Suppression of the gut microbiome ameliorates age-related arterial dysfunction and oxidative stress in mice.

KEY POINTS: Age-related arterial dysfunction, characterized by oxidative stress- and inflammation-mediated endothelial dysfunction and arterial stiffening, is the primary risk factor for cardiovascular diseases. To investigate whether age-related changes in the gut microbiome may mediate arterial dysfunction, we suppressed gut microbiota in young and old mice with a cocktail of broad-spectrum, poorly-absorbed antibiotics in drinking water for 3-4 weeks. In old mice, antibiotic treatment reversed endothelial dysfunction and arterial stiffening and attenuated vascular oxidative stress and inflammation. To provide insight into age-related changes in gut microbiota that may underlie these observations, we show that ageing altered the abundance of microbial taxa associated with gut dysbiosis and increased plasma levels of the adverse gut-derived metabolite trimethylamine N-oxide. The results of the present study provide the first proof-of-concept evidence that the gut microbiome is an important mediator of age-related arterial dysfunction and therefore may be a promising therapeutic target for preserving arterial function with ageing, thereby reducing the risk of cardiovascular diseases. ABSTRACT: Oxidative stress-mediated arterial dysfunction (e.g. endothelial dysfunction and large elastic artery stiffening) is the primary mechanism driving age-related cardiovascular diseases. Accumulating evidence suggests the gut microbiome modulates host physiology because dysregulation ('gut dysbiosis') has systemic consequences, including promotion of oxidative stress. The present study aimed to determine whether the gut microbiome modulates arterial function with ageing. We measured arterial function in young and older mice after 3-4 weeks of treatment with broad-spectrum, poorly-absorbed antibiotics to suppress the gut microbiome. To identify potential mechanistic links between the gut microbiome and age-related arterial dysfunction, we sequenced microbiota from young and older mice and measured plasma levels of the adverse gut-derived metabolite trimethylamine N-oxide (TMAO). In old mice, antibiotics reversed endothelial dysfunction [area-under-the-curve carotid artery dilatation to acetylcholine in young: 345 ± 16 AU vs. old control (OC): 220 ± 34 AU, P < 0.01; vs. old antibiotic-treated (OA): 334 ± 15 AU; P < 0.01 vs. OC] and arterial stiffening (aortic pulse wave velocity in young: 3.62 ± 0.15 m  s-1  vs. OC: 4.43 ± 0.38 m  s-1 ; vs. OA: 3.52 ± 0.35 m  s-1 ; P = 0.03). These improvements were accompanied by lower oxidative stress and greater antioxidant enzyme expression. Ageing altered the abundance of gut microbial taxa associated with gut dysbiosis. Lastly, plasma TMAO was higher with ageing (young: 2.6 ± 0.4 µmol  L-1   vs. OC: 7.2 ± 2.0 µmol  L-1 ; P < 0.0001) and suppressed by antibiotic treatment (OA: 1.2 ± 0.2 µmol  L-1 ; P < 0.0001 vs. OC). The results of the present study provide the first evidence for the gut microbiome being an important mediator of age-related arterial dysfunction and oxidative stress and suggest that therapeutic strategies targeting gut microbiome health may hold promise for preserving arterial function and reducing cardiovascular risk with ageing in humans.

Serum endotoxin, gut permeability and skeletal muscle metabolic adaptations following a short term high fat diet in humans.

