Rice-memolin, a novel peptide derived from rice bran, improves cognitive function after oral administration in mice

Many people eat polished rice, while rice bran, a by-product known to be rich in protein and expected to have potential functions for health benefits, has not been effectively utilized. In this study, we determined that orally administered Val-Tyr-Thr-Pro-Gly (VYTPG) derived from rice bran protein improved cognitive decline in mice fed a high-fat diet (HFD). It was demonstrated that VYTPG was released from model peptides corresponding to fragment sequences of original rice proteins (Os01g0941500, Os01g0872700, and allergenic protein) after treatment with thermolysin, a microorganism-derived enzyme often used in industrial scale processes. The thermolysin digest also improved cognitive decline after oral administration in mice. Because VYTPG (1.0 mg/kg) potently improved cognitive decline and is enzymatically produced from the rice bran, we named it rice-memolin. Next, we investigated the mechanisms underlying the cognitive decline improvement associated with rice-memolin. Methyllycaconitine, an antagonist for α7 nicotinic acetylcholine receptor, suppressed the rice-memolin-induced effect, suggesting that rice-memolin improved cognitive decline coupled to the acetylcholine system. Rice-memolin increased the number of 5-bromo-2’-deoxyuridine (BrdU)-positive cells and promoted the mRNA expression of EGF and FGF-2 in the hippocampus, implying that these neurotropic factors play a role in hippocampal neurogenesis after rice-memolin administration. Epidemiologic studies demonstrated that diabetes is a risk factor for dementia; therefore, we also examined the effect of rice-memolin on glucose metabolism. Rice-memolin improved glucose intolerance. In conclusion, we identified a novel rice-derived peptide that can improve cognitive decline. The mechanisms are associated with acetylcholine and hippocampal neurogenesis. Rice-memolin is the first rice-brain-derived peptide able to improve cognitive decline

Effects of Tripeptide IRW (Ile-Arg-Trp) on metabolism, mitochondrial biogenesis, and lifespan extension

Bioactive tripeptide IRW (Ile-Arg-Trp) was initially identified as an ACE (angiotensin-converting enzyme) inhibitory peptide from egg protein ovotransferrin. The pharmacological spectrum of IRW extends towards metabolic boosting and anti-diabetic properties as well. The overall objectives of this thesis were to understand the anti-aging effects of IRW via the study of its impact on the NAD axis, mitochondrial biogenesis, and lifespan extension. Firstly, treatment of muscle (L6) cells with IRW increased intracellular Nicotinamide phosphoribosyltransferase (NAMPT) protein levels and boosted Nicotinamide adenine dinucleotide (NAD+). Both immunoprecipitation and recombinant NAMPT assays indicated the possible NAMPT activating ability of IRW. Similarly, IRW increased NAMPT mRNA and protein levels in liver and muscle tissues of C57BL/6J mice fed a high-fat diet (HFD). A significant increased level of circulating NAD+ was also observed following IRW treatment. Dosing of Drosophila melanogaster with IRW elevated both D-NAAM (fly NAMPT) and NAD+ in vivo. However, IRW treatment did not boost NAMPT levels in Sirtuin 1 (SIRT1) KO cells, indicating a possible SIRT1-dependency for the pharmacological effect. Overall, these data indicate that IRW is a novel small peptide booster of the NAMPT pool. Next, IRW was also investigated for its impact on mitochondrial biogenesis. Results showed that IRW activates mitochondrial biogenesis resulting in increased mitochondrial DNA, ATP surge, improved metabolic and microbiome function. IRW activated the peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC1α), the master regulator of mitochondrial biogenesis, in multiple cell lines and tissues of C57BL/6J HFD mice. It also increased mitochondrial DNA in muscles of aged Drosophila fed with IRW for a week. The CRISPR-Cas9 experiments elucidated the underlying mechanism as Family with Sequence Similarity 120B (FAM120B) dependent, a constitutive activator of peroxisome proliferator activated receptor gamma (PPARγ). To the best of our knowledge, IRW is the first bioactive peptide to induce mitochondrial biogenesis in vitro and in vivo. Finally, the ability of IRW to extend lifespan was evaluated using the Drosophila model in vivo. Different fly lines, such as w, wy, and Wdah were fed regular fly medium supplemented with IRW. IRW treatment at 50 and 100 μM concentrations prolonged the median life span of white mutant (w) by 5.1 and 12.08% (respectively) and of yellow mutant (wy) by 12.1 and 22.9% (respectively). Likewise, midlife IRW feeding in Wdah flies improved lifespan significantly as well. Also, IRW treatment at these concentrations significantly improved the histone markers in flies and activated the expression of multiple gene pathways involved in sirtuins (SIRT1), antioxidant defense (SOD2), autophagy (ATG7), and insulin signaling (dInR). Together, our study identifies the first bioactive peptide with the ability to extend lifespan in vivo and suggests an important prospective role of IRW intake for healthy aging in humans. Overall, this research demonstrated the potential of IRW as an anti-aging functional food due to its role in promoting NAD+ levels and mitochondrial biogenesis leading to extension in lifespan

