The PPAR pan-agonist tetradecylthioacetic acid promotes redistribution of plasma cholesterol towards large HDL.

Tetradecylthioacetic acid (TTA) is a synthetic fatty acid with a sulfur substitution in the ß-position. This modification renders TTA unable to undergo complete ß-oxidation and increases its biological activity, including activation of peroxisome proliferator activated receptors (PPARs) with preference for PPARα. This study investigated the effects of TTA on lipid and lipoprotein metabolism in the intestine and liver of mice fed a high fat diet (HFD). Mice receiving HFD supplemented with 0.75% (w/w) TTA had significantly lower body weights compared to mice fed the diet without TTA. Plasma triacylglycerol (TAG) was reduced 3-fold with TTA treatment, concurrent with increase in liver TAG. Total cholesterol was unchanged in plasma and liver. However, TTA promoted a shift in the plasma lipoprotein fractions with an increase in larger HDL particles. Histological analysis of the small intestine revealed a reduced size of lipid droplets in enterocytes of TTA treated mice, accompanied by increased mRNA expression of fatty acid transporter genes. Expression of the cholesterol efflux pump Abca1 was induced in the small intestine, but not in the liver. Scd1 displayed markedly increased mRNA and protein expression in the intestine of the TTA group. It is concluded that TTA treatment of HFD fed mice leads to increased expression of genes involved in uptake and transport of fatty acids and HDL cholesterol in the small intestine with concomitant changes in the plasma profile of smaller lipoproteins.

Deactivating Fatty Acids: Acyl-CoA Thioesterase-Mediated Control of Lipid Metabolism.

The cellular uptake of free fatty acids (FFA) is followed by esterification to coenzyme A (CoA), generating fatty acyl-CoAs that are substrates for oxidation or incorporation into complex lipids. Acyl-CoA thioesterases (ACOTs) constitute a family of enzymes that hydrolyze fatty acyl-CoAs to form FFA and CoA. Although biochemically and biophysically well characterized, the metabolic functions of these enzymes remain incompletely understood. Existing evidence suggests regulatory roles in controlling rates of peroxisomal and mitochondrial fatty acyl-CoA oxidation, as well as in the subcellular trafficking of fatty acids. Emerging data implicate ACOTs in the pathogenesis of metabolic diseases, suggesting that better understanding their pathobiology could reveal unique targets in the management of obesity, diabetes, and nonalcoholic fatty liver disease.

The SCP2-thiolase-like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism.

Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cß2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Proteins 2016; 84:1075-1096. © 2016 Wiley Periodicals, Inc.

Fish oil and krill oil differentially modify the liver and brain lipidome when fed to mice.

BACKGROUND: Marine food is an important source of omega-3 fatty acids with beneficial health effects. Oils from marine organisms have different fatty acid composition and differ in their molecular composition. Fish oil (FO) has a high content of eicosapentaenoic and docosahexaenoic acids mainly esterified to triacylglycerols, while in krill oil (KO) these fatty acids are mainly esterified to phospholipids. The aim was to study the effects of these oils on the lipid content and fatty acid distribution in the various lipid classes in liver and brain of mice. METHODS: Mice were fed either a high-fat diet (HF), a HF diet supplemented with FO or with KO (n = 6). After six weeks of feeding, liver and brain lipid extracts were analysed using a shotgun and TAG lipidomics approach. Student t-test was performed after log-transformation to compare differences between study groups. RESULTS: Six weeks of feeding resulted in significant changes in the relative abundance of many lipid classes compared to control mice. In both FO and KO fed mice, the triacylglycerol content in the liver was more than doubled. The fatty acid distribution was affected by the oils in both liver and brain with a decrease in the abundance of 18:2 and 20:4, and an increase in 20:5 and 22:6 in both study groups. 18:2 decreased in all lipid classes in the FO group but with only minor changes in the KO group. Differences between the feeding groups were particularly evident in some of the minor lipid classes that are associated with inflammation and insulin resistance. Ceramides and diacylglycerols were decreased and cholesteryl esters increased in the liver of the KO group, while plasmalogens were decreased in the FO group. In the brain, diacylglycerols were decreased, more by KO than FO, while ceramides and lactosylceramides were increased, more by FO than KO. CONCLUSION: The changes in the hepatic sphingolipids and 20:4 fatty acid levels were greater in the KO compared to the FO fed mice, and are consistent with a hypothesis that krill oil will have a stronger anti-inflammatory action and enhances insulin sensitivity more potently than fish oil.

