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J Lipid Res. 2016 August; 57(8): 1541–1551.
PMCID: PMC4959869

Cholesteryl ester transfer protein alters liver and plasma triglyceride metabolism through two liver networks in female mice[S]

Abstract

Elevated plasma TGs increase risk of cardiovascular disease in women. Estrogen treatment raises plasma TGs in women, but molecular mechanisms remain poorly understood. Here we explore the role of cholesteryl ester transfer protein (CETP) in the regulation of TG metabolism in female mice, which naturally lack CETP. In transgenic CETP females, acute estrogen treatment raised plasma TGs 50%, increased TG production, and increased expression of genes involved in VLDL synthesis, but not in nontransgenic littermate females. In CETP females, estrogen enhanced expression of small heterodimer partner (SHP), a nuclear receptor regulating VLDL production. Deletion of liver SHP prevented increases in TG production and expression of genes involved in VLDL synthesis in CETP mice with estrogen treatment. We also examined whether CETP expression had effects on TG metabolism independent of estrogen treatment. CETP increased liver β-oxidation and reduced liver TG content by 60%. Liver estrogen receptor α (ERα) was required for CETP expression to enhance β-oxidation and reduce liver TG content. Thus, CETP alters at least two networks governing TG metabolism, one involving SHP to increase VLDL-TG production in response to estrogen, and another involving ERα to enhance β-oxidation and lower liver TG content. These findings demonstrate a novel role for CETP in estrogen-mediated increases in TG production and a broader role for CETP in TG metabolism.

Keywords: lipid and lipoprotein metabolism, estrogen, triglycerides, very low density lipoprotein, small heterodimer partner, estrogen receptor alpha

Elevated plasma TGs are a major risk factor for cardiovascular disease in women (1, 2). Incremental increases in plasma TGs elevate risk of myocardial infarction in women even after multifactorial adjustment for other risk factors, whereas the association between TGs and myocardial infarction is lost after multifactorial adjustment in men (1). Furthermore, estrogen replacement in postmenopausal women raises plasma TGs (3). This increase in plasma TG with estrogen replacement may counteract beneficial effects of estrogen, such as increased insulin sensitivity (4, 5), reduced LDL cholesterol, and increased HDL cholesterol (3). Several studies demonstrated that estrogen increases VLDL TG production in women (68), but the mechanisms behind this remain unknown.

In the fasting state, TGs are packaged into VLDL particles by the liver (9). In the fed state, intestinally absorbed TGs are packaged into chylomicrons (2). Both overproduction of VLDL and delayed clearance of chylomicrons can increase TG levels and increase risk of cardiovascular disease (2, 9). Once lipoproteins enter circulation, tissue lipases and transfer proteins, like cholesteryl ester transfer protein (CETP), modify the size and lipid content of lipoproteins. CETP facilitates lipid exchange between lipoproteins, resulting in TG enrichment of HDL (10). This CETP-mediated TG enrichment of HDL decreases HDL levels through increased HDL clearance (10, 11). Although CETP inhibitors were developed to raise HDL, CETP inhibitors have not reduced cardiovascular disease risk (12, 13). This may suggest that CETP has additional functions beyond regulation of HDL cholesterol levels. Currently, the role of CETP in regulating liver and plasma TG metabolism is unknown.

In this report, we show that transgenic expression of CETP in female mice is required for estrogen-mediated increases in TG production. Although mice naturally lack CETP, transgenic expression of CETP results in a human-like lipoprotein distribution (14). Previously, CETP was shown to improve HDL function in women, but not men (15). Additionally, we have shown that transgenic expression of CETP protected against insulin resistance in females (16), recapitulating how estrogen increases insulin sensitivity in women. This suggests that CETP may facilitate estrogen-specific functions. Here, we show that CETP expression also facilitates estrogen action on TG metabolism. Transgenic expression of CETP in female mice results in both estrogen-mediated increases in VLDL production and reduced liver TG content. We demonstrate that the effects of CETP expression require small heterodimer partner (SHP) or estrogen receptor α (ERα), two nuclear receptors governing liver TG metabolism. Liver SHP is required to increase VLDL-TG production in response to estrogen in CETP mice. Additionally, liver ERα is required for CETP expression to enhance β-oxidation and lower liver TG content.

