Protective Effects of Gnetin C from Melinjo Seed Extract against High-Fat Diet-Induced Hepatic Steatosis and Liver Fibrosis in NAFLD Mice Model.

Nonalcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease, can progress to hepatic steatosis, inflammation, and advanced fibrosis, increasing the risk of cirrhosis. Resveratrol, a natural polyphenol with antioxidant and anti-inflammatory properties, is beneficial in treating multiple metabolic diseases. Gnetin C, a resveratrol derivative obtained from Melinjo seed extract (MSE), shares similar health-promoting properties. We investigated the role of gnetin C in preventing NAFLD in a mouse model and compared it with resveratrol. Male C57BL/6J mice were fed a control diet (10% calories from fat), a high-fat choline-deficient (HFCD) diet (46% calories from fat) and HFCD diet supplemented with gnetin C (150 mg/kg BW·day-1) or resveratrol (150 mg/kg BW·day-1) for 12 weeks. Gnetin C supplementation reduced body and liver weight, and improved blood glucose levels and insulin sensitivity. Both gnetin C- and resveratrol reduced hepatic steatosis, with gnetin C also decreasing liver lipid content. Gnetin C and resveratrol ameliorated HFCD diet-induced hepatic fibrosis. The mRNA expression results, and western blot analyses showed that gnetin C and, to some extent, resveratrol downregulated fibrosis markers in the TGF-ß1 signaling pathway, indicating a possible safeguarding mechanism against NAFLD. These results suggest that gnetin C supplementation may protect against lipid deposition and hepatic fibrosis.

Fermented Rice Bran Supplementation Inhibits LPS-Induced Osteoclast Formation and Bone Resorption in Mice.

Fermented rice bran (FRB) is known to have numerous beneficial bioactivities, amongst which is its anti-inflammatory properties when used as a supplement. To determine its effects, we examined osteoclastogenesis and bone resorption caused by injections of lipopolysaccharide (LPS), using mice with and without FRB supplementation. The results were favorable: those that received FRB showed reduced osteoclast numbers and bone resorption compared to those with the control diet. Notably, receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-α (TNF-α) mRNA levels were shown to be lower in the LPS-treated animals with FRB supplementation. FRB's inhibitory effect on RANKL- and TNF-α-induced osteoclastogenesis was further confirmed in vitro. In culture, macrophages exhibited decreased TNF-α mRNA levels when treated with FRB extract and LPS versus treatment with LPS alone, but there was no significant change in RANKL levels in osteoblasts. We can conclude that FRB supplementation dampens the effect of LPS-induced osteoclastogenesis and bone resorption by controlling TNF-α expression in macrophages and the direct inhibition of osteoclast formation.

Biotin Enhances Testosterone Production in Mice and Their Testis-Derived Cells.

Late-onset hypogonadism, a male age-related syndrome characterized by a decline in testosterone production in the testes, is commonly treated with testosterone replacement therapy, which has adverse side effects. Therefore, an alternative treatment is highly sought. Supplementation of a high dosage of biotin, a water-soluble vitamin that functions as a coenzyme for carboxylases involved in carbohydrate, lipid, and amino acid metabolism, has been shown to influence testis functions. However, the involvement of biotin in testis steroidogenesis has not been well clarified. In this study, we examined the effect of biotin on testosterone levels in mice and testis-derived cells. In mice, intraperitoneal treatment with biotin (1.5 mg/kg body weight) enhanced testosterone levels in the serum and testes, without elevating serum levels of pituitary luteinizing hormone. To investigate the mechanism in which biotin increased the testosterone level, mice testis-derived I-10 cells were used. The cells treated with biotin increased testosterone production in a dose- and time-dependent manner. Biotin treatment elevated intracellular cyclic adenosine monophosphate levels via adenylate cyclase activation, followed by the activation of protein kinase A and testosterone production. These results suggest that biotin may have the potential to improve age-related male syndromes associated with declining testosterone production.

