CETP does not affect triglyceride production or clearance in APOE*3-Leiden mice.

The cholesteryl ester transfer protein (CETP) facilitates the bidirectional transfer of cholesteryl esters and triglycerides (TG) between HDL and (V)LDL. By shifting cholesterol in plasma from HDL to (V)LDL in exchange for VLDL-TG, CETP aggravates atherosclerosis in hyperlipidemic APOE*3-Leiden (E3L) mice. The aim of this study was to investigate the role of CETP in TG metabolism and high-fat diet-induced obesity by using E3L mice with and without the expression of the human CETP gene. On chow, plasma lipid levels were comparable between both male and female E3L and E3L.CETP mice. Further mechanistic studies were performed using male mice. CETP expression increased the level of TG in HDL. CETP did not affect the postprandial plasma TG response or the hepatic VLDL-TG and VLDL-apolipoprotein B production rate. Moreover, CETP did not affect the plasma TG clearance rate or organ-specific TG uptake after infusion of VLDL-like emulsion particles. In line with the absence of an effect of CETP on tissue-specific TG uptake, CETP also did not affect weight gain in response to a high-fat diet. In conclusion, the CETP-induced increase of TG in the HDL fraction of E3L mice is not associated with changes in the production of TG or with tissue-specific clearance of TG from the plasma.

midazolam (Roche), and 0.3125 mg/kg fentanyl (Janssen-Cilag). At t = 0 min, blood was taken via tail bleeding and mice were intravenously injected with 100 µL PBS containing 100 µCi Trans 35 S label to measure de novo total apolipoprotein B (apoB) synthesis. After 30 min, the animals received 500 mg tyloxapol/kg body weight (Triton WR-1339, Sigma-Aldrich) as a 10% (w/w) solution in sterile saline to prevent systemic lipolysis of newly secreted hepatic VLDL-TG ( 14 ). Additional blood samples were taken at t = 15, 30, 60, and 90 min after tyloxapol injection and used for determination of plasma TG concentration. At 120 min, the animals were euthanized and blood was collected by orbital puncture for isolation of VLDL by density gradient ultracentrifugation. 35 S-labeled total apoB content was measured in the VLDL fraction after precipitation with isopropanol (15)(16)(17).

In vivo clearance of VLDL-like emulsion particles
Glycerol tri [ 3 H]oleate-labeled VLDL-like emulsion particles (80 nm) were prepared as described by Rensen et al. ( 18 ). In short, radiolabeled emulsions were obtained by adding 200 µCi of glycerol tri[ 3 H]oleate (triolein, TO) to 100 mg of emulsion lipids before sonication (isotope obtained from GE Healthcare, Little Chalfont, UK). Mice were fasted 4 h, sedated as described above, and injected with the radiolabeled emulsion particles ( ( 19 ). Blood samples were taken using chilled paraoxon-coated capillaries by tail bleeding at 90 and 120 min of infusion to ensure that steady-state conditions had been reached. Subsequently, mice were euthanized and organs were quickly harvested and snap-frozen in liquid nitrogen. Analysis and calculations were performed as described ( 19 ).

Statistical analysis
Differences between groups were determined with the unpaired t -test for normally distributed data (GraphPad Prism 5 software, La Jolla, CA). A P -value < 0.05 was considered statistically signifi cant. Data are presented as means ± SEM.

Plasma lipids, lipoprotein profi les, and HL activity
To investigate the role of CETP in TG metabolism, male and female E3L and E3L.CETP mice were fasted overnight and plasma lipid levels were determined ( Table 1 ). Expression of CETP had no effect on total plasma lipid levels. Because we did not detect a difference between males and females, we decided to use only males for all subsequent experiments. Plasma lipoprotein profi les were determined on pooled plasma. Expression of CETP resulted in a shift of cholesterol from HDL to VLDL ( Fig. 1A ), as seen previously ( 3,20 ). Furthermore, a small amount of TG was detected in the HDL fraction upon expression of CETP ( Fig. VLDL-derived TG are lipolyzed in peripheral tissues by the enzyme LPL, whereas HDL-derived TG are presumably lipolyzed by HL and thus shunted to the liver. Therefore, we hypothesized that the CETP-mediated net transfer of TG from (V)LDL to HDL modulates the tissue-specifi c uptake of plasma TG and, as a consequence, affects the development of high-fat diet-induced obesity.

