Lunasin-Enriched Soy Extract (LunaRich XTM), in Combination with the Dietary Supplement Reliv Now, Reduces Free Fatty Acid by Increasing Plasma Leptin and Adiponectin Levels in LDL-Receptor Mutant Pigs
Alfredo F. Galvez1*, Hosea Matel1, Jan Ivey2 and Doug Bowles2
1Missouri Plant Science Center and 2University of Missouri, Columbia
Elevated levels of free fatty acids (FFA) are associated with obesity and insulin resistance and can lead to diabetes, metabolic syndrome and cardiovascular disease. Daily supplementation using 500 mg of lunasin-enriched soy extract (LES) and one serving (18.3 gm) of the dietary supplement Reliv Now® has been found to significantly reduce FFA levels by 74% and 65% after 4 and 6 weeks, respectively, on obese LDL-receptor mutant (Rapacz) pigs. The decrease in FFA levels can be explained by the coordinate increase in plasma levels of leptin by 52% and 64% and adiponectin by 20% and 60% after 4 and 6 weeks, respectively. The decrease in FFA and the increase in leptin and adiponectin correlates with slower weight gain in the obese pigs. Removal of LES and Reliv Now supplements from the basic diet of the pigs increases FFA and decreases leptin and adiponectin to the baseline, unsupplemented levels. These results suggest that supplementation with LES and Reliv Now has the potential to reduce FFA and increase leptin and adiponectin levels, which are associated with preventing obesity and insulin resistance and the related disease modalities arising from these health conditions. However, human clinical trials are needed to validate these preliminary results.
According to the US Centers for Disease Control (CDC), there is a rising trend in obesity levels today with a parallel increase in type 2 diabetes. Elevated levels of free fatty acids (FFA) have been associated with insulin resistance in obese patients  and can increase inflammation . Insulin resistance and pro-inflammatory response are clinically important because they can lead to several diseases like type 2 diabetes, hypertension and cardiovascular disease . Although there is a close association of obesity, insulin resistance and type 2 diabetes to elevated FFA levels, there is a lack of effective treatments to lower free fatty acids .
Lunasin is a bioactive component in soy with a novel chromatin-binding property and epigenetic effects on gene expression [4, 5]. The soy peptide is heat stable, water soluble and found in significant amounts in select soy protein preparations . It can get inside mammalian epithelial cells through its RGD cell adhesion motif, bind preferentially to deacetylated histones and inhibit histone H3 and H4 acetylation . There is growing evidence that responses to dietary and environmental effects involve epigenetic changes in gene expression, which are modulated by the reversible processes of DNA methylation-demethylation and histone acetylation-deacetylation [7, 8].
Lunasin has been shown to have an anti-mitotic effect when expressed inside mammalian cells  and prevents cancer formation when applied exogenously in cell culture and in mice skin cancer model . The lunasin peptide has also been shown to have anti-inflammatory  and anti-oxidant effects , to improve innate immunity (unpublished results), and to prevent metastasis of colon cancer cells to the liver . The multiple health effects of the lunasin soy peptide have been attributed to its ability to bind to chromatin and affect gene expression associated with cellular health . The elucidation of its mechanism of action makes lunasin an important molecule for research studies to understand the emerging role of diet on epigenetic mechanisms that can impact the development of chronic diseases, such as obesity, diabetes and metabolic syndrome.
We have previously shown the effect of lunasin-enriched soy extract in lowering LDL cholesterol  and we now report a novel effect of the lunasin-enriched soy extract in combination with a soy- based dietary supplement, Reliv Now, on a supplement feeding study using a LDL-receptor mutant pig model . Reliv Now contains optimal blend of vitamins, minerals and herbal agents and is primarily used to lower risk factors for cardiovascular disease. Our study shows that supplementation with lunasin in combination with Reliv Now leads to a significant decrease in free fatty acid levels and the coordinated increase of leptin and adiponectin plasma levels in LDL-receptor mutant pigs
MATERIALS AND METHODS
We chose the Rapacz pig model  to test the biological effect of the lunasin-enriched soy extract (LES) extract on obesity, cardiovascular disease risk and metabolic syndrome because their weight and liver function are closer to humans than any other animal models. Also, the pigs have mutations to their LDL receptor gene that predispose them to high cholesterol, obesity and increased risk for heart disease . The 5 Rapacz pigs used for this study were approximately 1.5 years old and considered obese, weighing more than 20% from normal. The experiment was done with animal use permit at the Veterinary hospital at the University of Missouri, Columbia, MO.
In order to administer Reliv Now, the 18.3 gms powder was measured from a 29.6 cc scooper, mixed with 10 cc of water and formed into a dough ball that the pigs ate happily. The 500 mg dose of LES was selected based on previous study showing that 250 mg dose of LES was enough to reduce LDL cholesterol after 4 weeks in 1 -year old Rapacz pigs. We increased the dose to 500 mg to account for the increased weight of the pigs at the start of the study when they were 1.5 years of age. The 500 mg LES was put into two capsules of 250 mg each and was fed to the pigs by inserting them into a snack bar that the pigs like to eat.
To determine the bioavailability of lunasin after ingestion of 500 mg LES, we supplemented the diet of one pig with 500 mg LES and then blood draws were taken at 10 min, 30 min, 60 min and 24 hr after ingestion. The blood plasma was separated and the amount of lunasin was detected and quantitated by ELISA using a polyclonal lunasin antibody (Pacific BioLabs) and standard ELISA protocol.
To test the effects of the lunasin-enriched soy extract (LES) in combination with Reliv Now, we fed five Rapacz pigs daily with at least one kg of standard casein diet (BV233) and supplemented with 18.3 gm of Reliv Now and 500 mg of LES . The pigs were maintained on a soy-free diet (BV233 pig chow) throughout the treatment. Pigs were fed their regular diet (BV233) once per day in the morning at approximately 9:00 AM. Later in the afternoon, at approximately 4:00 PM, the Reliv Now and LES treatments were administered. Weight and blood draws were taken at pre-treatment (0), at 4 weeks, 6 weeks and 8 weeks after treatment had begun. At the end of 8 weeks, there was an additional 4 weeks washout period without treatment and weight and blood draws were also taken at the end of washout period (12 weeks). Blood draws were taken after an overnight fast (approximately 15h). Blood samples were tested for lipid panel including plasma levels of free fatty acids (FFA) by the Analytical Laboratory at the Veterinary School of the University of Missouri. Blood samples were collected into monoject tubes with 15% EDTA, centrifuged for 20 min at 3300 rpm to separate the blood plasma, which were transferred to 1 ml cryogenic vials and stored at -70oC to test for leptin and adiponectin levels. Plasma levels of leptin and adiponectin were determined using porcine leptin and adiponectin ELISA kits obtained from USCN Life Sciences, Inc. (Houston, TX USA). Standard protocols from the manufacturer were followed, including the use of 3 replicate measurements for each data point, to detect and quantify the amounts of leptin and adiponectin in the plasma.
Before conducting the supplementation study, we first determined whether lunasin can be detected in the blood plasma of a Rapacz pig at different time points after ingesting a 500 mg dose of lunasin-enriched soy extract (LES). Figure 1 shows that lunasin can be detected from the blood plasma as early as 10 min after ingestion and increased significantly after 30 and 60 min before peaking at 24h after ingestion. The total amount of lunasin detected in blood plasma after 24h was approximately 0.11 mg (Fig.1), which indicates that lunasin becomes bioavailable after ingestion of 500 mg LES.