Health Effects and Therapeutic Potential of the Gut Microbe Akkermansia muciniphila.

Akkermansia muciniphila is a bacterium commonly found in the human gastrointestinal tract that has received considerable interest as a potential probiotic for the improvement of gut health and overall metabolic function. A. muciniphila is enriched in the mucus layer of the intestinal lining, where it degrades mucin and plays a significant role in gut barrier maintenance and immune regulation. A higher abundance of A. muciniphila has been observed in the gut of healthy individuals relative to those with metabolic disorders, and multiple metabolic benefits, including improved glucose management, reduced body fat, and reduced inflammation have been linked to A. muciniphila. Current research on A. muciniphila primarily relies on mouse models, with limited human interventional studies available. While these animal studies offer valuable insights into the potential roles of A. muciniphila in health and disease, further clinical investigations in humans are needed to fully understand its impact. Here, we explore the current scope of A. muciniphila research and its potential as a therapeutic agent to improve gut and metabolic health while also emphasizing the need to optimize techniques to further improve studies of this organism

List of Publications 2019

textその他 / Othersothe

Bioactive Peptides from Milk

Milk is a major source of dietary energy, protein and fat. Due to their specific biological properties leading to health benefits, bioactive peptides (BAPs) derived from milk proteins have been a subject of intensive research during past two decades. These peptide sequences, encrypted within proteins, are liberated in vivo during gastrointestinal digestion or in vitro by fermentation with proteolytic starter cultures or using proteases. BAP generally comprises 2–20 amino acid (AA) residues. Isolation and characterization of BAP of different bioactivities from milk protein hydrolysates of cow, buffalo, camel, goat, mare, sheep, donkey and yak milks have been reported. Bioactivities of BAP, which depend on constituent AAs and the sequence, include mineral binding, opioid, angiotensin-converting enzyme (ACE) inhibition, immunomodulatory, cytotoxicity, antibacterial and antithrombotic. This chapter focuses on the methodologies adopted to produce BAPs and their prospective role in health enhancing nutraceuticals/pharmaceuticals

