Bezafibrate at Clinically Relevant Doses Decreases Serum/Liver Triglycerides via Down-Regulation of Sterol Regulatory Element-Binding Protein-1c in Mice: A Novel Peroxisome Proliferator-Activated Receptor -Independent Mechanism

The triglyceride-lowering effect of bezafibrate in humans has been attributed to peroxisome proliferator-activated receptor (PPAR) activation based on results from rodent studies. However, the bezafibrate dosages used in conventional rodent experiments are typically higher than those in clinical use ( 50 versus 10 mg/kg/day), and thus it remains unclear whether such data can be translated to humans. Furthermore, because bezafibrate is a pan-PPAR activator, the actual contribution of PPAR to its triglyceride-lowering properties remains undetermined. To address these issues, bezafibrate at clinically relevant doses (10 mg/kg/day; low) was administered to wild-type and Ppara-null mice, and its effects were compared with those from conventionally used doses (100 mg/kg/day; high). Pharmacokinetic analyses showed that maximum plasma concentration and area under the concentration-time curve in bezafibrate-treated mice were similar to those in humans at low doses, but not at high doses. Low-dose bezafibrate decreased serum/liver triglycerides in a PPAR -independent manner by attenuation of hepatic lipogenesis and triglyceride secretion. It is noteworthy that instead of PPAR activation, down-regulation of sterol regulatory element-binding protein (SREBP)-1c was observed in mice undergoing low-dose treatment. High-dose bezafibrate decreased serum/liver triglycerides by enhancement of hepatic fatty acid uptake and -oxidation via PPAR activation, as expected. In conclusion, clinically relevant doses of bezafibrate exert a triglyceride-lowering effect by suppression of the SREBP-1c-regulated pathway in mice and not by PPAR activation. Our results may provide novel information about the pharmacological mechanism of bezafibrate action and new insights into the treatment of disorders involving SREBP-1c. Bezafibrate and other fibrate drugs are clinically used as hypolipidemic agents to preferentially lower serum triglyceride (TG) levels. Several large-scale clinical trials have demonstrated a relationship between the TG-lowering effect of fibrates and a reduction in the risk of cardiovascular events in patients with dyslipidemia, type 2 diabetes mellitus, and metabolic syndrome (BIP Study Group, 2000; Keech et al., 2005; Tenenbaum et al., 2005). The mechanisms accounting for the hypolipidemic effect of fibrates in humans are explained mainly as an increase in the lipolysis of TG-rich lipoproteins, such as very-low-density lipoprotein (VLDL), This research was supported by the Intramural Research Program of the National Institutes of Health National Cancer Institute. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.052928. ABBREVIATIONS: TG, triglyceride; Apo, apolipoprotein; ACC, acetyl-CoA carboxylase; AUC, area under the plasma concentration-time curve; Cmax, maximum plasma concentration; CPT, carnitine palmitoyl-CoA transferase; FAS, fatty acid synthase; FAT, fatty acid translocase; FATP, fatty acid transport protein; FFA, free fatty acid; FXR, farnesoid X receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPAT, glycerol-3phosphate acyltransferase; LC/MS/MS, high-performance liquid chromatography/tandem mass spectrometry; LPL, lipoprotein lipase; LXR, liver X receptor; MCAD, medium-chain acyl-CoA dehydrogenase; MTP, microsomal TG transfer protein; PCR, polymerase chain reaction; PDK, pyruvate dehydrogenase kinase; PMP70, 70-kDa peroxisomal membrane protein; PPAR, peroxisome proliferator-activated receptor; SCAP, SREBP cleavage-activating protein; SCD, stearoyl-CoA desaturase; SHP, short heterodimer partner; SREBP, sterol regulatory element-binding protein; Tmax, time to reach Cmax; VLDL, very-low-density lipoprotein. 0026-895X/09/7504-782–792 MOLECULAR PHARMACOLOGY Vol. 75, No. 4 U.S. Government work not protected by U.S. copyright 52928/3447440 Mol Pharmacol 75:782–792, 2009 Printed in U.S.A. 782 at A PE T Jornals on O cber 8, 2017 m oharm .aspeurnals.org D ow nladed from and a decrease in VLDL secretion from the liver (Staels et al., 1998). These effects are believed to be mediated by activation of the nuclear receptor peroxisome proliferator-activated receptor (PPAR) . PPAR is highly expressed in the liver, heart, and kidney and functions as a central regulator of fatty acid and TG metabolism (Aoyama et al., 1998; Nakajima et al., 2004; Kamijo et al., 2007). A number of studies using mice lacking the PPAR gene (Ppara-null mice) have revealed the direct contribution of PPAR to the TG-lowering effect of fibrates; for example, PPAR activation by bezafibrate suppresses the transcriptional expression of apolipoprotein (Apo) CIII (Peters et al., 2003), an endogenous inhibitor of lipoprotein lipase (LPL) and hepatic TG lipase, leading to an enhancement of lipoprotein lipolysis. Fibrate-induced PPAR activation also increases hepatic expression of fatty acid transporters and -oxidation enzymes (Aoyama et al., 1998; Motojima et al., 1998). These experimental results from rodent models have helped to understand the possible mechanisms of fibrate action in humans. However, two important issues arise when rodent data on the TG-reducing effect of bezafibrate are used to explain the mechanism of this drug in humans. One is the uncertainty about the actual contribution of PPAR to its effect, because bezafibrate can activate other PPAR subtypes [ ( ) and ] (Brown et al., 1999) and lower serum TG levels even in Ppara-null mice (Peters et al., 2003). Another issue is the dosage discrepancy between previous rodent studies (typically 50 mg/kg/day) and human clinical practice ( 10 mg/ kg/day) (Peters et al., 2003; Hays et al., 2005; Nagasawa et al., 2006). As such, it remains unclear whether the earlier experimental conditions used in rodents accurately reflect the conditions in bezafibrate-treated humans. In the present study, the contribution of PPAR to the TG-lowering effect of bezafibrate was examined using wildtype and Ppara-null mice administered the drug at clinically relevant doses (10 mg/kg/day), and its effects were compared with those at conventional experimental doses (100 mg/kg/ day). We were surprised to find that the mechanism of bezafibrate TG reduction differed according to dose and at low doses was independent of PPAR . Materials and Methods Mice and Bezafibrate Treatment. All experiments were conducted in accordance with animal care guidelines approved by the Shinshu University School of Medicine. Ppara-null mice on a Sv/129 genetic background were described elsewhere (Lee et al., 1995; Akiyama et al., 2001). The mice were housed in a temperatureand light-controlled environment (25°C; 12-h light/dark cycle) and maintained with tap water ad libitum and a 7% fat-containing standard