Acyl Coenzyme Research Articles

Xuezhitong capsule, an extract of Allium macrostemon Bunge, exhibits reverse cholesterol transport and accompanies high-density lipoprotein levels to protect against hyperlipidemia in ApoE-/- mice.

Xuezhitong capsules (XZT) are derived from Xie Bai and used for abnormal lipid homeostasis treatment through maintained metabolic balance. However, their mechanisms are largely unknown. Here, we mainly assessed the contribution of reverse cholesterol transport (RCT) and the accompanying increase in the high-density lipoprotein (HDL) effects of XZT to cholesterol dysfunction amelioration in mice. We assessed serum lipids by using enzymatic kits. We observed atherosclerotic plaque formation by hematoxylin-eosin (HE) and Oil Red O staining. We studied the lipid metabolism, fatty acid synthase (FAS), HDL, low-density lipoprotein receptor (LDLR), triglyceride (TG) metabolic enzyme expression levels, and RCT function in various tissues upon stimulation with high-fat diet, XZT, and some positive drugs by ELISA. After 34 weeks of high-fat diet administration, blood lipids levels increased because attenuated by XZT treatment (800 and 1,600 mg/kg, i.g.). XZT improved the lipid metabolism instability, induced RCT activation, and subsequently increased the HDL levels in hyperlipidemic mice (P<0.05). FAS (P<0.05) and LDLR (P<0.01) levels also remarkably improved. The effects of XZT were closely associated with RCT activation and the accompanying increase in the HDL levels, as characterized by XZT-induced preservation in ATP-binding cassette transporter member 1 (ABCA1), scavenger receptor class B type 1 (SRB1), acyl coenzyme A: cholesterol acyltransferase (ACAT), lecithin cholesterol acyltransferase (LCAT), apolipoprotein A I (ApoA1) and apolipoprotein B (ApoB). However, XZT showed no effect on high fat diet-activated TG metabolic enzyme expression levels (P>0.05). XZT are promising drugs in balancing the cholesterol dysfunction from hyperlipidemia through RCT activation and accompanying increase in HDL levels.

The effect of a physiological increase in temperature on mitochondrial fatty acid oxidation in rat myofibers.

We investigated the effect of temperature increase on mitochondrial fatty acid (FA) and carbohydrate oxidation in the slow-oxidative skeletal muscles (soleus) of rats. We measured mitochondrial respiration at 35°C and 40°C with the physiological substrates pyruvate + 4 mM malate (Pyr) and palmitoyl-CoA (PCoA) + 0.5 mM malate + 2 mM carnitine in permeabilized myofibers under nonphosphorylating () or phosphorylating () conditions. Mitochondrial efficiency was calculated by the respiratory control ratio (RCR = /). We used guanosine triphosphate (GTP), an inhibitor of uncoupling protein (UCP), to study the mechanisms responsible for alterations of mitochondrial efficiency. We measured hydrogen peroxide (H2O2) production under nonphosphorylating and phosphorylating conditions at both temperatures and substrates. We studied citrate synthase (CS) and 3-hydroxyl acyl coenzyme A dehydrogenase (3-HAD) activities at both temperatures. Elevating the temperature from 35°C to 40°C increased PCoA- and decreased PCoA-RCR, corresponding to the uncoupling of oxidative phosphorylation (OXPHOS). GTP blocked the heat-induced increase of PCoA-. Rising temperature moved toward a Pyr- increase, without significance. Heat did not alter H2O2 production, resulting from either PCoA or Pyr oxidation. Heat induced an increase in 3-HAD but not in CS activities. In conclusion, heat induced OXPHOS uncoupling for PCoA oxidation, which was at least partially mediated by UCP and independent of oxidative stress. The classically described heat-induced glucose shift may actually be mostly due to a less efficient FA oxidation. These findings raise questions concerning the consequences of heat-induced alterations in mitochondrial efficiency of FA metabolism on thermoregulation.NEW & NOTEWORTHY Ex vivo exposure of skeletal myofibers to heat uncouples substrate oxidation from ADP phosphorylation, decreasing the efficiency of mitochondria to produce ATP. This heat effect alters fatty acids (FAs) more than carbohydrate oxidation. Alteration of FA oxidation involves uncoupling proteins without inducing oxidative stress. This alteration in lipid metabolism may underlie the preferential use of carbohydrates in the heat and could decrease aerobic endurance.

