Vitamin D Receptor Promotes Healthy Microbial Metabolites

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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-specifc deletion of VDR in intestinal epithelial cells or myeloid cells. Conditional VDR deletion severely changed metabolites specifcally produced from carbohydrate, protein, lipid, and bile acid metabolism. Eighty-four out of 765 biochemicals were signifcantly altered due to the Vdr status, and 530 signifcant 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 diferences in metabolites generated from energy expenditure, tri-carboxylic acid cycle, tocopherol, polyamine metabolism, and bile acids. The efect of HFD was more pronounced in female mice after VDR deletion. Interestingly, the expression levels of farnesoid X receptor in liver and intestine were signifcantly increased after intestinal epithelial VDR deletion and were further increased by the high-fat diet. Our study highlights the gender diferences, tissue specifcity, 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 metabolome — 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., infammatory bowel disease and obesity)2,3. Te human Vdr gene is the frst gene identifed as a vital host factor that shapes the gut microbiome at the genetic level4. In mice lacking VDR, we observed signifcant shifs 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. Tose 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 content, Alistipes and Odoribacter were signifcantly reduced whereas Eggerthella was increased6. Intestinal specifc 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 specifc tissues) modulate microbial metabolites and the microbiome, thus contributing to the high risk of metabolic diseases. We used intestinal epithelium-specifc VDR knock out (VDRΔIEC) mice and myeloid cell-specifc 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 efect of high-fat diet (HFD) on VDRΔIEC mice as compared to control chow diet-fed mice. We also correlated the altered metabolite profles to specifc mechanisms that lead to the observed changes in the host and microbiome.

1Division 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: [email protected]; [email protected]

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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 profle of metabolites.

Results Deletion of intestinal epithelial VDR impacted the overall metabolite profle. First, we examined the efects of intestinal epithelial VDR on the metabolite profle. Among named biochemical compounds, VDRΔIEC mice exhibited alterations in 68 metabolites (of which 35 increased and 33 decreased) with P ≤ 0.05 signifcance level and 55 biochemicals with 0.05 < P < 0.1 signifcance 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 diferent 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 signifcant diferentially expressed biochemicals. Te 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 infuencing host functions9. Te most signifcant 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 bacteria ferment nondigestible complex-carbohydrates and release glycogen, amino-sugars, and pentoses10. Tese are considered major factors in shaping the composition and physiology of the microbiome. As shown in Fig. 1B, VDRΔIEC had a signifcant (P ≤ 0.05) increase in amino-sugar metabolite N-acetylmuramate compared to the VDRLoxP control mice. Conversely, N-acetylglucosaminylasparagine, glucose, and galactonate were downregulated in the VDRΔIEC group (Fig. 1B). Tese results suggest that the loss of intestinal epithelial VDR in host modi- fes the carbohydrate metabolism, thus afecting 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 signifcant surge in polyamine, 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 mechanisms, 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 defciency in the intestinal epithelium altered the levels of many xenobiotics. RF analysis of animals showed that naringenin, a favonoid displays strong anti-infammatory and antioxidant function, among the pool of top 30 metabolites (Fig. 1A). Further ANOVA analysis indicates that most xenobiotics were signifcantly 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-specifc VDR knockdown contributes to the defnitive alteration of metabolites indicating the tissue-specifc role of host VDR. VDR is known to have tissue and cell-specifc roles15. Hence, we examined our myeloid cell-specifc VDR KO model. In the VDRΔlyz mice, 100 known biochemicals were found to be signifcantly altered with P-value ≤0.05 (of which 56 increased and 44 decreased) and 58 chemicals showed with signifcance 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 signifcantly changed (P ≤ 0.05) (black box, Table 2) and another 49 fell in

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A Maltose Maltotriose Ceramide (d18:1/20:0, d16:1/22:0, d20... Lactosyl-N-palmitoyl-sphingosine (d18... Taurohyodeoxycholic acid Diacetylspermidine* 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) Increasing Importance to group Separation N-alpha-acetylornithine 1-stearoyl-2-oleoyl-GPE (18:0/18:1) N2-acetyllysine 2-oleoylglycerol (18:1) Color Pathway Dihydrobiopterin Quinolinate AminoAcids 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

2 N1,N12-diacetylspermine 1.0 1.5 0.0 0.5 N('1)-acetylspermidine FC ratio ΔIEC LoxP Diacetylspermidine VDR /VDR N2-acetyllysine N-acetylglutamate

