RESUMO
Cholesteryl ester transfer protein (CETP) plays a key role in high-density lipoprotein (HDL) cholesterol metabolism, but normal mice are deficient in CETP. In this study, transgenic mice expressing both human apolipoprotein B 100 (ApoB-100) and human CETP (hApoB100/hCETP) were used to characterize the effects of CETP inhibition and peroxisome proliferator-activated receptor alpha (PPARalpha) agonism on lipid profiles. Torcetrapib (3, 10, and 30 mg/kg), a CETP inhibitor, fenofibrate (30 mg/kg), a weak PPARalpha agonist, and GW590735 (3 and 10 mg/kg), a potent and selective PPARalpha agonist were given orally for 14 days to hApoB100/hCETP mice and lipid profiles were assessed. The average percentages of HDL, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) cholesterol fractions in hApoB100/hCETP mice were 34.8%, 61.6%, and 3.6%, respectively, which is similar to those of normolipidemic humans. Both torcetrapib and fenofibrate significantly increased HDL cholesterol and reduced LDL cholesterol, and there was a tendency for torcetrapib to reduce VLDL cholesterol and triglycerides. GW590735 significantly increased HDL cholesterol, decreased LDL and VLDL cholesterol, and significantly reduced triglycerides. Maximal increases in HDL cholesterol were 37%, 53%, and 84% with fenofibrate, torcetrapib, and GW590735, respectively. These results, in mice that exhibit a more human-like lipid profile, demonstrate an improved lipid profile with torcetrapib, fenofibrate, and GW590735, and support the use of selective PPARalpha agonism for the treatment of lipid disorders. In addition, these data demonstrate the use of hApoB100/hCETP transgenic mice to identify, characterize, and screen compounds that increase HDL cholesterol.
Assuntos
Anticolesterolemiantes/farmacologia , Apolipoproteína B-100/genética , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , HDL-Colesterol/sangue , LDL-Colesterol/sangue , PPAR alfa/agonistas , Animais , Proteínas de Transferência de Ésteres de Colesterol/genética , VLDL-Colesterol/sangue , Relação Dose-Resposta a Droga , Fenofibrato/farmacologia , Humanos , Camundongos , Camundongos Transgênicos , Propionatos/farmacologia , Quinolinas/administração & dosagem , Quinolinas/farmacologia , Tiazóis/farmacologiaRESUMO
Liver X receptor (LXR) activation promotes reverse cholesterol transport (RCT) in rodents but has major side effects (increased triglycerides and LDL-cholesterol levels) in species expressing cholesteryl ester transfer protein (CETP). In the face of dyslipidemia, it remains unclear whether LXR activation stimulates RCT in CETP species. We therefore used a hamster model made dyslipidemic with a 0.3% cholesterol diet and treated with vehicle or LXR agonist GW3965 (30 mg/kg bid) over 10 days. To investigate RCT, radiolabeled (3)H-cholesterol macrophages or (3)H-cholesteryl oleate-HDL were then injected to measure plasma and feces radioactivity over 72 or 48 h, respectively. The cholesterol-enriched diet increased VLDL-triglycerides and total cholesterol levels in all lipoprotein fractions and strongly increased liver lipids. Overall, GW3965 failed to improve both dyslipidemia and liver steatosis. However, after (3)H-cholesterol labeled macrophage injection, GW3965 treatment significantly increased the (3)H-tracer appearance by 30% in plasma over 72 h, while fecal (3)H-cholesterol excretion increased by 156% (P < 0.001). After (3)H-cholesteryl oleate-HDL injection, GW3965 increased HDL-derived cholesterol fecal excretion by 64% (P < 0.01 vs. vehicle), while plasma fractional catabolic rate remained unchanged. Despite no beneficial effect on dyslipidemia, LXR activation promotes macrophage-to-feces RCT in dyslipidemic hamsters. These results emphasize the use of species with a more human-like lipoprotein metabolism for drug profiling.
