Vitamin D Receptor Promotes Healthy Microbial Metabolites and Microbiome

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com/scientificreports
OPEN Vitamin D receptor promotes

healthy microbial metabolites and
microbiome
Ishita Chatterjee1, Rong Lu1, Yongguo Zhang1, Jilei Zhang1, Yang Dai 2, Yinglin Xia1 ✉ &
Jun Sun 1 ✉
Microbiota derived metabolites act as chemical messengers that elicit a profound impact on host
physiology. Vitamin D receptor (VDR) is a key genetic factor for shaping the host microbiome. However,
it remains unclear how microbial metabolites are altered in the absence of VDR. We investigated
metabolites from mice with tissue-specific deletion of VDR in intestinal epithelial cells or myeloid cells.
Conditional VDR deletion severely changed metabolites specifically produced from carbohydrate,
protein, lipid, and bile acid metabolism. Eighty-four out of 765 biochemicals were significantly altered
due to the Vdr status, and 530 significant changes were due to the high-fat diet intervention. The
impact of diet was more prominent due to loss of VDR as indicated by the differences in metabolites
generated from energy expenditure, tri-carboxylic acid cycle, tocopherol, polyamine metabolism, and
bile acids. The effect of HFD was more pronounced in female mice after VDR deletion. Interestingly,
the expression levels of farnesoid X receptor in liver and intestine were significantly increased after
intestinal epithelial VDR deletion and were further increased by the high-fat diet. Our study highlights
the gender differences, tissue specificity, and potential gut-liver-microbiome axis mediated by VDR that
might trigger downstream metabolic disorders.
Metabolites are the language between microbiome and host1. To understand how host factors modulate the
microbiome and consequently alter molecular and physiological processes, we need to understand the metabo-
lome — the collection of interacting metabolites from the microbiome and host.
Vitamin D/VDR signaling contributes to the genetic, environmental, immune, and microbial aspects of
human diseases (e.g., inflammatory bowel disease and obesity)2,3. The human Vdr gene is the first gene identified
as a vital host factor that shapes the gut microbiome at the genetic level4. In mice lacking VDR, we observed
significant shifts in the microbiota relative to control mice. In humans, correlations between the microbiota and
serum measurements of selected bile acids and fatty acids were detected4. Those metabolites include known lig-
ands and downstream metabolites of VDR5. Moreover, we have demonstrated that VDR knock out (KO) (Vdr−/−)
mice have depleted Lactobacillus and enriched Clostridium and Bacteroides in feces. Notably, in the cecal con-
tent, Alistipes and Odoribacter were significantly reduced whereas Eggerthella was increased6. Intestinal specific
deletion of VDR (VDRΔIEC) leads to microbial dysbiosis due to a decrease in the butyrate-producing bacteria7,8.
However, it is unclear how the loss of VDR impacts microbial metabolites.
In the current study, we hypothesize that host factors (e.g., VDR status in specific tissues) modulate microbial
metabolites and the microbiome, thus contributing to the high risk of metabolic diseases. We used intestinal
epithelium-specific VDR knock out (VDRΔIEC) mice and myeloid cell-specific VDR KO (VDRΔlyz) mice to assess
whether the microbiome-associated metabolic changes linked with conditional loss of VDR in a particular tissue.
Because the majority of metabolic syndromes are multifactorial, we further evaluated the effect of high-fat diet
(HFD) on VDRΔIEC mice as compared to control chow diet-fed mice. We also correlated the altered metabolite
profiles to specific mechanisms that lead to the observed changes in the host and microbiome.
1
Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago,
USA. 2Department of Bioengineering, University of Illinois at Chicago, Chicago, USA. ✉e-mail: yxia@uic.edu;
junsun7@uic.edu
Scientific Reports | (2020) 10:7340 | https://doi.org/10.1038/s41598-020-64226-7 1

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Statistical Comparisons
VDRΔIEC/VDRLoxP
ANOVA contrasts Chow
Total biochemicals p ≤ 0.05 68
Biochemicals (↑↓) 35 33
Total biochemicals 0.05 < p < 0.10 55
Biochemicals (↑↓) 25 30
Table 1. Intestinal epithelial VDR on the profile of metabolites.
Results
Deletion of intestinal epithelial VDR impacted the overall metabolite profile. First, we exam-
ined the effects of intestinal epithelial VDR on the metabolite profile. Among named biochemical compounds,
VDRΔIEC mice exhibited alterations in 68 metabolites (of which 35 increased and 33 decreased) with P ≤ 0.05 sig-
nificance level and 55 biochemicals with 0.05 < P < 0.1 significance level (of which 25 increased and 30 decreased)
(Table 1).
Random Forest (RF) analysis of metabolites among chow diet-fed animals revealed the impact of different
metabolites. Figure 1A shows a list of the top 30 biochemicals that contribute to maximum importance. Of these,
maltose, maltotriose, and ceremide are among the top three most significant differentially expressed biochem-
icals. The action of the intestinal microbiome is responsible for the generation of several metabolites derived
from carbohydrates, amino acids, bile acids, heme, and other dietary sources. Several of these metabolites are
reabsorbed in the gut and can bind to cellular receptors, thus potentially influencing host functions9. The most
significant alterations in metabolites were generated by four main super-pathways including (A) carbohydrate,
(B) protein/ amino acids, (C) lipid, and (D) xenobiotics metabolism.
Carbohydrate metabolism. Carbohydrates are the primary source of energy for gut microbiota. Colonic bacte-
ria ferment nondigestible complex-carbohydrates and release glycogen, amino-sugars, and pentoses10. These are
considered major factors in shaping the composition and physiology of the microbiome. As shown in Fig. 1B,
VDRΔIEC had a significant (P ≤ 0.05) increase in amino-sugar metabolite N-acetylmuramate compared to the
VDRLoxP control mice. Conversely, N-acetylglucosaminylasparagine, glucose, and galactonate were downregu-
lated in the VDRΔIEC group (Fig. 1B). These results suggest that the loss of intestinal epithelial VDR in host modi-
fies the carbohydrate metabolism, thus affecting mainly glycolysis, gluconeogenesis, pyruvate, fructose, mannose
galactose, and amino-sugar metabolism pathways.
Protein/Amino-acid metabolism. Gut bacteria produce a range of metabolites by synthesizing proteinogenic

amino acids via protein fermentation11. These metabolites are known to exert beneficial or harmful effects
on the host. VDRΔIEC mice showed increased levels of N1,N12-diacetylspermine, N(‘1)-acetylspermidine,
N-acetylglutamate, N2-acetyllysine, and diacetylspermidine (Fig. 1C), indicating a significant surge in polyam-
ine, glutamate, and lysine metabolism. A decrease in tryptophan and methionine metabolism in the VDRΔIEC
group was indicated by decreased taurine, kynurine, and other related metabolites (Fig. 1C).
Lipid metabolism. Gut bacteria affect lipid metabolism through multiple direct and/or indirect mecha-
nisms, including bile acid metabolism, cholesterol transport, and energy expenditure12,13. We found a signif-
icant increase of octadecanedioate, 1-stearoyl-2-oleoyl-GPE, 1-palmitoyl-2-linoleoyl-galactosylglycerol,
1-palmitoyl-galactosylglycerol, and 1-oleoyl-GPG in VDRΔIEC mice compared to those in the VDRLoxP mice. In
contrast, the levels of valerylglycine, trimethylamine N-oxide, glycerophosphoserine, glycerophosphoinositol,
and 2-hydroxyheptanoate were decreased in the VDRΔIEC mice (Fig. 1D), indicating that fatty acid and phospho-
lipid metabolism was altered in the VDRΔIEC mice.
Xenobiotics/Others. Xenobiotics are the extrinsic molecules ingested by the host from the environment and
are subsequently metabolized by microorganisms and transformed into hundreds of metabolites14. VDR defi-
ciency in the intestinal epithelium altered the levels of many xenobiotics. RF analysis of animals showed that
naringenin, a flavonoid displays strong anti-inflammatory and antioxidant function, among the pool of top 30
metabolites (Fig. 1A). Further ANOVA analysis indicates that most xenobiotics were significantly downregulated
in the VDRΔIEC mice (Fig. 1E). Ergothioneine (ET), an anti-oxidant sulfur-containing derivative of the histidine
(amino acid) was the only xenobiotic found to be upregulated in VDRIEC (Fig. 1E).
Myeloid cell-specific VDR knockdown contributes to the definitive alteration of metabolites

indicating the tissue-specific role of host VDR. VDR is known to have tissue and cell-specific roles15.
Hence, we examined our myeloid cell-specific VDR KO model. In the VDRΔlyz mice, 100 known biochemicals
were found to be significantly altered with P-value ≤0.05 (of which 56 increased and 44 decreased) and 58 chem-
icals showed with significance level 0.05 < P < 0.10 (of which 28 increased and 30 decreased) (Table 2), compared
to chow-fed VDRLoxP control animals.
We compared the change in metabolites derived from VDRΔIEC and VDRΔlyz mice by Welch’s two-sample
t-test and found 118 metabolites were significantly changed (P ≤ 0.05) (black box, Table 2) and another 49 fell in

A Maltose
Maltotriose
Ceramide (d18:1/20:0, d16:1/22:0, d20...
Lactosyl-N-palmitoyl-sphingosine (d18...
Taurohyodeoxycholic acid
Diacetylspermidine*
Increasing Importance to group Separation

1-oleoyl-GPG (18:1)*
3-hydroxylaurate
1-palmitoyl-GPG (16:0)*
10-hydroxystearate
1,2-dilinoleoyl-GPE (18:2/18:2)*
1-palmitoyl-2-linoleoyl-galactosylgly...
N-acetylglutamate
Naringenin
Eicosanoylsphingosine (d20:1)*
Lignoceroyl ethanolamide (24:0)*
Pheophytin A
1-oleoylglycerol (18:1)
Ribulose/xylulose
Behenoyl ethanolamide (22:0)*
1-palmitoyl-GPI (16:0)
N-alpha-acetylornithine
1-stearoyl-2-oleoyl-GPE (18:0/18:1)
N2-acetyllysine
2-oleoylglycerol (18:1)
Color Pathway
Dihydrobiopterin
Quinolinate Amino Acids
Beta-sitosterol Carbohydrate
Cofactors
Arachidate (20:0) Lipids
D-urobilin Xenobiotics
10 15 20 25 30 35 40
Mean Decrease Accuracy
B Carbohydrate Metabolites C Amino Acid Metabolites