Animals
Human CETP transgenic mice that express CETP under control of its natural fl anking regions (strain 5203) ( 11 ) were obtained from Jackson Laboratories (Bar Harbor, ME) and crossbred with E3L mice ( 12 ) in our local animal facility to obtain heterozygous E3L.CETP mice ( 3 ). Mice (12-16 weeks old) were housed in a temperature-and humidity-controlled environment and were fed a standard chow diet with free access to water. Mice 12 weeks of age were fed a high-fat diet (60% energy derived from bovine fat; D 12492, Research Diet Services, Wijk bij Duursted e, The Netherlands) for 12 weeks to induce obesity. Body weight was measured during the intervention and the delta was calculated. All animal experiments were approved by the Animal Ethics Committee from the Leiden University Medical Center and The Netherlands Organization for Applied Scientifi c Research, Leiden, The Netherlands.

Plasma parameters
Plasma was obtained after overnight fasting (unless indicated otherwise) via tail vein bleeding in chilled paraoxon-coated capillary tubes to prevent ex vivo lipolysis and assayed for TG and total cholesterol using commercially available kits 1488872 and 236691 from Roche Molecular Biochemicals (Indianapolis, IN), respectively. Plasma CETP mass was analyzed using the CETP ELISA kit from ALPCO Diagnostics (Salem, NH). FFA were measured using NEFA C kit from Wako Diagnostics (Instruchemie, Delfzijl, The Netherlands). HL activity in plasma was determined by measuring plasma triacylglycerol hydrolase activity as described earlier ( 13 ).

Lipoprotein profi ling
To determine the lipid distribution over plasma lipoproteins, lipoproteins were separated using fast protein liquid chromatography. Plasma was pooled per group, and 50 l of each pool was injected onto a Superose 6 PC 3.2/30 column (Äkta System, Amersham Pharmacia Biotech, Piscataway, NJ) and eluted at a consta nt fl ow rate of 50 l/min in PBS, 1 mM EDTA, pH 7.4. Fractions of 50 l were collected and assayed for cholesterol and TG as described above.

Postprandial response
Mice were fasted overnight with food withdrawn at 6:00 PM the day before the experiment. Mice received an intragastric olive oil load (Carbonell, Cordoba, Spain) of 200 µL. Prior to the bolus and 1, 2, 3, 4, 6, and 10 h after the bolus, blood samples (30 µL) were drawn via tail bleeding for TG determination as described above. The circulating levels were corrected for the levels of TG prior to the bolus and the area under the curve (AUC) was calculated over the period of 0-10 h using GraphPad software.

Hepatic VLDL-TG and VLDL-apolipoprotein B production
Mice were fasted for 4 h with food withdrawn at 5:00 AM prior to the start of the experiment. During the experiment, mice were sedated with 6.25 mg/kg acepromazine (Alfasan), 6.25 mg/kg

VLDL-like emulsion TG clearance
Clearance of TG from circulation is also a major determinant of TG metabolism and therefore we examined the effect of CETP on TG clearance from VLDL-like emulsions, which have previously been shown to mimic the metabolic behavior of TG-rich lipoproteins ( 18,21 ). After 4 h fasting, mice were injected with a bolus of [ 3 H]TOlabele d VLDL-like emulsion particles. The decay of [ 3 H] TO in plasma was not affected by the expression of CETP ( Fig. 4 ) ( Fig. 5A ). Also, the uptake of [ 3 H]TO-derived radioactivity by liver, muscle, white adipose tissue, and brown adipose tissue was not altere d due to the expression of CETP ( Fig.  5B ). The serum half-life and organ-specifi c uptake of [ 14 C] FA were also not changed upon expression of CETP (data not shown).

