Activated hepatic stellate cells (HSCs) play crucial roles in liver fibrosis. In the course of liver injury, HSCs, which reside in perisinusoidal spaces and lose lipid droplets, morphologically change into a myofibroblastic phenotype and acquire an increased proliferation activity in what is known as the activated state. We have investigated therapeutic strategies for liver fibrosis by promoting spontaneous reversion or inducing apoptosis in activated HSCs. Vitamin E consists of four tocopherols and four tocotrienols, all of which are well-known antioxidants. In this study, the antiproliferative and proapoptotic effects of a tocol, which lacks methyl groups attached to the chromanol ring, and four tocopherols were investigated using activated HSCs. δ-Tocopherol and tocol exhibited relatively high proliferation inhibitory and proapoptotic abilities. However, they did not show proliferation inhibition ability on primary hepatocytes or HepG2 cells. Significant cell detachment was also observed in δ-tocopherol- and tocol-treated HSCs. Decreased protein expressions of α-smooth muscle actin and β1 integrin were observed in a dose-dependent manner. These results indicate that δ-tocopherol and tocol induce anoikis in activated HSCs.
We previously found that 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (γCEHC), a metabolite of the vitamin E isoforms γ-tocopherol or γ-tocotrienol, accumulated in the rat small intestine. The aim of this study was to evaluate tissue distribution of vitamin E metabolites. A single dose of α-tocopherol, γ-tocopherol or a tocotrienol mixture containing α- and γ-tocotrienol was orally administered to rats. Total amounts of conjugated and unconjugated metabolites in the tissues were measured by HPLC with an electrochemical detector, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) was used as an internal standard. Twenty-four hours later, the vitamin E isoforms were detected in most tissues and in the serum. However, 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (αCEHC), a metabolite of α-tocopherol or α-tocotrienol, and γCEHC accumulated in the serum and in some tissues including the liver, small intestine and kidney. Administration of α-tocopherol increased the γCEHC concentration in the small intestine, suggesting that α-tocopherol enhances γ-tocopherol catabolism. In contrast, ketoconazole, an inhibitor of cytochrome P450 (CYP)-dependent vitamin E catabolism, markedly decreased the γCEHC concentration. These data indicate that vitamin E metabolite accumulates not only in the liver but also in the small intestine and kidney. We conclude that some dietary vitamin E is catabolized to carboxyethyl-hydroxychroman in the small intestine and is secreted into the circulatory system.
The purpose of this study was to clarify the effects of nutrients on the gonadal development of male rats kept under constant darkness as a model of disturbed daily rhythm. In the present study we examined fat-soluble vitamins and their interactions in this test population. Four fat-soluble vitamins (vitamin A (V.A), vitamin D (V.D), vitamin E (V.E) and vitamin K (V.K)) were selected as experimental factors, and the dietary content of these vitamins was normal (AIN-93G) or three times the normal content. Lighting conditions (constant darkness or normal lighting) were also added as a factor. Four-week-old rats (Fischer 344 strain) were kept under constant darkness or normal lighting (12-h light/dark cycle) for 4 wk. The lighting condition and V.E, and the interactions between the lighting condition and V.E and between V.A and V.D were observed to affect the testes and epididymides weights. There was an influence of the lighting condition only on the seminal vesicles and prostate weights and the serum testosterone concentration. Among the constant darkness groups (D-groups), the highest value for testes weight was observed under the normal-V.A, normal-V.D and high-V.E diet. The interaction between lighting condition and V.E showed the testes weight increased slightly in response to changing to a high-V.E diet from a normal-V.E diet under normal lighting (N-group) but was greatly increased in response to this change in the D-group. It became clear that the amount of dietary V.E necessary for the gonadal development of rats increases when rats are kept under constant darkness.
We examined the effects of raw Chinese yam (Dioscorea opposita), containing resistant starch (RS), on lipid metabolism and cecal fermentation in rats. Raw yam (RY) and boiled yam (BY) contained 33.9% and 6.9% RS, respectively. Male Sprague-Dawley rats were fed a cholesterol-free, control (C) diet supplemented with or without 15 and 30 g of RY or BY/100 g for 3 wk. Plasma total cholesterol concentrations in the tail vein of rats fed the 30% RY diet were significantly lower than in the C group throughout the feeding period. Compared with the C group, non-HDL concentrations in arterial plasma in the 30% RY group was significantly reduced. Liver cholesterol concentration in rats fed the 30% RY diet was significantly higher compared with those fed the C diet. Hepatic cholesterol 7α-hydroxylase mRNA and fecal bile acid excretion were significantly higher in the BY, but not the RY group, compared with the C group. Fecal cholesterol excretion in the 30% RY group was greater compared with the C group. Hepatic microsomal triacylglycerol transfer protein mRNA was significantly lower in the 30% RY group compared with the C group. Cecal pools of acetate, propionate and butyrate were 113-257%, 181-476% and 410-789% greater in the RY group compared with the C group. These results suggest raw yam is effective as a source of RS and facilitates production of short chain fatty acid (SCFA), especially butyrate, in the rat cecum. In addition, RY has a plasma-cholesterol lowering effect, possibly due to the inhibited release of VLDL.
