Levels of lipid-derived gut microbial metabolites differ among plant matrices in an in vitro model of colon fermentation.

This study explored differences in microbial lipid metabolites among sunflower seeds, soybeans, and walnuts. The matrices were subjected to in vitro digestion and colonic fermentation. Defatted digested materials and fiber/phenolics extracted therefrom were added to sunflower oil (SO) and also fermented. Targeted and untargeted lipidomics were employed to monitor and tentatively identify linoleic acid (LA) metabolites. Walnut fermentation produced the highest free fatty acids (FFAs), LA, and conjugated LAs (CLAs). Defatted digested walnuts added to SO boosted FFAs and CLAs production; the addition of fibre boosted CLAs, whereas the addition of phenolics only increased 9e,11z-CLA and 10e,12z-CLA. Several di-/tri-hydroxy-C18-FAs, reported as microbial LA metabolites for the first time, were annotated. Permutational multivariate analysis of variance indicated significant impacts of food matrix presence and type on lipidomics and C18-FAs. Our findings highlight how the food matrices affect CLA production from dietary lipids, emphasizing the role of food context in microbial lipid metabolism.

Human gut microbiota composition and its predicted functional properties in people with western and healthy dietary patterns.

PURPOSE: Some dietary habits cluster together, and for this reason it is advised to study the impact of entire dietary patterns on human health, rather than that of individual dietary habits. The main objective of this study was to evaluate differences in gut microbiota composition and their predicted functional properties between people with a healthy (HDP) and western (WDP) dietary pattern. METHODS: A cross-sectional, observational study was carried out on 200 participants enrolled 2017-2018 in Poznan, Poland, equally distributed into HDP and WDP groups. Diet was estimated using 3-day food records and information on stool transit times was collected. Fecal microbiota composition was assessed by 16S rRNA gene sequencing and its functional properties were predicted by the PICRUSt2 workflow. RESULTS: The α-diversity did not differ between people with WDP and HDP, but ß-diversity was associated with dietary pattern. People with HDP had higher relative abundances (RA) of Firmicutes and Faecalibacterium and lower RA of Bacteroidota and Escherichia-Shigella than participants with WDP. Only a small proportion of the variance in microbiota composition (1.8%) and its functional properties (2.9%) could be explained by dietary intake (legumes, simple sugars and their sources, like fruit, soft drinks) and stool transit characteristics. CONCLUSION: Gut microbiota composition and predicted metabolic potential is shaped by overall diet quality as well as the frequency of defecation; however, the cumulative effect of these explain only a relatively low proportion of variance.

The effects of sulfated secondary bile acids on intestinal barrier function and immune response in an inflammatory in vitro human intestinal model.

Dysbiosis-related perturbations in bile acid (BA) metabolism were observed in inflammatory bowel disease (IBD) patients, which was characterized by increased levels of sulfated BAs at the expense of secondary BAs. However, the exact effects of sulfated BAs on the etiology of IBD are not investigated yet. Therefore, we aimed to investigate the effects of sulfated deoxycholic acid (DCA), sulfated lithocholic acid (LCA) and their unsulfated forms on intestinal barrier function and immune response. To this end, we first established a novel in vitro human intestinal model to mimic chronic intestinal inflammation as seen during IBD. This model consisted of a co-culture of Caco-2 and HT29-MTX-E12 cells grown on a semi-wet interface with mechanical stimulation to represent the mucus layer. A pro-inflammatory environment was created by combining the co-culture with LPS-activated dendritic cells (DCs) in the basolateral compartment. The presence of activated DCs caused a decrease in transepithelial electrical resistance (TEER), which was slightly restored by LCA and sulfated DCA. The expression of genes related to intestinal epithelial integrity and the mucus layer were slightly, but not significantly increased. These results imply that sulfated BAs have a minor effect on intestinal barrier function in Caco-2 and HT29-MTX-E12 cells. When exposed directly to DCs, our results point towards anti-inflammatory effects of secondary BAs, but to a minor extent for sulfated secondary BAs. Future research should focus on the importance of proper transformation of BAs by bacterial enzymes and the potential involvement of BA dysmetabolism in IBD progression.

Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation.

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.

Bacterial folate biosynthesis and colorectal cancer risk: more than just a gut feeling.

