Prolyl-hydroxyproline, a collagen-derived dipeptide, enhances hippocampal cell proliferation, which leads to antidepressant-like effects in mice.

Depression has been a mental health issue worldwide. We previously reported that ginger-degraded collagen hydrolysate (GDCH) suppressed depression-like behavior in mice. Furthermore, prolyl-hydroxyproline (PO) and hydroxyprolyl-glycine (OG) were detected in the circulating blood after the oral administration of GDCH. In the present study, PO, but not OG, was detected in the cerebrospinal fluid of rats after the oral administration of GDCH, suggesting that PO is transported from blood to the brain. We then investigated the effects of PO and OG on the depression-like behavior of mice. The oral administration of PO significantly decreased depression-like behavior in the forced swim test. OG had no antidepressant-like effect. In addition, proline and hydroxyproline, components of PO, also had no antidepressant-like effect after their oral administration. PO significantly increased the gene expression of brain-derived neurotrophic factor and nerve growth factor in the hippocampus, and promoted the proliferation of neural progenitor cells in vivo and in vitro. PO also increased the dopamine concentration in the prefrontal cortex. Thus, PO-dependent regulation of neurotrophic function and neurotransmitter may be the mechanism for antidepressant-like behavior. Together, these results demonstrate that PO is an antidepressant bioactive peptide accompanying the proliferation of hippocampal neural progenitor cells.

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.

The daidzein- and estradiol- induced anorectic action in CCK or leptin receptor deficiency rats.

We investigated the effect of daidzein feeding and estradiol treatment on food intake in cholecystokinin-1 receptor (CCK1R) deficiency, leptin receptor (ObRb) deficiency rats and their wild-type rats. These rats underwent an ovariectomy or a sham operation. For the 5 week experiment, each rat was divided in three groups: control, daidzein (150 mg/kg diet), and estradiol (4.2 µg/rat/day) groups. In both CCK1R+ and CCK1R- rats, daidzein feeding and estradiol treatment significantly decreased food intake. Daidzein feeding significantly reduced food intake in ovariectomized ObRb- rats, although not in ObRb+ rats. Estradiol treatment significantly lowered food intake in ovariectomized ObRb+ and ObRb- rats. In the ovariectomized rats, estradiol treatment significantly increases uterine weight, while daidzein feeding did not change it, suggesting that daidzein might have no or weak estrogenic effect in our experiment. These results suggest that CCK1R and ObRb signalings were not essential for the daidzein- and estradiol-induced anorectic action.

Fish protein intake induces fast-muscle hypertrophy and reduces liver lipids and serum glucose levels in rats.

In our previous study, fish protein was proven to reduce serum lipids and body fat accumulation by skeletal muscle hypertrophy and enhancing basal energy expenditure in rats. In the present study, we examined the precise effects of fish protein intake on different skeletal muscle fiber types and metabolic gene expression of the muscle. Fish protein increased fast-twitch muscle weight, reduced liver triglycerides and serum glucose levels, compared with the casein diet after 6 or 8 weeks of feeding. Furthermore, fish protein upregulated the gene expressions of a fast-twitch muscle-type marker and a glucose transporter in the muscle. These results suggest that fish protein induces fast-muscle hypertrophy, and the enhancement of basal energy expenditure by muscle hypertrophy and the increase in muscle glucose uptake reduced liver lipids and serum glucose levels. The present results also imply that fish protein intake causes a slow-to-fast shift in muscle fiber type.

Dynamics of appetite-mediated gene expression in daidzein-fed female rats in the meal-feeding method.

We previously found that daidzein decreased food intake in female rats. The present study aimed to elucidate the relationship between dynamics of appetite-mediated neuropeptides and the anorectic effect of daidzein. We examined appetite-mediated gene expression in the hypothalamus and small intestine during the 3 meals per day feeding method. Daidzein had an anorectic effect specifically at the second feeding. Neuropeptide-Y (NPY) and galanin mRNA levels in the hypothalamus were significantly higher after feeding in the control but not in the daidzein group, suggesting that daidzein attenuated the postprandial increase in NPY and galanin expression. The daidzein group had higher corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamus after feeding, and increased cholelcystokinin (CCK) mRNA levels in the small intestine, suggesting that CCK is involved in the hypothalamic regulation of this　anorectic effect. Therefore, daidzein may induce anorexia by suppressing expression of NPY and galanin and increasing expression of CRH in the hypothalamus.

