A new metabolite derived from the microbiota linked to cardiovascular disease

A new metabolite derived from the microbiota linked to cardiovascular disease

OVERVIEW

  • Metabolomic screening has identified a new metabolite associated with cardiovascular disease in the blood of people with type 2 diabetes.
  • This metabolite, phenylacetylglutamine (PAGln), is produced by the intestinal microbiota and the liver, from the amino acid phenylalanine from dietary proteins.
  • PAGln binds to adrenergic receptors expressed on the surface of blood platelets, which results in making them hyper-responsive.
  • A beta blocker drug widely used in clinical practice (Carvedilol) blocks the prothrombotic effect of PAGln.

A research group from the Cleveland Clinic in the United States recently identified a new metabolite of the microbiota that is clinically and mechanistically linked to cardiovascular disease (CVD). This discovery was made possible by the use of a metabolomic approach (i.e. the study of metabolites in a given organism or tissue), a powerful and unbiased method that identified, among other things, trimethylamine oxide (TMAO) as a metabolite promoting atherosclerosis and branched-chain amino acids (BCAAs) as markers of obesity.

The new metabolomic screening has identified several compounds associated with one or more of these criteria in the blood of people with type 2 diabetes: 1) association with major adverse cardiovascular events (MACE: myocardial infarction, stroke or death) in the past 3 years; 2) heightened levels of type 2 diabetes; 3) poor correlation with indices of glycemic control. Of these compounds, five were already known: two which are derived from the intestinal microbiota (TMAO and trimethyllysine) and three others that are diacylglycerophospholipids. Among the unknown compounds, the one that was most strongly associated with MACE was identified by mass spectrometry as phenylacetylglutamine (PAGln).

In summary, here is how PAGln is generated (see the left side of Figure 1):

  • The amino acid phenylalanine from dietary proteins (animal and plant origin) is mostly absorbed in the small intestine, but a portion that is not absorbed ends up in the large intestine.
  • In the large intestine, phenylalanine is first transformed into phenylpyruvic acid by the intestinal microbiota, then into phenylacetic acid by certain bacteria, particularly those expressing the porA
  • Phenylacetic acid is absorbed and transported to the liver via the portal vein where it is rapidly metabolized into phenylacetylglutamine or PAGln.

Figure 1. Schematic summary of the involvement of PAGln in the increase in platelet aggregation, athero-thrombosis and major adverse cardiovascular events. From Nemet et al., 2020.

Researchers have shown that PAGln increases the effects associated with platelet activation and the potential for thrombosis in whole blood, on isolated platelets and in animal models of arterial damage.

PAGln binds to cell sites in a saturable manner, suggesting specific binding to membrane receptors. The researchers then demonstrated that PAGln binds to G-protein coupled adrenergic receptors, expressed on the surface of the platelet cell membrane. The stimulation of these receptors by PAGln causes the hyperstimulation of the platelets, which then become hyper-responsive and accelerate the platelet aggregation and the thrombosis process.

Finally, in a mouse thrombus model, it has been shown that a beta blocker drug widely used in clinical practice (Carvedilol) blocks the prothrombotic effect of PAGln. This result is particularly interesting because it suggests that the beneficial effects of beta blockers may be partly caused by reversing the effects of high PAGln levels. The identification of PAGln could lead to the development of new targeted and personalized strategies for the treatment of cardiovascular diseases.

Effectiveness of exercise to prevent and mitigate diabetes: An important role of the gut microbiota

Effectiveness of exercise to prevent and mitigate diabetes: An important role of the gut microbiota

OVERVIEW

  • In overweight, prediabetic and sedentary men, exercise induced changes in the gut microbiota that are correlated with improvements in blood sugar control and insulin sensitivity.
  • The microbiota of the participants who are “responders” to exercise had a greater ability to produce short chain fatty acids (SCFAs) and to eliminate branched-chain amino acids (BCAAs). Conversely, the microbiota of non-responders was characterized by an increased production of metabolically harmful compounds.
  • Transplantation of the fecal microbiota of responders into obese mice produced roughly the same beneficial effects of exercise on insulin resistance. Such effects were not observed after transplanting the microbiota of non-responders.
Regular exercise has beneficial effects on blood glucose control and insulin sensitivity, and is therefore an interesting strategy to prevent and mitigate type 2 diabetes. Unfortunately, in some people, exercise does not cause a favourable metabolic response, a phenomenon called “exercise resistance”. The causes of this phenomenon have not been clearly established, although some researchers have suggested that genetic predispositions and epigenetic changes may contribute to this.