Folate is a B-vitamin with an important role in health and disease. The optimal folate status with regard to human health remains controversial. A low intake of natural folate as well as excessive intake of synthetic folic acid, were previously linked to an increased risk of colorectal cancer or with aberrant molecular pathways related to carcinogenesis in some studies. Importantly, most studies conducted so far, solely focused on dietary intake or circulating levels of folate in relation to cancer risk. Notably, diet or dietary supplements are not the only sources of folate. Several bacteria in the gastrointestinal tract can synthesize B-vitamins, including folate, in quantities that resemble dietary intake. The impact of bacterial folate biosynthesis concerning human health and disease remains unexplored. This review highlights current insights into folate biosynthesis by intestinal bacteria and its implications for processes relevant to cancer development, such as epigenetic DNA modifications and DNA synthesis. Moreover, we will reflect on the emerging question whether food-grade or intestinal bacteria can be considered a potential target to ensure sufficient levels of folate in the gastrointestinal tract and, hence the relevance of bacterial folate biosynthesis for disease prevention or treatment.

Akkermansia muciniphila ameliorates the age-related decline in colonic mucus thickness and attenuates immune activation in accelerated aging Ercc1 -/Δ7 mice.

BACKGROUND: The use of Akkermansia muciniphila as potential therapeutic intervention is receiving increasing attention. Health benefits attributed to this bacterium include an improvement of metabolic disorders and exerting anti-inflammatory effects. The abundance of A. muciniphila is associated with a healthy gut in early mid- and later life. However, the effects of A. muciniphila on a decline in intestinal health during the aging process are not investigated yet. We supplemented accelerated aging Ercc1 -/Δ7 mice with A. muciniphila for 10 weeks and investigated histological, transcriptional and immunological aspects of intestinal health. RESULTS: The thickness of the colonic mucus layer increased about 3-fold after long-term A. muciniphila supplementation and was even significantly thicker compared to mice supplemented with Lactobacillus plantarum WCFS1. Colonic gene expression profiles pointed towards a decreased expression of genes and pathways related to inflammation and immune function, and suggested a decreased presence of B cells in colon. Total B cell frequencies in spleen and mesenteric lymph nodes were not altered after A. muciniphila supplementation. Mature and immature B cell frequencies in bone marrow were increased, whereas B cell precursors were unaffected. These findings implicate that B cell migration rather than production was affected by A. muciniphila supplementation. Gene expression profiles in ileum pointed toward a decrease in metabolic- and immune-related processes and antimicrobial peptide production after A. muciniphila supplementation. Besides, A. muciniphila decreased the frequency of activated CD80+CD273- B cells in Peyer's patches. Additionally, the increased numbers of peritoneal resident macrophages and a decrease in Ly6Cint monocyte frequencies in spleen and mesenteric lymph nodes add evidence for the potentially anti-inflammatory properties of A. muciniphila. CONCLUSIONS: Altogether, we show that supplementation with A. muciniphila prevented the age-related decline in thickness of the colonic mucus layer and attenuated inflammation and immune-related processes at old age. This study implies that A. muciniphila supplementation can contribute to a promotion of healthy aging.

Integrative analysis of gut microbiota composition, host colonic gene expression and intraluminal metabolites in aging C57BL/6J mice.

The aging process is associated with diminished colonic health. In this study, we applied an integrative approach to reveal potential interactions between determinants of colonic health in aging C57BL/6J mice. Analysis of gut microbiota composition revealed an enrichment of various potential pathobionts, including Desulfovibrio spp., and a decline of the health-promoting Akkermansia spp. and Lactobacillus spp. during aging. Intraluminal concentrations of various metabolites varied between ages and we found evidence for an increased gut permeability at higher age. Colonic gene expression analysis suggested that during the early phase of aging (between 6 and 12 months), expression of genes involved in epithelial-to-mesenchymal transition and (re)organization of the extracellular matrix were increased. Differential expression of these genes was strongly correlated with Bifidobacterium spp. During the later phase of aging (between 12 and 28 months), gene expression profiles pointed towards a diminished antimicrobial defense and were correlated with an uncultured Gastranaerophilales spp. This study demonstrates that aging is associated with pronounced changes in gut microbiota composition and colonic gene expression. Furthermore, the strong correlations between specific bacterial genera and host gene expression may imply that orchestrated interactions take place in the vicinity of the colonic wall and potentially mediate colonic health during aging.