Three differently generated salmon protein hydrolysates reveal opposite effects on hepatic lipid metabolism in mice fed a high-fat diet.

This study investigates the effects of salmon peptide fractions, generated using different enzymatic hydrolyzation methods, on hepatic lipid metabolism. Four groups of mice were fed a high-fat diet with 20% casein (control group) or 15% casein and 5% of peptide fractions (treatment groups E1, E2 and E4) for 6weeks. Weight gain was reduced in mice fed E1 and E4-diets compared to control, despite a similar feed intake. Reduced plasma and liver triacylglycerol levels in E1 and E4-mice were linked to reduced fatty acid synthase (FAS) activity and hepatic expression of lipogenic genes. By contrast, plasma and liver lipids increased in the E2 group, concomitant with increased hepatic FAS activity and Δ9 desaturase gene expression. Shotgun lipidomics showed that MUFAs were significantly reduced in the E1 and E4 groups, whereas PUFAs were increased, and the opposite was observed in the E2 group. In conclusion, bioactive peptides with distinctive properties could potentially be isolated from salmon hydrolysates.

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities.

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nß2-Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

Regulation of peroxisomal lipid metabolism: the role of acyl-CoA and coenzyme A metabolizing enzymes.

Peroxisomes are nearly ubiquitous organelles involved in a number of metabolic pathways that vary between organisms and tissues. A common metabolic function in mammals is the partial degradation of various (di)carboxylic acids via α- and ß-oxidation. While only a small number of enzymes catalyze the reactions of ß-oxidation, numerous auxiliary enzymes have been identified to be involved in uptake of fatty acids and cofactors required for ß-oxidation, regulation of ß-oxidation and transport of metabolites across the membrane. These proteins include membrane transporters/channels, acyl-CoA thioesterases, acyl-CoA:amino acid N-acyltransferases, carnitine acyltransferases and nudix hydrolases. Here we review the current view of the role of these auxiliary enzymes in peroxisomal lipid metabolism and propose that they function in concert to provide a means to regulate fatty acid metabolism and transport of products across the peroxisomal membrane.

Acyl-CoA thioesterase 9 (ACOT9) in mouse may provide a novel link between fatty acid and amino acid metabolism in mitochondria.

Acyl-CoA thioesterase (ACOT) activities are found in prokaryotes and in several compartments of eukaryotes where they hydrolyze a wide range of acyl-CoA substrates and thereby regulate intracellular acyl-CoA/CoA/fatty acid levels. ACOT9 is a mitochondrial ACOT with homologous genes found from bacteria to humans and in this study we have carried out an in-depth kinetic characterization of ACOT9 to determine its possible physiological function. ACOT9 showed unusual kinetic properties with activity peaks for short-, medium-, and saturated long-chain acyl-CoAs with highest V max with propionyl-CoA and (iso) butyryl-CoA while K cat/K m was highest with saturated long-chain acyl-CoAs. Further characterization of the short-chain acyl-CoA activity revealed that ACOT9 also hydrolyzes a number of short-chain acyl-CoAs and short-chain methyl-branched CoA esters that suggest a role for ACOT9 in regulation also of amino acid metabolism. In spite of markedly different K ms, ACOT9 can hydrolyze both short- and long-chain acyl-CoAs simultaneously, indicating that ACOT9 may provide a novel regulatory link between fatty acid and amino acid metabolism in mitochondria. Based on similar acyl-CoA chain-length specificities of recombinant ACOT9 and ACOT activity in mouse brown adipose tissue and kidney mitochondria, we conclude that ACOT9 is the major mitochondrial ACOT hydrolyzing saturated C2-C20-CoA in these tissues. Finally, ACOT9 activity is strongly regulated by NADH and CoA, suggesting that mitochondrial metabolic state regulates the function of ACOT9.

The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism.

