Chronic Intake of a Meal Including Alaska Pollack Protein Increases Skeletal Muscle Mass and Strength in Healthy Older Women: A Double-Blind Randomized Controlled Trial.

BACKGROUND: In animal studies, a meal containing Alaska pollack protein (APP) induces fast-twitch muscle hypertrophy. To our knowledge, no interventional studies have examined the benefits of APP intake on muscle mass and muscle weakness and the prevention of sarcopenia in older individuals. OBJECTIVES: We evaluated the effects of APP intake on skeletal muscle mass, muscle strength, and physical performance among healthy community-dwelling older Japanese women. METHODS: In this double-blind randomized controlled trial, healthy women ≥ 65 y old were allocated to an APP or whey protein control (CON) group. Participants ingested test protein meals (5.0-5.1 g protein/serving) daily for 24 wk. Between-group differences in the change of skeletal muscle mass index (SMI) as the primary outcome and muscle strength as a secondary outcome were tested using multifrequency BIA and a handheld dynamometer, respectively, at baseline, and 4, 12, and 24 wk. The mean changes in the measured primary and secondary outcome variables from baseline to 4, 12, and 24 wk were compared using unpaired t tests. RESULTS: There were no between-group differences in nutritional status, food intake, or total energy and protein intakes at baseline, 12 wk, or 24 wk. The change in SMI was 0.12 kg/m2 (95% CI: 0.01, 0.23 kg/m2) and 0.11 kg/m2 (95% CI: 0.03, 0.19 kg/m2) greater in the APP group than in the CON group at 12 wk and 24 wk (P ≤ 0.03) and knee extension strength was 0.07 Nm/kg BW (95% CI: 0.02, 0.12 Nm/kg BW) and 0.05 Nm/kg BW (95% CI: 0.00, 0.09 Nm/kg BW) higher in the APP group than in the CON group at these times (P ≤ 0.015), respectively. The groups did not differ at 4 wk. CONCLUSIONS: Daily intake of a meal containing APP compared with whey protein increases skeletal muscle mass and lower-extremity muscle strength in healthy older women, suggesting that an APP-containing meal may be useful in the prevention of sarcopenia in this group.This trial was registered at as UMIN000035718.

Suppressive Effect of Lactococcus lactis subsp. cremoris YRC3780 on a Murine Model of Japanese Cedar Pollinosis.

Accumulating evidence suggests that Lactococcus lactis subsp. cremoris YRC3780 isolated from kefir has the potential to alleviate allergic responses. Herein, we investigated the effect of YRC3780 on a murine model of Japanese cedar pollinosis (JCP). BALB/c mice immunized with cedar pollen extract (CPE) exhibited an increase in serum immunoglobulin E and developed nasal inflammatory responses including sneezing, nasal hyperresponsiveness, and nasal eosinophil accumulation upon intranasal allergen challenge. These responses were suppressed by the oral administration of YRC3780, although the effects on CPE-induced sneezing response and eosinophil infiltration were not statistically significant. Total fecal microbiota diversity was not affected by allergen immunization and challenge or by YRC3780 administration. However, the abundances of Bifidobacteriales, Veillonellaceae, Lactococcus, and Lactococcus lactis were larger and that of Bacteroides was smaller in YRC3780-treated mice compared with those in CPE-challenged and YRC3780-untreated mice. Our findings suggest the usefulness of YRC3780 for alleviating JCP.

Fish Protein Promotes Skeletal Muscle Hypertrophy via the Akt/mTOR Signaling Pathways.