A growing body of data indicates that an imbalance in the gut microbiota (dysbiosis) plays an important role in the development of insulin resistance and type 2 diabetes. Several different mechanisms are involved, including an increase in intestinal permeability and increased endotoxemia, changes in the production of certain short chain fatty acids and branched-chain amino acids, and disturbances in bile acid metabolism. Changes in the composition and function of the gut microbiota have been observed in people with type 2 diabetes and prediabetics. One study also showed that transplanting a healthy person’s microbiota into the intestines of people with metabolic syndrome results in increased microbial diversity and improved blood sugar control as well as sensitivity to insulin.

The intestinal microbiota (formerly intestinal flora) is a complex ecosystem of bacteria, archaea (small microorganisms without nuclei), eukaryotic microorganisms (fungi, protists) and viruses, which has evolved with human beings for several thousands of years. A human gut microbiota, which can weigh up to 2 kg, is absolutely necessary for digestion, metabolic function, and resistance to infection. The human gut microbiota has an enormous metabolic capacity, with more than 1,000 different species of bacteria and 3 million unique genes (the microbiome).

Recent data indicate that exercise modulates the gut microbiota in humans as well as in other species of animals. For example, it has been found that the gut microbiota of professional athletes is more diverse and has a healthier metabolic capacity than the microbiota of sedentary people. However, it is still unclear how these exercise-induced changes in the microbiota are involved in the metabolic benefits (see figure below).

Figure. Changes in the gut microbiota and intestinal epithelium through exercise and health benefits. BDNF: Brain-derived neurotrophic factor (growth factor). From: Mailing et al., 2019.

A study published in Cell Metabolism tried to answer this question by performing an intervention in overweight, prediabetic and sedentary men. Study participants were randomly assigned to a control group (sedentary) or to a 12-week supervised training program. Blood and fecal samples were collected before and after the procedure. After the 12 weeks, modest but significant weight loss and fat loss were observed in people who exercised, with improvements in several metabolic parameters, such as insulin sensitivity, favourable lipid profiles, improved cardiorespiratory capacity and levels of adipokines (signalling molecules secreted by adipose tissues) which are functionally associated with insulin sensitivity. The researchers observed that there was a high interpersonal variability in the results. After classifying the participants as “non-responders” and “responders”, according to their insulin sensitivity score, the researchers analyzed the composition of each participant’s microbiota.

Among responders, exercise altered the concentration of more than 6 species of bacteria belonging to the genera Firmicutes, Bacteroidetes, and Probacteria. Among these bacteria, those belonging to the genus Bacteroidetes are involved in the metabolism of short chain fatty acids (SCFAs). Among the most striking differences between the microbiota of responders and non-responders, the researchers noted a 3.5-fold increase in the number of Lanchospiraceae bacterium, a butyrate producer (a SCFA), which is an indicator of intestinal health. The bacterium Alistipes shahii, which has already been associated with inflammation and is present in higher amounts in obese people, decreased by 43% in responders, while it increased 3.88 times in non-responders. The Prevotella copri bacteria proliferated at a reduced rate in the responders; it is one of the main bacteria responsible for the production of branched-chain amino acids (BCAAs) in the gut and contributes to insulin resistance.

The researchers then transplanted the fecal microbiota of responders and non-responders into obese mice. The fecal microbiota transplantation (FMT) of the responders had the effect in mice of reducing blood sugar and insulin as well as improving insulin sensitivity, while such favourable effects were not observed in mice that received a FMT from non-responders.

Mice saw their blood levels of SCFAs increase significantly, while the levels of BCAAs (leucine, isoleucine, valine) and aromatic amino acids (phenylalanine, tryptophan) decreased after receiving the microbiota from responders. In contrast, mice that received the microbiota from non-responders saw opposite changes in the levels of these same metabolites. BCAA supplementation attenuated the beneficial effects of FMT from responders on blood sugar regulation and insulin sensitivity, while SCFA supplementation in mice that received the microbiota of non-responders partially corrected the defect in blood glucose regulation and insulin sensitivity.