Phenylacetyltaurine
N-acetylmuramate Taurine
N-acetylglucosaminylasparagine Cysteine sulfinic acid
Galactonate 3-hydroxyanthranilate
Glucose Kynurenine
N1,N12-diacetylspermine
1.0
1.5
2
0.5
0.0
N('1)-acetylspermidine
FC ratio
VDR
ΔIEC LoxP Diacetylspermidine
/VDR
N2-acetyllysine
N-acetylglutamate
0.5
0.0
2.0
1.0
1.5
FC ratio
ΔIEC LoxP
VDR /VDR
D Lipid Metabolites E Xenobiotics Metabolites
Succinimide
2-hydroxyheptanoate* 2-aminophenol sulfate
Diosmin (diosmetin 7-rutinoside)
Glycerophosphoinositol Tartronate (hydroxymalonate)
Glycerophosphoserine Diosmetin
Soyasaponin III
Trimethylamine N-oxide Soyasaponin II
Soyasaponin I
Valerylglycine Sinapet
Pheophytin A
Pheophorbide A
Naringenin
1-oleoyl-GPG Mannonate*
Homostachydrine*
1-palmitoyl-galactosylglycerol Glycitein
1-palmitoyl-2-linoleoyl-galactosylglycerol Ferulic acid 4-sulfate
Ergothioneine
Equol sulfate
1-stearoyl-2-oleoyl-GPE Equol
Octadecanedioate Cinnamoylglycine
Genistein
2,4,6-trihydroxybenzoate
0.0
1.0
2.0
0.5
1.5
2-hydroxyhippurate (salicylurate)
Hippurate
FC ratio 0.6 1.2 1.8
0
ΔIEC LoxP FC ratio
VDR /VDR ΔIEC LoxP
VDR /VDR
Figure 1. Impact of intestinal epithelial VDR deletion on metabolite profile: (A) Random Forest (RF) analysis
showing the top 30 most important metabolites resulting from using biochemical data derived from VDRLoxP,
VDR∆IEC and VDR∆lyz mice fecal samples. The variables are ordered top-to-bottom as most-to-least important
in categorizing between VDR deleted (VDR∆IEC and VDR∆lyz) and VDRLoxP groups. Different color indicates
specific metabolites resulting from different super-pathways like light blue = amino acid; green = carbohydrate,
purple = cofactors, blue = lipids, Teal= Xenobiotics. Fold change (FC) ratios of the average concentrations
of metabolites between VDRΔIEC mice to that in the VDRLoxP. Graph denotes only those biochemicals which
displayed maximum fold change. Metabolites are listed as their origin of metabolic pathways: (B) carbohydrate
(C) amino acid (D) lipid and (E) xenobiotic. Differences are assessed by the Mann–Whitney U test. VDRΔIEC
(N = 17) & VDRLoxP (N = 16) mice. Significance is established at adjusted 0.05 < P < 0.1.

Statistical Comparison
Chow
VDRΔLYZ/VDRLoxP VDRΔLYZ/VDRΔIEC
Welch’s Two-Sample t-Test All Female Male All Female Male
Total biochemicals p ≤ 0.05 100 74 147 118 44 122
Biochemicals (↑↓) 56|44 52|22 46|101 59|59 26|18 43|79
Total biochemicals
58 45 105 49 31 64
0.05 < p < 0.10
Biochemicals (↑↓) 28|30 24|21 20|85 26|23 19|12 15|49
Table 2. Myeloid cell-specific VDR contributes to the alteration of metabolites different from the Intestinal
epithelial VDR.
A Carbohydrate Metabolites B Amino Acid Metabolites
N-acetylmuramate N-acetyl-cadaverine
Diacetylchitobiose Histidine
Fucose
Matose Proline
Maltotriose Citrulline
Arabinose Ornithine
Xylose
Ribulose/xylulose
N-acetylproline
5.0
0.0
3.0
4.0
1.0
2.0
Cysteine
FC ratio Asparagine
lyz LoxP
VDR / VDR N-methylalanine
2.0
0.0
0.5
1.5
1.0
2.5
FC ratio
lyz LoxP
VDR / VDR
C Lipid Metabolites
D Xenobiotics Metabolites
Behenoyl ethanolamide (22:0)

γ-tocopherol/β-tocopherol
Arachidoyl ethanolamide (20:0)
gamma-tocotrienol
Octadecanedioylcarnitine
Alpha-tocotrienol
Dihomo-linolenoylcarnitine
Alpha-tocopherol
Hexadecasphingosine Nicotinamide
Quinolinate
2-linoleoylglycerol
0 0.4 0.8 1.2 1.6
1,2-dilinoleoyl-GPE FC ratio
lyz LoxP
2-oleoylglycero VDR / VDR
Oleoyl-linoleoyl-glycerol
Palmitoyl-linoleoyl-glycerol
3.5
1.5
2.5
0.5
0
1.0
3.0
FC ratio
lyz LoxP
VDR / VDR
Figure 2. Overall alteration in the metabolites following myeloid cell-specific VDR deletion: Fold change ratio
generated by (A) carbohydrate (B) amino acids (C) lipid and (D) xenobiotics metabolism in VDR∆lyz mice. The
graph represents only those biochemicals showing maximum alterations among known detectable metabolites.
Differences are assessed by the Mann–Whitney U test. VDR∆lyz (N = 10) & VDRLoxP (N = 16). Significance is
established at adjusted 0.05 < P < 0.1.
the significant level 0.05 < P < 0.1. Here, we focused on the major changes in the carbohydrate, amino acids, lipid,
and xenobiotics metabolites in VDR∆lyz model.
Loss of VDR in myeloid cells in the VDR∆lyz group showed altered carbohydrate metabolism and increased
levels of the amino sugar metabolite N-acetylmuramate (Fig. 2A), similar to the VDRΔIEC mice. Unlike the
VDRΔIEC group, VDR∆lyz mice showed an increase in pentose and glycogen metabolism in conjunction with
elevated amounts of ribulose/xylulose, xylose, arabinose, maltotriose, maltose, and fucose (Fig. 2A). A promi-
nent decrease in diacetylchitobiose in VDRΔlyz mice was observed. The VDR∆lyz group also showed decreased
histidine, proline, and citrulline, which was accompanied by a rise in N-acetyl proline and asparagine levels,

compared to VDRLoxP (Fig. 2B). These data suggest a reduction in urea-arginine-proline metabolism and elevated
alanine and aspartate metabolism in VDR∆lyz mice.
In contrast to VDRLoxP mice, VDR∆lyz mice demonstrated a significant increase in palmitoyl-linoleoyl-glycerol
and a substantial decrease in sphingosines and fatty acid metabolism, indicated by metabolites like hexadecas-
phingosine and dihomo-linolenoylcarnitine (Fig. 2C). Remarkably, among the different xenobiotic metabolism
pathways, the VDRΔlyz group displayed a significant downregulation of quinolinate and tocopherol pathway
derived metabolites and an increase in nicotinamide (Fig. 2D). Thus, these data indicate the different roles of
VDR in intestinal epithelial cells and myeloid cells.
VDR deletion significantly impacted bile acid profile, especially in females. VDR is known to
function as a bile acid sensor in the intestine and loss of VDR is known to disquiet the bile acid homeostasis16,17.
Here, we assessed modifications in metabolites from secondary bile acid metabolism pathways. The microbi-
ota converts primary bile acids to secondary bile acids, which are then reabsorbed and can affect diverse bio-
logical processes18. Among different secondary bile acids, lithocholate, and deoxycholate, were increased due
to loss of VDR in VDRΔIEC and in VDR∆lyz mice (Fig. 3A). When comparing VDRΔIEC females to VDRLoxP
females, we found an increase in deoxycholic acid 3 sulphate and in deoxycholate (Fig. 3B). A decrease in
7,12-diketolithocholate, was specifically observed in VDR∆lyz female mice (Fig. 3C).
Both VDR∆IEC and VDR∆lyz female mice showed a significant increase in deoxycholate, 3-dehydrodeoxychlate,
lithocholate, 12-ketolithocholate, de-hydrolithocholate, and 3b-hydroxy-5-cholenoic acid, compared to control
females (Fig. 3B,D). Tauroursodeoxycholic acid sulfate was decreased in the VDR∆IEC and VDR∆lyz female mice.
Overall, our results indicate that VDR deletion significantly influences bile acid metabolism in a gender-specific
manner.
VDR deficiency resulted in significant alterations in the polyamines levels. Polyamines have been
shown to play a role in facilitating a switch between different coactivator complexes that bind to nuclear recep-
tors such as VDR19. Here, we observed a significant elevation in polyamines, such as N1, N12-diacetylspermine
(Fig. 4A), diacetylspermidine (Fig. 4B), and N (‘1)- acetylspermidine (Fig. 4C) in chow-fed VDRΔIEC animals,
compared with the VDRLoxP mice indicating accumulation of polyamines in VDR deficient animals. As noted,
increases in polyamine levels were observed in male VDRΔIEC mice.
Long-chain fatty acids (LCFAS) and acylcarnitines were significantly elevated in VDR defi-
cient mice indicating perturbations with β-oxidation. Fatty acid beta-oxidation is one of the main
energy-yielding metabolic processes. An earlier study has shown that Vitamin D/VDR plays an important role
in the composition of fatty acids via direct regulation of Elovl3 (an FA elongase enzyme) expression20. Hence, we
evaluated the effect of VDR deletion on fatty acid metabolites. We found that carnitines were significantly ele-
vated in fecal samples from VDRΔIEC and VDR∆lyz mice, compared to the VDRLoxP, including myristoylcarnitine
(C14), palmitoylcarnitine (C16), oleoylcarnitine (C18:1) (Fig. 5A–C). This increase is accompanied by an eleva-
tion in long-chain fatty acids (Fig. 5A–C) that were mostly observed in VDRΔIEC and VDR∆lyz females, compared
to VDRLoxP mice (Table 3). Defects in the beta-oxidation of fatty acids can be evaluated based on acylcarnitines
(AC). Substantial increase in acylcarnitines and long-chain fatty acids could be potential indicators of elevated
beta-oxidation in VDR deficient animals. However, there is no significant change in 3-hydroxybutyrate (BHBA).
HFD intervention altered metabolite profile in VDRLoxP and VDRΔIEC mice. Studies have shown
that obese humans and mice have microbiomes very different from their lean controls21–24. We further evaluated
how diet impacted the metabolites in mice with tissue-specific VDR deletion. The 30-top ranking biochemicals in
the importance plot suggests key differences in peptides, lipid metabolism, cofactors, vitamins, and amino acids
with maximum impact on threonyl phenylalanine (Fig. 6A). RF-classification using named metabolites detected
in VDRLoxP and VDRΔIEC with HFD gave a predictive accuracy of 100%.
Principal Component Analysis (PCA) for fecal samples showed clear separation based on the diet (Fig. 6B).
Microbiome-derived metabolites had divergent trends following HFD feeding. Aromatic amino acids like phe-
nyl lactate (PLA), phenethylamine, 3-hydroxyphenylacetate, indole 3-carboxylate, indolelactate, and indole-
propionate were decreased in both VDRLoxP and VDRΔIEC HFD groups, as compared to the regular chow diet
(Fig. 7A). Alternatively, trans and cis-urocanate were significantly increased by HFD (Fig. 7B). Levels of equol, a
microbiota-derived metabolite known to exert epigenetic changes by inhibiting DNA methylation, histone mod-
ification, and regulating ncRNAs, was also found to be altered following VDR deletion and HFD.
Lower levels of tocopherol metabolism were associated with HFD intervention. Interestingly,
HFD-fed VDRΔIEC mice had lower levels of alpha-tocopherol (Fig. 7C) in contrast to VDRLoxP and was noted
important in RF analysis (Fig. 6A). Additional decreases were observed in levels of alpha-tocopherol and gamma
tocopherol/betatocopherol (Fig. 7C) in HFD fed animals. Lower levels of tocopherols in HFD fed VDR defi-
cient animals might be indicative of increased risk for colon cancer. Alternatively, polyamines levels in HFD fed
VDRΔIEC animals were also impacted greatly. Here, we observed significant elevation in levels of polyamines,
such as spermidine, N1, N2-diacetylspermine, in all VDRΔIEC animals especially after HFD feeding, as compared
to VDRLoxP group (Fig. 7D). These data suggest an accumulation of polyamines in HFD fed VDR deficient ani-
mals. Because polyamines have strong anti-inflammatory functions25, these changes may impact aspects of host
immunity.