The importance of peroxisomes in lipid metabolism is now well established and peroxisomes contain approximately 60 enzymes involved in these lipid metabolic pathways. Several acyl-CoA thioesterase enzymes (ACOTs) have been identified in peroxisomes that catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. A number of acyltransferase enzymes, which are structurally and functionally related to ACOTs, have also been identified in peroxisomes, which conjugate (or amidate) bile acid-CoAs and acyl-CoAs to amino acids, resulting in the production of amidated bile acids and fatty acids. The function of ACOTs is to act as auxiliary enzymes in the α- and ß-oxidation of various lipids in peroxisomes. Human peroxisomes contain at least two ACOTs (ACOT4 and ACOT8) whereas mouse peroxisomes contain six ACOTs (ACOT3, 4, 5, 6, 8 and 12). Similarly, human peroxisomes contain one bile acid-CoA:amino acid N-acyltransferase (BAAT), whereas mouse peroxisomes contain three acyltransferases (BAAT and acyl-CoA:amino acid N-acyltransferases 1 and 2: ACNAT1 and ACNAT2). This review will focus on the human and mouse peroxisomal ACOT and acyltransferase enzymes identified to date and discuss their cellular localizations, emerging structural information and functions as auxiliary enzymes in peroxisomal metabolic pathways.

Localization of peroxisomal matrix proteins by photobleaching.

The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.

The nudix hydrolase 7 is an Acyl-CoA diphosphatase involved in regulating peroxisomal coenzyme A homeostasis.

Coenzyme A (CoASH) is an obligate cofactor for lipids undergoing beta-oxidation in peroxisomes. Although the peroxisomal membrane appears to be impermeable to CoASH, peroxisomes contain their own pool of CoASH. It is believed that CoASH enters peroxisomes as acyl-CoAs, but it is not known how this pool is regulated. The mouse nudix hydrolase 7 (NUDT7alpha) was previously identified in peroxisomes as a CoA-diphosphatase, and therefore suggested to be involved in regulation of peroxisomal CoASH levels. Here we show that mouse NUDT7alpha mainly acts as an acyl-CoA diphosphatase, with highest activity towards medium-chain acyl-CoAs, and much lower activity with CoASH. Nudt7alpha mRNA is highly expressed in liver, brown adipose tissue and heart, similar to enzymes involved in peroxisomal lipid degradation. Nudt7alpha mRNA is down-regulated by Wy-14,643, a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand, in a PPARalpha-dependent manner in mouse liver. In highly purified peroxisomes, nudix hydrolase activity is highest with C(6)-CoA and is decreased by fibrate treatment. Under certain conditions, such as treatment with peroxisome proliferators or fasting, an increase in peroxisomal CoASH levels has been reported, which is in line with a decreased expression/activity of NUDT7alpha. Taken together these data suggest that NUDT7alpha function is tightly linked to peroxisomal CoASH/acyl-CoA homeostasis.

Novel functions of acyl-CoA thioesterases and acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism.

Peroxisomes are single membrane bound organelles present in almost all eukaryotic cells, and to date have been shown to contain approximately 60 identified enzymes involved in various metabolic pathways, including the oxidation of a variety of lipids. These lipids include very long-chain fatty acids, methyl branched fatty acids, prostaglandins, bile-acid precursors and xenobiotics that are either beta-oxidized or alpha-oxidized in peroxisomes. The recent identification of several acyl-CoA thioesterases and acyltransferases in peroxisomes has revealed their various functions in acting as auxiliary enzymes in alpha- and beta-oxidation in this organelle. To date, 9 functional acyl-CoA thioesterases and acyltransferases have been identified in mouse and 4 functional acyl-CoA thioesterases and acyltransferases in human, thus these enzymes make up a substantial portion of peroxisomal proteins. This review will therefore focus on new and emerging roles for these enzymes in assisting with the oxidation of various lipids, amidation of lipids for excretion from peroxisomes, and in controlling coenzyme A levels in peroxisomes.

PPARalpha is a key regulator of hepatic FGF21.