Nutraceutical Antioxidants and Their Therapeutic Potential in Neurodegeneration

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease that affects motor neurons of the brain and spinal cord. Many studies indicate that mitochondrial oxidative stress (MOS) is a principal mechanism underlying the pathophysiology of this and other devastating neurodegenerative diseases. Here, we investigated a unique whey protein supplement (Immunocal®) to determine its neuroprotective efficacy in several in vitro models of MOS and in an in vivo mouse model of ALS. This non-denatured whey supplement contains cystine which is an oxidized form of cysteine, an essential precursor for synthesis of the endogenous antioxidant, glutathione (GSH). In primary cultured rat cerebellar granule neurons (CGNs), pre- incubation with Immunocal® completely protected against MOS induced by HA14-1, an inhibitor of the pro-survival Bcl-2 protein. This effect was prevented by co-incubation with the gamma-glutamyl cysteine ligase inhibitor, buthionine sulfoximine, demonstrating that the de novo synthesis of GSH underlies the neuroprotective mechanism of Immunocal®. Additionally, Immunocal® displayed significant protection against an array of MOS-inducing agents, including sodium nitroprusside, copper, and aluminum, supporting its ability to upregulate mitochondrial antioxidant capacity. In accordance with these findings in CGNs, Immunocal® decreased cell death due to both H2O2 and glutamate toxicity in NSC34 motor neuron-like cells. Immunocal® also significantly protected CHO cells from MOS evoked by overexpression of amyloid precursor protein (APP). Immunocal® treatment in NSC34 motor neuron-like cells decreased cell death caused by both H2O2 and glutamate glycine. Most compelling are our findings in the hSOD1G93A mouse model of ALS. These mice were given Immunocal® (3.33% solution in drinking water) ad libitum, beginning at 60-days-old. Although no effect on overall survival was observed, Immunocal®-treated mice displayed a significant (7 ±1.08 day) delay in disease onset, compared to mutant control mice. Importantly, Immunocal®-treated mice showed a highly significant decrease in the rate of decline in grip strength. Finally, using HPLC-ECD we found that whole blood and lumbar spinal cord GSH levels were each depleted by nearly 50% in end-stage hSOD1G93A mice, and these reductions were essentially prevented in mutant mice receiving Immunocal®. These findings suggest that sustaining GSH by supplementation with Immunocal® may help to mitigate the progression of ALS through suppression of MOS

Functional and bioactive properties of flaxseed proteins and peptides

The aim of this thesis was to determine flaxseed protein functionality and confirm the bioactive properties of the enzymatic protein hydrolysates using in vitro and in vivo methods. Flaxseed albumins and globulins were extracted using NaCl and then separated by membrane dialysis. SDS-PAGE analyses showed that the globulin fraction consisted of polypeptides in the 10-50 kDa range while the albumin fraction mainly contained a 10 kDa polypeptide. Amino acid analysis revealed significantly (p<0.05) higher levels of hydrophobic amino acids in the globulin when compared to the albumin, which also corresponded to globulin’s higher surface hydrophobicity. A systematic evaluation of methods to determine the protein content of enzymatic protein hydrolysates suggests HPLC amino acid analysis as the most accurate. Subsequent hydrolysis of flaxseed proteins was carried out using thermoase GL-30, a food grade protease, in order to produce flaxseed protein hydrolysates (FPH), which were then fractionated by means of ultrafiltration into peptide fractions ranging in size from <1-10 kDa. When tested for in vitro antihypertensive and antioxidant properties, the FPH and its membrane fractions were found to scavenge free radicals, chelate metal ions, and reduce ferric iron, in addition to inhibiting the activities of angiotensin converting enzyme and renin. The flaxseed proteins also reduced systolic blood pressure in spontaneously hypertensive rats after oral administration by up to 37 mmHg within 8 h of administration. Finally, this work demonstrated the capacity of calmodulin-dependent phosphodiesterase (CaMPDE)-inhibitory flaxseed-derived peptides to be transported across the intestinal epithelium, and to influence activity of the target enzyme following absorption. Initial cell culture experiments using Caco-2 cells confirmed cell membrane permeation by the peptides in addition to absence of cytotoxicity. After individual oral administration of six CaMPDE-inhibitory peptides, two were detected in the blood of Wistar rats up to 90 min while all six peptides each inhibited plasma CaMPDE activity with AGA being the most effective. In addition to the discovery of several novel antioxidant flaxseed peptide sequences, this work demonstrated the antihypertensive properties of flaxseed protein hydrolysates, and provided novel information on the bioavailability and in vivo efficacy of flaxseed-derived CaMPDE-inhibitory peptides.February 201