Isoflavone consumption reduces inflammation through modulation of phenylalanine and lipid metabolism

doi:10.21203/rs.3.rs-1871117/v1

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Isoflavone consumption reduces inflammation through modulation of phenylalanine and lipid metabolism

https://doi.org/10.21203/rs.3.rs-1871117/v1

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Introduction

Phytoestrogen found in soy, fruits, peanuts, and other legumes, has been identified as a metabolite capable of providing beneficial effect in multiple pathological conditions due to its ability to mimic endogenous estrogen. Interestingly, the health promoting effect of phytoestrogens such as isoflavone is dependent on the presence of gut bacteria with ability to metabolize dietary phytoestrogen into metabolite such as equol which have higher affinity for endogenous estrogen receptors. We have previously shown that patients with multiple sclerosis (MS), a neuroinflammatory disease lack gut bacteria with the ability to metabolize phytoestrogen. We have validated the importance of phytoestrogen and gut bacteria in disease protection utilizing an animal model of MS. Specifically, we have shown that an isoflavone rich diet can protect from neuroinflammatory diseases and that protection was dependent on the ability of gut bacteria to metabolize isoflavone into equol. Additionally, mice on a diet with isoflavone showed an anti-inflammatory response compared to the mice on a phyto-free diet. However, it is unknown how isoflavone and/or equol mediates its protective effect especially the effect of isoflavone on host metabolomics.

Objectives

In this study we utilized untargeted metabolomics to identify the metabolites modulated by the presence of dietary isoflavone.

Results

We found that the consumption of isoflavone increases anti-inflammatory monounsaturated fatty acids and beneficial polyunsaturated fatty acids while reducing pro-inflammatory glycerophospholipids, sphingolipids, phenylalanine metabolism, and arachidonic acid derivatives.

Conclusion

Isoflavone consumption alters the systemic metabolic landscape through resultant increase in monounsaturated fatty acids and beneficial polyunsaturated fatty acids while reducing proinflammatory metabolites and pathways. This highlights a potential mechanism by which an isoflavone diet modulate immune mediated disease.

phytoestrogen

isoflavone

inflammation

untargeted metabolomics

microbiome

multiple sclerosis

Recently, diet has emerged as an important regulator of human health. Aside from nutrition it provides numerous bioactive molecules such as polyphenols, flavonoids, phytoestrogens, carotenoids, and vitamins necessary for maintaining a healthy state (Sun et al., 2019). Trillions of bacteria (microbiota) living in our gut play an important role in mediating the beneficial effects of dietary compounds as they help in breaking down original plant compounds into more potent metabolites (Jensen et al., 2021). Thus, consuming a healthy diet full of vegetables and fruits will enrich the growth of healthy gut microbiota, which in turn will help produce beneficial metabolites necessary for maintaining health. For example, plant-based foods containing phytoestrogen compounds are thought to be beneficial for human health due to their structural similarity with estrogens which allow them to bind and activate endogenous estrogen receptors (ERs). Phytoestrogen compounds present in plant-based food have weak affinity for ERs, but numerous bacteria present in the human gut have enzymes that can metabolize dietary phytoestrogens into secondary metabolites to enhance their ability to bind and activate endogenous ERs (Aboushanab et al., 2021). Certain gut bacteria such as Parabacteroides, Adlercreutzia, Slackia, Lactobacillus and Bifidobacterium genera can metabolize the isoflavone daidzein into S-equol which has higher activity than daidzein itself (Mayo et al., 2019). For simplicity S-equol will be referred to as equol throughout the rest of this work. The loss of these bacteria and absence of equol may create a void in which non-commensal bacteria may establish themselves and predispose to disease by inducing the production of pro-inflammatory mediators.

Therefore, it is not surprising that multiple human diseases are linked with the loss of beneficial metabolites and gut microbiota (dysbiosis) which is characterized by an increase in pro-inflammatory mediators plus pathogenic gut bacteria and a decrease in anti-inflammatory mediators (Mangalam et al., 2021). Interestingly, we have previously shown that people with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system lack gut bacteria with the ability to metabolize dietary phytoestrogen (Chen et al., 2016). Besides MS, isoflavone has been associated with protection against other diseases such as cardiovascular disease, osteoporosis, prostate cancer, breast cancer, atherosclerosis, and diabetic conditions (Pabich et. al, 2019; Sathyapalan et al., 2018). Isoflavones are also being studied as a treatment strategy for atrophy, menopause, and postmenopausal symptoms (Pabich et al., 2019).