The metabolic regulator fibroblast growth factor 21 (FGF21) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF21 is regulated by the peroxisome proliferator-activated receptor alpha (PPARalpha). Fasting or treatment of mice with the PPARalpha agonist Wy-14,643 induced FGF21 mRNA by 10-fold and 8-fold, respectively. In contrast, FGF21 mRNA was low in PPARalpha deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF21. Obese ob/ob mice, known to have increased PPARalpha levels, displayed 12-fold increased hepatic FGF21 mRNA levels. The potential importance of PPARalpha for FGF21 expression also in human liver was shown by Wy-14,643 induction of FGF21 mRNA in human primary hepatocytes, and PPARalpha response elements were identified in both the human and mouse FGF21 promoters. Further studies on the mechanisms of regulation of FGF21 by PPARalpha in humans will be of great interest.

Peroxisomes contain a specific phytanoyl-CoA/pristanoyl-CoA thioesterase acting as a novel auxiliary enzyme in alpha- and beta-oxidation of methyl-branched fatty acids in mouse.

Phytanic acid and pristanic acid are derived from phytol, which enter the body via the diet. Phytanic acid contains a methyl group in position three and, therefore, cannot undergo beta-oxidation directly but instead must first undergo alpha-oxidation to pristanic acid, which then enters beta-oxidation. Both these pathways occur in peroxisomes, and in this study we have identified a novel peroxisomal acyl-CoA thioesterase named ACOT6, which we show is specifically involved in phytanic acid and pristanic acid metabolism. Sequence analysis of ACOT6 revealed a putative peroxisomal targeting signal at the C-terminal end, and cellular localization experiments verified it as a peroxisomal enzyme. Subcellular fractionation experiments showed that peroxisomes contain by far the highest phytanoyl-CoA/pristanoyl-CoA thioesterase activity in the cell, which could be almost completely immunoprecipitated using an ACOT6 antibody. Acot6 mRNA was mainly expressed in white adipose tissue and was co-expressed in tissues with Acox3 (the pristanoyl-CoA oxidase). Furthermore, Acot6 was identified as a target gene of the peroxisome proliferator-activated receptor alpha (PPARalpha) and is up-regulated in mouse liver in a PPARalpha-dependent manner.

Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart.

Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of protein expression of MTE-I and UCP3 or about MTE-I activity; thus, we investigated the effects of diabetes and treatment with a PPAR alpha agonist on these parameters. Rats were either made diabetic with streptozotocin (55 mg/kg ip) and maintained for 10-14 days or treated with the PPAR alpha agonist fenofibrate (300 mg/kg/day) for 4 weeks. MTE-I and UCP3 protein expression, MTE-1 activity, palmitate export, and oxidative phosphorylation were measured in isolated cardiac mitochondria. Diabetes and fenofibrate increased cardiac MTE-I mRNA, protein, and activity ( approximately 4-fold compared with controls). This increase in activity was matched by a 6-fold increase in palmitate export in fenofibrate-treated animals, despite there being no effect in either group on UCP3 protein expression. Both diabetes and fenofibrate caused significant decreases in state III respiration of isolated mitochondria with pyruvate + malate as the substrate, but only diabetes reduced state III rates with palmitoylcarnitine. Both diabetes and specific PPAR alpha activation increased MTE-I protein, activity, and palmitate export in the heart, with little effect on UCP3 protein expression.

A peroxisomal acyltransferase in mouse identifies a novel pathway for taurine conjugation of fatty acids.

A wide variety of endogenous carboxylic acids and xenobiotics are conjugated with amino acids, before excretion in urine or bile. The conjugation of carboxylic acids and bile acids with taurine and glycine has been widely characterized, and de novo synthesized bile acids are conjugated to either glycine or taurine in peroxisomes. Peroxisomes are also involved in the oxidation of several other lipid molecules, such as very long chain acyl-CoAs, branched chain acyl-CoAs, and prostaglandins. In this study, we have now identified a novel peroxisomal enzyme called acyl-coenzyme A:amino acid N-acyltransferase (ACNAT1). Recombinantly expressed ACNAT1 acts as an acyltransferase that efficiently conjugates very long-chain and long-chain fatty acids to taurine. The enzyme shows no conjugating activity with glycine, showing that it is a specific taurine conjugator. Acnat1 is mainly expressed in liver and kidney, and the gene is localized in a gene cluster, together with two further acyltransferases, one of which conjugates bile acids to glycine and taurine. In conclusion, these data describe ACNAT1 as a new acyltransferase, involved in taurine conjugation of fatty acids in peroxisomes, identifying a novel pathway for production of N-acyltaurines as signaling molecules or for excretion of fatty acids.

