Tryptamine, a Microbial Metabolite in Fermented Rice Bran Suppressed Lipopolysaccharide-Induced Inflammation in a Murine Macrophage Model.

Fermentation is thought to alter the composition and bioavailability of bioactive compounds in rice bran. However, how this process affects the anti-inflammatory effects of rice bran and the bioactive compounds that might participate in this function is yet to be elucidated. This study aimed to isolate bioactive compounds in fermented rice bran that play a key role in its anti-inflammatory function. The fermented rice bran was fractionated using a succession of solvent and solid-phase extractions. The fermented rice bran fractions were then applied to lipopolysaccharide (LPS)-activated murine macrophages to evaluate their anti-inflammatory activity. The hot water fractions (FRBA), 50% ethanol fractions (FRBB), and n-hexane fractions (FRBC) were all shown to be able to suppress the pro-inflammatory cytokine expression from LPS-stimulated RAW 264.7 cells. Subsequent fractions from the hot water fraction (FRBF and FRBE) were also able to reduce the inflammatory response of these cells to LPS. Further investigation revealed that tryptamine, a bacterial metabolite of tryptophan, was abundantly present in these extracts. These results indicate that tryptamine may play an important role in the anti-inflammatory effects of fermented rice bran. Furthermore, the anti-inflammatory effects of FRBE and tryptamine may depend on the activity of the aryl hydrocarbon receptor.

Geranylgeraniol Inhibits Lipopolysaccharide-Induced Inflammation in Mouse-Derived MG6 Microglial Cells via NF-κB Signaling Modulation.

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K2 sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1ß, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/ß, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.

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.

The Effect of Liver Hydrolysate on Chronic Ethanol-Induced Hepatic Injury in Normal Rats.

The mechanism underlying the improvement in hepatic function by liver hydrolysate (LH) after ethanol-induced hepatic injury is unclear. Therefore, we investigated the effects of LH administration on chronic ethanol-induced hepatic injury in normal rats and the mechanism underlying the improvement of its symptoms by LH. LH attenuated liver damage and reduced oxidative stress after chronic ethanol-induced hepatic injury in normal rats. LH treatment reduced hepatic injury biomarkers of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT). LH treatment also decreased levels of 8-hydroxy-deoxyguanosine (8-OHdG) as oxidative stress marker. LH may prove beneficial to prevent the liver damage of chronic ethanol, at least in part, by alleviating oxidative stress.

Cysteine Sulfoxides Enhance Steroid Hormone Production via Activation of the Protein Kinase A Pathway in Testis-Derived I-10 Tumor Cells.

Testosterone plays an important role in male sexual characteristics and maturation, and decreased testosterone levels increase the risk of several diseases. Recently, onion extract rich in cysteine sulfoxides, which are amino acids unique to onions, has been reported to alleviate age-related symptoms resulting from decreased testosterone levels in males. However, the mechanism underlying the suppression of low testosterone levels by cysteine sulfoxides has not been elucidated. In this study, we found that onion extract containing cysteine sulfoxides enhanced progesterone, a precursor of testosterone, in mouse testis-derived I-10 tumor cells. Furthermore, cysteine sulfoxides activated protein kinase A (PKA) and cyclic adenosine monophosphate response element-binding protein, which are key factors in steroidogenesis. These results suggest that cysteine sulfoxides enhance steroid hormone production via activation of the PKA signaling pathway.

Menaquinone-4 Suppresses Lipopolysaccharide-Induced Inflammation in MG6 Mouse Microglia-Derived Cells by Inhibiting the NF-κB Signaling Pathway.

The overactivation of microglia is known to trigger inflammatory reactions in the central nervous system, which ultimately induce neuroinflammatory disorders including Alzheimer's disease. However, increasing evidence has shown that menaquinone-4 (MK-4), a subtype of vitamin K2, can attenuate inflammation in the peripheral system. Whereas it was also observed at high levels within the brain, its function in this organ has not been well characterized. Therefore, we investigated the effect of MK-4 on microglial activation and clarified the underlying mechanism. Mouse microglia-derived MG6 cells were exposed to lipopolysaccharide (LPS) either with or without MK-4 pretreatment. Cell responses with respect to inflammatory cytokines (Il-1ß, Tnf-α, and Il-6) were measured by qRT-PCR. We further analyzed the phosphorylation of TAK1, IKKα/ß, and p65 of the NF-κB subunit by Western blotting. We observed that in LPS-induced MG6 cells, MK-4 dose-dependently suppressed the upregulation of inflammatory cytokines at the mRNA level. It also significantly decreased the phosphorylation of p65, but did not affect that TAK1 and IKKα/ß. Furthermore, the nuclear translocation of NF-κB in LPS-induced MG6 cells was inhibited by MK-4. These results indicate that MK-4 attenuates microglial inflammation by inhibiting NF-κB signaling.