0.0 0.5 1.0 1.5 2.0 FC ratio ΔIEC LoxP VDR /VDR

D Lipid Metabolites E Xenobiotics Metabolites Succinimide 2-hydroxyheptanoate* 2-aminophenol sulfate Diosmin(diosmetin7-rutinoside) Glycerophosphoinositol Tartronate (hydroxymalonate) Glycerophosphoserine Diosmetin Soyasaponin III Trimethylamine N-oxide Soyasaponin II Soyasaponin I Valerylglycine Sinapet PheophytinA PheophorbideA Naringenin 1-oleoyl-GPG Mannonate* Homostachydrine* 1-palmitoyl-galactosylglycerol Glycitein Ferulic acid 4-sulfate 1-palmitoyl-2-linoleoyl-galactosylglycerol Ergothioneine Equol sulfate 1-stearoyl-2-oleoyl-GPE Equol Octadecanedioate Cinnamoylglycine Genistein 2,4,6-trihydroxybenzoate 0.0 0.5 1.0 1.5 2.0 2-hydroxyhippurate (salicylurate) Hippurate FC ratio 0 0.6 1.2 1.8 ΔIEC LoxP FC ratio VDR /VDR ΔIEC LoxP VDR /VDR

Figure 1. Impact of intestinal epithelial VDR deletion on metabolite profle: (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. Te variables are ordered top-to-bottom as most-to-least important in categorizing between VDR deleted (VDR∆IEC and VDR∆lyz) and VDRLoxP groups. Diferent color indicates specifc metabolites resulting from diferent 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. Diferences are assessed by the Mann–Whitney U test. VDRΔIEC (N = 17) & VDRLoxP (N = 16) mice. Signifcance is established at adjusted 0.05 < P < 0.1.

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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-specifc VDR contributes to the alteration of metabolites diferent from the Intestinal epithelial VDR.

ABCarbohydrate Metabolites Amino Acid Metabolites

N-acetylmuramate N-acetyl-cadaverine Diacetylchitobiose Histidine Fucose Matose Proline Maltotriose Citrulline Arabinose Ornithine Xylose Ribulose/xylulose N-acetylproline

0.0 1.0 2.0 3.0 4.0 5.0 Cysteine FC ratio Asparagine lyz LoxP VDR / VDR N-methylalanine

0.0 0.5 1.0 1.5 2.0 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

0 0.5 1.0 1.5 2.5 3.0 3.5 FC ratio lyz LoxP VDR / VDR

Figure 2. Overall alteration in the metabolites following myeloid cell-specifc VDR deletion: Fold change ratio generated by (A) carbohydrate (B) amino acids (C) lipid and (D) xenobiotics metabolism in VDR∆lyz mice. Te graph represents only those biochemicals showing maximum alterations among known detectable metabolites. Diferences are assessed by the Mann–Whitney U test. VDR∆lyz (N = 10) & VDRLoxP (N = 16). Signifcance is established at adjusted 0.05 < P < 0.1.

the signifcant 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 prominent decrease in diacetylchitobiose in VDRΔlyz mice was observed. Te VDR∆lyz group also showed decreased histidine, proline, and citrulline, which was accompanied by a rise in N-acetyl proline and asparagine levels,

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compared to VDRLoxP (Fig. 2B). Tese 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 signifcant increase in palmitoyl-linoleoyl-glycerol and a substantial decrease in sphingosines and fatty acid metabolism, indicated by metabolites like hexadecasphingosine and dihomo-linolenoylcarnitine (Fig. 2C). Remarkably, among the diferent xenobiotic metabolism pathways, the VDRΔlyz group displayed a signifcant downregulation of quinolinate and tocopherol pathway derived metabolites and an increase in nicotinamide (Fig. 2D). Tus, these data indicate the diferent roles of VDR in intestinal epithelial cells and myeloid cells.

VDR deletion signifcantly impacted bile acid profle, 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 modifcations in metabolites from secondary bile acid metabolism pathways. Te microbiota converts primary bile acids to secondary bile acids, which are then reabsorbed and can afect diverse biological processes18. Among diferent 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 specifcally observed in VDR∆lyz female mice (Fig. 3C). Both VDR∆IEC and VDR∆lyz female mice showed a signifcant 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 signifcantly infuences bile acid metabolism in a gender-specifc manner.

VDR defciency resulted in signifcant alterations in the polyamines levels. Polyamines have been shown to play a role in facilitating a switch between diferent coactivator complexes that bind to nuclear receptors such as VDR19. Here, we observed a signifcant 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 defcient 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 efect of VDR deletion on fatty acid metabolites. We found that carnitines were signifcantly elevated in fecal samples from VDRΔIEC and VDR∆lyz mice, compared to the VDRLoxP, including myristoylcarnitine (C14), palmitoylcarnitine (C16), oleoylcarnitine (C18:1) (Fig. 5A–C). Tis increase is accompanied by an elevation 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 defcient animals. However, there is no signifcant change in 3-hydroxybutyrate (BHBA).

