Masashi Morifuji

Comparison of different sources and degrees of hydrolysis of dietary protein: effect on plasma amino acids, dipeptides, and insulin responses in human subjects.

The effect of protein fractionation on the bioavailability of amino acids and peptides and insulin response and whether the protein source influences these effects in humans are poorly understood. This study compared the effects of different sources and degrees of hydrolysis of dietary protein, independent of carbohydrate, on plasma amino acid and dipeptide levels and insulin responses in humans. Ten subjects were enrolled in the study, with five subjects participating in trials on either soy or whey protein and their hydrolysates. Protein hydrolysates were absorbed more rapidly as plasma amino acids compared to nonhydrolyzed protein. Whey protein also caused more rapid increases in indispensable amino acid and branched-chain amino acid concentrations than soy protein. In addition, protein hydrolysates caused significant increases in Val-Leu and Ile-Leu concentrations compared to nonhydrolyzed protein. Whey protein hydrolysates also induced significantly greater stimulation of insulin release than the other proteins. Taken together, these results demonstrate whey protein hydrolysates cause significantly greater increases in the plasma concentrations of amino acids, dipeptides, and insulin.

Hydroxyproline-containing dipeptides and tripeptides quantified at high concentration in human blood after oral administration of gelatin hydrolysate.

Abstract Several hydroxyproline (Hyp)-containing food-derived collagen peptides were identified in human blood after oral ingestion of gelatin hydrolysates. However, these types of peptides were not quantified in human plasma. In this report, a sensitive LC-MS/MS method was introduced for simultaneous quantitative analysis of Hyp-containing peptides. All peptide concentrations were determined accurately, with all coefficients of determination (r2) >0.999. The method achieved detection and quantification limits of 0.01 pmol/ml and 12.5−1,000 pmol/ml in plasma, respectively. Concentrations were quantified for nine Hyp-containing peptides in human plasma by this method, identifying Pro-Hyp (Cmax = 60.65 ± 5.74 nmol/ml) as the major constituent of food-derived collagen peptides, while the minor components were Ala-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp, Phe-Hyp, Leu-Hyp, Ile-Hyp, Gly-Pro-Hyp, and Pro-Hyp-Gly (Cmax from 23.84 to 0.67 nmol/ml). Thus a total of nine Hyp-containing peptides in human plasma were successfully quantified by this approach. The concentration of Hyp-containing peptides is substantially higher than that following oral administration of other peptides.

Dietary whey protein increases liver and skeletal muscle glycogen levels in exercise-trained rats

We investigated the effect of different types of dietary protein on glycogen content in liver and skeletal muscle of exercise-trained rats. Twenty-four male Sprague-Dawley rats (approximately 100 g; n 6 per group) were divided into sedentary or exercise-trained groups with each group being fed either casein or whey protein as the source of dietary protein. Rats in the exercised groups were trained during 2 weeks using swimming exercise for 120 min/d, 6 d/week. Exercise training resulted in an increase in the skeletal muscle glycogen content. Furthermore, the whey protein group significantly increased the skeletal muscle glycogen content compared with the casein group. The increase in glycogen content in liver was significantly greater in rats fed the whey protein diet compared with those fed the casein diet. We also found that the whey protein diet increased the activity of liver glucokinase, whereas it decreased the activities of 6-phosphofructokinase and pyruvate kinase compared with the casein diet. However, hepatic total glycogen synthase activity and mRNA expression were similar with the two diets. In the skeletal muscle, whey protein decreased only 6-phosphofructokinase activity compared with casein. Total glycogen synthase activity in the skeletal muscle in the whey protein group was significantly higher than that in the casein group. The present study is the first to demonstrate that a diet based on whey protein may increase glycogen content in liver and skeletal muscle of exercise-trained rats. We also observed that whey protein regulated glycogen metabolism in these two tissues by different mechanisms.

Dietary soya protein intake and exercise training have an additive effect on skeletal muscle fatty acid oxidation enzyme activities and mRNA levels in rats.

