This cross-sectional study focused on the metabotype profile associated with a healthy lifestyle. The LIFE and CON groups were of similar age, education, and sex distribution, but differed significantly in body composition and exercise and dietary patterns. The proteomics dataset previously published from this cross-sectional study showed strong group differences for 39 proteins supporting a lower innate immune activation signature and greater lipoprotein metabolism and HDL remodeling in the LIFE group (30). In this analysis, untargeted metabolomics of more than 10,000 metabolite peaks revealed a distinct difference in the plasma metabolome between the LIFE and CON groups. Multivariate LOO modeling confirmed that group status (LIFE vs. CON) was strongly predicted by the metabolite signature and exceeded the prediction model from the proteomics data (30). A total of 118 metabolite peaks library matched to metabolites were identified that most significantly differentiated LIFE and CON groups. An enriched pathway analysis using Mummichog indicated group differences for 16 metabolic pathways highlighted by contrasts in bile acid and amino acid metabolism.
The reduced plasma bile acid signature in the LIFE vs. CON group is a novel and important finding from this cross-sectional study. Plasma hydroxycholesterol, a cholesterol precursor in primary bile acid metabolism, and more than 10 primary and secondary bile acids were significantly lower in the LIFE versus CON groups. Other studies indicate that plasma bile acid concentrations vary widely between individuals and that this variance is due to lifestyle, gut microbial, and genetic factors (47). Normally enterohepatic circulation of bile acids is very efficient and only a small proportion of bile acids escape into the systemic circulation (48). Circulating bile acids at normal low concentrations have regulatory functions and exert signaling functions in peripheral tissues and organs through specific nuclear receptors including the farnesoid X receptor (FXR) and the Takeda G protein-coupled receptor 1 (TGR5) (46, 47). Emerging data indicate that individuals with obesity and various diseases including type 2 diabetes mellitus have elevated plasma bile acid concentrations in the fasted state (48, 49). One study showed that even in young and relatively healthy adults, plasma bile acid levels were associated with cardiometabolic and inflammatory disease risk biomarkers (50). A 14-week exercise and weight loss intervention study demonstrated that total fasting bile acids decreased by 30% accompanied by a 55% increase in serum levels of the rate-limiting enzyme cholesterol 7 alpha-hydroxylase (CYP7A1) (51). Limited data suggest that aerobic capacity influences bile acid metabolism (52) and that intake of dietary fiber and polyphenols from whole plant foods have a significant effect on the gut microbiome and bile acid metabolism and related signaling pathways (53, 54). Additional human systems biology-based studies using a variety of multiomics approaches will broaden current understandings regarding the specific and combined lifestyle relationships of body composition and dietary and exercise patterns on bile acid metabolism (47, 48).
LIFE versus CON group differences were found for seven of 20 standard amino acids, with higher histidine and lower glutamic acid, glutamine, β-alanine, phenylalanine, tyrosine, and proline. This LIFE-related amino acid signature was spread across seven different metabolic pathways including histidine, lysine, pyrimidine, amino sugars, β-alanine, tyrosine, and butanoate metabolism. Some aspects of this LIFE versus obese-CON-related amino acid signature have been reported by others, but the literature is far from consistent (18, 26). There is agreement that amino acid metabolism is extensively altered in various disease states and influenced by body composition and lifestyle habits (55). In a cross-sectional study with obese and non-obese women serum amino acids including histidine, arginine, threonine, glycine, lysine, and serine were found to be significantly lower in obese women as compared to non-obese controls, similar to our results (56). In our study, the most important LIFE versus CON contrast was for glutamic acid, an acidic, non-essential amino acid that is involved in numerous metabolic pathways. The plasma concentration of glutamic acid levels is inversely related to visceral adipose tissue and may be influenced by obesity-induced changes in the gut microbiota (57). Pathway enrichment for LIFE versus CON identified the histidine metabolic pathway as most affected with higher levels of histidine, 4-imidazoleacetic acid, and L-formiminoglutamic acid and lower levels of glutamic acid in the LIFE group. Histidine is an essential amino acid and has been positively associated with insulin sensitivity, obesity, liver and kidney disease, and heart failure, and inversely related to inflammation and oxidative stress (56, 58). The gut microbiome appears to play a key role in regulating diet histidine bioavailability (59). Plasma levels of branched chain amino acids (BCAAs) did not differ between LIFE and CON groups in contrast to other studies that have noted elevated plasma BCAA levels in obese groups (18, 26). The literature is mixed, however, regarding plasma BCAA levels and associations with adiposity, longevity, sarcopenia, and diabetes (60).
