Dietary Phospholipids Prepared From Scallop Internal Organs Attenuate the Serum and Liver Cholesterol Contents by Enhancing the Expression of Cholesterol Hydroxylase in the Liver of Mice

In this study, we successfully prepared scallop oil (SCO), which contains high levels of phospholipids (PL) and eicosapentaenoic acid (EPA), from the internal organs of the Japanese giant scallop (Patinopecten yessoensis), one of the most important underutilized fishery resources in Japan. The intake of SCO lowers the serum and liver cholesterol contents in mice; however, whether the fatty acids (FA) composition or PL of SCO exhibits any cholesterol-lowering effect remains unknown. To elucidate whether the cholesterol-lowering function is due to FA composition or PL of SCO, and investigate the cholesterol-lowering mechanism by SCO, in the present study, mice were fed SCO's PL fraction (SCO-PL), triglyceride (TG)-type oil with almost the same FA composition as SCO-PL, called SCO's TG fraction (SCO-TG), soybean oil (SOY-TG), and soybean's PL fraction (SOY-PL). Male C57BL/6J mice (5-week-old) were fed high-fat and cholesterol diets containing 3% (w/w) experimental oils (SOY-TG, SOY-PL, SCO-TG, and SCO-PL) for 28 days. The SCO-PL diet significantly decreased the serum and liver cholesterol contents compared with the SOY-TG diet, but the intake of SOY-PL and SCO-TG did not show this effect. This result indicated that the serum and liver cholesterol-lowering effect observed in the SCO intake group was due to the effect of SCO-PL. The cholesterol-lowering effect of SCO-PL was in part related to the promotion of liver cholesterol 7α-hydroxylase (CYP7A1) expression, which is the rate-limiting enzyme for bile acid synthesis. In contrast, the expression levels of the ileum farnesoid X receptor (Fxr) and fibroblast growth factor 15 (Fgf15), which inhibit the expression of liver CYP7A1, were significantly reduced in the SCO-PL group than the SOY-TG group. From these results, the increase in the liver CYP7A1 expression by dietary SCO-PL was in part through the reduction of the ileum Fxr/Fgf15 regulatory pathway. Therefore, this study showed that SCO-PL may be a health-promoting component as it lowers the serum and liver cholesterol contents by increasing the liver CYP7A1 expression, which is not seen in SOY-PL and SCO-TG.

FC 071ENHANCED DEGRADATION OF 3-HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE (HMGCR) MAY CONTRIBUTE TO THE LOWERING OF LDL CHOLESTEROL SEEN WITH ROXADUSTAT IN CHRONIC KIDNEY DISEASE PATIENTS WITH ANEMIA

Background and Aims Roxadustat is an oral hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor approved in China and Japan for the treatment of anemia in patients with chronic kidney disease (CKD). In Phase 3 clinical studies, roxadustat was shown to increase hemoglobin levels in both dialysis-dependent (DD) and non-dialysis-dependent (NDD) CKD patients with anemia. This increase in hemoglobin and the accompanying decrease in hepcidin, a hormone that sequesters iron in intracellular stores, is consistent with the known role that HIF plays in the regulation of erythropoiesis. The HIF pathway may also influence cholesterol metabolism; at high altitude, total and low-density lipoprotein cholesterol (LDL-C) decrease in healthy individuals. The cholesterol biosynthesis pathway is well characterized and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) has long been recognized as the rate-limiting enzyme. Multiple feedback mechanisms are known to regulate HMGCR activity, one of which involves degradation of HMGCR via interaction with insulin-induced genes 1 and 2 (INSIG1 and INSIG2), two closely related endoplasmic reticulum membrane proteins. Here, we report clinical trial data showing a reduction in LDL-C in patients with anemia of CKD who were treated with roxadustat, and describe the results of investigation into the mechanism of this cholesterol-lowering effect. Method We studied the effect of roxadustat on LDL-C levels in human subjects and examined the potential underlying mechanisms via cell culture experiments. Clinical data were pooled from six pivotal phase 3 studies, three in patients with NDD-CKD and three in patients with DD-CKD, including those with incident dialysis (ID; on dialysis <4 months at randomization). Mean changes from baseline (CFB) in LDL-C (regardless of statin use) averaged over weeks 12–28 were analyzed using a mixed ANCOVA model of repeated measures. We investigated the effect of roxadustat on HMGCR activity in a cell-free enzyme assay and on HMGCR protein levels in cultured Hep3B cells. We explored involvement of the HIF pathway in regulation of HMGCR protein levels in Hep3B cells by means of HIF-1α/HIF-2α small interfering (si)RNA knockdown. Protein expression levels were assessed by SDS-PAGE and Western blot analysis or electrochemiluminescent immunoassays. Gene expression levels were evaluated by real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Results In patients with NDD-CKD, there was a 17.2% reduction in LDL-C averaged over weeks 12–28 in the roxadustat group (n=1994) vs. a 1.4% increase in the placebo group (n=1430) (least-squares mean [LSM] [SE] treatment difference = -19.83 mg/dL [1.186], p<0.0001) (Table). In patients with DD-CKD, LDL-C was reduced by 18.5% in the roxadustat group (n=1650) vs. 1.7% in the epoetin alfa group (n=1741) (LSM treatment difference = -15.80 mg/dL, p<0.0001) (Table). In the ID-DD patients, LDL-C dropped by 21.5% in the roxadustat group (n=680) vs. 4.6% in the epoetin alfa group (n=691) (LSM treatment difference = -17.50 mg/dL, p<0.0001) (Table). After confirming that roxadustat does not directly inhibit HMGCR enzyme activity, we found that the increase in HMGCR protein levels caused by cholesterol depletion in cells was suppressed by roxadustat. However, roxadustat had no effect on HMGCR mRNA expression, indicating that the suppression of HMGCR protein levels by roxadustat was the result of an effect on protein turnover. Subsequent siRNA studies revealed that the regulation of HMGCR protein levels by roxadustat was HIF dependent and mediated via upregulation of INSIG2. Conclusion These data show that treatment with roxadustat vs. placebo or epoetin alfa lowered LDL-C in patients with NDD-CKD and DD-CKD, respectively, and highlight a potential HIF-dependent mechanism for this cholesterol-lowering effect.