MATERIALS and METHODS

An expanded methods section is available in the online data supplement. Primers for quantitative RT-PCR (qRT-PCR) are listed in supplemental Table S1.

Animals

CETP transgenic mice were purchased from Jackson Laboratories [C57BL/6-Tg(CETP)UCTP20Pnu/J, Stock No: 001929]. Nontransgenic littermates were used as WT controls. CETP mice were bred with ERαflox/flox mice with Cre recombinase under control of the albumin promoter [liver-specific knockout of ERα (LKO-ERα), ERαflox/flox;ALB-Cre+/−, described previously (17)] to generate LKO-ERα CETP (ERαflox/flox;ALB-Cre+/−;CETP+/−) and LKO-ERα littermates. CETP mice were bred with SHPflox/flox mice with Cre recombinase under control of the albumin promoter [LKO-SHP, SHPflox/flox;ALB-Cre+/−, described previously (18)] to generate LKO-SHP CETP (SHPflox/flox;ALB-Cre+/−;CETP+/−) and LKO-SHP littermates. All strains were backcrossed at least 10 generations onto the C57BL/6 background. Females were ovariectomized to remove the contribution of endogenous ovarian hormones. After recovering for 6–7 days, mice were injected subcutaneously with vehicle (sesame oil) or estrogen (1 μg/g, β-estradiol-3-benzoate; Sigma). Mice were euthanized 24 h after estrogen treatment to prevent changes associated with long-term estrogen replacement, such as reduced adiposity, reduced insulin, and increased free fatty acids (19). All animals were euthanized between 8 and 11 AM.

Lipid and lipoprotein analysis

Blood was collected in EDTA tubes (Sarstedt). Plasma TGs and cholesterol were measured using colorimetric kits (Infinity). Plasma lipoproteins were separated using fast-performance liquid chromatography (FPLC) on a Superose6 column (GE Healthcare). Liver TG and total cholesterol content were determined by the Vanderbilt Hormone Assay Core. Plasma estradiol was measured by colorimetric ELISA (Calbiotech). Plasma β-hydroxybutyrate was measured following 18 h fasting and 5 h refeeding using a colorimetric kit (Cayman Chemical). Liver protein disulfide isomerase (PDI) activity was measured from homogenates made in RIPA buffer with protease and phosphatase inhibitors (ThermoFisher) using a fluorescence kit (EnzoLife Sciences).

In vivo TG clearance and production

TG clearance was measured in 12 h fasted mice after oral gavage with olive oil (200 μl/mouse). Plasma TGs were measured from tail blood sampling over 5 h. TG production was measured in 3 h fasted mice after intravenous administration of Triton WR-1339 (500 mg/kg). Plasma TGs were measured over 2 h.

Statistical analysis

Data are summarized using mean and standard error. Statistical tests between two groups were analyzed by unpaired Student’s t-test. Data with more than one group were analyzed by one-way ANOVA with Bonferroni post hoc comparisons of selected columns. Repeated measures one-way ANOVA was used for measures of plasma TG over time with Bonferroni posttest comparisons. Genotype effects were determined by two-way ANOVA. P values <0.05 were considered statistically significant. Animal numbers are indicated in figure legends.

