Plasma Cholesterol-Lowering Activity of Soybean Germ Phytosterols

Soybean germ phytosterols (SGP) largely exist in soybean germ oil. Our previous study demonstrated that soybean germ oil was effective in reducing plasma cholesterol. However, it remains unknown if its phytosterols are the active ingredients responsible for the plasma cholesterol-lowering activity. The present study aimed to test the effect of SGP on plasma cholesterol and to investigate its associated underlying mechanisms using hamsters as animal model. Male hamsters (n = 40) were randomly divided into five groups (n = 8/group) and fed one of the five diets: a non-cholesterol diet (NCD), a high cholesterol diet (HCD), a HCD diet containing 0.5% cholestyramine (PC), and two HCD diets containing 0.1% (LP) and 0.2% (HP) SGP, respectively, for six weeks. Results showed that SPG reduced plasma cholesterol level in a dose-dependent manner, whereas it dose-dependently increased the excretion of both fecal neutral and acidic sterols. SGP was also effective in displacing cholesterol from micelles. It was concluded that SGP possessed hypocholesterolemic activity, likely by inhibiting cholesterol absorption in the intestine and promoting fecal sterol excretion.

Abstract 390: The Absence of Abcg5 Abcg8 Reveals a Sexually Dimorphic Adaption to Impaired Biliary Cholesterol Secretion

Objective: The ABCG5 ABCG8 (G5G8) sterol transporter is the primary mechanism for biliary cholesterol secretion, but mice maintain fecal sterol excretion in its absence. The mechanism by which mice maintain sterol excretion in the absence of this pathway is not known. Transintestinal cholesterol excretion (TICE) is an alternative pathway to hepatobiliary secretion. We investigated the impact of G5G8 deficiency on TICE in the absence of Sitosterolemia. Methods and Results: We compared both hepatobiliary and transintestinal cholesterol excretion rates in wild-type (WT) and G5G8 deficient mice of both sexes. WT and G5G8 were maintained on a plant-sterol free diet from the time of weaning to prevent the development of secondary phenotypes associated with Sitosterolemia. Biliary and intestinal cholesterol secretion rates were determined by biliary diversion with simultaneous perfusion of the proximal 10 cm of the small bowel. Among WT mice, biliary cholesterol secretion was greater in female mice compared to males. Conversely, male mice exhibited greater rates of TICE than females. As expected, WT mice had higher biliary cholesterol secretion rates than their G5G8 deficient littermates. However, the decline in biliary cholesterol secretion was far less in male mice compared to females in the absence of G5G8. In female mice, the absence of G5G8 resulted in a two-fold increase in TICE, whereas males were unaffected. Conclusion: Female mice are more dependent upon the biliary pathway for cholesterol excretion, whereas males are more dependent upon TICE. G5G8 independent pathways are present for both biliary and intestinal cholesterol secretion. Female and male mice differ in their adaptation to G5G8 deficiency in order to maintain fecal sterol excretion.

Abstract 106: Transintestinal Cholesterol Excretion and Macrophage Reverse Cholesterol Transport are not Stimulated in Hepatic ABCG8 Knockdown Mice Treated with an LXR Agonist

Transintestinal cholesterol excretion (TICE) is a recently discovered pathway by which cholesterol travels from plasma to the small intestine for direct excretion into the feces. Hallmarks of animal models with TICE include severely diminished biliary cholesterol secretion but near normal levels of hepatic cholesterol and fecal neutral sterol excretion. Using an ATP binding cassette transporter G8 (ABCG8) antisense oligonucleotide (ASO) to knock down ABCG8 specifically in liver (G8 HKD ), we created a novel mouse model with significantly decreased biliary cholesterol excretion but a 658% increase in hepatic cholesterol accumulation and a 78% reduction in fecal neutral sterol excretion, indicating a dysfunction in the TICE pathway. LXR agonists have previously been shown to stimulate the TICE pathway. In order to more definitively prove the TICE pathway was disfunctional in G8 HKD mice, we treated wild type (WT) and G8 HKD mice with the LXR agonist T0901317 and measured markers of TICE stimulation. As expected, in WT mice, T0901317 doubled biliary cholesterol concentrations. A similar effect was seen in G8 HKD mice treated with T0901317, but biliary cholesterol concentrations remained significantly less than their WT counterparts. These levels of biliary cholesterol closely mirrored hepatic ABCG8 mRNA expression. T0901317 stimulated fecal neutral sterol excretion by >1000% in wild type mice but only by 190% in G8 HKD mice. These data indicate that TICE is disfunctional in G8 HDK mice since the pathway was not stimulated to the same extent in WT and G8 HKD mice by an LXR agonist. Some controversy remains over whether the TICE pathway transports macrophage derived cholesterol. In order to address this issue, we performed a macrophage RCT assay on WT and TICE disfunctional G8 HKD mice. T0901317 stimulated macrophage RCT (fecal neutral sterol 3H dpm) by >2300% in wild type mice but only by 370% in G8 HKD mice. T0901317 increased fecal acidic sterol 3H count by 65-75% in both wild type and G8 HKD mice. These results indicate that macrophage RCT is impaired when the TICE pathway is decreased. In sum, our data shows that hepatic ABCG8 plays a key role in the TICE pathway and that impairing the TICE pathway through hepatic ABCG8 knockdown causes decreased macrophage RCT.

Abstract 396: Acute Hepatic ACAT2 Knockdown Transiently Increases Plasma and Hepatic Free Cholesterol and Fecal Neutral Sterol Excretion

In spite of the advent of statins, atherosclerotic coronary vascular disease (ASCVD) remains the number one killer of Americans. A way to reduce LDL cholesterol, the primary risk factor of ASCVD, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both biliary and non-biliary pathways. The lipoprotein that delivers cholesterol from the liver through the plasma to the small intestine for transintestinal cholesterol excretion (TICE) is not yet known. We have previously shown that chronic knockdown in mice of hepatic acyl-CoA cholesterol acyltransferase 2 (ACAT2), a cellular enzyme that converts free cholesterol (FC) into cholesteryl ester (CE), appeared to cause the formation of hepatic apoB-containing lipoproteins that preferentially trafficked cholesterol to the small intestine for TICE. We tested the hypothesis that the plasma concentration of TICE-competent, apoB-containing lipoproteins, could be increased by preloading the liver with cholesterol and then acutely depleting the cholesterol by knocking down hepatic ACAT2 with antisense oligonucleotides (ASO). After feeding a high cholesterol (0.2% wt/wt) diet for six weeks, C57BL/6 mice were treated with control non-targeting ASO or ACAT2 ASO for one or two weeks. After only one week of ACAT2 knockdown (ACAT2KD) hepatic ACAT2 protein expression was decreased nearly 80%. This translated into a 50% decrease in hepatic CE concentration in conjunction with a rarely seen 2-fold increase in hepatic FC concentration. Acute hepatic ACAT2KD increased plasma FC levels by 25%, which subsided after two weeks of treatment. The increased plasma FC was primarily associated with large and small LDL. After one week of hepatic ACAT2KD mice had a minor, non-significant increase in biliary cholesterol levels but had a 2-fold increase in fecal neutral sterol (FNS) excretion. In summary our data show that when CE is rapidly cleared from the liver, FNS levels dramatically increase with a transient increase in hepatic and plasma FC levels. We believe that in the ACAT2KD mice the increased plasma FC associated with LDL is feeding into TICE thus resulting in increased FNS.