High-fat diet-induced obesity
We and others have previously shown that modulation of tissue-specifi c TG-derived FA delivery can have a major impact on the development of high-fat diet-induced obesity [reviewed in ( 22 )]. To exclude the possibility that CETP expression results in a minor change in tissue-specifi c TG-derived FA uptake that over a prolonged period would affect the development of obesity, E3L and E3L. CETP mice were fed a high-fat diet (60% energy in the form of fat) for 12 weeks, and body weight was measured over time. The high-fat diet did not affect plasma CETP levels in E3L.CETP mice (3.8 ± 0.4 µg/ml on chow and 3.6 ± 0.3 µg/ml on high-fat diet). Furthermore, the highfat diet resulted in a similar decrease in plasma TG in both E3L and E3L.CETP mice (1.04 ± 0.11 and 0.92 ± 0.14 mmol/L, respectively). CETP did not affect the high-fat diet-induced body weight gain at any time point during the 12 weeks ( Fig. 6 ).

DISCUSSION
Novel drugs that inhibit CETP activity as therapy to increase HDL-C levels are in various stages of development 1B, insert). HL activity did not differ between E3L and E3L.CETP mice (3.9 ± 1.5 vs. 3.2 ± 1.3 µmol FFA/h/ml, respectively). This indicates that there is exchange of TG from the VLDL to the HDL fraction by CETP, which may be indicative of changes in TG metabolism.

Postprandial TG clearance
To examine whether CETP-mediated transfer of TG from VLDL to HDL infl uences plasma TG metabolism, we determined postprandial TG response. After an overnight fast, mice received an intragastric gavage of olive oil. Plasma TG concentrations were measured over a 10 h period and the AUC was calculated. Expression of CETP in E3L mice did not affect the postprandial TG changes in plasma ( Fig. 2 ).

