Fatty acid amides present in Camembert cheese improved cognitive decline after oral administration in mice.

Herein, we investigated the effects of Camembert cheese (CC) and its fatty acid contents on cognitive function in mice by employing the object recognition test to evaluate hippocampus-dependent memory. Orally administered CC improved the cognitive decline induced by a high-fat diet. Next, we focused on myristamide (MA), oleamide, and stearamide, which are fatty acid amides produced during the fermentation process of CC. We found that oral administration of MA improved cognitive decline. Notably, an improvement was not observed using myristic acid, a free fatty acid that is not amidated. Thus, fatty acid amidation may contribute to the physiological activity. Moreover, we investigated changes in gene expression related to neurogenesis in the hippocampus. After MA administration, mRNA expression analysis indicated that MA increased hippocampal brain-derived neurotrophic factor expression.

Human milk-specific fat components enhance the secretion of ghrelin by MGN3-1 cells.

Triacylglycerols (TAGs) are a major fat component in human milk. Since gastric lipase produces 1,2-diacylglycerol from TAGs, we focused on the bioactivity of human milk-derived diacylglycerols in stomach cells. Ghrelin is produced in the stomach and acts as an important regulator of growth hormone secretion and energy homeostasis. In this study, we showed that 1-oleoyl-2-palmitoylglycerol (OP) increased ghrelin secretion, whereas 1,3-dioleoyl-2-palmitoylglycerol (OPO), a major component of human milk TAGs, did not increase ghrelin secretion in the ghrelin-secreting cell line, MGN3-1. Therefore, diacylglycerol OP may directly contribute to the regulation of ghrelin secretion. We also found that 2-palmitoylglycerol and 1- and 2-oleoylglycerol increased ghrelin secretion. Finally, we demonstrated that intracellular cAMP levels and preproghrelin and ghrelin O-acyl transferase expression levels were enhanced by OP treatment in MGN3-1 cells. This may represent an example of a novel mother-infant interaction mediated by fat components derived from human breast milk.

Characterization of rice endosperm-derived antidepressant-like peptide (REAP): An orally active novel tridecapeptide derived from rice protein.

It is ideal to ingest bioactive substances from daily foods to stay healthy. Rice is the staple food for almost half of the human population. We found that an orally administered enzymatic digest of rice endosperm protein exhibits antidepressant-like effects in the tail suspension test (TST) using mice. A comprehensive peptide analysis of the digest using liquid chromatography-tandem mass spectrometry was performed, and a tridecapeptide QQFLPEGQSQSQK, detected in the digest, was chemosynthesized. Oral administration of the tridecapeptide exhibited antidepressant-like effects at a low dose comparable to classical antidepressant in the TST. This also exhibited anti-depressant-like effect in the forced swim test. We named it rice endosperm-derived antidepressant-like peptide (REAP). Intriguingly, intraperitoneal administration had no effect. Orally administered REAP(8-13) but not REAP(1-7) exhibited antidepressant-like activity, suggesting that the C-terminal structure is important for the antidepressant-like effect. We confirmed the presence of REAP, corresponding to rice glutelin type B4(130-142) and B5(130-142), in the digest. The effects of REAP were blocked by both dopamine D1 and D2 antagonists. These results suggest that it exerts its antidepressant-like activity through activation of the dopamine system. Taken together, oral administration of a novel tridecapeptide exhibited antidepressant-like effects via the dopamine system. This is the first report of a rice-derived peptide that exhibits antidepressant-like effects.

The Effect of the Rice Endosperm Protein Hydrolysate on the Subjective Negative Mood Status in Healthy Humans: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial.