5-hydroxylysine was increased and other lysine metabolites were decreased in LIFE versus CON groups. Limited data indicate that obesity may be related to enhanced lysine degradation via the saccharopine pathway (61). Lysine is subjected to diverse enzyme-catalyzed post-translational modifications (PTMs), including methylation, acetylation, crotonylation, ubiquitination, and SUMOylation. Acetyllysine (or acetylated lysine) is an acetyl-derivative of the amino acid lysine. In proteins, the acetylation of lysine residues is an important mechanism of epigenetics. Free trimethyllysine (TML) is involved in the carnitine biosynthesis pathway, where it acts as the first intermediate in a series of four enzymatic reactions to generate L-carnitine (62). TML is an important post-translationally modified amino acid with functions in carnitine biosynthesis and regulation of key epigenetic processes. The dataset from this cross-sectional study support lower levels of lysine degradation in the LIFE group, and the clinical significance of this finding remains to be determined. In contrast, pipecolic acid, an L-alpha amino acid metabolite product of lysine microbiome catabolism and a marker of dry bean intake (63) was elevated in the LIFE group with a high VIP value of 2. There is increasing evidence that pipecolic acid is an important regulator of immunity in both plants and humans (64).
Metabolites from the pyrimidine metabolism pathway including uracil, uridine, thymine, and 5-methylcytosine were higher in LIFE versus CON groups, with lower levels of glutamine, cytidine, cytosine, and pseudouridine. Uridine is an uracil nucleoside that is involved in a variety of biological functions including RNA and DNA biosynthesis, glucose and lipid metabolism, glycogen deposition, insulin sensitivity, energy homeostasis, protein and lipid glycosylation, extracellular matrix biosynthesis, and detoxification of xenobiotics (65). Limited human data indicate that plasma uridine levels are inversely related to obesity (66). In mice, uridine supplementation attenuates HFD-induced obesity and NAFLD (67). A high uridine to pseudouridine ratio (as shown in the LIFE group) has been linked to a reduced risk for stroke (68). Uridine decreases oxidative stress and inflammation in vitro and was linked to lower levels of aging indicators in mice (69). Thus, alterations in plasma metabolites related to the pyrimidine pathway may serve as important and novel biomarkers of lifestyle habits and reduced disease risk.
Lifestyle habits had a positive influence on vitamin D3 (cholecalciferol) metabolism with higher plasma calcifediol (25(OH)D3) and calcitriol (1,25(OH)2D3) and lower 1,24,25-trihydroxyvitamin D3 (a 1,25(OH)2D3 catabolism metabolite) in the LIFE versus CON groups. A poor vitamin D status has been linked to obesity and numerous clinical conditions including the metabolic syndrome, type 2 diabetes mellitus, systemic inflammation, autoimmune disorders, and neurodegenerative diseases (70–73). Underlying mechanisms for low vitamin D status in obese populations are unclear but may be related in part to reduced outdoor physical activity and volumetric dilution due to greater volumes of adipose tissue (71).
The N-glycan degradation pathway analysis indicated reduced plasma levels in the LIFE group for mannose, galactose, N-acetylglucosamine, N-acetylneuraminic acid, and fucose. N-glycans (oligosaccharide-protein molecules) are basic components of cell membranes and secreted proteins and help regulate multiple physiological processes. In humans, N-glycosylation involves collections of mannose, galactose, fucose, and sialic acids including N-acetylneuraminic acid and N-acetylglucosamine. Sialic acids are acidic sugars typically located at the terminal positions of glycoproteins (74, 75). The amino sugars N-acetyl-D-mannosamine and N-acetyl-D-glucosamine (lower plasma levels in the LIFE group) are essential precursors of sialic acids. Plasma N-glycans and sialic acid levels are rather stable in healthy individuals over time but can be altered due to physiological, pathological, or lifestyle changes (74, 75). For example, elevated plasma levels of N-acetylneuraminic acid and N-acetylglucosamine have emerged as potential metabolic markers for inflammation, coronary artery disease progression, and a variety of other diseases (76, 77). Elevated plasma mannose has been reported in obese adults and is now considered a biomarker for future risk of several chronic diseases (18, 78, 79). Increased L-fucose in serum and urine is a potential biomarker for cancer, diabetes, cardiovascular disease, cirrhosis, alcoholic liver disease and gastric ulcers (80, 81). The markedly lower plasma levels of N-glycan degradation metabolites in the LIFE group supports the interpretation of reduced chronic disease risk due to positive lifestyle habits. N-acetylglucosamine when polymerized with glucuronic acid forms heparin sulfate and is distributed throughout connective, neural, and epithelial tissues. Lower levels of plasma N-acetylglucosamine support the pathway analysis finding of a lowered degradation of heparan sulfate in the LIFE group (82).
