Efficacy of Long-Term Feeding of α-Glycerophosphocholine for Aging-Related Phenomena in Old Mice.

α-Glycerophosphocholine (GPC) is a natural source of choline. It reportedly prevents aging-related decline in cognitive function, but the underlying mechanism remains unclear. Although it is understood that aging influences taste sensitivity and energy regulation, whether GPC exerts antiaging effects on such phenomena requires further elucidation. Here, we used old C57BL/6J mice that were fed a GPC-containing diet, to investigate the molecular mechanisms underlying the prevention of a decline in cognitive function associated with aging and examine the beneficial effects of GPC intake on aging-related phenomena, such as taste sensitivity and energy regulation. We confirmed that GPC intake reduces the aging-related decline in the expression levels of genes related to long-term potentiation. Although we did not observe an improvement in aging-related decline in taste sensitivity, there was a notable improvement in the expression levels of ß-oxidation-associated genes in old mice. Our results suggest that the prevention of aging-related decline in cognitive function by GPC intake may be associated with the improvement of gene expression levels of long-term potentiation. Furthermore, GPC intake may positively influence lipid metabolism.

Anti-diabetic effect of S-adenosylmethionine and α-glycerophosphocholine in KK-Ay mice.

Six-week-old male KK-Ay mice received drinking water with S-adenosylmethionine (SAM), α-glycerophosphocholine (GPC), or SAM+GPC for 10 weeks. The serum glucose of SAM+GPC at 15 weeks old, total cholesterol of GPC at 12 weeks old, and triglyceride of GPC at 15 weeks old and of SAM at 16 weeks old were reduced. SAM+GPC reduced serum leptin and food intake. Abbreviations: SAM: S-adenosylmethionine; GPC: α-glycerophosphocholine.

Nutritional characterization of sake cake (sake-kasu) after heat-drying and freeze-drying.

Sake cake contains rice-derived components, as well as cell components and metabolites of Aspergillus oryzae and Saccharomyces cerevisiae. In this study, the effect of food processing on sake cake (sake-kasu) ingredients was investigated. Sake cake, obtained through brewing liquefied rice, was heat-dried (HD) or freeze-dried (FD) and analyzed. There were no differences in the amounts of proteins, lipids, carbohydrates, vitamin B6, choline, betaine, nicotinic acid, ß-glucan and resistant proteins in HD and FD. There was also no difference in the amount of hydrolyzed amino acids in HD and FD, but many free amino acids were observed in HD. S-adenosyl methionine (SAM) was found to be abundant in FD. Meanwhile, nucleic acid-related components were found to be increased in HD, which seems to be due to the degradation of microbial metabolites. When considering the health benefits of sake cake, it is necessary to pay attention to the effects of processing method. Abbreviations CE-TOFMS: capillary electrophoresis time-of-flight mass spectrometry.

Sake cake (sake-kasu) ingestion increases branched-chain amino acids in the plasma, muscles, and brains of senescence-accelerated mice prone 8.

To examine metabolic effects of sake cake ingestion, plasma and tissues were analyzed in senescence-accelerated mice prone 8 (SAMP8) fed a sake cake diet. As a result, branched-chain amino acids (BCAA) were found to be significantly higher in the plasma, gastrocnemius muscles and brains of the sake cake group than in the control group. Mice in the sake cake group showed stronger grip strength than the control group. High levels of circulating BCAA have been reported to be associated with pathological states, such as metabolic diseases, but the parameters of glucose and lipid metabolism were not affected between the two groups. Otherwise, pyridoxal was significantly higher and nicotinamide as well as 1-methylnicotinamide showed a tendency to be higher in the plasma of the sake cake group than in the control group. These findings indicate that intake of sake cake increases the levels of BCAA, vitamin B6, and vitamin B3. Abbreviation: CE-TOFMS: capillary electrophoresis time-of-flight mass spectrometry.

The delaying effect of alpha-glycerophosphocholine on senescence, transthyretin deposition, and osteoarthritis in senescence-accelerated mouse prone 8 mice.

