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Comparative Study
. 2009 Apr;75(4):782-92.
doi: 10.1124/mol.108.052928. Epub 2009 Jan 5.

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 alpha-independent mechanism

Takero Nakajima  1 , Naoki Tanaka, Hiroki Kanbe, Atsushi Hara, Yuji Kamijo, Xiaowei Zhang, Frank J Gonzalez, Toshifumi Aoyama
Affiliations
Comparative Study

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 alpha-independent mechanism

Takero Nakajima et al. Mol Pharmacol. 2009 Apr.

Abstract

The triglyceride-lowering effect of bezafibrate in humans has been attributed to peroxisome proliferator-activated receptor (PPAR) alpha activation based on results from rodent studies. However, the bezafibrate dosages used in conventional rodent experiments are typically higher than those in clinical use (> or =50 versus < or =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 PPARalpha 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 PPARalpha-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 beta-oxidation via PPARalpha 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 PPARalpha 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.

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Figures

Fig. 1.
Fig. 1.
Plasma pharmacokinetics of bezafibrate in wild-type (+/+) and Ppara-null (-/-) mice. Seven-day pharmacokinetics of bezafibrate at 10, 30, 60, or 100 mg/kg/day in wild-type (A) and Ppara-null (B) mice. Plasma concentration of bezafibrate was measured using the LC/MS/MS technique. Data are expressed as mean ± S.D. (n = 3 at each time point).
Fig. 2.
Fig. 2.
Histological appearance of hematoxylin-and-eosin-stained liver sections from bezafibrate-treated wild-type (+/+) and Ppara-null (-/-) mice. Bezafibrate at high (100 mg/kg/day) or low (10 mg/kg/day) doses was orally administered to wild-type and Ppara-null mice for 7 days. No steatosis was found in the livers of wild-type mice. Hepatic steatosis in Ppara-null mice was markedly ameliorated by low-dose bezafibrate. Hepatic inflammation or hepatocyte degeneration was not detected in any group. Scale bars in the upper and lower panels of control wild-type mice indicate 200 and 50 μm, respectively.
Fig. 3.
Fig. 3.
Effects of bezafibrate on fatty acid β-oxidation and uptake in the livers of wild-type (+/+) and Ppara-null (-/-) mice. Hepatic expression of mRNAs encoding peroxisomal (A) and mitochondrial (B) fatty acid β-oxidation enzymes, proteins related to lipoprotein lipolysis (C), and fatty acid transporters (D) was analyzed by quantitative real-time PCR and normalized to that of GAPDH mRNA. Relative mRNA levels are shown as -fold changes of those of control wild-type mice. Data are expressed as mean ± S.D. (n = 6 in each group). White bar, control group; gray bar, low-dose bezafibrate (10 mg/kg/day) group; black bar, high-dose bezafibrate (100 mg/kg/day) group. *, P < 0.05; **, P < 0.01; ***, P < 0.001, between treated and untreated mice of the same genotype.
Fig. 4.
Fig. 4.
Effects of bezafibrate on de novo lipogenesis and TG secretion in the livers of wild-type (+/+) and Ppara-null (-/-) mice. A and C, hepatic expression of mRNAs encoding lipogenic enzymes (A) and proteins associated with VLDL secretion (C) was analyzed by quantitative real-time PCR and normalized to that of GAPDH mRNA. Relative mRNA levels are shown as -fold changes of those of control wild-type mice. Data are expressed as mean ± S.D. (n = 6 in each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 between treated and untreated mice of the same genotype. B and D, immunoblot analysis of lipogenic enzymes (B) and MTP (D). Fifty micrograms of whole-liver lysate proteins from each mouse were loaded into each well. Actin was used as a loading control. Band intensity was quantified densitometrically and normalized to that of actin. Relative protein levels are shown as -fold changes of those of control wild-type mice. Data are expressed as mean ± S.D. (n = 6 in each group). *, P < 0.05; **, P < 0.01, between treated and untreated mice of the same genotype. White bars, control group; gray bars, low-dose bezafibrate (10 mg/kg/day) group; black bars, high-dose bezafibrate (100 mg/kg/day) group.
Fig. 5.
Fig. 5.
