Long-chain Fatty Acyl Research Articles

Interaction of Resveratrol with Lipid Membranes

Background: Resveratrol is a phytoalexin synthesized by plants. It has antioxidant properties and is a popular nutritional supplement. Beneficial properties of resveratrol, such as, anticancer and anti-inflammatory properties have been reported. Resveratrol is a constituent of red wine and found in the skin of red grapes. In order to understand the interaction between resveratrol and biological membranes, we evaluated the effect of resveratrol on model lipid membranes using differential scanning calorimetry (DSC) and computational studies. Methods: Phospholipids such as, Dilauroylphosphatidylcholine (DLPC), Dimyristoyphosphatidylcholine (DMPC), Dipalmitoylphosphatidyl choline (DPPC), Distearoylphosphatidylcholine (DSPC), and 1-palmitoyl-2-oleyl phosphatidylcholine (POPC) were purchased from Avanti Polar Lipids (Alabaster, Alabama). Each phospholipid was mixed with resveratrol at different molar ratios, phospholipid: resveratrol, 10:1, 10:3 and 10:5. Computational simulations were also performed to evaluate the interactions of DSPC and resveratrol. Results: Resveratrol abolishes only the transition of the DLPC, which is the shortest phospholipid of those tested. It reduces the transition temperature for all the other phospholipids even at the lowest ratio tested, phospholipid: resveratrol, 10:1. Resveratrol reduces the transition temperature of DSPC from 55 °C to 51 °C. Furthermore, using DSC, we also observed another transition, a sharp exothermic peak above 275 °C in the interaction of resveratrol with DSPC. We performed computational simulations of the DSPC membrane at different temperatures with and without resveratrol. The simulation indicates that resveratrol affects the transition temperature of the DSPC, which is in agreement with our DSC data. In conclusion, our data indicate that resveratrol abolishes the transition of DLPC and acts as a plasticizer for phospholipids with longer fatty acyl chains.

The complex role of SIRT6 in carcinogenesis.

SIRT6, a member of the mammalian sirtuins family, functions as a mono-ADP-ribosyl transferase and NAD(+)-dependent deacylase of both acetyl groups and long-chain fatty acyl groups. SIRT6 regulates diverse cellular functions such as transcription, genome stability, telomere integrity, DNA repair, inflammation and metabolic related diseases such as diabetes, obesity and cancer. In this review, we will discuss the implication of SIRT6 in the biology of cancer and the relevance to organism homeostasis and lifespan.

N-Acylethanolamines Bind to SIRT6.

Sirtuin 6 (SIRT6) is an NAD+-dependent histone deacetylase enzyme that is involved in multiple molecular pathways related to aging. Initially, it was reported that SIRT6 selectively deacetylated H3K9Ac and H3K56Ac; however, it has more recently been shown to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups in vitro. Subsequently, fatty acids were demonstrated to increase the catalytic activity of SIRT6. In this study, we investigated whether a series of N-acylethanolamines (NAEs), quercetin, and luteolin could regulate SIRT6 activity. NAEs increased SIRT6 activity, with oleoylethanolamide having the strongest activity (EC50 value of 3.1 μm). Quercetin and luteolin were demonstrated to have dual functionality with respect to SIRT6 activity; namely, they inhibited SIRT6 activity with IC50 values of 24 and 2 μm, respectively, and stimulated SIRT6 activity more than sixfold (EC50 values of 990 and 270 μm, respectively).

Alteration of substrate selection of antibiotic acylase from β-lactam to echinocandin.

