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

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. 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. Elevated plasma TGs are a major risk factor for cardiovascular disease in women (1Nordestgaard B.G. Benn M. Schnohr P. Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women.J. Am. Med. Assoc. 2007; 298: 299-308Crossref PubMed Scopus (1613) Google Scholar, 2Bansal S. Buring J.E. Rifai N. Mora S. Sacks F.M. Ridker P.M. Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women.J. Am. Med. Assoc. 2007; 298: 309-316Crossref PubMed Scopus (1240) Google Scholar). 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 (1Nordestgaard B.G. Benn M. Schnohr P. Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women.J. Am. 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Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women.J. Am. Med. Assoc. 2007; 298: 309-316Crossref PubMed Scopus (1240) Google Scholar). Both overproduction of VLDL and delayed clearance of chylomicrons can increase TG levels and increase risk of cardiovascular disease (2Bansal S. Buring J.E. Rifai N. Mora S. Sacks F.M. Ridker P.M. Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women.J. Am. Med. Assoc. 2007; 298: 309-316Crossref PubMed Scopus (1240) Google Scholar, 9Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.A. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the Cholesterol and Recurrent Events (CARE) trial.Circulation. 2000; 102: 1886-1892Crossref PubMed Scopus (412) Google Scholar). 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 (10Charles M.A. Kane J.P. New molecular insights into CETP structure and function: a review.J. Lipid Res. 2012; 53: 1451-1458Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This CETP-mediated TG enrichment of HDL decreases HDL levels through increased HDL clearance (10Charles M.A. Kane J.P. New molecular insights into CETP structure and function: a review.J. Lipid Res. 2012; 53: 1451-1458Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 11Lamarche B. Uffelman K.D. Carpentier A. Cohn J.S. Steiner G. Barrett P.H. Lewis G.F. Triglyceride enrichment of HDL enhances in vivo metabolic clearance of HDL apo A-I in healthy men.J. Clin. Invest. 1999; 103: 1191-1199Crossref PubMed Scopus (207) Google Scholar). Although CETP inhibitors were developed to raise HDL, CETP inhibitors have not reduced cardiovascular disease risk (12Schwartz G.G. Olsson A.G. Abt M. Ballantyne C.M. Barter P.J. Brumm J. Chaitman B.R. Holme I.M. Kallend D. Leiter L.A. et al.dal-OUTCOMES Investigators. Effects of dalcetrapib in patients with a recent acute coronary syndrome.N. Engl. J. Med. 2012; 367: 2089-2099Crossref PubMed Scopus (1560) Google Scholar, 13Barter P.J. Caulfield M. Eriksson M. Grundy S.M. Kastelein J.J.P. Komajda M. Lopez-Sendon J. Mosca L. Tardif J. Waters D.D. et al.Effects of torcetrapib in patients at high risk for coronary events.N. Engl. J. Med. 2007; 357: 2109-2122Crossref PubMed Scopus (2610) Google Scholar). 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 (14Marotti K.R. Castle C.K. Murray R.W. Rehberg E.F. Polites H.G. Melchior G.W. The role of cholesteryl ester transfer protein in primate apolipoprotein-a-I metabolism. Insights from studies with transgenic mice.Arterioscler. Thromb. Vasc. Biol. 1992; 12: 736-744Crossref Google Scholar). Previously, CETP was shown to improve HDL function in women, but not men (15Villard E.F. El Khoury P. Duchene E. Bonnefont-Rousselot D. Clement K. Bruckert E. Bittar R. Le Goff W. Guerin M. Elevated CETP activity improves plasma cholesterol efflux capacity from human macrophages in women.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2341-2349Crossref PubMed Scopus (32) Google Scholar). Additionally, we have shown that transgenic expression of CETP protected against insulin resistance in females (16Cappel D.A. Palmisano B.T. Emfinger C.H. Martinez M.N. McGuinness O.P. Stafford J.M. Cholesteryl ester transfer protein protects against insulin resistance in obese female mice.Mol. Metab. 2013; 2: 457-467Crossref PubMed Scopus (19) Google Scholar), 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. An expanded methods section is available in the online data supplement. Primers for quantitative RT-PCR (qRT-PCR) are listed in supplemental Table S1. 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 (17Zhu L. Brown W.C. Cai Q. Krust A. Chambon P. McGuinness O.P. Stafford J.M. Estrogen treatment after ovariectomy protects against fatty liver and may improve pathway-selective insulin resistance.Diabetes. 