δ-Opioid receptor activation stimulates normal diet intake but conversely suppresses high-fat diet intake in mice.

The central opioid system is involved in a broadly distributed neural network that regulates food intake. Here, we show that activation of central δ-opioid receptor not only stimulated normal diet intake but conversely suppressed high-fat diet intake as well. [D-Pen(2,5)]-enkephalin (DPDPE), an agonist selective for the δ-receptor, increased normal diet intake after central administration to nonfasted male mice. The orexigenic activity of DPDPE was inhibited by blockade of cyclooxygenase (COX)-2, lipocalin-type prostaglandin D synthase (L-PGDS), D-type prostanoid receptor 1 (DP(1)), and neuropeptide Y (NPY) receptor type 1 (Y1) for PGD(2) and NPY, respectively, suggesting that this was mediated by the PGD(2)-NPY system. In contrast, DPDPE decreased high-fat diet intake in mice fed a high-fat diet. DPDPE-induced suppression of high-fat diet intake was blocked by antagonists of melanocortin 4 (MC(4)) and corticotropin-releasing factor (CRF) receptors but not by knockout of the L-PGDS gene. These results suggest that central δ-opioid receptor activation suppresses high-fat diet intake via the MC-CRF system, independent of the orexigenic PGD(2) system. Furthermore, orally administered rubiscolin-6, an opioid peptide derived from spinach Rubisco, suppressed high-fat diet intake. This suppression was also blocked by centrally administered naltrindole, an antagonist for the δ-receptor, suggesting that rubiscolin-6 suppressed high-fat diet intake via activation of central δ-opioid receptor.

Characterization of Tyr-Leu-Gly, a novel anxiolytic-like peptide released from bovine αS-casein.

We found previously that dipeptide YL exhibits orally active anxiolytic activity comparable to diazepam. The YL sequence is often observed in the primary structure of natural food proteins. In the present study, we investigated whether YL and YL analogues are released from bovine αS-casein by gastrointestinal proteases. YLG, corresponding to αS1-casein (aa 91-93), was more effectively released from αS-casein than YL by pepsin-pancreatin digestion, mimicking gastrointestinal enzymatic conditions. Using the synthetic model peptide, we determined that trypsin cleaved the N terminus of YLG, and elastase and carboxypeptidase contributed to cleave the C-terminus. YLG exhibited orally active anxiolytic-like activity in the elevated plus maze and open-field tests in mice. The anxiolytic-like activity of YLG was inhibited by WAY100135, SCH23390 or bicuculline, antagonists of serotonin 5-HT1A, dopamine D1, and GABA(A) receptors, respectively; however, YLG had no affinity for these receptors. The pepsin-pancreatin digest of αS-Casein also exhibited anxiolytic-like activity. Meanwhile, anxiolytic-like activity of α-casozepine, an αS1-casein-derived decapeptide with YL sequence in the N terminus, was blocked by WAY100135, SCH23390, or bicuculline, equally to YLG and YL; however, it was not detected in the pepsin-pancreatic digest. Taken together, we found that YLG is released after pepsin-pancreatic digestion of αS-casein and exhibits potent anxiolytic-like activity via activation of serotonin, dopamine, and the GABA receptor system.

The vagotomy alleviates the anorectic effect of an excess amount of dietary leucine on rats fed a low-protein diet.

It is well known that large dose of leucine reduces the food intake and causes growth retardation in experimental animals when leucine is given with a low-protein diet. However, the mechanism for the anorectic effect of leucine has not yet been clarified. We demonstrate here that the anorectic effect of leucine was significantly reduced in a vagotomized rat.

Increased levels of extracellular dopamine in the nucleus accumbens and amygdala of rats by ingesting a low concentration of a long-chain Fatty Acid.

Changes in the extracellular concentration of dopamine (DA) in the nucleus accumbens (NAc) shell and the basolateral amygdala (BLA) resulting from the voluntary ingestion of either corn oil, mineral oil, or 1% linoleic acid diluted with mineral oil as a vehicle were measured in rats by using in vivo microdialysis after they had been trained to establish a preference for corn oil. Ingesting the mineral oil caused no significant change in DA level in the NAc shell, whereas corn oil ingestion significantly increased the DA level during 0-15 min of the test session, reaching the maximum level of 129.8 ± 6.2% compared with the baseline after 10 min. Ingesting linoleic acid also resulted in a significant increase in DA level during 0-20 min, reaching 125.9 ± 9.0% after 10 min. Similar results were obtained in the BLA. Despite its very low calorie content, a low concentration of non-esterified fatty acid increased the DA levels equivalent to those resulting from corn oil in the brain's reward system.