Taken together, these results suggest that the gut microbiota and its metabolites are involved in the beneficial metabolic effects caused by exercise. In addition, this study indicates that poor adaptation of the gut microbiota is partly responsible for the lack of a favourable metabolic response in people who do not respond to exercise.

Eggs: To consume with moderation

Eggs: To consume with moderation

The old debate over whether egg consumption is detrimental to cardiovascular health has been revived since the recent publication of a study that finds a significant, albeit modest, association between egg or dietary cholesterol consumption and the incidence of cardiovascular disease (CVD) and all-cause mortality. Eggs are an important food source of cholesterol: a large egg (≈50 g) contains approximately 186 mg of cholesterol. The effect of eggs and dietary cholesterol on health has been the subject of much research over the last five decades, but recently it has been assumed that this effect is less important than previously thought. For example, the guidelines of medical and public health organizations have in recent years minimized the association between dietary cholesterol and CVD (see the 2013 AHA/ACC Lifestyle Guidelines and the 2015–2020 Dietary Guidelines for Americans). In 2010, the American guidelines recommended consuming less than 300 mg of cholesterol per day; however, the most recent recommendations (2014–2015) do not specify a daily limit. This change stems from the fact that cholesterol intake from eggs or other foods has not been shown to increase blood levels of LDL-cholesterol or the risk of CVD, as opposed to the dietary intake of saturated fat that significantly increases LDL cholesterol levels, a significant risk of CVD.

Some studies have reported that dietary cholesterol increases the risk of CVD, while others reported a decrease in risk or no effect with high cholesterol consumption. In 2015, a systematic review and meta-analysis of prospective studies was unable to draw conclusions about the risk of CVD associated with dietary cholesterol, mainly because of heterogeneity and lack of methodological rigour in the studies. The authors suggested that new carefully adjusted and rigorously conducted cohort studies would be useful in assessing the relative effects of dietary cholesterol on the risk of CVD.

What distinguishes the study recently published in JAMA from those published previously is its great methodological rigour, in particular a more rigorous categorization of the components of the diet, which makes it possible to isolateindependent relationships between the consumption of eggs or cholesterol from other sources and the incidence of CVD. The cohorts were also carefully harmonized, and several fine analyses were performed. The data came from six U.S. cohorts with a total of 29,615 participants who were followed for an average of 17.5 years.

The main finding of the study is that greater consumption of eggs or dietary cholesterol (including eggs and meat) is significantly associated with a higher risk of CVD and premature mortality. This association has a dose-response relationship: for every additional 300 mg of cholesterol consumed daily, the risk of CVD increases by 17% and that of all-cause mortality increases by 18%. Each serving of ½ egg consumed daily is associated with an increased risk of CVD of 6% and an increased risk of all-cause mortality of 8%. On average, an American consumes 295 mg of cholesterol every day, including 3 to 4 eggs per week. The model used to achieve these results took into account the following factors: age, gender, race/ethnicity, educational attainment, daily energy intake, smoking, alcohol consumption, level of physical activity, use of hormone therapy. These adjustments are very important when you consider that egg consumption is commonly associated with unhealthy behaviours such as smoking, physical inactivity and unhealthy eating. These associations remain significant after additional adjustments to account for CVD risk factors (e.g. body mass index, diabetes, blood pressure, lipidemia), consumption of fat, animal protein, fibre and sodium.

A review of this study suggests that the association between cholesterol and the incidence of CVD and mortality may be due in part to residual confounding factors. The authors of this review believe that health-conscious people reported eating fewer eggs and cholesterol-containing foods than they actually did. Future studies should include “falsification tests” to determine whether a “health consciousness” factor is the cause of the apparent association between dietary cholesterol and CVD risk.

Eggs, TMAO and atherosclerosis
A few years ago, a metabolomic approach identified a compound in the blood, trimethylamine-N-oxide (TMAO), which is associated with increased cardiovascular risks. TMAO is formed from molecules from the diet: choline, phosphatidylcholine (lecithin) and carnitine. Bacteria present in the intestinal flora convert these molecules into trimethylamine (TMA), then the TMA is oxidized to TMAO by liver enzymes called flavin monooxygenases. The main dietary sources of choline and carnitine are red meat, poultry, fish, dairy products and eggs (yolks). Eggs are an important source of choline (147 mg/large egg), an essential nutrient for the liver, muscles and normal foetal development, among others.