Assuntos
Benzoatos/farmacologia , Benzilaminas/farmacologia , Colesterol/metabolismo , Dislipidemias/metabolismo , Receptores Nucleares Órfãos/agonistas , Animais , Benzoatos/uso terapêutico , Benzilaminas/uso terapêutico , Ácidos e Sais Biliares/análise , Transporte Biológico/efeitos dos fármacos , Doenças Cardiovasculares/tratamento farmacológico , Linhagem Celular , Colesterol/administração & dosagem , Colesterol/sangue , Cricetinae , Modelos Animais de Doenças , Dislipidemias/sangue , Dislipidemias/etiologia , Fígado Gorduroso/etiologia , Fezes/química , Regulação da Expressão Gênica/efeitos dos fármacos , Absorção Intestinal/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas/sangue , Lipoproteínas HDL/administração & dosagem , Lipoproteínas HDL/sangue , Lipoproteínas HDL/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado , Macrófagos/metabolismo , Mesocricetus , Camundongos , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Stearoyl-CoA Desaturase 1 (SCD1) is a central enzyme that catalyzes the biosynthesis of monounsaturated fatty acids from saturated fatty acids. SCD1 is an emerging target in obesity and insulin resistance due to the improved metabolic profile obtained when the enzyme is genetically inactivated. Here, we have investigated if the pharmacological inhibition of SCD1 could elicit the same profile. We have identified a small molecule, GSK993 and characterized it as a potent and orally available SCD1 inhibitor. In Zucker(fa/fa) rats, GSK993 exerted a marked reduction in hepatic lipids as well as a significant improvement of glucose tolerance. Furthermore, in a diet-induced insulin resistant rat model, GSK993 induced a very strong reduction in Triton-induced hepatic Very Low Density Lipoprotein-Triglyceride production. In addition, following a hyperinsulinemic-euglycemic clamp in GSK993-treated animals, we observed an improvement in the whole body insulin sensitivity as reflected by an increase in the glucose infusion rate. Taken together, these findings demonstrate that the pharmacological inhibition of SCD1 translates into improved lipid and glucose metabolic profiles and raises the interest of SCD1 inhibitors as potential new drugs for the treatment of insulin resistance.
Assuntos
Inibidores Enzimáticos/farmacologia , Resistência à Insulina , Insulina/farmacologia , Isoquinolinas/farmacologia , Pirazóis/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Bovinos , Linhagem Celular Tumoral , Dieta/efeitos adversos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Glucose/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , RatosRESUMO
We describe the discovery of novel potent inhibitors of 2,3-oxidosqualene:lanosterol cyclase inhibitors (OSCi) from a focused pharmacophore-based screen. Optimization of the most tractable hits gave a series of compounds showing inhibition of cholesterol biosynthesis at 2mg/kg in the rat with distinct pharmacokinetic profiles. Two compounds were selected for toxicological study in the rat for 21 days in order to test the hypothesis that low systemic exposure could be used as a strategy to avoid the ocular side effects previously described with OSCi. We demonstrate that for this series of inhibitors, a reduction of systemic exposure is not sufficient to circumvent cataract liabilities.
Assuntos
Catarata/enzimologia , Dislipidemias/enzimologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Olho/efeitos dos fármacos , Transferases Intramoleculares/antagonistas & inibidores , Animais , Anticolesterolemiantes/efeitos adversos , Anticolesterolemiantes/síntese química , Anticolesterolemiantes/farmacocinética , Catarata/induzido quimicamente , Catarata/tratamento farmacológico , Linhagem Celular Tumoral , Dislipidemias/induzido quimicamente , Inibidores Enzimáticos/efeitos adversos , Olho/metabolismo , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Oxazóis/farmacocinética , Oxazóis/uso terapêutico , Piperazinas/efeitos adversos , Piperazinas/síntese química , Piperazinas/farmacocinética , Piperidinas/farmacocinética , Piperidinas/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
The alkaloid drug berberine (BBR) was recently described to decrease plasma cholesterol and triglycerides (TGs) in hypercholesterolemic patients by increasing expression of the hepatic low density lipoprotein receptor (LDLR). Using HepG2 human hepatoma cells, we found that BBR inhibits cholesterol and TG synthesis in a similar manner to the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Significant increases in AMPK phosphorylation and AMPK activity were observed when the cells were incubated with BBR. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK, correlated with a subsequent increase in fatty acid oxidation. All of these effects were abolished by the mitogen-activated protein kinase kinase inhibitor PD98059. Treatment of hyperlipidemic hamsters with BBR decreased plasma LDL cholesterol and strongly reduced fat storage in the liver. These findings indicate that BBR, in addition to upregulating the LDLR, inhibits lipid synthesis in human hepatocytes through the activation of AMPK. These effects could account for the strong reduction of plasma TGs observed with this drug in clinical trials.
Assuntos
Adenilato Quinase/metabolismo , Berberina/farmacologia , Hipolipemiantes/farmacologia , Lipídeos/biossíntese , Acetil-CoA Carboxilase/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Linhagem Celular Tumoral , Cricetinae , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Fosforilação/efeitos dos fármacos , Receptores de LDL/metabolismoRESUMO
Plasma levels of low-density lipoprotein (LDL) cholesterol are considered to be a major risk factor for the development of cardiovascular diseases. The LDL receptor is the key component in the maintenance of cholesterol homeostasis in the body, playing a pivotal role by regulating the hepatic catabolism of LDL cholesterol. Many clinical studies using statins, which up-regulate the LDL receptor expression via a feedback mechanism, have demonstrated that the reduction of LDL cholesterol levels lowers the incidence of cardiovascular events in both primary and secondary prevention. In this context, new strategies designed to increase hepatic LDL receptor activity can be considered as attractive opportunities for future therapy. Several potential new drugs have been described in the last decade to up-regulate LDL receptor expression in vitro and in vivo, thus allowing the identification of new transcriptional and post-transcriptional mechanisms.