RESULTS

Estrogen raises plasma TGs in CETP females but not WT females

Estrogen treatment raised plasma estrogen concentration and uterine weight equally in both WT and CETP females after ovariectomy (OVX) (Fig. 1A, B). Estrogen treatment increased plasma TGs by 50% in CETP mice (55.2 ± 4.9 vs. 83.6 ± 6.1 mg/dl, P < 0.01; Fig. 1C) but did not alter plasma TGs in WT mice (55.1 ± 4.2 vs. 61.9 ± 6.7 mg/dl; Fig. 1C). Estrogen treatment modestly, but nonsignificantly, increased plasma cholesterol in CETP females (Fig. 1D). In CETP mice, estrogen treatment enriched the TG content of VLDL as measured by FPLC (Fig. 1E, F). The increase in cholesterol in CETP mice treated with estrogen was distributed in VLDL, LDL, and HDL (Fig. 1G, H). VLDL apoB levels were significantly higher in estrogen-treated CETP mice relative to WT mice, suggesting a higher number of VLDL particles after estrogen treatment in CETP mice (Fig. 1I). Thus, transgenic expression of CETP in females causes increased plasma TGs in VLDL in response to estrogen.

Fig. 1.
Estrogen treatment raises plasma TGs in CETP but not WT female mice. A–D: Ovariectomized female mice with transgenic expression of CETP and nontransgenic, WT littermates were given vehicle (Veh; sesame oil) or estrogen (E2; 1 μg/g, β-estradiol-3-benzoate) ...

Estrogen treatment raises TG production in CETP females

To determine how estrogen raises plasma TGs and VLDL-TG content in CETP females, we measured plasma clearance and production of TG. Estrogen treatment did not alter postprandial plasma TG concentration after an oral olive oil bolus in either WT or CETP females (Fig. 2A, B). Because estrogen treatment did not significantly alter postprandial TG concentrations, vehicle- and estrogen-treated data were pooled within each genotype. CETP expression resulted in a greater postprandial TG excursion relative to WT females (1,397.0 ± 157.5 vs. 1,029.0 ± 61.3 mg·dl−1·h, P < 0.05; Fig. 2C). Because postprandial plasma TG concentration is a balance of intestinal production of chylomicron TGs and clearance from plasma, we measured chylomicron TG production in vehicle- and estrogen-treated WT and CETP female mice. Neither estrogen treatment nor CETP expression significantly altered chylomicron TG production (supplemental Fig. S1), indicating the increased postprandial TG excursion in CETP mice is likely due to impaired TG clearance. TG production was measured in fasted mice after administration of the lipoprotein lipase inhibitor Triton WR-1339. In WT females, estrogen treatment modestly, but nonsignificantly, lowered TG production (Fig. 2D). In CETP females, however, estrogen treatment raised TG production (Fig. 2E). Estrogen treatment did not alter plasma apoB protein levels after administration of Triton WR-1339 (supplemental Fig. S2), indicating that estrogen may alter TG content of VLDL particles without affecting apoB production. TG production was markedly lower in vehicle-treated CETP females relative to vehicle-treated WT females (179 ± 107.8 vs. 360.1 ± 94.71 μmol−1·kg−1·h−1 CETP vehicle vs. WT vehicle, P < 0.01; Fig. 2D, E). Plasma free fatty acid levels were not different between WT and CETP females regardless of estrogen treatment (Fig. 2F). Thus, plasma TGs were not different between vehicle-treated CETP and WT females due to the net effect of reduced VLDL-TG production and delayed TG clearance in CETP females. Estrogen treatment, however, raised plasma TGs through enhanced TG production in CETP females but not in WT females.

Fig. 2.
Estrogen treatment raises TG production in CETP mice via enhanced expression and activity of genes involved in VLDL synthesis and assembly. Plasma TGs following oral TG bolus (200 µl/mouse) in 12 h fasted WT (A) and CETP (B) females treated with ...