Skeletal muscle is the largest organ in the body and has a broad range of plasticity, undergoing atrophy in response to aging or disease and hypertrophy in response to nutritional supplements or exercise. Loss of skeletal muscle mass and force increases the risk of falls, impairs mobility, and leads to reduced quality of life. In a previous study, we demonstrated that taking in Alaska pollock protein (APP) for only 7 d increased the gastrocnemius muscle mass in rats. This study was conducted to identify hypertrophic myofibers and analyze how hypertrophy occurs within them. Twenty male rats were randomly divided into two groups and administered a diet of casein or APP for 7 d. The expression of each myosin heavy chain (MyHC) isoform in a cross-sectional area was then measured. MyHC IIb and IIx isoforms exhibited hypertrophic features in the gastrocnemius muscles of the APP-fed rats. Furthermore, comprehensive proteomic analyses were conducted to identify changes in protein expression due to muscle hypertrophy. Our results, evaluated by pathway analyses, indicated that the activity of the growth factor signaling pathway was significantly impacted by APP consumption. Moreover, APP could promote protein synthesis by activating the protein kinase B/mechanistic target of the rapamycin signaling pathway, which is also promoted by exercise.

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.

Effects of Lactococcus lactis subsp. cremoris YRC3780 daily intake on the HPA axis response to acute psychological stress in healthy Japanese men.

BACKGROUND: Lactococcus lactis subsp. cremoris (YRC3780), which is isolated from kefir, has been associated with anti-allergic effects in humans. However, it remains unknown whether daily intake of YRC3780 attenuates the response to psychological stress in humans in parallel with changes to the gut microbiome. We examined the fundamental role of YRC3780 in the gut microbiome, stress response, sleep, and mental health in humans. METHODS: Effects of daily intake of YRC3780 on the hypothalamic-pituitary-adrenal (HPA) axis response to acute psychological stress were investigated in a double-blind, placebo-controlled clinical trial involving 27 healthy young men (mean age and body mass index: 23.5 years and 21.5 kg/m2) who were randomly assigned to placebo (n = 13) or YRC3780 (n = 14) groups. The HPA axis response to acute psychological stress, the diurnal rhythm of HPA axis activity, and gut microbiome were assessed and compared between the two groups. RESULTS: The results showed that daily intake of YRC3780 significantly lowered morning salivary cortisol levels compared with placebo. In addition, salivary cortisol levels following a social stress test significantly decreased +40 min after beginning the TSST in the YRC3780-treated group compared to placebo. There were no significant differences between the two groups in terms of actigraphy-based sleep quality, but the subjective sleep quality and mental health were significantly improved in the YRC3780-treated group compared to placebo. CONCLUSIONS: Our study suggests that daily intake of YRC3780 improves the HPA axis response to acute psychological stress, which might be associated with a decrease in morning cortisol levels.

Fish Protein Ingestion Induces Neural, but Not Muscular Adaptations, Following Resistance Training in Young Adults.

Purpose: Nutritional supplementation in conjunction with exercise is of interest for the prevention or improvement of declines in motor performances in older adults. An understanding of the effects on both young and older adults contributes to its effective application. We investigated the effect of fish protein ingestion with resistance training on neural and muscular adaptations in young adults using interventions and assessments that have already been tested in older adults. Methods: Eighteen young adults underwent 8 weeks of isometric knee extension training. During the intervention, nine participants ingested 5 g of fish protein (n = 9, Alaska pollack protein, APP), and the other nine participants ingested casein as a control (n = 9, CAS) in addition to daily meals. Before, during, and after the intervention, the isometric knee extension force, lower extremity muscle mass, and motor unit firing pattern of knee extensor muscles were measured. Results: Maximum voluntary contraction (MVC) was significantly increased in both APP and CAS groups from 0 weeks to 4, 6, and 8 weeks of intervention (p < 0.001), but there were no significant differences between the groups (p = 0.546-0.931). Muscle mass was not significantly changed during the intervention in either group (p = 0.250-0.698). Significant changes in motor unit firing rates (p = 0.02 and 0.029 for motor units recruited at 20-40% of MVC and at 40-60%) were observed following the intervention in the APP but not CAS (p = 0.120-0.751) group. Conclusions: These results suggest that dietary fish protein ingestion changes motor unit adaptations following resistance training in young adults.

Modulation of Neural and Muscular Adaptation Processes During Resistance Training by Fish Protein Ingestions in Older Adults.