Assuntos
Receptores de LDL/metabolismo , Animais , Hormônios/farmacologia , Humanos , Fígado/metabolismo , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Proteínas Quinases/metabolismo , Receptores de LDL/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
Imidazoline-like drugs are centrally-acting antihypertensive agents that inhibit the activity of the sympathetic nervous system by interacting with the alpha2-adrenoreceptor and also with a non-adrenergic imidazoline binding site called the imidazoline 1 receptor. Recently, these molecules were proposed to play an additional role in cardiovascular diseases by acting on glucose and lipid metabolism. We used S23515, a potent imidazoline-like molecule acting selectively on blood pressure through the imidazoline 1 receptor, to decipher the effects of these drugs on lipid metabolism. We found that S23515 inhibited specifically and dose-dependently cholesterol synthesis in cultured rodent and primate hepatocytes. This hypocholesterolemic effect was likely due to the inhibition of the oxido:lanosterol cyclase (OSC), a rate-limiting enzyme in the cholesterol biosynthetic pathway. Partial OSC inhibition induced by S23515 led to the generation of 24(S),25-epoxycholesterol, a potent ligand for the liver X receptor (LXR). Furthermore, S23515 increased in human macrophages the expression of both ABCA1 and G1, the 2 ATP binding cassette transporters, which play a pivotal role in the reverse cholesterol transport. Thus, these results suggest that S23515, and potentially other imidazoline-like drugs, could exert hypolipidemic effects in addition to their hypotensive activities.
Assuntos
Inibidores Enzimáticos/farmacologia , Hepatócitos/enzimologia , Transferases Intramoleculares/antagonistas & inibidores , Metabolismo dos Lipídeos , Oxazóis/farmacologia , Animais , Atorvastatina , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Primers do DNA , Hepatócitos/efeitos dos fármacos , Ácidos Heptanoicos/farmacologia , Cinética , Neoplasias Hepáticas , Reação em Cadeia da Polimerase , Pirróis/farmacologia , RatosRESUMO
Up-regulation of low-density lipoprotein receptor (LDLr) is a key mechanism to control elevated plasma LDL-cholesterol levels. In the present paper, we compare the ability of four distinct pharmacological drugs to up-regulate LDLr expression in human hepatocytes. HepG2 cells were treated with the steroidal analog GW707, the oxidosqualene cyclase inhibitor U18666A, the 3beta-hydroxysterol Delta(7)-reductase inhibitor AY-9944 and the vacuolar-type ATPase inhibitor bafilomycin A1. We found that the four compounds induced sequestration of free cholesterol in the endosomal/lysosomal compartment leading to a positive filipin staining pattern and a complete inhibition of cholesteryl ester synthesis. As a consequence of the sequestration of cholesterol, the expression and the activity of LDLr were strongly induced resulting from a transcriptional effect which was measured by a reporter gene assay. These effects were fully abolished when an exogenous water soluble cholesterol analog was added to the cells. These findings have led to the identification of a common mechanism to up-regulate LDLr expression in human hepatocytes and may represent an interesting alternative approach to identify new hypolipidemic drugs.
Assuntos
Colesterol/metabolismo , Endossomos/metabolismo , Hepatócitos/metabolismo , Lisossomos/metabolismo , Receptores de LDL/biossíntese , Androstenos/farmacologia , Anticolesterolemiantes/farmacologia , Colesterol/biossíntese , Endossomos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Macrolídeos/farmacologia , Esteroides/farmacologia , Células Tumorais Cultivadas , Regulação para Cima , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano/farmacologiaRESUMO
Recently, a new class of lipid-lowering agents has been described that upregulate LDL receptor (LDLr) activity. These agents are proposed to activate sterol-regulated gene expression through binding to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Here, we show that the steroidal LDLr upregulator, GW707, induces accumulation of lysosomal free cholesterol and inhibits LDL-stimulated cholesterol esterification, similar to that observed in U18666A-treated cells and in Niemann-Pick type C1 (NPC1) mutants. Moreover, we demonstrate that induction of the NPC-like phenotype by GW707 is independent of SCAP function. We find that treatment with GW707 does not increase SREBP-dependent gene expression above that observed in lipoprotein-starved cells. Rather, we show that the apparent increase in SREBP-dependent activity in GW707-treated cells is attributable to a failure to appropriately suppress sterol-regulated gene expression, as has been shown previously for U18666A-treated cells and NPC mutant fibroblasts. We further demonstrate that cells treated with either GW707 or U18666A fail to appropriately generate 27-hydroxycholesterol in response to LDL cholesterol. Taken together, these findings support a mechanism in which GW707 exerts its hypolipidemic effects through disruption of late endosomal/lysosomal sterol trafficking and subsequent stimulation of LDLr activity.