A polyphenol-rich green Mediterranean diet enhances epigenetic regulatory potential: the DIRECT PLUS randomized controlled trial.

BACKGROUND: The capacity of a polyphenol-enriched diet to modulate the epigenome in vivo is partly unknown. Given the beneficial metabolic effects of a Mediterranean (MED) diet enriched in polyphenols and reduced in red/processed meat (green-MED), as previously been proven by the 18-month DIRECT PLUS randomized controlled trial, we analyzed the effects of the green-MED diet on methylome and transcriptome levels to highlight molecular mechanisms underlying the observed metabolic improvements. METHODS: Our study included 260 participants (baseline BMI = 31.2 kg/m2, age = 5 years) of the DIRECT PLUS trial, initially randomized to one of the intervention arms: A. healthy dietary guidelines (HDG), B. MED (440 mg polyphenols additionally provided by walnuts), C. green-MED (1240 mg polyphenols additionally provided by walnuts, green tea, and Mankai: green duckweed shake). Blood methylome and transcriptome of all study subjects were analyzed at baseline and after completing the 18-month intervention using Illumina EPIC and RNA sequencing technologies. RESULTS: A total of 1573 differentially methylated regions (DMRs; false discovery rate (FDR) < 5 %) were found in the green-MED compared to the MED (177) and HDG (377) diet participants. This corresponded to 1753 differentially expressed genes (DEGs; FDR < 5 %) in the green-MED intervention compared to MED (7) and HDG (738). Consistently, the highest number (6 %) of epigenetic modulating genes was transcriptionally changed in subjects participating in the green-MED intervention. Weighted cluster network analysis relating transcriptional and phenotype changes among participants subjected to the green-MED intervention identified candidate genes associated with serum-folic acid change (all P < 1 × 10-3) and highlighted one module including the KIR3DS1 locus, being negatively associated with the polyphenol changes (e.g. P < 1 × 10-4), but positively associated with the MRI-assessed superficial subcutaneous adipose area-, weight- and waist circumference- 18-month change (all P < 0.05). Among others, this module included the DMR gene Cystathionine Beta-Synthase, playing a major role in homocysteine reduction. CONCLUSIONS: The green-MED high polyphenol diet, rich in green tea and Mankai, renders a high capacity to regulate an individual's epigenome. Our findings suggest epigenetic key drivers such as folate and green diet marker to mediate this capacity and indicate a direct effect of dietary polyphenols on the one­carbon metabolism.

The effect of high-polyphenol Mediterranean diet on visceral adiposity: the DIRECT PLUS randomized controlled trial.

BACKGROUND: Mediterranean (MED) diet is a rich source of polyphenols, which benefit adiposity by several mechanisms. We explored the effect of the green-MED diet, twice fortified in dietary polyphenols and lower in red/processed meat, on visceral adipose tissue (VAT). METHODS: In the 18-month Dietary Intervention Randomized Controlled Trial PoLyphenols UnproceSsed (DIRECT-PLUS) weight-loss trial, 294 participants were randomized to (A) healthy dietary guidelines (HDG), (B) MED, or (C) green-MED diets, all combined with physical activity. Both isocaloric MED groups consumed 28 g/day of walnuts (+ 440 mg/day polyphenols). The green-MED group further consumed green tea (3-4 cups/day) and Wolffia globosa (duckweed strain) plant green shake (100 g frozen cubes/day) (+ 800mg/day polyphenols) and reduced red meat intake. We used magnetic resonance imaging (MRI) to quantify the abdominal adipose tissues. RESULTS: Participants (age = 51 years; 88% men; body mass index = 31.2 kg/m2; 29% VAT) had an 89.8% retention rate and 79.3% completed eligible MRIs. While both MED diets reached similar moderate weight (MED: - 2.7%, green-MED: - 3.9%) and waist circumference (MED: - 4.7%, green-MED: - 5.7%) loss, the green-MED dieters doubled the VAT loss (HDG: - 4.2%, MED: - 6.0%, green-MED: - 14.1%; p < 0.05, independent of age, sex, waist circumference, or weight loss). Higher dietary consumption of green tea, walnuts, and Wolffia globosa; lower red meat intake; higher total plasma polyphenols (mainly hippuric acid), and elevated urine urolithin A polyphenol were significantly related to greater VAT loss (p < 0.05, multivariate models). CONCLUSIONS: A green-MED diet, enriched with plant-based polyphenols and lower in red/processed meat, may be a potent intervention to promote visceral adiposity regression. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03020186.