Due to limitations in the curation of metabolites in the library of the Mummichog analysis modules, metabolites including those related to gut microbiome catabolism of food substrates and environmental contaminants were not included in the pathway analysis. Several gut microbiome metabolites reflecting a higher intake of plant-based foods and enhanced gut microbiome alpha diversity were elevated in the LIFE versus CON group including hippuric acid, cinnamoylglycine, cinnamic acids, 3,4-dimethoxyphenylacetic acid, 3-phenylpropanoic acid, and 2-phenylpropionate. An elevated gut microbial metabolite signature in adults with higher lifestyle scores has been reported previously (83). Citric acid cycle metabolites generated from the butanoate metabolism pathway differed between LIFE and CON groups, with higher levels of succinic acid. The butanoate metabolism pathway involves short chain fatty acids (SCFA) produced by bacterial fermentation of undigested carbohydrates (including dietary fiber) and proteins. SCFAs are precursors for numerous metabolites including succinic acid that helps regulate cellular nutrient metabolism and white adipose tissue deposition, muscle fiber remodeling during recovery from exercise, and immune system function (84). Plasma levels of two disaccharides, lactose and sucrose, are indicators of a leaky gut syndrome and were lower in the LIFE versus CON group.
Plasma levels of numerous environmental contaminants were lower in the LIFE versus CON groups including propham (a potato herbicide), fenoxycarb (carbamate-based insecticide metabolite), monocyclohexyl phthalate and (5-Carboxy-2-ethylpentyl)phthalate (plasticizer metabolites), prometon (an herbicide), 3-hydroxycarbofuran (a pesticide carbofuran metabolite), furalaxyl and propamocarb free base (fungicides), and 9-hydroxyfluorene (insecticide and algaecide). Two other cross-sectional studies showed lower levels of blood persistent organochlorine pesticides (POPs) in lean or physically active compared to obese or sedentary adults (85, 86). Dietary, lifestyle, and environmental exposures are still being investigated, but some of the environmental contaminants identified in this study tend to accumulate in the fatty tissues of commonly consumed livestock. Thus, a higher intake of red meat fat in the CON group may have increased the body-exposure burden of environmental contaminants (87).
Pathway enrichment identified LIFE versus CON differences in the glycosphingolipid biosynthesis and metabolism pathway, with higher levels of the key metabolite phosphorylcholine. Glycosphingolipids (GSLs) are a specialized class of membrane lipids that support various cellular functions. Phosphorylcholine (PC) is the hydrophilic polar head group of some phospholipids and is a component of the platelet-activating factor and the phospholipids phosphatidylcholine and sphingomyelin (88). Non-pathogenic antibodies against PC are naturally occurring and present in healthy adults. About 5–10% of circulating immunoglobulin M (IgM) consists of IgM anti-PC. IgM anti-PC is negatively associated with several chronic inflammatory conditions, including atherosclerosis, CVD, rheumatic diseases and chronic kidney disease (CKD) (88).
Other metabolites of importance that were elevated in the LIFE group included beneficial fatty acids such as γ-linolenic acid, docosahexaenoic acid (DHA) and eicosatetraenoic acid (EPA). Lower levels of beneficial fatty acids have been reported in obese populations (18). The reduced form of glutathione was significantly elevated in the LIFE group and is an indicator of reduced oxidative stress (89). Tryptamine, 2-hydroxyethyl)indole, and serotonin are gut microbial catabolites of tryptophan and were elevated in the LIFE group. These metabolites play roles in the gut-brain axis, immune surveillance, and inflammation regulation (90). Two other gut microbial catabolites of tryptophan were decreased in the LIFE group including indole-3-methyl acetate and indole-3-propionic acid. Plasma betaine and lutein levels were higher in the LIFE group. Betaine is a methyl donor, regulates osmotic pressure, has positive effects on intestinal and kidney health, and exerts anti-inflammatory and anti-oxidative effects (91). Lutein is a common carotenoid in plant foods. Several metabolites related to pain relief medications were elevated in the CON group, with higher levels of acetaminophen higher in the LIFE group. Plasma nicotine and cotinine were higher in the LIFE group and may indicate a higher prevalence of vaping.