Lipid Luminations: From Microbes to Foam Cells

Atherosclerosis is a lipid-driven disorder and the main underlying cause of cardiovascular disease (CVD).1 Microbiology has long been a critical focal area of medicine in terms of the effect of bacterial organisms on health and disease. What is the contemporary view of the relationship between these two aspects of clinical medicine?

The role of the intestinal microbiome in the production of bioactive compounds derived from nutrient metabolism represents a previously neglected interface. Advances in the sciences of metagenomics and metabolomics (see definitions on page 19) have allowed exploration of how these compounds contribute to pathologic states.2,3

The gut’s microbial ecosystem could be described as the largest endocrine organ in the body. Biologically active compounds are produced through nutrient metabolism and carried to the circulatory system, where they exert their effects elsewhere in the body. The microbial symbionts in the gut have been added to the growing list of complex determinants of atherogenesis.4,5

A review of the literature published in the last five years is replete with studies suggesting a mechanistic association between the gut microbiome as shaped by host genetics, and lipid metabolism, thereby influencing development of CVD.6 This microenvironment has been found to actively participate in the development of complex metabolic disorders such as obesity, insulin resistance, and diabetes, as well as hypertension, sleep apnea, and heart failure.

The two techniques of molecular 16S ribosomal RNA surveys, and direct sequencing of DNA are used to profile the fecal microbiota, as opposed to older, more cumbersome culture methods. This approach has shown the gut microbiome to be a complex community of 100 trillion Archaean and bacterial cells, representing >1000 species.7,8

Microbial composition in the gut includes five bacterial phyla, predominantly Bacteriodetes and Firmicutes, and 1 Archaea (Euryarchaeota). The Firmicutes phylum contains genera such as Clostridium and Lactobacillus, several strains of which function as probiotics and butyrate producers. Bacteriodetes genera, including Bacteroides and Prevotella, among others, are involved in colonic degradation of complex lipopolysaccharides and peptidoglycans.

Euryarchaeota are involved in the process of intestinal methanogenesis.4,7,8 Shifts in the composition of the microbiome during the transitions between infancy, adulthood, and old age are being defined. Aging, for example, is associated with a reduction in the abundance of Bifidobacterium, associated with an upregulation of the inflammatory response in the gut epithelium. This dysbiosis may enhance inflammatory responses that occur more in the aged.8 Comparisons of the gut microbiota of aboriginal tribes in Venezuela and westernized cultures reveals a marked decrease in ecological diversity.9

In this article, we will briefly explore three recent peer-reviewed journal manuscripts elucidating this process.

Microbial Processing

Our gut microbiota act as a filter of our largest environmental exposure (our diet) and, thus, influences the bioavailability of dietary constituents in the host. Conversely, diet modulates the composition of the human gut microbiome, leading to a predilection toward certain diseases, such as diabetes and CVD.

Products of the microbial metabolism of nutrients may act as signaling molecules and thus influence the host metabolism in terms of intestinal function as well as affecting liver, brain, muscle, and adipose tissue. Three major areas of effect include the fermentation of lipopolysaccharides, bile acid metabolism, and metabolism of choline from the diet.4

Wang, et al., published their findings from case-controlled studies (comprised of an initial learning cohort, an independent validation cohort, and a larger validation cohort of 1,876 subjects presenting for cardiac stress testing) in 2011 in the journal Nature. They identified three metabolites of dietary lipid phosphatidylcholine (choline, betaine, and trimethylamine N-oxide [TMAO]) and showed their dose-dependent associations with cardiovascular disease.2

Choline is important for lipid metabolism, including synthesis of very-low-density lipoproteins (VLDL) in the liver. Choline deficiency is associated with hepatic steatosis and neural abnormalities. It is found in most animal products and some plant products and is particularly abundant in egg yolk, meats, liver, and high-fat dairy products.10,11 Betaine is a known oxidation product of choline. TMAO arises from the bacterial metabolism of choline via an intermediate precursor, trimethylamine, which subsequently is oxidized in the liver by flavin monooxygenase isoform 3 (FMO3). TMAO originally was thought to be an inert nitrogenous waste product of protein degradation that was excreted in urine. However, TMAO has been shown to have direct biological activity that facilitates atherogenesis, thus adversely affecting cardiovascular disease risk. In gnotobiotic mouse models, TMAO supplementation fostered enhanced macrophage foam cell formation in the arterial subendothelium by upregulation of macrophage scavenger receptors implicated in their formation.12