Exercise training and regular physical activity increase oxidation of fat. Enhanced oxidation of fat is important for preventing lifestyle diseases such as hypertension and obesity. The aim of the present study in rats was to determine whether intake of dietary soya protein and exercise training have an additive effect on the activity and mRNA expression of enzymes involved in skeletal muscle fatty acid oxidation. Male Sprague-Dawley rats (n 32) were assigned randomly into four groups (eight rats per group) and then divided further into sedentary or exercise-trained groups fed either casein or soya protein diets. Rats in the exercise groups were trained for 2 weeks by swimming for 120 min/d, 6 d/week. Exercise training decreased hepatic triacylglycerol levels and retroperitoneal adipose tissue weight and increased skeletal muscle carnitine palmitoyltransferase 1 (CPT1) activity and mRNA expression of CPT1, beta-hydroxyacyl-CoA dehydrogenase (HAD), acyl-CoA oxidase, PPARgamma coactivator 1alpha (PGC1alpha) and PPARalpha. Soya protein significantly decreased hepatic triacylglycerol levels and epididymal adipose tissue weight and increased skeletal muscle CPT1 activity and CPT1, HAD, acyl-CoA oxidase, medium-chain acyl-CoA dehydrogenase, PGC1alpha and PPARalpha mRNA levels. Furthermore, skeletal muscle HAD activity was the highest in exercise-trained rats fed soya protein. We conclude that exercise training and soya protein intake have an important additive role on induction of PPAR pathways, leading to increased activity and mRNA expression of enzymes involved in fatty acid oxidation in skeletal muscle and reduced accumulation of body fat.

Collagen hydrolysate intake improves the loss of epidermal barrier function and skin elasticity induced by UVB irradiation in hairless mice.

Ultraviolet B (UVB) irradiation induces serious damage to the skin. Collagen hydrolysate and collagen‐derived peptides have effects on skin function in vivo and in vitro. However, few studies have investigated changes in the epidermal barrier or dermal elasticity caused by UVB. Here, we investigated the loss of epidermal barrier function and skin elasticity induced by UVB irradiation in hairless mice fed collagen hydrolysate.

Preexercise ingestion of carbohydrate plus whey protein hydrolysates attenuates skeletal muscle glycogen depletion during exercise in rats

OBJECTIVE Depletion of glycogen stores is associated with fatigue during both sprint and endurance exercises and therefore it is considered important to maintain adequate tissue stores of glycogen during exercise. The aims of the present study in rats were therefore to investigate the effects of preexercise supplementation with carbohydrate and whey protein hydrolysates (WPH) on glycogen content, and phosphorylated signaling molecules of key enzymes that regulate glucose uptake and glycogen synthesis during exercise. METHODS Male SD rats were used in the study (n=7/group). Prior to exercise, one group of rats was sacrificed, whereas the other groups were given either water, glucose, or glucose plus WPH solutions. After ingestion of the test solutions, glycogen-depleting exercise was carried out for 60 min. The rats were then sacrificed and the triceps muscles excised quickly. RESULTS Compared to water or glucose only, preexercise ingestion of glucose plus WPH caused a significant attenuation of muscle glycogen depletion during the postexercise period. Coingestion of glucose and WPH also significantly lowered phosphorylated glycogen synthase levels compared to ingestion of water only. In the glucose plus WPH group, the levels of phosphorylated Akt were increased significantly compared to the group ingesting water only, while the levels of phosphorylated PKC were significantly higher than in the groups ingesting only water or glucose. CONCLUSION Taken together, these results indicate that, compared to ingestion of glucose or water only, preexercise ingestion of carbohydrate plus WPH activates skeletal muscle proteins of key enzymes that regulate glucose uptake and glycogen synthesis during exercise, thereby attenuating exercise-induced glycogen depletion.

A novel mechanism for improvement of dry skin by dietary milk phospholipids: Effect on epidermal covalently bound ceramides and skin inflammation in hairless mice

BACKGROUND Dietary milk phospholipids (MPLs) increase hydration of the stratum corneum and reduced transepidermal water loss (TEWL) in hairless mice fed a standard diet. However, the mechanism by which MPLs improve skin barrier functions has yet to be established. OBJECTIVE This study was designed to examine the mechanism by which MPLs may affect covalently bound ceramides and markers of skin inflammation and improve the skin barrier defect in hairless mice fed a magnesium-deficient (HR-AD) diet. METHODS Four-week-old female hairless mice were randomized into four groups (n=10/group), and fed a standard (control) diet, the HR-AD diet, the HR-AD diet supplemented with either 7.0 g/kg MPLs (low [L]-MPL) or 41.0 g/kg MPLs (high [H]-MPL). RESULTS Dietary MPLs improved the dry skin condition of hairless mice fed the HR-AD diet. MPLs significantly increased the percentage of covalently bound ω-hydroxy ceramides in the epidermis, and significantly decreased both thymus and activation-regulated chemokine (TARC) mRNA and thymic stromal lymphopoietin (TSLP) mRNA levels in skin, compared with the HR-AD diet. Furthermore, the MPL diets significantly decreased serum concentrations of immunoglobulin-E, TARC, TSLP, and soluble P-selectin versus the HR-AD diet. CONCLUSION Our study showed for the first time that dietary MPLs may modulate epidermal covalently bound ceramides associated with formation of lamellar structures and suppress skin inflammation, resulting in improved skin barrier function.

