Carnitine palmitoyltransferase I (CPT-I)

In H9c2 cells, etomoxir facilitates the distinct metabolic channeling of fatty acid and glycerol precursors into cardiolipin[2]. Although etomoxir reduces the incorporation of [1-14C]palmitic acid or [1-14C]oleic acid into cardiolipin, it has no effect on the activity of the enzymes involved in the biosynthesis and remodeling of cardiolipin[2]. Cardiolipin's incorporation of [1,3-3H]glycerol is increased by etomoxir. The process involves a 33% rise in glycerol kinase activity, which causes an increase in glycerol flux via the cardiolipin biosynthesis de novo pathway [2].

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When etomoxir binds irreversibly to CPT-1's catalytic site, it inhibits CPT-1's activity while simultaneously increasing transplantation oxidase. Etomoxir was created as a probe for the outer mitochondrial membrane-localized mitochondrial carnitine scaffold amplification enzyme-1 (CPT-1). Etomoxir stimulates DNA synthesis and myocardial development in the myocardium by acting as an oxisome proliferator. Consequently, etomoxan is regarded as a PPARalpha agonist in addition to a CPT1. Etomoxir has been proposed as a target for activation of cardiac mutations. It is a member of the ethylene oxide kinase carnitine template transferase I family. Carnitine template transferase I activity is irreversibly transcribed upon activation of Etomoxir therapy. Consequently, there is a decrease in base import as mitochondria and beta-oxidation, leading to an increase in cytoplasmic accumulation and oxidation. Etomoxir's long delays (24 hours) have even distinct impacts on the expression of enzymes.

Etomoxir suppresses the reduction of BMSCs-differentiated osteoblasts and significantly inhibits the decrease of bone mineral density (BMD) and bone breaking strength in mice fed high fat (HF) and db/db diets[3]. In mice fed HF and db/db, etopoxir inhibits the increase in mitochondrial ROS generation in osteoblasts and mice[3]. The in vivo partial inhibition of carnitine palmitoyltransferase-I (CPT-I) caused by etomoxir does not change the rates of cardiac long-chain fatty acid uptake and oxidation[4].

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This study evaluated the association between free fatty acid (FFA), ROS generation, mitochondrial dysfunction and bone mineral density (BMD) in type 2 diabetic patients and investigated the molecular mechanism. db/db and high fat (HF)-fed mice were treated by Etomoxir, an inhibitor of CPT1, MitoQ, and PFT-α, an inhibitor of P53. Bone metabolic factors were assessed and BMSCs were isolated and induced to osteogenic differentiation. FFA, lipid peroxidation and mtDNA copy number were correlated with BMD in T2DM patients. Etomoxir, MitoQ and PFT-α significantly inhibited the decrease of BMD and bone breaking strength in db/db and HF-fed mice and suppressed the reduction of BMSCs-differentiated osteoblasts. Etomoxir and MitoQ, but not PFT-α, inhibited the increase of mitochondrial ROS generation in db/db and HF-fed mice and osteoblasts. In addition, Etomoxir, MitoQ and PFT-α significantly inhibited mitochondrial dysfunction in osteoblasts. Moreover, mitochondrial apoptosis was activated in osteoblasts derived from db/db and HF-fed mice, which was inhibited by Etomoxir, MitoQ and PFT-α. Furthermore, mitochondrial accumulation of P53 recruited Bax and initiated molecular events of apoptotic events. These results demonstrated that fatty acid oxidation resulted in ROS generation, activating P53/Bax-mediated mitochondrial apoptosis, leading to reduction of osteogenic differentiation and bone loss in T2DM.[3]

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Although CPT-I (carnitine palmitoyltransferase-I) is generally regarded to present a major rate-controlling site in mitochondrial beta-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall LCFA (long-chain fatty acid) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg of body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity, were not altered in the hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and oxidation, and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. It is likely that sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases[4].

shRNA-GFP lentiviral plasmid construction and infection[1]
The puromycin resistance gene in 5 individual shRNA lentiviral plasmids against CPT1A was replaced with GFP cDNA using standard molecular biology techniques. Briefly, a 1.4 kb GFP cDNA insert was subcloned from the lentiviral PELPS-GFP plasmid into KpnΙ and BamHΙ sites in the pLKO.1 shRNA plasmid. The efficacy of each individual lentiviral plasmid was determined by immunoblot analysis. Lentiviral infections were performed as described36. Lymphocytes expressing shRNA against CPT1A were compared with the corresponding control plasmid that we also engineered to express GFP instead of puromycin resistance. The control plasmid encoded a scrambled shRNA sequence from the human β-actin gene. The efficiency of lentiviral infection ranged from 60–90% across experiments. In assessments of cell proliferation, enumeration was performed using bead-based counting methods following gating on GFP + cells. The titers for scramble-GFP and shRNA-CPT1A-GFP viral supernatants were 9.45e6 and 7.34e6 TU/ml, respectively.[1]