Direct dietary exposure to TMAO or its precursors, including choline and L-carnitine such as that found in eggs and red meat, respectively, caused reductions in reverse cholesterol transport and altered cholesterol and sterol metabolism in the endothelium, the liver, and the intestine. TMAO levels are affected by hepatic FMO3, as stated, and this molecule is regulated in part by the farnesoid X receptor (FXR), which is an important bileactivated nuclear receptor.5

In 2013, an even larger cohort of subjects with stable cardiovascular disease was evaluated for this exposure. Tang WH, et al., published their findings of elevated levels of TMAO being predictive of major adverse cardiac events (MACE), such as death, nonfatal myocardial infarction, and stroke in the New England Journal of Medicine.13

Tang’s study group first performed a metabolomic analysis of the plasma of stable CVD patients (without evidence of acute coronary syndrome) and identified choline, betaine, and TMAO levels. They subsequently showed dose-dependent associations with cardiovascular disease in a large clinical cohort of >4,000 patients undergoing elective cardiac catheterization. Over the indicated threeyear follow-up period, participants who went on to have MACE had higher baseline levels of TMAO compared with those who did not have MACE. Those in the highest quartile of TMAO levels had a significantly increased risk of an event, with a hazard ratio of 2.54 (confidence interval 1.96 to 3.28; P<0.001). Even after adjustment for traditional cardiovascular risk factors, the relationship between higher levels of TMAO and MACE remained and was associated with an increased risk of death, nonfatal myocardial infarction, or stroke. The inclusion of TMAO as a covariate resulted in a significantly better risk assessment over traditional risk factors alone. Their results also confirmed earlier observations in gnotobiotic mice that TMA, the precursor of TMAO, is produced by gut microbiota.

Effect of Gut Microbiota on Variation in Blood Lipids

Recent data published by Fu and colleagues of the Netherlands in 20151 in Circulation Research provided evidence that variations in the gut microbiome significantly alters blood lipids. In their study of 893 human subjects in the Netherlands, they investigated the impact of gut microbiome diversity on body mass index (BMI) and blood lipid levels. They observed a strong association between gut microbial composition and a variance in BMI and blood lipid levels, specifically triglycerides and high-density lipoprotein cholesterol (HDL-C). A much weaker association with total and low-density lipoprotein cholesterol (LDL-C) was seen. Measures of microbial richness and diversity (defined by operational taxonomic units) clearly indicated that an inverse correlation exists between microbial diversity, BMI, and triglycerides (TG), along with a positive correlation with HDL-C. Also, certain microbial taxa had a proportionate impact on lipids alone, separate from BMI, based on a risk score calculated from host genetic variants identified from genome-wide association studies (GWAS).

These observations support the idea that increased diversity of the gut microbiome is associated with a more favorable cardiovascular risk profile.14

Conclusion

Substantial evidence suggests the gut microbiome plays a central role in the development of coronary artery disease. It is associated with CVD risk factors including diabetes, obesity, and lipid abnormalities. The metabolite TMAO formed in liver from a gut microbiota derived precursor is linked to atherogenesis.

Causality is yet to be determined, but the better we understand the interface between diet, the metagenome, and disease, the greater likelihood there is of targeted interventions directed toward identification of at-risk patients, prevention, and treatment. A clear understanding of the role of gut microbial diversity in the journey from infancy to old age, and how that diversity is influenced by our diet, will better equip us to affect population health. The messages we have been providing our patients for decades in regards to the importance of nutrition and lifestyle change make even greater sense.

Disclosure statement: Dr. Willard is on the Speakers Bureau for Regeneron and Sanofi-Aventis.

Terminology

Gnotobiotics is the study of animals living in a microbiologically defined environment, either germ-free or having been colonized with known
bacteria.

Metagenome is the total DNA that can be extracted from an environment. The human metagenome is the aggregate of host DNA and the
microbiota. The terms “metagenome” and “microbiome” may be used interchangeably.

Metagenomics is the study of the metagenome and can either be targeted (usually 16S ribosomal RNA) or untargeted (shotgun sequencing).
Microbiota is the total microbial community inhabiting a specific environment. Cellular density increases along the length of the gut. The
colonic microbiota is the densest and most diverse community in the human body.

Microbiome is the collective genomic content of a microbiota.

Metabolomics is the systematic study of the unique chemical “fingerprints” left behind by small molecules (metabolites) in specific cellular
processes.

Probiotics are defined as live microorganisms — such as lactobacillus or Bifidobacterium — which, when administered in adequate
amounts, confer a health benefit for the host.

Prebiotics are non-digestible food ingredients such as inulin and transgalactooligosaccarides. When consumed in adequate quantities, they
selectively stimulate the growth, activity or both of one or a limited number of microbial genera or species in the gut microbiota, thereby
conferring health benefits to the host.