The effect of a high-polyphenol Mediterranean diet (Green-MED) combined with physical activity on age-related brain atrophy: the Dietary Intervention Randomized Controlled Trial Polyphenols Unprocessed Study (DIRECT PLUS).

BACKGROUND: The effect of diet on age-related brain atrophy is largely unproven. OBJECTIVES: We aimed to explore the effect of a Mediterranean diet (MED) higher in polyphenols and lower in red/processed meat (Green-MED diet) on age-related brain atrophy. METHODS: This 18-mo clinical trial longitudinally measured brain structure volumes by MRI using hippocampal occupancy score (HOC) and lateral ventricle volume (LVV) expansion score as neurodegeneration markers. Abdominally obese/dyslipidemic participants were randomly assigned to follow 1) healthy dietary guidelines (HDG), 2) MED, or 3) Green-MED diet. All subjects received free gym memberships and physical activity guidance. Both MED groups consumed 28 g walnuts/d (+440 mg/d polyphenols). The Green-MED group consumed green tea (3-4 cups/d) and Mankai (Wolffia-globosa strain, 100 g frozen cubes/d) green shake (+800 mg/d polyphenols). RESULTS: Among 284 participants (88% men; mean age: 51 y; BMI: 31.2 kg/m2; APOE-ε4 genotype = 15.7%), 224 (79%) completed the trial with eligible whole-brain MRIs. The pallidum (-4.2%), third ventricle (+3.9%), and LVV (+2.2%) disclosed the largest volume changes. Compared with younger participants, atrophy was accelerated among those ≥50 y old (HOC change: -1.0% ± 1.4% compared with -0.06% ± 1.1%; 95% CI: 0.6%, 1.3%; P < 0.001; LVV change: 3.2% ± 4.5% compared with 1.3% ± 4.1%; 95% CI: -3.1%, -0.8%; P = 0.001). In subjects ≥ 50 y old, HOC decline and LVV expansion were attenuated in both MED groups, with the best outcomes among Green-MED diet participants, as compared with HDG (HOC: -0.8% ± 1.6% compared with -1.3% ± 1.4%; 95% CI: -1.5%, -0.02%; P = 0.042; LVV: 2.3% ± 4.7% compared with 4.3% ± 4.5%; 95% CI: 0.3%, 5.2%; P = 0.021). Similar patterns were observed among younger subjects. Improved insulin sensitivity over the trial was the parameter most strongly associated with brain atrophy attenuation (P < 0.05). Greater Mankai, green tea, and walnut intake and less red and processed meat were significantly and independently associated with reduced HOC decline (P < 0.05). Elevated urinary concentrations of the polyphenols urolithin-A (r = 0.24; P = 0.013) and tyrosol (r = 0.26; P = 0.007) were significantly associated with lower HOC decline. CONCLUSIONS: A Green-MED (high-polyphenol) diet, rich in Mankai, green tea, and walnuts and low in red/processed meat, is potentially neuroprotective for age-related brain atrophy.This trial was registered at clinicaltrials.gov as NCT03020186.

Effect of green-Mediterranean diet on intrahepatic fat: the DIRECT PLUS randomised controlled trial.