Because VLDL production by the liver is the main source of TGs in the fasted state, we sought to understand if estrogen treatment altered expression of genes of VLDL synthesis and assembly in WT and CETP mice [for review of VLDL assembly, see Sundaram and Yao (20)]. Liver mRNA expression of apoB (encoded by Apob) and microsomal triglyceride-transfer protein (MTP; encoded by Mttp) were increased in CETP females relative to WT but did not change with estrogen treatment (Fig. 2G). Liver MTP activity was lower in CETP females relative to WT but did not significantly change with estrogen treatment (supplemental Fig. S3). PDI (encoded by P4hb, Pdia3, and Pdia4) is a critical subunit of MTP (21). Overexpression of PDI is sufficient to facilitate TG export even when MTP levels are low (22). Estrogen increased expression of several isoforms of PDI (P4hb, Pdia3, and Pdia4) in CETP females, but not in WT females (Fig. 2G). Corresponding with increased mRNA expression of PDI with estrogen treatment, liver PDI activity increased 4-fold with estrogen treatment in CETP females, but not in WT females (Fig. 2H). Taken together, these data indicate that CETP raises plasma TGs with estrogen treatment by increasing VLDL-TG production and increasing expression and activity of PDI in the liver.

CETP lowers liver TG content through enhanced β-oxidation

Because liver TG is the source for VLDL-TG, we sought to understand if CETP altered liver TG content in these female mice after OVX. Estrogen treatment reduced liver TG content by 70% in WT females (5.82 ± 0.81 vs. 1.64 ± 0.56 μg/mg liver; Fig. 3A). Surprisingly, expression of CETP reduced liver TG content by nearly 60% relative to WT mice (2.46 ± 0.77 vs. 5.82 ± 0.81 μg/mg liver, CETP vehicle vs. WT vehicle; Fig. 3A). Estrogen treatment did not further reduce liver TG content in CETP females (Fig. 3A). Liver cholesterol content did not change with estrogen treatment in either WT or CETP females (Fig. 3B). Thus, expression of CETP substantially reduced liver TG content. Because liver TG content is a major determinant of VLDL production, this reduced liver TG content likely explains why TG production rates were lower in CETP females compared with WT females.

Fig. 3.
Transgenic expression of CETP lowers liver TG content. A, B: Liver TG (A) and cholesterol (B) content in WT and CETP females treated with Veh or E2. *** P < 0.001 (ANOVA, n = 5–7/group). C: Plasma β-hydroxybutyrate in 18 h fasted ...

To determine how CETP reduced liver TG content, we examined markers of β-oxidation, TG synthesis, and TG uptake. During prolonged fasting, the liver produces ketone bodies through β-oxidation of fatty acids. Therefore, plasma ketone bodies serve as an in vivo index of liver β-oxidation. After an 18 h fast, CETP females had more than twice the levels plasma β-hydroxybutyrate, the most abundant plasma ketone, compared with WT females (Fig. 3C). Following 5 h of refeeding, plasma β-hydroxybutyrate levels decreased to similar levels in both WT and CETP females (Fig. 3C). In vehicle-treated mice, CETP expression raised mRNA levels of several genes involved in β-oxidation in liver (Ppara, Cpt2, Acox1, and Acadm; Fig. 3D), which cumulatively increased β-oxidation in vivo as indicated by increased plasma β-hydroxybutyrate levels (Fig. 3C). Estrogen treatment reduced expression of several β-oxidation targets similarly in WT and CETP mice (Fig. 3D). Expression of CETP did not substantially reduce expression of genes involved in TG synthesis (supplemental Fig. S4A) or TG uptake and storage (supplemental Fig. S4B), suggesting that these pathways are unlikely to contribute to the reduction in liver TG seen in CETP females. Surprisingly, CETP expression not only blunted the estrogen response of certain TG metabolic genes (i.e., Fasn, supplemental Fig. S4A; Cd36, supplemental Fig. S4B), but also promoted new responses to estrogen in other TG metabolic targets (i.e., Ppara, Fig. 3D; Srebf2, supplemental Fig. S4B) that are not seen in WT females. CETP expression did not alter tissue delivery of estrogen to muscle, white adipose, or liver (supplemental Fig. S5). Thus, CETP expression caused a differential response to estrogen treatment in several pathways involved in liver TG metabolism without affecting delivery of estrogen to tissues. Furthermore, CETP expression increased liver β-oxidation, which likely explains how CETP expression reduces liver TG content.

