Reduction of VLDL secretion decreases cholesterol excretion in niemann-pick C1-like 1 hepatic transgenic mice

Abstract

An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE) is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL) support TICE, antisense oligonucleotides (ASO) were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg) mice, which predominantly excrete cholesterol via TICE, and wild type (WT) littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG) and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.

Figure 1. Hepatic and intestinal MTP expression following MTP ASO treatment.

Liver and small intestine were collected from L1Tg and WT mice following 6 weeks of treatment with control ASO or MTP ASO. Quantitation of MTP mRNA in liver (A) and proximal small intestine (C) was conducted by real-time PCR using individual RNA samples (n = 5 per treatment group). Western blot analysis of MTP and β-actin in liver (B) and proximal small intestine (D). Data in graphs represent the means ± SEM, and means not sharing a common superscript differ significantly (p<0.05).

Figure 2. Liver lipid levels in mice with hepatic MTP knockdown.

After 6 weeks of control ASO or MTP ASO treatment, fasting liver samples were collected and analyzed for the concentrations of cholesteryl ester (A), free cholesterol (B), triglyceride (C) and phospholipid (D). All hepatic lipid values were normalized to the protein content of the extracted tissue, and represent the means ± SEM (9–10 mice per treatment group). Means not sharing a common superscript differ significantly (p<0.05).

Figure 3. Biliary lipid levels and fecal cholesterol excretion in mice with hepatic MTP knockdown.

After 6 weeks of control ASO or MTP ASO treatment, gallbladder bile was collected and analyzed for the concentration of cholesterol (A), bile acids (B), and phospholipids (C). For 3 days prior to euthanasia, feces were quantitatively collected for analysis of fecal cholesterol excretion (D). Data represent the means ± SEM (n = 7–10 per treatment group), and means not sharing a common superscript differ significantly (p<0.05).

Figure 4. Hepatic secretion of lipid and apoB with MTP knockdown.

Following 6 weeks of treatment with control ASO or MTP ASO, L1Tg and WT mice were fasted for 4-orbitally with tyloxapol (500 mg/kg) and [35S]Met/Cys. Blood samples were periodically collected and the plasma was analyzed for TG (A) and TC (C) concentration. The hepatic secretion rates of TG (B) and TC (D) were determined by linear regression analysis. Secretion of newly synthesized apoB100 and apoB48 (E) was measured by autoradiography of radiolabeled apoB that had been immunoprecipitated from plasma collected 3 hrs post-tyloxapol injection. The autoradiography data in panel E show samples that were separated on one gel and were exposed for the same time to the same piece of X ray film. Data represent the means ± SEM (n = 5 per treatment group), and means not sharing a common superscript differ significantly (p<0.05).

Figure 5. Plasma lipoprotein cholesterol and apoliporotein distribution following MTP knockdown.

After treatment for 6 weeks with control ASO or MTP ASO, fasting plasma was collected from WT and L1Tg mice and analyzed for total cholesterol (A) and lipoprotein cholesterol distribution, which were used to calculate the cholesterol concentration in VLDL (B), LDL (C), transition lipoprotein [TL] (D) and HDL (E). Data represent the means ± SEM (n = 9–10 mice per treatment group), and means not sharing a common superscript differ significantly (p<0.05). An equal volume of pooled plasma from 4–5 mice per treatment group was separated by FPLC (F & G) and fractions containing VLDL (H), LDL (I), and TL (J) were collected. Following SDS-PAGE, the lipoprotein fractions were immunoblotted to determine the content of apoB and apoE.

Figure 6. Liver expression of LDLR and ABCA1 protein in mice with hepatic MTP knockdown.

Following 6 weeks of control ASO or MTP ASO treatment, liver was collected for immunoblot analysis of LDLR and ABCA1. To quantify protein expression, band intensity for LDLR (A) and ABCA1 (B) was measured by densitometry and normalized to the band intensity of β-actin. Data represent the means ± SEM (n = 4–5 mice per treatment group), and means not sharing a common superscript differ significantly (p<0.05).