Flaxseed-Derived Peptide, IPPF, Inhibits Intestinal Cholesterol Absorption and Hepatic Cholesterol Synthesis in Caco-2 and HepG2 Cells

Background：Flaxseed peptide (FPs) showed serum cholesterol-lowering activity in SD rats fed a high-cholesterol diet, but the cholesterol-lowering amino acid sequences and mechanism of FPs were still unclear. Methods: FPs were separated via ultrafiltration, and the amino acid sequences of the selected fractions were determined via high-performance liquid chromatography- Electrospray Ionisation - Orbitrap- Mass spectrometry (HPLC-ESI-Orbitrap MS). These peptides then were synthesized by solid-phase synthesis (SPPS). IPPF with the highest CMSR was determined to exist in flaxseed protein by specific antibodies. The effects of IPPF on intestinal cholesterol absorption and hepatic cholesterol metabolism were investigated in Caco-2 cells and HepG2 cells.Results：1 kDa FP5 fraction had the highest cholesterol micelle solubility inhibition rate (CMSR) 72.39% compared with the other ultrafiltration fractions. Then Eleven peptides were identified from FP5. Ile-Pro-Pro-Phe (IPPF), with the highest CMSR 93.47%, was selected to research the cholesterol-lowering mechanism in Caco-2 and HepG2 cells. IPPF significantly reduces the amount of cholesterol transported in Caco2 cells and the amount of total cholesterol in HepG2 cells. IPPF significantly modulated the protein levels of NCP1L1 and ABCG5/8 in Caco2 cells and significantly reduced the mRNA levels of Srebp-2 and Hmgcr in HepG2 cells. Conclusion: IPPF inhibits cholesterol intestinal absorption through modulating the expression of cholesterol transporters in Caco-2 cells and reduces hepatic cholesterol synthesis through inhibiting the SREBP2-regulated mevalonate (HMGCR) pathway in HepG2 cells. IPPF is a new food-derived inhibitor of intestinal cholesterol absorption and hepatic cholesterol synthesis without side effects and provides a nutritional therapy component for hypercholesterolemia.

Murine deficiency of peroxisomal L-bifunctional protein (EHHADH) causes medium-chain 3-hydroxydicarboxylic aciduria and perturbs hepatic cholesterol homeostasis

Peroxisomes play an essential role in the β-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA β-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA β-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA β-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA β-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA β-oxidation is a regulator of hepatic cholesterol biosynthesis.

Metabolic Effects of Hydrophobic Alginate Derivative and Tetrahydrolipstatin in Rats Fed Diet Supplemented With Palm Fat and Cholesterol

The effects of octadecylamide of alginic acid (amidated alginate) and tetrahydrolipstatin on serum and hepatic cholesterol, and the faecal output of fat and sterols were compared in rats. Amidated alginate is a sorbent of lipids, tetrahydrolipstatin is inhibitor of pancreatic lipase. Rats were fed diets containing cholesterol and palm fat at 10 and 70 g/kg, respectively. Palm fat was provided by coconut meal. Amidated alginate at 40 g/kg diet significantly decreased serum total cholesterol, LDL and hepatic cholesterol, hepatic lipids, and increased faecal output of fat and coprostanol. Tetrahydrolipstatin at 300 mg/kg diet significantly decreased LDL cholesterol, hepatic lipids and increased faecal output of fat. The intake of feed was not significantly influenced, however the weight gains in rats fed amidated alginate was lower than in rats of the control group. Both amidated alginate and tetrahydrolipstatin modified fatty acid profile in excreta lipids. Concentrations of saturated fatty acids were decreased and that of unsaturated fatty acids were increased. Despite different mode of action, amidated alginate and tetrahydrolipstatin were equally efficient in the removing dietary fat from the body.