Lifelong calorie restriction affects indicators of colonic health in aging C57Bl/6J mice.

Diminished colonic health is associated with various age-related pathologies. Calorie restriction (CR) is an effective strategy to increase healthy lifespan, although underlying mechanisms are not fully elucidated. Here, we report the effects of lifelong CR on indicators of colonic health in aging C57Bl/6J mice. Compared to an ad libitum control and moderate-fat diet, 30% energy reduction was associated with attenuated immune- and inflammation-related gene expression in the colon. Furthermore, expression of genes involved in lipid metabolism was higher upon CR, which may point towards efficient regulation of energy metabolism. The relative abundance of bacteria considered beneficial to colonic health, such as Bifidobacterium and Lactobacillus, increased in the mice exposed to CR for 28 months as compared to the other diet groups. We found lower plasma levels of interleukin-6 and lower levels of various metabolites, among which are bile acids, in the colonic luminal content of CR-exposed mice as compared to the other diet groups. Switching from CR to an ad libitum moderate-fat diet at old age (24 months) revealed remarkable phenotypic plasticity in terms of gene expression, microbiota composition and metabolite levels, although expression of a subset of genes remained CR-associated. This study demonstrated in a comprehensive way that CR affects indicators of colonic health in aging mice. Our findings provide unique leads for further studies that need to address optimal and feasible strategies for prolonged energy deprivation, which may contribute to healthy aging.

Plasticity of lifelong calorie-restricted C57BL/6J mice in adapting to a medium-fat diet intervention at old age.

Calorie restriction (CR) is a dietary regimen that supports healthy aging. In this study, we investigated the systemic and liver-specific responses caused by a diet switch to a medium-fat (MF) diet in 24-month-old lifelong, CR-exposed mice. This study aimed to increase the knowledge base on dietary alterations of gerontological relevance. Nine-week-old C57BL/6J mice were exposed either to a control, CR, or MF diet. At the age of 24 months, a subset of mice of the CR group was transferred to ad libitumMF feeding (CR-MF). The mice were sacrificed at the age of 28 months, and then, biochemical and molecular analyses were performed. Our results showed that, despite the long-term exposure to the CR regimen, mice in the CR-MF group displayed hyperphagia, rapid weight gain, and hepatic steatosis. However, no hepatic fibrosis/injury or alteration in CR-improved survival was observed in the diet switch group. The liver transcriptomic profile of CR-MF mice largely shifted to a profile similar to the MF-fed animals but leaving ~22% of the 1,578 differentially regulated genes between the CR and MF diet groups comparable with the expression of the lifelong CR group. Therefore, although the diet switch was performed at an old age, the CR-MF-exposed mice showed plasticity in coping with the challenge of a MF diet without developing severe liver pathologies.

Association between DNA methylation profiles in leukocytes and serum levels of persistent organic pollutants in Dutch men.

Consumption of polluted fish may lead to high levels of persistent organic pollutants (POPs) in humans, potentially causing adverse health effects. Altered DNA methylation has been suggested as a possible contributor to a variety of adverse health effects. The aim of this study was to evaluate the relationship between serum POP levels (dioxins, polychlorobiphenyls, and perfluoroctane sulphonate) and DNA methylation. We recruited a total of 80 Dutch men who regularly consumed eel from either low- or high-polluted areas, and subsequently had normal or elevated POP levels. Clinical parameters related to e.g. hormone levels and liver enzymes were measured as biomarkers for adverse health effects. The Infinium 450K BeadChip was used to assess DNA methylation in a representative subset of 34 men. We identified multiple genes with differentially methylated regions (DMRs; false discovery rate <0.05) related to POP levels. Several of these genes are involved in carcinogenesis (e.g. BRCA1, MAGEE2, HOXA5), the immune system (e.g. RNF39, HLA-DQB1), retinol homeostasis (DHRS4L2), or in metabolism (CYP1A1). The DMRs in these genes show mean methylation differences up to 7.4% when comparing low- and high-exposed men, with a mean difference up to 14.4% for single positions within a DMR. Clinical parameters were not significantly associated with serum POP levels. This is the first explorative study investigating extensive DNA methylation in relation to serum POP levels among men. We observed that elevated POP levels are associated with aberrant DNA methylation profiles in adult men who consumed high-polluted eel. These preliminary findings warrant further confirmation in other populations.

