Citrus limonoid nomilin inhibits osteoclastogenesis in vitro by suppression of NFATc1 and MAPK signaling pathways.

BACKGROUND: Animal experiment studies have revealed a positive association between intake of citrus fruits and bone health. Nomilin, a limonoid present in citrus fruits, is reported to have many biological activities in mammalian systems, but the mechanism of nomilin on bone metabolism regulation is currently unclear. PURPOSE: To reveal the mechanism of nomilin on osteoclastic differentiation of mouse primary bone marrow-derived macrophages (BMMs) and the mouse RAW 264.7 macrophage cell line into osteoclasts. STUDY DESIGN: Controlled laboratory study. Effects of nomilin on osteoclastic differentiation were studied in in vitro cell cultures. METHODS: Cell viability of RAW 264.7 cells and BMMs was measured with the Cell Counting Kit. TRAP-positive multinucleated cells were counted as osteoclast cell numbers. The number and area of resorption pits were measured as bone-resorbing activity. Osteoclast-specific genes expression was evaluated by quantitative real-time PCR; and proteins expression was evaluated by western blot. RESULTS: Nomilin significantly decreased TRAP-positive multinucleated cell numbers compared with the control, and exhibited no cytotoxicity. Nomilin decreased bone resorption activity. Nomilin downregulated osteoclast-specific genes, NFATc1 and TRAP mRNA levels. Furthermore, nomilin suppressed MAPK signaling pathways. CONCLUSION: This study demonstrates clearly that nomilin has inhibitory effects on osteoclastic differentiation in vitro. These findings indicate that nomilin-containing herbal preparations have potential utility for the prevention of bone metabolic diseases.

Fish oil prevents excessive hepatic lipid accumulation without inducing oxidative stress.

We examined the effects of fish oil (FO) on high-cholesterol diet-induced hepatic lipid accumulation and oxidative stress. Female C57BL/6J mice were fed diets consisting of safflower oil (SO), 1 en% FO (1FO), 2 en% FO (2FO), or 20 en% FO (20FO) with or without 2 weight% (wt%) cholesterol (SO/CH, 1FO/CH, 2FO/CH, and 20FO/CH groups, respectively) for 8 weeks. The hepatic triacylglyceride levels were significantly lower in the 2FO/CH and 20FO/CH groups than in the SO/CH group. The hepatic mRNAs of fatty acid oxidation-related genes were upregulated and the fatty acid synthesis-related genes were downregulated by the FO feeding. Adverse effects were not observed in the plasma levels of indicators of oxidative stress in response to the consumption of FO up to 20 en%. These results suggest that FO consumption in the range of 2-20 en% prevents hepatic lipid accumulation, thus improving lipid metabolism without causing oxidative stress.

Effects of dietary fat energy restriction and fish oil feeding on hepatic metabolic abnormalities and insulin resistance in KK mice with high-fat diet-induced obesity.

We investigated the effects of dietary fat energy restriction and fish oil intake on glucose and lipid metabolism in female KK mice with high-fat (HF) diet-induced obesity. Mice were fed a lard/safflower oil (LSO50) diet consisting of 50 energy% (en%) lard/safflower oil as the fat source for 12 weeks. Then, the mice were fed various fat energy restriction (25 en% fat) diets - LSO, FO2.5, FO12.5 or FO25 - containing 0, 2.5, 12.5, or 25 en% fish oil, respectively, for 9 weeks. Conversion from a HF diet to each fat energy restriction diet significantly decreased final body weights and visceral and subcutaneous fat mass in all fat energy restriction groups, regardless of fish oil contents. Hepatic triglyceride and cholesterol levels markedly decreased in the FO12.5 and FO25 groups, but not in the LSO group. Although plasma insulin levels did not differ among groups, the blood glucose areas under the curve in the oral glucose tolerance test were significantly lower in the FO12.5 and FO25 groups. Real-time polymerase chain reaction analysis showed fatty acid synthase mRNA levels significantly decreased in the FO25 group, and stearoyl-CoA desaturase 1 mRNA levels markedly decreased in the FO12.5 and FO25 groups. These results demonstrate that body weight gains were suppressed by dietary fat energy restriction even in KK mice with HF diet-induced obesity. We also suggested that the combination of fat energy restriction and fish oil feeding decreased fat droplets and ameliorated hepatic hypertrophy and insulin resistance with suppression of de novo lipogenesis in these mice.

Effect of concomitantly used fish oil and cholesterol on lipid metabolism.