OBJECTIVE: To examine the effectiveness of green-Mediterranean (MED) diet, further restricted in red/processed meat, and enriched with green plants and polyphenols on non-alcoholic fatty liver disease (NAFLD), reflected by intrahepatic fat (IHF) loss. DESIGN: For the DIRECT-PLUS 18-month randomized clinical trial, we assigned 294 participants with abdominal obesity/dyslipidaemia into healthy dietary guidelines (HDG), MED and green-MED weight-loss diet groups, all accompanied by physical activity. Both isocaloric MED groups consumed 28 g/day walnuts (+440 mg/day polyphenols provided). The green-MED group further consumed green tea (3-4 cups/day) and Mankai (a Wolffia globosa aquatic plant strain; 100 g/day frozen cubes) green shake (+1240 mg/day total polyphenols provided). IHF% 18-month changes were quantified continuously by proton magnetic resonance spectroscopy (MRS). RESULTS: Participants (age=51 years; 88% men; body mass index=31.3 kg/m2; median IHF%=6.6%; mean=10.2%; 62% with NAFLD) had 89.8% 18-month retention-rate, and 78% had eligible follow-up MRS. Overall, NAFLD prevalence declined to: 54.8% (HDG), 47.9% (MED) and 31.5% (green-MED), p=0.012 between groups. Despite similar moderate weight-loss in both MED groups, green-MED group achieved almost double IHF% loss (-38.9% proportionally), as compared with MED (-19.6% proportionally; p=0.035 weight loss adjusted) and HDG (-12.2% proportionally; p<0.001). After 18 months, both MED groups had significantly higher total plasma polyphenol levels versus HDG, with higher detection of Naringenin and 2-5-dihydroxybenzoic-acid in green-MED. Greater IHF% loss was independently associated with increased Mankai and walnuts intake, decreased red/processed meat consumption, improved serum folate and adipokines/lipids biomarkers, changes in microbiome composition (beta-diversity) and specific bacteria (p<0.05 for all). CONCLUSION: The new suggested strategy of green-Mediterranean diet, amplified with green plant-based proteins/polyphenols as Mankai, green tea, and walnuts, and restricted in red/processed meat can double IHF loss than other healthy nutritional strategies and reduce NAFLD in half. TRIAL REGISTRATION NUMBER: NCT03020186.

Genetically programmed changes in transcription of the novel progranulin regulator.

Progranulin is a glycoprotein marking chronic inflammation in obesity and type 2 diabetes. Previous studies suggested PSRC1 (proline and serine rich coiled-coil 1) to be a target of genetic variants associated with serum progranulin levels. We aimed to identify potentially functional variants and characterize their role in regulation of PSRC1. Phylogenetic module complexity analysis (PMCA) prioritized four polymorphisms (rs12740374, rs629301, rs660240, rs7528419) altering transcription factor binding sites with an overall score for potential regulatory function of Sall > 7.0. The effects of these variants on transcriptional activity and binding of transcription factors were tested by luciferase reporter and electrophoretic mobility shift assays (EMSA). In parallel, blood DNA promoter methylation of two regions was tested in subjects with a very high (N = 100) or a very low (N = 100) serum progranulin. Luciferase assays revealed lower activities in vectors carrying the rs629301-A compared with the C allele. Moreover, EMSA indicated a different binding pattern for the two rs629301 alleles, with an additional prominent band for the A allele, which was finally confirmed with the supershift for the Yin Yang 1 transcription factor (YY1). Subjects with high progranulin levels manifested a significantly higher mean DNA methylation (P < 1 × 10-7) in one promoter region, which was in line with a significantly lower PSRC1 mRNA expression levels in blood (P = 1 × 10-3). Consistently, rs629301-A allele was associated with lower PSRC1 mRNA expression (P < 1 × 10-7). Our data suggest that the progranulin-associated variant rs629301 modifies the transcription of PSRC1 through alteration of YY1 binding capacity. DNA methylation studies further support the role of PSRC1 in regulation of progranulin serum levels. KEY MESSAGES: PSRC1 (proline and serine rich coiled-coil 1) SNPs are associated with serum progranulin levels. rs629301 regulates PSRC1 expression by affecting Yin Yang 1 transcription factor (YY1) binding. PSRC1 is also epigenetically regulated in subjects with high progranulin levels.

The Effect of Wolffia globosa Mankai, a Green Aquatic Plant, on Postprandial Glycemic Response: A Randomized Crossover Controlled Trial.