BACKGROUND: Our previous work demonstrated that a short-term high fat diet (HFD) increased fasting serum endotoxin, altered postprandial excursions of serum endotoxin, and led to metabolic and transcriptional responses in skeletal muscle in young, healthy male humans. PURPOSE: The purpose of the present study was to determine if a short-term high fat diet: 1) increases intestinal permeability and, in turn, fasting endotoxin concentrations and 2) decreases postprandial skeletal muscle fat oxidation. METHODS: Thirteen normal weight young adult males (BMI 23.1 ±â€¯0.8 kg/m2, age 22.2 ±â€¯0.4 years) were fed a control diet (55% carbohydrate, 30% fat, 9% of which was saturated, 15% protein) for two weeks, followed by 5 days of an isocaloric HFD (30% carbohydrate, 55% fat, 25% of which was saturated, 15% protein, isocaloric to the control diet). Intestinal permeability (via four sugar probe test) was assessed in the fasting state. Both before and after the HFD, a high fat meal challenge (HFM, 820 kcal, 25% carbohydrate, 63% fat, 26% of which was saturated, and 12% protein) was administered. After an overnight fast, blood samples were collected before and every hour for 4 h after the HFM to assess endotoxin, and other serum blood measures. Muscle biopsies were obtained from the vastus lateralis before and 4 h after the HFM in order to assess substrate oxidation (glucose, fatty acid and pyruvate) using radiolabeled techniques. Insulin sensitivity was assessed via intravenous glucose tolerance test. Intestinal permeability, blood samples and muscle biopsies were assessed in the same manner before and following the HFD. MAIN FINDINGS: Intestinal permeability was not affected by HFD (p > 0.05), but fasting endotoxin increased two fold following the HFD (p = 0.04). Glucose oxidation and fatty acid oxidation in skeletal muscle homogenates significantly increased after the HFM before the HFD (+97%, and +106% respectively) but declined after the HFM following 5 days of the HFD (-24% and +16% respectively). Fatty acid suppressibility of pyruvate oxidation increased significantly after the HFM (+32%) but this physiological effect was abolished following 5 days of the HFD (+7%). Insulin sensitivity did not change following the HFD. CONCLUSION: These findings demonstrate that in healthy young men, consuming an isocaloric HFD for 5 days increases fasting endotoxin, independent of changes in gut permeability. These changes in endotoxin are accompanied by a broad effect on skeletal muscle substrate metabolism including increases in postprandial fat oxidation. Importantly, the latter occurs independent of changes in body weight and whole-body insulin sensitivity.

Monomeric cocoa catechins enhance ß-cell function by increasing mitochondrial respiration.

A hallmark of type 2 diabetes (T2D) is ß-cell dysfunction and the eventual loss of functional ß-cell mass. Therefore, mechanisms that improve or preserve ß-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on ß-cell function. INS-1 832/13-derived ß-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract and with oligomeric or polymeric procyanidin-rich fraction demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in ß-cell fuel utilization. Mitochondrial complex III, IV and V components are up-regulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with nuclear factor, erythroid 2 like 2 (Nrf2) nuclear localization and expression of Nrf2 target genes including nuclear respiratory factor 1 (Nrf1) and GA binding protein transcription factor alpha subunit (GABPA), essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and up-regulation of genes critical for mitochondrial respiration, glucose-stimulated insulin secretion and ultimately improved ß-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients.

Mechanisms by which cocoa flavanols improve metabolic syndrome and related disorders.

Dietary administration of cocoa flavanols may be an effective complementary strategy for alleviation or prevention of metabolic syndrome, particularly glucose intolerance. The complex flavanol composition of cocoa provides the ability to interact with a variety of molecules, thus allowing numerous opportunities to ameliorate metabolic diseases. These interactions likely occur primarily in the gastrointestinal tract, where native cocoa flavanol concentration is high. Flavanols may antagonize digestive enzymes and glucose transporters, causing a reduction in glucose excursion, which helps patients with metabolic disorders maintain glucose homeostasis. Unabsorbed flavanols, and ones that undergo enterohepatic recycling, will proceed to the colon where they can exert prebiotic effects on the gut microbiota. Interactions with the gut microbiota may improve gut barrier function, resulting in attenuated endotoxin absorption. Cocoa may also positively influence insulin signaling, possibly by relieving insulin-signaling pathways from oxidative stress and inflammation and/or via a heightened incretin response. The purpose of this review is to explore the mechanisms that underlie these outcomes, critically review the current body of literature related to those mechanisms, explore the implications of these mechanisms for therapeutic utility, and identify emerging or needed areas of research that could advance our understanding of the mechanisms of action and therapeutic potential of cocoa flavanols.

Analysis of Cocoa Proanthocyanidins Using Reversed Phase High-Performance Liquid Chromatography and Electrochemical Detection: Application to Studies on the Effect of Alkaline Processing.

Flavan-3-ols and proanthocyanidins play a key role in the health beneficial effects of cocoa. Here, we developed a new reversed phased high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method for the analysis of flavan-3-ols and proanthocyanidins of degree of polymerization (DP) 2-7. We used this method to examine the effect of alkalization on polyphenol composition of cocoa powder. Treatment of cocoa powder with NaOH (final pH 8.0) at 92 °C for up to 1 h increased catechin content by 40%, but reduced epicatechin and proanthocyanidins by 23-66%. Proanthocyanidin loss could be modeled using a two-phase exponential decay model (R(2) > 0.7 for epicatchin and proanthocyanidins of odd DP). Alkalization resulted in a significant color change and 20% loss of total polyphenols. The present work demonstrates the first use of HPLC-ECD for the detection of proanthocyanidins up to DP 7 and provides an initial predictive model for the effect of alkali treatment on cocoa polyphenols.

