The Association between Cerebral Small Vessel Disease and the Gut Microbiome: A Cross-Sectional Analysis.

BACKGROUND: Recent studies have demonstrated an association between the gut microbiome and cognitive function. However, the associations between the gut microbiome and brain parenchyma damage, and their underlying mechanisms, remain unclear. MATERIALS AND METHODS: We performed a cross-sectional sub-analysis using data from our prospective cohort study to determine the association between the gut microbiome and cerebral small vessel disease (SVD). We assessed patient demographics, risk factors, cognitive function, brain imaging, voxel-based specific regional analysis system for Alzheimer's Disease (VSRAD, indicating brain atrophy), and the gut microbiome as indicated by enterotypes and faecal microbiome metabolites. We then analysed the associations between total SVD scores, cognitive function, and the gut microbiome. RESULTS: We analysed data from 87 patients without dementia or a history of stroke, 64 of whom exhibited mild cognitive impairment. Higher total SVD scores were associated with cognitive decline and behavioural and psychological symptoms. Compared with all other patients, patients with enterotype I (Bacteroides >30%) were more likely to have cognitive decline (median scores: Mini-Mental State Examination, 25 vs. 27, P = 0.047; Clinical Dementia Rating-Sum of Boxes, 1.5 vs. 0.5, P = 0.002) and present with cerebral SVD and high VSRAD scores (1.01 vs. 0.57, P = 0.012). Furthermore, faecal metabolites were significantly higher in patients with higher total SVD scores compared with those with lower scores. Multivariable logistic regression analyses indicated that certain gut microbiomes may double the risk of white matter hyperintensity. CONCLUSIONS: The gut microbiome is associated with cerebral SVD.

Mushroom consumption and incident risk of prostate cancer in Japan: A pooled analysis of the Miyagi Cohort Study and the Ohsaki Cohort Study.

In vivo and in vitro evidence has shown that mushrooms have the potential to prevent prostate cancer. However, the relationship between mushroom consumption and incident prostate cancer in humans has never been investigated. In the present study, a total of 36,499 men, aged 40-79 years, who participated in the Miyagi Cohort Study in 1990 and in the Ohsaki Cohort Study in 1994 were followed for a median of 13.2 years. Data on mushroom consumption (categorized as <1, 1-2 and ≥3 times/week) was collected using a validated food frequency questionnaire. Cox proportional hazards regression analysis was used to estimate multivariate hazard ratios (HRs) and 95% confidence intervals (CIs) for prostate cancer incidence. During 574,397 person-years of follow-up, 1,204 (3.3%) cases of prostate cancer were identified. Compared to participants with mushroom consumption <1 time/week, frequent mushroom intake was associated with a decreased risk of prostate cancer (1-2 times/week: HRs [95% CIs] = 0.92 [0.81, 1.05]; ≥3 times/week: HRs [95% CIs] = 0.83 [0.70, 0.98]; p-trend = 0.023). This inverse relationship was especially obvious among participants aged ≥50 years and did not differ by clinical stage of cancer and intake of vegetables, fruit, meat and dairy products. The present study showed an inverse relationship between mushroom consumption and incident prostate cancer among middle-aged and elderly Japanese men, suggesting that habitual mushroom intake might help to prevent prostate cancer.

NAPE-PLD controls OEA synthesis and fat absorption by regulating lipoprotein synthesis in an in vitro model of intestinal epithelial cells.