Several cross-sectional studies in Pima Indians and Caucasians have indicated that obese individuals with type 2 diabetes have a higher basal metabolic rate (BMR) than healthy, obese individuals. However, no study has investigated this comparison in Japanese subjects, who are known to be susceptible to type 2 diabetes due to genetic characteristics. Thirty obese Japanese adults with pre-type 2 diabetes (n=7) or type 2 diabetes (n=13) or without diabetes (n=10) participated in this study. BMR was measured using indirect calorimetry. The relationships between residual BMR (calculated as measured BMR minus BMR adjusted for fat-free mass, fat mass, age, and sex) and biomarkers including fasting glucose, glycosylated hemoglobin (HbA1c), fasting insulin, homeostasis model assessment of insulin resistance (HOMA-R), triglycerides, and free fatty acids were examined using Pearson's correlation. BMR in diabetic subjects adjusted for fat-free mass, fat mass, age, and sex was 7.1% higher than in non-diabetic subjects. BMR in diabetic subjects was also significantly (p<0.05) higher than in non-diabetic subjects. There was a significant correlation between residual BMR and fasting glucose (r=0.391, p=0.032). These results indicate that in the Japanese population, obese subjects with type 2 diabetes have higher BMR compared with obese non-diabetic subjects. The fasting glucose level may contribute to these differences.
To investigate the causes why pups of dams fed a low-fat high-carbohydrate diet (LFD) showed a strong preference for fat, three groups of dams were fed one of three diets during pregnancy and lactation: the LFD, a control diet (CTD) or a high-fat low-carbohydrate diet (HFD). After weaning, pups of each of the three groups were divided into two equal subgroups (Pair 1 and Pair 2), for a total of six pup subgroups. Each subgroup was placed on a two-choice diet program of the LFD and the HFD (Pair 1), or the LFD and a HFDLE (with cellulose added to maintain the same energy concentration as the LFD) (Pair 2), for 3 wk. Although the energy intake of dams fed the LFD during the nursing period was lower than that of the HFD group, no significant difference in body weight was observed among the three groups. At weaning, the body weight of pups nursed by dams fed the LFD was lower than that of the other groups. In Pair 1, the HFD intake ratio of the LFD and the HFD groups during the self-selection period was higher than that of the CTD group. In Pair 2, the HFDLE intake ratio of the LFD and the CTD groups was lower than that of the HFD group. At the end of the self-selection period, no significant difference in body weight was observed among the three groups of Pair 1. However, in Pair 2, the body weight of the LFD group was lower than that of the other groups. Therefore, it was supposed that pups of dams fed the LFD showed strong preference for the HFD containing high energy in order to achieve optimal growth.
Conjugated linoleic acid (CLA) is a collective term used for fatty acids with a conjugated double bond that are geometrical and positional isomers of linoleic acid. Anti-obesity and anti-cancer properties, an immunopotentiation effect, and promotion of bone formation by CLA have been shown in cell culture and animal studies. A mixture of 9c11t- and 10t12c-CLA is now used as a health food supplement after testing in clinical trials. These trials focused on improvement of lipid metabolism by CLA, whereas few studies have examined absorption and metabolism of CLA in humans. In addition, there is no report concerning absorption and metabolism of CLA in Japanese. This study was designed to examine CLA concentration in blood, the elimination rate of CLA, and metabolic differences between 9c11t-CLA and 10t12c-CLA in blood in Japanese who ingested CLA (about 2 g/d, equal weights of 9c11t-CLA and 10t12c-CLA) for 3 wk. Blood samples were collected 1 wk before the 3-wk period, on the first and last days of the period, and 1 wk after the end of the period, and the CLA concentration and distribution in blood were investigated. The CLA concentration in blood was significantly increased by CLA ingestion and reached 36 μmol/L. The CLA concentration in blood one week after the intake period was significantly lower than that at the end of CLA intake. The 10t12c-CLA level in plasma decreased faster than that of 9c11t-CLA. This suggests faster metabolism (fatty acid β oxidation) of 10t12c-CLA compared with 9c11t-CLA.
The aim of the present study was to evaluate the validity and reproducibility of a food frequency questionnaire (FFQ) for use among young Japanese children. Forty-seven mothers of children aged 6 y completed two 3-d diet records and two FFQs over a 6-mo period. The FFQ asked the mothers how often, on average, their children consumed each of the 162 food items listed and what the usual serving size of each item was during the 6 mo prior to the study. Intakes of macro- and micronutrients were estimated by multiplying the frequency by the serving size for each food item. The validity of the FFQ was assessed by comparison of the two 3-d diet records. The reproducibility of the FFQ was based on the first and second FFQ administrations. The validity correlation ranged from 0.05 for α-tocopherol to 0.59 for retinol. The median correlation was 0.40. The reproducibility correlation was higher than 0.50 for all nutrients. For most nutrients, FFQ had acceptable reproducibility, whereas validity showed low to moderate correlations. Our FFQ could reasonably rank individuals according to dietary intake for epidemiologic studies, although the validity of the questionnaire is limited to specific nutrients.
α-Lipoic acid (LA) is a naturally occurring disulfide-containing compound used as an antioxidant supplement which also has been used as a medicine for diabetic neuropathy in Europe. Physiologically LA acts as a coenzyme of mitochondrial multienzyme complex in its protein bound form but it is not yet clear how the externally administrated LA is incorporated into other proteins in the same protein-bound form or why the bound form is active as an antioxidant. The binding and cleavage of LA to or from the protein is mediated by lipoamidase and thus determines LA distribution in tissues. We have developed a simple sensitive assay for lipoamidase using a fluorescent substrate, dansyl-α-lipoyllysine (DLL). Lipoamidase in tissues cleaves the amide bond between LA and the ε-amino-lysine moiety to release dansylated lysine (DL). A HPLC comparison of the fluorescence intensity between DLL and DL was used to quantify the enzyme activity. The hydrolytic reaction did not occur when the tissue was heat-treated before incubation with DLL and was inhibited by free LA, especially by the R-enantiomer of LA (physiologically active form). Nε-Acetyl-L-lysine did not compete with DLL in the cleavage reaction. The method was applied for the determination of lipoamidase activity levels in various rat tissues. It was revealed the spleen had the highest activity followed by the kidney, heart, lung and liver. The activity in the brain was below the detection limit of the assay.