Intermittent calorie restriction largely counteracts the adverse health effects of a moderate-fat diet in aging C57BL/6J mice.

SCOPE: Calorie restriction (CR) has been shown to extend life- and health-span in model species. For most humans, a life-long CR diet is too arduous to adhere to. The aim of this study was to explore whether weekly intermittent CR can (1) provide long-term beneficial effects and (2) counteract diet-induced obesity in male aging mice. METHODS AND RESULTS: In this study, we have exposed C57Bl/6J mice for 24 months to an intermittent (INT) diet, alternating weekly between CR of a control diet and ad libitum moderate-fat (MF) feeding. This weekly intermittent CR significantly counteracted the adverse effects of the MF diet on mortality, body weight, and liver health markers in 24-month-old male mice. Hepatic gene expression profiles of INT-exposed animals appeared much more comparable to CR- than to MF-exposed mice. At 12 months of age, a subgroup of MF-exposed mice was transferred to the INT diet. Gene expression profiles in the liver of the 24-month-old diet switch mice were highly similar to the INT-exposed mice. However, a small subset of genes was consistently changed by the MF diet during the first phase of life. CONCLUSION: Weekly intermittent CR largely, but not completely, reversed adverse effects caused by a MF diet.

Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.

Ubiquitous persistent organic pollutants (POPs) can accumulate in humans where they might influence differentiation of adipocytes. The aim of this study was to investigate whether DNA methylation is one of the underlying mechanisms by which POPs affect adipocyte differentiation, and to what extent DNA methylation can be related to gene transcription. Adipocyte differentiation was induced in two human cell models with continuous exposure to different POPs throughout differentiation. From the seven tested POPs, perfluorooctanesulfonic acid (PFOS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased lipid accumulation, while tributyltin (TBT) increased lipid accumulation. In human mesenchymal stem cells (hMSCs), TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, possibly related to the phenotypic outcome, but without concomitant gene expression changes. Differential methylation was predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrates that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in human differentiating adipocytes.

Maternal exposure to a Western-style diet causes differences in intestinal microbiota composition and gene expression of suckling mouse pups.

SCOPE: The long-lasting consequences of nutritional programming during the early phase of life have become increasingly evident. The effects of maternal nutrition on the developing intestine are still underexplored. METHODS AND RESULTS: In this study, we observed (1) altered microbiota composition of the colonic luminal content, and (2) differential gene expression in the intestinal wall in 2-week-old mouse pups born from dams exposed to a Western-style (WS) diet during the perinatal period. A sexually dimorphic effect was found for the differentially expressed genes in the offspring of WS diet-exposed dams but no differences between male and female pups were found for the microbiota composition. Integrative analysis of the microbiota and gene expression data revealed that the maternal WS diet independently affected gene expression and microbiota composition. However, the abundance of bacterial families not affected by the WS diet (Bacteroidaceae, Porphyromonadaceae, and Lachnospiraceae) correlated with the expression of genes playing a key role in intestinal development and functioning (e.g. Pitx2 and Ace2). CONCLUSION: Our data reveal that maternal consumption of a WS diet during the perinatal period alters both gene expression and microbiota composition in the intestinal tract of 2-week-old offspring.

Accumulation of persistent organic pollutants in consumers of eel from polluted rivers compared to marketable eel.