Oleoylethanolamide (OEA), a fatty acid ethanolamide (FAE), is a lipid mediator that controls food intake and lipid metabolism. Accumulating data imply the importance of intestinal OEA in controlling satiety in addition to gastrointestinal peptide hormones. Although the biochemical pathway of FAE production has been illustrated, the enzymes responsible for the cleavage of OEA from its precursor N-acyl-phosphatidylethanolamine (NAPE) must be identified among reported candidates in the gut. In this study, we assessed the involvement of NAPE-specific phospholipase D (NAPE-PLD), which can directly release FAEs from NAPE, in intestinal OEA synthesis and lipid metabolism. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPER-associated protein 9 (Cas9)-mediated deletion of the NAPE-PLD gene in intestinal epithelial-like Caco-2 cells reduced OEA levels, regardless of their differentiation states. Transcriptome analysis revealed that deletion of NAPE-PLD activates a transcriptional program for nutrient transportation, including lipids and lipoproteins, and inactivates cell-cycle or mitosis-related genes in Caco-2 cells. In addition, the basolateral secretion of lipoproteins was increased in NAPE-PLD-deleted cells although lipoprotein size was not affected. By contrast, cellular lipid levels were reduced in NAPE-PLD-deleted cells. Overall, these results indicate that NAPE-PLD plays important roles in OEA synthesis and fat absorption by regulating lipoprotein production in the intestinal epithelial cells.-Igarashi, M., Watanabe, K., Tsuduki, T., Kimura, I., Kubota, N. NAPE-PLD controls OEA synthesis and fat absorption by regulating lipoprotein synthesis in an in vitro model of intestinal epithelial cells.

The 1975 type Japanese diet improves the gut microbial flora and inhibits visceral fat accumulation in mice.

In this study, the 1975 type Japanese diet was prepared and its effects and related mechanism were examined in mice. Mice were assigned to three experimental groups, the CD group fed a control diet, the MD group fed a modern Japanese diet (MD), and the JD group fed the 1975 type Japanese diet (JD) for 4 weeks. MD and JD were low protein, high fat, and high carbohydrate diets compared to the CD. Total white adipose tissue weights were significantly increased in the MD group compared to those in the CD group and were decreased in the JD group compared to those in the MD group. In the JD group, adipocyte hypertrophy was inhibited and Hsl mRNA expression was enhanced in epididymal adipose tissue and the number of bacteria associated with the production of short chain fatty acids was increased. Therefore, the JD inhibits lipid accumulation in white adipose tissue. ABBREVIATIONS: Actb: ß-actin; ALT: alanine aminotransferase; ANOVA: analyses of variance; AST: aspartate aminotransferase; Fas: fatty acid synthase; G6pdx: glucose 6-phosphate dehydrogenase; HE: hematoxylin and eosin; HOMA-IR: Homeostatic model assessment for insulin resistance; Hsl: hormone-sensitive lipase; JD: 1975 type Japanese diet; Leptin: leptin; MD: modern Japanese diet; Me: malic enzyme; NEFA: non-esterified fatty acids; PL: phospholipids; Pparδ: peroxisome proliferator-activated receptor delta; Pparγ: peroxisome proliferator-activated receptor gamma; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; SAMP8: senescence-accelerated prone 8; SEM: standard error of the mean; Srebp1c: Sterol regulatory element binding protein 1c; TBARS: thiobarbituric acid reactive substance; TC: total cholesterol; TG: Triacylglycerol; V3: variable regions 3.

Sphingoid bases of dietary ceramide 2-aminoethylphosphonate, a marine sphingolipid, absorb into lymph in rats.

Various functions of dietary sphingolipids have been reported; however, little is known about marine sphingolipids. Ceramide 2-aminoethylphosphonate (CAEP), an abundant sphingolipid in marine mollusks, frequently has a unique triene type of sphingoid base [2-amino-9-methyl-4,8,10-octadecatriene-1,3-diol (d19:3)]. We previously reported that dietary CAEP prepared from the skin of squid was digested in the intestinal mucosa of mice via ceramides to yield free sphingoid bases. How dietary CAEP is then used in the body remains unclear. Here, we investigated the absorption of dietary CAEP using a lipid absorption assay on the lymph collected from rats with thoracic duct cannulation. Our results reveal that sphingoid bases derived from CAEP, including d16:1, d18:1, and d19:3, were detected in the lymph after administration of CAEP. Lymphatic recovery of d19:3 was lower than that of other sphingoid bases. A large fraction of the absorbed sphingoid bases was present as complex sphingolipids, whereas a smaller portion was present in the free form. Fatty acids in ceramide moieties found in the lymph were partially different from dietary CAEP, which indicates that sphingoid bases derived from CAEP could be, at least in part, resynthesized into complex sphingolipids. Future studies should elucidate the metabolism of sphingoid bases derived from CAEP.

