Acyl-CoA:cholesterol acyltransferase (ACAT)

Lecimibide (DuP 128) (10 μM, 24 h) can block 85% of the HepG2 cells' cellular esterification reaction [1]. Researchers observed a reduction in cholesterol synthesis following incubation of HepG2 cells with the ACAT inhibitors Lecimibide/DuP 128 and CI-1011. This may explain the reduced hepatic HMG-CoA reductase activities observed in rats consuming glycosylated forms of naringenin and hesperetin.

In pigs fed high fat and frozen 36% and 31%, respectively, Lecimibide (DuP 128)(iv, 2.2 mg/kg/day) significantly decreased total triglyceride and very low-density lipoprotein (VLDL) triglyceride concentrations. Total cholesterol, VLDL cholesterol, LDL cholesterol, HDL cholesterol, and LDL apoB content were not significantly affected [2].

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To further test the hypothesis that newly synthesized cholesteryl esters regulate hepatic apolipoprotein B (apoB) secretion into plasma, apoB kinetic studies were carried out in seven control miniature pigs and in seven animals after 21 days intravenous administration of the acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor Lecimibide/DuP 128 (2.2 mg/kg/day). Pigs were fed a fat (34% of calories; polyunsaturated/monounsaturated/saturated ratio, 1:1:1) and cholesterol (400 mg/day; 0.1%; 0.2 mg/kcal) containing pig chow based diet. DuP 128 significantly reduced total plasma triglyceride and very low density lipoprotein (VLDL) triglyceride concentrations by 36 and 31%, respectively (P<0.05). Autologous 131I-VLDL and 125I-LDL were injected simultaneously into each pig and apoB kinetic data was analyzed using multicompartmental analysis (SAAM II). The VLDL apoB pool size decreased by 26% (0.443 vs. 0.599 mg/kg; P<0. 001) which was due entirely to a 28% reduction in VLDL apoB production or secretion rate (1.831 vs. 2.548 mg/kg/h; P=0.006). The fractional catabolic rate (FCR) for VLDL apoB was unchanged. The LDL apoB pool size and production rate were unaffected by DuP 128 treatment. Hepatic microsomal ACAT activity decreased by 51% (0.44 vs. 0.90 nmol/min/mg; P<0.001). Although an increase in hepatic free cholesterol and subsequent decrease in both LDL receptor expression and LDL apoB FCR might be expected, this did not occur. The concentration of hepatic free cholesterol decreased 12% (P=0.008) and the LDL apoB FCR were unaffected by Lecimibide/DuP 128 treatment. In addition, DuP 128 treatment did not alter the concentration of hepatic triglyceride or the activity of diacylglycerol acyltransferase, indicating a lack of effect of DuP 128 on hepatic triglyceride metabolism. In our previous studies, DuP 128 treatment of miniature pigs fed a low fat, cholesterol free diet, decreased VLDL apoB secretion by 65% resulting in a reduction in plasma apoB of 60%. We conclude that in miniature pigs fed a high fat, cholesterol containing diet, the inhibition of hepatic cholesteryl ester synthesis by Lecimibide/DuP 128 decreases apoB secretion into plasma, but the effect is attenuated relative to a low fat, cholesterol free diet [2].

Lipid synthesis and CE hydrolysis [1]
The incorporation of [1-14C]oleic acid or [1-14C]acetic acid into cellular lipids was determined as described previously. Radioactivity incorporated into CE, TG, and phospholipids (PL) was determined after separation of the lipid species by thin layer chromatography. Incorporation of [14C]oleic acid into CE was used as a measure of whole cell ACAT activity. CE hydrolysis was determined following a 24-h preincubation with [14C]oleic acid (in the absence of the flavonoids) to label an intracellular pool of CE. This was followed by incubations of 2–24 h in the presence of the flavonoids. The latter incubations were carried out in the presence of the specific ACAT inhibitor, Lecimibide/DuP 128 (10 μM), to inhibit cholesterol esterification. [14C]-oleic acid incorporation into cellular CE was determined as described above.

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ACAT1 and ACAT2 activities [1]
The direct effect of naringenin and hesperetin on ACAT1 and ACAT2 activities was determined in AC29 cells expressing either enzyme. In whole cells, the incorporation of [14C]oleic acid into cellular CE was measured in the presence or absence of naringenin or hesperetin over 5 h, essentially as described above. In further experiments, microsomes were isolated from AC29 cells expressing either ACAT. Cells were scraped from the plates and disrupted by sonication in ice-cold buffer containing 0.1 M phosphate, 0.25 M sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 μg/ml leupeptin at pH 7.4. Microsomes were isolated as in the method of Carr, Parks, and Rudel, and frozen at −80 °C until analysis. ACAT activity was determined in the presence of exogenous cholesterol, according to the methods of Billheimer, Tavani, and Nes, using 25–75 μg of microsomes as the source of ACAT. Naringenin and hesperetin were dissolved in DMSO (0.5% per assay) and added along with a cholesterol/triton WR-1339 buffer for 15 min at 37 °C prior to a 10-min incubation with the radiolabeled substrate. Results were compared with those obtained using 10 μM of the ACAT inhibitors Lecimibide/DuP 128 and CI-1011. Previously, we established in HepG2 cells that 10 μM of Lecimibide/DuP 128 or CI-1011 inhibited cellular esterification by 85% and 61%, respectively. Furthermore, Cases et al. reported that, in membranes from ACAT1 and ACAT2 baculovirus-infected insect cells, CI-1011 inhibited ACAT2 with an IC50 of 2.5 μM and ACAT1 with an IC50 of 10 μM.

