Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.     

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARalpha-dependent and -independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.     

A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus

Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the Δ12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.     

Metabolism of apical versus basolateral sn-2-monoacylglycerol and fatty acids in rodent small intestine

The metabolic fates of radiolabeled sn-2-monoacylglycerol (MG) and oleate (FA) in rat and mouse intestine, added in vivo to the apical (AP) surface in bile salt micelles, or to the basolateral (BL) surface via albumin-bound solution, were examined. Mucosal lipid products were quantified, and the results demonstrate a dramatic difference in the esterification patterns for both MG and FA, depending upon their site of entry into the enterocyte. For both lipids, the ratio of triacylglycerol to phospholipid (TG:PL) formed was approximately 10-fold higher for delivery at the AP relative to the BL surface. Further, a 3-fold higher level of FA oxidation was found for BL compared with AP substrate delivery. Incorporation of FA into individual PL species was also significantly different, with >2-fold greater incorporation into phosphatidylethanolamine (PE) and a 3-fold decrease in the phosphatidylcholine:PE ratio for AP- compared with BL-added lipid. Overnight fasting increased the TG:PL incorporation ratio for both AP and BL lipid addition, suggesting that metabolic compartmentation is a physiologically regulated phenomenon. These results support the existence of separate pools of TG and glycerolipid intermediates in the intestinal epithelial cell, and underscore the importance of substrate trafficking in the regulation of enterocyte lipid metabolism.     

Alterations in fatty acid oxidation in ischemic and reperfused myocardium

Summary The focus of this review centered on describing the effects of excess fatty acids on myocardial recovery during reperfusion following ischemic stress. Effects on mechanical function were modest in our studies and are likely to remain difficult/impossible to measure due to the independent phenomenon of stunning which obfuscates and no doubt dominates the influences of other mechanical determinants. Mitochondria appear capable of again using long-chain fatty acids as a preferred substrate and in the presence of restored oxygen delivery can produce normal levels of CO₂. These changes in oxidative metabolism are not mirrored by equal recoveries in mitochondrial energetics. Because of inefficiencies in electron transport and oxidative phosphorylation together with moderate uncoupling of electron transport from oxidative phosphorylation, ATP resynthesis is blunted. This explains in part the absolute decrease in contents of exchangeable nucleotides noted both in cytosol and mitochondria. Further impairments in recovery reside in the inability of the mitochondria to exchange adenine nucleotides into cytosol through the adenine nucleotide translocase antiport. These findings contribute to our understanding of mechanical stunning and may be of value in designing future strategies to optimize the handling of substrates during myocardial reperfusion.Visiting scientist from the Shang Hai Second Medical University, Peoples Republic of China.     

An HPLC-CAD/fluorescence lipidomics platform using fluorescent fatty acids as metabolic tracers

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. Here we describe a novel combined HPLC and LC-MS/MS method developed to characterize both total lipid profiles and the products of fluorescently labeled lipids. Using this approach, we found that lipids labeled with the fluorescent tags, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene [BODIPY(558/568)], and dipyrrometheneboron difluoride undecanoic acid (TopFluor) are all metabolized into varying arrays of polar and nonpolar fluorescent lipid products when they are fed to larval zebrafish. Quantitative metabolic labeling experiments performed in this system revealed significant effects of total dietary lipid composition on fluorescent lipid partitioning. We provide evidence that cholesterol metabolism in the intestine is important in determining the metabolic fates of dietary FAs. Using this method, we found that inhibitors of dietary cholesterol absorption and esterification both decreased incorporation of dietary fluorescent FAs into cholesterol esters (CEs), suggesting that CE synthesis in enterocytes is primarily responsive to the availability of dietary cholesterol. These results are the first to comprehensively characterize fluorescent FA metabolism and to demonstrate their utility as metabolic labeling reagents, effectively coupling quantitative biochemistry with live imaging studies.     