MiR-222 targets ACOX1, promotes triglyceride accumulation in hepatocytes

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver diseases. However, the exact pathogenesis of NAFLD remains to be elucidated. Despite the association with tumors and cardiovascular diseases, the role of miR-222 in NAFLD remains unclear. The present study was to investigate the role of miR-222 in NAFLD. MethodsWild-type C57BL/6 mice were fed a high-fat diet for 12 weeks to induce NAFLD. Normal human liver cell line (L02) was cultured with free fatty acid (FFA)-containing medium to stimulate cell steatosis. The mRNA levels of miR-222 and acyl Coenzyme A xidase 1 (ACOX1) were detected by quantitative-PCR (Q-PCR). The prediction of ACOX1 as the target gene for miR-222 was conducted via TargetScan. The overexpression or inhibition of miR-222 was mediated by miR-222 mimics or antagomir, and intracellular triglyceride levels were measured using a triglyceride kit. Luciferase reporter assays verified ACOX1 as the target gene for miR-222. ResultsmiR-222 was significantly elevated in both the in vivo and in vitro NAFLD models. Overexpression of miR-222 significantly increased triglyceride content in the L02 cells, while inhibition of miR-222 expression restricted the accumulation of triglyceride. Overexpression of miR-222 significantly inhibited ACOX1 expression. Transient transfection assays verified that ACOX1 3′-UTR luciferase reporter activity could be inhibited by miR-222 overexpression. ConclusionsThe present study suggested that miR-222 promotes the accumulation of triglycerides by inhibiting ACOX1.

Mutation of ACX1, a Jasmonic Acid Biosynthetic Enzyme, Leads to Petal Degeneration in Chinese Cabbage (Brassica campestris ssp. pekinensis).

Petal color, size, and morphology play important roles in protecting other floral organs, attracting pollinators, and facilitating sexual reproduction in plants. In a previous study, we obtained a petal degeneration mutant (pdm) from the ‘FT’ doubled haploid line of Chinese cabbage and found that the candidate gene for pdm, Bra040093, encodes the enzyme acyl-CoA oxidase1. In this study, we sought to examine the gene networks regulating petal development in pdm plants. We show that the mRNA and protein expression of Bra040093, which is involved in the jasmonic acid (JA) biosynthetic pathway, were significantly lower in the petals of pdm plants than in those of ‘FT’ plants. Similarly, the JA and methyl jasmonate (MeJA) contents of petals were significantly lower in pdm plants than in ‘FT’ plants and we found that exogenous application of these hormones to the inflorescences of pdm plants restored the ‘FT’ phenotype. Comparative analyses of the transcriptomes of ‘FT’, pdm and pdm + JA (pJA) plants revealed 10,160 differentially expressed genes (DEGs) with consistent expression tendencies in ‘FT’ vs. pdm and pJA vs. pdm comparisons. Among these DEGs, we identified 69 DEGs related to floral organ development, 11 of which are involved in petal development regulated by JA. On the basis of qRT-PCR verification, we propose regulatory pathways whereby JA may mediate petal development in the pdm mutant. We demonstrate that mutation of Bra040093 in pdm plants leads to reduced JA levels and that this in turn promotes changes in the expression of genes that are expressed in response to JA, ultimately resulting in petal degeneration. These findings thus indicate that JA is associated with petal development in Chinese cabbage. These results enhance our knowledge on the molecular mechanisms underlying petal development and lay the foundations for further elucidation of the mechanisms associated with floral organ development in Chinese cabbage.

The effect of acyl-CoA synthetase long-chain family member 5 on triglyceride synthesis in bovine preadipocytes.

Acyl-CoA synthetase long-chain family member 5 (ACSL5) is a member of the acyl coenzyme A (CoA) long-chain synthase families (ACSLs), and it plays a key role in fatty acid metabolism. In this study, we proved an association between the ACSL5 gene and triglyceride metabolism at the cellular level in cattle. pBI-CMV3-ACSL5 and pGPU6/GFP/Neo-ACSL5 plasmids were constructed and transfected into bovine preadipocytes by electroporation. The expression level of ACSL5 was detected by real-time quantitative PCR and western blot. The triglyceride content was detected by a triglyceride kit. The results indicated that the expression level of ACSL5 mRNA and protein in the pBI-CMV3-ACSL5-transfected group was significantly increased compared with those in the control group. Furthermore, the pGPU6/GFP/Neo-ACSL5-transfected group was significantly decreased compared with those in the control group. A cell triglyceride test showed that overexpression or silencing of the ACSL5 gene could affect synthesis of cellular triglycerides. This study investigated the mechanism of ACSL on bovine fat deposition, and also provides a new candidate gene for meat quality traits in beef cattle.