Miniature pigs weighing 22.3±0.7 kg were obtained from a local supplier. After being acclimatized for one week, animals were maintained on the experimental diet for 21 days before, and during the lipoprotein turnover studies. One week prior to the turnover study, an indwelling silicone elastomer (Silastic) catheter (1.96 mm internal diameter) was surgically implanted in an external jugular vein. Isoflurane USP was used as the anesthetic and ketamine USP as the preanesthetic. Catheters that were kept patent by filling with 7% EDTA-Na2, allowed for ease of sample injection, as well as blood sampling throughout each turnover study in unrestrained, unanesthetized animals. Pigs were studied in pairs, with each pair being same sex littermates. Seven animals received the ACAT inhibitor Lecimibide/DuP 128, at a dose of 50 mg/day, whereas seven control animals received the vehicle alone [2].

[1]. Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP. J Lipid Res. 2001 May;42(5):725-34.

[2]. Inhibition of cholesterol esterification by DuP 128 decreases hepatic apolipoprotein B secretion in vivo: effect of dietary fat and cholesterol. Biochim Biophys Acta. 1998 Jul 31;1393(1):63-79.

The citrus flavonoids, naringenin and hesperetin, lower plasma cholesterol in vivo. However, the underlying mechanisms are not fully understood. The ability of these flavonoids to modulate apolipoprotein B (apoB) secretion and cellular cholesterol homeostasis was determined in the human hepatoma cell line, HepG2. apoB accumulation in the media decreased in a dose-dependent manner following 24-h incubations with naringenin (up to 82%, P < 0.00001) or hesperetin (up to 74%, P < 0.002). Decreased apoB secretion was associated with reduced cellular cholesteryl ester mass. Cholesterol esterification was decreased, dose-dependently, up to 84% (P < 0.0001) at flavonoid concentrations of 200 microM. Neither flavonoid demonstrated selective inhibition of either form of acyl CoA:cholesterol acyltransferase (ACAT) as determined using CHO cells stably transfected with either ACAT1 or ACAT2. However, in HepG2 cells, ACAT2 mRNA was selectively decreased (- 50%, P < 0.001) by both flavonoids, whereas ACAT1 mRNA was unaffected. In addition, naringenin and hesperetin decreased both the activity (- 20% to - 40%, P < 0.00004) and expression (- 30% to - 40%, P < 0.02) of microsomal triglyceride transfer protein (MTP). Both flavonoids caused a 5- to 7-fold increase (P < 0.02) in low density lipoprotein (LDL) receptor mRNA, which resulted in a 1.5- to 2-fold increase in uptake and degradation of (125)I-LDL. We conclude that both naringenin and hesperetin decrease the availability of lipids for assembly of apoB-containing lipoproteins, an effect mediated by 1) reduced activities of ACAT1 and ACAT2, 2) a selective decrease in ACAT2 expression, and 3) reduced MTP activity. Together with an enhanced expression of the LDL receptor, these mechanisms may explain the hypocholesterolemic properties of the citrus flavonoids. [1]

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The reduction in VLDL apoB production did not translate into a reduction in either the conversion of VLDL apoB to LDL, or the LDL apoB pool size. Both the conversion of VLDL apoB to LDL apoB and percent of VLDL flux converted to LDL were increased. This did not appear to be due to VLDL particle composition or size distribution as shown in Table 4 and Fig. 1. A decrease in hepatic LDL receptor expression is associated with increased conversion of VLDL to LDL. However, Lecimibide/DuP 128 treatment did not alter either the LDL apoB FCR (largely determined by LDL receptor expression) or the VLDL apoB FCR. It is possible that the activities of lipoprotein lipase and/or hepatic lipase, both of which regulate VLDL conversion, were increased by Lecimibide/DuP 128; however, the activities of these enzymes were not measured in the present study.
The lack of an effect of Lecimibide/DuP 128 on LDL apoB concentrations was not due to a concomitant decrease in LDL clearance. Theoretically, the inhibition of ACAT would result in an increase in hepatic free cholesterol in a regulatory pool such that hepatic LDL receptor expression would be decreased. As in our previous studies, DuP 128 had no effect on LDL apoB FCR, and the hepatic free cholesterol concentration decreased with DuP 128 treatment rather than increased. This is consistent with the idea that the ACAT substrate pool is not tightly coupled to the free cholesterol pool that regulates LDL receptor expression [2].

C34H40F2N4OS

590.77

590.289

C, 69.12; H, 6.82; F, 6.43; N, 9.48; O, 2.71; S, 5.43

130804-35-2

71355

Typically exists as solid at room temperature

1.21g/cm3

1.613

9.802

2

5

16

42

751

0

CCCCCCCN(C(NC1=C(F)C=C(F)C=C1)=O)CCCCCSC2=NC(C3=CC=CC=C3)=C(N2)C4=CC=CC=C4

TVXOXGBTADZYCZ-UHFFFAOYSA-N

InChI=1S/C34H40F2N4OS/c1-2-3-4-5-13-22-40(34(41)37-30-21-20-28(35)25-29(30)36)23-14-8-15-24-42-33-38-31(26-16-9-6-10-17-26)32(39-33)27-18-11-7-12-19-27/h6-7,9-12,16-21,25H,2-5,8,13-15,22-24H2,1H3,(H,37,41)(H,38,39)

3-(2,4-difluorophenyl)-1-[5-[(4,5-diphenyl-1H-imidazol-2-yl)sulfanyl]pentyl]-1-heptylurea

DuP-128; Lecimibide; 130804-35-2; DuP128; lecimibida; Lecimibide [USAN:INN]; Lecimibide (USAN); UNII-A7T248B302; LECIMIBIDE [INN]; DuP 128; Lecimibide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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