OBJECTIVE: To compare the postprandial and overnight glycemic response using a novel green aquatic plant thought to provide a dietary source for high-quality protein, with an iso-carbohydrate/protein/caloric dairy shake. RESEARCH DESIGN AND METHODS: This is a randomized controlled crossover trial among 20 abdominally obese participants (age 51.4 years; fasting plasma glucose 110.9 mg/dL), who were allocated to replace dinner with either, first, a green shake containing Wolffia globosa duckweed (Mankai: specific-strain) or an iso-carbohydrate/protein/calorie yogurt shake. A 2-week flash glucose-monitoring system was used to assess postmeal glucose dynamics (6 net administration days; 97 observation days in total). We further obtained from each participant dietary/daily activity/satiety scale/sleep logs. Participants were recruited from the green-Mediterranean diet arm of the 18-month Dietary Intervention Randomized Controlled Trial-Polyphenols Unprocessed (DIRECT-PLUS) study. RESULTS: Wolffia globosa Mankai elicited a lower postprandial glucose peak compared with yogurt (∆peak = 13.4 ± 9.2 vs. 19.3 ± 15.1 mg/dL; P = 0.044), which occurred later (77.5 ± 29.2 vs. 59.2 ± 28.4 min; P = 0.037) and returned faster to baseline glucose levels (135.8 ± 53.1 vs. 197.5 ± 70.2 min; P = 0.012). The mean post-net incremental area under the curve (netAUC) was lower with Wolffia globosa up to 60 and 180 min (netAUC 60 min: 185.1 ± 340.1 vs. 441.4 ± 336.5 mg/dL/min, P = 0.005; netAUC 180 min: 707.9 ± 1,428.5 vs. 1,576.6 ± 1,810.1 mg/dL/min, P = 0.037). A Wolffia globosa-based shake replacing dinner resulted in lower next-morning fasting glucose levels (83.2 ± 0.8 vs. 86.6 ± 13 mg/dL; P = 0.041). Overall, postprandial glucose levels from the shake administration until the next morning were lower in the Wolffia globosa Mankai green shake compared with the yogurt shake (P < 0.001). Overnight sleep duration was similar (378.2 ± 22.4 vs. 375.9 ± 28.4 min; P = 0.72), and satiety rank was slightly higher for the Wolffia globosa shake compared with the yogurt shake (7.5 vs. 6.5; P = 0.035). CONCLUSIONS: Wolffia globosa Mankai duckweed may serve as an emerging alternative plant protein source with potential beneficial postprandial glycemic effects.

A Green-Mediterranean Diet, Supplemented with Mankai Duckweed, Preserves Iron-Homeostasis in Humans and Is Efficient in Reversal of Anemia in Rats.

BACKGROUND: Decreased dietary meat may deplete iron stores, as plant-derived iron bioavailability is typically limited. OBJECTIVES: We explored the effect of a low-meat Mediterranean (green-MED) diet, supplemented with Wolffia globosa duckweed (Mankai: rich in protein and iron) as a food source for humans, on iron status. We further examined the iron bioavailability of Mankai in rats. METHODS: Two hundred and ninety-four abdominally obese/dyslipidemic [mean age = 51.1 y; body mass index (kg/m2) = 31.3; 88% men] nonanemic participants were randomly assigned to physical activity (PA), PA + MED diet, or PA + green-MED diet. Both isocaloric MED groups consumed 28 g walnuts/d and the low-meat green-MED group further consumed green tea (800 mL/d) and Mankai (100 g green shake/d). In a complementary animal experiment, after 44 d of an iron deficiency anemia-inducing diet, 50 female rats (age = 3 wk; Sprague Dawley strain) were randomly assigned into: iron-deficient diet (vehicle), or vehicle + iso-iron: ferrous gluconate (FG) 14, Mankai 50, and Mankai 80 versions (1.7 mg · kg-1 · d-1 elemental iron), or FG9.5 and Mankai 50-C version (1.15 mg · kg-1 · d-1 elemental iron). The specific primary aim for both studies was changes in iron homeostasis parameters. RESULTS: After 6 mo of intervention, iron status trajectory did not differ between the PA and PA + MED groups. Hemoglobin modestly increased in the PA + green-MED group (0.23 g/dL) compared with PA (-0.1 g/dL; P < 0.001) and PA + MED (-0.1 g/dL; P < 0.001). Serum iron and serum transferrin saturation increased in the PA + green-MED group compared with the PA group (8.21 µg/dL compared with -5.23 µg/dL and 2.39% compared with -1.15%, respectively; P < 0.05 for both comparisons), as did folic acid (P = 0.011). In rats, hemoglobin decreased from 15.7 to 9.4 mg/dL after 44 d of diet-induced anemia. After depletion treatment, the vehicle-treated group had a further decrease of 1.3 mg/dL, whereas hemoglobin concentrations in both FG and Mankai iso-iron treatments similarly rebounded (FG14: +10.8 mg/dL, Mankai 50: +6.4 mg/dL, Mankai 80: +7.3 mg/dL; FG9.5: +5.1 mg/dL, Mankai 50-C: +7.1 mg/dL; P < 0.05 for all vs. the vehicle group). CONCLUSIONS: In humans, a green-MED low-meat diet does not impair iron homeostasis. In rats, iron derived from Mankai (a green-plant protein source) is bioavailable and efficient in reversal of anemia. This trial was registered at clinicaltrials.gov as NCT03020186.