Liver ERα is required for CETP-mediated lowering of liver TG content but not for estrogen-mediated increases in TG production

We next examined the molecular targets required for CETP to alter TG metabolism. ERα is the predominant estrogen receptor expressed in the liver (23) and regulates a number of lipid metabolic pathways in the liver (24, 25). To test the hypothesis that liver ERα is required for CETP expression to alter TG metabolism, we bred CETP transgenic mice onto a congenic strain with a hepatocyte-specific deletion of ERα (LKO-ERα) (17). Whereas CETP expression decreased liver TG nearly 60% relative to WT controls (Fig. 3A), deletion of liver ERα completely prevented CETP-mediated lowering of liver TG content relative to LKO-ERα controls (Fig. 4A). Additionally, in the absence of liver ERα, CETP failed to increase β-oxidation gene expression (Ppara, Cpt2, Acox1, and Acadm) with vehicle or estrogen treatment (Fig. 4B). Furthermore, CETP expression did not increase plasma levels of β-hydroxybutyrate in the absence of liver ERα (Fig. 4C). Liver cholesterol content was unaffected by estrogen treatment in LKO-ERα or LKO-ERα CETP mice (Fig. 4D). Thus, liver ERα is required for CETP expression to lower liver TG content and increase β-oxidation.

Fig. 4.
Liver ERα is required for CETP to lower liver TG content. A: Liver TG content in mice with LKO-ERα or mice with LKO-ERα with CETP (LKO-ERα CETP) females treated with Veh or E2 (ANOVA, n = 7–8/group). B: Liver mRNA ...

We next determined whether CETP expression also required liver ERα to increase plasma TGs and TG production in response to estrogen. Estrogen treatment did not raise plasma cholesterol in LKO-ERα or LKO-ERα CETP mice (Fig. 4E). Despite deletion of liver ERα, estrogen treatment raised plasma TGs in LKO-ERα CETP females (Fig. 4F), whereas estrogen treatment did not alter plasma TGs in LKO-ERα females (Fig. 4F). Estrogen did not alter TG production in LKO-ERα females (Fig. 4G). However, estrogen treatment raised TG production in LKO-ERα CETP females (Fig. 4H). Estrogen also raised liver PDI activity in LKO-ERα CETP but not LKO-ERα females (Fig. 4I). These data indicate that estrogen may raise VLDL production in mice expressing CETP via another estrogen receptor in liver, like the G protein-coupled estrogen receptor Gper1 (also known as Gpr30). To test the hypothesis that estrogen signals via Gper1 to raise VLDL production in CETP-expressing mice, we pretreated LKO-ERα and LKO-ERα CETP mice with a Gper1 antagonist prior to treatment with estrogen. Pretreatment with a Gper1 antagonist prevented estrogen from raising TG production in LKO-ERα CETP mice (supplemental Fig. S6), indicating that estrogen may signal through Gper1 to raise VLDL production in mice expressing CETP. Taken together, these data demonstrate that liver ERα is dispensable for estrogen-mediated increases in plasma TGs and TG production in CETP mice, but that liver ERα is required for CETP-mediated increases in β-oxidation and concomitant lowering of liver TG content.

CETP requires liver SHP to increase TG production with estrogen treatment

Because liver ERα was not required to raise TG production in response to estrogen treatment in CETP mice, we sought to determine additional factors required for this effect in CETP mice. Previously, we showed that CETP expression enhanced bile acid signaling to the nuclear receptor SHP in females (16). SHP regulates a number of metabolic pathways, including VLDL-TG production (26) and estrogen signaling (27, 28). Estrogen is also known to increase liver SHP expression in mice (28). We found that estrogen increased SHP mRNA in the liver of CETP mice (Fig. 5A). Estrogen also increased liver SHP mRNA in WT females, but this was not statistically significant. We also found that SHP regulates liver mRNA expression of several PDI isoforms (P4hb, Pdia3; Fig. 5B). Because estrogen induces expression of both SHP and PDI in CETP mice, we hypothesized that SHP may be required in CETP females to induce PDI and increase TG production in response to estrogen.