Loss of ACOT7 potentiates seizures and metabolic dysfunction.

Neurons uniquely antagonize fatty acid utilization by hydrolyzing the activated form of fatty acids, long chain acyl-CoAs, via the enzyme acyl-CoA thioesterase 7, Acot7. The loss of Acot7 results in increased fatty acid utilization in neurons and exaggerated stimulus-evoked behavior such as an increased startle response. To understand the contribution of Acot7 to seizure susceptibility, we generated Acot7 knockout (KO) mice and assayed their response to kainate-induced seizures. Acot7 KO mice exhibited potentiated behavioral and molecular indices of seizure severity following kainic acid administration, suggesting that fatty acid metabolism in neurons can be a critical regulator of neuronal activity. These data are consistent with the presentation of seizures in a human with genomic deletion of ACOT7 demonstrating the conservation of function across species. To further understand the metabolic complications arising from a deletion in Acot7, we subjected Acot7 KO mice to a high-fat diet. While the loss of Acot7 did not result in metabolic complications following a normal chow diet, a high-fat diet induced greater body weight gain, adiposity, and glucose intolerance in Acot7 KO mice. These data demonstrate that Acot7, a fatty acid metabolic enzyme highly enriched in neurons, regulates both brain-specific metabolic processes related to seizure susceptibility and the whole body response to dietary lipid.

Preservation of Acyl Coenzyme A Attenuates Pathological and Metabolic Cardiac Remodeling Through Selective Lipid Trafficking.

Metabolic remodeling in heart failure contributes to dysfunctional lipid trafficking and lipotoxicity. Acyl coenzyme A synthetase-1 (ACSL1) facilitates long-chain fatty acid (LCFA) uptake and activation with coenzyme A (CoA), mediating the fate of LCFA. The authors tested whether cardiac ACSL1 overexpression aids LCFA oxidation and reduces lipotoxicity under pathological stress of transverse aortic constriction (TAC). Mice with cardiac restricted ACSL1 overexpression (MHC-ACSL1) underwent TAC or sham surgery followed by serial in vivo echocardiography for 14 weeks. At the decompensated stage of hypertrophy, isolated hearts were perfused with 13C LCFA during dynamic-mode 13C nuclear magnetic resonance followed by in vitro nuclear magnetic resonance and mass spectrometry analysis to assess intramyocardial lipid trafficking. In parallel, acyl CoA was measured in tissue obtained from heart failure patients pre- and postleft ventricular device implantation plus matched controls. TAC-induced cardiac hypertrophy and dysfunction was mitigated in MHC-ACSL1 hearts compared with nontransgenic hearts. At 14 weeks, TAC increased heart weight to tibia length by 46% in nontransgenic mice, but only 26% in MHC-ACSL1 mice, whereas ACSL1 mice retained greater ejection fraction (ACSL1 TAC: 65.8±7.5%; nontransgenic TAC: 45.9±7.3) and improvement in diastolic E/E'. Functional improvements were mediated by ACSL1 changes to cardiac LCFA trafficking. ACSL1 accelerated LCFA uptake, preventing C16 acyl CoA loss post-TAC. Long-chain acyl CoA was similarly reduced in human failing myocardium and restored to control levels by mechanical unloading. ACSL1 trafficked LCFA into ceramides without normalizing the reduced triglyceride storage in TAC. ACSL1 prevented de novo synthesis of cardiotoxic C16- and C24-, and C24:1 ceramides and increased potentially cardioprotective C20- and C22-ceramides post-TAC. ACLS1 overexpression activated AMP activated protein kinase at baseline, but during TAC, prevented the reduced LCFA oxidation in hypertrophic hearts and normalized energy state (phosphocreatine:ATP) and consequently, AMP activated protein kinase activation. This is the first demonstration of reduced acyl CoA in failing hearts of humans and mice, and suggests possible mechanisms for maintaining mitochondrial oxidative energy metabolism by restoring long-chain acyl CoA through ASCL1 activation and mechanical unloading. By mitigating cardiac lipotoxicity, via redirected LCFA trafficking to ceramides, and restoring acyl CoA, ACSL1 delayed progressive cardiac remodeling and failure.

Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis.