Menaquinone-4 Amplified Glucose-Stimulated Insulin Secretion in Isolated Mouse Pancreatic Islets and INS-1 Rat Insulinoma Cells.

Vitamin K2 is indispensable for blood coagulation and bone metabolism. Menaquinone-4 (MK-4) is the predominant homolog of vitamin K2, which is present in large amounts in the pancreas, although its function is unclear. Meanwhile, ß-cell dysfunction following insulin secretion has been found to decrease in patients with type 2 diabetes mellitus. To elucidate the physiological function of MK-4 in pancreatic ß-cells, we studied the effects of MK-4 treatment on isolated mouse pancreatic islets and rat INS-1 cells. Glucose-stimulated insulin secretion significantly increased in isolated islets and INS-1 cells treated with MK-4. It was further clarified that MK-4 enhanced cAMP levels, accompanied by the regulation of the exchange protein directly activated by the cAMP 2 (Epac2)-dependent pathway but not the protein kinase A (PKA)-dependent pathway. A novel function of MK-4 on glucose-stimulated insulin secretion was found, suggesting that MK-4 might act as a potent amplifier of the incretin effect. This study therefore presents a novel potential therapeutic approach for impaired insulinotropic effects.

Geranylgeraniol Suppresses the Expression of IRAK1 and TRAF6 to Inhibit NFκB Activation in Lipopolysaccharide-Induced Inflammatory Responses in Human Macrophage-Like Cells.

Geranylgeraniol (GGOH), a natural isoprenoid found in plants, has anti-inflammatory effects via inhibiting the activation of nuclear factor-kappa B (NFκB). However, its detailed mechanism has not yet been elucidated. Recent studies have revealed that isoprenoids can modulate signaling molecules in innate immune responses. We found that GGOH decreased the expression of lipopolysaccharide (LPS)-induced inflammatory genes in human macrophage-like THP-1 cells. Furthermore, we observed that the suppression of NFκB signaling proteins, in particular interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), occurred in GGOH-treated cells prior to LPS stimulation, suggesting an immunomodulatory effect. These results indicate that GGOH may modulate and help prevent excessive NFκB activation that can lead to numerous diseases.

Development of novel silanol-based human pregnane X receptor (PXR) agonists with improved receptor selectivity.

Pregnane X receptor (PXR) is a ligand-dependent transcription factor that is considered to be a potential therapeutic target for multiple diseases. Herein, we report the development and structure-activity relationship studies of a new series of hPXR agonists. Focusing on our recently developed silanol-sulfonamide scaffold, we developed the potent hPXR agonist 28, which shows good selectivity over hLXRα and ß, hFXR, and hRORα and γ. Examination of the structure-activity relationship suggested a possible strategy to manipulate the selectivity. Docking simulation indicated the presence of an additional binding cavity and polar contacts in the ligand-binding pocket of hPXR. This information should be helpful for the future development of more potent and selective hPXR ligands.

A novel function of geranylgeraniol in regulating testosterone production.

Isoprenoids play widely differing roles in various physiological processes in animals and plants. Geranylgeraniol (GGOH) is an isoprenoid found in plants, and is an important metabolic derivative in the isoprenoid/cholesterol synthesis pathway. Earlier studies focused on GGOH's ability to improve the side effects of bisphosphonate therapy by regulating the mevalonate pathway. More recently, the mevalonate pathway-independent effects of GGOH have been described, including anti-inflammatory, anti-tumorigenic, and neuroprotective activities. It is noteworthy that GGOH regulates the steroidogenesis pathway in testis-derived I-10 tumor cells. Testosterone is a hormone produced via steroidogenesis in testicles and plays a role in fetal development and the male reproductive system. GGOH enhanced testosterone and progesterone (its precursor) levels in I-10 cells by activating adenylate cyclase via cAMP/PKA signaling, without altering phosphodiesterase activity. These findings highlight the potential benefits of GGOH as a therapeutic agent for low testosterone levels, such as late-onset hypogonadism in men.