Administration of alpha-glycerophosphocholine (GPC), a choline compound in food, is expected to contribute to human health. In this study, we evaluated its effect on aging in senescence-accelerated mouse prone 8 (SAMP8) mice. Male SAMP8 mice had free access to a commercial stock diet and drinking water with or without GPC (0.07 mg/ml). Mice in the GPC group had significantly lower total senescence grading score than that of the control group at 36 weeks of age. Administration of GPC decreased the deposition of transthyretin (TTR), an amyloidogenic protein, in the brain. Aggregated TTR activated microglia and led to neuroinflammation. Thus, GPC would protect the brain by reducing TTR deposition and preventing neuroinflammation. In a histological study of knee joints, it was found that SAMP8 mice administered GPC showed decreased joint degeneration. These results suggest that GPC delays the aging process and may be a useful compound in anti-aging functional food development.

Preliminary detection method for added rice- and sugarcane-derived brewer's alcohol in bulk samples of sake by measurement of hydrogen, oxygen, and carbon isotopes.

The δD and δ(18)O of sake without ethanol extraction as well as the δ(13)C of ethanol were determined in order to detect rice-derived brewer's alcohol. Brewing tests demonstrated that the δD decreases upon addition of brewer's alcohol (both rice- and sugarcane-derived), while the δ(18)O has little response to either, and the δ(13)C increases only for added sugarcane-derived alcohol.

Lignin is linked to ethyl-carbamate formation in ume (Prunus mume) liqueur.

Ethyl carbamate concentrations in oak barrel-aged ume (Prunus mume) liqueurs were measured, and possible explanations for elevated levels were examined. The average concentration was 0.30 mg/L, significantly higher than in ume liqueurs not aged in oak (0.08 mg/L). Oak powder extracts were prepared from both untoasted and toasted oak powder by extraction with aqueous ethanol, and these were used to make ume liqueurs. Relative to a no-oak control, the ethyl carbamate concentrations were 3.8 and 11 times higher in the ume liqueur made with the untoasted and toasted oak powder extracts respectively. The extracts were loaded onto a C18 column, washed with water, and eluted with methanol. The (13)C-NMR spectra for the main constituents of the methanol elution fractions were consistent with those for lignin or fragments thereof. The methanol fractions were added to ume liqueur which was stored for 3 months. Relative to a control, the ethyl carbamate concentrations in the 3-month old liqueurs were found to be 1.2 and 4.6 higher for the untoasted oak-powder and the toasted oak-powder respectively. Ethyl carbamate was formed when lignin was added to a 40% aqueous ethanol solution that contained potassium cyanide. These observations suggest that lignin or fragments thereof promote the formation of ethyl carbamate.

The concentration of ethyl carbamate in commercial ume (Prunus mume) liqueur products and a method of reducing it.

The ethyl carbamate concentration of commercial ume liqueur products was studied, and a method of reducing it was examined from the viewpoint of antioxidation. The average ethyl carbamate concentration across 38 ume liqueur products was 0.12 mg/l (0.02-0.33 mg/l). When potassium metabisulfite was added to a concentration of 0-1,000 ppm during production, the generation of ethyl carbamate was reduced in a concentration-dependent manner, but when the amount of potassium metabisulfite added was below the maximum level allowed under the Japanese Food Sanitation Act, the reduction was only 27%. When ume liqueurs were produced under deoxygenated conditions created using an oxygen absorber, the ethyl carbamate concentration was reduced by up to 47% as compared with the control group, probably due mainly to a reduction in free hydrogen cyanide. When ume liqueur was produced in an oxygen atmosphere, the ethyl carbamate concentration increased by up to 50% as compared with the control group. Thus, oxygen may be involved in the generation of ethyl carbamate in ume liqueur production.

Low-Dose Ethanol Has Impacts on Plasma Levels of Metabolites Relating to Chronic Disease Risk in SAMP8 mice.