A
5.0 Lithocholate Deoxycholate
4.0
4.0
3.0
3.0
2.0
2.0
1.0
1.0
0.0 0.0
F M F M F M F M F M F M
LoxP ΔIEC ΔIEC Δlyz
VDR
R VDR
VD VDR Δlyz VDR LoxP VDR VDR
B Secondary bile acid metabolism in female
Tauroursodeoxycholic acid sulfate (2)

3b-hydroxy-5-cholenoic acid
Dehydrolithocholate
12-ketolithocholate
Lithocholate
6-beta-hydroxylithocholate
3-dehydrodeoxycholate
Deoxycholic acid 3-sulfate
Deoxycholate
0 4 8 12 16
FC ratio
ΔIEC LoxP
VDR / VDR
C Secondary bile acid metabolism D Secondary bile acid metabolism in female
Tauroursodeoxycholic acid sulfate (2)

7,12-diketolithocholate
7-ketodeoxycholate
Lithocholate
Taurocholenate sulfate*
6-oxolithocholate
Deoxycholate
Dehydrolithocholate
0.0 0.4 0.8 1.2 1.6
FC ratio 12-ketolithocholate
Δlyz
VDR / VDR LoxP Lithocholate
Deoxycholate
0 2 4 6
FC ratio
Δlyz
VDR / VDR LoxP
Figure 3. VDR deletion altered bile acid (BA) metabolism: Box-plot diagrams displaying changes in secondary
bile acid (A) lithocholate and deoxycholate in control VDRLoxP group as compared to VDRΔIEC and VDR∆lyz
mice. (B) Specific changes in secondary bile acid metabolites as noted in female VDR∆IEC mice. (C) Collective
changes demonstrated by VDR∆lyz mice. (D) Definite variations in secondary bile acid metabolites displayed
by female VDR∆lyz mice. The data presented as the fold change (FC) ratios of the average concentrations of
identified BA species in respective groups. VDRΔIEC group (N = 17; F = 8, M = 9), VDR∆lyz (N = 10; F = 5,
M = 5) & VDRLoxP (N = 16; F = 6, M = 10). Differences are assessed by the Mann–Whitney U test. Significance
is established at an adjusted 0.05 < P < 0.1.
Gender-specific changes in HFD fed mice following VDR deletion. When metabolites were ana-
lyzed based on gender, there was a separation between males and females that were fed HFD (Supplementary
Fig. 1A,B), but in animals fed chow diet this effect was less significant (Fig. 6B).
Intestinal VDR deficiency extensively alters primary and secondary bile acid metabolites and bile acids
are known to shape the gut microbiome especially in obesity. As anticipated, VDR deficiency along with HFD
immensely altered the bile acid levels in fecal samples. Specifically, levels of the bile acids, taurolithocho-
late 3 sulphate, and taurocholenate sulphate were raised in VDR deficient animals (Fig. 8A). Metabolites like
N-acetyltyrosine, N-formylphenylalanine, and indolepropionate were significantly changed in the VDR ΔIEC,

A 8.0
6.0
4.0
2.0
0.0
F M F M F M
Chow Chow Chow
LoxP ΔIEC Δlyz
VDR VDR VDR
Diacetylspermidine*
B 5.0
4.0
3.0
2.0
1.0
0.0 F M F M F M
Chow Chow Chow
LoxP ΔIEC Δlyz
VDR VDR VDR
C 10.0
8.0
6.0
4.0
2.0
0.0
F M F M F M
Chow Chow Chow
VDR LoxP VDR ΔIEC VDR Δlyz
Figure 4. VDR deficiency results in significant alterations in the levels of polyamines: Box-plot diagrams
showing increased levels of polyamines metabolites namely, (A) N1, N12-diacetylspermine, (B)
diacetylspermidine, (C) N (‘1) acetylspermidine were noted following VDR deletion (in VDRΔIEC & VDR∆lyz)
in mice. This data is represented as the BOX-Plot diagram showing maximum and minimum variation among
the group. VDRΔIEC group (N = 17; F = 8, M = 9), VDR∆lyz (N = 10; F = 5, M = 5) & VDRLoxP (N = 16;
F = 6, M = 10). The ratio of fold-change differences are assessed by the Mann–Whitney U test. Significance is
established at adjusted 0.05 < P < 0.1.
compared to VDRLoxP males; they remain unaltered after HFD feeding, suggesting that diet might impact changes
that resulted from VDR deficiency in males (Fig. 8B).
Metabolites and microbiome regulated by VDR. Using Hierarchical clustering analysis (HCA)
(Fig. 9A), a stepwise clustering method that groups metabolically similar samples close to one another, we found
fecal samples did not show primary clustering by genotype (VDRLoxP, VDRΔIEC, and VDR∆lyz). Because the dele-
tion of intestinal epithelial cell-specific VDR impacted metabolites differently than myeloid cell-specific VDR
deletion, we further checked whether these specific metabolite profiles are linked to changes in the microbiome.
Microbial analysis showed VDRLoxP fecal samples contain Lactobacillus, Butyricimonas, Lactococcus, while
VDRΔIEC samples contain Clostridium, Eubacterium, Bacteroides, Tannerella, and Prevotella taxa. The difference in
the microbial communities might be related to differences in tryptophan, polyamine, and tocopherol metabolism
observed in this study. The abundance of Parabacteroides affected by VDR signaling in both human and mouse

A Myristoleoylcarnitine Myristate
12.0 3.0
10.0
8.0 2.0
6.0
4.0 1.0
2.0
0.0 0.0
LoxP ΔIEC Δlyz LoxP ΔIEC Δlyz
VDR VDR VDR VDR VDR VDR
B
Palmitoleoylcarnitine Palmitoleate
8.0 2.4
2.0
6.0
1.6
4.0
1.2
2.0
0.8
0.0 0.4
F M F M F M
LoxP ΔIEC Δlyz F M F M F M
VDR VDR VDR LoxP ΔIEC Δlyz
VDR VDR VDR
C
Oleoylcarnitine Oleate/vaccenate
5.0 3.0
4.0 2.5
2.0
3.0
1.5
2.0
1.0
1.0 0.5
0.0 0.0
LoxP ΔIEC Δlyz LoxP ΔIEC Δlyz
Figure 5. VDR deficiency in mice increased long-chain fatty acids and acylcarnitines: Fecal samples
derived from VDRΔIEC & VDR∆lyz animals showed increased levels of carnitines (A) myristoylcarnitine, (B)
palmitoylcarnitine, (C) oleoylcarnitine, as compared to controls. This surge was accompanied by elevated
levels of long-chain fatty acids (LCFAs) (A) myristate, (B) palmitoleate, (C) oleate. This data is represented as
BOX-Plot diagram showing maximum and minimum variation among the group. This data is represented as
BOX-Plot diagram showing maximum and minimum variation among the group. VDRΔIEC group (N = 17;
F = 8, M = 9), VDR∆lyz (N = 10; F = 5, M = 5) & VDRLoxP (N = 16; F = 6, M = 10). Significance is established at
adjusted 0.05 < P < 0.1.
samples are reported to alter secondary bile acids in obesity4. Interestingly, we found dysregulation of secondary
bile acids after VDR deletion followed by HFD intervention.
VDRΔIEC mice showed an increasing trend in E. coli, and a decreasing trend of Prevotella dentalis and A.
Muciniphila populations compared to VDRLoxP mice (Fig. 9B). A significant decrease in Parabacteroides sp. CT06
and Parabacteroides distasonis were noted in VDRΔIEC mice. However, VDRΔlyz mice did not show similar changes
(Fig. 9C). Alterations in maltose metabolism in VDR deficient mice (Fig. 1A) could be related to the abundance of
E. coli in those mice, as shown in our previous studies7,8.
Changes in VDR and FXR in liver and colon with or without HFD. Two nuclear receptors, VDR
and farnesoid X receptor (FXR) interact with each other in a Vitamin D3-independent manner26. To verify the
changes of VDR and related pathways in addition to microbiome and metabolites, we investigated the protein
expression of VDR and FXR in mice with or without HFD. Western blot analysis of FXR indicated a ~ 4-fold and
~5-fold increase in FXR in the colon (Fig. 10A,B) and liver (Fig. 10C,D) of HFD fed VDRΔIEC mice, respectively.
Our metabolite analysis indicated that VDR status significantly impacts bile acid metabolism. Hence, we wanted