Genomic insights into the origin of farming in the ancient Near East.

We report genome-wide ancient DNA from 44 ancient Near Easterners ranging in time between ~12,000 and 1,400 bc, from Natufian hunter-gatherers to Bronze Age farmers. We show that the earliest populations of the Near East derived around half their ancestry from a 'Basal Eurasian' lineage that had little if any Neanderthal admixture and that separated from other non-African lineages before their separation from each other. The first farmers of the southern Levant (Israel and Jordan) and Zagros Mountains (Iran) were strongly genetically differentiated, and each descended from local hunter-gatherers. By the time of the Bronze Age, these two populations and Anatolian-related farmers had mixed with each other and with the hunter-gatherers of Europe to greatly reduce genetic differentiation. The impact of the Near Eastern farmers extended beyond the Near East: farmers related to those of Anatolia spread westward into Europe; farmers related to those of the Levant spread southward into East Africa; farmers related to those of Iran spread northward into the Eurasian steppe; and people related to both the early farmers of Iran and to the pastoralists of the Eurasian steppe spread eastward into South Asia.

Exenatide-induced reduction in energy intake is associated with increase in hypothalamic connectivity.

OBJECTIVE: Glucagon-like peptide-1 receptor agonists such as exenatide are known to influence neural activity in the hypothalamus of animals and to reduce energy intake. In humans, however, significant weight loss has been observed in only a subgroup of patients. Why only some individuals respond with weight loss and others do not remains unclear. In this functional magnetic resonance imaging (fMRI) study, we investigated differences in hypothalamic connectivity between "responders" (reduction in energy intake after exenatide infusion) and "nonresponders." RESEARCH DESIGN AND METHODS: We performed a randomized, double-blinded, placebo-controlled, cross-over fMRI study with intravenous administration of exenatide in obese male volunteers. During brain scanning with continuous exenatide or placebo administration, participants rated food and nonfood images. After each scanning session, energy intake was measured using an ad libitum buffet. Functional hypothalamic connectivity was assessed by eigenvector centrality mapping, a measure of connectedness throughout the brain. RESULTS: Responders showed significantly higher connectedness of the hypothalamus, which was specific for the food pictures condition, in the exenatide condition compared with placebo. Nonresponders did not show any significant exenatide-induced changes in hypothalamic connectedness. CONCLUSIONS: Our results demonstrate a central hypothalamic effect of peripherally administered exenatide that occurred only in the group that showed an exenatide-dependent anorexigenic effect. These findings indicate that the hypothalamic response seems to be the crucial factor for the effect of exenatide on energy intake.

Sequence variants at CYP1A1-CYP1A2 and AHR associate with coffee consumption.