The Japanese Dietary Pattern Is Associated with Longer Disability-Free Survival Time in the General Elderly Population in the Ohsaki Cohort 2006 Study.

BACKGROUND: Epidemiologic observations have raised expectations that the Japanese dietary pattern could promote longer disability-free survival (DFS) times among the Japanese population; however, no previous study has examined this issue. OBJECTIVE: The aim of this study was to investigate the association between the Japanese dietary pattern and DFS time in the elderly Japanese population. METHODS: We analyzed follow-up data covering a 10-y period for 9456 elderly Japanese individuals (aged ≥65 y) participating in a community-based prospective cohort study. Dietary habits were assessed using a food-frequency questionnaire. Based on previous studies, we used 9 food items to calculate the Japanese Diet Index (JDI) score: rice, miso soup, fish and shellfish, green and yellow vegetables, seaweed, pickled vegetables, green tea (1 point for each item if the consumption value was more than or equal to the median, and 0 points otherwise), beef and pork, and coffee (0 points for each item if the consumption value was more than or equal to the median, and 1 point otherwise). Differences in median age at incident disability or death [50th percentile differences (PDs)] according to quartiles (Q1-Q4) of the JDI score were estimated using Laplace regression. RESULTS: During the follow-up period, 4233 (44.8%) incident disability or death events occurred. In addition, a higher JDI score was significantly associated with longer DFS time: compared with the lowest quartile of JDI scores (Q1), the multivariate-adjusted 50th PD (95% CI) was 7.1 (1.8, 12.4) mo longer for Q4. Each 1-SD increase of the JDI score was associated with 3.7 (1.7, 5.7) additional months of life without disability (P-trend < 0.01). No differences were seen in sex or chronic condition (no or ≥1 chronic condition) at baseline. A post hoc analysis showed a larger effect on DFS time when using a modified JDI score without coffee. CONCLUSION: These results suggest that the Japanese dietary pattern is associated with improved DFS time in the general elderly population.

Carbohydrate-restricted diet promotes skin senescence in senescence-accelerated prone mice.

This study used senescence-accelerated prone mice (SAMP8) to examine the effects of a carbohydrate-restricted diet on aging and skin senescence, to determine how long-term carbohydrate restriction affects the aging process. Three-week-old male SAMP8 mice were divided into three groups after 1 week of preliminary feeding: one was given a controlled diet, the other was given a high-fat diet, and the third was given a carbohydrate-restricted diet. Ad libitum feeding was administered until the mice reached 50 weeks of age. Before the end of the test period, a grading test was used to evaluate visible aging in the mice. After the test period, serum and skin samples in mice were obtained and submitted for analysis. As a result, the grading test demonstrated that there was significant progression of visible aging in the carbohydrate-restricted group, as well as a decreased survival rate. Histological examination of the skin revealed that the epidermis and dermis in the carbohydrate-restricted group had become thinner. Analysis of the mechanisms involved demonstrated an increase in serum interleukin-6, aggravated skin senescence, inhibition of skin autophagy and activation of skin mTOR. Therefore, this study proved that a carbohydrate-restricted diet promoted skin senescence in senescence-accelerated mice.

The effect of dietary lipid on gut microbiota in a senescence-accelerated prone mouse model (SAMP8).

Gut microbiota change with aging and diet. In a previous study, it was shown that a moderate-fat diet enriched with fish oil had beneficial effects for elderly patients, so we examined the effect of this diet on aging-related changes in gut microbiota in this study. We used 3-month-old male senescence-accelerated prone mice (SAMP8). The mice were fed a normal diet containing 4 g soybean oil/100 g of diet for 6 months and then divided into 4 groups: (1) the Baseline group, ended breeding at 6 months old; (2) the Control group, continued on a normal diet until 15 months old; (3) the MF group, switched to a moderate-fat diet until 15 months old; and (4) the MF + FO group, switched to a moderate-fat diet enriched with fish oil until 15 months old. When mice were 6 or 15 months old, fecal samples were collected and gut microbiota analysis was performed. Gut microbiota analysis at the genus level showed that bacteria known to increase in association with fatty liver and intestinal inflammation increased with aging. However, this alteration was largely inhibited by the moderate-fat diet enriched with fish oil. On the other hand, there was a decrease with aging in the bacteria that play a role in energy consumption, but this alteration was inhibited by the moderate-fat diet enriched with fish oil. These results suggest that a moderate-fat diet enriched with fish oil has beneficial effects on gut microbiota in aging.