Characterization of ovolin, an orally active tryptic peptide released from ovalbumin with anxiolytic-like activity.

We found that tryptic digest of ovalbumin after oral (p.o.) and intraperitoneal (i.p.) administration exhibited anxiolytic-like activity in mice, and then searched for orally active low-molecular-weight peptides with anxiolytic-like activity in the tryptic digest. Val-Tyr-Leu-Pro-Arg, named ovolin, corresponding to ovalbumin (280-284), mimicked the anxiolytic-like activity after p.o. and i.p. administration. The anxiolytic-like activity of ovolin was inhibited by indomethacin, a cyclooxygenase (COX) inhibitor, or BWA868C, an antagonist of the DP1 receptor for prostaglandin (PG) D2 . Ovolin-induced anxiolytic-like activity was also blocked by SCH58261 or bicuculline, antagonists of the adenosine A2A and GABAA receptors, respectively. Ovolin has no affinity for the DP1 , A2A and GABAA receptors. Taken together, ovolin may exhibit anxiolytic-like activity in a manner dependent on the PGD2 -DP1 system coupled to the A2A and GABAA receptors.

Complement C5a exhibits anxiolytic-like activity via the prostaglandin D2-DP1 receptor system coupled to adenosine A2A and GABAA receptors.

We have recently found that central PGD(2) exhibits anxiolytic-like activity. Here we show that complement C5a exhibits anxiolytic-like activity via the PGD(2) system. Centrally administered C5a had anxiolytic-like activity at a dose of 0.3 pmol/mouse in the elevated plus-maze test in mice. C5a-induced anxiolytic-like activity was inhibited by indomethacin, a cyclooxygenase inhibitor, or BWA868C, an antagonist of DP(1) receptor for PGD(2), respectively. The anxiolytic effect of C5a was also blocked by SCH58261 or bicuculline, antagonists of adenosine A(2A) and GABA(A) receptors, respectively, which were activated downstream of PGD(2)-DP(1) receptor. These results suggest that C5a exhibits anxiolytic-like activity via the PGD(2)-DP(1) receptor system coupled to the activation of adenosine A(2A) and GABA(A) receptors.

Mechanism of Soy Isoflavone Daidzein-Induced Female-Specific Anorectic Effect.

Epidemiological studies suggest that regular intake of soy isoflavone exerts a preventive effect on postmenopausal obesity and other forms of dysmetabolism. Estrogens inhibit eating behavior. Soy isoflavones may act as estrogen agonist in estrogen-depleted conditions, whereas they may either act as an estrogen antagonist or be ineffective in estrogen-repleted conditions. We investigated the effects of dietary soy isoflavone on food intake under various estrogen conditions using male, ovariectomized (OVX), and non-OVX female rats, and compared the effects with those of estradiol. We found that soy isoflavones reduced food intake in females specifically, regardless of whether ovariectomy had been performed, whereas subcutaneous implantation of estradiol pellet did not reduce food intake in intact female rats, but did so in OVX female and male rats. Contrary to this hypothesis, the reduction in food intake may not be caused by the estrogenic properties of soy isoflavones. It is of great interest to understand the mechanisms underlying the anorectic effects of soy isoflavones. In this non-systematic review, we summarize our recent studies that have investigated the bioactive substances of anorectic action, pharmacokinetic properties of soy isoflavones, and the modification of central and peripheral signals regulating appetite by soy isoflavones, and selected studies that were identified via database mining.

Dietary Alaska Pollack Protein Induces Acute and Sustainable Skeletal Muscle Hypertrophy in Rats.