Fold of Change
ANOVA Contrasts Welch’s 2-Sample t-Test
VDR∆IECVDR Chow
LoxPLoxP VDR∆IECVDR LoxP
Chow HFD VDRlyz VDR LoxP
Sub Pathway Biochemical Name Chow HFD Female Male Female Male All Female Male
myristate (14:0) 1.24 1.23 1.44 1.07 1.52 1 1.19 1.33 1.05
pentadecanoate (15:0) 1.33 0.87 1.81 0.98 1.07 0.71 1.39 1.97 0.96
palmitate (16:0) 1.19 1.41 1.33 1.07 2.28 0.87 1.46 1.49 1.42
Long Chain Saturated Fatty Acid margarate (17:0) 1.36 1.5 1.81 1.02 2.69 0.83 1.57 2.26 1.07
stearate (18:0) 1.15 1.48 1.31 1 2.5 0.88 1.25 1.42 1.12
nonadecanoate (19:0) 1.3 1.56 1.83 0.92 2.78 0.88 1.27 1.76 0.95
arachidate (20:0) 1.31 1.7 1.72 1 3 0.96 1.28 1.48 1.14
palmitoleate (16:1n7) 1.19 1.05 1.38 1.02 1.06 1.04 1.12 1.28 1.02
10-heptadecenoate (17:1n7) 1.3 1.09 1.51 1.11 1.24 0.95 1.3 1.59 1.07
Long Chain Monounsaturated oleate/vaccenate (18:1) 1.37 1.19 1.66 1.13 1.92 0.73 1.59 1.76 1.45
Fatty Acid 10-nonadecenoate (19:1n9) 1.32 1.4 1.68 1.03 2.13 0.92 1.65 2.06 1.39
eicosenoate (20:1) 1.51 1.6 2.23 1.02 2.67 0.96 1.32 1.82 1.08
erucate (22:1n9) 1.56 1.7 2.74 0.88 2.73 1.05 0.87 1.48 0.66
myristoylcarnitine (C14) 0.91 2.89 1.34 0.63 1.71 4.9 0.53 1.08 0.33
palmitoylcarnitine (C16) 1.15 2.6 2.11 0.63 1.9 3.56 1.06 2.34 0.67
Fatty Acid Metabolism margaroylcarnitine (C17)* 1.03 1.93 1.56 0.68 1.5 2.48 0.94 1.67 0.65
(Acyl Carnitine, Long Chain
Saturated) arachidoylcarnitine (C20)* 1.18 1 1.5 0.93 0.7 1.42 0.83 1.3 0.61
behenoylcarnitine (C22)* 1.2 0.84 1.53 0.93 0.54 1.32 0.9 1.3 0.72
lignoceroylcarnitine (C24)* 1.12 0.94 1.4 0.89 0.73 1.21 1.06 1.35 0.91
myristoleoylcarnitine (C14:1)* 0.86 2.77 1.28 0.57 3.27 2.35 0.47 0.86 0.35
Fatty Acid Metabolism (Acyl palmitoleoylcarnitine (C16:1)* 0.81 2.7 1.47 0.45 2.16 3.37 0.55 1.2 0.35
Carnitine, Monounsaturated) oleoylcarnitine (C18:1) 1.18 2.49 2.1 0.66 1.78 3.49 1.01 2.21 0.6
eicosenoylcarnitine (C20:1)* 1.45 1.74 2.22 0.94 1.23 2.45 1.06 1.98 0.66
linoleoylcarnitine (C18:2)* 0.92 2.1 1.71 0.5 1.44 3.07 0.69 1.64 0.43
linolenoylcarnitine (C18:3)* 1.61 1.02 2.92 0.89 0.63 1.65 1.25 2.92 0.72
Fatty Acid Metabolism (Acyl
Carnitine, Polyunsaturated) dihomo-linoleoylcarnitine (C20:2)* 1.15 1.62 1.42 0.94 1.04 2.53 0.78 1.32 0.52
(Acyl Carnitine, Hydroxy)
arachidonoylcarnitine (C20:4) 0.97 1.39 2.52 0.37 0.55 3.53 0.49 1.47 0.21
(S)-3-hydroxybutyrylcarnitine 1.59 0.49 3.33 0.76 0.37 0.65 1.35 2.79 0.84
Table 3. Long-chain fatty acids and acylcarnitines elevated in VDR deficient mice.
to check whether hepatic FXR was altered. Immunohistochemical staining (IHC) of liver sections showed a sim-
ilar increase in FXR, consistent with the observation by western blots.
Discussion
In the current study, we have demonstrated that targeted deletion of VDR in intestinal or myeloid cells distinc-
tively transformed metabolite profiles and the gut microbiome, leading to an increased risk of obesity. We found
that 84 identifiable biochemicals that were significantly altered due to the VDR status. When challenged with a
HFD, 530 metabolites showed discrete changes. These changes were observed due to variations in carbohydrate,
protein, lipid, and xenobiotic pathways. The deletion of VDR mainly impacted bile acid, LCFA, polyamine, and
tocopherol metabolism. VDRΔIEC mice challenged with HFD diet had the most dramatic changes in metabolites
generated from energy expenditure, TCA cycle, tocopherol, and polyamine metabolism. Interestingly, HFD along
with loss of VDR influenced female mice more than the males. At the protein level, we found that FXR expres-
sion was increased after VDR deletion and that HFD further elevated FXR in the colon and liver of VDRΔIEC
mice. It is known that microbiota-mediated changes in bile acid profiles signal through FXR. FXR contributes
directly to diet-induced obesity by promoting increased adiposity and altering the microbiota composition27. A
study on HFD fed rats showed an increased expression of FXR28. Our results clearly indicate that the gut micro-
biota actively participates in host metabolism by regulating metabolites generated from bile acid metabolism
via VDR-FXR signaling. VDR deficiency alters the metabolite profile and FXR expression in the host. Our data
suggest the tissue specificity and gender differences of VDR in regulating metabolites and impacting gut-liver
axis (Fig. 10F).
Our study highlights the importance of carbohydrate, lipid, and amino acid metabolism following VDR dele-
tion indicating changes in glycogen metabolism as well as lipid and amino acid super pathways, whereas RF
analysis using data derived from HFD fed VDR loxP and VDR∆IEC mice pointed to peptides, lipid metabolism,
cofactors, vitamins (e.g., alpha-tocopherol), and amino acid metabolism. Loss of intestinal VDR increased taurine
and kynurenine levels. The amino acid metabolite taurine is known to be protective against inflammation, apop-
tosis, and oxidative stress29. The kynurenine pathway is associated with inflammatory neurological disorders30.

A
Threonylphenylalanine
Lysylleucine
Lanosterol
1-methinicotinamide
Hepatodecasphingosine
Indolepropionate
Ceramide
Increasing Importance to group Separation

Valylleucine
N-acetylputrescine
Alanylleucine
Palmitoylcarnitine
7-methylguanine
Margaroycarnitine
Oleoyl ethanolamide
Sphingosine
3-ureidopropionate
Oleoylcarnitine
Putrescine
Docosadienoate
Eicosenoate
N-Stearoyl-sphingosine
Cysteine-S-sulfate
Sphinganine Color by Super_Pathway
Thymidine Amino Acid
3-hydroxyanthranilate Cofactors &Vitamins
1-palmitoyl-GPG Lipids
1-linoleoyl-GPG Nucleotide
alpha-tocopherol Peptide
Cystine
14 15 16 17 18 19 20 21 22 23
Mean Decrease Accuracy
30
B
20
Comp.2 [13.82%]
10
-10
LoxP
VDR + Chow
-20 LoxP
VDR + HFD
ΔIEC
VDR + Chow
ΔIEC
-30 VDR + HFD
Comp.1 [33.89%]
Figure 6. Alteration in the metabolites following VDR Deletion and HFD intervention: (A) Random forest
(RF) analysis showing the top 30 metabolites responsible for classification as mean decrease accuracy values.
Most vital biochemicals are listed from data derived from fecal samples of VDRLoxP, VDR∆IEC mice fed with
HFD or chow diet. Different colors indicate different biochemicals like, light blue = amino acid, purple = co-
factors &vitamins, blue = lipids, orange = nucleotide, red = peptide. (B) Principal Component Analysis (PCA)
on metabolite level Correlations. Color code: light blue= VDRLoxP + chow, orange = VDRLoxP + HFD, purple=
VDR∆IEC chow, red = VDR∆IEC + HFD. VDRΔIEC group (Chow diet: N = 17; HFD: N = 7), & VDRLoxP (Chow
Diet: N = 16; HFD: N = 6).
Microbial action in the gut is responsible for the generation of several metabolites derived from bile acids,
amino acids, heme, and dietary sources. Bile acids are reabsorbed in the intestine by enterohepatic recirculation

A Phenyllactate (PLA) 3-hydroxyphenylacetate

Indole-3 carboxylate 6.0 5.0
2.0
4.0
1.5
4.0 3.0
1.0
2.0
2.0
0.5 1.0
0.0 0.0 0.0

F M F M F M F M F M F M F M F M F M F M F M F M
Chow HFD Chow
Ch HFD D Ch
C
Chow HFD D Chow HF
HFD
FD Chow
Ch
Ch HFD
HFF
FD
Chow HFD
LoxP/LoxP ΔIEC VDR
LoxP/LoxP
VDR
ΔIEC
VDR
LoxP/LoxP VDR ΔIEC
VDR VDR
Cis-Uroconate
B 6.0
Trans-Uroconate
5.0 6.0
4.0
3.0 4.0
2.0
2.0
1.0
0.0
F M F M F M F M 0.0
Chow HFD Chow HFD F M F M F M F M
LoxP ΔIEC Chow
Cho
ow HFD
HF D Chow
Ch
how
o HFD
H FD
VDR VDR LoxP
L ΔIEC
VDR VDR
Alpha-tocopherol
C 1.4 2.0
Gamma-tocopherol/beta-tocopherol
1.2
1.6
1.0
1.2
0.8
0.6 0.8
0.4
0.4
F M F M F M F M F M F M F M F M
Chow HFD Chow HFD Chow HFD Chow HFD
LoxP ΔIEC
VDR VDR LoxP ΔIEC
VDR VDR
D 8.0
6.0
4.0
2.0
0.0
F M F M F M F M
Chow HFD Chow HFD
ΔIEC
VDR LoxP VDR
Figure 7. HFD altered the metabolite profile: Intestinal specific VDR deletion along with HFD diet decreased
(red arrow) the levels of (A) indole 3 carboxylate, PLA, 3-hydroxyphenylacetate and increase in (B) trans
and cis-uroconate. Altered expression of biochemicals resulting from (C) tocopherol and (D) polyamine
metabolism after HFD feeding. These data are represented as the BOX-Plot diagram showing maximum and
minimum variation among the group. VDRΔIEC group (Chow diet: N = 17; HFD: N = 7) & VDRLoxP (Chow
Diet: N = 16; HFD:N = 6). Significance is established at adjusted 0.05 < P < 0.1.
and can affect diverse biological processes31. In our study, deletion of VDR altered secondary bile acids, signifying
the crucial role of VDR in assembling the bile acid pool. Alterations in the bile acid profile were more obvious
in females with VDR deletion, as well as in those receiving HFD. Previous studies have shown that the deletion
of VDR alters metabolic responses in female mice32,33. Vdr gene polymorphisms are associated with PCOS and
osteoporosis34. Bile acids are also considered significant factors in shaping the microbiome of diet-induced obese
mice35. The dysfunction of the VDR-associated bile acid pathway observed in our study further explains the risk
of HFD-induced obesity without the protection of Vitamin D/VDR.
Excessive accumulation of lipids such as long chain acylcarnitines (LCACs), ceramides, and other metabolites
are implicated in cell stress and inflammation. Our study demonstrates that long-chain fatty acids (LCFAs) and
acylcarnitines are significantly elevated in VDR deficient animals, potentially due to perturbations in β-oxidation.
In the absence of vitamin, polyunsaturated fats can be oxidized in the intestines to produce mutagens and sub-
sequently, inflammatory cells in close proximity to the colon can produce reactive oxygen species36. As a result,
VDR deficiency may cause lower levels of tocopherols, which may be indicative of an increased risk for colon
cancer.