We validated the importance of phytoestrogen metabolizing bacteria in MS utilizing experimental autoimmune encephalomyelitis (EAE), an animal model of MS (Jensen et al., 2021). In this study we showed that mice on an isoflavone diet developed milder disease compared to those on a phytoestrogen-free (phyto-free) diet. Most importantly, the isoflavone diet-mediated disease protection was dependent on the presence of phytoestrogen metabolizing gut bacteria specifically those lacking in patients with MS (pwMS). We also observed that compared to mice on a phyto-free diet, mice on an isoflavone diet demonstrated lower inflammatory responses as measured by antigen-specific T cell proliferation and cytokine levels (Jensen et al., 2021). In summary, published studies and our data established an important role of an isoflavone diet in suppressing inflammatory diseases (Aboushanab et al., 2021; Chen et al., 2016; Jensen et al., 2021; Mayo et al., 2019; Pabich et al., 2019; Sathyapalan et al., 2018).

Due to structural similarity of equol to endogenous estrogen, it can interact with ERs which are expressed in majority of mammalian cells and tissue (Warner et al., 2017). Thus, equol could have systemic effect beyond the gut. Further, recent studies have also highlighted thousands of metabolites present in the human blood play an important role in maintaining the overall health by regulating numerous physiological processes (François et al., 2021; Poisson et al., 2015; Rangel-Huerta et al., 2017; Zhang et al., 2015). Based on these data, we hypothesized that isoflavone and its metabolite equol may mediate its disease-protective effect through modulation of the host metabolome specifically the balance between pro and anti-inflammatory metabolites.

Therefore, in this study we investigated whether mice on a diet with isoflavone had a distinct metabolite profile compared to mice on diet lacking isoflavone (and all other phytoestrogens). We observed significant differences in the metabolome compositions between the two diets. In fact, we observed enrichment of metabolites linked with a healthy state in mice on a diet with isoflavone and a consequent increase in metabolites linked with disease state in mice on a phyto-free diet. Ours is the first study to profile the metabolome using untargeted metabolomics in mice on a diet with or without isoflavone. These findings give us an insight into how isoflavone might provide protection from diseases through alteration of specific metabolites which can be harnessed for development of future therapy for MS as well as other inflammatory diseases.

2.1 Experimental mice and diet treatment

C57BL/6J male mice (4–6 weeks old) were purchased from the Jackson Laboratories (Bar Harbor, ME) and allowed to adjust to their new environment for 1–2 weeks. Mice (n = 10) were then switched to a diet with isoflavone [genistein (0.24 g/kg of diet) and daidzein (0.22 g/kg of diet)]] or on a diet without any phytoestrogen (Envigo, Indianapolis, IN) ad libitum for 6 weeks as described previously (Jensen et al., 2021).

2.2 Serum sample collection

At the end of the six-week diet, blood was collected, and the serum was separated by standard blood clotting method from all mice. These serum samples were stored at -80℃ and shipped to Metabolon (Morrisville, NC) for metabolite identification.

2.3 Untargeted metabolomics

Metabolic profiling was performed by Metabolon Inc. (Morrisville, NC) using proprietary methods as previously described by our group (Mangalam et al., 2013). In brief Metabolon Inc. extracts proteins and small molecules from the sample utilizing methanol and intense shaking followed by centrifugation. For metabolite identification, Metabolon then utilizes reverse-phase ultra-high performance chromatography mass spectrometry with negative and positive ion mode electrospray ionization and hydrophilic interaction ultra-performance liquid chromatography mass spectrometry. Metabolon also ran several controls along with our samples to assure quality measurements. Compounds were then identified based on their retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (MS/MS spectral data) by comparison to a library of compounds. The output of the workflow was a data table containing the abundance of each metabolite present in each sample.

