Tadateru Hamada

Cholesterol esterase accelerates intestinal cholesterol absorption

Mechanisms of acceleration of cholesterol absorption by cholesterol esterase were investigated in various experimental conditions. Lymphatic recovery of cholesterol intubated as a micellar solution containing phosphatidylcholine (PC) into the duodenum was enhanced by the co-administration of cholesterol esterase in rats drained of bile and pancreatic juice. However, no accelerated incorporation was observed when cholesterol was solubilized in PC-depleted micelles. Cholesterol esterase dose-dependently accelerated the incorporation of cholesterol into differentiated Caco-2 cells, only when cholesterol was solubilized in PC-containing micelles. The accelerated incorporation of cholesterol into Caco-2 cells by cholesterol esterase disappeared when the enzyme was preincubated with a suicide inhibitor of cholesterol esterase. Cholesterol esterase has an activity as phospholipase A(2). When 10% of PC in bile salt micelles was replaced by lysophosphatidylcholine (lysoPC), the incorporation of cholesterol into Caco-2 cells was significantly accelerated. Cholesterol esterase enhanced the incorporation of micellar cholesterol into brush border membranes prepared from the rat jejunum. The addition of cholesterol esterase to bile salt micelles accelerated the release of micellar cholesterol in a dose-dependent manner, only when the micelles contained PC. These observations strongly suggest that cholesterol esterase hydrolyzes PC in bile salt micelles and thereby, accelerating the release of cholesterol from bile salt micelles. This may be a major cause of the acceleration of cholesterol absorption by cholesterol esterase.

Solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats

Intestinal absorption of various plant sterols was investigated in thoracic duct-cannulated normal rats. Lymphatic recovery was the highest in campesterol, intermediate in brassicasterol and sitosterol, and the lowest in stigmasterol and sitostanol. Higher solubility in the bile salt micelle was observed in sitosterol, campesterol, and sitostanol than in brassicasterol and stigmasterol. The solubility of the latter two sterols was extremely low. When the affinity of plant sterols for the bile salt micelle was compared in an in vitro model system, which assessed sterol transfer from the micellar to the oil phase, the transfer rate was the highest in brassicasterol, intermediate in campesterol and stigmasterol, and lowest in sitosterol and sitostanol. Although no significant correlations between lymphatic recovery of plant sterols and their micellar solubility or transfer rate from the bile salt micelle were observed, highly positive correlation was obtained between the lymphatic recovery and the multiplication value of the micellar solubility and the transfer rate. These observations strongly suggest that both solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats.

Hydrolysis of Micellar Phosphatidylcholine Accelerates Cholesterol Absorption in Rats and Caco-2 Cells

Lymphatic recovery of cholesterol infused into the duodenum as bile salt micelles containing phosphatidylcholine (PC) was accelerated by the co-administration of phospholipase A2 in bile and pancreatic juice diverted rats. Previously we observed that cholesterol esterase, which has the ability to hydrolyze PC, caused the same effect under a similar experimental condition (Ikeda et al., Biochim. Biophys. Acta, 1571, 34–44 (2002)). Accelerated cholesterol absorption was also observed when a part of micellar PC was replaced by lysophosphatidylcholine (LysoPC) and oleic acid. Phospholipase A2 facilitated the incorporation of micellar cholesterol into Caco-2 cells in a dose-dependent manner. There was a highly negative correlation between the incorporation of cholesterol into Caco-2 cells and the content of micellar PC remaining in the culture medium. The release of cholesterol as a monomer from bile salt micelles was enhanced when a part of micellar PC was replaced with LysoPC and oleic acid. These results strongly suggest that the release of monomer cholesterol from bile salt micelles is accelerated by hydrolysis of PC in bile salt micelles and hence that cholesterol absorption is enhanced.

Missense Mutation in Abcg5 in SHRSP Rats Does Not Accelerate Intestinal Absorption of Plant Sterols: Comparison with Wistar Rats

Stroke-prone spontaneously hypertensive rats (SHRSP) deposit plant sterols in their bodies and have a mutation in ATP binding cassette transporter G5 (Abcg5). Lymphatic recovery rates of campesterol and sitosterol in SHRSP rats were comparable to those in Wistar rats, a strain that does not deposit plant sterols in the body and has no mutation in Abcg5. Higher absorption of stigmasterol and sitostanol was observed in SHRSP rats than in Wistar rats, but the differences between SHRSP and Wistar rats were quite small, because the absorbed amounts of these two sterols were much lower than those of campesterol and sitosterol. The in situ uptake of 3H-sitosterol and 14C-cholesterol solubilized in the bile salt micelle into intestinal mucosa was comparable between SHRSP and Wistar rats. These observations suggest that a mutation in Abcg5 does not greatly influence intestinal absorption of plant sterols in SHRSP rats, at least in comparison with Wistar rats.

Missense mutation of Abcg5 in stroke-prone spontaneously hypertensive rats does not influence lymphatic sitosterol absorption regardless of the dose: comparison with Wistar rats.

The lymphatic recovery of radiolabeled sitosterol administered in various amounts to the stomach was almost the same between stroke-prone spontaneously hypertensive rats (SHRSPs), a strain having a missense mutation in ATP binding cassette transporter g5 (Abcg5), and Wistar rats, a normal strain. The results suggest that the mutation of Abcg5 in SHRSPs, compared with Wistar rats, did not influence the ability for intestinal sitosterol absorption regardless of the dose.

Cholesterol Esterase Bound to Intestinal Brush Border Membranes Does Not Accelerate Incorporation of Micellar Cholesterol into Absorptive Cells

We confirmed that cholesterol esterase accelerated the incorporation of unesterified cholesterol solubilized in bile salt micelles into differentiated Caco-2 cells under various experimental conditions. Rat pancreatic juice and bovine cholesterol esterase increased the incorporation of micellar cholesterol into rat intestinal brush border membranes. The incorporation of micellar cholesterol was not changed in the brush border membranes enriched in and depleted of cholesterol esterase. The results suggest that the accelerated incorporation of micellar cholesterol by cholesterol esterase into absorptive cells is not mediated by the enzyme bound to the brush border membranes.

Effect of a Liver X Receptor Agonist on Deposition and Lymphatic Absorption of Plant Sterols in Stroke-Prone Spontaneously Hypertensive Rats Having a Mutation in ATP-Binding Cassette Transporter G5

The effects were compared of T0901317, a liver X receptor agonist, on deposition in the liver and serum and lymphatic absorption of plant sterols in stroke-prone spontaneously hypertensive rats (SHRSPs) having a missense mutation in Abcg5, which codes for ATP-binding cassette transporter (ABC) G5, with those in Wistar rats. Both strains were pair-fed for 7 d with a 0.5% plant sterol diet with or without 5 mg/kg of body weight of T0901317. The deposition of plant sterols in the liver and serum was higher in SHRSPs than in Wistar rats. A significant reduction of plant sterol deposition was observed in Wistar rats, but not in SHRSPs when T0901317 was given. Both strains were then fed for 7 d with a control diet with or without T0901317. The lymphatic absorption of plant sterols was reduced to almost half the normal level by the T0901317 treatment. However, no difference in absorption was apparent between SHRSPs and Wistar rats regardless of the T0901317 treatment. These results suggest that the plant sterol deposition in SHRSPs was not necessarily caused by the increased absorption of plant sterols.