Fig. 5.
Liver SHP is required for CETP to raise TG production with estrogen treatment. A: CETP enhances estrogen-mediated induction of liver Shp mRNA expression. WT or CETP female mice were given Veh or E2, and mRNA expression of Shp was measured using qRT-PCR. ...

To test the hypothesis that CETP requires liver SHP to raise plasma TG production in response to estrogen treatment, we bred CETP transgenic mice onto a congenic strain with a hepatocyte-specific deletion of SHP (LKO-SHP; Fig. 5B). Estrogen did not alter plasma cholesterol levels in either LKO-SHP or LKO-SHP CETP females (Fig. 5C). In the absence of liver SHP, estrogen treatment failed to raise plasma TGs in females with CETP (Fig. 5D). Additionally, estrogen treatment also failed to raise TG production in females expressing CETP in the absence of liver SHP (Fig. 5E, F). In fact, TG production decreased with estrogen treatment in both LKO-SHP and LKO-SHP CETP mice. This decrease in TG production may be due to estrogen-mediated increases in liver bile acid levels (29), which are known to reduce liver TG production (30). In addition to the effects on plasma TG metabolism, liver SHP deletion prevented estrogen-mediated increases in mRNA expression of genes of VLDL synthesis and assembly in CETP females (Fig. 5G, compared with Fig. 2G). Finally, deletion of liver SHP prevented estrogen-mediated increases in PDI activity in CETP females (Fig. 5H, compared with ). Thus, liver SHP expression is required for estrogen to increase plasma TGs, TG production, and mRNA expression and activity of genes involved in VLDL assembly in CETP females.

We next tested if liver SHP was required for CETP expression to alter liver TG content. Similar to a previous report (27), deletion of liver SHP prevented estrogen-mediated reductions in liver TG content in females (supplemental Fig. S7A). Whereas CETP expression previously lowered liver TG content by 60% relative to WT mice (Fig. 3A), expression of CETP in the absence of liver SHP actually raised liver TG content by 35% relative to LKO-SHP females (4.70 ± 0.74 vs. 3.46 ± 0.50 μg/mg, LKO-SHP CETP vehicle vs. LKO-SHP vehicle, P < 0.05; supplemental Fig. S7A). Because liver fat content is a determinate of VLDL production, this increase in liver TG content likely contributed to raising TG production in LKO-SHP CETP to similar levels as LKO-SHP mice, while CETP previously reduced TG production relative to WT mice (Fig. 2D, E). This increased liver TG content was also associated with reduced mRNA levels of Acox1, a gene involved in liver β-oxidation (supplemental Fig. S7B), but not mRNA levels of other β-oxidation genes. Despite higher liver TG content, liver mRNA expression of lipoprotein uptake receptors Ldlr and Lrp1 were reduced in LKO-SHP CETP relative to LKO-SHP females (supplemental Fig. S7C). Additionally, estrogen treatment lowered liver TG content in LKO-SHP CETP females (supplemental Fig. S7A). Estrogen treatment did not alter liver cholesterol content in either LKO-SHP or LKO-SHP CETP females (supplemental Fig. S7D). Thus, in the absence of liver SHP, CETP expression did not reduce liver TG content. In fact, CETP expression raised liver TG content in the absence of liver SHP, which was subsequently reduced in response to estrogen. Taken together, these data indicate that liver SHP is required for CETP to raise plasma TGs and TG production in response to estrogen treatment.

