Fenofibrate increases very low density lipoprotein triglyceride production despite reducing plasma triglyceride levels in APOE*3-Leiden.CETP mice

Abstract

The peroxisome proliferator-activated receptor alpha (PPARalpha) activator fenofibrate efficiently decreases plasma triglycerides (TG), which is generally attributed to enhanced very low density lipoprotein (VLDL)-TG clearance and decreased VLDL-TG production. However, because data on the effect of fenofibrate on VLDL production are controversial, we aimed to investigate in (more) detail the mechanism underlying the TG-lowering effect by studying VLDL-TG production and clearance using APOE*3-Leiden.CETP mice, a unique mouse model for human-like lipoprotein metabolism. Male mice were fed a Western-type diet for 4 weeks, followed by the same diet without or with fenofibrate (30 mg/kg bodyweight/day) for 4 weeks. Fenofibrate strongly lowered plasma cholesterol (-38%) and TG (-60%) caused by reduction of VLDL. Fenofibrate markedly accelerated VLDL-TG clearance, as judged from a reduced plasma half-life of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles (-68%). This was associated with an increased post-heparin lipoprotein lipase (LPL) activity (+110%) and an increased uptake of VLDL-derived fatty acids by skeletal muscle, white adipose tissue, and liver. Concomitantly, fenofibrate markedly increased the VLDL-TG production rate (+73%) but not the VLDL-apolipoprotein B (apoB) production rate. Kinetic studies using [(3)H]palmitic acid showed that fenofibrate increased VLDL-TG production by equally increasing incorporation of re-esterified plasma fatty acids and liver TG into VLDL, which was supported by hepatic gene expression profiling data. We conclude that fenofibrate decreases plasma TG by enhancing LPL-mediated VLDL-TG clearance, which results in a compensatory increase in VLDL-TG production by the liver.

FIGURE 1.

Fenofibrate increases the clearance of VLDL-like emulsion particles in E3L.CETP mice. Mice received a Western-type diet without or with fenofibrate (0.03% w/w). After 4 h of fasting, mice were injected with [³H]TO and [¹⁴C]CO-labeled VLDL-like emulsion particles (1 mg of TG), and plasma samples were taken at indicated time points to determine the plasma clearance of [³H]TO (A) and [¹⁴C]CO (C). At 30 min after injection, the uptake of ³H-activity (B) and ¹⁴C activity (D) was determined in liver, heart, skeletal muscle, and gonadal white adipose tissue (gWAT). Data are means ± S.E. (n = 5). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

Fenofibrate increases hepatic lipase and lipoprotein lipase activity in postheparin plasma of E3L.CETP mice. Mice received a Western-type diet without or with fenofibrate (0.03% w/w). After 4 h of fasting, heparin was injected, and postheparin plasma was collected. Plasma was incubated with a [³H]TO-containing substrate mixture in the absence or presence of 1 m NaCl, to estimate both the HL and LPL activity. Data are means ± S.E. (n = 8). ****, p < 0.0001.

FIGURE 3.

Fenofibrate increases hepatic VLDL-TG production in E3L.CETP mice. Mice received a Western-type diet without or with fenofibrate (0.03% w/w). After 4 h of fasting, mice were consecutively injected with Trans³⁵S label (t = −30 min) and tyloxapol (t = 0 min), and blood samples were drawn up to 90 min after tyloxapol injection. Plasma TG concentrations were determined and plotted as the increase in plasma TG as compared with baseline (A). The rate of TG production was calculated from the slopes of the curves from the individual mice (B). After 120 min, the total VLDL fraction was isolated by ultracentrifugation and the rate of newly synthesized VLDL-³⁵S-apoB (C) as well as the amount of triglycerides (TG), total cholesterol (TC), and phospholipids (PL) per mg VLDL protein (D) was measured. Data are means ± S.E. (n = 9). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 4.

Fenofibrate decreases hepatic lipid content in E3L.CETP mice. Mice received a Western-type diet without or with fenofibrate (0.03% w/w). Livers were collected after a 4-h fast, and lipids were extracted. TG (A), free cholesterol (FC) (B), and cholesteryl esters (CE) (C) were quantified. Data are means ± S.E. (n = 6). **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Fenofibrate equally increases the incorporation of both plasma FA and liver TG in VLDL-TG in E3L.CETP mice. Mice received a Western-type diet without or with fenofibrate (0.03%, w/w). Mice were continuously infused with [³H]palmitic acid after 2 h of fasting and received tyloxapol after 4 h of fasting, and the increase in plasma TG (A) and [³H]TG (B) was subsequently measured. According to the equations as schematically represented (C), the relative contribution of re-esterified plasma FA to VLDL-TG production was calculated (D). Data are means ± S.E. (n = 5). *, p < 0.05. V₁, incorporation rate of plasma FA in VLDL-TG; V₂, incorporation rate of liver TG in VLDL-TG; pTG, VLDL precursor pool.