A 7.0
Taurolithocholate 3-sulfate
12.0
Taurocholenate sulfate*
6.0 10.0
5.0 8.0
4.0
6.0
3.0
4.0
2.0
2.0
1.0
0.0 0.0
F M F M F M F M
F M F M F M F M Chow HFD Chow HFD
Chow HFD Chow HFD ΔIEC
LoxP VDR
VDR LoxP VDR ΔIEC VDR
N-acetyltyrosine Indolepropionate
B 5.0 7.0
6.0
4.0
5.0
3.0 4.0
2.0 3.0
2.0
1.0
1.0
0.0 0.0
F M F M F M F M F M F M F M F M
Chow HFD Chow HFD Chow HFD Chow HFD
LoxP ΔIEC LoxP ΔIEC
VDR VDR VDR VDR
Figure 8. Gender-specific alteration in metabolites displayed following intestinal VDR deletion and HFD
intervention. HFD fed female VDRΔIEC mice showed increased (A) taurolithocholate 3-sulphate and
taurocholenate sulphate. Variations in the biochemicals namely (B) N-acetyltyrosine and indolepropionate.
Data are expressed as Boxplot diagram. Differences were assessed by the Mann–Whitney U test. VDRΔIEC group
(Chow diet: N = 17; HFD: N = 7), & VDRLoxP (Chow Diet: N = 16; HFD: N = 6). Significance is established at
adjusted 0.05 < P < 0.1.
VDR plays an important role in regulating several physiological functions through host-microbiome interac-
tions4. It is crucial for inflammation and immune responses15,37,38. Dysbiosis has emerged as a key risk factor for
developing a myriad of metabolic diseases including obesity, atherosclerosis, cardiovascular disease, and Type 2
diabetes39–41. A shift in the metabolic capacitance of the microbiota is also associated with the severity of nonal-
coholic fatty liver disease (NAFLD)42. Our previous studies have shown that VDR knock out (VDR−/−) mice have
depleted Lactobacillus and enriched Clostridium and Bacteroides in feces. Notably, in the cecal content, Alistipes
and Odoribacter population were significantly down, and Eggerthella were increased. Intestinal specific deletion of
VDR leads to dysbiosis due to increased E. coli and Bacteroides and decreased butyrate-producing bacteria7. This
imbalance resulted in defective autophagy in colitis8. In the current study, we found that VDR deletion contributes
to the variation of microbial contents, supporting the changes in metabolite profile. Accordingly, the percentage of
abundance of Parabacteroides distasonis (PD) significantly dropped in VDRΔIEC mice. For example, PD is known
to modulate host metabolism via FXR pathway by producing secondary bile acids43. N-acetylmuramate released
by L. acidophilus has an anti-inflammatory effect on LPS-induced inflammation. Decreased N-acetylmuramate
in VDR∆lyz mice might be associated with loss of L. acidophilus as reported in our previous study44. Consistent
with the recent report that indicated that high-fat diet depletes the indole-3-carboxylate and other tryptophan
derived microbial metabolites, which are known to attenuate weight gain in rats45. Because polyamines have
strong anti-inflammatory functions46, these changes may impact aspects of immunity. Previous studies from
our lab have indicated a correlation between the short-chain fatty acid (SCFA) butyrate and VDR8,15. Lack of
1,25(OH)2D3 or VDR deficiency results in microbial dysbiosis, leading to greater susceptibility to colitis, which
might be important for patients with IBD8,47–49. Loss of SCFA and VDR is also connected to a higher risk of colon
cancer50. Probiotic treatments could potentially exert beneficial effects depending on the VDR status51.
Gut microbiota controls neurobehavior via modulating brain insulin sensitivity and metabolism of trypto-
phan, the precursor of serotonin52. Increased influx of tryptophan into the brain by HFD could be related to
increased blood insulin levels. VDRΔlyz group displayed significant downregulation of quinolinate and tocopherol
pathway derived metabolites and increase in nicotinamide. Quinolinic acid acts as a neurotoxin, proinflamma-
tory mediator, and prooxidant molecule53. Loss of intestinal VDR also increased kynurenine, a pathway associ-
ated with inflammatory neurological disorder30. These changes indicate the role of VDR in neurophysiology. The
role of VDR/vitamin D in the gut-brain axis needs further investigation in future research.
We have demonstrated gender differences in metabolites that may be regulated by VDR status. Female mice
were shown to be more affected by VDR deletion than male mice. Higher circulating bile acids were observed in
obese and type-2 diabetes54. Thus, significant elevation of secondary bile acids in VDR deficient females indicates

