A new method for the preparation of a purified glucosylceramide and ceramide from shiitake mushroom.

Ingestion of plant and fungal glucosylceramides is known to reduce colon carcinogenesis and skin barrier damage in mice and humans. However, such effects in animal experiments have not been revealed for plant and fungal ceramides because the content of ceramides contained in plants and fungi is so low that the large amount required for animal experiments is difficult to obtain. Noting that the fungus shiitake mushroom (Lentinula edodes) is rich in a glucosylceramide, (4E,8E)-N-d-2'-hydroxypalmitoyl-1-O-ß-d-glucopyranosyl-9-methyl-4,8-sphingadienine [Glc-d19:2(4E,8E,9Me)-h16:0], we developed a new method to purify this fungal glucosylceramide using ethanol precipitation and high-performance liquid chromatography. We also developed a new method to produce large amounts of a ceramide [d19:2(4E,8E,9Me)-h16:0] from this purified glucosylceramide using human glycoside hydrolase family 30 glucocerebrosidase (imiglucerase). These methods will be useful for elucidating the physiological function by ingestion of fungal ceramides in animal experiments.

Differential effects of black currant anthocyanins on diffuser- or negative lens-induced ocular elongation in chicks.

PURPOSE: To compare the inhibitory effects of 4 different types of black currant anthocyanins (BCAs) on ocular elongation in 2 different chick myopia models. METHODS: In the first model, diffusers were used to induce form vision deprivation. In the second model, negative (-8D) spherical lenses were used to create a defocused retinal image. Either the diffusers or the -8D lenses were placed on the right eyes of 8-day-old chicks for 4 days. Ocular biometric components were measured using an A-scan ultrasound instrument on the third day after application of either the diffusers or -8D lenses. Interocular differences (globe component dimensions of the right diffuser or eyes covered with -8D lenses minus those of the open left eyes) were considered to evaluate the effect of BCAs. The BCAs used were cyanidin-3-glucoside (C3G), cyanidin-3-rutinoside (C3R), delphinidin-3-rutinoside (D3R), and delphinidin-3-glucoside (D3G). Each anthocyanin was administered intravenously at a dose of 0.027 µmol/kg once a day for 3 days. RESULTS: Compared to the vehicle treatment, C3G and C3R treatments significantly reduced both differential increases (positive values of interocular differences) of the ocular axial length induced by diffusers or -8D lenses (diffusers; C3G, C3R, and control: 0.32±0.051 mm, P<0.05; 0.25±0.034 mm, P<0.01; and 0.52±0.047 mm, -8D lenses; C3G, C3R, and control: 0.25±0.049 mm, P<0.01; 0.17±0.049 mm, P<0.001; and 0.50±0.056 mm). In contrast, compared to vehicle treatment, D3R treatment significantly decreased the differential increases in the ocular axial length only in chicks with myopia induced by -8D lenses (D3R and control: 0.17±0.049 mm and 0.50±0.056 mm, P<0.001). D3G did not inhibit the differential increase in the ocular axial length induced by either diffusers or -8D lenses. CONCLUSIONS: This study showed that the 4 tested BCAs had different effects on the 2 different experimental models of myopia.

The effect of high-intensity intermittent swimming on post-exercise glycogen supercompensation in rat skeletal muscle.

A single bout of prolonged endurance exercise stimulates glucose transport in skeletal muscles, leading to post-exercise muscle glycogen supercompensation if sufficient carbohydrate is provided after the cessation of exercise. Although we recently found that short-term sprint interval exercise also stimulates muscle glucose transport, the effect of this type of exercise on glycogen supercompensation is uncertain. Therefore, we compared the extent of muscle glycogen accumulation in response to carbohydrate feeding following sprint interval exercise with that following endurance exercise. In this study, 16-h-fasted rats underwent a bout of high-intensity intermittent swimming (HIS) as a model of sprint interval exercise or low-intensity prolonged swimming (LIS) as a model of endurance exercise. During HIS, the rats swam for eight 20-s sessions while burdened with a weight equal to 18% of their body weight. The LIS rats swam with no load for 3 h. The exercised rats were then refed for 4, 8, 12, or 16 h. Glycogen levels were almost depleted in the epitrochlearis muscles of HIS- or LIS-exercised rats immediately after the cessation of exercise. A rapid increase in muscle glycogen levels occurred during 4 h of refeeding, and glycogen levels had peaked at the end of 8 h of refeeding in each group of exercised refed rats. The peak glycogen levels during refeeding were not different between HIS- and LIS-exercised refed rats. Furthermore, although a large accumulation of muscle glycogen in response to carbohydrate refeeding is known to be associated with decreased insulin responsiveness of glucose transport, and despite the fact that muscle glycogen supercompensation was observed in the muscles of our exercised rats at the end of 4 h of refeeding, insulin responsiveness was not decreased in the muscles of either HIS- or LIS-exercised refed rats compared with non-exercised fasted control rats at this time point. These results suggest that sprint interval exercise enhances muscle glycogen supercompensation in response to carbohydrate refeeding as well as prolonged endurance exercise does. Furthermore, in this study, both HIS and LIS exercise prevented insulin resistance of glucose transport in glycogen supercompensated muscle during the early phase of carbohydrate refeeding. This probably led to the enhanced muscle glycogen supercompensation after exercise.