Fermented rice bran supplementation attenuates chronic colitis-associated extraintestinal manifestations in female C57BL/6N mice.

Patients with inflammatory bowel disease (IBD) have higher incidence of extraintestinal manifestations (EIM), including liver disorders, sarcopenia, and neuroinflammation. Fermented rice bran (FRB), generated from rice bran (RB), is rich in bioactive compounds, and exhibits anti-colitis activity. However, its role in EIM prevention is still unclear. Here, for the first time, we investigated whether EIM in female C57Bl/6N mice is attenuated by FRB supplementation. EIM was induced by repeated administration of 1.5% dextran sulfate sodium (DSS) in drinking water (4 d) followed by drinking water (12 d). Mice were divided into 3 groups-control (AIN93M), 10% RB, and 10% FRB. FRB ameliorated relapsing colitis and inflammation in muscle by significantly lowering proinflammatory cytokines Tnf-α and Il-6 in serum and advanced glycation end product-specific receptor (Ager) in serum and muscle when compared with the RB and control groups. As FRB reduced aspartate aminotransferase levels and oxidative stress, it might prevent liver disorders. FRB downregulated proinflammatory cytokine and chemokine transcripts responsible for neuroinflammation in the hippocampus and upregulated mRNA expression of G protein coupled receptors (GPRs), Gpr41 and Gpr43, in small and large intestines, which may explain the FRB-mediated protective mechanism. Hence, FRB can be used as a supplement to prevent IBD-associated EIM.

Effect of Vitamin K-Mediated PXR Activation on Drug-Metabolizing Gene Expression in Human Intestinal Carcinoma LS180 Cell Line.

The pregnane X receptor (PXR) is the key regulator of our defense mechanism against foreign substances such as drugs, dietary nutrients, or environmental pollutants. Because of increased health consciousness, the use of dietary supplements has gradually increased, and most of them can activate PXR. Therefore, an analysis of the interaction between drugs and nutrients is important because altered levels of drug-metabolizing enzymes or transporters can remarkably affect the efficiency of a co-administered drug. In the present study, we analyzed the effect of vitamin K-mediated PXR activation on drug metabolism-related gene expression in intestine-derived LS180 cells via gene expression studies and western blotting analyses. We demonstrated that menaquinone 4 (MK-4), along with other vitamin Ks, including vitamin K1, has the potential to induce MDR1 and CYP3A4 gene expression. We showed that PXR knockdown reversed MK-4-mediated stimulation of these genes, indicating the involvement of PXR in this effect. In addition, we showed that the expression of MDR1 and CYP3A4 genes increased synergistically after 24 h of rifampicin and MK-4 co-treatment. Our study thus elucidates the importance of drug-nutrient interaction mediated via PXR.

Fermented Rice Bran Supplementation Prevents the Development of Intestinal Fibrosis Due to DSS-Induced Inflammation in Mice.

Fermented rice bran (FRB) is known to protect mice intestines against dextran sodium sulfate (DSS)-induced inflammation; however, the restoration of post-colitis intestinal homeostasis using FRB supplementation is currently undocumented. In this study, we observed the effects of dietary FRB supplementation on intestinal restoration and the development of fibrosis after DSS-induced colitis. DSS (1.5%) was introduced in the drinking water of mice for 5 days. Eight mice were sacrificed immediately after the DSS treatment ended. The remaining mice were divided into three groups, comprising the following diets: control, 10% rice bran (RB), and 10% FRB-supplemented. Diet treatment was continued for 2 weeks, after which half the population of mice from each group was sacrificed. The experiment was continued for another 3 weeks before the remaining mice were sacrificed. FRB supplementation could reduce the general observation of colitis and production of intestinal pro-inflammatory cytokines. FRB also increased intestinal mRNA levels of anti-inflammatory cytokine, tight junction, and anti-microbial proteins. Furthermore, FRB supplementation suppressed markers of intestinal fibrosis. This effect might have been achieved via the canonical Smad2/3 activation and the non-canonical pathway of Tgf-ß activity. These results suggest that FRB may be an alternative therapeutic agent against inflammation-induced intestinal fibrosis.

