Soybean Bioactive Peptides and Their Functional Properties.

Soy consumption has been associated with many potential health benefits in reducing chronic diseases such as obesity, cardiovascular disease, insulin-resistance/type II diabetes, certain type of cancers, and immune disorders. These physiological functions have been attributed to soy proteins either as intact soy protein or more commonly as functional or bioactive peptides derived from soybean processing. These findings have led to the approval of a health claim in the USA regarding the ability of soy proteins in reducing the risk for coronary heart disease and the acceptance of a health claim in Canada that soy protein can help lower cholesterol levels. Using different approaches, many soy bioactive peptides that have a variety of physiological functions such as hypolipidemic, anti-hypertensive, and anti-cancer properties, and anti-inflammatory, antioxidant, and immunomodulatory effects have been identified. Some soy peptides like lunasin and soymorphins possess more than one of these properties and play a role in the prevention of multiple chronic diseases. Overall, progress has been made in understanding the functional and bioactive components of soy. However, more studies are required to further identify their target organs, and elucidate their biological mechanisms of action in order to be potentially used as functional foods or even therapeutics for the prevention or treatment of chronic diseases.

The α' subunit of ß-conglycinin and various glycinin subunits of soy are not required to modulate hepatic lipid metabolism in rats.

PURPOSE: This study examined the effect of soy proteins with depletion of different subunits of the two major storage proteins, ß-conglycinin and glycinin, on hepatic lipids and proteins involved in lipid metabolism in rats, since the bioactive component of soy responsible for lipid-lowering is unclear. METHODS: Weanling Sprague Dawley rats were fed diets containing either 20% casein protein in the absence (casein) or presence (casein + ISF) of isoflavones or 20% alcohol-washed soy protein isolate (SPI) or 20% soy protein concentrates derived from a conventional (Haro) or 2 soybean lines lacking the α' subunit of ß-conglycinin and the A1-3 (1TF) or A1-5 (1a) subunits of glycinin. After 8 weeks, the rats were necropsied and liver proteins and lipids were extracted and analysed. RESULTS: The results showed that soy protein diets reduced lipid droplet accumulation and content in the liver compared to casein diets. The soy protein diets also decreased the level of hepatic mature SREBP-1 and FAS in males, with significant decreases in diets 1TF and 1a compared to the casein diets. The effect of the soy protein diets on female hepatic mature SREBP-1, FAS, and HMGCR was confounded since casein + ISF decreased these levels compared to casein alone perhaps muting the decrease by soy protein. A reduction in both phosphorylated and total STAT3 in female livers by ISF may account for the gender difference in mechanism in the regulation and protein expression of the lipid modulators. CONCLUSIONS: Overall, soy protein deficient in the α' subunit of ß-conglycinin and A1-5 subunits of glycinin maintain similar hypolipidemic function compared to the conventional soy protein. The exact bioactive component(s) warrant identification.

P2X receptors regulate adenosine diphosphate release from hepatic cells.

Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2-4 h. Nucleotide release from hepatic cells is stimulated by the Ca(2+) ionophore, ionomycin, and by the P2 receptor agonist, 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (BzATP). Ionomycin (10 µM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10-100 µM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 µM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca(2+) levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

Hepatic lipase release is inhibited by a purinergic induction of autophagy.

BACKGROUND/AIMS: We have shown that extracellular adenosine diphosphate (ADP) affects lipoprotein secretion from liver cells by stimulating cellular autophagic degradation. In this study, we investigated the effect of ADP and cellular autophagy on hepatic lipase (HL) release from human liver cells. METHODS/RESULTS: Depletion of media serum stimulates an autophagic response in liver cells, which parallels an 8-fold increase in the release of ADP into the media and a complete inhibition of HL release. Treatment of cells with exogenous ADP stimulates cellular autophagy and also blocks HL release. Treatment with the autophagic stimulant and proteasomal inhibitor, ALLN (25 µM), reduces cellular HL levels and blocks HL release at 4h. In contrast, treatment with the autophagy inhibitor, 3-methyladenine (3-MA) (5 mM), increases cellular HL levels and stimulates HL release. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy. siRNA-targeted reduction in P2Y13 protein expression stimulates the release of HL by 5 to 8-fold, while overexpression of P2Y13 blocks HL release. HL release from liver cells is therefore inhibited by a purinergic induction of autophagy. To evaluate the effect of extracellular ADP on the processing of HL, we expressed a V5-epitope tag-labeled HL (HL-V5) and then measured secretion, uptake and degradation. Two isoforms of HL-V5, at 62 and 68 kDa, are released from HepG2 cells, but only the 62 kDa protein undergoes reuptake / internalization. The 62 kDa HL-V5 isoform progressively accumulates in the cell over 24h, with no detectible modification or degradation. Treatment of liver cells with ADP has no effect on HL-V5 internalization or degradation at 30 min and 4h. CONCLUSION: These studies show that extracellular nucleotides act to prevent HL accumulation in the media by stimulating cellular autophagic degradation and blocking HL release.