Dietary whey hydrolysate with exercise alters the plasma protein profile: A comprehensive protein analysis

OBJECTIVE It has been shown that dietary whey protein accelerates glucose uptake by altering glycoregulatory enzyme activity in skeletal muscle. In the present study, we investigated the effect of dietary whey protein on endurance and glycogen resynthesis and attempted to identify plasma proteins that reflected the physical condition by a comprehensive proteomics approach. METHODS Male c57BL/6 mice were divided into four groups: sedentary, sedentary with whey protein hydrolysate, exercise, and exercise with whey protein hydrolysate. The mice in the exercise groups performed treadmill running exercise five times per week for 4 wk. Protein profiling of plasma sample obtained from individuals was performed, as were measurements of endurance performance and the glycogen content of gastrocnemius muscle. RESULTS After the training period, the endurance of mice fed the whey diet was improved compared with that of mice fed the control diet. Muscle glycogen content was significantly increased after 4 wk of exercise, and intake of whey protein led to a further increase in glycogen. Apolipoproteins A-II and C-I and β(2)-glycoprotein-1 were found to be altered by training combined with the intake of whey protein, without significant changes induced by exercise or whey protein alone. CONCLUSION Results of the present study suggest that these three proteins may be potential biomarkers of improved endurance and glycogen resynthesis and part of the mechanism that mediates the benefits of whey protein.

Development and Validation of a Liquid Chromatography Tandem Mass Spectrometry Method for Simultaneous Determination of Four Anthocyanins in Human Plasma after Black Currant Anthocyanins Ingestion

Black currant anthocyanins consist of delphinidin-3-glucoside (1), delphinidin-3-rutinoside (2), cyanidin-3-glucoside (3), and cyanidin-3-rutinoside (4). A liquid chromatography tandem mass spectrometry method for simultaneous determination of four anthocyanins in human plasma was developed and validated. Samples were prepared using solid phase extraction, followed by chromatographic separation with a reverse phase C(18) column with gradient elution using mobile phases containing water, acetonitrile, and formic acid. The quantification of four anthocyanins was determined by multiple reaction monitoring using electrospray ionization. The method showed good selectivity, sensitivity (limits of quantification for four anthocyanins were 0.2 nmol/L), linearity (0.2-20 nmol/L; r > 0.999), intra- and interday precision, accuracy (<14%), and recovery (62.5-85.7%). Analyte stability was investigated in detail. This method was successfully applied to the determination of delphinidin-3-glucoside (1), delphinidin-3-rutinoside (2), cyanidin-3-glucoside (3), and cyanidin-3-rutinoside (4) concentrations in human plasma after ingestion of a single dose of black currant anthocyanins (87.9 micromol (58.8 mg) total anthocyanins).

Dietary whey protein hydrolysates increase skeletal muscle glycogen levels via activation of glycogen synthase in mice.

Previously, we have shown that consuming carbohydrate plus whey protein hydrolysates (WPHs) replenished muscle glycogen after exercise more effectively than consuming intact whey protein or branched-chain amino acids (BCAAs). The mechanism leading to superior glycogen replenishment after consuming WPH is unclear. In this 5 week intervention, ddY mice were fed experimental diets containing WPH, a mixture of whey amino acids (WAAs), or casein (control). After the intervention, gastrocnemius muscle glycogen levels were significantly higher in the WPH group (4.35 mg/g) than in the WAA (3.15 mg/g) or control (2.51 mg/g) groups. In addition, total glycogen synthase (GS) protein levels were significantly higher in the WPH group (153%) than in the WAA (89.2%) or control groups, and phosphorylated GS levels were significantly decreased in the WPH group (51.4%). These results indicate that dietary WPH may increase the muscle glycogen content through increased GS activity.