S-allyl Cysteine Enhances Testosterone Production in Mice and Mouse Testis-Derived I-10 Cells.

Hypogonadism, associated with low levels of testosterone synthesis, has been implicated in several diseases. Recently, the quest for natural alternatives to prevent and treat hypogonadism has gained increasing research interest. To this end, the present study explored the effect of S-allyl cysteine (SAC), a characteristic organosulfur compound in aged-garlic extract, on testosterone production. SAC was administered at 50 mg/kg body weight intraperitoneally into 7-week-old BALB/c male mice in a single-dose experiment. Plasma levels of testosterone and luteinizing hormone (LH) and testis levels of proteins involved in steroidogenesis were measured by enzymatic immunoassay and Western blot, respectively. In addition, mouse testis-derived I-10 cells were also used to investigate the effect of SAC on steroidogenesis. In the animal experiment, SAC significantly elevated testosterone levels in both the plasma and the testis without changing the LH level in plasma and increased phosphorylated protein kinase A (p-PKA) levels. Similar results were also observed in I-10 cells. The findings demonstrating the increasing effect of SAC on p-PKA and mRNA levels of Cyp11a suggest that SAC increases the testosterone level by activating the PKA pathway and could be a potential target for hypogonadism therapeutics.

Supplementation with Fermented Rice Bran Attenuates Muscle Atrophy in a Diabetic Rat Model.

Fermented rice bran (FRB), a prospective supplement, has been proven to ameliorate certain medical conditions. However, its nutraceutical effect on muscle atrophy has never been investigated. The present study aimed to evaluate the effect of FRB on muscle atrophy in a streptozotocin (STZ)-induced diabetic rat model. Three groups of Sprague-Dawley rats, namely the control, STZ, and FRB groups, were treated as follows. The diabetic groups (STZ and FRB) were injected intraperitoneally with STZ (40 mg/kg BW), whereas the control group was injected with the vehicle. The STZ and control groups were fed the AIN93M diet, and the FRB group was fed 10% of FRB based on the AIN93M diet. The diabetic groups had reduced muscle size compared to the control group; however, these changes were alleviated in the FRB group. Moreover, the FRB group had a significantly lower expression of FBXO32/Atrogin-1 and TRIM63/MuRF1 (p < 0.05) due to blocked NF-κB activation. In conclusion, the anti-inflammatory effect of FRB may be beneficial for ameliorating muscle atrophy in diabetic conditions.

Dietary supplementation with geranylgeraniol suppresses lipopolysaccharide-induced inflammation via inhibition of nuclear factor-κB activation in rats.

PURPOSE: The isoprenoid geranylgeraniol (GGOH) inhibits nuclear factor-kappa B (NF-κB) activation in the liver, yet the mechanism remains unclear. We investigated the modulation and inhibition of lipopolysaccharide (LPS)-induced NF-κB signaling in the liver of rats fed a GGOH-supplemented diet. METHODS: Rats were fed a diet supplemented with or without GGOH for 10 days. Rats were then intraperitoneally injected with 0.5 mg/kg LPS or vehicle (sterilized saline) and fasted for 18 h. Plasma levels of the inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, and the liver damage indicators alanine and aspartate aminotransferases (ALT and AST) were assessed. Liver mRNA and proteins were assayed for changes in NF-κB target genes and signal transduction genes. RESULTS: Rats fed a high-dose, GGOH-supplemented diet showed significantly lower levels of plasma inflammatory cytokines and ALT and AST activities. In the liver, GGOH significantly suppressed NF-κB activation and mRNA expression of its pro-inflammatory target genes. Furthermore, GGOH supplementation substantially suppressed mRNA expression of signal transducer genes upstream of the IκB kinase complex. Western blotting of liver extracts further demonstrated the substantial decrease in total IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6), leading to lower signal transduction and inhibition of NF-κB after LPS. CONCLUSION: A 10-day, high-dose, GGOH-supplemented diet was sufficient to inhibit LPS-induced inflammation and activation of NF-κB in rat livers. GGOH significantly modulated NF-κB signaling molecules, inhibiting its signal transduction and activation in the liver, thus protecting against liver damage.