2.4 Statistical analysis of metabolites

Both MetaboAnalyst 5.0 (Pang et al., 2021)d (R Core Team, 2022) (version 4.1.1) were utilized for statistical analyses of the metabolites observed in the serum samples from both groups. Overall data was first filtered based on missing values. If a given metabolite had missing values for more than 50% of the samples in each group, it was removed. All other missing values were filled with ⅕ the minimum value for the given metabolite. Data were then normalized by sum scaling and log (base 10) transforming. For univariate testing we utilized a Wilcoxon Rank Sum test and the p-values were adjusted by utilizing Benjamini-Hochberg procedure. Results were considered significant if the p-value < 0.05 and the adjusted p-value was < 0.1. Univariate tests were further visualized utilizing ggplot2 (Hadley Wickham, 2016). Pathway analysis utilized Holm-Bonferroni for a stricter p-value adjustment. As this correction is stricter, we consider a Holm’s p < 0.20 significant.

3.1 Isoflavone alters the metabolome

First, we generated a Principal Coordinate Analysis (PCA) plot to determine whether isoflavone affected the overall composition of the metabolome (Fig. 1). Based on the PCA, the first and second PC components explained 36.2% and 15.8% of the variance, respectively, between the two diets. The mice on a diet with isoflavone clustered separately from mice on a phyto-free diet suggesting the metabolite composition was distinct between the two diets.

We next performed hierarchical clustering based on the Euclidean distance of the top 25 features (Fig. 2). The heatmap generated by this analysis showed distinct groupings of metabolites based on diet. Specifically, 8 of the 25 metabolites were increased and 17 were decreased in the isoflavone diet group compared to the phyto-free diet. The metabolites found to be increased in the isoflavone group were equol sulfate, 4-ethylphenyl sulfate, linolenate (18:3n3), palmitoleate (16:1n7), linoleate (18:2n6), 10-heptadecenoate, oleate/vaccenate (18:1), and palmitate (16:0). The metabolites found to be decreased in isoflavone were p-cresol sulfate, 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0), hydroxyproline, 2-amino-p-cresol sulfate, phenylalanine, 1-stearoyl-2-docosahexaenoyl-GPE, sphingosine 1-phosphate, 1-oleoyl-2-docosahexaenoyl-GPE, 1-oleoyl-2-arachidonoyl-GPE, sphingomyelin (d18:1/20:0, d16:1/22:0), sphingomyelin (d18:1/22:1, d18:2/22:0, d161/24:1), 1-2-dipalmitoyl-GPC, stearoyl sphingomyelin, adenosine, sphingomyelin (d18:2/24:1/18:0), 1-palmitoyl-2-docosahexaenoyl-GPC, 1-stearoyl-2-linoleoyl-GPCF. Thus, hierarchical clustering-based analysis suggested that presence of isoflavone in diet had an effect on host metabolites as highlighted by enrichment or depletion of specific metabolites.

3.2 Individual metabolites are altered on an isoflavone diet

To identify metabolite-based biomarkers specific to the isoflavone diet, we used the nonparametric Wilcoxon Rank Sum test. While only considering metabolites significant if their p-value was < 0 .05 and their adjusted p-value (FDR) was < 0.10, we found 132 significantly different metabolites. 109 of these metabolites were significantly increased while 23 were decreased in the isoflavone diet compared to the phyto-free diet.

All 8 metabolites found to be increased in the heatmap were also identified by univariate test as significantly increased: equol sulfate, 4-ethylphenyl sulfate, linolenate (18:3n3), palmitoleate (16:1n7), linoleate (18:2n6), 10-heptadecenoate, oleate/vaccenate (18:1), and palmitate (16:0) (Fig. 3A-H).

The univariate test identified 8 of the 17 metabolites previously found in the heatmap analysis as significantly decreased in the isoflavone-consuming mice. These metabolites include p-cresol sulfate, 2-amino-p-cresol sulfate, 1-stearoyl-2-docosahexaenoyl-GPE, sphingomyelin (d18:1/20:0, d16:1/22:0), 1-oleoyl-2-docosahexaenoyl-GPE, stearoyl sphingomyelin, adenosine, and 1-oleoyl-2-arachidonoyl-GPE (18:1/20:4) (Fig. 4A-H). Significant values are in Table 1. Similar to hierarchical clustering-based analysis, univariate test also suggested that dietary isoflavone influence overall host metabolite composition with modulation of specific metabolites.