Assessments of both neural and muscular adaptations during interventions would provide valuable information for developing countermeasures to age-related muscle dysfunctions. We investigated the effect of fish protein ingestion on training-induced neural and muscular adaptations in older adults. Twenty older adults participated 8 weeks of isometric knee extension training intervention. The participants were divided into two groups who took fish protein (n = 10, Alaska pollack protein, APP) or casein (n = 10, CAS). Maximal muscle strength during knee extension, lower extremity muscle mass (body impedance method), and motor unit firing pattern of knee extensor muscle (high-density surface electromyography) were measured before, during, and after the intervention. Muscle strength were significantly increased in both CAS (124.7 ± 5.8%) and APP (117.1 ± 4.4%) after intervention (p < .05), but no significant differences between the groups were observed (p > .05). Significant increases in lower extremity muscle mass from 0 to 8 weeks were demonstrated only for APP (102.0 ± 3.2, p < .05). Greater changes in motor unit firing pattern following intervention were represented in CAS more than in APP. These results suggest that nutritional supplementations could modulate neural and muscular adaptations following resistance training and fish protein ingestion preferentially induces muscular adaptation without the detectable neural adaptation in older adults.

Dietary Alaska pollack protein improves skeletal muscle weight recovery after immobilization-induced atrophy in rats.

The promotion of muscle recovery after immobilization is important to preserve an optimum health status. Here, we examined the effect of dietary Alaska pollack protein (APP) on skeletal muscle weight after atrophy induced by hind limb immobilization using plaster immobilization technique. Rat left limb was casted with a wetted plaster cast under anesthesia. After 2 weeks of feeding, the cast was removed and the rats were divided into three groups, namely, a baseline group, high-fat casein diet group, and high-fat APP diet group. After 3 weeks of feeding, the skeletal muscles (soleus, extensor digitorum longus [EDL], and gastrocnemius) were sampled. The estimated weight gains of soleus, gastrocnemius, and EDL muscle in the immobilized limbs were significantly larger in the rats fed with APP diet as compared with those fed with casein diet. In soleus muscle, dietary APP increased the expression of Igf1 and Myog genes in the immobilized limbs after the recovery period.

Investigation of the Lipid Changes That Occur in Hypertrophic Muscle due to Fish Protein-feeding Using Mass Spectrometry Imaging.

Alaska pollack protein (APP) was previously shown to reduce serum triacylglycerol and the atherogenic index and significantly increase gastrocnemius muscle mass in rats. To determine which myofibers are involved in this observed hypertrophy, we stained the gastrocnemius muscle with fast and slow fiber-specific antibodies and measured the muscle fiber diameter. We observed muscle hypertrophy in both the fast and slow fibers of APP-fed rats. Although muscle hypertrophy leads to drastic lipid changes, the amount of lipids did not differ significantly between casein-fed and APP-fed rats. To determine the lipid changes at the molecular species level and their localization, we performed matrix-assisted laser desorption/ionization mass spectrometry imaging to visualize lipids in the gastrocnemius muscles. We determined that lipid molecules were significantly changed due to APP feeding. Thus, APP feeding changes muscle lipid metabolism, and these metabolic changes might be related to hypertrophy.

Hypothalamic-pituitary-adrenal axis differentially responses to morning and evening psychological stress in healthy subjects.

AIM: The hypothalamic-pituitary-adrenal (HPA) axis responds to changing environmental demands including psychological stressors. The aim of the present study was to assess whether the time of day effects on the acute response of HPA axis activity to acute psychological stress. METHOD: We studied 27 healthy young subjects. The subjects were participated two experiments as follows. In the first experiment, subjects were instructed to keep their regular sleep schedule for 2 weeks which were measured by using a wrist-worn activity monitor. Afterward, to evaluate a diurnal rhythm of salivary cortisol, eight saliva samples were collected during waking period every 2 hours from when the subjects woke up. In the second experiment, the subjects were randomly assigned to two groups. The Trier Social Stress Test (TSST) was performed either in the morning (n = 14) or in the evening (n = 13). We measured diurnal rhythm of salivary cortisol and stress response of salivary cortisol and heart rate by the TSST. Morning and evening tests were started at 2 hours and 10 hours after woke up, respectively. RESULTS: All subjects showed a normal diurnal rhythm of salivary cortisol concentration, with a peak in the morning immediately after awaking and a minimum in the evening. The salivary cortisol response after the TSST was significantly increased from the prestress level in the morning but not in the evening. CONCLUSION: The HPA response to acute psychological stress was more pronounced in the morning than in the evening, correlating with the circadian regulation of cortisol synthesis.