Cholesterol-lowering effect of bile salt hydrolase from a Lactobacillus johnsonii strain mediated by FXR pathway regulation

Hypercholesterolemia is a major risk factor for cardiovascular diseases worldwide. In this study, recombinant bile salt hydrolase (BSH) from the strain L. johnsonii 334 with high cholesterol reduction ability was...

The Influence of Galactooligosaccharide Addition to a Plant Sterol-Enriched Beverage upon Plant Sterol Colonic Metabolization: A Clinical Trial

The consumption of milk-based fruit beverages enriched with plant sterols (PSs) has previously showed a cholesterol-lowering effect in postmenopausal women [1]. The addition of galactooligosaccharides (GOSs) to these kinds of beverages could enhance their functionalities; however, their effect on the colonic metabolism of PSs is yet unknown. To shed light on this, a randomized, double blind, crossover study with postmenopausal women (n = 42, 58 ± 4 years) was carried out with the aim of evaluating GOS effects on PS colonic metabolism. Volunteers consumed 250 mL of a PS-enriched beverage (1%, w/v) daily with or without GOSs (1.8%, w/v) for 6 weeks, and feces samples were collected before and at the end of each intervention period. The contents of PS (sitosterol, sitostanol, campesterol, campestanol and stigmasterol) and its metabolites (ethylcoprostanol from sitosterol, methylcoprostanone from campesterol and ethylcoprostenol from stigmasterol) were measured by CG-MS [2]. Statistically significant increments (p < 0.05) in sterol concentrations (mg/g freeze-dry feces) were observed after the consumption of any of the beverages (with vs. without GOS addition) expressed as median (percentile 25; 75%): 8.29 (1.49; 17.27) vs. 10.79 (2.14; 19.30) for sitosterol, 12.95 (2.65; 20.66) vs. 14.47 (4.91; 21.56) for ethylcoprostanol, 2.84 (1.34; 4.91) vs. 3.16 (1.27; 4.80) for sitostanol, 1.09 (0.34; 2.03) vs. 1.41 (0.47; 2.11) for campesterol, 0.15 (0.03; 0.40) vs. 0.18 (0.03; 0.45) for methylcoprostanone, 0.46 (0.20; 0.80) vs. 0.44 (0.23; 0.82) for campestanol and 0.07 (0.00; 0.19) vs. 0.09 (0.02; 0.23) for stigmasterol. No significant changes were observed in ethylcoprostenol contents after the consumption of the beverage with or without GOSs (0.01 (−0.01; 0.02) vs. 0.002 (−0.02; 0.02)). No significant differences in net increments were observed between beverages. These results indicate that the presence of GOSs in PS-enriched beverages does not modify the colonic biotransformation of PSs.

Taurine Decreases Cellular Cholesterol Level in HepG2 Cells Partly through Upregulating Calcineurin

Objective: Taurine exerts cholesterol-lowering effect through inducing CYP7A1 and promoting the biotransformation of cholesterol into bile acids in livers, but its molecular mechanism remains unclear. Taurine also suppresses the expression of MCIP1, a calcineurin inhibitory protein. Here we aimed to explore whether calcineurin involves in the cholesterol-lowering effect and upregulation of CYP7A1 by taurine. Methods: High cellular cholesterol conditions were obtained by incubating with 0.2mM cholesterol contained DMEM in HepG2 cells. FK506, a calcineurin inhibitor, was used to depress cellular calcineurin. CnAb-/- cells are the HepG2 cells of which calcineurin was knocked down. Taurine was cultured in wild type, high-cholesterol conditions, calcineurin inhibition or deficiency HepG2 cells respectively for 24h or 48h. The levels of intracellular total cholesterol were determined by an enzymatic method and the expressions of CYP7A1, calcineurin, MEK1/2, c-Jun/p-c-Jun and SHP-1 were detected by western blotting. Results: High cellular cholesterol conditions in HepG2 cells were established and resulted in increased CYP7A1 and calcineurin expression. Taurine exhibited the decreasing-cholesterol effects on HepG2 cells regardless of whether cells with high cholesterol conditions or inhibited / deleted intracellular calcineurin. However, the extent of decreasing cholesterol after calcineurin repression or deficiency was much less than that of controls. Taurine could induce the expression of CYP7A1 but this induction was abolished when the cellular calcineurin was inhibited or deleted. Taurine was able to suppress MEK1/2, p-c-Jun and SHP-1, which are several key molecules in one inhibitory pathway of CYP7A1 transcription, whereas this suppression on MEK1/2 but not p-c-Jun or SHP-1 was reversed after completely knocking down calcineurin. Conclusions: Calcineurin was found to be required partly in taurine-decreasing cholesterol effect through inhibiting MEK1/2 which resulted in CYP7A1 upregulation.