The rice endosperm protein (REP) hydrolysate containing the following rice endosperm protein derived oligopeptides QQFLPEGQSQSQK, LPEGQSQSQK, and pEQFLPEGQSQSQK (a N-terminal pyroglutamate residue-modified peptide) reportedly showed an antidepressant-like effect in an animal model. We investigated the effect of the REP hydrolysate on healthy humans who self-reported mental fatigue with subjectively low vigor. Seventy-six participants (age: 20-64 years) were randomly allocated to two groups. The influence of the REP hydrolysate on the mood state was evaluated in two studies: single intake (Study 1) and repeated intake over 4 weeks (Study 2). A salivary stress marker, Chromogranin A (CgA), was measured in Study 1. The single intake of the REP hydrolysate significantly improved the Profile of Mood Status 2nd edition for adults (POMS 2) subscale of Tension-Anxiety. Additionally, the salivary CgA concentrations were remarkably reduced after the single intake of the REP hydrolysate. Though a single intake of the REP hydrolysate did not significantly influence the other subscales and the TMD of the POMS 2 and the Euthymia Scale, both the subjective and objective results supported the possible effect of the REP hydrolysate on reducing anxiety and nervousness. No significant positive effects on the subjective mood state (Euthymia Scale and POMS 2) and sleep quality (Insomnia Severity Index) were observed in the trial setting employed for Study 2. In conclusion, a single intake of REP hydrolysate might help relax the subjective feelings of tension and anxiety. The effectiveness of repeated REP hydrolysate intake needs to be tested in a different clinical setting.

Soy-fortelin: A ghrelin sensitivity-enhancing peptide that stimulates food intake in aged mice.

Ghrelin sensitivity is known to decrease with aging in mice and humans, and the decrease contributes to anorexia with aging. In this study, we discovered novel ghrelin sensitivity-enhancing peptides. Ghrelin sensitivity was evaluated by examining whether dipeptide samples enhanced the calcium response to ghrelin in the growth hormone secretagogue receptor-transfected cell line. First, dipeptides were screened using a 336-dipeptide library and we revealed that Ser-Tyr (SY) potentiated ghrelin sensitivity in particular. Based on the structure-activity relationship determined using the dipeptide library and comprehensive analysis of peptides in the chymotrypsin digest of soy ß-conglycinin (ß-CG), which enhanced ghrelin sensitivity, candidate peptides were narrowed down. Among the chemosynthesized peptides, we discovered that an undecapeptide, SLVNNDDRDSY, corresponding to ß-CGα(267-277), stimulated ghrelin sensitivity in vitro. This peptide enhanced the orexigenic activity of ghrelin in C57BL/6 mice and stimulated food intake. Thus, we demonstrated that SLVNNDDRDSY stimulated ghrelin sensitivity in vitro and in vivo and named it "soy-fortelin". Moreover, orally administered soy-fortelin had a similar but smaller effect in the young C57BL/6 mice, whereas it strongly stimulated food intake in 2-year-old aged mice that exhibited high blood ghrelin levels and low ghrelin sensitivity. In conclusion, we discovered soy-fortelin as a novel peptide that enhances ghrelin sensitivity in vivo and in vitro and increases food intake in young and aged ghrelin-resistant mice. Soy-fortelin is the first food-derived peptide reported to enhance ghrelin sensitivity.

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.

Rice Endoplasmic Protein-Derived Peptides, Rice-Ghretropins A and B, Stimulate Ghrelin Release in MGN3-1 Cells and Increase Plasma Acylated Ghrelin and Food Intake in Mice.

In this study, we demonstrated that novel rice-derived bioactive peptides promote the secretion of ghrelin, an endogenous orexigenic hormone secreted from the stomach. The enzymatic digest of rice endosperm protein with subtilisin, a microorganism-derived enzyme, stimulated acylated ghrelin secretion in the ghrelin-releasing cell line MGN3-1 and increased food intake after oral administration in mice. By performing a comprehensive analysis based on structure-activity relationships, we selected candidate peptides from over 30,000 peptides in the rice digest. Among them, we found that QAFEPIRSV and TNPWHSPRQGSF, corresponding to the amino acid sequence of the rice endoplasmic proteins glutelin A1 or A2(52-60) and B1 or B2(31-42), respectively, stimulated acylated ghrelin release in MGN3-1 cells. We named them rice-ghretropins A and B. Pyroglutamate formation of rice-ghretropin A, [pyr1]-rice-ghretropin A, also promoted ghrelin secretion. Furthermore, oral administration of rice-ghretropins increased food intake, plasma ghrelin concentration, and small intestinal transit in mice. In addition, the subtilisin digest of the rice protein significantly increased food intake for 4 h in 9 month-old (control: 0.61 ± 0.049 g; digest: 0.83 ± 0.059 g) and 24 month-old mice (control: 0.52 ± 0.067 g; digest: 1.01 ± 0.064 g). In summary, we found that novel bioactive peptides, namely, rice-ghretropins, from the enzymatic digest of rice endosperm stimulated acylated ghrelin secretion and increased food intake. This is the first report of rice-derived exogenous bioactive peptides that increase acylated ghrelin secretion.