Orexigenic action of oral zinc: metabolomic analysis in the rat hypothalamus.

We previously reported an orexigenic action of oral zinc administration in male Sprague-Dawley (SD) rats during an early stage of feeding with a zinc-deficient diet, without decreased zinc concentrations in tissues. The overall conclusion was that orally but not intraperitoneally administered zinc stimulates food intake in short-term zinc-deficient-diet fed rats. We here investigate the mechanism of the orexigenic action of zinc using GC-MS/MS-targeted metabolomic analysis in the rat hypothalamus. Four-week-old, male SD/Slc rats were used, and after 2 days of feeding with a zinc-deficient diet, 3 mg of ZnSO4 in 5 mL saline solution were administered to each rat either orally or intraperitoneally. Three hours after administration, the rats were sacrificed and the hypothalamus were excised and analyzed. We found that the oral administration group showed increased concentrations of 3-aminopropanoic acid (ß-alanine), hypotaurine, dopamine, and biotin. In light of metabolomic analysis of these results, we indicate directions for further research.

Adenosine and adenosine-5'-monophosphate ingestion ameliorates abnormal glucose metabolism in mice fed a high-fat diet.

BACKGROUND: We have previously reported that ingestion of adenosine (ADN) and adenosine-5'-monophosphate (AMP) improves abnormal glucose metabolism in the stroke-prone spontaneously hypertensive rat model of non-obesity-associated insulin resistance. In this study, we investigated the effect of ADN and AMP ingestion on glucose metabolism in mice with high-fat diet-induced obesity. METHODS: Seven-week-old C57BL/6 J mice were administered distilled water (as a control), 10 mg/L ADN, or 13 mg/L AMP via their drinking water for 14 or 25 weeks, during which they were fed a high-fat diet. Oral glucose tolerance test (OGTT) was conducted on 21-week-old mice fasted for 16 h. Insulin tolerance test (ITT) was performed on 22-week-old mice fasted for 3 h. Blood and muscle were collected for further analysis of serum parameters, gene and protein expression levels, respectively. RESULTS: Glucose metabolism in the ADN and AMP groups was significantly improved compared with the control. OGTT and ITT showed that ADN and AMP groups lower than control group. Furthermore, phosphorylation of AMP-activated protein kinase (AMPK) and mRNA levels of genes involved in lipid oxidation were enhanced in the skeletal muscle of ADN- and AMP-treated mice. CONCLUSION: These results indicate that ingestion of ADN or AMP induces activation of AMPK in skeletal muscle and mitigates insulin resistance in mice with high-fat diet-induced diabetes.

Inhibitory effects of Kaempferia parviflora extract on monocyte adhesion and cellular reactive oxygen species production in human umbilical vein endothelial cells.

PURPOSE: The rhizome of Kaempferia parviflora (KP) is used in traditional Thai medicine. In this study, we investigated the effects of an ethanol KP extract and two of its components [5,7-dimethoxyflavone (DMF) and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (TMF)] on monocyte adhesion and cellular reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVECs), which provide an in vitro model of events relevant to the development and progression of atherosclerosis. METHODS: RAW264.7 mouse macrophage-like cells were incubated with various concentrations of KP extract or polymethoxyflavonoids and stimulated with lipopolysaccharide prior to measuring nitrite levels in the culture media. Monocyte adhesion was evaluated by measuring the fluorescently labeled human monocytic leukemia THP-1 cells that is attached to tumor necrosis factor-α (TNF-α)-stimulated HUVECs. Cellular ROS production was assessed by measuring cellular antioxidant activity using pyocyanin-stimulated HUVECs. RESULTS: KP extract and DMF reduced nitrite levels (as indicator of nitric oxide production) in LPS-stimulated RAW264.7 cells and also inhibited THP-1 cell adhesion to HUVECs. These treatments induced mRNA expression of endothelial nitric oxide synthase in TNF-α-stimulated HUVECs and downregulated that of various cell adhesion molecules, inflammatory mediators, and endothelial function-related genes. Angiotensin-converting enzyme activity was inhibited by KP extract in vitro. Furthermore, KP extract, DMF, and TMF inhibited the production of cellular ROS in pyocyanin-stimulated HUVECs. CONCLUSION: KP extract, DMF, and TMF showed potential anti-inflammatory and antioxidant effects in these in vitro models, properties that would inhibit the development and progression of atherosclerosis.