Table 1

**Individual metabolites altered in mice on an isoflavone diet.** This table contains the metabolites significantly altered in *both* the heatmap clustering and univariate analysis. The adjusted p-value was calculated utilizing the Benjamini-Hochberg procedure.
Metabolite	p-value	BH adjusted p-value	Isoflavone diet vs Phyto-free diet
Equol sulfate	.0079	.0804	Increased
4-ethylphenyl sulfate	.0079	.0804	Increased
Linolenate (18:3n3)	.0079	.0804	Increased
Linoleate (18:2n6)	.0079	.0804	Increased
10-heptadecenoate	.0079	.0804	Increased
oleate/vaccenate (18:1)	.0079	.0804	Increased
Palmitate (16:0)	.0079	.0804	Increased
Palmitoleate (16:1n7)	.0079	.0804	Increased
P-cresol sulfate	.0079	.0804	Decreased
2-amino-p-cresol sulfate	.0079	.0804	Decreased
1-stearoyl-2-docosahexaenoyl-GPE	.0079	.0804	Decreased
sphingomyelin (d18:1/20:0, d16:1/22:0)	.0079	.0804	Decreased
1-oleoyl-2-docosahexaenoyl-GPE,	.0079	.0804	Decreased
stearoyl sphingomyelin	.0079	.0804	Decreased
adenosine	.0079	.0804	Decreased
1-oleoyl-2-arachidonoyl-GPE (18:1/20:4)	.0159	.0962	Decreased

3.3 Metabolic pathways are altered on an isoflavone diet

As multiple metabolites varied between plasma from mice on a diet with or without isoflavone, we next investigated specific metabolic pathways affected by the diet. This analysis was performed with MetaboAnalyst, and the pathway analysis parameters included the Global Test for the enrichment methods and the Relative-betweenness Centrality for the topology analysis. The KEGG Mus musculus pathway library was chosen for identification. The pathways were considered significant if they had a p-value < 0.05 and a Holm’s p < 0.20. With these parameters we found 15 pathways significantly altered: Purine Metabolism, Selenocompound Metabolism, Tryptophan Metabolism, Thiamine Metabolism, Aminoacyl-tRNA Biosynthesis, Galactose Metabolism, Arachidonic Acid Metabolism, Linoleic Acid Metabolism, Alpha-Linolenic Acid Metabolism, Starch and Sucrose Metabolism, Phenylalanine Metabolism, Vitamin B6 Metabolism, Phosphatidylinositol Signaling System, Glutathione Metabolism, and Sphingolipid Metabolism. These pathways and their significant values can be seen in Table 2. Thus, the pathway analysis showed that presence of isoflavone in diet resulted in modulation of specific metabolic pathways.

Table 2

**Metabolic pathways altered in mice on an isoflavone diet.** This table contains the pathways significantly altered between isoflavone and phyto-free diet based on the Global Test. The adjusted p-value was calculated utilizing the Holm-Bonferroni method.
Pathway	p-value	Holm’s p-value
Purine Metabolism	. 00024	.0127
Selenocompound Metabolism	.00042	.0127
Tryptophan Metabolism	.0012	.024
Thiamine Metabolism	.0028	.0410
Aminoacyl-tRNA Biosynthesis	.0056	.0410
Galactose Metabolism	.0062	.0410
Arachidonic Acid Metabolism	.0068	.0410
Linoleic Acid Metabolism	.0068	.0410
Alpha-linoleic Acid Metabolism	.0068	.0410
Starch and Sucrose Metabolism	.0068	.0410
Phenylalanine Metabolism	.00836	.0456
Vitamin B6 Metabolism	.012	.062
Phosphatidylinositol Signaling System	.0324	.141
Glutathione Metabolism	.0328	.141
Sphingolipid Metabolism	.0453	.171

The significance of diet and gut microbiota in the pathobiology of MS is increasingly appreciated, specifically their potential as novel therapeutic agents (Bjørnevik et al., 2017; Yu et al., 2022; Zahoor et al., 2021). We and others have previously shown that MS patients have gut dysbiosis with depletion of beneficial gut bacteria, notably those able to metabolize phytoestrogens (Chen et al., 2016; Storm-Larsen et al., 2019). Further, using the animal model of MS, we have shown that an isoflavone (phytoestrogen) rich diet can protect mice from EAE, but this protection was dependent on the presence of specific gut bacteria and their ability to metabolize dietary phytoestrogen into equol (Jensen et al., 2021). In this study we performed untargeted metabolomics in the sera of mice on a diet with or without isoflavone and have shown enrichment or reduction of specific metabolites in mice on a diet with or without isoflavone.