Effect of the Japanese diet during pregnancy and lactation or post-weaning on the risk of metabolic syndrome in offspring.

We examined the effects on offspring of ingestion of the 1975 Japanese diet during pregnancy and lactation and after weaning in mice. Pregnant dams were divided into groups that were fed the Japanese diet or a control diet and raised until offspring were weaned. The offspring after weaning were further divided into groups that were raised on the Japanese diet or the control diet. Ingestion of the Japanese diet after weaning suppressed accumulation of visceral fat in offspring, and reduced the amount of lipids in serum and liver. This effect was weakened if the Japanese diet was only ingested during pregnancy and lactation. Therefore, it was suggested that ingestion of the Japanese diet of mothers during pregnancy and lactation weakens the lipid accumulation inhibitory effect of the Japanese diet in children.

The 1975 Japanese diet has a stress reduction effect in mice: search for physiological effects using metabolome analysis.

We aimed to find new physiological effects of the Japanese diet. First, to determine the key components in serum from mice fed the 1975 diet, serum from mice fed the 1960, 1975, 1990 or 2005 Japanese diet was analyzed using CE-TOFMS and LC-TOFMS. Based on these results, the key components were determined by principal component analysis. Among the identified compounds, GABA was included. Therefore, a stress reduction effect was inferred as a novel physiological effect of this diet. Next, we tested whether the 1975 diet had an actual stress reduction effect in mice. Mice were given the 1975 diet or a control diet for 4 weeks, after which they were divided into restraint stress and non-stress groups. Mice fed the 1975 diet had significantly decreased stress parameters compared with those fed the control diet. These results provide the first evidence that the 1975 Japanese diet has a stress reduction effect.

Standardisation of the Japanese diet for use in animal experiments.

The aim of this study was to develop a purified diet that mimics the characteristics of the Japanese diet using readily available materials with a simpler composition and a focus on quality, with the goal of facilitating performance of studies on the Japanese diet worldwide. The utility of the new diet was examined as a mimic of the standard Japanese diet for use in animal experiments. We examined whether a key characteristic of the Japanese diet of being less likely to cause obesity could be reproduced. The mimic diet had a balance of protein, fat and carbohydrate based on the 1975 Japanese diet, which is the least likely to cause obesity, and materials chosen with reference to the National Health and Nutrition Survey (NHNS). To examine similarities of the mimic diet with the model 1975 Japanese diet, we created a menu of the 1975 diet based on the NHNS and prepared the freeze-dried and powdered diet. The mimic diet, the 1975 Japanese diet, a control AIN-93G diet and a Western diet were fed to mice for 4 weeks. As a result, the mimic diet and the 1975 diet resulted in less accumulation of visceral fat and liver fat. Mice given these two diets showed similar effects. This indicates that the mimic diet used in this study has characteristics of the 1975 Japanese diet and could be used as a standard Japanese diet in animal experiments.

Intake of mulberry 1-deoxynojirimycin prevents colorectal cancer in mice.

The effect of 1-deoxynojirimycin, a caloric restriction mimetic, was examined in ICR mice with azoxymethane dextran sodium sulfate-induced colorectal cancer. Azoxymethane is a carcinogen (10 mg/kg body weight), and 2% dextran sodium sulfate (w/v) used as a colitis-inducing agent. Mice were separated into 5 groups: a group without colorectal cancer fed a normal diet (CO- group), and groups with colorectal cancer fed a normal diet (CO+ group), a calorie-restricted diet (caloric restriction group), and diets including 0.02% and 0.1% 1-deoxynojirimycin (l-1-deoxynojirimycin and H-1-deoxynojirimycin groups). The tumor incidence and number were reduced significantly in the caloric restriction group compared to the CO+ group, and were also suppressed in a dose-dependent manner by 1-deoxynojirimycin. mRNA for anti-apoptotic Bcl-2 was decreased and that for pro-apoptotic Bax was increased in the carcinoma tissue of CR, l-1-deoxynojirimycin and H-1-deoxynojirimycin groups. These results suggest that caloric restriction and 1-deoxynojirimycin inhibit growth of colorectal cancer by inducing apoptosis in an induced cancer model in mice.