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Immunoblotting[1]
CPT1A protein expression was assessed 5 days following shRNA lentiviral infection. Cells were lysed in RIPA-2 (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) containing protease and phosphatase inhibitors. Equal amounts of lysate were separated by SDS-PAGE and transferred electrophoretically to a PVDF membrane. Membranes were incubated in 5% nonfat milk in TBS (20 mM Tris, 135 mM NaCl) containing 0.1% Tween-20 (TBS-T) for 1 hr. After blocking, the membranes were probed with a 1:500 dilution of anti-CPT1A in 0.5% nonfat milk in TBS-T. After a series of washes in TBS-T, the membranes were incubated with followed by a 1:10,000 dilution of HRP-conjugated goat anti-rabbit IgG. Antibody binding was detected using West Femto SuperSignal chemiluminescent reagents. Relative protein loading was determined with a 1:2,000 dilution of mouse monoclonal antibody to β-Actin followed by a 1:5,000 dilution of HRP-conjugated sheep anti-mouse IgG.

Cell Viability Assay[2]
Cell Types: Rat heart H9c2 myoblastic cells
Tested Concentrations: 1-80 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: decreased the incorporation of [1-14C]fatty acids into CL and PtdGro in H9c2 cardiac myoblast cells but did not affect total incorporation of radioactivity into these cells.

Animal/Disease Models: 80 male C57BLKS/J lar-Leprdb/db mice[3]
Doses: 1 mg/kg
Route of Administration: Intraperitoneally injected; twice every week
Experimental Results: Serum alkaline phosphatase was increased in db/db mice, which event was Dramatically suppressed by Etomoxir. Serum level of osteocalcin, a marker of bone formation, was decreased in db/db mice and Etomoxir markedly inhibited the reduction of osteocalcin. Serum tartrate-resistant acid phosphatase was elevated in db/db mice which phenomenon was Dramatically suppressed by Etomoxir.

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Animal/Disease Models: Rats[4]
Doses: 20 mg/kg
Route of Administration: Injected daily; for 8 days
Experimental Results: Etomoxir-treated rats displayed a 44% decreased cardiac CPT-I activity.

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Male Lewis rats, weighing 150–200 g, were used in the present study. Animals were kept on a 12 h:12 h light/dark cycle and fed a Purina Chow diet and water ad libitum. The rats were divided into two groups: (1) control and (2) etomoxir. Etomoxir (20 mg/kg of body weight) was dissolved in 0.9% (w/v) NaCl and administered intraperitoneally for 8 days. Control rats received saline. The last injection was given 24 h before the experiment. Ethical approval for all experimental procedures was obtained from the Experimental Animal Committee of the Maastricht University, and the study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Animals were anaesthetized with an intraperitoneal injection of a nembutal and heparin (3:1) mixture. Subsequently, the heart was removed for LCFA uptake studies and for analyses of transporter protein contents. [4]

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80 male C57BLKS/J lar-Leprdb/db mice and 20 wild type littermates (8 week) were obtained from Model Animal Research Centre, Nanjing University, China. Mice were housed in cages in a limited access room, under temperature (23 ± 2 °C) and humidity (55 ± 5%) condition with a standard light (12 h light/dark) cycle and fed a regular diet. db/db mice were randomly divided into four groups: db/db group, Etomoxir group, MitoQ group, and PFT-α group. In the Etomoxir group, mice were intraperitoneally injected with 1 mg/kg Etomoxir twice every week. In the MitoQ group, 50 μmol/L MitoQ was given to the mice in water. Water bottles, containing either MitoQ, were covered with aluminum foil, and all bottles were refilled every 3 days. In the PFT-α group, mice were intraperitoneally injected with 1 mg/kg PFT-α twice every week. WT mice were administrated with vehicle instead. The experimental period is 8 weeks. At the end, peripheral blood samples and bone marrow cells were harvested for the assays. 100 C57BL/6 mice obtained from Experimental Animal Centre of Fourth Military Medical University. The mice were randomly divided into five groups: Control group, HF diet group, Etomoxir group, MitoQ group, and PFT-α group. Mice in HF diet, Etomoxir, MitoQ, and PFT-α groups were given high fat diet for 20 weeks and mice in Etomoxir, MitoQ, and PFT-α groups were administrated with Etomoxir, MitoQ, and PFT-α in the last 10 weeks. The administration of Etomoxir, MitoQ, and PFT-α were identical to the treatment in db/db mice. Control mice were administrated with vehicle instead [3].