The effects of low-dose alcohol on experimental animals are unclear. This study examined plasma metabolites in senescence-accelerated mice 8 (SAMP8) given low-dose ethanol, and compared them with aging progress and skeletal muscle strength. Male SAMP8 mice (10-wk-old) were given drinking water containing 0% (control), 1%, 2%, or 5% (v/v) ethanol for 14 wk. Compared with the control group, only mice who consumed 1% ethanol experienced a lower senescence score at 18 and 23 wk, as well as an increased limb grip strength at 21 wk. Plasma metabolites of control, 1% and 2% ethanol groups were analyzed by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS). Among the 7 metabolites affected by ethanol, notewhorthy is the positive association of the ethanol levels in drinking water with the levels of α-ketoglutarate (antioxidant and anti-inflammatory metabolite) and hippurate (antioxidant and microbial co-metabolite) (p<0.05). Intriguingly, the levels of 2-hydroxyisobutyrate (the biomarker of energy metabolism and microbial co-metabolite) were higher in the 1% ethanol group (p<0.05), but not in the 2% ethanol group as compared to the control. Furthermore, the levels of some of the metabolites affected were correlated with some variables in the grading score of senescence and muscle strength. This study provides a novel insight into how low-dose ethanol in SAMP8 mice modulates the levels of circulating metabolites relating to chronic disease risk.

Activation of heat shock genes is not necessary for protection by heat shock transcription factor 1 against cell death due to a single exposure to high temperatures.

Heat shock response, which is characterized by the induction of a set of heat shock proteins, is essential for induced thermotolerance and is regulated by heat shock transcription factors (HSFs). Curiously, HSF1 is essential for heat shock response in mammals, whereas in avian HSF3, an avian-specific factor is required for the burst activation of heat shock genes. Amino acid sequences of chicken HSF1 are highly conserved with human HSF1, but those of HSF3 diverge significantly. Here, we demonstrated that chicken HSF1 lost the ability to activate heat shock genes through the amino-terminal domain containing an alanine-rich sequence and a DNA-binding domain. Surprisingly, chicken and human HSF1 but not HSF3 possess a novel function that protects against a single exposure to mild heat shock, which is not mediated through the activation of heat shock genes. Overexpression of HSF1 mutants that could not bind to DNA did not restore the susceptibility to cell death in HSF1-null cells, suggesting that the new protective role of HSF1 is mediated through regulation of unknown target genes other than heat shock genes. These results uncover a novel role of vertebrate HSF1, which has been masked under the roles of heat shock proteins.

Effects of low dose of ethanol on the senescence score, brain function and gene expression in senescence-accelerated mice 8 (SAMP8).

Accumulating epidemiological evidence suggests light to moderate alcohol intake reduces risk of several chronic diseases. However, there is limited information regarding the effects of low alcohol intake in animal studies. This study investigated the effect of low ethanol dosage on senescence-accelerated mouse (SAMP8), an animal model of aging and neurodegenaration. Male SAMP8 mice (11 weeks old) had free access to a commercial stock diet with drinking water containing 0, 1 or 2% (v/v) ethanol for 15 weeks. The total grading score of senescence in the 1%-ethanol group was, in large part, the lowest among the three groups. Analysis using the open-field test revealed a significant elevation (+77%, P<0.05) in the rearing activity (index of seeking behavior) in the 1%-ethanol group, but not in the 2%-ethanol group. In addition, 2% ethanol elevated spontaneous locomotor activity (+75%, P<0.05), whereas 1% ethanol did not. Scrutiny of serum parameters indicated intake of 1% ethanol significantly decreased serum insulin levels (-13%, P<0.05), whereas 2% did not. Intake of 2% ethanol significantly elevated (2.5-fold, P<0.05) S100a8 mRNA (an inflammatory signal) in the brain, but that of 1% ethanol did not. Intriguingly, 1% ethanol intake remarkably elevated (10-fold, P<0.05) mRNA of brain alcohol dehydrogenase 1 (Adh1), which metabolizes lipid-peroxidation products and is involved in the synthesis of retinoic acid, a neuroprotective factor. Of note, 2%-ethanol intake did not exert this effect. Taken together, intake of 1% ethanol is likely to be beneficial for SAMP8 mice.

Direct activation of GABAA receptors by substances in the organic acid fraction of Japanese sake.

