Long-chain Fatty acyl-CoA Research Articles

MP31-04 ACSL4 IN PROSTATE CANCER GROWTH, INVASION AND HORMONAL RESISTANCE

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2014MP31-04 ACSL4 IN PROSTATE CANCER GROWTH, INVASION AND HORMONAL RESISTANCE Xinyu Wu, Yirong Li, Xinxin Du, Qinghu Ren, Max X. Kong, Jinhua wang, LingHang Wang, Yang Yang, Valerio Zhang, David Zhang, Fei Ye, Garrett Daniels, Fangming Deng, Jianjun Wei, Jonathan Melamed, Marie E. Monaco, and Peng Lee Xinyu WuXinyu Wu More articles by this author , Yirong LiYirong Li More articles by this author , Xinxin DuXinxin Du More articles by this author , Qinghu RenQinghu Ren More articles by this author , Max X. KongMax X. Kong More articles by this author , Jinhua wang Jinhua wang More articles by this author , LingHang WangLingHang Wang More articles by this author , Yang YangYang Yang More articles by this author , Valerio ZhangValerio Zhang More articles by this author , David ZhangDavid Zhang More articles by this author , Fei YeFei Ye More articles by this author , Garrett DanielsGarrett Daniels More articles by this author , Fangming DengFangming Deng More articles by this author , Jianjun WeiJianjun Wei More articles by this author , Jonathan MelamedJonathan Melamed More articles by this author , Marie E. MonacoMarie E. Monaco More articles by this author , and Peng LeePeng Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.913AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Previous studies have shown that the fatty acid biosynthetic enzymes are important in a variety of cancers including prostate cancer (PCa). However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established.The present study is to examine the role of the understudied lipid metabolic enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), in PCa. METHODS LNCaP, LNCaP-AI, PC3 and C4-2B cells were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression in cell proliferation, migration, invasion and apoptosis. Signal network proteins targeted by ACSL4 were analyzed by proteomic pathway array analysis. The effect of ACSL4 on tumor growth was examined in nude mice tumor xenograft models. RESULTS Forced expression of ACSL4 in LNCaP cells promotes cell proliferation and invasion, while ACSL4 knockdown by siRNA in PCa cells including LNCaP-AI, C4-2B and PC3, reduces cell proliferation. These effects were observed in both the absence and presence of androgens and/or estrogens, hormone-free media, although to varying degrees. The growth enhancing effect of ACSL4 is confirmed in vivo using nude mice xenografts to compare the growth of LNCaP vector control with LNCaP-ACSL4 cells. Consistently, ACSL4 expression is increased in human PCa compared to benign epithelial cells. Meanwhile, there is a reciprocal inverse relationship between ACSL4 and the androgen receptor (AR), with decreased AR expression in LNCaP-ACSL4 compared to LNCaP cells and increased AR levels in LNCaP-AI cells with ACSL4 knockdown using siRNA. Conversely, ACSL4 levels are decreased in PC3 cells with AR overexpression. Interestingly, there is an un-coupling of the inverse relationship between AR and ACSL4 in LNCaP-AI cells. Consistently, ACSL4 is also increased in human castrate resistant prostate cancer. Importantly, ACSL4 expression induces resistance to Casodex as evidenced by flow and apoptosis assays. The resistance to Casodex is mediated by BAX and Caspase 8. CONCLUSIONS Our study indicates that ACSL4 expression may serve as a biomarker with potential utility in the diagnosis and treatment of PCa. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e324 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Xinyu Wu More articles by this author Yirong Li More articles by this author Xinxin Du More articles by this author Qinghu Ren More articles by this author Max X. Kong More articles by this author Jinhua wang More articles by this author LingHang Wang More articles by this author Yang Yang More articles by this author Valerio Zhang More articles by this author David Zhang More articles by this author Fei Ye More articles by this author Garrett Daniels More articles by this author Fangming Deng More articles by this author Jianjun Wei More articles by this author Jonathan Melamed More articles by this author Marie E. Monaco More articles by this author Peng Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

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.