[1]. The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations.Sci Rep. 2018 Apr 19;8(1):6289.

[2]. Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells.J Lipid Res. 2003 Feb;44(2):415-23.

[3]. FFA-ROS-P53-mediated mitochondrial apoptosis contributes to reduction of osteoblastogenesis and bone mass in type 2 diabetes mellitus.Sci Rep. 2015 Jul 31;5:12724.

[4]. Etomoxir-induced partial carnitine palmitoyltransferase-I (CPT-I) inhibition in vivo does not alter cardiac long-chain fatty acid uptake and oxidation rates.Biochem J. 2009 Apr 15;419(2):447-55.

Etomoxir (ETO) is a widely used small-molecule inhibitor of fatty acid oxidation (FAO) through its irreversible inhibitory effects on the carnitine palmitoyl-transferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following costimulation through the CD28 receptor. We show that ETO has a moderate effect on T cell proliferation with no observable effect on memory differentiation, but a marked effect on oxidative metabolism. We show that this oxidative metabolism is primarily dependent upon glutamine rather than FAO. Using an shRNA approach to reduce CPT1a in T cells, we further demonstrate that the inhibition of oxidative metabolism in T cells by ETO is independent of its effects on FAO at concentrations exceeding 5 μM. Concentrations of ETO above 5 μM induce acute production of ROS with associated evidence of severe oxidative stress in proliferating T cells. In aggregate, these data indicate that ETO lacks specificity for CTP1a above 5 μM, and caution should be used when employing this compound for studies in cells due to its non-specific effects on oxidative metabolism and cellular redox[1].

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We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL. [2]

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This study evaluated the association between free fatty acid (FFA), ROS generation, mitochondrial dysfunction and bone mineral density (BMD) in type 2 diabetic patients and investigated the molecular mechanism. db/db and high fat (HF)-fed mice were treated by Etomoxir, an inhibitor of CPT1, MitoQ, and PFT-α, an inhibitor of P53. Bone metabolic factors were assessed and BMSCs were isolated and induced to osteogenic differentiation. FFA, lipid peroxidation and mtDNA copy number were correlated with BMD in T2DM patients. Etomoxir, MitoQ and PFT-α significantly inhibited the decrease of BMD and bone breaking strength in db/db and HF-fed mice and suppressed the reduction of BMSCs-differentiated osteoblasts. Etomoxir and MitoQ, but not PFT-α, inhibited the increase of mitochondrial ROS generation in db/db and HF-fed mice and osteoblasts. In addition, Etomoxir, MitoQ and PFT-α significantly inhibited mitochondrial dysfunction in osteoblasts. Moreover, mitochondrial apoptosis was activated in osteoblasts derived from db/db and HF-fed mice, which was inhibited by Etomoxir, MitoQ and PFT-α. Furthermore, mitochondrial accumulation of P53 recruited Bax and initiated molecular events of apoptotic events. These results demonstrated that fatty acid oxidation resulted in ROS generation, activating P53/Bax-mediated mitochondrial apoptosis, leading to reduction of osteogenic differentiation and bone loss in T2DM. [3]

C15H18CLO4.NA

320.74

320.079

C, 56.17; H, 5.66; Cl, 11.05; Na, 7.17; O, 19.95

828934-41-4

Etomoxir;124083-20-1; 828934-41-4 (sodium); 82258-36-4 (racemate)

57345784

Typically exists as white to off-white solids at room temperature

2.188

0

4

9

21

321

1

C([C@]1(OC1)C(=O)O)CCCCCOC1C=CC(Cl)=CC=1.[Na]

RPACBEVZENYWOL-XFULWGLBSA-M

InChI=1S/C15H19ClO4.Na/c16-12-5-7-13(8-6-12)19-10-4-2-1-3-9-15(11-20-15)14(17)18;/h5-8H,1-4,9-11H2,(H,17,18);/q;+1/p-1/t15-;/m1./s1

(2R)-2-[6-(4-chlorophenoxy)hexyl]-2-oxiranecarboxylic acid monosodium salt

B 80754; B 807-54; B80754; B807-54; ETOMOXIR; 124083-20-1; R-(+)-Etomoxir; Etomoxir [INN]; UNII-MSB3DD2XP6; MSB3DD2XP6; ethyl (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate; (R)-(+)-Etomoxir; B-807-54; B-80754

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (7.79 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.79 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.79 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 3.33 mg/mL (10.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)