DISCUSSION

It has become increasingly evident that women have a unique set of factors that contribute to risk of cardiovascular disease (31, 32). Understanding the unique aspects of cardiovascular disease risk in women may lead to discovery of novel therapeutics for women, as cardiovascular disease prevention has improved only modestly in women over the past 30 years when compared with men (33). Although estrogen replacement has been postulated to reduce cardiovascular disease risk relative to men, estrogen treatment actually raises VLDL-TG production (68), which may negate improvements in other cardiovascular disease risk factors. The mechanisms underlying estrogen-mediated increases VLDL-TG production in women remain poorly understood. We discovered that CETP may contribute to estrogen-mediated increases VLDL-TG production in females using a transgenic mouse model. In our efforts to understand the role of CETP in regulating VLDL production in response to estrogen, we also discovered several novel functions of CETP in females.

In the current study, we discovered that CETP has several novel effects on liver and plasma TG metabolism, some of which required treatment with estrogen. We found that CETP reduced liver TG content by a mechanism suggesting increased liver β-oxidation in females even when estrogen levels were low (CETP vehicle vs. WT vehicle after OVX; Fig. 3A). Although CETP lowered liver TG content when estrogen levels were low, the liver estrogen receptor ERα was required for CETP to mediate this effect. Additionally, CETP expression caused delayed clearance of postprandial TGs, which was unchanged by estrogen treatment. We also demonstrated that in response to estrogen CETP raises plasma TGs, increases TG production, and increases mRNA expression and activity of genes involved in VLDL synthesis and assembly. Although these effects of CETP on VLDL production required estrogen treatment, liver estrogen receptor ERα was dispensable for this effect. This suggests that estrogen may signal through another less highly expressed estrogen receptor or indirectly to raise VLDL in CETP females. We show that both Gper1 signaling and liver SHP were required for CETP to raise VLDL production in response to estrogen. These data indicate a potential novel signaling pathway involving Gper1, SHP, and PDI that is enhanced by the presence of CETP. A previous study showed that Gper1 can potentially regulate SHP in liver (34). Future work will determine how CETP interfaces either directly or indirectly with these novel signaling pathways. The present work establishes an important novel role for CETP in regulating liver TG metabolism through at least two distinct liver signaling networks, one involving liver ERα and a mechanism suggestive of enhanced β-oxidation to lower liver TG content, and another involving Gper1, SHP, and PDI in to raise VLDL production in response to estrogen.

Because we show that CETP has estrogen-specific effects on TG metabolism, CETP may underlie certain estrogen-specific responses to TG metabolism in humans. Hormone replacement therapy raises plasma TGs in postmenopausal women (3, 35). Moreover, estrogen contributes to elevations in TGs by increasing VLDL production (68). A true test of whether CETP facilitates estrogen-mediated increases in TGs in humans would be to compare women with and without CETP deficiency before and after estrogen replacement therapy. Genetic deficiencies in CETP, however, are extremely rare. One study, however, found that genetic polymorphisms in CETP modified the effect of hormone therapy on plasma lipoproteins (36). Two additional studies found sex-dependent effects of CETP polymorphisms on other aspects of TG metabolism. One found that CETP polymorphisms modified the effect of gender on postprandial TG clearance (37). Another study found that CETP polymorphisms increased risk of fatty liver disease in women but not in men (38). Taken together, our data along with human genetic data suggest that CETP may function more broadly in TG metabolism and may underlie certain sex-specific effects, especially in women with estrogen treatment.

Previous efforts to understand this pathway may have overlooked the important role of CETP in regulating TG metabolism in females because mice naturally lack CETP. The mouse models used in these studies had genetic presence or absence of transgenic CETP, whereas all humans have CETP. In humans, CETP activity varies 6- to 8-fold (39), likely due to effects of obesity (40, 41), insulin (42), and estrogen (43) on the regulation of CETP expression. Our transgenic CETP model allowed us to discover that CETP expression facilitates a hypertriglyceridemic response to estrogen, without the confounding effects of estrogen regulation on CETP expression. All females in this study were ovariectomized to remove the contribution of endogenous ovarian hormones and to reduce variability from natural estrus cycling. We examined the effects of estrogen 24 h after treatment to avoid long-term changes associated with estrogen treatment, such as reduced adiposity, reduced insulin, and increased plasma free fatty acids, all of which impact TG metabolism (19). Furthermore, although many studies use male mice, our study focused on the effect of estrogen on TG metabolism in females. In agreement with the present study, another group also found that CETP delayed clearance of plasma TGs in female mice (44). Another study found that CETP did not alter TG production or clearance in male ApoE*3-Leiden mice (45). Our results suggest that certain effects of CETP may be dependent on expression in females or treatment with estrogen. We previously showed that CETP can protect against insulin resistance in females, but not in males (16). Further understanding of how CETP alters TG metabolism in males will help identify general effects of CETP expression versus sex-specific or estrogen-specific functions of CETP.

