed a threefold reduction in reproduction rates. This reduced production of new nymphs reduces energy demand and would allow for accumulation of energy in the form of triacylglycerols. Therefore, the increased level of saturated fatty acids in aphids reared at low temperature is probably related to lower utilization of fatty acids rather than increased rates of biosynthesis.     

Carbon isotope dynamics during leaf litter decomposition with reference to lignin fractions

We studied the dynamics of the stable C isotope ratio (δ¹³C) of Swida controversa and Fagus crenata leaf litter during decomposition for 35 months with reference to the relative enrichment of the residue by lignin fraction-derived C. The study was carried out on upper and lower parts of a forest slope in a cool temperate forest in Japan. The enrichment of lignin fraction-C was associated with a decrease in the δ¹³C of S. controversa residue. The decrease in δ¹³C was greater at the lower than the upper site for S. controversa. In contrast, the relative increase in lignin fractions in F. crenata residue was not associated with the changes of δ¹³C. ¹³C nuclear magnetic resonance analysis, which revealed the relative decrease in alkyl-C and O-alkyl-C and the relative increase in aromatic-C and carbonyl-C, provided further evidence for the contribution of the selective preservation of lignin of plant origin to the increase in the lignin fraction.     

Protein dynamics by solid-state nuclear magnetic resonance spectroscopy: Peptide backbone of the coat protein in fd bacteriophage

¹³C and ¹⁵N chemical shift anisotropy and ¹⁵N¹H dipolar powder patterns from backbone sites of the coat protein in fd bacteriophage are not averaged by motion. This means that the polypeptide backbone of the protein has no large amplitude motions rapid compared to 10⁴ Hz. Relaxation studies on the ¹³C_α and ¹⁵N amide resonances indicate the presence of motions on the 10⁹ Hz timescale. These results are reconciled with a model where an otherwise rigid backbone undergoes small amplitude, rapid motions.     

Effects of palmitate and oleate on the respiratory quotient during acute feeding

Objective: Using tracers, we showed, over 9 hours, that palmitic acid (PA) is oxidized at a lower rate than oleic acid (OA). Our subsequent clinical trial showed that enriching the diet for 28 days with PA, relative to OA, lowered fatty acid (FA) oxidation. However, because this conclusion was based on indirect calorimetry for 7 hours after a test meal, transient differences in the kinetics of oxidation of OA and PA could explain these results. Thus, we hypothesized that increasing PA vs. OA would decrease FA oxidation during the first day of feeding the diets. Research Methods and Procedures: A double‐masked trial was conducted in 20 adults, who, after a baseline diet, were randomized to one of two experimental formula diets: high (HI) OA (PA = 1.7% kcal, OA = 31.4% kcal; N = 11) or HI PA (PA = 16.8% kcal, OA = 16.4% kcal; N = 9). Respiratory quotient (RQ) was measured over the first 14 hours of feeding the experimental diets (7:00 am to 9:00 pm ). To determine whether these subjects were representative of the subjects in the previous trial, we assessed RQ 28 days after beginning either diet. Results: During the first 14 hours of feeding the diets, time (p = 0.026) but not diet group had an effect on the difference between the RQ post‐feeding and the fasting pre‐value. However, RQ in the fed state was significantly higher in the HI PA group after 28 days of feeding. Discussion: Chronically increasing dietary PA for 28 days, but not acute meal feeding, lowers total FA oxidation.     

N-3 but not N-6 fatty acids reduce the expression of the combined adhesion and scavenger receptor CD36 in human monocytic cells

CD36, a multifunctional adhesion receptor e.g. for thrombospondin and collagen, as well as a scavenger receptor for oxidized low density lipoprotein, is expressed e.g. on platelets and monocytes. By this dual role it might be involved in early steps of atherosclerosis like the recruitment of monocytes and formation of foam cells. We therefore studied the effects of n-3 fatty acids on CD36 expression in human monocytic cells. Incorporation of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) into cellular phospholipids resulted in a significant reduction of CD36 expression at the mRNA and protein level, whereas arachidonic acid (AA, C20: 4n-6) and linoleic acid (LA, C18:2n-6) tended to increase CD36 expression compared to the control. This specific down-regulation of CD36 by n-3 fatty acids in cells involved in the initiation and progression of atherogenesis and inflammation, represents a further mechanism that may contribute to the beneficial effects of n-3 polyunsaturated fatty acids (PUFA) in these disorders.     

Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36

Transmembrane protein CD36 binds multiple ligands, including oxidized low-density lipoproteins (oxLDLs) and long-chain fatty acids (LCFAs). Our aim was to determine whether LCFAs compete with oxLDLs for binding to CD36. We addressed this issue by examining the inhibitory effect of LCFAs against the binding of Alexa-fluor-labeled oxLDLs (AFL-oxLDL) to a synthetic peptide representing the oxLDL-binding site on CD36 (3S-CD36_150–168). All of the unsaturated LCFAs tested, inhibited the binding of AFL-oxLDL to 3S-CD36_150–168, albeit to varying degrees. For instance, the concentrations required for 50% inhibition of binding for oleic, linoleic, and α-linolenic acids were 0.25, 0.97, and 1.2?mM, respectively. None of the saturated LCFAs tested (e.g. stearic acid) exhibited inhibitory effects. These results suggest that at least unsaturated LCFAs can compete with oxLDLs for binding to CD36. The study also provides information on the structural requirements of LCFAs for inhibition of oxLDLs–CD36 binding.