stearoyl
thioproline sphingomyelinacid
tauroursodeoxycholic
1-(1-enyl-palmitoyl)-2-arachidonoyl-G...
1-stearoyl-2-arachidonoyl-GPE
cis-urocanate
trans-urocanate
phenylalanylalanine
tyrosylglycine
gamma-glutamyl-epsilon-lysine
arginine
N-acetyl-beta-glucosaminylamine
valylglutamine (d18:1/18:0)
sulfate (2)
(18:0/2... -5 5
agmatine
asparagine
cysteine
lysine s-sulfatesulfate
N-(2-hydroxypalmitoyl)-sphingosine
indolin-2-one
ornithine
isoleucylglycine
citrulline
N-acetylmethionine
phenylacetate
5-methylthioadenosine
N-acetylarginine (MTA) (d18... (d...
deoxycarnitine
alpha-ketobutyrate
7-alpha-hydroxy-3-oxo-4-cholestenoate...
lactosyl-N-palmitoyl-sphingosine
3beta-hydroxy-5-cholestenoate
hydroxystearate
stearoylcarnitine
linolenoylcarnitine
12-ketolithocholate
6-oxolithocholate
dehydrolithocholate
3-hydroxybehenate* (C18)
(C18:3)* Group
eicosenoate
erucate
(16 (22:1n9)
2-palmitoylglycerol
stearate
margarate (18:0)
nonadecanoate
10-nonadecenoate
arachidate (20:1)
or 17)-methylstearate
18-methylnonadecanoate
2-hydroxyarachidate*
2-hydroxypalmitate
2-hydroxystearate
(17:0)
(20:0) (19:0)(16:0)
(19:1n9) (a19:0
(i20:0) or i...
palmitate
palmitoleate
docosadienoate(16:0)
dihomo-linoleate (16:1n7)
glycerophosphorylcholine
1-(1-enyl-oleoyl)-GPE
1-(1-enyl-palmitoyl)-GPC (20:2n6)
(22:2n6)
1-(1-enyl-palmitoyl)-2-linoleoyl-GPC
1-(1-enyl-palmitoyl)-2-arachidonoyl-G...
1-(1-enyl-palmitoyl)-2-oleoyl-GPC
1-(1-enyl-palmitoyl)-2-palmitoyl-GPC
arachidonoylcarnitine
dihomo-linolenoylcarnitine (GPC)
(P-18:1)*
(P-16:0)*
(C20:4) (C20:3n3 ...
(P-...
...
o...
LoxP
heptadecasphingosine
hexadecasphingosine
N-stearoyl-sphinganine
N-stearoyl-sphingosine
N-oleoyl-sphingosine
sphingosine
ceramide (d18:1/17:0,(d18:1/18:1)*
(d18:1/20:0,
(d18:1/14:0,
4-cholesten-3-one
eicosanoylsphingosine
sphinganine
desmosterol (d17:1)
(d16:1)*
(d18:0/18:0)*
(d18:1/18:0)*
d16:1/22:0,
d16:1/16:0)*
d17:1/18:0)*
(d20:1)* d20... VDR Chow
phytosphingosine
betaine
pyroglutamine*
mannitol/sorbitol
arabitol/xylitol
ribitol
mevalonolactone
N-acetyl-isoputreanine*
N-acetyl-beta-alanine
pantoate
3-amino-2-piperidone
N-acetylglutamine
N-acetylhistidine
hypotaurine
carnitine
creatine
urate
xanthurenate
N1-Methyl-2-pyridone-5-carboxamide
N1-Methyl-4-pyridone-3-carboxamide
1-methylhistamine
1-(3-aminopropyl)-2-pyrrolidone
5-hydroxyindoleacetate
N,N,N-trimethyl-alanylproline
succinylcarnitine
2,8-quinolinediol (C4-DC)
sulfate betaine...
LoxP
VDR Chow
glycitein
sulfate* sulfate
4-hydroxymandelate
2-hydroxyphenylacetate
N-formylanthranilic (2)
ribulonate/xylulonate/lyxonate*
vanillactate
4-hydroxybenzoate
3-(3-hydroxyphenyl)propionate
daidzein
hydroquinone
aconitate sulfate
2,4-dihydroxybutyrate
[cis (1)
sulfate
or trans] acid sulfate
citratesulfate
equol
homostachydrine*
anthranilate
stachydrine
trigonelline (N'-methylnicotinate)
N-glycolylneuraminate
quinate
N2,N5-diacetylornithine
propionylglycine
2-hydroxyoctanoate
p-cresol glucuronide*
N-carbamoylalanine
ΔIEC
VDR Chow
3-hydroxy-2-ethylpropionate
3-methyladipate
ethyl alpha-glucopyranoside
ethylmalonate
methylmalonate
hydantoin-5-propionate
N-formylmethionine
2'-O-methylcytidine
beta-guanidinopropanoate
biopterin
O-sulfo-L-tyrosine
1-methylnicotinamide (MMA) sulfate
7-methylguanine
N-acetyl-3-methylhistidine*
isobutyrylglycine
N-acetyl-2-aminooctanoate*
N-acetylpyrraline
3-hydroxypyridine
caffeic acid
6-hydroxyindole sulfate
C-glycosyltryptophan
equol
phenol glucuronide
tigloylglycine
glucuronide
2-amino-p-cresol sulfate
sulfate
sulfate VDR
ΔIEC
Chow
4-methylcatechol
phenylpropionylglycine
1,2,3-benzenetriol
indolepropionylglycine
2-acetamidophenol sulfate (2)
erythritol
2-butenoylglycine
4-methylhexanoylglycine
N-acetyl-1-methylhistidine*
p-cresol
allantoic sulfate
adipoylcarnitine
acid N-oxide
urea conjugate
p-hydroxybenzyl-glucosinolate
5-methylthioribose**
glycine
nicotinamide
hexanoylglycine
1-ribosyl-imidazoleacetate*
3-methylcrotonylglycine
orotidine
dihydrocaffeate
isocaproylglycine
4-hydroxycinnamate
4-vinylphenol
dihydrobiopterin
phenol sulfate ofsulfate
(C6-DC)
sulfate
S-(3-hydroxypropyl)mercapturic
sulfate C10H14O2
(2) sulfate
sulfate (1)* (...
acid Δlyz
3,4-dihydroxyphenylacetate
dihydroferulic
hippurate
allantoin
phenylacetylglycine
creatinine
guanidinoacetate
dimethylguanidino
ferulylglycine
isocitric lactone
N-(2-furoyl)glycine
ferulic acid
2-aminophenol acid
(1)
4-sulfate sulfate
valeric
sulfate acid (DMGV)* VDR Chow
2-hydroxyhippurate
cinnamoylglycine
trimethylamine
3-indoxyl
catechol
butyrylglycinesulfate
sulfate
isovalerylglycine
picolinoylglycine
indoleacetylglycine
3,4-methylene
4-hydroxyhippurate
gulonate*
valerylglycine N-oxide (salicylurate)
heptanoylglycine Gender
eicosapentaenoate
arachidonate
docosahexaenoate
2'-deoxycytidine
2'-deoxyguanosine
lignoceroylcarnitine
arachidoylcarnitine
behenoylcarnitine
margaroylcarnitine
oleoylcarnitine
palmitoylcarnitine (20:4n6)
(C18:1)
dihomo-linoleoylcarnitine
eicosenoylcarnitine (EPA;
(DHA;
(C24)*
(C20)*
(C22)*
(C17)*
(C16)
(C20:1)* 20:5n3)
22:6n3)
(C20:2)*
docosapentaenoate
1-stearoyl-GPI
2-stearoyl-GPE
N-acetylputrescine
putrescine
3-hydroxysuberate
cholate sulfate
myristoylcarnitine
myristoleoylcarnitine
linoleoylcarnitine
palmitoleoylcarnitine (18:0)
(18:0)*
glycerophosphoethanolamine
1-(1-enyl-palmitoyl)-GPE (C14)(n6
(C18:2)* DPA;
(C14:1)*
(C16:1)*(P-16:0)* 22:5n6) Female
1-(1-enyl-stearoyl)-GPE
1-palmitoyl-2-stearoyl-GPC
1-palmitoyl-GPC
1-stearoyl-GPC
1-stearoyl-GPE
margaroyl
palmitoleoyl
stearoyl
biliverdin
bilirubin (16:0)
(18:0)
ethanolamide*
ethanolamide*
ethanolamide (P-18:0)*
(16:0/18:0)
cholate (E,E)*
cholesterol
palmitoyl
cadaverine
ceramide
(Z,Z)
octadecanedioylcarnitine
octadecenedioylcarnitine
7,12-diketolithocholate
3-dehydrocholate
N-palmitoyl-sphinganine
N-palmitoyl-sphingosine
hydroxypalmitoyl
sphingomyelin sphingomyelin
(d17:1/16:0,
dihydrosphingomyelin
sphingomyelin
(d18:2/24:1,
(d18:1/24:1,
(3'-5')-adenylylguanosine*
(3'-5')-guanylyladenosine*
(C18-DC)*
(C18:1-DC)*
(d18:0/16:0) (d18:1...
d18:1/15:0...
(d18:1/16:0)
d18:1/24:2)* (d18:0...
d18:2/24:0)* Male
(3'-5')-guanylylcytidine
(3'-5')-guanylyluridine
5-aminovalerate
kynurenine
N-acetylglycine
succinimide
glycerophosphoserine*
cysteine
taurine sulfinic acid acid
N-acetylglucosaminylasparagine
phenylacetyltaurine
N-acetyltaurine
N-linoleoyltaurine*
N-oleoyltaurine
N-stearoyltaurine
guanine
N-acetylproline
taurodeoxycholate
taurohyodeoxycholic
taurochenodeoxycholate
taurocholate
tauro-beta-muricholate
tauroursodeoxycholate
prolylglycine
sarcosine
glycocholate sulfate Diet
3-hydroxyanthranilate
protoporphyrin
heptanoate
caprylate
I-urobilinogen
benzoate
caproate
isovalerate
valerate
nicotinamide (6:0)
(5:0) (7:0)
(8:0)
(i5:0)
N1,N12-diacetylspermine IX
1-palmitoyl-galactosylglycerol
1-stearoyl-GPG
1-oleoyl-GPG
1-palmitoyl-GPG
N-formylphenylalanine
N6-formyllysine
gamma-glutamylglycine
gamma-glutamylisoleucine*
gamma-glutamylvaline
1-pentadecanoylglycerol
tyrosol
5-methyl-2'-deoxycytidine
2'-deoxyinosine
2'-deoxyuridine
thymidine
1-methyl-5-imidazoleacetate (16:0)* (15:0)(16:0)*
(18:0)
(18:1)* Chow
spermidine
3-hydroxydecanoate
uridine-2',3'-cyclic
cytidine
hexadecenedioate
2'-deoxyadenosine
adenine
indolelactate
1,5-anhydroglucitol
2,3-dihydroxyisovalerate monophosphate
(C16:1-DC)*
(1,5-AG)
N6-succinyladenosine
pyridoxine
5-methylcytosine
imidazole
N-acetylserine (Vitamin
N-acetyl-cadaverine
lactate
N-acetylcitrulline
N-acetylthreonine
1-linoleoyl-GPG
1-oleoyl-GPE
1-linoleoyl-GPE
1-linoleoyl-GPI
1-oleoyl-GPI (18:1)
(18:2)*
(18:2)*
(18:1) B6)
1,2-dilinolenoyl-galactosylglycerol
1,2-dilinoleoyl-galactosylglycerol
1-oleoyl-GPC
1-palmitoyl-GPI
N,N-dimethylalanine
deoxymugineic
N-methyl-GABA
cystathionine
tartronate
dimethylglycine
kynurenate
naringenin
glycitein (18:1)
(16:0)
acid
(hydroxymalonate) (...
(1... HFD
soyasaponin
afromosin
daidzein
genistein sulfateIII
I
II
sulfate*
palmitoylcholine
linoleoylcholine*
oleoylcholine
formononetin (2)
gamma-tocopherol/beta-tocopherol
1-lignoceroyl-GPC
palmitoyl ethanolamide (24:0)
linoleoyl
oleoyl ethanolamide
ethanolamide
linolenoyl ethanolamide
alpha-tocotrienol
gamma-tocotrienol
oleoyl-linolenoyl-glycerol
linoleoyl-linolenoyl-glycerol
oleoyl-linoleoyl-glycerol
linolenoyl-linolenoyl-glycerol
linoleoyl-linoleoyl-glycerol
linoleoyl-linolenoyl-glycerol (18:1/18:3...
(18:2/1...
(18:1/18:2)...
(18:3/...
(18:2/18...
(18:2/1... Super Pathway
palmitoyl-linoleoyl-glycerol
D-urobilin
choline phosphate
1-palmitoyl-2-linoleoyl-GPC
1,2-dilinoleoyl-GPC
1-oleoyl-2-linoleoyl-GPC
12,13-DiHOME
glycerol
xylose 3-phosphate(hydrocinnamate)
glucose
galacturonate
arabinose
ribulose/xylulose (18:2/18:2) (16:0/18...
(16:0/18:2)
(18:1/18:2)*
3-phenylpropionate
enterolactone
N-acetylmuramate
N-acetylmethionine
1,2-dilinoleoyl-GPE
1-linoleoyl-2-linolenoyl-GPC
histamine
N-acetylhistamine
(3'-5')-adenylyladenosine*
(3'-5')-adenylylcytidine
(3'-5')-adenylyluridine
4-hydroxycinnamate
feruloylputrescine sulfoxide
(18:2/18:2)* (18:2/18...(16... Amino Acid
glycerate
stigmastadienone
1-palmitoyl-2-docosahexaenoyl-GPC
choline
1,2-dipalmitoyl-GPC
1-stearoyl-2-linoleoyl-GPC
1-palmitoyl-2-oleoyl-GPC
1-palmitoyl-2-palmitoleoyl-GPC
1-palmitoyl-2-arachidonoyl-GPE
fucose
N-acetylneuraminate
1-oleoyl-2-linoleoyl-GPE
adenosine (16:0/16:0) (18:0/18:2)*
(16:0/18:1)
(18:1/18:2)* (16:0/...
(16:0/...
3-hydroxydodecanedioate*
3-hydroxysebacate
alpha-tocopherol
lanosterol
ergosterol
mevalonate
1-linoleoylglycerol
1-oleoylglycerol
2-linoleoylglycerol
2-oleoylglycerol
stigmasterol
nicotianamine
hexadecatrienoate (18:1)
(18:2)
(16:3n3) Carbohydrate
tridecenedioate
hydroxymethylpyrimidine
delta-tocopherol
linoleate
linolenate
retinol
13-HODE (18:2n6)
[alpha
(Vitamin
beta-sitosterol
campesterol + 9-HODE
dehydrophytosphingosine*
pheophorbide
fucosterol A (C13:1-DC)*
5-(2-Hydroxyethyl)-4-methylthiazole
A) or gamma; (18:3n3 o...
equol
carotene
pheophytin
genistein
coumestrol
carotene
diosmin
adenosine diol
diol (1)
A (2)
2-oxindole-3-acetate
daidzein
beta-cryptoxanthin (3)
(diosmetin
(R)-3-hydroxybutyrylcarnitine
5'-monophosphate 7-rutinoside) (AMP) Co-factors &Vitamins
uridine
thymidine 5'-monophosphate
lignoceroyl 5'-monophosphate
2'-deoxyadenosine
2'-deoxycytidine
alpha-tocopherol ethanolamide
glycosyl-N-palmitoyl-sphingosine
sphingadienine
(S)-3-hydroxybutyrylcarnitine
octadecadienedioate
biotin
myo-inositol
quinolinate (UMP)
5'-monophosphate
5'-monophosphate
acetate (24:0)* (d18...
(C18:2-DC)*
3-ureidoisobutyrate
3-ureidopropionate
octadecanedioate
N-alpha-acetylornithine
10-hydroxystearate
diacetylspermidine*
N-methylalanine
nicotinate
arachidoyl
behenoyl
9,10-DiHOME ribonucleoside
ethanolamide
ethanolamide (C18-DC)
1-palmitoyl-digalactosylglycerol
N-delta-acetylornithine (20:0)*
(22:0)* (16:0)* Energy
2,8-quinolinediol
indole-3-carboxylate
(3'-5')-cytidylylcytidine*
(3'-5')-uridylylcytidine*
(3'-5')-cytidylyluridine*
(3'-5')-uridylyluridine
1,2-dipalmitoyl-GPE
1-palmitoyl-2-oleoyl-GPE
mannose
fructose
3,4-dihydroxybutyrate
sedoheptulose
pyridoxamine (16:0/16:0)*
(16:0/18:1)
N6-methyllysine
pantetheine
pantethine
gamma-glutamylphenylalanine
gamma-glutamyltyrosine
gamma-glutamylglutamine
gamma-glutamylthreonine
diaminopimelate
gamma-glutamyl-alpha-lysine
gamma-glutamylmethionine
anserine (C5-DC)
chiro-inositol
N6-carboxyethyllysine
imidazole propionate
2,3-dimethylsuccinate
3-methylglutarate/2-methylglutarate
5-hydroxyhexanoate
indoleacetate
indolepropionate
6-oxopiperidine-2-carboxylate
dihydroferulate
glutarate
2-hydroxyglutarate
2-methylcitrate/homocitrate
galactitol (dulcitol)
3-sulfo-L-alanine
Lipid
1-palmitoyl-2-linoleoyl-galactosylgly...
dodecanedioate
sebacate
undecanedioate
homoserine
cysteine (C10-DC)
3-aminoisobutyrate
cysteinylglycine
16-hydroxypalmitate
glycerophosphoglycerol
vanillate
gentisate
pyridoxal (C12-DC)
(C11-DC)
ribonate
2-hydroxyheptanoate*
beta-hydroxyisovalerate
pyrraline
maltose
maltotriose
1-palmitoyl-GPE
enterodiol
fumarate
malate (Vitamin
N-methylproline
N-methylpipecolate (16:0)
1-palmitoyl-2-linoleoyl-digalactosylg... Nucleotide
carboxyethyl-GABA
thiamin
oxalate
ferulate (ethanedioate)
azelate
suberate
cytidine (C9-DC)
(C8-DC)(18:2)
5'-monophosphateB1)
1-palmitoyl-2-linoleoyl-GPE
pyridoxate
3-dehydroshikimate
glycerol
sinapate
diosmetin
1-linoleoyl-GPC
glycerophosphoinositol*
2-hydroxyadipate
syringic
glucuronate
malonate acid
dimethylmalonic
3-methylglutaconate
2,6-dihydroxybenzoic
pimelate (C7-DC)
N6-carboxymethyllysine
succinate
trans-4-hydroxyproline
3-hydroxy-3-methylglutarate
methylsuccinate
picolinate acid acid (5'-CMP) (16:0/18:2) Partially Charecterized
2-isopropylmalate
salicylate
3-hydroxyhexanoate
N-acetyltryptophan
N-acetylphenylalanine
N-acetylvaline
N-acetylaspartate
N-carbamoylaspartate
gluconate
phenethylamine
2,4,6-trihydroxybenzoate (NAA)
N,N,N-trimethyl-5-aminovalerate
N,N-dimethyl-5-aminovalerate
lysylleucine
2-hydroxybutyrate/2-hydroxyisobutyrate
lactate
3-(4-hydroxyphenyl)lactate
phenyllactate (PLA)
alpha-hydroxyisovalerate
2-hydroxy-3-methylvalerate
alpha-hydroxyisocaproate
tryptamine
serotonin
tyramine
2-aminoadipate
3-formylindole Peptides
N6,N6,N6-trimethyllysine
1-methyl-4-imidazoleacetate
dimethylarginine
ectoine (C6-DC) (SDMA + ADMA)
3-hydroxyadipate*
N-acetylcysteine
1-methylguanine
1-methyladenine
pipecolate
5,6-dihydrouridine
pseudouridine
orotate
pantothenate
2-oxoarginine*
alpha-ketoglutaramate*
argininate*
galactonate
adipate
mannonate*
4-imidazoleacetate
arabonate/xylonate
fructosyllysine
N2,N6-diacetyllysine
4-guanidinobutanoate
erythronate*
threonate
Xenobiotics
2R,3R-dihydroxybutyrate
2S,3R-dihydroxybutyrate
N1-methyladenosine
5-methyluridine
uridine
guanosine
inosine
xanthosine (ribothymidine)
1-myristoyl-2-palmitoyl-GPC
1-palmitoyl-2-arachidonoyl-GPC
1-palmitoyl-2-dihomo-linolenoyl-GPC
1-stearoyl-2-arachidonoyl-GPC
1-stearoyl-2-oleoyl-GPC (14:0/16:0)
(18:0/18:1) (16:0/...
(18:0/2...(...
S-1-pyrroline-5-carboxylate
N-acetylglucosamine/N-acetylgalactosa...
1-(1-enyl-palmitoyl)-2-oleoyl-GPE
1-stearoyl-2-oleoyl-GPE
3-ketosphinganine
hyocholate
7-ketodeoxycholate
alpha-muricholate
beta-muricholate
3-hydroxylaurate
hexadecadienoate
tetradecadienoate
3-hydroxyoleate* (16:2n6)
(14:2)* (P-...
(12 or 13)-methylmyristate
3-hydroxypalmitate
hexadecasphinganine
N-palmitoyl-phytosphingosine
lithocholate
6-beta-hydroxylithocholate
deoxycholate
3b-hydroxy-5-cholenoic
deoxycholic
taurocholenate
taurolithocholate
adrenate acidsulfate*
(22:4n6)
dihomo-linolenate 3-sulfate
(20:3n3 (d16:0)*
acid (a15:0
or n6) or ...
(t18:0/1...
docosapentaenoate
oleate/vaccenate
3-hydroxystearate
myristate
pentadecanoate(14:0)
10-heptadecenoate
glutaminylleucine
threonylphenylalanine
valylleucine
alanylleucine
glycylleucine
aspartate
gamma-glutamylleucine (18:1)
(15:0) (n3
(17:1n7)DPA; 22:5n3)
leucylglycine
valylglycine
glycylisoleucine
glycylvaline
alanine
methionine
leucylalanine
leucylglutamine* sulfoxide
phenylalanylglycine
tryptophylglycine
glutamine
proline
histidine
methionine
glycine
threonine
serine
leucine
isoleucine
valine
tryptophan
phenylalanine
tyrosine
diacetylchitobiose
methionine
o-Tyrosine sulfone
2-hydroxydecanoate
methylphosphate
phosphate
2-hydroxynervonate*
2-hydroxybehenate
2-hydroxylignocerate*
L-urobilin
nicotinamide
hexadecanedioate
pterin adenine
2-hydroxyquinoline
hypoxanthine
thiamin riboside
monophosphate (C16-DC)
flavin mononucleotide
4-thiouracil
glutamate,
homocitrulline
formiminoglutamate
N6-acetyllysine
3-hydroxybutyrate
eicosenedioate
octadecenedioate
isohyodeoxycholate
ursocholate
cytosine dinucleotide
gamma-methyl (BHBA)
(C20:1-DC)* (FMN)
(C18:1-DC)* (FAD)
ester
N-acetyltyrosine
glutamate
ribose
uracil
xanthine
beta-alanine
nicotinate
thymine
2'-O-methyluridine
5-oxoproline
allo-threonine
ergothioneine
N-acetylglutamate
N2-acetyllysine
N-acetylasparagine
N-acetylalanine
riboflavin (Vitamin
4-methylthio-2-oxobutanoate
4-hydroxyphenylpyruvate
phenylpyruvate
pyruvate
N-acetylisoleucine
N-acetylleucine
alpha-ketoglutarate
3-methyl-2-oxobutyrate B2)
Group 3-methyl-2-oxovalerate
4-methyl-2-oxopentanoate
UICH-00155
UICH-00135
UICH-00147
UICH-00137
UICH-00133
UICH-00138
UICH-00162
UICH-00163
UICH-00170
UICH-00169
UICH-00171
UICH-00173
UICH-00175
UICH-00174
UICH-00176
UICH-00177
UICH-00172
UICH-00178
UICH-00168
UICH-00166
UICH-00167
UICH-00129
UICH-00153
UICH-00157
UICH-00123
UICH-00127
UICH-00158
UICH-00159
UICH-00131
UICH-00148
UICH-00139
UICH-00142
UICH-00124
UICH-00125
UICH-00126
UICH-00145
UICH-00132
UICH-00144
UICH-00146
UICH-00150
UICH-00143
UICH-00130
UICH-00152
UICH-00136
UICH-00149
UICH-00156
UICH-00128
UICH-00160
UICH-0016
UICH-001
UICH-00
UICH-0
UICH-
UICH
UIC
UIC
GENDER
ARAM_DIET
B Escherichia coli Prevotella dentalis C 30