2013; 62: 424-434Crossref PubMed Scopus (215) Google Scholar)] 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 (18Hartman H.B. Lai K. Evans M.J. Loss of small heterodimer partner expression in the liver protects against dyslipidemia.J. Lipid Res. 2009; 50: 193-203Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar)] 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 (19D'Eon T.M. Souza S.C. Aronovitz M. Obin M.S. Fried S.K. Greenberg A.S. Estrogen regulation of adiposity and fuel partitioning. Evidence of genomic and non-genomic regulation of lipogenic and oxidative pathways.J. Biol. Chem. 2005; 280: 35983-35991Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). All animals were euthanized between 8 and 11 AM. 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). 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. 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. 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. 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. 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 (20Sundaram M. Yao Z.M. Recent progress in understanding protein and lipid factors affecting hepatic VLDL assembly and secretion.Nutr. Metab. (Lond.). 2010; 7: 35Crossref PubMed Scopus (120) Google Scholar)]. 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 (21Wetterau J.R. Aggerbeck L.P. Laplaud P.M. Mclean L.R. Structural-properties of the microsomal triglyceride-transfer protein complex.Biochemistry. 1991; 30: 4406-4412Crossref PubMed Scopus (77) Google Scholar). Overexpression of PDI is sufficient to facilitate TG export even when MTP levels are low (22Wang S. Chen Z.J. Lam V. Han J. Hassler J. Finck B.N. Davidson N.O. Kaufman R.J. IRE1 alpha-XBP1s induces PDI expression to increase MTP activity for hepatic VLDL assembly and lipid homeostasis.Cell Metab. 2012; 16: 473-486Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). 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. 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. 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. We next examined the molecular targets required for CETP to alter TG metabolism. ERα is the predominant estrogen receptor expressed in the liver (23Couse J.F. Lindzey J. Grandien K. Gustafsson J.A. Korach K.S. Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse.Endocrinology. 1997; 138: 4613-4621Crossref PubMed Scopus (767) Google Scholar) and regulates a number of lipid metabolic pathways in the liver (24Villa A. Torre S. Stell A. Cook J. Brown M. Maggi A. Tetradian oscillation of estrogen receptor alpha is necessary to prevent liver lipid deposition.Proc. Natl. Acad. Sci. USA. 2012; 109: 11806-11811Crossref PubMed Scopus (69) Google Scholar, 25Han S.I. Komatsu Y. Murayama A. Steffensen K.R. Nakagawa Y. Nakajima Y. Suzuki M. Oie S. Parini P. Vedin L.L. et al.Estrogen receptor ligands ameliorate fatty liver through a nonclassical estrogen receptor/liver X receptor pathway in mice.Hepatology. 2014; 59: 1791-1802Crossref PubMed Scopus (57) Google Scholar). 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) (17Zhu L. Brown W.C. Cai Q. Krust A. Chambon P. McGuinness O.P. Stafford J.M. Estrogen treatment after ovariectomy protects against fatty liver and may improve pathway-selective insulin resistance.Diabetes. 2013; 62: 424-434Crossref PubMed Scopus (215) Google Scholar). 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. 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. 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 (16Cappel D.A. Palmisano B.T. Emfinger C.H. Martinez M.N. McGuinness O.P. Stafford J.M. Cholesteryl ester transfer protein protects against insulin resistance in obese female mice.Mol. Metab. 2013; 2: 457-467Crossref PubMed Scopus (19) Google Scholar). SHP regulates a number of metabolic pathways, including VLDL-TG production (26Huang J. Iqbal J. Saha P.K. Liu J. Chan L. Hussain M.M. Moore D.D. Wang L. Molecular characterization of the role of orphan receptor small heterodimer partner in development of fatty liver.Hepatology. 2007; 46: 147-157Crossref PubMed Scopus (120) Google Scholar) and estrogen signaling (27Wang X.L. Lu Y. Wang E. Zhang Z.J. Xiong X.L. Zhang H.J. Lu J.L. Zheng S. Yang J. Xia X.F. et al.Hepatic estrogen receptor alpha improves hepatosteatosis through upregulation of small heterodimer partner.J. Hepatol. 2015; 63: 183-190Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 28Lai K. Harnish D.C. Evans M.J. Estrogen receptor alpha regulates expression of the orphan receptor small heterodimer partner.J. Biol. Chem. 2003; 278: 36418-36429Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Estrogen is also known to increase liver SHP expression in mice (28Lai K. Harnish D.C. Evans M.J. Estrogen receptor alpha regulates expression of the orphan receptor small heterodimer partner.J. Biol. Chem. 2003; 278: 36418-36429Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 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. 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 (