A Novel Aromatic Carboxylic Acid Inhibits Luciferase Enzymatic Activity in Mammalian Cells by Acylation of an Active Regulatory Lysine Residue

Firefly luciferase (Luc) is widely used as a reporter enzyme in cell-based assays for gene expression. A novel aromatic carboxylic acid, F-53 substantially inhibited the enzymatic activity of Luc in a Luc reporter screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MS/MS) analyses showed that F-53 modified Luc at lysine-529 via amidation of the F-53 carboxyl group. The lysine-529 residue of Luc, which plays a regulatory catalytic role, can be acetylated. Luc also has a long-chain fatty acyl-CoA synthase activity. An in vitro assay that involved both recombinant Luc and mouse liver microsomes identified F-53-CoA as the reactive form produced from F-53. However, whereas the inhibitory effect of F-53 is observed in Hela cells that transiently expressed Luc, it is not observed in an in vitro assay that involves recombinant Luc alone. Therefore, insights into the activities of certain mammalian transferases can be translated to better understand the acylation by F-53.The purification of the interacting proteins with F-53 in mammalian cells was carried out using F-53-immobilized Magnetic FG beads as bait and those were analyzed by using nano-Liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight hybrid mass spectrometry (nano-LC/ESI-QTOF-MS).

Long Chain Fatty Acyl-CoA Synthetase 4 Is a Biomarker for and Mediator of Hormone Resistance in Human Breast Cancer

The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.

A Novel Aromatic Carboxylic Acid Inactivates Luciferase by Acylation of an Enzymatically Active Regulatory Lysine Residue

Firefly luciferase (Luc) is widely used as a reporter enzyme in cell-based assays for gene expression. A novel aromatic carboxylic acid, F-53, reported here for the first time, substantially inhibited the enzymatic activity of Luc in a Luc reporter screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MS/MS) analyses showed that F-53 modifies Luc at lysine-529 via amidation of the F-53 carboxyl group. The lysine-529 residue of Luc, which plays a regulatory catalytic role, can be acetylated. Luc also has a long-chain fatty acyl-CoA synthase activity. An in vitro assay that involved both recombinant Luc and mouse liver microsomes identified F-53-CoA as the reactive form produced from F-53. However, whereas the inhibitory effect of F-53 is observed in Hela cells that transiently expressed Luc, it is not observed in an in vitro assay that involves recombinant Luc alone. Therefore, insights into the activities of certain mammalian transferases can be translated to better understand the acylation by F-53. The insights from this study about the novel inhibitory modification mechanism might help not only to avoid misinterpretation of the results of Luc-based reporter screening assays but also to explain the pharmacological and toxicological effects of carboxylic acid-containing drugs.

Divergence between human and murine peroxisome proliferator-activated receptor alpha ligand specificities

Peroxisome proliferator-activated receptor α (PPARα) belongs to the family of ligand-dependent nuclear transcription factors that regulate energy metabolism. Although there exists remarkable overlap in the activities of PPARα across species, studies utilizing exogenous PPARα ligands suggest species differences in binding, activation, and physiological effects. While unsaturated long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) function as ligands for recombinant mouse PPARα (mPPARα), no such studies have been conducted with full-length human PPARα (hPPARα). The objective of the current study was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for hPPARα or whether there exist species differences for ligand specificity and affinity. Both hPPARα and mPPARα bound with high affinity to LCFA-CoA; however, differences were noted in LCFA affinities. A fluorescent LCFA analog was bound strongly only by mPPARα, and naturally occurring saturated LCFA was bound more strongly by hPPARα than mPPARα. Similarly, unsaturated LCFA induced transactivation of both hPPARα and mPPARα, whereas saturated LCFA induced transactivation only in hPPARα-expressing cells. These data identified LCFA and LCFA-CoA as endogenous ligands of hPPARα, demonstrated species differences in binding specificity and activity, and may help delineate the role of PPARα as a nutrient sensor in metabolic regulation.