The antibiotic acylases belonging to the N-terminal nucleophile hydrolase superfamily are key enzymes for the industrial production of antibiotic drugs. Cephalosporin acylase (CA) and penicillin G acylase (PGA) are two of the most intensively studied enzymes that catalyze the deacylation of β-lactam antibiotics. On the other hand, aculeacin A acylase (AAC) is known to be an alternative acylase class catalyzing the deacylation of echinocandin or cyclic lipopeptide antibiotic compounds, but its structural and enzymatic properties remain to be explored. In the present study, 3D homology models of AAC were constructed, and docking simulation with substrate ligands was performed for AAC, as well as for CA and PGA. The docking models of AAC with aculeacin A suggest that AAC has the deep narrow binding pocket for the long-chain fatty acyl group of the echinocandin molecule. To confirm this, CA mutants have been designed to form the binding pocket for the long acyl chain. Experimentally synthesized mutant enzymes exhibited lower enzymatic activity for cephalosporin but higher activity for aculeacin A, in comparison with the wild-type enzyme. The present results have clarified the difference in mechanisms of substrate selection between the β-lactam and echinocandin acylases and demonstrate the usefulness of the computational approaches for engineering the enzymatic properties of antibiotic acylases.

Relationship of Circulating Fatty Acid Profile to Metabolic Disorders Associated with Insulin Resistance

Relationship of Circulating Fatty Acid Profile to Metabolic Disorders Associated with Insulin Resistance

Role of SIRT7 in hepatic lipid metabolism

Sirtuins are evolutionarily conserved enzymes that regulate a wide variety of biological processes, such as aging, genomic stability, tumorigenesis, and metabolism [1]. To date, seven sirtuins have been identified in mammals (SIRT1–SIRT7), which share a highly conserved NADbinding and catalytic core domain, but have distinct flanking Nand C-terminal domains. The divergent Nand C-termini of sirtuins are responsible for the different functions and subcellular localizations of these enzymes. Although sirtuins were originally described as NAD-dependent histone deacetylases, these enzymes are now known to act not only on histones, but also on numerous transcription factors and enzymes. SIRT1 controls the acetylation of proliferator-activated receptor-c co-activator 1a (PGC1a), p53, and forkhead box O, among other targets, whereas SIRT3 is a major deacetylase in mitochondria [2, 3]. In addition, a few sirtuins have either weak or undetectable deacetylase activity. For example, SIRT4 is reported to act as an ADP-ribosyltransferase [4], and SIRT5 has both demalonylase and desuccinylase activities [5]. SIRT6 has been shown to deacetylate acetylated lysine 9 of histone H3 (H3K9Ac) and was also recently reported to preferentially hydrolyze long-chain fatty acyl groups on acylated protein targets [6]. SIRT7 mRNA is ubiquitously expressed in all tissues examined to date, with the exception of skeletal muscle [7]. Human (NP_057622.1), mouse (NP_694696.2), and rat (NP_001100543.1) SIRT7 proteins consist of 400, 402, and 402 amino acids, respectively. Human SIRT7 contains a conserved NAD-binding and catalytic core domain (amino acids 90–331) as well as flanking N-terminal (amino acids 1–89) and C-terminal (332–400) regions (Fig. 1a). The histidine residue at position 187 (H187) (corresponding to mouse H188) is highly conserved among sirtuins and is reported to be important for binding with NAD [8]. The N-terminal region of SIRT7 contains a nuclear localization signal (NLS) (LQGRSRRREGLKRRQE, amino acids 61–76), and the C-terminal region contains a nucleolar localization signal (NoLS) (KRTKRKKVT, amino acids 392–400) [9]. SIRT7 is enriched in the nucleolus, but is also present in the nucleoplasm (Fig. 1b). In contrast to SIRT1 to SIRT6, the enzymatic activity and physiological functions of SIRT7 were poorly defined until recently. SIRT7 was first reported to promote ribosomal RNA transcription by interacting with RNA polymerase I (Pol I) and the transcription factor UBF [7, 10]. PAF53, a subunit of Pol I that is required for rDNA transcription, was recently identified as a target of SIRT7 [11]. SIRT7 was also shown to function as NAD-dependent deacetylase with high selectivity for acetylated lysine 18 of histone H3 (H3K18Ac) [12]. This deacetylase activity plays a role in the gene-specific transcriptional repression of a select subset of H3K18Ac-containing promoters, such as RPS20 and NME1 [12]. H3K18Ac-specific deacetylation by SIRT7 is important for maintaining the phenotype and stabilizing the tumorigenicity of human cancer cells [12]. Our group also demonstrated that Myb-binding protein 1a (Mybbp1a) binds to SIRT7, thereby inhibiting the deacetylation of H3K18 [13]. & Kazuya Yamagata k-yamaga@kumamoto-u.ac.jp

Efficient demyristoylase activity of SIRT2 revealed by kinetic and structural studies.