An orally active plant Rubisco-derived peptide increases neuronal leptin responsiveness.

Nutrient excess, such as the intake of a high-fat diet, reduces hypothalamic responses to exogenously administered leptin and induces dietary obesity; however, orally active components that attenuate neural leptin dysregulation have yet to be identified. We herein demonstrated that YHIEPV, derived from the pepsin-pancreatin digestion of the green leaf protein Rubisco, increased the leptin-induced phosphorylation of STAT3 in ex vivo hypothalamic slice cultures. We also showed that YHIEPV mitigated palmitic acid-induced decreases in leptin responsiveness. Furthermore, orally administered YHIEPV promoted leptin-induced reductions in body weight and food intake in obese mice. In addition, dietary-induced body weight gain was significantly less in mice orally or centrally administered YHIEPV daily than in saline-control mice. Cellular leptin sensitivity and the levels of proinflammatory-related factors, such as IL1ß and Socs-3, in the hypothalamus of obese mice were also restored by YHIEPV. YHIEPV blocked cellular leptin resistance induced by forskolin, which activates Epac-Rap1 signaling, and reduced the level of the GTP-bound active form of Rap1 in the brains of obese mice. Collectively, the present results demonstrated that the orally active peptide YHIEPV derived from a major green leaf protein increased neural leptin responsiveness and reduced body weight gain in mice with dietary obesity.

Rap1 in the VMH regulates glucose homeostasis.

The hypothalamus is a critical regulator of glucose metabolism and is capable of correcting diabetes conditions independently of an effect on energy balance. The small GTPase Rap1 in the forebrain is implicated in high-fat diet-induced (HFD-induced) obesity and glucose imbalance. Here, we report that increasing Rap1 activity selectively in the medial hypothalamus elevated blood glucose without increasing the body weight of HFD-fed mice. In contrast, decreasing hypothalamic Rap1 activity protected mice from diet-induced hyperglycemia but did not prevent weight gain. The remarkable glycemic effect of Rap1 was reproduced when Rap1 was specifically deleted in steroidogenic factor-1-positive (SF-1-positive) neurons in the ventromedial hypothalamic nucleus (VMH) known to regulate glucose metabolism. While having no effect on body weight regardless of sex, diet, and age, Rap1 deficiency in the VMH SF1 neurons markedly lowered blood glucose and insulin levels, improved glucose and insulin tolerance, and protected mice against HFD-induced neural leptin resistance and peripheral insulin resistance at the cellular and whole-body levels. Last, acute pharmacological inhibition of brain exchange protein directly activated by cAMP 2, a direct activator of Rap1, corrected glucose imbalance in obese mouse models. Our findings uncover the primary role of VMH Rap1 in glycemic control and implicate Rap1 signaling as a potential target for therapeutic intervention in diabetes.

Wheat-ghretropins: novel ghrelin-releasing peptides derived from wheat protein.

Ghrelin is an endogenous orexigenic hormone mainly produced by stomach cells and is reported to influence appetite, gastrointestinal motility and growth hormone secretion. We observed that enzymatic digest of wheat gluten stimulated ghrelin secretion from mouse ghrelinoma 3-1, a ghrelin-releasing cell line. Further on, we characterized the ghrelin-releasing peptides present in the digest by comprehensive peptide analysis using liquid chromatography-mass spectrometry and structure-activity relationship. Among the candidate peptides, we found that SQQQQPVLPQQPSF, LSVTSPQQVSY and YPTSL stimulated ghrelin release. We then named them wheat-ghretropin A, B and C, respectively. In addition, we observed that wheat-ghretropin A increased plasma ghrelin concentration and food intake in mice after oral administration. Thus, we demonstrated that wheat-ghretropin stimulates ghrelin release both in vitro and in vivo. To the best of our knowledge, this is the first report of a wheat-derived exogenous bioactive peptide that stimulates ghrelin secretion.