We observed that isoflavone diet affected host metabolism as highlighted by a distinct metabolite composition in mice on a diet with or without isoflavone. Several studies have shown that the beneficial effects of a diet or a dietary supplement are associated with global change in host metabolome (Díaz-Rubio et al., 2015; Rangel-Huerta et al., 2017; Torres Santiago et al., 2019). Healthy adults consuming antioxidant rich juice (n = 28) (Díaz-Rubio et al., 2015) or consuming orange juice (n = 30) (Rangel-Huerta et al., 2017) showed a distinct metabolic profile compared to the baseline. Similarly, Santiago et al. reported a change in urinary and fecal metabolite in Wistar rats consuming oral resveratrol compared to the water group (Torres Santiago et al., 2019). Thus, our data showing a distinct metabolic profile in mice on an isoflavone diet is in accordance with previous studies reporting that dietary supplement can have a global effect on host metabolites.

Heat map clustering analysis, which measures the samples’ Euclidean distances from one another, showed modulation of the top 25 metabolites; with 8 being increased and 17 being decreased in mice on the isoflavone diet compared to the phyto-free diet. Using Wilcoxon Signed Rank Test for a univariate analysis, we identified 132 metabolites to be significantly different between these two groups. Specifically, there were 110 metabolites significantly increased and 22 metabolites significantly decreased on the isoflavone diet compared to the phyto-free diet. Of note, equol sulfate was the most increased of any metabolite, and this is expected as isoflavone is broken down into equol which is then converted into equol sulphate. This finding serves as a positive control that our diets did in fact differ by presence of isoflavone which was metabolized into equol by gut bacteria (Díaz-Rubio et al., 2015). Analysis of specific pathways showed that several pathways were different between the two groups, specifically phenylalanine metabolism, linoleic acid metabolism, alpha-linolenic acid metabolism, sphingomyelin metabolism, and arachidonic acid metabolism.

Numerous significantly altered metabolites were linked to phenylalanine (Phe) metabolism. Phe is an aromatic amino acid, which is essential for the host and obtained from diet as the host does not naturally produce it. Dietary Phe is broken down into Tyrosine (Tyr) or 4-hydroxyphenylpyruvic acid (Gryp et al., 2017). Both of these metabolites can further be broken down into 4-ethylphenyl sulfate, p-cresol sulfate, and p-cresol glucuronide (Gryp et al., 2017). Phenylalanine was decreased in the isoflavone diet along with its secondary metabolites p-cresol sulfate (PCS) and p-cresol glucuronide (PCG). PCS and PCG are broken down from Phe and Tyr through anaerobic intestinal bacteria (Wishart et al., 2022). Interestingly, pwMS have higher levels of PCS in plasma as well as cerebrospinal fluids and PCS can induce axonal damage in cultured neurons (Ntranos et al., 2022). Additionally, pwMS treated with dimethyl fumarate showed decreased levels of PCS compared to untreated group (Ntranos et al., 2022). PCS had also been proposed as a biomarker for autism spectrum disorder (ASD) as its levels are elevated in the urine and feces of children with ASD (el Aidy et al., 2021). The current understanding is that PCS passes the blood-brain-barrier and affects microglia and neurons to induce neuroinflammation and axonal damages in neurological diseases such as MS and ASD. Thus, isoflavone may reduce neuroinflammation by modulation of phenylalanine metabolism specifically by reduction in the level of pro-inflammatory PCS.

Further investigation of the significant metabolites revealed isoflavone had altered fatty acid metabolism. Firstly, we found palmitoleate, oleate, linoleate, and linolenate were all significantly increased in isoflavone diet. Palmitoleate is the salt form of the monounsaturated fatty acid, palmitoleic acid, and is modulated in several inflammatory diseases but depending on the disease, palmitoleate levels had been shown to be either increased or decreased (Akazawa et al., 2021). Oleate is also the salt form of a monounsaturated fatty acid, oleic acid, which had shown to possess anti-inflammatory activity. In fact, fatty tissue from pwMS lack oleic acid and it is suggested to be responsible for regulatory T cell (Treg) dysfunction (Pompura et al., 2021). The exposure of Tregs from pwMS and healthy controls (HC) with oleic acid in culture restored suppressive function of these Tregs (Pompura et al., 2021). Additionally, presence of both palmitoleate and oleate had been linked with reduced inflammation in skeletal muscle cells (Coll et al., 2008). Thus, isoflavone may also mediate its anti-inflammatory effect through induction of the monounsaturated fatty acids palmitoleate and oleate both of which had been linked with the healthy state of the host.