Sirtuins are a class of enzymes originally identified as nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacetylases. Among the seven mammalian sirtuins, SIRT1-7, only SIRT1-3 possess efficient deacetylase activity in vitro, whereas SIRT4-7 possess very weak in vitro deacetylase activity. Several sirtuins that exhibit weak deacetylase activity have recently been shown to possess more efficient activity for the removal other acyl lysine modifications, such as succinyl lysine and palmitoyl lysine. Here, we demonstrate that even the well-known deacetylase SIRT2 possesses efficient activity for the removal of long-chain fatty acyl groups. The catalytic efficiency (kcat/Km) for the removal of a myristoyl group is slightly higher than that for the removal of an acetyl group. The crystal structure of SIRT2 in complex with a thiomyristoyl peptide reveals that SIRT2 possesses a large hydrophobic pocket that can accommodate the myristoyl group. Comparison of the SIRT2 acyl pocket to those of SIRT1, SIRT3, and SIRT6 reveals that the acyl pockets of SIRT1-3 are highly similar, and to a lesser degree, similar to that of SIRT6. The efficient in vitro demyristoylase activity of SIRT2 suggests that this activity may be physiologically relevant and warrants future investigative studies.

Alternate deacylating specificities of the archaeal sirtuins Sir2Af1 and Sir2Af2.

Sirtuins were originally shown to regulate a wide array of biological processes such as transcription, genomic stability, and metabolism by catalyzing the NAD+-dependent deacetylation of lysine residues. Recent proteomic studies have revealed a much wider array of lysine acyl modifications in vivo than was previously known, which has prompted a reevaluation of sirtuin substrate specificity. Several sirtuins have now been shown to preferentially remove propionyl, succinyl, and long-chain fatty acyl groups from lysines, which has changed our understanding of sirtuin biology. In light of these developments, we revisited the acyl specificity of several well-studied archaeal and bacterial sirtuins. We find that the Archaeoglobus fulgidus sirtuins, Sir2Af1 and Sir2Af2, preferentially remove succinyl and myristoyl groups, respectively. Crystal structures of Sir2Af1 bound to a succinylated peptide and Sir2Af2 bound to a myristoylated peptide show how the active site of each enzyme accommodates a noncanonical acyl chain. As compared to its structure in complex with an acetylated peptide, Sir2Af2 undergoes a conformational change that expands the active site to accommodate the myristoyl group. These findings point to both structural and biochemical plasticity in sirtuin active sites and provide further evidence that sirtuins from all three domains of life catalyze noncanonical deacylation.PDB Code(s): 4TWI; 4TWJ

Thioesterase superfamily member 2 (Them2) and phosphatidylcholine transfer protein (PC-TP) interact to promote fatty acid oxidation and control glucose utilization.

Thioesterase superfamily member 2 (Them2) is a mitochondrion-associated long-chain fatty acyl coenzyme A (CoA) thioesterase that is highly expressed in the liver and oxidative tissues. Them2 activity in vitro is increased when it interacts with phosphatidylcholine transfer protein (PC-TP), a cytosolic lipid binding protein. Them2-/- and Pctp-/- mice exhibit enhanced hepatic insulin sensitivity and increased adaptive thermogenesis, and Them2-/- mice are also resistant to diet-induced hepatic steatosis. Although we showed previously that a Them2-PC-TP complex suppresses insulin signaling, the enzymatic activity of Them2 suggests additional direct involvement in regulating hepatic nutrient homeostasis. Here we used cultured primary hepatocytes to elucidate biochemical and cellular mechanisms by which Them2 and PC-TP regulate lipid and glucose metabolism. Under conditions simulating fasting, Them2-/- and Pctp-/- hepatocytes each exhibited decreased rates of fatty acid oxidation and gluconeogenesis. In results indicative of Them2-dependent regulation by PC-TP, chemical inhibition of PC-TP failed to reproduce these changes in Them2-/- hepatocytes. In contrast, rates of glucose oxidation and lipogenesis in the presence of high glucose concentrations were decreased only in Them2-/- hepatocytes. These findings reveal a primary role for Them2 in promoting mitochondrial oxidation of fatty acids and glucose in the liver.