Lipid droplets (LDs) serve as cytoplasmic reservoirs for energy-rich fatty acids (FAs) stored in the form of triacylglycerides (TAGs). During nutrient stress, yeast LDs cluster adjacent to the vacuole/lysosome, but how this LD accumulation is coordinated remains poorly understood. The ER protein Mdm1 is a molecular tether that plays a role in clustering LDs during nutrient depletion, but its mechanism of function remains unknown. Here, we show that Mdm1 associates with LDs through its hydrophobic N-terminal region, which is sufficient to demarcate sites for LD budding. Mdm1 binds FAs via its Phox-associated domain and coenriches with fatty acyl-coenzyme A ligase Faa1 at LD bud sites. Consistent with this, loss of MDM1 perturbs free FA activation and Dga1-dependent synthesis of TAGs, elevating the cellular FA level, which perturbs ER morphology and sensitizes yeast to FA-induced lipotoxicity. We propose that Mdm1 coordinates FA activation adjacent to the vacuole to promote LD production in response to stress, thus maintaining ER homeostasis.

Coupled biosynthesis and esterification of 1,2,4-butanetriol to simplify its separation from fermentation broth.

1,2,4-Butanetriol (BT) is a valuable chemical with versatile applications in many fields and can be produced through biosynthetic pathways. As a trihydric alcohol, BT possesses good water solubility and is very difficult to separate from fermentation broth, which does complicate the production process and increase the cost. To develop a novel method for BT separation, a biosynthetic pathway for 1,2,4-butanetriol esters with poor water solubility was constructed. Wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase (Atf) from Acinetobacter baylyi, Mycobacterium smegmatis, and Escherichia coli were screened, and the acyltransferase from A. baylyi (AtfA) was found to have higher capability. The BT producing strain with AtfA overexpression produced 49.5 mg/L BT oleate in flask cultivation. Through enhancement of acyl-CoA production by overexpression of the acyl-CoA synthetase gene fadD and deleting the acyl coenzyme A dehydrogenase gene fadE, the production was improved to 64.4 mg/L. Under fed-batch fermentation, the resulting strain produced up to 1.1 g/L BT oleate. This is the first time showed that engineered E. coli strains can successfully produce BT esters from xylose and free fatty acids.

Peroxisome-derived lipids regulate adipose thermogenesis by mediating cold-induced mitochondrial fission.

Peroxisomes perform essential functions in lipid metabolism, including fatty acid oxidation and plasmalogen synthesis. Here, we describe a role for peroxisomal lipid metabolism in mitochondrial dynamics in brown and beige adipocytes. Adipose tissue peroxisomal biogenesis was induced in response to cold exposure through activation of the thermogenic coregulator PRDM16. Adipose-specific knockout of the peroxisomal biogenesis factor Pex16 (Pex16-AKO) in mice impaired cold tolerance, decreased energy expenditure, and increased diet-induced obesity. Pex16 deficiency blocked cold-induced mitochondrial fission, decreased mitochondrial copy number, and caused mitochondrial dysfunction. Adipose-specific knockout of the peroxisomal β-oxidation enzyme acyl-CoA oxidase 1 (Acox1-AKO) was not sufficient to affect adiposity, thermogenesis, or mitochondrial copy number, but knockdown of the plasmalogen synthetic enzyme glyceronephosphate O-acyltransferase (GNPAT) recapitulated the effects of Pex16 inactivation on mitochondrial morphology and function. Plasmalogens are present in mitochondria and decreased with Pex16 inactivation. Dietary supplementation with plasmalogens increased mitochondrial copy number, improved mitochondrial function, and rescued thermogenesis in Pex16-AKO mice. These findings support a surprising interaction between peroxisomes and mitochondria regulating mitochondrial dynamics and thermogenesis.

Preventive or Curative Administration of Taurine Regulates Lipid Metabolism in the Liver of Rats with Alcoholic Liver Disease.