Fish protein hydrolysate exhibits anti-obesity activity and reduces hypothalamic neuropeptide Y and agouti-related protein mRNA expressions in rats.

Fish protein is a source of animal protein that is consumed worldwide. Although it has been reported that the intake of Alaska pollack protein (APP) reduces body fat accumulation and increases muscle weight in rats, the mechanisms underlying these effects are poorly understood. As a possibility, peptides released from APP in the gastrointestinal tract are important to the functions of APP. In the present study, we examined the effects of APP hydrolysate digested artificially with pepsin and pancreatin on white adipose tissue and skeletal muscle. We found that APP hydrolysate group shows significantly lower weight of white adipose tissue and higher weight of soleus muscle than the control group. We also found that APP hydrolysate group reduces food intake and mRNA expressions of neuropeptide Y and agouti-related protein in the hypothalamus compared with the control group. These results may imply that APP hydrolysate exhibits anti-obesity activity by the reduction of appetite and the enhancement of basal energy expenditure by skeletal muscle hypertrophy in rats. The downregulation of orexigenic gene by APP hydrolysate in the hypothalamus may contribute to the reduction of appetite. These results suggest that the effect of APP on anti-obesity and muscle hypertrophy may be induced by peptides released from APP in the gastrointestinal tract.

Diagnostic performance of a semiconductor gamma-camera system as studied by multicenter registry.

BACKGROUND: Despite the introduction of the novel cadmium-zinc-telluride (CZT) single-photon emission computed tomography (SPECT) in Japan; its diagnostic value in clinical practice remains largely unknown. METHODS: The Semiconductor SPECT Study group is a multicenter Japanese registry which registered 1000 patients to evaluate the diagnostic utility of the CZT camera system (Discovery NM530c; GE Healthcare, Haifa, Israel). The patients underwent stress myocardial SPECT and coronary angiography within a 3-month interval. A significant stenosis was defined as ≥75% diameter narrowing based on the American Heart Association classification. RESULTS: Technetium (99mTc) radiotracer (555-1110MBq) was used in 71% and thallium-201 (201Tl) (74-148MBq) in 29%. The scan times with 99mTc-radiotracer were 5-10min for stress and 3-10min for rest, whereas those with 201Tl were 5-9min for stress and 8-10min for rest. To detect individual coronary stenosis, the respective sensitivities, specificities, and accuracies were 74%, 85%, and 81% for left anterior descending coronary artery (LAD) stenosis, 76%, 89%, and 85% for left circumflex stenosis, and 72%, 86%, and 82% for right coronary artery stenosis. However, 66% sensitivity and 91% specificity for LAD stenosis were observed with 99mTc-radiotracer, whereas 88% sensitivity and 63% specificity were found with 201Tl. CONCLUSIONS: The novel CZT SPECT system facilitated a short scan time with reduced radiotracer dose, yielding an acceptable diagnostic performance for angiographical coronary artery disease, although the low sensitivity for LAD detection with 99mTc-radiotracer needs to be refined.

Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling.