Escherichia coli
abundance (%)
1.0
Percentage of
40 20
abundance (%)
Percentage of
30 0.8
abundance (%)
Percentage of
0.6 10
20
10 0.4 0
0 0.2 -10
LoxP Δlyz
-10 0.0 LoxP ΔIEC
VDR VDR
LoxP ΔIEC VDR VDR
VDR VDR
Parabacteroides sp. CT06 Akkermansia muciniphila Parabacteroides sp. CT06

0.5 0.08 0.5
*
abundance (%)
abundance (%)
abundance (%)
0.4
Percentage of
Percentage of
0.4
Percentage of
0.06
0.3 0.3
0.04
0.2 0.2
0.1 0.02 0.1
0.0 0.00 0.0
LoxP ΔIEC LoxP ΔIEC LoxP Δlyz
Parabacteroides distasonis Parabacteroides distasonis

0.20 * 0.20
abundance (%)
abundance (%)
Percentage of
0.15
Percentage of
0.15
0.10 0.10
0.05 0.05
0.00 0.00
LoxP ΔIEC LoxP Δlyz
VDR VDR VDR VDR
Figure 9. (A) Heat map of a global metabolomic study comparing fecal samples from VDR deficient animals to
the control group as well as that were kept on HFD or a chow diet. Different group, gender, diet, super-pathways
are indicated by colors as indicated in the right panel. Variations in explicit microbial content following targeted
VDR deletion in intestine in (B) VDRΔIEC mice and (C) myeloid cell-specific VDRΔlyz mice. Each dot indicates
percentage of abundance in each mice sample. Significance is established at adjusted P < 0.05.
that VDR deficiency plays a critical role in bile acid accumulation. The same increase was not observed in males,
suggesting that sex hormones might also play a role in bile acid accumulation. This might be the reason why
Vitamin D deficiency makes females more vulnerable to metabolic disorders, including obesity55. Polyamines
facilitate a switch between different coactivator complexes that bind to nuclear receptors, such as VDR19.