Carnitine supplementation attenuates myocardial lipid accumulation in long‐chain acyl‐CoA dehydrogenase knockout mice

Elevation of long-chain acylcarnitine levels is a hallmark of long-chain mitochondrial β-oxidation (FAO) disorders, and can be accompanied by secondary carnitine deficiency. To restore free carnitine levels, and to increase myocardial export of long-chain fatty acyl-CoA esters, supplementation of L-carnitine in patients has been proposed. However, carnitine supplementation is controversial, because it may enhance the potentially lipotoxic buildup of long-chain acylcarnitines in the FAO-deficient heart. In this longitudinal study, we investigated the effects of carnitine supplementation in an animal model of long-chain FAO deficiency, the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. Cardiac size and function, and triglyceride (TG) levels were quantified using proton magnetic resonance imaging (MRI) and spectroscopy ((1)H-MRS) in LCAD KO and wild-type (WT) mice. Carnitine was supplemented orally for 4 weeks starting at 5 weeks of age. Non-supplemented animals served as controls. In vivo data were complemented with ex vivo biochemical assays. LCAD KO mice displayed cardiac hypertrophy and elevated levels of myocardial TG compared to WT mice. Carnitine supplementation lowered myocardial TG, normalizing myocardial TG levels in LCAD KO mice. Furthermore, carnitine supplementation did not affect cardiac performance and hypertrophy, or induce an accumulation of potentially toxic long-chain acylcarnitines in the LCAD KO heart. This study lends support to the proposed beneficial effect of carnitine supplementation alleviating toxicity by exporting acylcarnitines out of the FAO-deficient myocardium, rather than to the concern about a potentially detrimental effect of supplementation-induced production of lipotoxic long-chain acylcarnitines.

Metabolome Response to Glucose in the β-Cell Line INS-1 832/13

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mM glucose and in response to increasing concentrations of glucose in the INS-1 832/13 β-cell line. U-[(13)C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.

Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Identification and characterization of Rv0494: a fatty acid-responsive protein of the GntR/FadR family from Mycobacterium tuberculosis

Escherichia coli FadR, a member of the GntR family of transcription factors, plays dual roles in fatty acid metabolism. FadR-DNA binding is inhibited by fatty acyl-CoAs, and thus FadR acts as a sensor of the fatty acid level in bacteria. We have identified FadR-binding sites in the upstream regions of genes showing altered expression after the disruption of fatty acid biosynthesis in Mycobacterium tuberculosis. A FadR homologue in M. tuberculosis, Rv0494, was identified, which binds to its operator in the upstream region of the kas operon. We have shown that FadRMt (Rv0494) directly binds to long-chain fatty acyl-CoA and that binding quenches the intrinsic fluorescence of the purified protein. FadR-DNA binding can be impaired by long-chain fatty acyl-CoA compounds. Overexpression of Rv0494 in Mycobacterium bovis BCG reduced the basal level expression of kas operon genes, thereby suggesting the repressor nature of this protein in fatty acid synthase II regulation. This is the first report, to the best of our knowledge, of a GntR/FadR family protein acting as a fatty acid-responsive transcriptional regulator in M. tuberculosis, suggesting a possible role for this protein in mycolic acid biosynthesis.

Fatty Acid Transport Protein 4 (FATP4) Prevents Light-Induced Degeneration of Cone and Rod Photoreceptors by Inhibiting RPE65 Isomerase

Although rhodopsin is essential for sensing light for vision, it also mediates light-induced apoptosis of photoreceptors in mouse. RPE65, which catalyzes isomerization of all-trans retinyl fatty acid esters to 11-cis-retinol (11cROL) in the visual cycle, controls the rhodopsin regeneration rate and photoreceptor susceptibility to light-induced degeneration. Mutations in RPE65 have been linked to blindness in affected children. Despite such importance, the mechanism that regulates RPE65 function remains unclear. Through unbiased expression screening of a bovine retinal pigment epithelium (RPE) cDNA library, we have identified elongation of very long-chain fatty acids-like 1 (ELOVL1) and fatty acid transport protein 4 (FATP4), which each have very long-chain fatty acid acyl-CoA synthetase (VLCFA-ACS) activity, as negative regulators of RPE65. We found that the VLCFA derivative lignoceroyl (C24:0)-CoA inhibited synthesis of 11cROL, whereas palmitoyl (C16:0)-CoA promoted synthesis of 11cROL. We further found that competition of FATP4 with RPE65 for the substrate of RPE65 was also involved in the mechanisms by which FATP4 inhibits synthesis of 11cROL. FATP4 was predominantly expressed in RPE, and the FATP4-deficient RPE showed significantly higher isomerase activity. Consistent with these results, the regeneration rate of 11-cis-retinaldehyde and the recovery rate for rod light sensitivity were faster in FATP4-deficient mice than wild-type mice. Moreover, FATP4-deficient mice displayed increased accumulation of the cytotoxic all-trans retinaldehyde and hypersusceptibility to light-induced photoreceptor degeneration. Our findings demonstrate that ELOVL1, FATP4, and their products comprise the regulatory elements of RPE65 and play important roles in protecting photoreceptors from degeneration induced by light damage.