Physiological effects and tissue distribution from large doses of tocotrienol in rats.

Supplementation to an AIN93G-based diet of tocotrienol (T3) for 13 weeks administered to Fischer 344/slc rats showed a safety profile with no side effects. Dose-dependent T3 levels were detected in many tissues. Under the present experimental conditions, a continuous intake of the T3 concentrate would be safe in the rats as long as the T3 content was less than 0.20% of the dietary intake.

Dietary vitamin K alleviates the reduction in testosterone production induced by lipopolysaccharide administration in rat testis.

Vitamin K is essential for the posttranslational modifications of blood coagulation factors and proteins present in the bone matrix. Vitamin K is distributed not only in the liver and bones but is also abundant in the brain, kidney, and gonadal tissues. However, the function of extra-hepatic/bone vitamin K has not been fully elucidated. Previously, we observed that dietary supplementation with vitamin K suppresses inflammation, and vitamin K deficiency decreases testicular testosterone production in rats. Here, we examined whether the dietary vitamin K state affects testicular steroidogenesis in lipopolysaccharide (LPS)-treated rats because vitamin K has anti-inflammatory activity. Male Wistar rats were fed either vitamin K-free or control diets for 35 d, and then intraperitoneally administered LPS (0.5 mg kg(-1) body weight) to induce inflammation for 6 h. Vitamin K deficiency symptoms were not observed in the vitamin K-free diet group; however, the vitamin K levels in the testis were significantly lower in the vitamin K-free diet group than in the control diet group. After LPS treatment, plasma testosterone levels were significantly reduced in the vitamin K-free diet group compared with the control diet group. Testicular mRNA and protein levels of Cyp11a, a rate-limiting enzyme in steroidogenesis, corresponded to plasma testosterone levels. However, plasma luteinizing hormone levels were unaffected by diet and LPS. Phosphorylated nuclear factor κB p65 in the testis was significantly increased in the LPS-treated, vitamin K-free diet group compared with control. These results indicate that dietary vitamin K affects testicular vitamin K levels and ameliorates the LPS-induced reduction in testicular testosterone synthesis. Testicular vitamin K might facilitate the inhibition of inflammation signal transduction and maintain steady levels of testosterone.

Menaquinone-4 enhances testosterone production in rats and testis-derived tumor cells.

BACKGROUND: Vitamin K is essential for the posttranslational modification of various Gla proteins. Although it is widespread in several organs, including the testis, the function of vitamin K in these organs is not well characterized. In this study, we investigated the function of vitamin K in the testis and analyzed its role in steroidogenesis. METHODS: Eight-week-old male Wistar rats were fed a diet supplemented with menaquinone-4 (MK-4, 75 mg/kg diet), one of the predominant K2 vitamins present in the testis, for 5 weeks. In vivo testosterone levels of the rats' plasma and testes were measured by enzyme-linked immunosorbent assay, and in vitro testosterone levels of testis-derived tumor cells (I-10 cells) maintained in Ham's F-10 medium with 10% fetal bovine serum were measured following treatment with MK-4 (0 to 100 µM) at several time points. Testosterone and cellular protein levels were analyzed with respect to their effects on steroidogenesis. RESULTS: Testosterone levels in the plasma and testes of MK-4-fed rats were significantly increased compared to those of control rats, with no obvious differences in plasma luteinizing hormone levels. Secreted testosterone levels from I-10 cells were elevated by MK-4, but not by vitamin K1, in a dose-dependent manner independent of cAMP treatment. Western blot analysis revealed that expression of CYP11A, the rate-limiting enzyme in steroidogenesis, and phosphorylation levels of protein kinase A (PKA) and the cAMP response element-binding protein were all stimulated by the presence of MK-4. Enhancement of testosterone production was inhibited by H89, a specific inhibitor of PKA, but not by warfarin, an inhibitor of γ-glutamylcarboxylation. CONCLUSIONS: MK-4 stimulates testosterone production in rats and testis-derived tumor cells via activation of PKA. MK-4 may be involved in steroidogenesis in the testis, and its supplementation could reverse the downregulation of testosterone production in elders.