Although cholesterol plays various important roles in the body, when overconsumed, it causes atherosclerosis and results in ischemic heart disease. On the other hand, dietary fish oils contain n-3 fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid, which prevent ischemic heart disease. This effect of n-3 fatty acids mainly results from the combined effects of inhibiting lipogenesis via a decrease of the mature form of sterol regulatory element-binding proteins (SREBPs) and stimulating fatty acid oxidation via peroxisome proliferator-activator receptor (PPAR) alpha activation in the liver. In this study, we examined the interactive effects on lipid metabolism of dietary 2% cholesterol (w/w) and 20% or 50% energy fish oil. In a safflower oil diet with 2% cholesterol, hepatic lipids accumulated. On the other hand, hepatic lipids did not accumulate in the fish oil diets with cholesterol. Furthermore, in the groups with fish oil energy ratios of 20%, the negative feedback control of cholesterol affected SREBP-2, and the actions of fish oil and cholesterol were equivalent, but this was not observed in the cases with fish oil energy ratios of 50%. The results of this study suggest that differences in lipid accumulation in the body are due to differences in lipid source and energy ratios which differentially impact the control of transcription factors by cholesterol.

Anti-obesity effect of fish oil and fish oil-fenofibrate combination in female KK mice.

AIM: The aim of our study is to elucidate the effects of EPA- or DHA-rich fish oil, and of the latter plus fenofibrate, on lipid metabolism in female KK mice. METHODS: Female KK mice were fed purified experimental diets containing lard/safflower oil (4:6, Lard/SO), EPA-rich fish oil (EPA), DHA-rich fish oil (DHA), or DHA-rich fish oil plus 0.2% (w/w) fenofibrate (DHA+FF) for 8 weeks. At the end of the experiments, we measured levels of plasma lipids, hepatic triglycerides, and cholesterol, as well as the hepatic mRNA expression of lipogenic and lipidolytic genes. RESULTS: The final body weight of EPA- and DHA-fed groups was significantly lower than that of the Lard/SO-fed group, and that of the DHA+FF-fed group was the lowest. All three fish oil treatments significantly reduced plasma insulin levels. Hepatic lipid levels significantly decreased in all three of these groups compared with the Lard/SO-fed group. Plasma adiponectin increased in both the EPA-and DHA-fed groups, but the increase was suppressed in the DHA+FF-fed group. Hepatocytes of Lard/SO-fed mice were filled with numerous fat droplets, but fat accumulation was inhibited in both EPA- and DHA-fed mice and was significantly prevented by fenofibrate treatment. SREBP-1c mRNA levels were decreased by about half in EPA- and DHA-fed mice compared with Lard/SO-fed mice. FAS, Insig-1, HMG-CoA reductase, and LDL-receptor mRNA levels also markedly decreased in both EPA- and DHA-fed mice, but there was no additional decrease in DHA+FF fed mice. Fenofibrate treatment significantly induced mRNA expression of AOX and UCP-2, but not of PPARalpha. CONCLUSION: These data suggest that fish oil inhibited body weight gain and exhibited an anti-obesity effect through the inhibition of lipid synthesis in female KK mice. Furthermore, fenofibrate treatment markedly inhibited body weight gain by the induction of fatty acid oxidation. Plasma adiponectin levels did not increase in mice fed DHA-rich fish oil with fenofibrate, although white adipose tissue (WAT) weight significantly decreased. We considered that adiponectin sensitivity increased more in mice fed DHA-rich fish oil with fenofibrate than in mice fed DHA-rich fish oil alone.

Interaction of fenofibrate and fish oil in relation to lipid metabolism in mice.

AIM: The aim of our study is to elucidate the interactive effects on lipid metabolism of fenofibrate and two fish oils with EPA and DHA contents in mice. METHODS: Female C57BL/6J mice were fed purified experimental diets containing safflower oil (SO), EPA-rich menhaden oil (MO) or DHA-rich tuna oil (TO) with or without 0.1% fenofibrate for 8 weeks. At the end of the experiments, we measured plasma lipids and hepatic triglycerides and cholesterol, and the hepatic mRNA expression of lipogenic and lipidolytic genes. RESULTS: Plasma TG levels fell in the group fed MO or TO alone and fell significantly in all fenofibrate-treated groups. Although plasma total cholesterol levels fell significantly in fish oil-fed groups, fenofibrate treatments increased significantly plasma total cholesterol levels in these fish oil groups, but not in the group fed SO alone; however, hepatic triglyceride and total cholesterol levels markedly decreased in MO-or TO-fed mice. In lipid synthesis, the hepatic mRNA level of SREBP-1c was not reduced in either fish oil group; however, Insig-1 mRNA decreased in MO and TO feeding groups by about half and FAS or SCD-1 mRNA decreased significantly in MO and TO feeding groups, compared with the SO feeding group. In both fish oil groups, SREBP-2 mRNA decreased significantly and HMG-CoA reductase mRNA also decreased with/without fenofibrate. On the other hand, fenofibrate supplementation significantly induced the mRNA expression of AOX and UCP-2, which play a role in lipid catabolism, in all diets. CYP7A1 mRNA increased markedly in mice fed MO diet with fenofibrate, compared with TO diet with fenofibrate. CONCLUSION: These data suggest that differences in dietary contents of EPA and DHA do not influence the inhibition of lipogenesis, and that fenofibrate supplementation stimulates fatty acid oxidation, regardless of the oil type; however, cholesterol catabolism was induced by a combination of EPA-rich fish oil and fenofibrate, which suggests that EPA has a greater synergistic ability for cholesterol catabolism induction by fenofibrate than DHA.