Imaging mass spectrometry reveals fiber-specific distribution of acetylcarnitine and contraction-induced carnitine dynamics in rat skeletal muscles

Carnitine is well recognized as a key regulator of long-chain fatty acyl group translocation into the mitochondria. In addition, carnitine, as acetylcarnitine, acts as an acceptor of excess acetyl-CoA, a potent inhibitor of pyruvate dehydrogenase. Here, we provide a new methodology for accurate quantification of acetylcarnitine content and determination of its localization in skeletal muscles. We used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI–IMS) to visualize acetylcarnitine distribution in rat skeletal muscles. MALDI–IMS and immunohistochemistry of serial cross-sections showed that acetylcarnitine was enriched in the slow-type muscle fibers. The concentration of ATP was lower in muscle regions with abundant acetylcarnitine, suggesting a relationship between acetylcarnitine and metabolic activity. Using our novel method, we detected an increase in acetylcarnitine content after muscle contraction. Importantly, this increase was not detected using traditional biochemical assays of homogenized muscles. We also demonstrated that acetylation of carnitine during muscle contraction was concomitant with glycogen depletion. Our methodology would be useful for the quantification of acetylcarnitine and its contraction-induced kinetics in skeletal muscles.

Are long chain acyl CoAs responsible for suppression of mitochondrial metabolism in hibernating 13-lined ground squirrels?

Hibernation in 13-lined ground squirrels (Ictidomys tridecemlineatus) is associated with a substantial suppression of whole-animal metabolism. We compared the metabolism of liver mitochondria isolated from torpid ground squirrels with those from interbout euthermic (IBE; recently aroused from torpor) and summer euthermic conspecifics. Succinate-fuelled state 3 respiration, calculated relative to mitochondrial protein, was suppressed in torpor by 48% and 44% compared with IBE and summer, respectively. This suppression remains when respiration is expressed relative to cytochrome c oxidase activity. We hypothesized that this suppression was caused by inhibition of succinate transport at the dicarboxylate transporter (DCT) by long-chain fatty acyl CoAs that may accumulate during torpor. We predicted, therefore, that exogenous palmitoyl CoA would inhibit respiration in IBE more than in torpor. Palmitoyl CoA inhibited respiration ~70%, in both torpor and IBE. The addition of carnitine, predicted to reverse palmitoyl CoA suppression by facilitating its transport into the mitochondrial matrix, did not rescue the respiration rates in IBE or torpor. Liver mitochondrial activities of carnitine palmitoyl transferase did not differ among IBE, torpor and summer animals. Although palmitoyl CoA inhibits succinate-fuelled respiration, this suppression is likely not related exclusively to inhibition of the DCT, and may involve additional mitochondrial transporters such as the adenine-nucleotide transporter.

Activation of the Protein Deacetylase SIRT6 by Long-chain Fatty Acids and Widespread Deacylation by Mammalian Sirtuins

Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD(+)-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ε-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6.

Hepatic SREBP-2 and cholesterol biosynthesis are regulated by FoxO3 and Sirt6

Cholesterol homeostasis is crucial for cellular function and organismal health. The key regulator for the cholesterol biosynthesis is sterol-regulatory element binding protein (SREBP)-2. The biochemical process and physiological function of SREBP-2 have been well characterized; however, it is not clear how this gene is epigenetically regulated. Here we have identified sirtuin (Sirt)6 as a critical factor for Srebp2 gene regulation. Hepatic deficiency of Sirt6 in mice leads to elevated cholesterol levels. On the mechanistic level, Sirt6 is recruited by forkhead box O (FoxO)3 to the Srebp2 gene promoter where Sirt6 deacetylates histone H3 at lysines 9 and 56, thereby promoting a repressive chromatin state. Remarkably, Sirt6 or FoxO3 overexpression improves hypercholesterolemia in diet-induced or genetically obese mice. In summary, our data suggest an important role of hepatic Sirt6 and FoxO3 in the regulation of cholesterol homeostasis.