Properties of Zip4 accumulation during zinc deficiency and its usefulness to evaluate zinc status: a study of the effects of zinc deficiency during lactation.

Systemic and cellular zinc homeostasis is elaborately controlled by ZIP and ZnT zinc transporters. Therefore, detailed characterization of their expression properties is of importance. Of these transporter proteins, Zip4 functions as the primarily important transporter to control systemic zinc homeostasis because of its indispensable function of zinc absorption in the small intestine. In this study, we closely investigated Zip4 protein accumulation in the rat small intestine in response to zinc status using an anti-Zip4 monoclonal antibody that we generated and contrasted this with the zinc-responsive activity of the membrane-bound alkaline phosphatase (ALP). We found that Zip4 accumulation is more rapid in response to zinc deficiency than previously thought. Accumulation increased in the jejunum as early as 1 day following a zinc-deficient diet. In the small intestine, Zip4 protein expression was higher in the jejunum than in the duodenum and was accompanied by reduction of ALP activity, suggesting that the jejunum can become zinc deficient more easily. Furthermore, by monitoring Zip4 accumulation levels and ALP activity in the duodenum and jejunum, we reasserted that zinc deficiency during lactation may transiently alter plasma glucose levels in the offspring in a sex-specific manner, without affecting homeostatic control of zinc metabolism. This confirms that zinc nutrition during lactation is extremely important for the health of the offspring. These results reveal that rapid Zip4 accumulation provides a significant conceptual advance in understanding the molecular basis of systemic zinc homeostatic control, and that properties of Zip4 protein accumulation are useful to evaluate zinc status closely.

Altered activity profile of a tertiary silanol analog of multi-targeting nuclear receptor modulator T0901317.

We report the design, synthesis, and physicochemical/biological evaluation of novel silanol derivative 6 (sila-T) as a silanol analog of multi-target nuclear receptor modulator T0901317 (5). Compound 6 showed intermediate hydrophobicity between the corresponding alcohol 13 and perfluoroalcohol 5. While 5 exhibited potent activities toward liver X receptor α and ß, farnesoid X receptor, pregnane X receptor (PXR) and retinoic acid receptor-related orphan receptor (ROR)γ, silanol 6 exhibited activity only toward PXR and RORs. Incorporation of silanol instead of perfluoroalcohol is a promising option for developing novel target-selective, biologically active compounds.

Geranylgeraniol enhances testosterone production via the cAMP/protein kinase A pathway in testis-derived I-10 tumor cells.

Testosterone levels in men decrease with age; this decline has been linked to various diseases and can shorten life expectancy. Geranylgeraniol (GGOH) is an isoprenoid found in plants that plays an important role in several biological processes; however, its role in steroidogenesis is unknown. Here, we report that GGOH enhances the production of testosterone and its precursor progesterone in testis-derived I-10 tumor cells. GGOH induced protein kinase A (PKA) activity and increased cAMP levels and was found to regulate cAMP/PKA signaling by activating adenylate cyclase without altering phosphodiesterase activity. GGOH also stimulated mRNA and protein levels of steroidogenic acute regulatory protein, a downstream effector in the cAMP/PKA pathway. These results demonstrate that GGOH enhances steroidogenesis in testis-derived cells by modulating cAMP/PKA signaling. Our findings have potential applications for the development of therapeutics that increase testosterone levels in aging men.

Fermented rice bran supplementation mitigates metabolic syndrome in stroke-prone spontaneously hypertensive rats.