Low-dose dioxins alter gene expression related to cholesterol biosynthesis, lipogenesis, and glucose metabolism through the aryl hydrocarbon receptor-mediated pathway in mouse liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental contaminant. TCDD binds and activates the transcription factor aryl hydrocarbon receptor (AHR), leading to adverse biological responses via the alteration of the expression of various AHR target genes. Although small amounts of TCDD are consumed via contaminated daily foodstuffs and environmental exposures, the effects of low-dose TCDD on gene expression in animal tissues have not been clarified, while a number of genes affected by high-dose TCDD were reported. In this study, we comprehensively analyzed gene expression profiles in livers of C57BL/6N mice that were orally administered relatively low doses of TCDD (5, 50, or 500 ng/kg body weight (bw) day(-1)) for 18 days. The hepatic TCDD concentrations, measured by gas chromatography-mass spectrometry, were 1.2, 17, and 1063 pg toxicity equivalent quantity (TEQ)/g, respectively. The mRNA level of the cytochrome P450 CYP1A1 was significantly increased by treatment with only TCDD 500 ng/kg bw day(-1). DNA microarray and quantitative RT-PCR analyses revealed changes in the expression of genes involved in the circadian rhythm, cholesterol biosynthesis, fatty acid synthesis, and glucose metabolism in the liver with at all doses of TCDD employed. However, repression of expression of genes involved in energy metabolism was not observed in the livers of Ahr-null mice that were administered the same dose of TCDD. These results indicate that changes in gene expression by TCDD are mediated by AHR and that exposure to low-dose TCDD could affect energy metabolism via alterations of gene expression.

Vitamin K deficiency reduces testosterone production in the testis through down-regulation of the Cyp11a a cholesterol side chain cleavage enzyme in rats.

Vitamin K (K) is an essential factor for the posttranslational modification of blood coagulation factors as well as proteins in the bone matrix (Gla proteins). It is known that K is not only distributed in the liver and bones but also abundantly distributed in the brain, kidney, and gonadal tissues. However, the role of K in these tissues is not well clarified. In this study, we used DNA microarray and identified the genes whose expression was affected in the testis under the K-deficient (K-def) state. The expression of genes involved in the biosynthesis of cholesterol and steroid hormones was decreased in the K-def group. The mRNA levels of Cyp11a - a rate-limiting enzyme in testosterone synthesis - positively correlated with the menaquinone-4 (MK-4) concentration in the testis. Moreover, as compared to the control (Cont) and K-supplemented (K-sup) groups, the K-def group had decreased testosterone concentrations in the plasma and testis. These results suggested that K is involved in steroid production in the testis through the regulation of Cyp11a.

Vitamin K suppresses lipopolysaccharide-induced inflammation in the rat.

Vitamin K (K) is essential for blood coagulation and bone metabolism in mammals. K acts as a cofactor in the posttranslational synthesis of gamma-carboxyglutamic acid from glutamic acid residues. In addition to the liver and bone, K is found in the brain, heart, kidney and gonadal tissue. However, the physiological role of K in these various organs is not yet fully understood. It is likely that K has functions other than its role as a cofactor of protein gamma-glutamyl carboxylation. We used in this study the DNA microarray technique to identify the effect of K status on gene expression in the rat liver. The expression of genes involved in the acute inflammation response was enhanced in rats fed with a K-deficient diet relative to the control and K1-supplemented diet groups. Moreover, dietary supplementation with K1 suppressed the inflammation induced by lipopolysaccharide administration. These results indicate that orally administrated K1 suppressed inflammation in the rat.