Figure 10. Western blot results showing increased FXR expression in the colonic epithelium (A) as well as in
liver hepatic cells (B) following VDR deletion and HFD. (C) IHC staining of FXR (in brown color) in hepatic
sections indicated increased expression (N = 3–6). (D) A working model showing role of VDR in regulating
microbiome and metabolite and obesity. The absence of VDR leads to altered metabolites, which contribute to
the disease state. Significance was established at adjusted P < 0.05.
The increase in polyamines was more significant in male VDRΔIEC mice, indicating the gender differences in
metabolite-VDR interactions.
One of the limitations of our study is that it does not provide information on urolithin and that certain bio-
chemicals did not reach levels of significance. However, we did clearly demonstrate that in the absence of VDR,

altered intestinal homeostasis occurred which could drive an altered intestinal metabolism. We need to deter-
mine the potential function of the microbiota by functional and taxonomic annotation of the microbiota, using
sample-matched data. Other future directions could include plasma samples from these mice, which will further
improve our understanding of the role of intestinal homeostasis to host metabolism. As a path forward, we need
to validate changes seen in this dataset in various disease models specifically related to metabolic syndrome and
in human cohorts (representing both diets and genders).
Conclusion
VDR is crucial for maintaining a healthy microbiome and metabolome. Our study reports how VDR defi-
ciency not only drives derangements in gut-microbiome but also significantly alters the metabolite profile in a
tissue- and gender-specific manner. We have demonstrated the role of nuclear receptors (e.g., VDR and FXR)
in regulating host physiology and microbial metabolites in health and obesity. These findings may potentially
inform strategies for the prevention and management of metabolic diseases by elevating VDR and restoring
host-microbiome-metabolites.
Methods
Experimental animals and design. VDRLoxP mice were formerly developed by Dr. Geert Carmeliet.
VDRΔIEC mice were obtained by crossing the VDRLoxP mice with villin-cre mice and VDRΔlyz mice were obtained
by crossing Lyz-cre mice. Both Vilin-cre and Lyz-cre mice purchased from Jackson Laboratories. VDRΔIEC mice
were derived from heterozygous mating pairs so that wild type and conditional KO mice came from the same
litter. The same breeding method was used for the VDRLoxP mice.
Six-week old VDRΔIEC (8 female and 9 male), VDRΔlyz (5 female and 5 male), and VDRLoxP (6 female and 10
male) mice were received normal chow (10% fat calories) diet. All mice were housed in specific pathogen-free
environments under a controlled condition of 12 h light/12 h dark cycle at 20–22 °C and 45 ± 5% humidity, with
free access to food and ultrapure water. All animal work was approved by the University of Illinois at Chicago
Committee on Animal Resources. All experiments were performed in accordance with relevant guidelines and
regulations.
This was a three-way (Genotype, Diet Treatment, and Gender) study design. To further evaluate the diet effect
of VDRΔIEC versus VDRLoxP, an additional 7 VDRΔIEC (3 female and 4 male) and 6 VDRLoxP (4 female and 2 male)
mice were fed with high-fat diet (45% fat calories) for 16 weeks. The body weights and food intake of all animals
were observed once a week during the experiments. Fecal contents of mice were carefully collected in separate
Eppendorf tubes, labeled with unique identification number and stored at −80 °C until sipped. Samples were
transported to Metabolon Inc, NC, USA in dry ice by overnight shipment for analysis.
Sample preparation. Fecal samples were prepared using the automated MicroLab STAR system from ®
Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC
purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein
matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous
shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided
into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion
mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for
analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples
®
were placed briefly on a TurboVap (Zymark) to remove the organic solvent. The sample extracts were stored
overnight under nitrogen before preparation for analysis.
Western blotting. Colonic mucosa and liver tissues from mice were isolated and sonicated in lysis buffer (1%
Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA pH 8.0, 0.2 mmol/L
sodium ortho-vanadate, and protease inhibitor cocktail), as previously described8. Primary antibodies to mouse
VDR, β-actin (Sigma-Aldrich, Milwaukee, WI, USA), and FXR (Santacruz, CA, USA) were used. WB was finally
visualized using an ECL kit. The relative abundance of protein was determined using Image-J (NIH) software.
The gels/blots used in figures are checked their compliance with the digital image and integrity policies in Nature
publisher.
Immunohistochemical staining (IHC). Immunohistochemistry (IHC) was performed using sections

of paraffin-embedded liver tissue as previously described6. Sections were incubated in primary antibody FXR
(Santacruz, CA, USA) 1:50 diluted for in blocking buffer) at 4 °C overnight. Then washed three times with 0.1%
Tween in PBS, and incubated with biotin-conjugated secondary antibody at room temperature for 1 h, washed,
incubated with ABC reagent at RT for 1 h (Vector lab PK-6100 standard), washed, visualized with DAB kit (Vector
lab SK-4100) and counterstained with hematoxylin. Images were captured by B21 fluorescence microscope.
Shotgun sequencing. Fecal samples were used for shotgun sequencing. genomic DNA was fragmented into
relatively small pieces (generally 250-600 bp fragments) prior to sequencing. Subsequently, the known sequences
are used to manipulate the DNA by way of PCR amplification (to increase the total amount of DNA but without
selecting for any specific sequences) and for the initiation of the sequencing reaction, again without selection for
any specific sequence from the source genomic DNA. Millions to hundreds of millions of short sequences (gen-
erally 150 bases, in pairs) are generated using Illumina sequencing platforms. These data were analyzed by (a)
searching for lineage-specific marker genes; (b) high-throughput BLAST analysis of individual sequences against
a reference sequence; (c) assembly of larger DNA sequences (“contigs”) from the short-read data. Ultimately, the
annotated data can be used to characterize the gene content of microbial communities, measure diversity, and

identify differences in the relative abundance of microbial features (i.e., taxa, genes, and pathways) between dif-
ferent groups of samples. We used the UIC genomic facility for our studies.
Statistical analysis. Raw data were extracted, peak-identified and QC processed using Metabolon’s hard-
ware and software. Compounds were identified by comparison to library entries of purified standards or recur-
rent unknown entities. Furthermore, biochemical identifications are based on three standard criteria. A variety of
procedures were carried out to ensure that a high-quality data set was made available for statistical analysis and
data interpretation. Metabolites were quantified and data were normalized as necessary.
The analysis data were presented as a fold change ratio of treatment vs. control. All the tests were two-sided.
The numbers of biochemicals were summarized as statistical significance at P ≤ 0.05 and 0.05 < P < 0.10 levels.
Following log transformation and imputation of missing values as appropriate, a two-way ANOVA with contrasts
and Welch’s two-sample t-test were used to identify biochemicals that differed significantly between genotypes
and treatment groups. Three-way ANOVA was further conducted to identify biochemicals exhibiting significant
interaction and main effects for experimental parameters of genotype, diet, and gender. An estimate of the false
discovery rate (q-value) was calculated to take into account the multiple comparisons that normally occur in
metabolomic-based studies. The matched pairs t-test is used to test whether two unknown means are different
from paired observations taken on the same sample. To present the high-level overview of data structure for
experimental parameters of genotype, diet, and gender, principal component analysis (PCA) along with hierar-
chical clustering analysis (HCA) as well as random forest (RF) analysis were conducted to highlight biochemical
alterations between fecal samples collected from VDRΔIEC, VDR∆lyz, and VDR LoxP, mice that were kept either on
HFD or chow diet as well as gender differences.
Ethics approval and consent to participate. No human study. All animal studies were performed fol-
lowing ACC guidelines at the University of Illinois at Chicago (UIC), IL, USA.
Data availability
Data and material will be available by request.
Received: 24 October 2019; Accepted: 3 January 2020;

Published: xx xx xxxx
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Acknowledgements
We would like to thank Dr. Joann Romano-Keeler, Shari Garrett and Jason Xia for helping with proofreading.
We would like to acknowledge the support from UIC Cancer Center, the NIDDK grant R01 DK105118,
R01DK114126, and DOD CDMRP log No BC160450P1 to Jun Sun.
Author contributions
Jun Sun obtained funds, designed the study, and directed the project. Yinglin Xia designed the study and directed
the project for the statistical analysis of microbiome and other data. Ishita Chatterjee performed Western blots,
IHC, and detailed analysis, prepared for figures and draft, and analyzed the metabolite data. Rong Lu and
Yongguo Zhang the performed animal studies; Jilei Zhang helped with the metagenomic analysis of microbiome.
Yang Dai helped with microbiome and bioinformatics analysis of the data. All authors contributed to the writing
of the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-64226-7.
Correspondence and requests for materials should be addressed to Y.X. or J.S.
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© The Author(s) 2020

Vitamin D Receptor Promotes Healthy Microbial Metabolites and Microbiome

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OPEN Vitamin D receptor promotes

Scientific Reports | (2020) 10:7340 | https://doi.org/10.1038/s41598-020-64226-7 1

Table 1. Intestinal epithelial VDR on the profile of metabolites.

Protein/Amino-acid metabolism. Gut bacteria produce a range of metabolites by synthesizing proteinogenic

Myeloid cell-specific VDR knockdown contributes to the definitive alteration of metabolites

Scientific Reports | (2020) 10:7340 | https://doi.org/10.1038/s41598-020-64226-7 2

Increasing Importance to group Separation

B Carbohydrate Metabolites C Amino Acid Metabolites

D Lipid Metabolites E Xenobiotics Metabolites

Scientific Reports | (2020) 10:7340 | https://doi.org/10.1038/s41598-020-64226-7 3

A Carbohydrate Metabolites B Amino Acid Metabolites

Behenoyl ethanolamide (22:0)

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B Secondary bile acid metabolism in female

Tauroursodeoxycholic acid sulfate (2)

C Secondary bile acid metabolism D Secondary bile acid metabolism in female

Tauroursodeoxycholic acid sulfate (2)

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Increasing Importance to group Separation

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A Phenyllactate (PLA) 3-hydroxyphenylacetate

0.0 0.0 0.0

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B Escherichia coli Prevotella dentalis C 30

Parabacteroides sp. CT06 Akkermansia muciniphila Parabacteroides sp. CT06

Parabacteroides distasonis Parabacteroides distasonis

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Immunohistochemical staining (IHC). Immunohistochemistry (IHC) was performed using sections

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Received: 24 October 2019; Accepted: 3 January 2020;

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© The Author(s) 2020

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