Purinergic signaling, dyslipidemia and inflammatory disease.

Metabolic syndrome is a compound obesity disorder, wherein the abnormal metabolism of glucose and lipid is associated with the development of chronic inflammatory diseases. The prevalence of this disease is increasing in the developed world, but the causative linkage between these metabolic disorders has remained obscure. Metabolic disease may be associated with chronic nucleotide secretion, purinergic signaling and activation of inflammatory pathways. Purinergic signaling has been implicated in impaired glucose metabolism and inflammatory disease and may contribute to dyslipidemia. Our research shows that purinergic signaling disrupts hepatic lipoprotein metabolism by blocking insulin receptor signaling and by activating cellular autophagic pathways. Chronic stimulation of purinergic signaling may therefore be causative to glucose and lipid metabolic disorders and associated with the development of cardiovascular disease.

Extracellular nucleotides inhibit insulin receptor signaling, stimulate autophagy and control lipoprotein secretion.

Hyperglycemia is associated with abnormal plasma lipoprotein metabolism and with an elevation in circulating nucleotide levels. We evaluated how extracellular nucleotides may act to perturb hepatic lipoprotein secretion. Adenosine diphosphate (ADP) (>10 µM) acts like a proteasomal inhibitor to stimulate apoB100 secretion and inhibit apoA-I secretion from human liver cells at 4 h and 24 h. ADP blocks apoA-I secretion by stimulating autophagy. The nucleotide increases cellular levels of the autophagosome marker, LC3-II, and increases co-localization of LC3 with apoA-I in punctate autophagosomes. ADP affects autophagy and apoA-I secretion through P2Y(13). Overexpression of P2Y(13) increases cellular LC3-II levels by ~50% and blocks induction of apoA-I secretion. Conversely, a siRNA-induced reduction in P2Y(13) protein expression of 50% causes a similar reduction in cellular LC3-II levels and a 3-fold stimulation in apoA-I secretion. P2Y(13) gene silencing blocks the effects of ADP on autophagy and apoA-I secretion. A reduction in P2Y(13) expression suppresses ERK1/2 phosphorylation, increases the phosphorylation of IR-ß and protein kinase B (Akt) >3-fold, and blocks the inhibition of Akt phosphorylation by TNFα and ADP. Conversely, increasing P2Y(13) expression significantly inhibits insulin-induced phosphorylation of insulin receptor (IR-ß) and Akt, similar to that observed after treatment with ADP. Nucleotides therefore act through P2Y(13), ERK1/2 and insulin receptor signaling to stimulate autophagy and affect hepatic lipoprotein secretion.

Hepatic lipase, high density lipoproteins, and hypertriglyceridemia.

Hepatic lipase (HL) is a lipolytic enzyme that contributes to the regulation of plasma triglyceride (TG) levels. Elevated TG levels may increase the risk of developing coronary heart disease, and studies suggest that mutations in the HL gene may be associated with elevated TG levels and increased risk of coronary heart disease. Hepatic lipase facilitates the clearance of TG from the very low density lipoprotein (VLDL) pool, and this function is governed by the composition and quality of high density lipoprotein (HDL) particles. In humans, HL is a liver resident enzyme regulated by factors that release it from the liver and activate it in the bloodstream. HDL regulates the release of HL from the liver and HDL structure controls HL transport and activation in the circulation. Alterations in HDL-apolipoprotein composition can perturb HL function by inhibiting the release and activation of the enzyme. HDL structure may therefore affect plasma TG levels and coronary heart disease risk.

HDL-ApoE content regulates the displacement of hepatic lipase from cell surface proteoglycans.