A milk-derived pentapeptide reduces blood pressure in advanced hypertension in a CCK system-dependent manner.

We recently found that a peptide that activates the cholecystokinin (CCK) system effectively reduces blood pressure in spontaneously hypertensive rats (SHR) after the development of hypertension, after which hypotensive drugs are sometimes less effective. In this study, we investigated the vasorelaxation and antihypertensive effects of a peptide derived from a milk protein in SHR with advanced hypertension. The vasorelaxing activity was measured using the mesenteric artery isolated from SHR and the systemic blood pressure was measured by the tail-cuff method. KFWGK was released from bovine serum albumin (BSA) and the model peptide after subtilisin digestion. KFWGK relaxed the mesenteric artery and this vasorelaxation activity was inhibited by lorglumide, an antagonist of the CCK1 receptor. KFWGK more potently relaxed the artery from advanced-stage SHR than that from early-stage SHR. Orally administered KFWGK exhibited potent and long-lasting antihypertensive effects in SHR after the development of hypertension (the minimum effective dose was 5 µg kg-1). The KFWGK-induced antihypertensive effects were also blocked by a CCK antagonist, suggesting that it activates the CCK system. In conclusion, KFWGK, a CCK-dependent vasorelaxant peptide, exhibited potent antihypertensive effects in SHR after the development of hypertension.

Anti-Hypertensive Effects of Peptides Derived from Rice Bran Protein.

Hypertension is one of the major risk factors for arteriosclerosis. Anti-hypertensive peptides derived from animal proteins, such as milk, eggs and fish, are well studied. Anti-hypertensive peptides have also been identified from plant proteins such as soybeans. Rice bran, a byproduct of white rice polishing, is rich in protein and its high protein efficiency ratio is well known. This review discusses the anti-hypertensive peptides identified from rice bran protein and their mechanisms. In addition, we describe protease-digested rice bran from which functional peptides have not been isolated.

Dipeptide tyrosyl-leucine exhibits antidepressant-like activity in mice.

Depression is a worldwide health problem. In the present study, we found that a dipeptide, tyrosyl leucine (Tyr-Leu, YL), administered orally, intracerebroventricularly, or intraperitoneally exhibited a potent antidepressant-like activity in the forced swim and tail suspension tests in naïve mice. YL increased the amount of cells expressing c-Fos, a marker for neuronal activity, in the dentate gyrus of the hippocampus. YL increased bromo-2'-deoxyuridine-positive cells and doublecortin expression in the dentate gyrus of the hippocampus, suggesting that YL enhanced the proliferation of hippocampal progenitor cells in vivo and in vitro. YL did not affect hippocampal mRNA and protein expression of BDNF, which is a regulatory factor of both neurogenesis and depression-like behavior. Intriguingly, YL suppressed activation of the hypothalamo-pituitary-adrenal axis by forced swim stress. Moreover, other aromatic amino acid-leucines, Phe-Leu and Trp-Leu, also exhibited antidepressant-like activities, suggesting that the structure of aromatic amino acid-leucine may be important for antidepressant activity. In addition, bovine milk casein-derived peptide, Tyr-Leu-Gly (YLG), an anxiolytic peptide, exhibited an antidepressant-like activity. Our findings demonstrate that YL exhibits an antidepressant-like effect, moderates the stress response, and induces hippocampal neuronal proliferation through a signal pathway independent of BDNF.

Ser-Tyr and Asn-Ala, vasorelaxing dipeptides found by comprehensive screening, reduce blood pressure via different age-dependent mechanisms.

To understand the changes in physiological responses due to aging, a number of bioactive probes based on different signal transduction pathways are necessary. In this study, we comprehensively and systematically investigated changes in blood vessel function with age using a 336-dipeptide library. In the early stage of hypertension, the most potent vasorelaxant dipeptide was Ser-Tyr (SY) in the mesenteric artery isolated from spontaneously hypertensive rats (SHR). SY-induced vasorelaxation and anti-hypertensive effects were blocked by L-NAME, an inhibitor of nitric oxide synthase (NOS), suggesting that SY activates the NO system. On the other hand, the patterns of dipeptides with vasorelaxation activity in early and advanced stages of hypertension were different. In the advanced stage, the most potent vasorelaxing dipeptide was Asn-Ala (NA). Orally administered NA (1.5 mg/kg) reduced the blood pressure in the advanced stage, at which drugs were sometimes less effective, and the anti-hypertensive effects lasted for 6 hr. The NA-induced vasorelaxation and anti-hypertensive activity was blocked by lorglumide, an antagonist of the cholecystokinin CCK1 receptor, suggesting that NA activated the CCK system. Taken together, in the early and advanced stages of hypertension, SY and NA exhibited vasorelaxing and anti-hypertensive effects via the NO and CCK systems, respectively.