A low degree of fatty acid unsaturation leads to high resistance to lipid peroxidation in mitochondria and microsomes of different organs of quail (Coturnix coturnix japonica)

Birds – particularly long-lived species – have special adaptations for preventing tissue damage caused by reactive oxygen species. The objective of the present study was to analyse the fatty acid composition and non-enzymatic lipid peroxidation of mitochondria and microsomes obtained from liver, heart and brain of quail (Coturnix coturnix japonica), a short-lived bird. Fatty acids located in total lipids of rat liver, heart and brain mitochondria and microsomes were determined using gas chromatography and lipid peroxidation was evaluated using a chemiluminescence assay. The unsaturated fatty acid content found in mitochondria and microsomes of all tissue examined was approximately 50 and 40%, respectively with a prevalence of C18:1 n9. The C18:2 n6 content in brain mitochondria was significantly lower as compared to liver and heart mitochondria. Whereas the C20:4 n6 content in mitochondria from all tissues examined and brain microsomes was approximately 6%, liver and heart microsomes exhibited lower values. C22:6 n3 was absent in liver mitochondria, very low content in liver microsomes and heart organelles (between 0.5 and 1%) and high content in brain organelles, with mitochondria having the highest value (11%). Whereas liver and heart organelles were not affected when subjected to lipid peroxidation, brain mitochondria were highly affected, as indicated by the increase in chemiluminescence and a considerable decrease of C20:4 n6 and C22:6 n3. These results indicate that a low degree of fatty acid unsaturation in liver and heart organelles of quail, a short-lived bird, may confer advantage by decreasing their sensitivity to lipid peroxidation process.     

Origin of fatty acid synthesis: Thermodynamics and kinetics of reaction pathways

Summary The primitiveness of contemporary fatty acid biosynthesis was evaluated by using the thermodynamics and kinetics of its component reactions to estimate the extent of its dependence on powerful and selective catalysis by enzymes. Since this analysis indicated that the modern pathway is not primitive because it requires sophisticated enzymatic catalysis, we here propose an alternative pathway of primitive fatty acid synthesis that uses glycolaldehyde as a substrate. In contrast to the modern pathway, this primitive pathway is not dependent on an exogenous source of phosphoanhydride energy (ATP). Furthermore, the chemical spontaneity of its reactions suggests that it could have been readily catalyzed by the rudimentary biocatalysts available at an early stage in the origin of life.     

Regulation of carbohydrate and fatty acid utilization by L-carnitine during cardiac development and hypoxia

This study is designed to investigate whether substrate preference in the myocardium during the neonatal period and hypoxia-induced stress is controlled intracellularly or by extracellular substrate availability. To determine this, the effect of exogenous L-carnitine on the regulation of carbohydrate and fatty acid metabolism was determined during cardiac stress (hypoxia) and during the postnatal period. The effect of L-carnitine on long chain (palmitate) and medium chain (octonoate) fatty acid oxidation was studied in cardiac myocytes isolated from less than 24 h old (new born; NB), 2 week old (2 week) and hypoxic 4 week old (HY) piglets. Palmitate oxidation was severely decreased in NB cells compared to those from 2 week animals (0.456 ± 0.04 vs. 1.207 ± 0.52 nmol/mg protein/30 min); surprisingly, cells from even older hypoxic animals appeared shifted toward the new born state (0.695 ± 0.038 nmol/mg protein/30 min). Addition of L-carnitine to the incubation medium, which stimulates carnitine palmitoyl-transferase I (CPTI) accelerated palmitate oxidation 3 fold in NB and approximately 2 fold in HY and 2 week cells. In contrast, octanoate oxidation which was greater in new born myocytes than in 2 week cells, was decreased by L-carnitine suggesting a compensatory response. Furthermore, oxidation of carbohydrates (glucose, pyruvate, and lactate) was greatly increased in new born myocytes compared to 2 week and HY cells and was accompanied by a parallel increase in pyruvate dehydrogenase (PDH) activity. The concentration of malonyl-CoA, a potent inhibitor of CPTI was significantly higher in new born heart than at 2 weeks. These metabolic data taken together suggest that intracellular metabolic signals interact to shift from carbohydrate to fatty acid utilization during development of the myocardium. The decreased oxidation of palmitate in NB hearts probably reflects decreased intracellular L-carnitine and increased malonyl-CoA concentrations. Interestingly, these data further suggest that the cells remain compliant so that under stressful conditions, such as hypoxia, they can revert toward the neonatal state of increased glucose utilization.     