BACKGROUND: Previous study shown that enzyme treated-rice bran effectively improved hypertension and glucose intolerance in stroke-prone spontaneously hypertensive rat (SHRSP). However, dual fermentation of rice bran's efficacy against metabolic syndrome in SHRSP is still unknown. METHODS: Fermented rice bran (FRB) was prepared by dual fermentation of rice bran using fungi and lactic acid bacteria. The effect of FRB on metabolic syndrome in stroke-prone spontaneously hypertensive rats (SHRSP) was investigated by single and chronic supplementation. RESULTS: Dual fermentation of rice bran enriches the functional value of rice bran. Single-dose oral administration of FRB (2 g/kg body weight) reduced systolic blood pressure; however, chronic supplementation with 5 % FRB (4 weeks) significantly reduced both systolic and diastolic blood pressure. FRB supplementation improved leptin impairment and increased serum adiponectin levels and angiotensin-converting enzyme inhibitory activity. Furthermore, FRB supplementation improved glucose tolerance and insulin sensitivity as well as serum insulin levels. Lipid profiles were also improved by the regulation of 5' adenosine monophosphate-activated protein kinase activation. Moreover, supplementation with FRB reduced the expressions of hepatic transcription factors such as liver X receptor alpha, sterol regulatory element-binding protein 1c, and carbohydrate-responsive element-binding protein alpha, as well as their target genes. In conclusion, dietary supplementation with FRB may lower hypertension and alleviate metabolic syndrome. CONCLUSION: Metabolic syndrome was better alleviated with FRB supplementation. We therefore suggest FRB as an alternative medicine to reduce the risks of lifestyle-related diseases.

Systematic synthesis and anti-inflammatory activity of ω-carboxylated menaquinone derivatives--Investigations on identified and putative vitamin K2 metabolites.

Vitamin K is an essential nutrient for blood coagulation and bone homeostasis, and also functions in many physiological processes including inflammation and cancer progression. However, the nature and activities of its metabolites remain unclear. We report here systematic synthesis of ω-carboxylated derivatives of menaquinone (vitamin K2), including previously identified metabolites 5, K acid I (10), and K acid II (12), and evaluation of their inhibitory activity toward LPS-stimulated induction of inflammatory cytokines. These results should contribute to an improved understanding of the biochemistry and pharmacology of vitamin K.

Fermented barley extract supplementation ameliorates metabolic state in stroke-prone spontaneously hypertensive rats.

We studied the effects of fermented barley extract P (FBEP) in stroke-prone spontaneously hypertensive rats (SHRSP). Male 10-week-old SHRSP were divided into three groups that were fed: an AIN-93M diet (control), a low dose of FBEP (4 g/kg; FBEP1), and a high dose of FBEP (20 g/kg; FBEP2) for three weeks. Hypertension was significantly improved by the use of FBEP supplementation. The FBEP diet improved plasma triglyceride, insulin sensitivity, enhanced plasma catalase, and superoxide dismutase activities, and decreased plasma 8-hydroxy-2'-deoxyguanosine levels. In addition, the FBEP diet upregulated hepatic antioxidative genes and modulated Nrf2 protein levels in the liver. Furthermore, a single oral dose of FBEP (2 g/kg body weight) was able to lower blood pressure in SHRSP. In conclusion, our data suggest that increased expression of hepatic antioxidative genes and modulation of Nrf2 may play a role in the regulation of metabolic diseases in SHRSP consuming a FBEP diet.

Impact of fasting time on hepatic lipid metabolism in nutritional animal studies.

Many animal studies on improvement of lipid metabolism, using dietary components, fast the animals on the final day of the feeding. Although fasting has a significant impact on lipid metabolism, its time-dependent influence is not fully understood. We examined the effects of several fasting times on lipid metabolism. Rats fed with a semisynthetic diet for 2 wk were killed after 0 (9:00 am), 6 (7:00 am-1:00 pm), 9 (0:00 am-9:00 am), and 13 h (8:00 pm-9:00 am) of fasting. Compared to the 0 h group, marked reduction of liver weight and hepatic triacylglycerol content was observed in the 9 and 13 h groups. Activities of hepatic enzymes involved in fatty acid synthesis gradually decreased during fasting. In contrast, drastic time-dependent reduction of gene expression, of the enzymes, was observed. Expression of carnitine palmitoyltransferase mRNA was higher in the fasting groups than in the 0 h group. Our study showed that fasting has a significant impact on several parameters related to lipid metabolism in rat liver.