Orally administered milk-derived tripeptide improved cognitive decline in mice fed a high-fat diet.

The functions of the brain, which is thought of as an organ highly independent from the periphery, are often affected by the peripheral environment. Indeed, epidemiologic studies demonstrated that diabetes was a risk factor for dementia. It was also reported that the intake of dairy products, such as milk, reduces the risk of developing dementia. We found that mice on a short-term high-fat diet (HFD) for 1 wk had reduced cognitive function. Thus, using this acute model, we investigated the effects of milk-derived peptide on cognitive decline induced by HFD. Tyr-Leu-Gly (YLG), a tripeptide derived from αS1-casein, a major bovine milk protein, is released by gastrointestinal proteases. We found that orally administered YLG improved cognitive decline induced by 1-wk HFD intake in the object recognition test. YLG also improved cognitive decline in the object location test. Thus, we found that YLG improved cognitive decline induced by HFD. Next, we examined the effects of YLG on the hippocampus, a brain area essential for cognitive function. HFD intake decreased the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells, and this decrease was improved by YLG administration. HFD intake decreased nerve growth factor (NGF) and glial cell line-derived neurotrophic factor, whereas YLG increased NGF and ciliary neurotrophic factor, suggesting that these neurotropic factors play a role in hippocampal neurogenesis after YLG administration. In conclusion, we demonstrated that 1-wk HFD reduced cognitive function. Furthermore, we found that YLG, a milk-derived tripeptide, improved cognitive decline in mice on HFD. The HFD reduced neural stem cell proliferation, and YLG improved this reduction. YLG is the first reported milk peptide to improve cognitive decline induced by HFD intake.-Nagai, A., Mizushige, T., Matsumura, S., Inoue, K., Ohinata, K. Orally administered milk-derived tripeptide improved cognitive decline in mice fed a high-fat diet.

Comprehensive analysis of a dipeptide library to identify ghrelin release-modulating peptides.

We performed a comprehensive analysis of ghrelin release-modulating activity of a dipeptide library using MGN3-1, a ghrelin-producing cell line. We found that most dipeptides suppress ghrelin secretion, whereas the N-terminal Ser-containing dipeptides and a few others stimulate it. N-terminal amino acid residues, but not C-terminal residues, play a dominant role in the effects of dipeptides. Among dipeptides, Leu-Ile (LI) and Ser-Val (SV) most strongly suppress and stimulate ghrelin secretion, respectively. LI activates Gi signaling and SV acts via the MAPK pathway. Orally administered LI and SV reduce and increase plasma ghrelin levels and food intake in mice, respectively. In conclusion, LI and SV, found based on the comprehensive screening of a dipeptide library, modulate ghrelin secretion in vitro and in vivo.

Vasorelaxant and Antihypertensive Effects That Are Dependent on the Endothelial NO System Exhibited by Rice Bran-Derived Tripeptide.

We recently identified a novel, potent antihypertensive peptide, Leu-Arg-Ala (LRA; minimum effective dose = 0.25 mg/kg), from rice bran protein. In this study, we found that LRA potently relaxed mesenteric arteries isolated from spontaneously hypertensive rats (SHRs) (EC50 = 0.1 µM). In contrast, the vasorelaxant activity of each amino acid that constitutes the LRA tripeptide was remarkably attenuated. The LRA-induced vasorelaxant activity was inhibited by N(G)-nitro-l-arginine methyl ester (L-NAME; NO synthase [NOS] inhibitor) but not by an antagonist of bradykinin B2 and Mas receptors or by a phosphoinositide 3-kinase inhibitor. The antihypertensive effect induced after the oral administration of LRA was inhibited by L-NAME. LRA also induced the phosphorylation of endothelial NOS in human umbilical vein endothelial cells. Taken together, LRA may exhibit antihypertensive effects via NO-mediated vasorelaxation. LRA is the first example of a NO-dependent vasorelaxant peptide identified from rice bran protein.