A comparison of heart and liver fatty acid-binding proteins: interactions with fatty acids and possible functional differences studied with fluorescent fatty acid analogues

Summary Fatty acid-binding proteins (FABP) are distinct but related gene products which are found in many mammalian cell types. They are generally present in high abundance, and are found in those tissues where free fatty acid (ffa) flux is high. The function(s) of FABP is unknown. Also not known is whether all FABP function similarly in their respective cell types, or whether different FABP have unique functions. The purpose of these studies was to assess whether different members of the FABP family exhibit different structural and functional properties. Two fluorescent analogues of ffa were used to compare the liver (L-FABP) and heart (H-FABP) binding proteins. The propionic acid derivative of diphenylhexatriene (PADPH) was used to examine the physical properties of the ffa binding site on L- and H-FABP, as well as the relative distribution of ffa between FABP and membranes. An anthroyloxy-derivative of palmitic acid, 2AP, was used to monitor the transfer kinetics of ffa from liver or heart FABP to acceptor membranes, using a resonance energy transfer assay. The results demonstrate that the ffa binding sites of both FABP are hydrophobic in nature, although the L-FABP site is more nonpolar than the H-FABP site. Equilibration of PADPH between L-FABP and phosphatidylcholine (PC) bilayers resulted in a molar partition preference of > 20: 1, L-FABP : PC. Similar studies with H-FABP resulted in a PADPH partition preference of only 3:1, H-FABP : PC. Finally, the transfer of 2AP from H-FABP to acceptor membranes was found to be 50-fold faster than transfer from L-FABP. These studies demonstrate that important structural and functional differences exist between different members of the FABP family, and therefore imply that the roles of different FABP may be unique.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Liver FABP - H-FABP Heart FABP - SUV Small Unilamellar Vesicle - PADPH 3-[p-(6-Phenyl)-1,3,5-Hexatrienyl]-phenylpropionic acid - 2AP 2-(9-Anthroyloxy)Palmitic acid - Q Quantum yield - _F Fluorescence lifetime     

Metabolism of ricinoleic acid into gamma-decalactone: beta-oxidation and long chain acyl intermediates of ricinoleic acid in the genus Sporidiobolus sp

In order to study differences in gamma-decalactone production in yeast, four species of Sporidiobolus were cultivated with 5% of methyl ricinoleate as the lactone substrate. In vivo studies showed different time courses of intermediates of ricinoleic acid breakdown between the four species. In vitro studies of the beta-oxidation system were conducted with crude cell extracts of Sporidiobolus spp. and with ricinoleyl-CoA (RCoA) as substrate. The beta-oxidation was detected by measuring acyl-CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase activities, and acetyl-CoA production. The time courses of the CoA esters resulting from RCoA breakdown by crude extract of Sporidiobolus spp. permit the proposal of different metabolic models in the yeast. These models explained the differences observed during in vivo studies.     

Preliminary studies about novel strategies to reverse chemoresistance to adriamycin regarding glutathione metabolism,peroxisomal and extraperoxisomal hydroperoxide and valproic acid metabolic pathways

Summary— The present work was aimed at defining novel strategies to reverse chemoresistance to anticancer drugs, especially by interfering with cellular glutathione metabolism, peroxisomal and/or extraperoxisomal hydroperoxide metabolic pathways. Preliminary results are presented about molecules we demonstrated to be capable of interfering with hydrogen peroxide metabolism in cells. Prior to describing these molecules, a short overview of glutathione and free radical metabolic pathways is presented as well as a rapid presentation of the characteristics of chemo-sensitivity and -resistance towards the anticancer drug adriamycin, with special emphasis on hydrogen peroxide metabolism. The strategies currently developed to reverse chemoresistance are further presented. in subsequent sections, our own strategy to achieve inhibition of hydrogen peroxide breakdown and stimulation of peroxisomal hydrogen peroxide production is illustrated on the basis of molecular modelling studies and biochemical investigations on extraperoxisomal and peroxisomal metabolic pathways. Preliminary studies on cultured cells have been initiated. The perspective for future studies is presented as well as other possible models of chemoresistance as target for the design of hydrogen peroxide metabolism-interfering pharmacomolecules.     