Role of patatin-like phospholipase domain-containing 3 on lipid-induced hepatic steatosis and insulin resistance in rats

Genome-wide array studies have associated the patatin-like phospholipase domain-containing 3 (PNPLA3) gene polymorphisms with hepatic steatosis. However, it is unclear whether PNPLA3 functions as a lipase or a lipogenic enzyme and whether PNPLA3 is involved in the pathogenesis of hepatic insulin resistance. To address these questions we treated high-fat-fed rats with specific antisense oligonucleotides to decrease hepatic and adipose pnpla3 expression. Reducing pnpla3 expression prevented hepatic steatosis, which could be attributed to decreased fatty acid esterification measured by the incorporation of [U-13C]-palmitate into hepatic triglyceride. While the precursors for phosphatidic acid (PA) (long-chain fatty acyl-CoAs and lysophosphatidic acid [LPA]) were not decreased, we did observe an ∼20% reduction in the hepatic PA content, ∼35% reduction in the PA/LPA ratio, and ∼60%-70% reduction in transacylation activity at the level of acyl-CoA:1-acylglycerol-sn-3-phosphate acyltransferase. These changes were associated with an ∼50% reduction in hepatic diacylglycerol (DAG) content, an ∼80% reduction in hepatic protein kinase Cε activation, and increased hepatic insulin sensitivity, as reflected by a 2-fold greater suppression of endogenous glucose production during the hyperinsulinemic-euglycemic clamp. Finally, in humans, hepatic PNPLA3 messenger RNA (mRNA) expression was strongly correlated with hepatic triglyceride and DAG content, supporting a potential lipogenic role of PNPLA3 in humans. Conclusion: PNPLA3 may function primarily in a lipogenic capacity and inhibition of PNPLA3 may be a novel therapeutic approach for treatment of nonalcoholic fatty liver disease-associated hepatic insulin resistance. ((Hepatology 2013;57:1763-1772))

Plasma Ceramides Target Skeletal Muscle in Type 2 Diabetes

Type 2 diabetes (T2DM) is characterized by hyperglycemia, and the underlying pathophysiology includes insulin resistance and pancreatic β-cell failure. The precise cellular and molecular regulators of insulin resistance and impaired insulin secretion are under intense investigation, but a unifying theory remains elusive. The role of lipids in insulin resistance and β-cell failure has been the subject of considerable empirical investigation, and intermediates of lipid metabolism including diacylglycerols, long-chain fatty acyl-CoAs, and ceramides have all been examined to varying degrees and with varying levels of success. There is good evidence that diacylglycerols and fatty acyl-CoAs directly contribute to dysregulation of cellular insulin signaling through incomplete fatty acid oxidation and/or phosphorylation/dephosphorylation of specific serine and threonine sites on insulin receptor substrate-1 (1,2). Evidence regarding the role of ceramides in insulin resistance has been less consistent. Ceramides are members of the sphingolipid family of lipids and are integral to the structure of the lipid bilayer that makes up all cell membranes (3). They also exert biological effects through cellular proliferation, differentiation, and cell death and interact with several pathways involved in insulin resistance, oxidative stress, inflammation, and apoptosis, all of which are linked to T2DM (4–6). Several lines of evidence suggest that the liver is the major source of plasma ceramides in animals and humans (7,8). In a hamster model, de novo synthesis of ceramides in the liver is induced in response to …

Insights into the Substrate Specificity of a Thioesterase Rv0098 of Mycobacterium Tuberculosis through X-ray Crystallographic and Molecular Dynamics Studies

The crystal structure of Rv0098, a long-chain fatty acyl-CoA thioesterase from Mycobacterium tuberculosis with bound dodecanoic acid at the active site provided insights into the mode of substrate binding but did not reveal the structural basis of substrate specificities of varying chain length. Molecular dynamics studies demonstrated that certain residues of the substrate binding tunnel are flexible and thus modulate the length of the tunnel. The flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. A combination of crystallographic and molecular dynamics studies thus explained the structural basis of accommodating long chain substrates by Rv0098 of M. tuberculosis.