The role of CETP in atherosclerotic cardiovascular disease remains unclear despite several decades of work in this area. CETP activity or mass has correlated both positively (46) and negatively (39, 47) with cardiovascular disease. Genetic polymorphisms in CETP have been associated with cardiovascular disease in targeted approaches (48) but have not been associated with cardiovascular disease in genome-wide association studies (4951). Mouse models show that CETP improves (52) or worsens (53) measures of atherosclerosis. Whether CETP inhibition reduces cardiovascular disease risk remains to be determined. Although two clinical trials of CETP inhibitors did not reduce cardiovascular disease outcomes (12, 13), and a third was recently halted due to inefficacy, CETP inhibition may ultimately remain a viable therapeutic strategy because of its LDL-lowering properties. Our data demonstrate that CETP inhibits clearance of TG and apoB-containing chylomicrons. Inhibition of CETP should therefore increase clearance of TG and apoB particles and lower plasma TGs. Recently, a CETP inhibitor was shown to increase clearance of apoB-containing lipoproteins and lowered plasma TGs in humans (54). The failure of several CETP inhibitors may also suggest that CETP has additional functions beyond regulating HDL cholesterol levels.

In conclusion, our work demonstrates that CETP is required for estrogen to increase VLDL-TG production and that CETP has a broader function in TG metabolism in a transgenic mouse model expressing CETP. While CETP-mediated reductions in liver TG content should lower risk of atherosclerosis (55), CETP-mediated impairment of postprandial TG clearance and increases in VLDL production in response to estrogen might increase risk of atherosclerosis (2, 9). Thus, CETP may have both beneficial and harmful effects on cardiovascular disease risk. Furthermore, the dual effect of CETP may explain why CETP inhibitors have not been effective in reducing cardiovascular disease risk. Additional understanding how CETP alters TG metabolism may foster development of more effective therapies to treat cardiovascular disease in humans.

Supplementary Material

Supplemental Data:

Acknowledgments

The authors acknowledge the helpful assistance of the Vanderbilt Hormone Assay Core (supported by National Institutes of Health Grant DK020593 to the Vanderbilt Diabetes Research Center) and excellent support by the Vanderbilt Mouse Metabolic Phenotyping Core (supported by National Institutes of Health Grant DK59637).

Footnotes

Abbreviations:

CETP
cholesteryl ester transfer protein
ERα
estrogen receptor alpha
FPLC
fast-performance liquid chromatography
Gper1
G protein-coupled estrogen receptor
LKO-ERα
liver-specific knockout of ERα LKO-SHP, liver-specific knockout of SHP
MTP
microsomal triglyceride-transfer protein
OVX
ovariectomy
PDI
protein disulfide isomerase
qRT-PCR
quantitative RT-PCR
SHP
small heterodimer partner

B. T. Palmisano was supported by the Vanderbilt Medical Scientist Training Program (T32GM07347) and the National Institutes of Health (F30DK104514). Y. K. Lee was supported by the National Institutes of Health (R01DK093774). J. M. Stafford was supported by the Center for Integrated Healthcare, U.S. Department of Veterans Affairs (BX002223) and the National Institutes of Health (R01DK109102). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures: none.

[S]The online version of this article (available at http://www.jlr.org) contains a supplement.

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