Our previous studies suggested that Alaska pollack protein (APP) intake increases skeletal muscle mass and that it may cause a slow-to-fast shift in muscle fiber type in rats fed a high-fat diet after 56 days of feeding. In this study, we explored whether dietary APP induces acute and sustainable skeletal muscle hypertrophy in rats fed a normal-fat diet. Male 5-week-old Sprague-Dawley rats were divided into four groups and fed a purified ingredient-based high-fat diet or a purified ingredient-based normal-fat diet with casein or APP, containing the same amount of crude protein. Dietary APP significantly increased gastrocnemius muscle mass (105~110%) after 2, 7 days of feeding, regardless of dietary fat content. Rats were separated into two groups and fed a normal-fat diet with casein or APP. Dietary APP significantly increased gastrocnemius muscle mass (110%) after 56 days of feeding. Dietary APP significantly increased the cross-sectional area of the gastrocnemius skeletal muscle and collagen-rich connective tissue after 7 days of feeding. It decreased the gene expression of Mstn /Myostatin, Trim63/MuRF1, and Fbxo32/atrogin-1, but not other gene expression, such as serum IGF-1 after 7 days of feeding. No differences were observed between casein and APP groups with respect to the percentage of Type I, Type IIA, and Type IIX or IIB fibers, as determined by myosin ATPase staining after 7 days of feeding. In the similar experiment, the puromycin-labeled peptides were not different between dietary casein and APP after 2 days of feeding. These results demonstrate that APP induces acute and sustainable skeletal muscle hypertrophy in rats, regardless of dietary fat content. Dietary APP, as a daily protein source, may be an approach for maintaining or increasing muscle mass.

Neuromodulatory peptides: Orally active anxiolytic-like and antidepressant-like peptides derived from dietary plant proteins.

Mental disorders are a severe health problem, and the number of patients is growing worldwide. Increased anxiety and decreased motivation due to excessive mental stress further accelerated the severity of the problem. Enzymatic digestion of food proteins produces bioactive peptides with various physiological functions, some of which exhibit neuromodulatory effects with oral administration. Recently, studies reported that some peptides produced from plant proteins such as soybeans, leaves, and grains exhibit emotional regulatory functions such as strong anxiolytic-like and antidepressant-like effects comparable to pharmaceuticals. Conventionally, researchers investigated bioactive peptides by fractionation of protein hydrolysates and structure-activity relationship. As a novel methodology for analyzing bioactive peptides, the information obtained by peptidomics simultaneous analysis of the digested fractions of proteins using mass spectrometry has been effectively utilized. Some small-sized peptides such as dipeptides and tripeptides released food-derived proteins show emotional regulating effects. Moreover, some middle-sized peptides produced after intestinal digestion may exhibit the emotional regulating effect via the vagus nerve, and the importance of the gut-brain axis is also focused. As the central mechanism of emotional regulation, it has been found that these plant-derived peptides regulated monoamine neurotransmitter signaling and hippocampal neurogenesis.

Lipid Dynamics due to Muscle Atrophy Induced by Immobilization.

Muscle atrophy refers to skeletal muscle loss and dysfunction that affects glucose and lipid metabolism. Moreover, muscle atrophy is manifested in cancer, diabetes, and obesity. In this study, we focused on lipid metabolism during muscle atrophy. We observed that the gastrocnemius muscle was associated with significant atrophy with 8 days of immobilization of hind limb joints and that muscle atrophy occurred regardless of the muscle fiber type. Further, we performed lipid analyses using thin layer chromatography, liquid chromatography-mass spectrometry, and mass spectrometry imaging. Total amounts of triacylglycerol, phosphatidylserine, and sphingomyelin were found to be increased in the immobilized muscle. Additionally, we found that specific molecular species of phosphatidylserine, phosphatidylcholine, and sphingomyelin were increased by immobilization. Furthermore, the expression of adipose triglyceride lipase and the activity of cyclooxygenase-2 were significantly reduced by atrophy. From these results, it was revealed that lipid accumulation and metabolic changes in specific fatty acids occur during disuse muscle atrophy. The present study holds implications in validating preventive treatment strategies for muscle atrophy.

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.

Colocalization of GPR120 with phospholipase-Cbeta2 and alpha-gustducin in the taste bud cells in mice.

A recent study has demonstrated that the G-protein coupled receptor GPR120 is expressed in the taste bud cells in rats. In this study, we have identified the types of taste cell that express GPR120 in C57/BL6 mice. Double immunostaining for GPR120 and the markers of type II taste cells (phospholipase-Cbeta2 and alpha-gustducin) revealed that the majority of the GPR120-positive taste cells are type II taste cells. In contrast, it was observed that GPR120 was rarely colocalized with the marker of type III cells (neuronal cell adhesion molecule). These results suggested that GPR120 is mainly expressed in the type II taste cells and might function as a sensor for dietary fat.