Excessive consumption causes alcoholic liver disease (ALD), which injures hepatocytes and induces imbalance of lipid metabolism. Taurine is known to protect the liver from various liver injuries, and relieve lipid profile. Our previous studies also found that taurine can prevent or cure ALD, reduce fat deposition, but the mechanism remains unclear. In the present study, ALD rat model was established by administration of alcohol, pyrazole and high fat diet. Two percent taurine was administered at the same time or after ALD model establishment. Serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), serum and hepatic TC, TG, HDL-C and LDL-C were analyzed. Real-Time RT-PCR was conducted to detect the mRNA expressions of fatty acid synthetase (FAS), acetyl-CoA catboxylase (ACC), carnitine palmitoyl transferase 1 (CPT-1), 3-Hydroxy-3-methyl glutaric acid acyl Coenzyme A reductase (HMGCR), peroxisome proliferators activated receptor α (PPARα) and sterol regulatory element-binding protein 1c (SREBP-1c). The results showed that serum ALT, AST, serum and hepatic TC, TG and LDL-C were higher, while HDL-C in ALD model rats was lower than normal rats, the changes of which can be significantly relieved by taurine administration. mRNA expressions of ACC, FAS, CPT-1, HMGCR, PPARα and SREBP-1c which were significantly changed by ethanol can also be regulated by taurine. The results indicated that taurine can prevent and repair hepatic injury of ALD rats and balance lipid metabolism indexes in the liver, the mechanisms may involves in the regulation of related enzymes and transcriptional regulators participated in lipid metabolism.

Complete Genome Sequence of Saccharospirillum mangrovi HK-33T Sheds Light on the Ecological Role of a Bacterium in Mangrove Sediment Environment.

We present the genome sequence of Saccharospirillum mangrovi HK-33T, isolated from a mangrove sediment sample in Haikou, China. The complete genome of S. mangrovi HK-33T consisted of a single-circular chromosome with the size of 3,686,911bp as well as an average G + C content of 57.37%, and contained 3,383 protein-coding genes, 4 operons of 16S-23S-5S rRNA genes, and 52 tRNA genes. Genomic annotation indicated that the genome of S. mangrovi HK-33T had many genes related to oligosaccharide and polysaccharide degradation and utilization of polyhydroxyalkanoate. For nitrogen cycle, genes encoding nitrate and nitrite reductase, glutamate dehydrogenase, glutamate synthase, and glutamine synthetase could be found. For phosphorus cycle, genes related to polyphosphate kinases (ppk1 and ppk2), the high-affinity phosphate-specific transport (Pst) system, and the low-affinity inorganic phosphate transporter (pitA) were predicted. For sulfur cycle, cysteine synthase and type III acyl coenzyme A transferase (dddD) coding genes were searched out. This study provides evidence about carbon, nitrogen, phosphorus, and sulfur metabolic patterns of S. mangrovi HK-33T and broadens our understandings about ecological roles of this bacterium in the mangrove sediment environment.

Current status and new approaches in prostate cancer diagnosis

Abstract Prostate adenocarcinoma, remaining among top most common cancers, is a heterogeneous group of tumors with a diverse morphological structure. Basing on the histological architecture of cancer tissue, individual cases can be classified into different therapeutic groups. Current diagnosis of prostate cancer brings many challenges. The major problem is the lack of effective and accessible diagnostic methods that would eliminate incidences of overdiagnosis and prevent unnecessary treatments of many patients. There are many efforts to determine favorable and unfavorable molecular prognostic factors. The basic marker currently used in this field is prostate-specific antigen (PSA). Increased level of PSA may suggest the presence of prostate cancer although its level is not specific for the disease and can be elevated also in certain benign hyperplastic or inflammatory conditions as well as after irritation or rectal examination. Clinical symptoms such as dysuria or hematuria are often uncharacteristic and benign prostatic diseases which cannot be confirmed on the basis of physical examination alone. Also, we often deal with the situation of false negative results of prostate needle biopsy, which require many tests to determine the final correct diagnosis. Moreover, prostate cancer can also be present in patients with non-elevated serum PSA level. Due to such difficulties, the search for new molecular markers that could be used for diagnostic purposes is underway. Evaluation of survivin level in prostate cancer tissue may serve as a new diagnostic indicator of prostate cancer progression. Other useful molecular biomarkers with good potential in prostate cancer diagnosis are AMACR (Alpha Methyl Acyl Coenzyme A Racemase), p-63 or Ki-67 or microRNAs present in body fluids.

Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism.