Research on food and nutrition characteristics of conjugated fatty acids.

In this study, the physiological effects of fatty acids with conjugated double bonds were widely examined in vitro and in vivo. Initially, a method for determination of conjugated fatty acids in food and biological samples was established. I then clarified that the oxidative stability of conjugated fatty acids was improved by the form of triacylglycerol and addition of an antioxidant, and the influence of this effect on the metabolism and pharmacokinetics of conjugated fatty acids was clarified in vivo. In addition, antitumor, anti-angiogenesis, and antiobesity effects of conjugated fatty acids were found for the first time, thus demonstrating the usefulness of conjugated fatty acids. This communication mainly outlines the data obtained for conjugated linolenic acid. In addition, this review summarizes my research on conjugated fatty acid.

High-fat diet intake from senescence inhibits the attenuation of cell functions and the degeneration of villi with aging in the small intestine, and inhibits the attenuation of lipid absorption ability in SAMP8 mice.

We examined the effect of a high-fat diet from senescence as a means of preventing malnutrition among the elderly. The senescence-accelerated mouse P8 was used and divided into three groups. The 6C group was given a normal diet until 6 months old. The 12N group was given a normal diet until 12 months old. The 12F group was given a normal diet until 6 months old and then a high-fat diet until 12 months old. In the oral fat tolerance test, there was a decrease in area under the curve for serum triacylglycerol level in the 12N group and a significant increase in the 12F group, suggesting that the attenuation of lipid absorption ability with aging was delayed by a high-fat diet from senescence. To examine this mechanism, histological analysis in the small intestine was performed. As a result, the degeneration of villi with aging was inhibited by the high-fat diet. There was also a significant decrease in length of villus in the small intestine in the 12N group and a significant increase in the 12F group. The high-fat diet from senescence inhibited the degeneration of villi with aging in the small intestine, and inhibited the attenuation of lipid absorption ability.

Decreased lipid absorption due to reduced pancreatic lipase activity in aging male mice.

Malnutrition due to aging is partly caused by decreased absorption of nutrients by the gastrointestinal tract. However, the underlying mechanism is unclear and changes in lipid absorption with aging are poorly understood. In this study, changes in lipid absorption with aging were examined in mice aged 3 and 25 months. After overnight fasting, blood samples were collected from snipped tails and then soybean oil was administered orally. Three hours later, mice were sacrificed by decapitation and the liver, pancreas, small intestine and blood were collected. The increase in serum triacylglycerol after soybean oil administration was significantly lower in the older mice, indicating a decrease in lipid absorption with aging. Measurement of mRNA levels for triacylglycerol absorption-related genes showed that mRNA for pancreatic lipase tended to decrease in 25-month-old mice. There was no significant difference in the protein level of pancreatic lipase, but the enzyme activity showed a significant decrease in the older mice. To examine this mechanism, expression levels of mRNA for protein turnover-related genes in the pancreas were measured. The level of a proteasomal mRNA showed a significant decrease in 25-month-old mice. This suggests that the ability to degrade unfolded protein decreases in the aging pancreas, and that this leads to reduction of pancreatic lipase activity and a decrease in lipid absorption.

Jacaric acid, a linolenic acid isomer with a conjugated triene system, has a strong antitumor effect in vitro and in vivo.

In this study, we compared the cytotoxic effects of natural conjugated linolenic acids (CLnAs) on human adenocarcinoma cells (DLD-1) in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. The antitumor effect of the CLnA with the strongest cytotoxic effect was then examined in mice. The results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with jacaric acid (JA) having the strongest effect. Examination of the mechanism of cell death showed that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. The intracellular levels of incorporated CLnAs were measured to examine the reason for differences in cytotoxic effects. These results showed that JA was taken into cells efficiently. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo in nude mice into which DLD-1 cells were transplanted. These results suggest that JA can be used as a dietary constituent for prevention of cancer.