A prospective study indicated that elevated plasma concentrations of TMAO were associated with a risk of major cardiac events (myocardial infarction, stroke, death), independent of traditional risk factors for cardiovascular disease, markers of inflammation, and renal function. It has been proposed that TMAO promotes atherosclerosis by increasing the number of macrophage scavenger receptors, which carry oxidized LDL (LDLox) to be degraded within the cell, and by stimulating macrophage foam cells (i.e. filled with LDLox fat droplets), which would lead to increased inflammation and oxidation of cholesterol that is deposited on the atheroma plaques. A randomized controlled study indicates that the consumption of 2 or more eggs significantly increases the TMAO in blood and urine, with a choline conversion rate to TMAO of approximately 14%. However, this study found no difference in the blood levels of two markers of inflammation, LDLox and C-reactive protein (hsCRP).

Not all experts are convinced that TMAO contributes to the development of CVD. A major criticism is focused on fish and seafood, foods that may contain significant amounts of TMAO, but are associated with better cardiovascular health. For example, muscle tissue in cod contains 45–50 mmol TMAO/kg. For comparison, the levels of choline, a precursor of TMAO, are 24 mmol/kg in eggs and 10 mmol/kg in red meat. The only sources of choline that are equivalent to that in TMAO in marine species are beef and chicken liver. TMAO contained in fish and seafood is therefore significantly more important quantitatively than TMAO that can be generated by the intestinal flora from choline and carnitine from red meat and eggs. This was also measured: plasma levels of TMAO are much higher in people who have a fish-based diet (> 5000 μmol / L) than in people who eat mostly meat and eggs (139 μmol / L). In their response to this criticism, the authors of the article point out that not all fish contain the same amounts of TMAO and that many (e.g. sea bass, trout, catfish, walleye) do not contain any. Fish that contain a lot of TMAO are mainly deep-sea varieties (cod, haddock, halibut). The TMAO content of other fish, including salmon, depends on the environment and when they are caught.

Other experts believe this could be a case of reverse causality: the reduction in renal function associated with atherosclerosis could lead to an accumulation of TMAO, which would mean that this metabolite is a marker and not the cause of atherosclerosis. To which the authors of the hypothesis counter that the high concentration of TMAO is associated with a higher risk of cardiovascular events even when people have completely normal kidney function.

Diabetes and insulin resistance
People who are overweight (BMI> 25) and obese (BMI> 50) are at higher risk of becoming insulin resistant and having type 2 diabetes and metabolic syndrome, conditions that can, independently or in combination, lead to the development of cardiovascular disease. There is evidence that dietary cholesterol may be more harmful to diabetics. Intestinal absorption of cholesterol is impaired in diabetics, i.e. it is increased. However, in a randomized controlled trial, when diabetic patients consumed 2 eggs per day, 6 times per week, their lipid profile was not altered when their diet contained mono- and polyunsaturated fatty acids. Other studies (mostly subsidized by the egg industry) suggest that eggs are safe for diabetics.

Dr. J. David Spence of the Stroke Prevention & Atherosclerosis Research Center believes that people at risk for CVD, including diabetics, should avoid eating eggs (see also this more detailed article). This expert in prevention argues that it is the effects of lipids after a meal that matter, not fasting lipid levels. Four hours after a meal high in fat and cholesterol, harmful phenomena such as endothelial dysfunction, vascular inflammation and oxidative stress are observed. While egg whites are unquestionably a source of high-quality protein, egg yolks should not be eaten by people with cardiovascular risks or genetic predispositions to heart disease.

The association between the consumption of eggs or foods containing cholesterol and the risk of CVD is modest. But since this risk increases with the amount consumed, people who eat a lot of eggs or foods containing cholesterol have a significant risk of harming their cardiovascular health. For example, according to the study published in JAMA, people who consume two eggs per day instead of 3 or 4 per week have a 27% higher risk of CVD and a 34% higher risk of premature mortality. It is therefore prudent to minimize the consumption of eggs (less than 3 or 4 eggs per week) and meat in order to limit the high intake of cholesterol and choline and avoid promoting atherosclerosis.