Pan-colonic pharmacokinetics of catechins and procyanidins in male Sprague-Dawley rats.

Poor absorption and bioavailability of procyanidins from the upper gastrointestinal tract result in the majority of the dose reaching the colon. During colonic transit, progressive microbial metabolism likely produces gradients of procyanidins and microbial metabolites along the length of the colon, suggesting that proximal and distal regions are exposed to different profiles of procyanidins and metabolites. However, previous studies have largely treated the colon as a single organ or looked at fecal profiles, and differences in the profiles of native and metabolite compounds between regions have not been observed. The metabolism kinetics of procyanidins larger than trimers and formation of metabolites in the colon have not been well characterized. Therefore, the objective of this study was to determine the kinetics of delivery and microbial metabolism of monomeric, dimeric and oligomeric procyanidins in the cecum and proximal, mid and distal colon. Sprague-Dawley rats were gavaged grape seed extract and sacrificed over 18 h. Analysis of luminal contents showed distinct native and metabolite profiles for each region. Procyanidins had maximum concentrations at approximately 3h postgavage for all sections. Metabolites reached maximum concentrations from 3 to 18 h postgavage. The appearance of metabolites was highly dependent on species: larger metabolites were found at earlier times in the more proximal segments, and smaller metabolites were found at later times in more distal regions. This study allowed for the observation of regions in the lower gastrointestinal tract, giving insight into the distribution and delivery of procyanidins and their microbial metabolites throughout the colon.

Short-term high-fat diet increases postprandial trimethylamine-N-oxide in humans.

The gut microbiota plays an obligatory role in the metabolism of nutrients containing trimethylamine moieties, such as L-carnitine and choline, leading to the production of the proatherogenic trimethylamine-N-oxide (TMAO). We hypothesized that a short-term, high-fat diet would increase fasting and postprandial plasma concentrations of TMAO in response to a high-fat meal challenge. Following a 2-week eucaloric control diet, 10 nonobese men (18-30 years) consumed a eucaloric, high-fat diet (55% fat) for 5 days. Plasma TMAO was measured after a 12-hour fast and each hour after for 4 hours following a high-fat meal (63% fat) at baseline and after the high-fat diet using ultraperformance liquid chromatography/ tandem mass spectrometry. Fasting plasma TMAO did not increase significantly following the high-fat diet (1.83 ± 0.21 vs 1.6 ± 0.24 µmol/L). However, plasma TMAO was higher at hour 1 (2.15 ± 0.28 vs 1.7 ± 0.30 µmol/L), hour 2 (2.3 ± 0.29 vs 1.8 ± 0.32 µmol/L), hour 3 (2.4 ± 0.34 vs 1.58 ± 0.19 µmol/L), and hour 4 (2.51 ± 0.33 vs 1.5 ± 0.12 µmol/L) (all P < .05) following the high-fat diet as compared with the baseline postprandial response. In conclusion, a short-term, high-fat diet does not increase fasting plasma TMAO concentrations but appears to increase postprandial TMAO concentrations in healthy, nonobese, young men. Future studies are needed to determine the mechanisms responsible for these observations.

Probiotic supplementation and trimethylamine-N-oxide production following a high-fat diet.

OBJECTIVE: The objective of this study was to test the hypothesis that the multi-strain probiotic VSL#3 would attenuate the increase in fasting plasma concentrations of trimethylamine-N-oxide (TMAO) following a high-fat diet. METHODS: Nineteen healthy, non-obese males (18-30 years) participated in the present study. Following a 2-week eucaloric control diet, subjects were randomized to either VSL#3 (900 billion live bacteria) or placebo (cornstarch) during the consumption of a hypercaloric (+1,000 kcal day(-1) ), high-fat diet (55% fat) for 4 weeks. Plasma TMAO, L-carnitine, choline, and betaine (UPLC-MS/MS) were measured at baseline and following a high-fat diet. RESULTS: Plasma TMAO significantly increased 89% ± 66% vs. 115% ± 61% in both the VSL#3 and placebo groups, respectively; however, the magnitude of change in plasma TMAO was not different (P > 0.05) between them. Plasma L-carnitine, choline, and betaine concentrations did not increase following the high-fat diet in either group. CONCLUSIONS: A high-fat diet increases plasma TMAO in healthy, normal-weight, young males. However, VSL#3 treatment does not appear to influence plasma TMAO concentrations following a high-fat diet. Future studies are needed to determine whether other therapeutic strategies can attenuate the production of TMAO.