Reduced Virulence of the Vibrio cholerae fadD Mutant Is Due to Induction of the Extracytoplasmic Stress Response

Vibrio cholerae, an important human intestinal pathogen, is responsible for the diarrheal disease cholera. The pathogenesis of V. cholerae is a highly coordinated process that involves diverse regulatory factors. It has recently been demonstrated that disruption of the V. cholerae fadD gene, encoding a long-chain fatty acyl coenzyme A (acyl-CoA) ligase, drastically reduces expression of the major virulence genes and in vivo lethality of this important human pathogen. This effect was due to reduced membrane localization of the central virulence regulator TcpP. In this study, the reason for the impaired membrane localization of TcpP in the fadD mutant was investigated. We demonstrate that extracytoplasmic stress is induced in the V. cholerae ΔfadD strain. In response to the extracytoplasmic stress, the integral membrane protease RseP is activated and degrades the membrane-localized TcpP in the fadD mutant strain. Indeed, disruption of the rseP gene in a fadD mutant background restored membrane localization of TcpP and expression of the downstream virulence genes toxT, ctxA, and tcpA. Increased expression of the σ(E) regulon genes in ethanol-treated wild-type V. cholerae indicated that ethanol exposure could induce an extracytoplasmic stress response in V. cholerae. Ethanol treatment also led to activation of the RseP protease activity and resulted in degradation of membrane-localized TcpP and subsequent reduction in expression of the virulence genes. Taken together, these results suggest that extracytoplasmic stress response per se reduces virulence of V. cholerae by impairing membrane localization of TcpP.

SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine.

The Sir2 family of enzymes or sirtuins are known as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases1 and have been implicated in the regulation of transcription, genome stability, metabolism, and lifespan2, 3. However, four of the seven mammalian sirtuins have very weak deacetylase activity in vitro. Here we show that human Sirt6 efficiently removes long chain fatty acyl groups, such as myristoyl, from lysine residues. The crystal structure of Sirt6 reveals a large hydrophobic pocket that can accommodate long chain fatty acyl groups. We demonstrate further that Sirt6 promotes the secretion of tumor necrosis factor α (TNFα) by removing the fatty acyl modification on K19 and K20 of TNFα. Protein lysine fatty acylation has been known to occur in mammalian cells, but the function and regulatory mechanisms of this modification were unknown. Our data suggest that protein lysine fatty acylation is a novel mechanism that regulates protein secretion. The discovery of Sirt6 as an enzyme that controls protein lysine fatty acylation provides new opportunities to investigate the physiological function of the previously ignored protein posttranslational modification.

Sirtuin on a high-fat diet

It emerges that the sirtuin enzyme SIRT6 preferentially removes long-chain fatty-acyl, rather than acetyl, protein modifications. This activity regulates secretion of the inflammation-associated protein TNF-α. See Letter p.110 Enzymes of the sirtuin family attract a lot of interest because of they regulate ageing, transcription, apoptosis and metabolism. They are often described as NAD-dependent deacetylases, but in fact some of them have only very weak deacetylase activity in vitro and two were shown previously to preferentially use alternative substrates. Here, human SIRT6, an enzyme important for DNA repair and transcription, is shown to remove long-chain fatty acyl groups, such as myristoyl, from lysine residues and to have a function in promoting TNF-α secretion. Its previously described histone deacetylase activity may account for only part of its function. The identification of SIRT6's new activity points to protein lysine fatty acylation as a potentially important area for study.