Additionally, linoleate and linolenate, both polyunsaturated fatty acids (PUFAs) and the salt form of linoleic acid and linolenic acid, respectively, were increased in the mice on an isoflavone diet. Metabolism of linoleic acid and alpha-linolenic acid (ALA) were also found to be pathways significantly modulated based on our pathway analysis. PUFAs have been linked to protection from many inflammatory diseases including MS. A study evaluating effects of dietary intake of PUFA and MS risk showed that among all PUFA tested, only ALA showed inverse association with MS risk (Bjørnevik et al., 2017). In a follow-up study, the group reported that the higher serum levels of ALA were associated with lower disease activity in pwMS (Bjornevik et al., 2019). Additionally, a recent study had shown that pwMS with high ALA levels showed reduced risk of relapse and longer time to convert from clinically isolated syndrome to clinically definite multiple sclerosis (Munger et al., 2022). Utilizing an animal model of MS, we have also identified metabolites linked with PUFA, such as ALA and linoleic acid as significant biomarkers of disease (Mangalam et al., 2013; Poisson et al., 2015). Additionally, we showed that resolvin D1 (RvD1), a downstream metabolite of ALA, ameliorated disease in a chronic EAE model (Poisson et al., 2015; Zahoor et al., 2021). Thus, increased levels of beneficial PUFAs in mice on a diet with isoflavone suggest that PUFAs may have a role in creating an anti-inflammatory environment due to the consumption of isoflavone.

As for fatty acids that were depleted on an isoflavone diet compared to phyto-free, glycerophospholipids and sphingolipids from Phosphatidylcholine (PC) metabolism and ceramide metabolism were significantly decreased. Specifically, the glycerophospholipids involved in PC metabolism that were depleted in the isoflavone group include 1-stearoyl-2-docosahexaenoyl-GPE, 1-palmitoyl-2-docosahexaenoyl-GPC, 1-2-dipalmitoyl-GPC, 1-stearoyl-2-linoleoyl-GPC, and 1-oleoyl-2-docosahexaenoyl-GPE (Wishart et al., 2022). A study on the cerebral spinal fluid of MS and non-MS patients found that 4 of the 6 glycerophospholipids analyzed were significantly increased in MS patients (Nogueras et al., n.d.). Suggesting that glycerophospholipid composition alteration contributes to the disease state and thus the suppression of these metabolites on a diet with isoflavone supports an anti-inflammatory role of isoflavone.

PC also contributes to the production of sphingolipids with the help of ceramides (Li et al., 2008). Ceramides are lipid molecules that are metabolized into sphingolipids, namely sphingomyelin and sphingosine, which can be further broken down into sphingosine-1-phosphate (Hannun et al., 2010). Many structures of sphingomyelin, stearoyl sphingomyelin, and sphingosine-1-phosphate were found to be significantly decreased on the isoflavone diet. Additionally, sphingolipid metabolism was found to be a distinguishing pathway between our two diets based on our pathway analysis. Sphingomyelins are found in animal cell membranes and most abundantly in the myelin sheath protecting nerve cell axons (Jana & Pahan, 2010). Sphingosine-1-phosphate is a signaling molecule also found in animal cell membranes are present during inflammation (Spiegel et al., 2011). In fact, sphingosine 1-phosphate is activated by pro-inflammatory cytokines and is involved in T-cell trafficking. One MS treatment involves the use of sphingosine 1-phosphate receptor modulators to mediate its beneficial effect in pwMS (Mcginley et al., 2021). Due to the reduction in PC metabolism and these ceramide derivatives, our results suggest isoflavone promotes an anti-inflammatory environment by reducing the metabolism of glycerophospholipids and sphingolipids.