Analysis of the mouse and human acyl-CoA thioesterase (ACOT) gene clusters shows that convergent, functional evolution results in a reduced number of human peroxisomal ACOTs.

The maintenance of cellular levels of free fatty acids and acyl-CoAs, the activated form of free fatty acids, is extremely important, as imbalances in lipid metabolism have serious consequences for human health. Acyl-coenzyme A (CoA) thioesterases (ACOTs) hydrolyze acyl-CoAs to the free fatty acid and CoASH, and thereby have the potential to regulate intracellular levels of these compounds. We previously identified and characterized a mouse ACOT gene cluster comprised of six genes that apparently arose by gene duplications encoding acyl-CoA thioesterases with localizations in cytosol (ACOT1), mitochondria (ACOT2), and peroxisomes (ACOT3-6). However, the corresponding human gene cluster contains only three genes (ACOT1, ACOT2, and ACOT4) coding for full-length thioesterase proteins, of which only one is peroxisomal (ACOT4). We therefore set out to characterize the human genes, and we show here that the human ACOT4 protein catalyzes the activities of three mouse peroxisomal ACOTs (ACOT3, 4, and 5), being active on succinyl-CoA and medium to long chain acyl-CoAs, while ACOT1 and ACOT2 carry out similar functions to the corresponding mouse genes. These data strongly suggest that the human ACOT4 gene has acquired the functions of three mouse genes by a functional convergent evolution that also provides an explanation for the unexpectedly low number of human genes.

The identification of a succinyl-CoA thioesterase suggests a novel pathway for succinate production in peroxisomes.

Dicarboxylic acids are formed by omega-oxidation of fatty acids in the endoplasmic reticulum and degraded as the CoA ester via beta-oxidation in peroxisomes. Both synthesis and degradation of dicarboxylic acids occur mainly in kidney and liver, and the chain-shortened dicarboxylic acids are excreted in the urine as the free acids, implying that acyl-CoA thioesterases (ACOTs), which hydrolyze CoA esters to the free acid and CoASH, are needed for the release of the free acids. Recent studies show that peroxisomes contain several acyl-CoA thioesterases with different functions. We have now expressed a peroxisomal acyl-CoA thioesterase with a previously unknown function, ACOT4, which we show is active on dicarboxylyl-CoA esters. We also expressed ACOT8, another peroxisomal acyl-CoA thioesterase that was previously shown to hydrolyze a large variety of CoA esters. Acot4 and Acot8 are both strongly expressed in kidney and liver and are also target genes for the peroxisome proliferator-activated receptor alpha. Enzyme activity measurements with expressed ACOT4 and ACOT8 show that both enzymes hydrolyze CoA esters of dicarboxylic acids with high activity but with strikingly different specificities. Whereas ACOT4 mainly hydrolyzes succinyl-CoA, ACOT8 preferentially hydrolyzes longer dicarboxylyl-CoA esters (glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA). The identification of a highly specific succinyl-CoA thioesterase in peroxisomes strongly suggests that peroxisomal beta-oxidation of dicarboxylic acids leads to formation of succinate, at least under certain conditions, and that ACOT4 and ACOT8 are responsible for the termination of beta-oxidation of dicarboxylic acids of medium-chain length with the concomitant release of the corresponding free acids.

A revised nomenclature for mammalian acyl-CoA thioesterases/hydrolases.

Acyl-CoA thioesterases, also known as acyl-CoA hydrolases, are a group of enzymes that hydrolyze CoA esters such as acyl-CoAs (saturated, unsaturated, branched-chain), bile acid-CoAs, CoA esters of prostaglandins, etc., to the corresponding free acid and CoA. However, there is significant confusion regarding the nomenclature of these genes. In agreement with the HUGO Gene Nomenclature Committee and the Mouse Genomic Nomenclature Committee, a revised nomenclature for mammalian acyl-CoA thioesterases/hydrolases has been suggested for the 12 member family. The family root symbol is ACOT, with human genes named ACOT1-ACOT12, and rat and mouse genes named Acot1-Acot12. Several of the ACOT genes are the result of splicing events, and these splice variants are cataloged.