Ganglioside-binding specificities of E. coli enterotoxin LT-IIc: Importance of long-chain fatty acyl ceramide

Bacterial heat-labile (LT) enterotoxins signal through tightly regulated interactions with host cell gangliosides. LT-IIa and LT-IIb of Escherichia coli bind preferentially to gangliosides with a NeuAcα2-3Galβ1-3GalNAc terminus, with key distinctions in specificity. LT-IIc, a newly discovered E. coli LT, is comprised of an A polypeptide with high homology, and a B polypeptide with moderate homology, to LT-IIa and LT-IIb. LT-IIc is less cytotoxic than LT-IIa and LT-IIb. We theorized that LT-IIc-host cell interaction is regulated by specific structural attributes of immune cell ganglioside receptors and designed experiments to test this hypothesis. Overlay immunoblotting to a diverse array of neural and macrophage gangliosides indicated that LT-IIc bound to a restrictive range of gangliosides, each possessing a NeuAcα2-3Galβ1-3GalNAc with a requisite terminal sialic acid. LT-IIc did not bind to GM1a with short-chain fatty acyl ceramides. Affinity overlay immunoblots, constructed to a diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIc bound to GM1a comprised of long-chain fatty acyl ceramides. Findings were confirmed with LT-IIc also binding to GM1a of RAW264.7 cells, comprised of a long-chain fatty acyl ceramide. Thus, LT-IIc-ganglioside binding differs distinctly from that of LT-IIa and LT-IIb. LT-IIc binding is not just dependent on carbohydrate composition, but also upon the orientation of the oligosaccharide portion of GM1a by the ceramide moiety. These studies are the first demonstration of LT-ganglioside dependence upon ceramide composition and underscore the contribution of long-chain fatty acyl ceramides to host cell interactions.

Acyl-sulfamates target the essential glycerol-phosphate acyltransferase (PlsY) in Gram-positive bacteria

PlsY is the essential first step in membrane phospholipid synthesis of Gram-positive pathogens. PlsY catalyzes the transfer of the fatty acid from acyl-phosphate to the 1-position of glycerol-3-phosphate to form the first intermediate in membrane biogenesis. A series of non-metabolizable, acyl-sulfamate analogs of the acyl-phosphate PlsY substrate were prepared and evaluated as inhibitors of Staphylococcus aureus PlsY and for their Gram-positive antibacterial activities. From this series phenyl (8-phenyloctanoyl) sulfamate had the best overall profile, selectively inhibiting S. aureus phospholipid biosynthesis and causing the accumulation of both long-chain fatty acids and acyl–acyl carrier protein intermediates demonstrating that PlsY was the primary cellular target. Bacillus anthracis was unique in being more potently inhibited by long chain acyl-sulfamates than other bacterial species. However, it is shown that Bacillus anthracis PlsY is not more sensitive to the acyl-sulfamates than S. aureus PlsY. Metabolic profiling showed that B. anthracis growth inhibition by the acyl-sulfamates was not specific for lipid synthesis illustrating that the amphipathic acyl-sulfamates can also have off-target effects in Gram-positive bacteria. Nonetheless, this study further advances PlsY as a druggable target for the development of novel antibacterial therapeutics, through the discovery and validation of the probe compound phenyl (8-phenyloctanoyl) sulfamate as a S. aureus PlsY inhibitor.

Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

Determination of phospholipase activity of PAF acetylhydrolase

This article presents a radiometric assay to determine the enzymatic activity of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2 and phospholipase A2 group 7A. The method is based on the release of radioactively labeled acetate from sn-2-labeled PAF and separation of substrate and product using reversed-phase column chromatography on octadecyl silica gel cartridges. The assay is fast, convenient, reproducible, sensitive, and inexpensive. The instrumentation required includes standard laboratory equipment and a liquid scintillation counter. The assay is also useful to determine the activity of intracellular PAF-AH (PAF-AH II), provided that a few modifications are included. The enzymatic activity determined using PAF as the substrate is a direct indication of the ability of plasma samples, purified preparations, and cellular and tissue lysates to hydrolyze short- and medium-chain phospholipids that may or may not harbor oxidized functionalities. In addition, the assay can be used to test the suitability of other phospholipids, including species containing oxidized, long-chain sn-2 fatty acyl groups, as PAF-AH substrates. This versatile assay can be used to accurately determine PAF-AH activity in biological samples and preliminarily assess affinity and efficiency of the hydrolysis of potential substrates present in complex mixtures.