Acyl coenzyme A (CoA)-binding protein (ACBP) can bind acyl-CoAs with high specificity and affinity, thus playing multiple roles in cellular functions. Mitochondria of the apicomplexan parasite Toxoplasma gondii have emerged as key organelles for lipid metabolism and signaling transduction. However, the rationale for how this parasite utilizes acyl-CoA-binding protein to regulate mitochondrial lipid metabolism remains unclear. Here, we show that an ankyrin repeat-containing protein, TgACBP2, is localized to mitochondria and displays active acyl-CoA-binding activities. Dephosphorylation of TgACBP2 is associated with relocation from the plasma membrane to the mitochondria under conditions of regulation of environmental [K+]. Under high [K+] conditions, loss of ACBP2 induced mitochondrial dysfunction and apoptosis-like cell death. Disruption of ACBP2 caused growth and virulence defects in the type II strain but not in type I parasites. Interestingly, mitochondrial association factor-1 (MAF1)-mediated host mitochondrial association (HMA) restored the growth ability of ACBP2-deficient type II parasites. Lipidomics analysis indicated that ACBP2 plays key roles in the cardiolipin metabolism of type II parasites and that MAF1 expression complemented the lipid metabolism defects of ACBP2-deficient type II parasites. In addition, disruption of ACBP2 caused attenuated virulence of Prugniuad (Pru) parasites for mice. Taking the results collectively, these data indicate that ACBP2 is critical for the growth and virulence of type II parasites and for the growth of type I parasites under high [K+] conditions.IMPORTANCEToxoplasma gondii is one of the most successful human parasites, infecting nearly one-third of the total world population. T. gondii tachyzoites residing within parasitophorous vacuoles (PVs) can acquire fatty acids both via salvage from host cells and via de novo synthesis pathways for membrane biogenesis. However, although fatty acid fluxes are known to exist in this parasite, how fatty acids flow through Toxoplasma lipid metabolic organelles, especially mitochondria, remains unknown. In this study, we demonstrated that Toxoplasma expresses an active ankyrin repeat containing protein TgACBP2 to coordinate cardiolipin metabolism. Specifically, HMA acquisition resulting from heterologous functional expression of MAF1 rescued growth and lipid metabolism defects in ACBP2-deficient type II parasites, manifesting the complementary role of host mitochondria in parasite cardiolipin metabolism. This work highlights the importance of TgACBP2 in parasite cardiolipin metabolism and provides evidence for metabolic association of host mitochondria with T. gondii.

Crystal Structure of PigA: A Prolyl Thioester-Oxidizing Enzyme in Prodigiosin Biosynthesis.

Prodigiosin is an intensely red pigment comprising three pyrroles. The biosynthetic pathway includes a two-step proline oxidation catalyzed by phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PigA), with flavin adenine dinucleotide (FAD) as its cofactor. The enzyme is crystallized in the apo form and in complex with FAD and proline. As an acyl coenzyme A dehydrogenase (ACAD) family member, the protein folds into a β-sheet flanked by two α-helical domains. PigA forms a tetramer, which is consistent with analytical ultracentrifugation results. FAD binds to PigA in a similar way to that in the other enzymes of the ACAD family. The variable conformations of loop β4-β5 and helix αG correlate well with the structural flexibility required for substrate entrance to the Re side of FAD. Modeling with PigG, the acyl carrier protein, suggests a reasonable mode of interaction with PigA. The structure helps to explain the proline oxidation mechanism, in which Glu244 plays a central role by abstracting the substrate protons. It also reveals a plausible pocket for oxygen binding to the Si side of FAD.

Abstract 3532: Bitter melon extract suppresses triple-negative breast cancer growth by inhibiting ACAT1

Abstract Breast cancer is the most common malignant cancer diagnosed in women worldwide each year. Triple-negative breast cancers (TNBC), defined by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), account for approximately 15% of breast cancers. Moreover, TNBC is usually more aggressive and metastatic with a worse prognosis. Bitter melon (Momordica charantia) is well known as a dietary supplement. Recently, we have shown an anti-cancer activity of bitter melon extract (BME) in breast cancer mouse models, although the mechanism is poorly understood. Furthermore, we became interested to understand the role of BME on cholesterol and lipid metabolism in TNBC. Our mass spectrometry data suggested that stored as a cholesteryl esters (CE) in Lipid droplets (LD) were much lower in BME treated TNBC cells. We found that not only CE but also LD was reduced in BME treated TNBC cells. The acyl-CoA:cholesterol acyl transferase-1 (ACAT-1) catalyzes the formation of CE from free cholesterols together with long-chain fatty acyl coenzyme A. Therefore, inhibition of ACAT-1 resulted in downregulation of CE, and upregulation free cholesterols. The attenuated CE is known to reduce cancer proliferation. Subsequent study demonstrated that BME treatment highly inhibited ACAT-1 in TNBC cells. The upregulated free cholesterol negatively regulates sterol regulatory element-binding proteins (SREBPs), which regulates lipogenesis protein such as fatty acid synthase (FASN) associated with cancer cell proliferation. We also observed that SREBP-1 and -2 were barely detectable, and FASN and low-density lipoprotein receptor (LDLR) were reduced in BME treated TNBC cells as compared to control cells. To our knowledge, this is the first report demonstrating that BME suppresses TNBC cell growth through ACAT-1 inhibition. BME supplement may potentially be an additional regimen in human breast cancer treatment. Citation Format: So Hee Shim, Robert Steele, Ratna Ray. Bitter melon extract suppresses triple-negative breast cancer growth by inhibiting ACAT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3532.

Salmonella Tol-Pal Reduces Outer Membrane Glycerophospholipid Levels for Envelope Homeostasis and Survival during Bacteremia.

ABSTRACTSalmonellae regulate membrane lipids during infection, but the exact proteins and mechanisms that promote their survival during bacteremia remain largely unknown. Mutations in genes encoding the conserved Salmonella enterica serovar Typhimurium (S. Typhimurium) Tol-Pal apparatus caused the outer membrane (OM) sensor lipoprotein, RcsF, to become activated. The capsule activation phenotype for the mutants suggested that Tol-Pal might influence envelope lipid homeostasis. The mechanism involves reducing OM glycerophospholipid (GPL) levels, since the mutant salmonellae similarly accumulated phosphatidylglycerols (PGl) and phosphatidylethanolamines (PE) within the OM in comparison to the wild type. The data support the Escherichia coli model, whereby Tol-Pal directs retrograde GPL translocation across the periplasm. The S. Typhimurium mechanism involves contributions from YbgC, a cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase, and CpoB, a periplasmic TolA-binding protein. The functional relationship between Tol-Pal and YbgC and CpoB was previously unresolved. The S. Typhimurium Tol-Pal proteins contribute similarly toward promoting OM-GPL homeostasis and Rcs signaling inactivity but differently toward promoting bacterial morphology, rifampin resistance, survival in macrophages, and survival in mice. For example, tolQ, tolR, tolA, and cpoB mutants were significantly more attenuated than ybgC, tolB, and pal mutants in a systemic mouse model of disease. Therefore, key roles exist for TolQ, TolR, TolA, and CpoB during murine bacteremia, which are independent of maintaining GPL homeostasis. The ability of TolQR to channel protons across the inner membrane (IM) is necessary for S. Typhimurium TolQRA function, since mutating conserved channel-facing residues rendered TolQ ineffective at rescuing deletion mutant phenotypes. Therefore, Tol-Pal promotes S. Typhimurium survival during bacteremia, in part, by reducing OM GPL concentrations, while TolQRA and CpoB enhance systemic virulence by additional mechanisms.

Acyl-group specificity of AHL synthases involved in quorum-sensing in Roseobacter group bacteria.

N-Acylhomoserine lactones (AHLs) are important bacterial messengers, mediating different bacterial traits by quorum sensing in a cell-density dependent manner. AHLs are also produced by many bacteria of the marine Roseobacter group, which constitutes a large group within the marine microbiome. Often, specific mixtures of AHLs differing in chain length and oxidation status are produced by bacteria, but how the biosynthetic enzymes, LuxI homologs, are selecting the correct acyl precursors is largely unknown. We have analyzed the AHL production in Dinoroseobacter shibae and three Phaeobacter inhibens strains, revealing strain-specific mixtures. Although large differences were present between the species, the fatty acid profiles, the pool for the acyl precursors for AHL biosynthesis, were very similar. To test the acyl-chain selectivity, the three enzymes LuxI1 and LuxI2 from D. shibae DFL-12 as well as PgaI2 from P. inhibens DSM 17395 were heterologously expressed in E. coli and the enzymes isolated for in vitro incubation experiments. The enzymes readily accepted shortened acyl coenzyme A analogs, N-pantothenoylcysteamine thioesters of fatty acids (PCEs). Fifteen PCEs were synthesized, varying in chain length from C4 to C20, the degree of unsaturation and also including unusual acid esters, e.g., 2E,11Z-C18:2-PCE. The latter served as a precursor of the major AHL of D. shibae DFL-12 LuxI1, 2E,11Z-C18:2-homoserine lactone (HSL). Incubation experiments revealed that PgaI2 accepts all substrates except C4 and C20-PCE. Competition experiments demonstrated a preference of this enzyme for C10 and C12 PCEs. In contrast, the LuxI enzymes of D. shibae are more selective. While 2E,11Z-C18:2-PCE is preferentially accepted by LuxI1, all other PCEs were not, except for the shorter, saturated C10–C14-PCEs. The AHL synthase LuxI2 accepted only C14 PCE and 3-hydroxydecanoyl-PCE. In summary, chain-length selectivity in AHLs can vary between different AHL enzymes. Both, a broad substrate acceptance and tuned specificity occur in the investigated enzymes.

Insulin promotes progression of colon cancer by upregulation of ACAT1

BackgroundInsulin resistant and the progression of cancer is closely related. The aim of this study was to investigate the effect of insulin on the proliferation and migration of colon cancer cells and its underlying mechanism.MethodsColon carcinoma tissues from the 80 cases of colon cancer patients were collected. Immunohistochemistry was used to detect the expression of acyl coenzyme A: cholesterol acyltransferase1 (ACAT1), and we analyzed the correlation between hyperglycemia and ACAT1, hyperglycemia and metastasis. CCK8 assay and transwell assay were used to investigate the effect of different concentrations of insulin and ACAT1siRNA on human colon cancer cell line HT-29. ACAT1 mRNA expression and protein level in HT-29 cells were determined by real-time quantitative PCR and western blotting, respectively.ResultsBiopsies from patients with colon carcinoma showed hyperglycemia links ACAT1, lymph nodes metastasis and distant metastasis. Insulin markedly promoted cell proliferation and migration in human colon cancer HT-29 cells. Moreover, ACAT1mRNA expression and protein level were increased by insulin. ACAT1siRNA resulted in a complete inhibition of the ACAT1 mRNA expression. Consequently insulin-triggered cell proliferation and migration on colon cancer cells were inhibited.ConclusionThe progression of colon cancer has a positive correlation with hyperinsulinemia. Insulin-triggered cell proliferation and metastatic effects on colorectal cancer cells are mediated by ACAT1. Therefore, insulin could promote colon cancer progression by upregulation of ACAT1, which maybe is a potential therapeutic target for colon cancer.

ATR-101, a selective ACAT1 inhibitor, decreases ACTH-stimulated cortisol concentrations in dogs with naturally occurring Cushing\u2019s syndrome

BackgroundCushing’s syndrome in humans shares many similarities with its counterpart in dogs in terms of etiology (pituitary versus adrenal causes), clinical signs, and pathophysiologic sequelae. In both species, treatment of pituitary- and adrenal-dependent disease is met with limitations. ATR-101, a selective inhibitor of ACAT1 (acyl coenzyme A:cholesterol acyltransferase 1), is a novel small molecule therapeutic currently in clinical development for the treatment of adrenocortical carcinoma, congenital adrenal hyperplasia, and Cushing’s syndrome in humans. Previous studies in healthy dogs have shown that ATR-101 treatment led to rapid, dose-dependent decreases in adrenocorticotropic hormone (ACTH) stimulated cortisol levels. The purpose of this clinical study was to investigate the effects of ATR-101 in dogs with Cushing’s syndrome.MethodsATR-101 pharmacokinetics and activity were assessed in 10 dogs with naturally-occurring Cushing’s syndrome, including 7 dogs with pituitary-dependent disease and 3 dogs with adrenal-dependent disease. ATR-101 was administered at 3 mg/kg PO once daily for one week, followed by 30 mg/kg PO once daily for one (n = 4) or three (n = 6) weeks. Clinical, biochemical, adrenal hormonal, and pharmacokinetic data were obtained weekly for study duration.ResultsATR-101 exposure increased with increasing dose. ACTH-stimulated cortisol concentrations, the primary endpoint for the study, were significantly decreased with responders (9 of 10 dogs) experiencing a mean ± standard deviation reduction in cortisol levels of 50 ± 17% at study completion. Decreases in pre-ACTH-stimulated cortisol concentrations were observed in some dogs although overall changes in pre-ACTH cortisol concentrations were not significant. The compound was well-tolerated and no serious drug-related adverse effects were reported.ConclusionsThis study highlights the potential utility of naturally occurring canine Cushing’s syndrome as a model for human disease and provides proof of concept for ATR-101 as a novel agent for the treatment of endocrine disorders like Cushing’s syndrome in humans.