Carnitine palmitoyltransferase I (CPT-I)

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 [1]. 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 [2].

Etomoxir is a transcription factor that is involved in free equilibrium (FFA) oxidation and is linked to the important enzyme CPT1. Further supporting the direct response of P53 and Bax, as well as the role of FAO-mediated catalytic ROS production in the db/db model, is the fact that P53 directly responds to Bax, and Bax is blocked by etomoxir [3]. At a dose of 20 mg/kg body weight, etomoxir was injected once daily for eight days, resulting in a 44% reduction in specific CPT-I activity. The catalyst CPT-I activity was 44% lower in the catalyst treated with etomoxir. Lewis's blood glucose levels remained unchanged after 8 days of treatment with 20 mg/kg Etomoxir, in line with previous Etomoxir feeding trials. Similarly, etomoxir feeding had no effect on harvest mass in the hindlimb or on general growth traits like weight gain. However, in those treated with etomoxir, both liver and heart mass grew significantly by 11% [4].

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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].

Biochemical measurement[3]
Blood samples were centrifuged at 3000 rpm for 10 minutes at 4 °C. Serum FFA was measured using an ELISA kit according to the manufacturer’s instructions. Serum triglyceride (TG), HDL and LDL were detected by commercial assay kits. Serum alkaline phosphatase (ALP), osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) were measured to assess osteogenic and osteoclastic activity using the Elisa assay kits.

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Measurement of lipid peroxidation[3]
PBMCs were isolated and purified by isopycnic centrifugation using Histopaque-1119 and Histopaque-1077. Lipid peroxidation in PBMCs and differentiated osteoblasts were evaluated by the measurement of thiobarbituric acid-reactive substances (TBARS) levels using a commercial kit according to the manufacturer’s instructions.

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].

Animal treatment[3]
All animal experiments were performed according to the procedures approved by Fourth Military Medical University Animal Care and Use Committee and were carried out in accordance with the approved guidelines. 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.

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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]

[1]. The use of partial fatty acid oxidation inhibitors for metabolic therapy of angina pectoris and heart failure. Herz. 2002 Nov;27(7):621-36.

[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.

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

(2R)-2-[6-(4-chlorophenoxy)hexyl]-2-epoxyethylene carboxylate is an aromatic ether.

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Some fatty acid oxidation inhibitors have attracted considerable interest because they hold promise for combating gene expression dysregulation in hypertrophic cardiomyocytes. Some of these compounds have been developed for the treatment of non-insulin-dependent diabetes mellitus and stable angina. Shifting from fatty acid oxidation to glucose oxidation reduces gluconeogenesis and improves cardiac efficiency. The following enzyme inhibitors can increase glucose oxidation: etomoxifenoxine, oxyphenidyl palmitate, S-15176, metoprolol, amiodarone, piperacillin (carnitine palmitoyltransferase-1); aminocarnitine, piperacillin (carnitine palmitoyltransferase-2); hydrazone (carnitine-acylcarnitine translocase). MET-88 (γ-butyryl betaine hydroxylase); 4-bromocrotonic acid, trimetazidine, and possibly ranolazine (thiolase); hypoglycine (butyryl-CoA dehydrogenase); dichloroacetic acid (pyruvate dehydrogenase kinase). Clinical trials of trimetazidine and ranolazine have shown that this substrate oxidation shift has antianginal effects. Etomoxicillin and MET-88 improve overloaded cardiac function by increasing the density of the sarcoplasmic reticulum calcium pump (SERCA2). The promoters of SERCA2 and α-myosin heavy chain contain sequences that are expected to respond to transcription factors of glucose metabolites and/or peroxisome proliferator-activated receptor (PPAR) agonists. Further elucidation of novel compounds that upregulate SERCA2 expression is closely related to the characterization of SERCA2 promoter regulatory sequences. [1] We investigated the effect of etomoxicillin treatment on de novo synthesis of cardiolipin (CL) in H9c2 cardiomyocytes. Etomoxicillin treatment did not affect the activity of CL synthesis and remodeling enzymes, but it led to a decrease in the amount of [1-14C]palmitic acid or [1-14C]oleic acid incorporated into CL. The mechanism was that, due to a 35% increase in membrane phosphatidylphosphatase activity (P < 0.05), lipid synthesis shifted to the use of 1,2-diacyl-sn-glycerol, thereby reducing the flux of fatty acids via the de novo CL synthesis pathway. Conversely, etomoxicillin treatment increased the amount of [1,3-3H]glycerol incorporated into CL. This mechanism manifested as a 33% increase in glycerol kinase activity (P < 0.05), resulting in an increased glycerol flux via the de novo cardiolipin (CL) synthesis pathway. Etomoxicillin treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thus diverting glycerol and fatty acids from the synthesis of 1,2,3-triacyl-sn-glycerol to the synthesis of phosphatidylcholine and phosphatidylethanolamine. Conversely, etomoxib inhibited inositol-[3H] incorporation into phosphatidylinositol by inhibiting inositol uptake. Etomoxib did not affect the uptake of [3H]serine, but led to an increase in the production of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxib treatment has different effects on the de novo synthesis of glycerides from various metabolic precursors. Furthermore, etomoxib mediates a unique and differentiated metabolic pathway for glycerol and the fatty acid precursor centripetal phospholipid (CL). [2]