Targeted deletion of thioesterase superfamily member 1 promotes energy expenditure and protects against obesity and insulin resistance

Mammalian acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoAs to form free fatty acids plus CoA, but their metabolic functions remain undefined. Thioesterase superfamily member 1 (Them1; synonyms Acot11, StarD14, and brown fat inducible thioesterase) is a long-chain fatty acyl-CoA thioesterase that is highly expressed in brown adipose tissue and is regulated by both ambient temperature and food consumption. Here we show that Them1(-/-) mice were resistant to diet-induced obesity despite greater food consumption. Them1(-/-) mice exhibited increased O(2) consumption and heat production, which were accompanied by increased rates of fatty acid oxidation in brown adipose tissue and up-regulation of genes that promote energy expenditure. Them1(-/-) mice were also protected against diet-induced inflammation in white adipose tissue, as well as hepatic steatosis, and demonstrated improved glucose homeostasis. The absence of Them1 expression in vivo and in cell culture led to markedly attenuated diet- or chemically induced endoplasmic reticulum stress responses, providing a mechanism by which Them1 deficiency protects against insulin resistance and lipid deposition. Taken together, these data suggest that Them1 functions to decrease energy consumption and conserve calories. In the setting of nutritional excess, the overproduction of free fatty acids by Them1 provokes insulin resistance that is associated with inflammation and endoplasmic reticulum stress.

In Vivo Clinical Measures of Intermediary Metabolism Are Inadequate

The heart derives most of its energy via mitochondrial oxidative metabolism. The heart's fuels or energy substrates include fatty acids, lactate, glucose, and ketones. Substrate concentrations vary during the day, driven mainly by postprandial status. Entry into mitochondrial oxidation requires metabolism to acetyl-coenzyme A (CoA), the fuel of the tricarboxylic acid (TCA) cycle. Acetyl-CoA oxidation generates reduced Nicotinamide adenine dinucleotide (NADH), which drives oxidative phosphorylation, the reduction of O2 to H2O, and, ultimately, the synthesis of ATP, which is essential for myocardial mechanics. The heart uses this highly regulated series of enzyme-catalyzed reactions to convert chemical energy into mechanical energy.1 Metabolism and function in the heart are inextricably linked. Article see p 201 The pathological states of the heart, such as ischemia, hypertrophy, and failure, modify metabolism. Available technologies to measure intermediary metabolism have been inadequate. Magnetic resonance spectroscopy (MRS) can be used to spatially map or at least locally measure metabolism in vivo. Measurement of endogenous triglycerides in patients using 1H MRS has demonstrated that cardiac steatosis precedes the onset of type 2 diabetes mellitus and left ventricular systolic dysfunction.2 In the failing human heart and in preclinical heart failure models, 31P MRS has shown that flux through the creatinine kinase reaction is altered.3,4 Also, ATP and phosphocreatine are reduced after myocardial infarction due to myocyte loss.5 Myocardial energy substrate can be measured with 13C MRS. It has the chemical specificity, but its inherently low sensitivity limits the spatial and temporal resolution necessary for clinical application. In isolated hearts, 13C MRS has to be used with labeled substrate molecules to define TCA cycle kinetics. The metabolites of the TCA cycle have not been measured directly in these experiments. The content of the TCA cycle metabolites is …

Regulation of Lipid Metabolism by Dicer Revealed through SILAC Mice

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems.

Host Acyl Coenzyme A Binding Protein Regulates Replication Complex Assembly and Activity of a Positive-Strand RNA Virus

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.

Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2.

Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may be required for its enzymatic activity and/or the E. coli protein is misfolded.