Globally, many river sediments are seriously contaminated with persistent organic pollutants (POPs) known to accumulate in aquatic food. In the Netherlands, toxicological risks of human exposure to dioxins and dioxin-like compounds led to a ban on eel fishing in the Rhine-Meuse delta. The aim of this study is to investigate differences in serum POP levels in consumers of eel from high-polluted areas and consumers of eel from low-polluted areas or aquaculture. In total 80 Dutch men were included, aged 40-70 years, with a habitual eel consumption of at least one portion (150 g) per month. Total levels of dioxins and dioxin-like compounds were measured in serum of all participants with the DR CALUX bioassay, validated with GC-MS. For a subgroup of 38 participants extensive POP measurements were performed. We revealed that consumption of eel from polluted rivers resulted in 2.5 and up to 10 times increased levels of dioxins and polychlorinated biphenyls (PCBs) respectively compared to controls. The highest PCB levels were detected for PCB 153, with a median level of 896 ng/g lipid and a maximum level of 5000 ng/g lipid in the high-exposed group. Furthermore, hydroxylated PCB metabolites (OH-PCBs: sum of 4-OH-CB107, 4-OH-CB146, 4'-OH-CB172, and 4-OH-CB187) were 8 times higher in men who consumed eel from polluted areas, and detected at levels (median 4.5 ng/g ww) reported to cause adverse health effects. Also, the majority of the perfluoroalkyl substances (PFASs) were significantly higher in consumers of eel from pullulated areas. In conclusion, this study is the first to reveal that (past) consumption of eel from polluted rivers resulted in high body burdens of dioxins, PCBs, OH-PCBs and PFASs. We confirmed the predictions made in a former risk assessment, and the high levels of dioxins and dioxin-like compounds as well as the OH-PCBs are of health concern.

Fibroblast growth factor 21 reflects liver fat accumulation and dysregulation of signalling pathways in the liver of C57BL/6J mice.

Fibroblast growth factor 21 (Fgf21) has emerged as a potential plasma marker to diagnose non-alcoholic fatty liver disease (NAFLD). To study the molecular processes underlying the association of plasma Fgf21 with NAFLD, we explored the liver transcriptome data of a mild NAFLD model of aging C57BL/6J mice at 12, 24, and 28 months of age. The plasma Fgf21 level significantly correlated with intrahepatic triglyceride content. At the molecular level, elevated plasma Fgf21 levels were associated with dysregulated metabolic and cancer-related pathways. The up-regulated Fgf21 levels in NAFLD were implied to be a protective response against the NAFLD-induced adverse effects, e.g. lipotoxicity, oxidative stress and endoplasmic reticulum stress. An in vivo PPARα challenge demonstrated the dysregulation of PPARα signalling in the presence of NAFLD, which resulted in a stochastically increasing hepatic expression of Fgf21. Notably, elevated plasma Fgf21 was associated with declining expression of Klb, Fgf21's crucial co-receptor, which suggests a resistance to Fgf21. Therefore, although liver fat accumulation is a benign stage of NAFLD, the elevated plasma Fgf21 likely indicated vulnerability to metabolic stressors that may contribute towards progression to end-stage NAFLD. In conclusion, plasma levels of Fgf21 reflect liver fat accumulation and dysregulation of metabolic pathways in the liver.

Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α-glucosidase inhibitor or a Nrf2-inducer.

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin - the latter with and without rapamycin, and two drugs previously examined: 17-α-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-α-estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male-specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α-glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.

Comprehensive DNA Methylation and Gene Expression Profiling in Differentiating Human Adipocytes.

Insight into the processes controlling adipogenesis is important in the battle against the obesity epidemic and its related disorders. The transcriptional regulatory cascade involved in adipocyte differentiation has been extensively studied, however, the mechanisms driving the transcription activation are still poorly understood. In this study, we explored the involvement of DNA methylation in transcriptional regulation during adipocyte differentiation of primary human mesenchymal stem cells (hMSCs). Genome-wide changes in DNA methylation were measured using the Illumina 450K BeadChip. In addition, expression of 84 adipogenic genes was determined, of which 43 genes showed significant expression changes during the differentiation process. Among these 43 differentially expressed genes, differentially methylated regions (DMRs) were detected in only three genes. By comparing genome-wide DNA methylation profiles in undifferentiated and differentiated adipocytes 793 significant DMRs were detected. Pathway analysis revealed the adipogenesis pathway as the most statistically significant, although only a small number of genes were differentially methylated. Genome-wide DNA methylation changes for single probes were most often located in intergenic regions, and underrepresented close to the transcription start site. In conclusion, DNA methylation remained relatively stable during adipocyte differentiation, implying that changes in DNA methylation are not the underlying mechanism regulating gene expression during adipocyte differentiation. J. Cell. Biochem. 117: 2707-2718, 2016. © 2016 Wiley Periodicals, Inc.

The effects of long-term daily folic acid and vitamin B12 supplementation on genome-wide DNA methylation in elderly subjects.