We investigated the effect of substances present in Japanese sake on the response of ionotropic γ-aminobutyric acid (GABA)A receptors expressed in Xenopus oocytes. Sake was fractionated by ion-exchange chromatography. The fraction containing organic acids (OA fraction) showed agonist activities on the GABAA receptor. OA fractions from sake were analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Of the 64 compounds identified, 13 compounds showed GABAA receptor agonist activities. Especially, l-lactic acid showed high agonist activity and its EC50 value was 37µM. Intraperitoneal injections of l-lactic acid, gluconic acid, and pyruvic acid (10, 10, and 5mg/kg BW, respectively), which showed agonistic activity on the GABAA receptor, led to significant anxiolytic effects during an elevated plus-maze test in mice.

Comparison of the neurotoxicities between volatile organic compounds and fragrant organic compounds on human neuroblastoma SK-N-SH cells and primary cultured rat neurons.

These are many volatile organic compounds (VOCs) that are synthesized, produced from petroleum or derived from natural compounds, mostly plants. Fragrant and volatile organic compounds from plants have been used as food additives, medicines and aromatherapy. Several clinical and pathological studies have shown that chronic abuse of VOCs, mainly toluene, causes several neuropsychiatric disorders. Little is known about the mechanisms of neurotoxicity of the solvents. n-Octanal, nonanal, and 2-ethyl-1-hexanol, which are used catalyzers or intermediates of chemical reactions, are released into the environment. Essential oils have the functions of self-defense, sterilization, and antibiosis in plants. When volatile organic compounds enter the body, there is the possibility that they will pass through the blood-brain barrier (BBB) and affect the central nervous system (CNS). However, the direct effects of volatile organic compounds on neural function and their toxicities are still unclear. We compared the toxicities of n-octanal, nonanal and 2-ethyl-1-hexanol with those of five naturally derived fragrant organic compounds (FOCs), linalool, cis-3-hexen-1-ol, isoamyl alcohol, n-propyl alcohol and n-phenethyl alcohol. MTT assay of human neuroblastoma SK-N-SH cells showed that the IC50 values of linalool, cis-3-hexen-1-ol, isoamyl alcohol, n-propyl alcohol and phenethyl alcohol were 1.33, 2.3, >5, >5, and 2.39 mM, respectively, and the IC50 values of toluene, n-octanal, nonanal and 2-ethyl-1-hexanol were 850, 37.2, 8.31 and 15.1 µM, respectively. FOCs showed lower toxicities than those of VOCs. These results indicate that FOCs are safer than other compounds.

Hsp25, a member of the Hsp30 family, promotes inclusion formation in response to stress.

Protein aggregates are oligomeric complexes of misfolded proteins, and serve as the seeds of inclusion bodies termed aggresomes in the cells. Heat shock proteins (Hsps) prevent misfolding and aggregate formation. Here, we found that only avian Hsp25 dominantly accumulated in the aggresomes induced by proteasome inhibition. Molecular cloning of chicken Hsp25 (cHsp25) revealed that it belongs to the Hsp30 family, which is a subfamily of the alpha-crystallin/small Hsp gene family. Unexpectedly, overexpression of cHsp25 into HeLa cells promoted inclusion formation whereas overexpression of mouse Hsp27 and its chicken homologue did not. These results suggest that cHsp25 acts differently from other small Hsps on protein aggregates.

Feeding induces expression of heat shock proteins that reduce oxidative stress.

Heat shock proteins (Hsps) are induced in response to various kinds of environmental and physiological stresses. However, it is unclear whether Hsps play roles in protecting cells in the digestive organs against xenobiotic chemicals. Here, we found that feeding induces expression of a set of Hsps specifically in the mouse liver and intestine by activating heat shock transcription factor 1 (HSF1). In the liver, HSF1 is required to suppress toxic effects of electrophiles, which are xenobiotic chemicals causing oxidative stress. We found that overexpression of Hsp27, which elevates cellular glutathione level, promotes survival of culture cells exposed to electrophiles. These results suggest a novel mechanism of cell protection against xenobiotic chemicals in the food.

Heat shock transcription factor HSF1 is required for survival of sensory hair cells against acoustic overexposure.