乳酸菌胆盐水解酶和共轭脂肪酸产生及对宿主脂代谢影响的研究进展

乳酸菌是一类影响宿主脂代谢的人体肠道益生菌。乳酸菌对脂代谢的影响作用与其产生胆盐水解酶(bile salt hydrolase,EC3.5.1.24,BSH)及共轭转化多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)关系密切。菌株差异、菌群分布和饮食差异是影响BSH及共轭脂肪酸产生的重要因素。本文重点阐述了两类物质对宿主脂代谢的影响机制,以期为后续研究提供借鉴。BSH能够降解肝脏分泌的胆汁酸(bile acids,BAs),降低脂类物质的吸收。BAs的降解产物胆汁酸脱氧胆酸(deoxycholic acid,DCA)和石胆酸(lithocholic acid,LCA)能够通过机体信号通路法尼类X受体(farnesoid X receptor,FXR)、小异二聚体伴侣(small heterodimer partner,SHP)及肝脏X受体(liver X receptor,LXR)等信号通路进行调控,促进胆固醇转运及向BAs转化。此外,BSH还能够通过下调固醇调节元件结合蛋白1c (sterol regulatory element binding protein 1c,SREBP-1c)、上调5ʹ-腺苷单磷酸激活蛋白激酶α(5ʹ-AMP activated protein kinase,AMPKα)和过氧化物酶体增殖物激活受体α (peroxisome proliferator-activated receptor α,PPARα)抑制脂质合成,促进脂质的分解。PUFAs可被乳酸菌转化产生共轭脂肪酸,如共轭亚油酸(conjugated linoleic acid,CLA)和共轭亚麻酸(conjugated linolenic acid,CLNA),CLA/CLNA能够促进机体产生瘦素(leptin,LP),抑制食欲、促进能量消耗;CLA/CLNA还可以通过激活PPARα进行调控,促进人体脂质的氧化分解。乳酸菌通过以上多种途径共同作用调节宿主的脂代谢,对深入理解乳酸菌调控脂代谢机制及临床应用有着重要意义。     

A novel role for fatty acid transport protein 1 in the regulation of tricarboxylic acid cycle and mitochondrial function in 3T3-L1 adipocytes

Fatty acid transport proteins (FATPs) are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. Whereas some FATP1 translocates to the plasma membrane in response to insulin, the majority of FATP1 remains within intracellular structures and bioinformatic and immunofluorescence analysis of FATP1 suggests the protein primarily resides in the mitochondrion. To evaluate potential roles for FATP1 in mitochondrial metabolism, we used a proteomic approach following immunoprecipitation of endogenous FATP1 from 3T3-L1 adipocytes and identified mitochondrial 2-oxoglutarate dehydrogenase. To assess the functional consequence of the interaction, purified FATP1 was reconstituted into phospholipid-containing vesicles and its effect on 2-oxoglutarate dehydrogenase activity evaluated. FATP1 enhanced the activity of 2-oxoglutarate dehydrogenase independently of its acyl-CoA synthetase activity whereas silencing of FATP1 in 3T3-L1 adipocytes resulted in decreased activity of 2-oxoglutarate dehydrogenase. FATP1 silenced 3T3-L1 adipocytes exhibited decreased tricarboxylic acid cycle activity, increased cellular NAD⁺/NADH, increased fatty acid oxidation, and increased lactate production indicative of altered mitochondrial energy metabolism. These results reveal a novel role for FATP1 as a regulator of tricarboxylic acid cycle activity and mitochondrial function.     

A novel role for central ACBP/DBI as a regulator of long‐chain fatty acid metabolism in astrocytes

Acyl‐CoA‐binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl‐CoA esters. Several studies have suggested that ACBP acts as an acyl‐CoA pool former and regulates long‐chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam‐Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABA_A receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism‐related gene expression using ACBP‐deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA‐CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes.