Inhibitor Of Long-chain acyl-CoA Synthetase Research Articles

Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

BackgroundGlioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST).ResultsUsing CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity.ConclusionsOur results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.

Palmitate induces cardiomyocyte death via inositol requiring enzyme-1 (IRE1)-mediated signaling independent of X-box binding protein 1 (XBP1)

Overloading of the saturated fatty acid (SFA) palmitate induces cardiomyocyte death. The purpose of this study is to elucidate signaling pathways contributing to palmitate-induced cardiomyocyte death.Palmitate-induced cardiomyocyte death was induced in Toll-like receptor 2/4 double-knockdown cardiomyocytes to a similar extent as wild-type cardiomyocytes, while cardiomyocyte death was canceled out by triacsin C, a long-chain acyl-CoA synthetase inhibitor. These results indicated that palmitate induced cytotoxicity after entry and conversion into palmitoyl-CoA. Palmitoyl-CoA is not only degraded by mitochondrial oxidation but also taken up as a component of membrane phospholipids. Palmitate overloading causes cardiomyocyte membrane fatty acid (FA) saturation, which is associated with the activation of endoplasmic reticulum (ER) unfolded protein response (UPR) signaling. We focused on the ER UPR signaling as a possible mechanism of cell death. Palmitate loading activates the UPR signal via membrane FA saturation, but not via unfolded protein overload in the ER since the chemical chaperone 4-phenylbutyrate failed to suppress palmitate-induced ER UPR. The mammalian UPR relies on three ER stress sensors named inositol requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Palmitate loading activated only IRE1 and PERK. Knockdown of PERK did not affect palmitate-induced cardiomyocyte death, while knockdown of IRE1 suppressed palmitate-induced cardiomyocyte death. However, knockdown of X-box binding protein 1 (XBP1), the downstream effector of IRE1, did not affect palmitate-induced cardiomyocyte death. These results were validated by pharmacological inhibitor experiments.In conclusion, we identified that palmitate-induced cardiomyocyte death was triggered by IRE1-mediated signaling independent of XBP1.

Production of conjugated linoleic acid by heterologous expression of linoleic acid isomerase in oleaginous fungus Mortierella alpina.

To increase the commercial value of oleaginous fungus Mortierella alpina by incorporation of trans-10,cis-12 conjugated linoleic acid (CLA) into the polyunsaturated fatty acids (PUFAs) of M. alpina via Propionibacterium acnes isomerase (PAI) conversion. The PAI gene and the codon optimization version were heterologously expressed in M. alpina via Agrobacterium tumefaciens-mediated transformation (ATMT). Coding usage modification significantly improved the translation of PAI transcripts and trans-10,cis-12 CLA was produced up to 1.2 mg l(-1), which corresponds to approx. 0.05% of the total fatty acid (TFA). Since PAI prefers free linoleic acid as a substrate rather than any other forms, 5 μM long-chain acyl CoA synthetase inhibitor was added and the trans-10,cis-12 CLA content increased approx. 24-fold to 29 mg l(-1), reaching up to 1.2% (w/w) of the TFA in M. alpina. Heterologous expression of PAI in M. alpina by ATMT methods is a practicable way in biosynthesis of CLA and this system may be a feasible platform for industrial production of CLA.

Modulation of Triglyceride and Cholesterol Ester Synthesis Impairs Assembly of Infectious Hepatitis C Virus

In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.

ACSL3 and GSK‐3β are essential for lipid upregulation induced by endoplasmic reticulum stress in liver cells

The endoplasmic reticulum (ER) is essential for lipid biosynthesis, and stress signals in this organelle are thought to alter lipid metabolism. Elucidating the mechanisms that underlie the dysregulation of lipid metabolism in hepatocytes may lead to novel therapeutic approaches for the treatment of lipid accumulation. We first tested the effects of several inhibitors on lipid dysregulation induced by tunicamycin, an ER stress inducer. Triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL) 1, 3, and 4, was the most potent among these inhibitors. We then analyzed the expression of the ACSL family during ER stress. The expression of ACSL3 was induced by ER stress in HuH-7 cells and in mice livers. ACSL3 shRNA, but not ACSL1 shRNA, inhibited the induction of lipid accumulation. GSK-3β inhibitors attenuated ACSL3 expression and the lipid accumulation induced by ER stress in HuH-7 cells. shRNA that target GSK-3β also inhibited the upregulation of ACSL3 and lipid accumulation in HuH-7 and HepG2 cells. The hepatitis B virus mutant large surface protein, which is known to induce ER stress, increased the lipid content of cells. Similarly, Triacsin C, and GSK-3β inhibitors abrogated the lipid dysregulation caused by the hepatitis B virus mutant large surface protein. Altogether, ACSL3 and GSK-3β represent novel therapeutic targets for lipid dysregulation by ER stress.

A Sequence Variation (I148M) in PNPLA3 Associated with Nonalcoholic Fatty Liver Disease Disrupts Triglyceride Hydrolysis

Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is associated with increased hepatic triglyceride content in humans. Here we examined the effect of the I148M substitution on the enzymatic activity and cellular location of PNPLA3. Structural modeling predicted that the substitution of methionine for isoleucine at residue 148 would restrict access of substrate to the catalytic serine at residue 47. In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. Expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content. Cell fractionation studies revealed that ∼90% of wild type PNPLA3 partitioned between membranes and lipid droplets; substitution of isoleucine for methionine at position 148 did not alter the subcellular distribution of the protein. These data are consistent with PNPLA3-I148M promoting triglyceride accumulation by limiting triglyceride hydrolysis.

AMPK activator, AICAR, inhibits palmitate-induced apoptosis in osteoblast

Osteoblast apoptosis reduces bone mineral density. Apoptosis can be induced in a variety of cells by palmitate, which is one of the most common saturated fatty acids in dietary fat. The AMPK activator, AICAR, has been shown to inhibit palmitate-induced apoptosis. However, the role of palmitate in osteoblast apoptosis is currently unknown. This study examined whether palmitate could induce apoptosis in osteoblasts, and if so, whether AICAR could alleviate palmitate-induced apoptosis. Palmitate reduced cell survival and induced apoptosis in a dose- and time-dependent manner in human fetal osteoblasts (hFOB) 1.19. While the long-chain acyl-CoA synthetase inhibitor, triacsin C, inhibited palmitate-induced apoptosis, anti-oxidants and ceramide synthesis inhibitors did not attenuate the apoptosis. AICAR prevented palmitate-induced apoptosis and the inhibition of AICAR-mediated increase in fatty acid oxidation by etomoxir did not affect the prevention of apoptosis by AICAR. Constitutively-active AMPK also inhibited palmitate-induced apoptosis. Treatment with an AMPK inhibitor (compound C) and a dominant-negative AMPK adenovirus suppressed the inhibitory effect of AICAR on apoptosis. Palmitate impaired the activation of ERK by fetal bovine serum, which was blocked by AICAR. Moreover, AICAR increased ERK activation, and ERK inhibitors, PD98059 and U0126, as well as a dominant-negative MEK1, abolished the inhibitory effect of AICAR on palmitate-induced apoptosis. AICAR also inhibited palmitate-induced apoptosis in osteoblastic differentiated cells from human bone marrow, which was accompanied by recovered ERK activity. These results suggest that palmitate induces apoptosis in osteoblasts through the impaired activation of ERK, and the activation of AMPK inhibits palmitate-induced apoptosis by activating ERK.

Mode of action of mood stabilizers: is the arachidonic acid cascade a common target?

Bipolar disorder is a major medical, social and economic burden worldwide. However, the mechanisms of action of effective antibipolar disorder drugs remain elusive. In this paper, we review studies using a neuropharmacological approach in unanesthetized rats, combined with kinetic, biochemical and molecular biology techniques, showing that chronic administration of three Food and Drug Administration-approved mood stabilizers (lithium, valproate and carbamazepine) at therapeutically relevant doses, selectively target the brain arachidonic acid (AA) cascade. Whereas chronic lithium and carbamazepine decrease the binding activity of activator protein-2 and in turn the transcription, translation and activity of its AA-selective calcium-dependent phospholipase A(2) gene product, valproate appears to be a non-competitive inhibitor of long-chain acyl-CoA synthetase. The net overlapping effects of the three drugs are decreased turnover of AA but not of docosahexaenoic acid in rat brain phospholipids, and decreased brain cyclooxygenase-2 and prostaglandin E(2). Although these observations support the hypothesis proposed by Rapoport and colleagues that the AA cascade is a common target of mood stabilizers, this hypothesis is not necessarily exclusive of other targets. Targeting the AA cascade with drugs or diet may be a useful therapeutic approach in bipolar disorder, and examining the AA cascade in patients might help in better understanding the disease.

Unsaturated Fatty Acids Phosphorylate and Destabilize ABCA1 through a Phospholipase D2 Pathway

Abnormal high density lipoprotein (HDL) metabolism among patients with diabetes and insulin resistance may contribute to their increased risk of atherosclerosis. ATP-binding cassette transporter ABCA1 mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Unsaturated fatty acids, which are elevated in diabetes, impair the ABCA1 pathway in cultured cells by destabilizing ABCA1 protein. Here we examined the cellular pathway that mediates the ABCA1 destabilizing effects of fatty acids. The long-chain acyl-CoA synthetase inhibitor triacsin C completely reversed fatty acid-induced ABCA1 destabilization, indicating that fatty acids need to be activated to their CoA derivatives to enhance ABCA1 degradation. Unsaturated but not saturated fatty acids stimulated phospholipase D (PLD) activity, the PLD inhibitor 1-butanol prevented the unsaturated fatty acid-induced reduction in ABCA1 levels, and the PLD2 activator mastoparan markedly reduced ABCA1 protein levels, implicating a role for PLD2 in the ABCA1 destabilizing effects of fatty acids. Unsaturated fatty acids and mastoparan increased phosphorylation of ABCA1 serines. PLD2 small interfering RNA abolished the ability of unsaturated fatty acids to inhibit lipid transport activity, to reduce protein levels, and to increase serine phosphorylation of ABCA1. The diacylglycerol analog oleoylacetylglycerol also reduced ABCA1 protein levels and increased its serine phosphorylation, suggesting that PLD2-generated diacylglycerols promote the destabilizing phosphorylation of ABCA1. These data provide evidence that intracellular unsaturated acyl-CoA derivatives destabilize ABCA1 by activating a PLD2 signaling pathway.

Triacsin C inhibits the formation of 1H NMR-visible mobile lipids and lipid bodies in HuT 78 apoptotic cells

Nuclear magnetic resonance-visible mobile lipids (ML) have been reported to accumulate during cell apoptosis in vitro and in vivo. The biogenesis, biochemical nature and structure of these lipids are still under debate. In this study, a human lymphoblastoid cell line, HuT 78, was induced to apoptosis by exposure to anti-Fas monoclonal antibodies (α-Fas mAb) followed by incubation for different time intervals (1–24 h, hypodiploid cell fraction, H, varying from 1% to over 60%) either in the presence or in the absence of 5.0 μM Triacsin C (TRC), specific inhibitor of long-chain acyl-CoA synthetase (ACS). The increase of ML in apoptotic cells correlated linearly with H and was associated with: (a) accumulation of intracellular lipid bodies, detected by confocal laser scanning microscopy in lipophilic dye-stained cells; (b) increases, detected by thin-layer chromatography in total lipid extracts, in the relative abundance of triacylglycerides (TAG) and cholesteryl esters (CE), with corresponding decreases of phospholipids (PL). TRC completely abolished both ML and lipid body formation in anti-Fas-treated apoptotic cells, with concomitant reversion of TAG, CE and PL to control levels, but did not alter cell viability nor did it inhibit apoptosis. ML signals detected during anti-Fas-induced apoptosis therefore appear to originate from neutral lipids assembled in intracellular lipid bodies, synthesised from cellular acyl-CoA pools.

Irreversible deacylation of plasma membrane phospholipids by the combined action of Mg 2+ and a long-chain acyl-CoA synthetase inhibitor in Saccharomyces cerevisiae

A long-chain acyl-CoA synthetase (Faap) inhibitor, adenosine 5'-hexadecylphosphate (AMPC16), caused lethal plasma membrane damage to Saccharomyces cerevisiae cells as reflected by the leakage of cytoplasmic K + into medium in which Mg 2+ was supplemented at 10 mM. AMPC16 did not interfere with the de novo synthesis of phospholipids using acetyl-CoA as a starting material, but the AMP analog accelerated the liberation of long- chain fatty acids from phospholipids with the aid of exogenous Mg 2+, suggesting that the role of this divalent cation is to maximize the rate of the acyl group turnover. This Mg 2+-dependent fungicidal effect of AMPC16 was similarly observed with a mutant lacking any of the phospholipase B (Plbp) isozymes, whereas it was mostly suppressed in a phospholipase C (Plc1p) deletion mutant in which the liberation of fatty acids from phospholipids was completely prevented. These results suggest that Plc1p is normally functional for phospholipid reconstitution in exponentially growing cells and enhancement of its activity by exogenous Mg 2+ could be a cause of the irreversible deacylation of plasma membrane phospholipids when their reacylation is blocked at the step of activation of long-chain fatty acids by one of the Faap isozymes.

Irreversible Deacylation of Plasma Membrane Phospholipids by the Combined Action of Mg2+ and a Long-Chain Acyl-CoA Synthetase Inhibitor in Saccharomyces cerevisiae

A long-chain acyl-CoA synthetase (Faap) inhibitor, adenosine 5'-hexadecylphosphate (AMPC16), caused lethal plasma membrane damage to Saccharomyces cerevisiae cells as reflected by the leakage of cytoplasmic K+ into medium in which Mg2+ was supplemented at 10 mM. AMPC16 did not interfere with the de novo synthesis of phospholipids using acetyl-CoA as a starting material, but the AMP analog accelerated the liberation of long- chain fatty acids from phospholipids with the aid of exogenous Mg2+, suggesting that the role of this divalent cation is to maximize the rate of the acyl group turnover. This Mg2+-dependent fungicidal effect of AMPC16 was similarly observed with a mutant lacking any of the phospholipase B (Plbp) isozymes, whereas it was mostly suppressed in a phospholipase C (Plc1p) deletion mutant in which the liberation of fatty acids from phospholipids was completely prevented. These results suggest that Plc1p is normally functional for phospholipid reconstitution in exponentially growing cells and enhancement of its activity by exogenous Mg2+ could be a cause of the irreversible deacylation of plasma membrane phospholipids when their reacylation is blocked at the step of activation of long-chain fatty acids by one of the Faap isozymes.

Engineering of Glycerol-stimulated Insulin Secretion in Islet Beta Cells: DIFFERENTIAL METABOLIC FATES OF GLUCOSE AND GLYCEROL PROVIDE INSIGHT INTO MECHANISMS OF STIMULUS-SECRETION COUPLING

Insulin secretion from beta cells in the islets of Langerhans can be stimulated by a number of metabolic fuels, including glucose and glyceraldehyde, and is thought to be mediated by metabolism of the secretagogues and an attendant increase in the ATP:ADP ratio. Curiously, glycerol fails to stimulate insulin secretion, even though it has been reported that islets contain abundant glycerol kinase activity and oxidize glycerol efficiently. We have reinvestigated this point and find that rat islets and the well differentiated insulinoma cell line INS-1 contain negligible glycerol kinase activity. A recombinant adenovirus containing the bacterial glycerol kinase gene (AdCMV-GlpK) was constructed and used to express the enzyme in islets and INS-1 cells, resulting in insulin secretion in response to glycerol. In AdCMV-GlpK-treated INS-1 cells a greater proportion of glycerol is converted to lactate and a lesser proportion is oxidized compared with glucose. The two fuels are equally potent as insulin secretagogues, despite the fact that oxidation of glycerol at its maximally effective dose (2-5 mM) occurs at a rate that is similar to the rate of glucose oxidation at its basal, nonstimulatory concentration (3 mM). We also investigated the possibility that glycerol may signal via expansion of the glycerol phosphate pool to allow enhanced fatty acid esterification and formation of complex lipids. Addition of Triacsin-C, an inhibitor of long-chain acyl-CoA synthetase, to AdCMV-GlpK-treated INS-1 cells did not inhibit glycerol-stimulated insulin secretion despite the fact that it blocked glycerol incorporation into cellular lipids. We conclude from these studies that glycerol kinase expression is sufficient to activate glycerol signaling in beta cells, showing that the failure of normal islets to respond to this substrate is due to a lack of this enzyme activity. Further, our studies show that glycerol signaling is not linked to esterification or oxidation of the substrate, but is likely mediated by its metabolism in the glycerol phosphate shuttle and/or the distal portion of the glycolytic pathway, either of which can lead to production of ATP and an increased ATP:ADP ratio.

Fatty acyl-coenzyme a is required for budding of transport vesicles from Golgi cisternae

We describe a new role for fatty acylation. Conditions were established under which vesicular transport from the cis to the medial Golgi compartment in vitro depends strongly upon the addition of a fatty acylcoenzyme A, e.g., palmitoyl-CoA. Using an inhibitor of long-chain acyl-CoA synthetase, we demonstrate that the fatty acid has to be activated by CoA to stimulate transport. A nonhydrolyzable analog of palmitoyl-CoA competitively inhibits transport. Electron microscopy and biochemical studies show that fatty acyl-CoA is required for budding of (non-clathrin-) coated transport vesicies from Golgi cisternae and that budding is inhibited by the nonhydrolyzable analog.