VLDL-TG and VLDL-apoB production
To further examine the effect of CETP on TG metabolism, we determined VLDL production. After 4 h of fasting, mice were injected with Trans 35 S and tyloxapol and the accumulation of endogenous VLDL-TG in plasma was measured over time. As is evident from Fig. 3A , the VLDL-TG production rate, as determined from the slope of the curve, was unchanged upon expression of CETP. Furthermore, the rate of VLDL-apoB production ( Fig. 3B ) as well as the ratio of TG-apoB ( Fig. 3C ), refl ecting the amount of TG per VLDL particle, did not differ between E3L and E3L.CETP mice. Plasma was obtained from overnight fasted male and female E3L and E3L.CETP mice on a chow diet (n = 12/group). Plasma TG, total cholesterol, FFA, and CETP levels were measured. n.d., not detected. Fig. 2. Postprandial plasma TG response. Overnight fasted mice received an intragastric olive oil gavage and blood samples were drawn up to 10 h (n = 6-9/group). Plasma TG concentrations were determined and AUC 0-10 was calculated. respon se, hepatic VLDL-TG production, clearance of TG from VLDL-like emulsion particles, or the development of high-fat diet-induced obesity. These fi ndings suggest that CETP-mediated transfer of TG from (V)LDL to HDL does not refl ect a substantial effect on overall plasma TG metabolism in E3L mice.
There is some controversy regarding the effects of CETP on TG metabolism in various mouse models. Studies in mice, expressing simian CETP, show that on an atherogenic diet, expression of CETP results in increased production and clearance of TG ( 24 ). Others have demonstrated that mice expressing human CETP when fed a regular chow diet show no alterations in VLDL-TG production ( 25,26 ). However, Salerno et al. ( 25 ) showed that CETP-expressing mice have an increased postprandial TG response and decreased clearance of TG from the circulation. This was attributed to a decrease in LPL activity and LPL gene transcription. We did not fi nd ( 5,6 ). The rationale for development of these drugs is based on the HDL-lowering effect of CETP due to the redistribution of cholesterol from HDL toward (V)LDL. Because CETP transfers both cholesterol and TG between lipoproteins, we focused here on the effect of CETP on TG metabolism. We studied the effect of CETP expression in E3L mice. The E3L mice display a human-like lipoprotein metabolism and are an established model for hyperlipidemia and atherosclerosis [as reviewed in ( 23 )]. We recently reported the HDL-lowering and proatherogenic properties of CETP expression on the E3L background ( 3 ). In this study, after 4 h fasting, plasma cholesterol was somewhat higher in the E3L.CETP mice. In the current study, we found no changes in plasma total cholesterol and TG after overnight fasting. We did fi nd a small increase in TG in the HDL fraction upon expression of CETP in E3L mice. Despite this relative increase in HDL-TG, CETP did not affect the postprandial TG Fig. 3. VLDL-TG and VLDL-apoB production. The 4 h fasted mice were consecutively injected with Trans 35 S label and tyloxapol and blood samples were drawn up to 90 min (n = 4-6/group). TG concentrations were determined in plasma and plotted as the increa se in plasma TG (A). After 120 min, the total VLDL fraction was isolated by ultracentrifugation and the rate of newly syn thesized VLDL-apoB (B) and the ratio of TG-apoB (C) was calculated.  3 H]TG activity in plasma was calculated based on the TG level, and the uptake of plasma TG by liver, skeletal muscle, white adipose tissue, and brown adipose tissue was calculated (B). changes in the postprandial TG response or in the clearance of TG from the circulation in E3L.CETP mice versus E3L mice. We also did not fi nd an effect of CETP in E3L mice on high-fat diet-induced obesity. Because modifi cation of tissue-specifi c FA delivery can signifi cantly affect high-fat diet-induced obesity, this further confi rms the absence of even a subtle effect of CETP on tissue-specifi c TG-derived FA uptake. It seems likely that the explanation for the discrepancy of our data with those of Salerno et al. ( 25 ) is associated with the more human-like lipoprotein metabolism on the E3L background as indicated by the presence of a substantial amount of apoB-containing lipoproteins.
Enrichment of HDL with TG has a major impact on HDL metabolism. In humans, it has been demonstrated that cholesterol and apoAI within TG-rich HDL are cleared more rapidly compared with those within TG-poor HDL ( 27 ). Similar observations have been made in various animal models ( 10,28,29 ). Thus, CETP-mediated TG enrichment of HDL has measurable effects on the kinetics of HDL-C and HDL-apoAI. Although these changes in HDL kinetics have the potential to have a substantial effect on TG metabolism, our results indicate that the CETPmediate d TG transfer does not alter the kinetics of TG clearance from the circulation. This may be explained by the apparently small contribution of HDL-TG to the overall fl ux of TG. Especially in the postprandial state, the amount of TG in chylomicrons exceeds the amount of TG in HDL by far, even when CETP activity is high. Alternatively, HDL-TG may be readily lipolyzed by HL and the fate of the resulting FA may not be quantitatively different from FA derived from VLDL-TG. During lipolysis of VLDL-TG by LPL, a signifi cant fraction of FA leaks to the circulation and is subsequently cleared by the liver ( 19 ). Because it has been postulated that HDL-TG-derived FA are also cleared by the liver ( 10 ), the fate of a substantial fraction of VLDL-TG-derived FA and HDL-TG-derived FA will thus be indistinguishable.
In conclusion, we show that expression of CETP does not affect overall TG metabolism and high-fat diet-induced obesity in E3L mice. This implicates that, at least under relatively normolipidemic conditions, pharmacological CETP inhibition is unlikely to disturb TG metabolism.
The authors are grateful to M.C. Maas and A.P. Tholens for excellent technical assistance.  6. High fat diet-induced obesity. Mice were fed a high-fat diet and body weight was measured during the dietary intervention and the increase in bodyweight was calculated (n = 12/group).