BACKGROUND: Folate and its synthetic form folic acid function as donor of one-carbon units and have been, together with other B-vitamins, implicated in programming of epigenetic processes such as DNA methylation during early development. To what extent regulation of DNA methylation can be altered via B-vitamins later in life, and how this relates to health and disease, is not exactly known. The aim of this study was to identify effects of long-term supplementation with folic acid and vitamin B12 on genome-wide DNA methylation in elderly subjects. This project was part of a randomized, placebo-controlled trial on effects of supplemental intake of folic acid and vitamin B12 on bone fracture incidence (B-vitamins for the PRevention Of Osteoporotic Fractures (B-PROOF) study). Participants with mildly elevated homocysteine levels, aged 65-75 years, were randomly assigned to take 400 µg folic acid and 500 µg vitamin B12 per day or a placebo during an intervention period of 2 years. DNA was isolated from buffy coats, collected before and after intervention, and genome-wide DNA methylation was determined in 87 participants (n = 44 folic acid/vitamin B12, n = 43 placebo) using the Infinium HumanMethylation450 BeadChip. RESULTS: After intervention with folic acid and vitamin B12, 162 (versus 14 in the placebo group) of the 431,312 positions were differentially methylated as compared to baseline. Comparisons of the DNA methylation changes in the participants receiving folic acid and vitamin B12 versus placebo revealed one single differentially methylated position (cg19380919) with a borderline statistical significance. However, based on the analyses of differentially methylated regions (DMRs) consisting of multiple positions, we identified 6 regions that differed statistically significantly between the intervention and placebo group. Pronounced changes were found for regions in the DIRAS3, ARMC8, and NODAL genes, implicated in carcinogenesis and early embryonic development. Furthermore, serum levels of folate and vitamin B12 or plasma homocysteine were related to DNA methylation of 173, 425, and 11 regions, respectively. Interestingly, for several members of the developmental HOX genes, DNA methylation was related to serum levels of folate. CONCLUSIONS: Long-term supplementation with folic acid and vitamin B12 in elderly subjects resulted in effects on DNA methylation of several genes, among which genes implicated in developmental processes.

Behavioural changes are a major contributing factor in the reduction of sarcopenia in caloric-restricted ageing mice.

BACKGROUND: In rodent models, caloric restriction (CR) with maintenance of adequate micronutrient supply has been reported to increase lifespan and to reduce age-induced muscle loss (sarcopenia) during ageing. In the present study, we further investigated effects of CR on the onset and severity of sarcopenia in ageing male C57BL/6 J mice. The aim of this study was to investigate whether CR induces changes in behaviour of the animals that could contribute to the pronounced health-promoting effects of CR in rodents. In addition, we aimed to investigate in more detail the effects of CR on the onset and severity of sarcopenia. METHODS: The mice received either an ad libitum diet (control) or a diet matching 70 E% of the control diet (C). Daily activity, body composition (dual energy X-ray absorptiometry), grip strength, insulin sensitivity, and general agility and balance were determined at different ages. Mice were killed at 4, 12, 24, and 28 months. Skeletal muscles of the hind limb were dissected, and the muscle extensor digitorum longus muscle was used for force-frequency measurements. The musculus tibialis was used for real-time quantitative PCR analysis. RESULTS: From the age of 12 months, CR animals were nearly half the weight of the control animals, which was mainly related to a lower fat mass. In the control group, the hind limb muscles showed a decline in mass at 24 or 28 months of age, which was not present in the CR group. Moreover, insulin sensitivity (oral glucose tolerance test) was higher in this group and the in vivo and ex vivo grip strength did not differ between the two groups. In the hours before food was provided, CR animals were far more active than control animals, while total daily activity was not increased. Moreover, agility test indicated that CR animals were better climbers and showed more climbing behaviours. CONCLUSIONS: Our study confirms earlier findings that in CR animals less sarcopenia is present. The mice on the CR diet, however, showed specific behavioural changes characterized by higher bursts of activity within a short time frame before consumption of a 70 E% daily meal. We hypothesize that the positive effects of CR on muscle maintenance in rodents are not merely a direct consequence of a lower energy intake but also related to a more active behaviour in a specific time frame. The burst of activity just before immediate start of eating, might lead to a highly effective use of the restricted protein sources available.