Coffee is the most commonly used stimulant and caffeine is its main psychoactive ingredient. The heritability of coffee consumption has been estimated at around 50%. We performed a meta-analysis of four genome-wide association studies of coffee consumption among coffee drinkers from Iceland (n = 2680), The Netherlands (n = 2791), the Sorbs Slavonic population isolate in Germany (n = 771) and the USA (n = 369) using both directly genotyped and imputed single nucleotide polymorphisms (SNPs) (2.5 million SNPs). SNPs at the two most significant loci were also genotyped in a sample set from Iceland (n = 2430) and a Danish sample set consisting of pregnant women (n = 1620). Combining all data, two sequence variants significantly associated with increased coffee consumption: rs2472297-T located between CYP1A1 and CYP1A2 at 15q24 (P = 5.4 · 10(-14)) and rs6968865-T near aryl hydrocarbon receptor (AHR) at 7p21 (P = 2.3 · 10(-11)). An effect of ∼0.2 cups a day per allele was observed for both SNPs. CYP1A2 is the main caffeine metabolizing enzyme and is also involved in drug metabolism. AHR detects xenobiotics, such as polycyclic aryl hydrocarbons found in roasted coffee, and induces transcription of CYP1A1 and CYP1A2. The association of these SNPs with coffee consumption was present in both smokers and non-smokers.

Serum vaspin concentrations are decreased after exercise-induced oxidative stress.

Elevated visceral adipose tissue-derived serpin (vaspin) serum concentrations are associated with impaired insulin sensitivity, but increase unexpectedly after long-term physical training. We therefore investigated the effect of an acute exercise bout and the effects of vitamin supplementation on chronic exercise effect and on serum vaspin concentrations. We measured serum vaspin and thiobarbituric acid-reactive substances (TBARS) concentrations in 80 individuals before and after a 1-hour acute exercise bout and independently in 40 healthy young men who were randomly assigned to either antioxidant (vitamin C (1,000 mg/day) and vitamin E (400 IU/day)) or to no supplementation after a standardized 4-week physical training program as a post hoc analysis. Serum vaspin concentrations significantly decreased after acute physical exercise as well as after 4 weeks of training in individuals without antioxidants. Changes in vaspin serum concentration correlate with increased TBARS serum concentrations both in response to a 1-hour exercise bout (r = -0.42, p < 0.01) and to the 4-week training (r = -0.31, p < 0.05). Interestingly, supplementation with antioxidants rather increased circulating vaspin levels in response to 4 weeks of exercise. In conclusion, vaspin serum concentrations are decreased by exercise-induced oxidative stress, but not by exercise-associated improvement in insulin sensitivity.

Antioxidants prevent health-promoting effects of physical exercise in humans.

Exercise promotes longevity and ameliorates type 2 diabetes mellitus and insulin resistance. However, exercise also increases mitochondrial formation of presumably harmful reactive oxygen species (ROS). Antioxidants are widely used as supplements but whether they affect the health-promoting effects of exercise is unknown. We evaluated the effects of a combination of vitamin C (1000 mg/day) and vitamin E (400 IU/day) on insulin sensitivity as measured by glucose infusion rates (GIR) during a hyperinsulinemic, euglycemic clamp in previously untrained (n = 19) and pretrained (n = 20) healthy young men. Before and after a 4 week intervention of physical exercise, GIR was determined, and muscle biopsies for gene expression analyses as well as plasma samples were obtained to compare changes over baseline and potential influences of vitamins on exercise effects. Exercise increased parameters of insulin sensitivity (GIR and plasma adiponectin) only in the absence of antioxidants in both previously untrained (P < 0.001) and pretrained (P < 0.001) individuals. This was paralleled by increased expression of ROS-sensitive transcriptional regulators of insulin sensitivity and ROS defense capacity, peroxisome-proliferator-activated receptor gamma (PPARgamma), and PPARgamma coactivators PGC1alpha and PGC1beta only in the absence of antioxidants (P < 0.001 for all). Molecular mediators of endogenous ROS defense (superoxide dismutases 1 and 2; glutathione peroxidase) were also induced by exercise, and this effect too was blocked by antioxidant supplementation. Consistent with the concept of mitohormesis, exercise-induced oxidative stress ameliorates insulin resistance and causes an adaptive response promoting endogenous antioxidant defense capacity. Supplementation with antioxidants may preclude these health-promoting effects of exercise in humans.