Fish oil changes the lifespan of Caenorhabditis elegans via lipid peroxidation.

Recently, we administered fish oil containing eicosapentaenoic acid and docosahexaenoic acid (DHA) to senescence-accelerated mice P8 (SAMP8), in order to investigate the effects on lifespan. Surprisingly, the lifespan of SAMP8 that were fed fish oil was shortened significantly, through a mechanism that likely involved lipid peroxidation. In this study, we investigated this phenomenon in further detail. To examine whether this phenomenon occurs only in SAMP8, we investigated the effect of fish oil on the lifespan of another organism species, Caenorhabditis elegans (C. elegans). C. elegans fed fish oil were cultured and the lifespan monitored. As a consequence of the provision of large amounts of fish oil the lifespan of C. elegans was shortened significantly, whereas an appropriate amount of fish oil extended their lifespan significantly. Lipid peroxide levels in C. elegans that were fed fish oil increased significantly in a dose-dependent manner. However, lipid peroxide levels in C. elegans were inhibited by the addition of fish oil and an antioxidant, α-tocopherol, and completely abrogated the changes in the lifespan. To further confirm whether the oxidation of n-3 polyunsaturated fatty acid in fish oil would change the lifespan of C. elegans, the effect of oxidized DHA was examined. Large amounts of oxidized DHA were found to shorten their lifespan significantly. Thus, fish oil changes the lifespan of C. elegans through lipid peroxidation.

Aging decreases antioxidant effects and increases lipid peroxidation in the Apolipoprotein E deficient mouse.

In this study, to study the effect of aging and Apolipoprotein E (ApoE) deficiency on antioxidant ability in mice, we examined whether lipid peroxidation is promoted by aging in ApoE deficient (ApoE(-/-)) mice, which have a shorter lifespan than normal mice. The levels of thiobarbituric acid-reactive substances (TBARS), a biomarker of lipid peroxidation, were measured in plasma and liver in ApoE(-/-) mice aged 12 weeks (young) and 52 weeks (early stage of senescence). TBARS in plasma and liver were significantly increased by aging. Next, we examined the reasons why lipid peroxidation was promoted by aging, based on measurement of protein and mRNA levels for antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in liver in ApoE(-/-) mice aged 12 and 52 weeks. The levels of superoxide dismutase 1 and 2 in liver were significantly decreased by aging. The mRNA level of catalase was also significantly decreased and the mRNA levels of superoxide dismutase 1, superoxide dismutase 2 and glutathione peroxidase 1 all showed a tendency to decrease with age. These results suggest that lipid peroxidation is caused by reduction of antioxidant activity with aging and that this promotes senescence and shortens lifespan in ApoE(-/-) mice.

Phosphatidylcholine hydroperoxide promotes VEGF-induced angiogenesis in endothelial cells and rat aorta ring cultures.

BACKGROUND: Phosphatidylcholine hydroperoxide (PCOOH) is a primary oxidation product of PC, and is markedly accumulated in blood plasma and arterial walls in atherosclerotic animals and humans. The role of PCOOH in the induction of angiogenesis is unknown. METHODS: In this study, we investigated whether PCOOH stimulated angiogenic responses (e.g., vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, and tube formation, and angiogenesis-related gene/protein expression) in human umbilical vein endothelial cells (HUVEC) and in an ex vivo rat aorta model. RESULTS: VEGF induced proliferation, migration, and tube formation of HUVEC, and these angiogenic responses were all enhanced by PCOOH but not by native (nonoxidized) PC. The angiogenic effects of PCOOH are considered to be mediated via generation of reactive oxygen species and activation of both PI3K/AKT and MAPK pathways. The angiogenic activities of PCOOH were also confirmed by the rat aortic ring assay. CONCLUSIONS: These results indicate that PCOOH can elicit several angiogenic responses. GENERAL SIGNIFICANCE: The present study implies an important role of PCOOH in atherosclerosis progression and plaque instability.