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This study assessed the association between free fatty acid (FFA), reactive oxygen species (ROS) production, mitochondrial dysfunction, and bone mineral density (BMD) in patients with type 2 diabetes and explored the molecular mechanisms involved. db/db mice and high-fat (HF) fed mice were treated with etomoxib (a CPT1 inhibitor), MitoQ, and the P53 inhibitor PFT-α, respectively. Bone metabolism factors were assessed, bone marrow mesenchymal stem cells (BMSCs) were isolated, and their differentiation into osteoblasts was induced. FFA, lipid peroxidation, and mitochondrial DNA copy number are associated with BMD in patients with type 2 diabetes. Etomoxir, MitoQ, and PFT-α significantly inhibited the decline in bone mineral density and fracture strength in db/db mice and high-fat diet-fed mice, and suppressed the reduction in the differentiation of bone marrow mesenchymal stem cells into osteoblasts. Etomoxir and MitoQ (but not PFT-α) inhibited the increase in mitochondrial reactive oxygen species (ROS) production in db/db mice, high-fat diet-fed mice, and osteoblasts. Furthermore, Etomoxir, MitoQ, and PFT-α significantly inhibited mitochondrial dysfunction in osteoblasts. Moreover, mitochondrial apoptosis was activated in osteoblasts derived from db/db mice and high-fat diet-fed mice, while Etomoxir, MitoQ, and PFT-α inhibited this apoptosis. In addition, the accumulation of p53 in mitochondria recruited Bax and initiated the molecular events of apoptosis. These results indicate that fatty acid oxidation leads to the generation of reactive oxygen species (ROS), which activates P53/Bax-mediated mitochondrial apoptosis, resulting in reduced osteogenic differentiation and bone loss in patients with type 2 diabetes mellitus (T2DM). [3] Although carnitine palmitoyltransferase I (CPT-I) is generally considered to be the main rate-limiting site of mitochondrial β-oxidation, it is not entirely clear whether CPT-I is the rate-limiting step for total long-chain fatty acid (LCFA) flux in the heart. Another important regulatory site for LCFA flux in the heart is the transmuscular LCFA transport mediated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we investigated the extent to which long-term pharmacological blockade of LCFA flux at the mitochondrial level affects LCFA uptake in the muscular membrane. Rats were injected daily with saline or the specific CPT-I inhibitor etomoxicillin at a dose of 20 mg/kg body weight for 8 consecutive days. Etomoxicillin-treated rats had a 44% reduction in cardiac CPT-I activity. Compared with the control group, the levels of CD36 and FABPpm in the myocardial membrane of rats treated with etoromoxib and the transport capacity of long-chain fatty acids (LCFA) were not changed. In addition, the rates of LCFA uptake and oxidation and glucose uptake in cardiomyocytes of rats treated with etoromoxib were not different from those of rats in the control group, whether under basal metabolic state or acute induced maximal metabolic demand. Finally, no triglyceride accumulation was observed in the hearts of rats treated with etoromoxib. Therefore, CPT-I does not appear to be the main rate-limiting site for total cardiac LCFA flux. The entry of myocardial long-chain fatty acids (LCFA) into cells, rather than mitochondrial long-chain fatty acid-coenzyme A (LCFA-CoA), may be a promising target for the normalization of LCFA flux in the treatment of cardiometabolic diseases. [4]

C17H23CLO4

326.82

326.128

C, 62.48; H, 7.09; Cl, 10.85; O, 19.58

124083-20-1

Etomoxir sodium salt;828934-41-4; 82258-36-4 (racemate) 124083-20-1 (free acid); 828934-40-3 (S-isomer); 132308-39-5 (potassium salt)

9840324

Colorless to light yellow solid (<32°C),or liquid (>34°C)

1.2±0.1 g/cm3

405.0±25.0 °C at 760 mmHg

142.6±22.2 °C

0.0±0.9 mmHg at 25°C

1.520

4.46

0

4

11

22

342

1

O=C(OCC)[C@@]1(OC1)CCCCCCOC2=CC=C(C=C2)Cl

DZLOHEOHWICNIL-QGZVFWFLSA-N

InChI=1S/C17H23ClO4/c1-2-20-16(19)17(13-22-17)11-5-3-4-6-12-21-15-9-7-14(18)8-10-15/h7-10H,2-6,11-13H2,1H3/t17-/m1/s1

Ethyl (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate

(R)-(+)-Etomoxir B 807-54 B80754 B-80754B807-54 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)

DMSO : ~100 mg/mL (~305.98 mM)

Solubility in Formulation 1: 3.5 mg/mL (10.71 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 35.0 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 2: ≥ 3.5 mg/mL (10.71 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 35.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2.5 mg/mL (7.65 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 ultrasonication.
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 4: 10 mg/mL (30.60 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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