Human hepatic lipase (HL) is an interfacial enzyme that must be liberated from cell surface proteoglycans to hydrolyze lipoprotein triglyceride. Both high-density lipoprotein (HDL) and apolipoprotein (apo)A-I can displace HL from cell surface proteoglycans, much like heparin. HL displacement is inhibited by HDL-apoE content. Postprandial HDL is approximately twofold better at displacing HL than is fasting HDL, but only has approximately one-half the apoE content. Enriching native HDL with triglyceride decreases HDL-apoE content and increases HL displacement. Incubation of HDL with the anti-apoE antibody, 6C5, also increases HL displacement. In contrast, enrichment of synthetic HDL with apoE significantly inhibits HL displacement. HDL from fasted female normolipidemic subjects displaces HL approximately twofold better than HDL from male subjects. HDL from female subjects also has significantly less apoE than HDL from males. Normolipidemic females have increased circulating HDL-bound HL. Hyperlipidemia has little effect on the HL displacement ability of HDL from men, whereas HDL from hypercholesterolemic females exhibits impaired HL displacement. HL displacement from liver heparan sulfate proteoglycans therefore appears to be linked to interlipoprotein apoE exchange. Decreased HL displacement is associated with higher HDL-apoE levels and may therefore affect vascular triglyceride hydrolysis.

Hepatic high-density lipoprotein secretion regulates the mobilization of cell-surface hepatic lipase.

HDL acts much like heparin to liberate hepatic lipase (HL) from cell surface proteoglycans and stimulate triglyceride clearance. Experiments were undertaken to evaluate the effects of factors that stimulate the secretion of HDL from the liver on the release of HL. Treatment of HepG2 cells with linoleic acid phospholipids (LAPL) (12 muM) promotes a similar increase in the accumulation of both HDL and HL in the cell media. LAPL also induce both apoA-I and HL release from primary human hepatocytes. Dilinoleoylphosphatidylcholine has a greater effect on both apoA-I secretion and HL release than palmitoyllinoleoylphosphatidylcholine. HL released from HepG2 cells is inactive and associated with a large HDL complex containing both apoA-I and apoA-II. Inclusion of the PPARalpha inhibitor, MK-886, or MAPK inhibitor, U0126, completely blocks the LAPL-induced apoA-I and HL accumulation in the media. LAPL-treated cell lysates, however, showed no change in HL protein expression nor HL mRNA. LAPL-induced HL release appears to be a consequence of the displacement ability of newly secreted HDL. Overexpression of pre-pro-apoA-I in HepG2 cells increased HL release, while siRNA inhibition of the apoA-I gene reduced HL in the media. The data show that factors that stimulate HDL secretion in hepatocytes act to also increase the release of HL. This may partly explain why HDL therapeutics often impact plasma triglyceride levels.

HDL composition regulates displacement of cell surface-bound hepatic lipase.

HDL is able to displace cell surface-bound hepatic lipase (HL) and stimulate vascular triglyceride (TG) hydrolysis, much like heparin. Displacement appears to be a result of a high-affinity association of HL and apoA-I. HDL varies in its ability to displace HL, and therefore experiments were undertaken to evaluate the impact of HDL composition and structure on HL displacement from cell surface proteoglycans. HDL apolipoprotein and lipid composition directly affect HL displacement. ApoA-II and apoC-I significantly increase HL displacement from the cell surface. While changes in HDL cholesteryl ester and fatty acid content have no effect on HL displacement, increases in HDL phospholipid and TG content significantly inhibit HL displacement. HDL fractions from hyperlipidemic patients are unable to displace HL from the cell surface. These results indicate that the structure and composition of HDL particles in plasma are central to regulation of HL displacement and the hydrolytic activity of HL.

Lipoprotein charge and vascular lipid metabolism.

Lipoproteins play a central role in transporting hydrophobic molecules through the bloodstream and between specific tissues. Lipoprotein molecules have a distinctive electrical charge and changes in electrostatic properties directly affect the metabolism of the lipoprotein. Lipoprotein charge controls interfacial interactions and determines the ability of the lipoprotein to interact with intravascular enzymes and cell surface proteins. Uniquely charged constituents of the lipoprotein thereby control the metabolism of lipoproteins by creating a regulatory system wherein the electrostatic properties of plasma lipoproteins determine the fate of intravascular lipids.