To drive lymphocyte proliferation and differentiation, common γ-chain (γc) cytokine receptors require hours to days of sustained stimulation. JAK1 and JAK3 kinases are found together in all γc-receptor complexes, but how their respective catalytic activities contribute to signaling over time is not known. Here we dissect the temporal requirements for JAK3 kinase activity with a selective covalent inhibitor (JAK3i). By monitoring phosphorylation of the transcription factor STAT5 over 20 h in CD4(+) T cells stimulated with interleukin 2 (IL-2), we document a second wave of signaling that is much more sensitive to JAK3i than the first wave. Selective inhibition of this second wave is sufficient to block cyclin expression and entry to S phase. An inhibitor-resistant JAK3 mutant (C905S) rescued all effects of JAK3i in isolated T cells and in mice. Our chemical genetic toolkit elucidates a biphasic requirement for JAK3 kinase activity in IL-2-driven T cell proliferation and will find broad utility in studies of γc-receptor signaling.

Mixed lactate and caffeine compound increases satellite cell activity and anabolic signals for muscle hypertrophy.

We examined whether a mixed lactate and caffeine compound (LC) could effectively elicit proliferation and differentiation of satellite cells or activate anabolic signals in skeletal muscles. We cultured C2C12 cells with either lactate or LC for 6 h. We found that lactate significantly increased myogenin and follistatin protein levels and phosphorylation of P70S6K while decreasing the levels of myostatin relative to the control. LC significantly increased protein levels of Pax7, MyoD, and Ki67 in addition to myogenin, relative to control. LC also significantly increased follistatin expression relative to control and stimulated phosphorylation of mTOR and P70S6K. In an in vivo study, male F344/DuCrlCrlj rats were assigned to control (Sed, n = 10), exercise (Ex, n = 12), and LC supplementation (LCEx, n = 13) groups. LC was orally administered daily. The LCEx and Ex groups were exercised on a treadmill, running for 30 min at low intensity every other day for 4 wk. The LCEx group experienced a significant increase in the mass of the gastrocnemius (GA) and tibialis anterior (TA) relative to both the Sed and Ex groups. Furthermore, the LCEx group showed a significant increase in the total DNA content of TA compared with the Sed group. The LCEx group experienced a significant increase in myogenin and follistatin expression of GA relative to the Ex group. These results suggest that administration of LC can effectively increase muscle mass concomitant with elevated numbers of myonuclei, even with low-intensity exercise training, via activated satellite cells and anabolic signals.

Covalent docking of large libraries for the discovery of chemical probes.

Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC ß-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).

Enhancement of Exopolysaccharide Production of Lactobacillus fermentum TDS030603 by Modifying Culture Conditions.

To optimize culture conditions that enhance production of a highly viscous exopolysaccharide of Lactobacillus fermentum TDS030603, a chemically defined medium was examined. The best yield was found to be 199 ± 23 mg/l when 48-hr cultivation was microaerobically performed at 30°C in the chemically defined medium supplemented with 5% glucose and 1% ammonium citrate without pH control. In response to the optimized exopolysaccharide production, the mRNA expression levels of epsB, epsE, and epsG elevated significantly. Our results indicated that the optimal C/N ratio and/or microaerobic condition can alter the expression levels of several exopolysaccharide biosynthesis-related genes promoting the exopolysaccharide production yield.

A preparation of cow's late colostrum fraction containing αs1-casein promoted the proliferation of cultured rat intestinal IEC-6 epithelial cells.

Colostrum is a complex mixture of bioactives that promotes neonate growth. Recently, we have found by in vivo study that skimmed, sterilized, and concentrated bovine late colostrum (SCBLC), obtained from a Holstein herd on days 6-7 after parturition, had an ability to maintain intestinal integrity. In the present study we investigated effects of SCBLC on rat intestinal IEC-6 cell proliferation in vitro. A fraction containing αs1-casein was found to have a robust stimulation effect as compared to other protein fractions from SCBLC and even the αs1-casein fraction from milk from other Holstein herds. Furthermore, the SCBLC αs1-casein molecule demonstrated not only slightly slower mobility on both SDS- and native-PAGE than other bovine milk αs1-caseins, but also a peculiar conformation reminiscent of moltenglobule in the circular dichroism spectrum. These findings may be of relevant to the competence of SCBLC to preserve intestinal integrity.