Wheat alkylresorcinols reduce micellar solubility of cholesterol in vitro and increase cholesterol excretion in mice

Abstract Epidemiological studies have shown that the consumption of whole grains can reduce risk for metabolic disorders. We recently showed that chronic supplementation with wheat alkylresorcinols (ARs) prevents glucose intolerance and insulin resistance with hepatic lipid accumulation induced in mice by a high-fat high-sucrose diet (HFHSD). This study examines the effects of ARs on the micellar solubility of cholesterol in vitro, as well as the effects of transient AR supplementation on faecal lipid excretion and plasma lipid levels in mice. We found that ARs formed bile micelles with taurocholate independently of phospholipids, and dose-dependently decreased the micellar solubility of cholesterol in a biliary micelle model. Transient AR supplementation with HFHSD increased faecal cholesterol and triglyceride contents and decreased plasma cholesterol concentrations. These suggest that one underlying mechanism through which ARs suppress diet-induced obesity is by interfering with the micellar cholesterol solubilisation in the digestive tract, which subsequently decreases cholesterol absorption.


Introduction
Epidemiological studies have suggested that wholegrain intake reduces the risk of metabolic disorders (He et al. 2010;Ye et al. 2012;McKeown et al. 2013). Whole grain comprises bran, germ and endosperm. Fibre is particularly concentrated in the bran fraction, and it is believed to contribute to the benefits conferred by whole grains on glucose homoeostasis (Fung et al. 2002;de Munter et al. 2007;Rosén et al. 2011). Jacobs et al. (2000) found that fibre from whole grain, but not from refined grain products, is inversely associated with all-cause mortality, suggesting that botanically linked fibre and phytochemicals in bran confer additional health benefits other than the effects of fibre alone (He et al. 2010).
Alkylresorcinols (ARs; 1,3-dihydroxy-5-alkylbenzene homologues with alkyl side chains containing 15-25 carbon atoms) represent a significant proportion of the phytochemicals found in wheat and rye (Ross et al. 2004). ARs are effectively absorbed in humans (landberg et al. 2006), rats (Kozubek et al. 1992) and mice (unpublished data), and their plasma concentrations serve as short-to-medium term biomarkers of the intake of wholegrain wheat and rye in humans. We recently showed that chronic supplementation with wheat AR prevents glucose intolerance and insulin resistance with hepatic lipid accumulation induced by a high-fat high-sucrose diet in mice by increasing faecal cholesterol content (Oishi et al. 2015); however, the mechanism(s) through which ARs increase faecal cholesterol concentrations has remained obscure. Several types of phytochemicals adjust cholesterol and triglyceride levels in the diabetic state (Baddar et al. 2011), or have cholesterol-lowering effects by decreasing the micellar solubility of cholesterol via their specific interactions with bile acids or phosphatidylcholine (PC), causing the increased faecal cholesterol excretion (Ngamukote et al. 2011;Kobayashi et al. 2014). Therefore, this study examines the effects of ARs on the micellar solubility of cholesterol in vitro and the effects of transient AR supplementation on faecal lipid excretion and plasma lipid levels in mice.

Results and discussion
We initially examined the binding of taurocholic acid to ARs with or without PC in vitro and found that it dose-dependently bound to ARs (Figure 1(A)). PC did not significantly affect taurocholic acid binding to ARs, suggesting that ARs directly interact with taurocholic acid in vitro. Figure 1(B) shows that ARs dose-dependently reduced the micellar solubility of cholesterol in the presence of PC in vitro, because cholesterol in the filtrate was dosedependently increased by ARs. We examined lipid profiles in plasma, liver and faeces in mice administered with ARs using a fast-feeding protocol to evaluate the transient effect of ARs in vivo (Table S1). Feeding with wheat ARs for 24 h reduced plasma cholesterol concentrations by 32% without affecting triglyceride concentrations, significantly increased faecal cholesterol and triglyceride concentrations without affecting bile acid concentrations. Wheat ARs also significantly increased the hepatic cholesterol concentration, which might have resulted from increased cholesterol synthesis as a result of a decrease in the return of cholesterol to the liver (Oishi et al. 2015).
The present findings showed that wheat ARs decreased the micellar solubility of cholesterol through direct binding to taurocholic acid in vitro. This suppresses the intestinal uptake of cholesterol. Thus, these actions of ARs might contribute to the increased faecal excretion of cholesterol found in mice supplemented with ARs.
As described above, a hypocholesterolaemic effect is generally ascribed to inhibited dietary cholesterol absorption. However, several studies have suggested a route for cholesterol excretion that does not involve the hepato-biliary system (van der Velde et al. 2010). Plant sterols reduce serum cholesterol by increasing cholesterol excretion via the intestinal transporter Abcg5/Abcg8 (Brufau et al. 2011). This study found that transient AR administration significantly reduced plasma cholesterol concentrations without affecting hepatic and faecal bile acid levels. These findings suggest that wheat ARs stimulate cholesterol excretion via a non-biliary route in addition to competitive suppression via binding to bile acids.

Conclusion
In this study, we found that feeding with wheat ARs transiently reduced plasma cholesterol concentrations in mice. We also found that the micellar solubility of cholesterol was decreased via wheat ARs binding to taurocholic acid in vitro, suggesting that ARs increase faecal cholesterol excretion by de-solubilising cholesterol-bile acid micelles in the small intestine.

Supplementary materials
Experimental details for this paper are available online, alongside Table S1. Values are means ± seM, n = 4 per group. significant differences from two-way or one-way aNoVa are shown in boxes. significant differences between 0 mg/ml and concentrations of taurocholic acid and cholesterol are indicated as *p < 0.05 and **p < 0.01.