To analyze the role of heat shock response in the cochleae, we induced major heat shock proteins, Hsp70, Hsp90, and Hsp27 by perfusion of hot saline into the middle ear cavity (called 'local heat shock') in guinea pigs. Hsps were induced in almost all of the cochlear cells including the sensory hair cells in the organ of Corti. We showed that loss of both the sensory hair cells and the auditory function induced by acoustic overexposure was inhibited by pretreatment of the inner ear with local heat shock. To examine the role of heat shock transcription factor 1(HSF), which activates heat shock genes in response to heat shock, in the protection of sensory hair cells, we analyzed acoustic injury in HSF1-null mice. We found that the loss of sensory hair cells was more significant in HSF1-null mice compared with that of wild-type mice when mice were subjected to acoustic overexposure. These results indicate that HSF1 is required for survival of the sensory hair cells against acoustic overexposure.

Beneficial effect of a low dose of ethanol on liver function and serum urate in rats fed a high-fat diet.

This study investigated the effects of the consumption of 1% or 2% (v/v) ethanol in drinking water for 12 wk on rats fed a high-fat diet. Body weight gain, food intake, and fluid intake were unaffected by ethanol intake. Adipose tissue weight, and serum glucose and lipids were unaffected. Compared to the control (no ethanol), 1% ethanol intake significantly reduced serum levels of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and ammonia (p<0.05), whereas 2% ethanol intake did so to a lesser extent. Serum urate was significantly lower in both the 1% and 2% ethanol groups than that in the control group (p<0.05). The results suggest a low dose of ethanol has beneficial effects on liver function and serum urate in rats fed a high-fat diet.

Hepatoprotective effects of a concentrate and components of sake against galactosamine (GalN)-induced liver injury in mice.

We investigated the hepatoprotective effects of a concentrate of sake (CS) and its components against D-galactosamine (GalN)-induced liver injury by measuring the plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in mice. CS significantly suppressed the GalN-induced elevation of ALT and AST activities. Each of four concentrated fractions extracted from sake (respectively consisting mainly of basic amino acids, neutral and acidic amino acids, organic acids and sugars) suppressed the GalN-induced elevation of ALT and AST activities. We focused on the sugar fraction containing glucose and ethyl alpha-D-glucoside (alpha-EG), which is a sake-specific sugar, as the major components and demonstrated that only alpha-EG showed significant suppression of the GalN-induced elevation of ALT and AST activities. We compared the effects of the alpha-EG analogues, methyl alpha-D-glucoside and ethyl beta-D-glucoside, on GalN-induced liver injury and confirmed that only alpha-EG significantly suppressed both the ALT and AST activities. Moreover, CS and alpha-EG suppressed the GalN-induced production of interleukin 6 (IL-6) and liver DNA fragmentation. Together these results show that CS and its component, alpha-EG, suppressed GalN-induced liver injury by inhibiting IL-6 production.

S-adenosylmethionine (SAM)-accumulating sake yeast suppresses acute alcohol-induced liver injury in mice.

The suppressive effects on acute alcoholic liver injury of S-adenosylmethionine (SAM) and the sake yeast, Saccharomyces cerevisiae Kyokai No. 9, have been shown previously. To enhance the suppression of acute alcoholic liver injury by sake yeast, we prepared SAM-accumulating sake yeast (SAM yeast). Male C57BL/6 mice that had been fed on a diet containing 0.25% SAM yeast or sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed on the SAM yeast, the ethanol-induced increases in both triglyceride (TG) and alanine aminotransferase (ALT) were significantly repressed. In addition, the SAM yeast-fed mice did not show an ethanol-induced decrease in hepatic SAM level, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by the SAM yeast. These results suggest that the SAM yeast had a stronger effect suppressing acute alcoholic liver injury in mice than the sake yeast.

Sake yeast suppresses acute alcohol-induced liver injury in mice.

Brewer's and baker's yeasts appear to have components that protect from liver injury. Whether sake yeast, Saccharomyces cerevisiae Kyokai no. 9, also has a hepatoprotective effect has not been examined. Here we show that sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by sake yeast. These results indicate that sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.