Identification of Rubisco anxiolytic-like peptides (rALPs) by comprehensive analysis of spinach green leaf protein digest.

Rubisco, an enzyme for photosynthetic carbon dioxide fixation, is a major green leaf protein and known as the most abundant protein on the Earth. We found that Rubisco digested mimicking gastrointestinal enzymatic conditions exhibited anxiolytic-like effects after oral administration in mice. Based on a comprehensive peptide analysis of the digest using nanoLC-Orbitrap-MS and the structure-activity relationship of known anxiolytic-like peptides, we identified SYLPPLTT, SYLPPLT and YHIEPV [termed Rubisco anxiolytic-like peptide (rALP)-1, rALP-1(1-7) and rALP-2, respectively], which exhibited potent anxiolytic-like effects after oral administration. The anxiolytic-like effects of rALP-1/rALP-1(1-7) were blocked by a serotonin 5-HT1A receptor antagonist, whereas rALP-2-induced effects were inhibited by a δ-opioid receptor antagonist. In conclusion, novel Rubisco-derived anxiolytic-like peptides, rALP-1/rALP-1(1-7) and rALP-2, act via independent neural pathways.

[Clarification of the Mechanism Involved in Orexigenic Action by Oral Zinc Ingestion].

We investigated the role of zinc in regulation of food intake using male SD rats during early-stage of zinc deficiency (the 3rd day of the feeding) without decreased zinc concentrations in tissues (hypothalamus and liver). As a result, we found that orally but not intraperitoneal administered zinc stimulates food intake in the short-term zinc-deficient rats. The mRNA expressions of hypothalamic peptides, such as orexin (OX) and neuropeptide Y (NPY), were increased after oral administration of zinc to increase food intake. Pretreatment with an antagonist for the NPY Y1 receptor or the orexin OX1 receptor blocked orexigenic activity by zinc administration. The stimulation of food intake by oral administration of zinc was also abolished by vagotomy. Taken together, our results indicate that zinc stimulates food intake in short-term zinc-deficient rats through the afferent vagus nerve followed by activating the hypothalamic peptide associated with food intake regulation. This study showed the first evidence that gastrointestinal zinc signal is indispensable for the food appetite induction in the experimentally anorexigenic rat. However, since it has not yet been clarified the mechanism involved in zinc sensing by the epithelial membrane of the gastrointestinal tract, further detailed investigations are necessary.

Enzymatic digest of whey protein and wheylin-1, a dipeptide released in the digest, increase insulin sensitivity in an Akt phosphorylation-dependent manner.

We investigated the effects of the enzymatic digest of ß-lactoglobulin, a major bovine milk whey protein, on glucose metabolism in KK-Ay mice, an animal model of type II diabetes. In the glucose tolerance test and insulin tolerance test (ITT), the thermolysin digest of ß-lactoglobulin decreased blood glucose levels, suggesting that it increases insulin sensitivity in diabetic KK-Ay mice. The digest also increased phosphorylation of Akt, an intracellular factor activated in response to the insulin receptor activation, in the liver and skeletal muscle. Next, we searched for a bioactive peptide present in the digest that increased the insulin sensitivity. Wheylin-1 is an anxiolytic-like dipeptide (Met-His) isolated from the thermolysin digest of ß-lactoglobulin. Wheylin-1 decreased blood glucose levels in the ITT test and increased hepatic Akt phosphorylation. Wheylin-1 also increased insulin-induced Akt phosphorylation in hepatic HepG2 cells and muscular C2C12 myotube cells. These results suggest that wheylin-1 increases insulin sensitivity in an Akt-dependent manner in vivo and in vitro. Taken together, we found that the thermolysin digest of bovine milk whey ß-lactoglobulin and wheylin-1 increase insulin sensitivity in an Akt system-dependent manner. Wheylin-1 is the first factor found that increases insulin sensitivity in association with Akt-phosphorylation.