Assessing palatability of long-chain fatty acids from the licking behavior of BALB/c mice.

Dietary oils such as corn oil, olive oil, and canola oil, which primarily contain triacylglycerol and small quantities of fatty acids, are highly palatable to animals. In a previous study, we examined the short-term (60 s) licking behavior of mice and observed that they exhibited a high licking response to a low concentration of fatty acid (linoleic acid), which is comparable to that observed for pure corn oil. This finding suggests that fatty acids contribute to the palatability of dietary oils. In order to supplement our knowledge of the fundamental features of fatty acid palatability in the oral cavity, we assessed the licking behavior of BALB/c mice to investigate the palatability of various types of long-chain fatty acids. The mice showed high licking responses to 1% unsaturated 16- and 18-carbon fatty acids (palmitoleic acid, 16:1; oleic acid, 18:1; linoleic acid, 18:2; and linolenic acid, 18:3), low licking responses to 16- and 20-carbon fatty acids (palmitic acid, 16:0 and arachidonic acid, 20:4), and no significant response to saturated fatty acids (stearic acid, 18:0 and arachidic acid, 20:0) or fatty acid derivatives (methyl linoleate and linole alcohol). Additionally, there were differences in the palatability of 18-carbon unsaturated fatty acids at very low concentrations. At fatty acid concentrations of 0.04% and 0.0625%, the mice showed significant preference for linoleic acid and linolenic acid, but not oleic acid, when compared with mineral oil. These results suggest that mice show high licking responses to 16- and 18-carbon unsaturated long-chain fatty acids at low concentrations. Further, we suggest that sensitivity to fatty acids is affected by the saturated state of the fatty acid, carbon chain length, and terminal carboxyl group.

Contribution of gustation to the palatability of linoleic acid.

We investigated the palatability of a low concentration of linoleic acid (LA) in short-term two-bottle choice tests and licking tests. To examine the contribution of gustation, mice were rendered anosmic with olfactory nerve transection surgery and test solutions were prepared using mineral oil (saturated long-chain hydrocarbon) to minimize textural effects. In the two-bottle choice tests between various pairs of different concentrations of corn oil and LA, both anosmic and the sham-operated mice constantly preferred a higher concentration of corn oil and LA. In the licking tests, the initial licking rate for 1% LA was higher than that for mineral oil in anosmic mice. In accordance with the results of the two-bottle choice test, the initial licking rate for corn oil and LA increased in a concentration-dependent manner in both anosmic and sham-operated mice in the licking test, and reached its peak at 100% corn oil and 1% LA. A preference comparison between 1% LA and 100% corn oil showed that anosmic mice preferred 1% LA over 100% corn oil. These results suggest that mice could recognize dietary fat and fatty acid solutions in the oral cavity without any olfactory or textural cues and the fatty acid recognition on their tongues might provide a pivotal cue to how dietary fat is recognized in the oral cavity.

Dietary daidzein induces accumulation of S-equol in enterohepatic circulation to far higher levels than that of daidzein in female rats with and without ovariectomy.

We previously found that daidzein decreased food intake in female rats. To understand the mechanism of anorectic action of dietary daidzein, it is necessary to determine distributions of daidzein and S-equol, a metabolite of intestinal bacterial conversion from daidzein, in the body. In the present study, we measured the concentrations of daidzein and S-equol in serum and bile in sham-operated and ovariectomized female rats fed a diet containing 150 mg/kg daidzein for 7 days. Dietary daidzein increased serum and bile concentrations of S-equol to far higher levels than those of daidzein. S-equol concentration was more than several hundred fold-higher in bile than in serum, regardless of ovariectomy. Moreover, to investigate whether accumulation of S-equol is facilitated by efficient enterohepatic circulation during continuous intake of daidzein and S-equol, female rats were fed diet containing daidzein or S-equol (both 150 mg/kg), or control diet for 1, 2, 3, or 5 days. Dietary daidzein significantly increased serum and bile concentrations of S-equol in a time-dependent manner, but not those of daidzein. These results indicated that substantial proportion of dietary daidzein was converted to S-equol, which underwent efficient enterohepatic circulation and predominantly accumulated there.