Effects of bezafibrate on SREBP-1c in the livers of wild-type (+/+) and Ppara-null (-/-) mice. A and C, hepatic expression of mRNAs encoding regulators of lipogenic enzymes (A) and SREBP-1c processing (C) was analyzed by quantitative real-time PCR and normalized to that of GAPDH mRNA. Relative mRNA levels are shown as -fold changes of those of control wild-type mice. Data are expressed as mean ± S.D. (n = 6 in each group). *, P < 0.05; **, P < 0.01, between treated and untreated mice of the same genotype. B and D, immunoblot analysis of nuclear SREBP-1c (B) and SCAP (D). For detection of nuclear SREBP-1c, hepatic nuclear fractions were prepared from each mouse, and 50 μg of nuclear protein was loaded into each well. For SCAP detection, 50 μg of whole-liver lysate proteins from each mouse were loaded into each well. Histone H1 and actin were used as loading controls for nuclear SREBP-1c and SCAP, respectively. Band intensity was quantified densitometrically and normalized to that of histone H1 or actin. Relative protein levels are shown as -fold changes of those of control wild-type mice. Data are expressed as mean ± S.D. (n = 6 in each group). *, P < 0.05; **, P < 0.01, between treated and untreated mice of the same genotype. White bars, control group; gray bars, low-dose bezafibrate (10 mg/kg/day) group; black bars, high-dose bezafibrate (100 mg/kg/day) group.
Fig. 6.
Fig. 6.
Effects of bezafibrate on PPARs in the livers of wild-type (+/+) and Ppara-null (-/-) mice. A and D, hepatic expression of mRNAs encoding PPARs (A) and PPARβ target genes (ACC2, PDK4, PDK2, and adipose differentiation-related protein) and PPARγ target gene (adipocyte fatty acid-binding protein) (D) was analyzed by quantitative real-time PCR and normalized to that of GAPDH mRNA. Relative mRNA levels are shown as -fold changes of those of control wild-type mice. Data are expressed as mean ± S.D. (n = 6 in each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001, between treated and untreated mice of the same genotype. B, immunoblot analysis of PMP70. Fifty micrograms of whole-liver lysate proteins from each mouse were loaded into each well. Actin was used as a loading control. Band intensity was quantified densitometrically and normalized to that of actin. Relative protein levels are shown as -fold changes of those of control mice. Data are expressed as mean ± S.D. (n = 6 in each group). ***, P < 0.001, between treated and untreated wild-type mice. C, cytochemical staining and morphometry of hepatic peroxisomes. Peroxisomes were detected as dark particles, and marked peroxisome proliferation was found under high-dose bezafibrate treatment only. Scale bars in the light (upper) and electron (lower) photomicrographs of control mice indicate 50 and 10 μm, respectively. Arrows indicate erythrocytes. The number of peroxisomes and area of each individual peroxisomal profile were measured in 10 electron photomicrographs from each mouse, and numerical and volume densities were calculated. Data are expressed as mean ± S.D. (n = 6 in each group). **, P < 0.01, between treated and untreated wild-type mice. White bars, control group; gray bars, low-dose bezafibrate (10 mg/kg/day) group; black bars, high-dose bezafibrate (100 mg/kg/day) group.
Fig. 7.
Fig. 7.
Effects of bezafibrate on PPARα and SREBP-1c at two intermediate doses in the livers of wild-type (+/+) mice. A and B, hepatic expression of mRNAs encoding PPARα and its targets (CPT-I and MCAD) (A) and SREBP-1c and its targets (ACC1 and FAS) (B) was analyzed by quantitative real-time PCR and normalized to that of GAPDH mRNA. Relative mRNA levels are shown as -fold changes of those of control mice. Data are expressed as mean ± S.D. (n = 6 in each group). C, immunoblot analysis of nuclear SREBP-1c, ACC1, and FAS. For detection of nuclear SREBP-1c, hepatic nuclear fractions (50 μg of protein) were loaded into each well. For analysis of ACC1 and FAS, whole liver lysates (50 μg of protein) were adopted. Histone H1 and actin were used as loading controls. Band intensity was quantified densitometrically and normalized to that of histone H1 or actin. Relative protein levels are shown as -fold changes of those of control mice. Data are expressed as mean ± S.D. (n = 6 in each group). *, P < 0.05, between treated and untreated mice. White bar, control group; gray bar, 30 mg/kg/day bezafibrate group; black bar, 60 mg/kg/day bezafibrate group. *P < 0.05, **P < 0.01, between treated and untreated mice.
Fig. 8.
Fig. 8.
Effects of fenofibrate and clofibrate at clinically relevant low doses on hepatic TG, PPARα, and SREBP-1c in wild-type (+/+) mice. A, hepatic TG content. Data are expressed as mean ± S.D. (n = 6 in each group). White bar, control group; gray bar, fenofibrate (5 mg/kg/day) group; black bar, clofibrate (15 mg/kg/day) group. *P < 0.05, between treated and untreated mice. B and C, hepatic expression of mRNAs encoding PPARα and its targets (CPT-I and MCAD) (B) and SREBP-1c and its targets (ACC1 and FAS) (C) was analyzed by quantitative real-time PCR and normalized to that of GAPDH mRNA. Relative mRNA levels are shown as fold changes of those of control mice. Data are expressed as mean ± S.D. (n = 6 in each group). Bars are identical to those in Fig. 8A. *, P < 0.05; **, P < 0.01, between treated and untreated mice.
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