Another lipid alteration found on the isoflavone diet was a reduction in the arachidonic acid derivative, 1-oleoyl-2-arachidonoyl-GPE. Arachidonic acid was also identified as a significant pathway in our pathway analysis. Arachidonic acid metabolism produces prostaglandins (Jewell et al., 2022), which are lipids that are produced in damaged and infected tissues to control inflammation, clotting, and overall blood flow (Ricciotti et al., 2011). The reduction of arachidonic acid metabolism in mice on isoflavone diet suggests that the isoflavone diet can reduce inflammation through suppression of pro-inflammatory arachidonic metabolism.

In conclusion, dietary isoflavone alters the host metabolome by promoting anti-inflammatory metabolites and suppressing pro-inflammatory metabolites. Specifically, we found that the major alterations were seen in phenylalanine metabolism and lipid metabolism. Several monounsaturated and beneficial PUFAs were increased on an isoflavone diet compared to phyto-free. Whereas several pro-inflammatory metabolites, including glycerophospholipids, sphingolipids, and fatty acids derived from arachidonic acid, were all depleted in the isoflavone diet. The pathways that were important in distinguishing the two diets directly relate to these findings as the metabolism of linoleic acid, ALA, phosphatidylcholine, ceramide, sphingomyelin, and arachidonic acid were all considered significant based on our pathway analysis. These pathways, and many of their metabolites, have been associated with inflammatory diseases as aforementioned. Overall, these results indicate that dietary isoflavone induces changes in the systemic metabolome potentially by alteration of gut microbiome and/or host response. Our study provides a rationale for future studies investigating the mechanism through which dietary isoflavone modulate host metabolic network specifically phenylalanine and lipid metabolism.

Acknowledgements and Funding: We acknowledge funding from the National Institutes of health/NIAID 1RO1AI137075 (A.K.M), Veteran Affairs Merit Award 1I01CX002212 (A.K.M), University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH P30 ES005605 (A.K.M), Gift from P. Heppelmann and M. Wacek to A.K.M and Carver Trust Pilot Grant (A.K.M). R.L.S. was supported by the Informatics Fellowship from the University of Iowa. S.N.J was supported on an institutional training grant (T32AI007485 to G. Bishop) and diversity supplement award to A.K.M on parent 1RO1AI137075.

Author Contributions: Conceptualizations, Nicole Cady, Samantha N. Jensen, and Ashutosh K. Mangalam; methodology, Rachel L. Shrode., Nicole Cady, Samantha N. Jensen, Nick Borcherding, and Ashutosh K. Mangalam; validation, Rachel L. Shrode , Nick Borcherding, and Ashutosh K. Mangalam; formal analysis, investigation, resources, and data curation, Rachel L. Shrode; writing – original draft preparations Rachel L. Shrode; writing – review and editing, Rachel L. Shrode, Nicole Cady, Samantha N. Jensen, Nick Borcherding, and Ashutosh K. Mangalam; supervision Nick Borcherding, and Ashutosh K. Mangalam, funding acquisitions Ashutosh K. Mangalam. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: All procedures were done according to the Institutional Animal Care and Use Committee (IACUC) guidelines at the University of Iowa.

Data Availability Statement: Metabolomic data utilized for analysis is located in the supplementary data as Table S1.

Conflicts of Interest: A.K.M. is one of the inventors of a technology claiming the use of Prevotella histicola to treat autoimmune diseases. A.K.M. received royalties from Mayo Clinic (paid by Evelo Biosciences). However, no fund or product from the patent were used in the present study. All other authors declare no commercial or financial relationships that could be a potential conflict of interest.

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Competing interest reported. A.K.M. is one of the inventors of a technology claiming the use of Prevotella histicola to treat autoimmune diseases. A.K.M. received royalties from Mayo Clinic (paid by Evelo Biosciences). However, no fund or product from the patent were used in the present study. All other authors declare no commercial or financial relationships that could be a potential conflict of interest.

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Isoflavone consumption reduces inflammation through modulation of phenylalanine and lipid metabolism

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Experimental mice and diet treatment

2.2 Serum sample collection

2.3 Untargeted metabolomics

2.4 Statistical analysis of metabolites

3. Results

3.1 Isoflavone alters the metabolome

3.2 Individual metabolites are altered on an isoflavone diet

3.3 Metabolic pathways are altered on an isoflavone diet

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

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