Molecular investigation of the mechanism of action through which lupin peptides induce hypocholesterolemic effects on HepG2 cells

State of art and aim: Lupin is a protein-rich grain legume, which has been domesticated long time ago and cultivated in different continents, either for animal or human nutrition. The seeds of these plants have some favorable features, in particular the protein percentage is comparable to that of soybean [1] and the content of indispensable amino acids is only slightly inferior. Besides these important nutritional features, lupin seed may also provide some health benefits, particularly in the area of dislipidemia prevention. Previous experiments in suitable animal models and in mild hypercholesterolemic individuals have shown that the consumption of lupin proteins may be useful for controlling total and low-density-lipoprotein (LDL) cholesterol levels [2-4]. With the objective of providing evidences that peptides generated by the digestion of lupin proteins may be responsible of the observed activities and for investigating the mechanism of action, HepG2 cells were treated with lupin peptides obtained either by pepsin (P) or trypsin (T) hydrolysis and molecular and functional investigations were performed on the LDL receptor / SREBP2 pathway. Results and discussion: Our findings indicate that peptides obtained from the hydrolysis of lupin proteins are able to interfere with the HMGCoAR activity. In particular, P peptides inhibit the enzyme with a statistical significance (-17%) only at the maximum tested dose (2.5 mg/ml). On the contrary, T peptides show a statistically significant reduction of the HMGCoAR activity by 37% at 0.25 mg/ml, by 57% at 0.5 mg/ml, and by 61% at 1.0 and 2.5 mg/ml. Immunoblotting experiments show that the treatments with lupin peptides induce an up-regulation of the SREBP2 protein level. In particular, P peptides up-regulate the SREBP2 protein level by 100%, 148%, and 162% vs. the control, while T peptides increase the SREBP2 protein level by 80%, 73%, and 44% vs. the control, after 0.5, 1.0, and 2.5 mg/mL treatments, respectively. In agreement with the increase of SREBP2 protein level, the up-regulation of the LDL receptor is detected. In particular, at 0.5, 1.0 2.5 mg/ml P peptides mediate a 147%, 136%, and 120% induction of the LDLR protein; whereas T peptides mediate a ~85% up-regulation at 0.5 and 1.0 mg/mL and a 61% up-regulation at 2.5 mg/ml vs. the control. From a functional point of view, the increase of LDLR proteins leads to an increase of the HepG2 cells ability to up-take LDL with final hypocholesterolemic effects. In particular, the treatment with P peptides at the concentration of 1.0 and 2.5 mg/ml increases the LDL uptake by 42% and 45%, respectively, vs. the control, whereas the LDL-uptake increase was not statistically significant at 0.5 mg/ml. On the other hand, at the concentration of 0.25, 0.5, and 1.0 mg/ml T peptides significantly raise the LDL-uptake by 52%, 50%, and 70%, respectively, vs. the control. The activation of LDLR/SREBP2 pathway is regulated by the activation of PI3K/Akt/GSK3β pathways.