Cacao polyphenols influence the regulation of apolipoprotein in HepG2 and Caco2 cells.

Cocoa powder is rich in polyphenols, such as catechins and procyanidins, and has been shown to inhibit low-density lipoprotein (LDL) oxidation and atherogenesis in a variety of models. Human studies have also shown daily intake of cocoa increases plasma high-density lipoprotein (HDL) and decreases LDL levels. However, the mechanisms responsible for these effects of cocoa on cholesterol metabolism have yet to be fully elucidated. The present study investigated the effects of cacao polyphenols on the production of apolipoproteins A1 and B in human hepatoma HepG2 and intestinal Caco2 cell lines. The cultured HepG2 cells or Caco2 cells were incubated for 24 h in the presence of cacao polyphenols such as (-)-epicatechin, (+)-catechin, procyanidin B2, procyanidin C1, and cinnamtannin A2. The concentration of apolipoproteins in the cell culture media was quantified using an enzyme-linked immunoassay, and the mRNA expression was quantified by RT-PCR. Cacao polyphenols increased apolipoprotein A1 protein levels and mRNA expression, even though apolipoprotein B protein and the mRNA expression were slightly decreased in both HepG2 cells and Caco2 cells. In addition, cacao polyphenols increased sterol regulatory element binding proteins (SREBPs) and activated LDL receptors in HepG2 cells. These results suggest that cacao polyphenols may increase the production of mature form SREBPs and LDL receptor activity, thereby increasing ApoA1 and decreasing ApoB levels. These results elucidate a novel mechanism by which HDL cholesterol levels become elevated with daily cocoa intake.

Post-exercise carbohydrate plus whey protein hydrolysates supplementation increases skeletal muscle glycogen level in rats.

Recent studies showed that a combination of carbohydrate and protein was more effective than carbohydrate alone for replenishing muscle glycogen after exercise. However, it remains to be unclear whether the source or degree of hydrolysis of dietary protein influences post-exercise glycogen accumulation. The aim of this study was to compare the effect of dietary protein type on glycogen levels in the post-exercise phase, and to investigate the effects of post-exercise carbohydrate and protein supplementation on phosphorylated enzymes of Akt/PKB and atypical PKCs. Male Sprague-Dawley rats, trained for 3 days, swam with a 2% load of body weight for 4 h to deplete skeletal muscle glycogen. Immediately after the glycogen-depleting exercise, one group was killed, whereas the other groups were given either glucose or glucose plus protein (whey protein, whey protein hydrolysates (WPH), casein hydrolysates or branched-chain amino acid (BCAA) solutions. After 2 h, the rats were killed, and the triceps muscles quickly excised. WPH caused significant increases in skeletal muscle glycogen level (5.01 +/- 0.24 mg/g), compared with whey protein (4.23 +/- 0.24 mg/g), BCAA (3.92 +/- 0.18 mg/g) or casein hydrolysates (2.73 +/- 0.22 mg/g). Post-exercise ingestion of glucose plus WPH significantly increased both phosphorylated Akt/PKB (131%) and phosphorylated PKCzeta (154%) levels compared with glucose only. There was a significant positive correlation between skeletal muscle glycogen content and phosphorylated Akt/PKB (r = 0.674, P < 0.001) and PKCzeta (r = 0.481, P = 0.017). Post-exercise supplementation with carbohydrate and WPH increases skeletal muscle glycogen recovery by activating key enzymes such as Akt/PKB and atypical PKCs.