HFD intervention altered metabolite profle in VDRLoxP and VDRΔIEC mice. Studies have shown that obese humans and mice have microbiomes very diferent from their lean controls21–24. We further evaluated how diet impacted the metabolites in mice with tissue-specifc VDR deletion. Te 30-top ranking biochemicals in the importance plot suggests key diferences in peptides, lipid metabolism, cofactors, vitamins, and amino acids with maximum impact on threonyl phenylalanine (Fig. 6A). RF-classifcation 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 indolepropionate were decreased in both VDRLoxP and VDRΔIEC HFD groups, as compared to the regular chow diet (Fig. 7A). Alternatively, trans and cis-urocanate were signifcantly increased by HFD (Fig. 7B). Levels of equol, a microbiota-derived metabolite known to exert epigenetic changes by inhibiting DNA methylation, histone mod- ifcation, 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 defcient 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 signifcant elevation in levels of polyamines, such as spermidine, N1, N2-diacetylspermine, in all VDRΔIEC animals especially afer HFD feeding, as compared to VDRLoxP group (Fig. 7D). Tese data suggest an accumulation of polyamines in HFD fed VDR defcient animals. Because polyamines have strong anti-infammatory functions25, these changes may impact aspects of host immunity.

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A Lithocholate DeoxycholateDeoxycholate 5.0 4.0

4.0 3.0

3.0 2.0 2.0

1.0 1.0

0.0 0.0 FMF M FM FM FMF M FFMM F M LoxPLoxP ΔIEC LoxP ΔIEC Δlyz VDR VDR VDR Δlyz VDR 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 Deoxycholicacid3-sulfate

Deoxycholate 0481216 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 3b-hydroxy-5-cholenoicacid 3b-hydroxy-5-cholenoic acid 7-ketodeoxycholate Lithocholate Taurocholenatesulfate* 3-dehydrodeoxycholate 6-oxolithocholate Deoxycholate Dehydrolithocholate 0.0 0.4 0.8 1.2 1.6 FC ratio 12-ketolithocholate Δlyz LoxP VDR / VDR Lithocholate 3-dehydrodeoxycholate Deoxycholate

0246 FC ratio Δlyz LoxP VDR / VDR

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) Specifc changes in secondary bile acid metabolites as noted in female VDR∆IEC mice. (C) Collective changes demonstrated by VDR∆lyz mice. (D) Defnite variations in secondary bile acid metabolites displayed by female VDR∆lyz mice. Te data presented as the fold change (FC) ratios of the average concentrations of identifed 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). Diferences are assessed by the Mann–Whitney U test. Signifcance is established at an adjusted 0.05 < P < 0.1.

Gender-specifc changes in HFD fed mice following VDR deletion. When metabolites were analyzed 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 efect was less signifcant (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 defciency along with HFD immensely altered the bile acid levels in fecal samples. Specifically, levels of the bile acids, taurolithocholate 3 sulphate, and taurocholenate sulphate were raised in VDR defcient animals (Fig. 8A). Metabolites like N-acetyltyrosine, N-formylphenylalanine, and indolepropionate were signifcantly changed in the VDRΔIEC,

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N1,N12-diacetylspermine A 8.0

6.0

4.0

2.0

0.0 F M FM FM Chow Chow Chow VDR LoxP VDR ΔIEC VDR Δlyz

Diacetylspermidine* B 5.0

4.0

3.0

2.0

1.0

0.0 FM F MFM Chow Chow Chow LoxP Δlyz VDR VDR ΔIEC VDR

N('1)-acetylspermidine C 10.0

8.0

6.0

4.0

2.0

0.0 F M FFM M Chow Chow Chow VDR LoxP VDR ΔIEC VDR Δlyz

Figure 4. VDR defciency results in signifcant 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. Tis 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). Te ratio of fold-change diferences are assessed by the Mann–Whitney U test. Signifcance is established at adjusted 0.05 < P < 0.1.

compared to VDRLoxP males; they remain unaltered afer HFD feeding, suggesting that diet might impact changes that resulted from VDR defciency 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 deletion of intestinal epithelial cell-specifc VDR impacted metabolites diferently than myeloid cell-specifc VDR deletion, we further checked whether these specifc metabolite profles 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. Te diference in the microbial communities might be related to diferences in tryptophan, polyamine, and tocopherol metabolism observed in this study. Te abundance of Parabacteroides afected by VDR signaling in both human and mouse

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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 F M F M F M F M FM F M ΔIEC LoxP ΔIEC Δlyz VDR LoxP VDR VDR Δlyz 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 FFFM F MFM 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 FFM M F M FM FFM M LoxP ΔIEC Δlyz ΔIEC VDR VDR VDR VDR LoxP VDR VDR Δlyz

Figure 5. VDR defciency 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. Tis surge was accompanied by elevated levels of long-chain fatty acids (LCFAs) (A) myristate, (B) palmitoleate, (C) oleate. Tis data is represented as BOX-Plot diagram showing maximum and minimum variation among the group. Tis 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). Signifcance 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 afer 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 signifcant 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 defcient 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 signifcantly impacts bile acid metabolism. Hence, we wanted

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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 defcient mice.

to check whether hepatic FXR was altered. Immunohistochemical staining (IHC) of liver sections showed a similar 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 profles and the gut microbiome, leading to an increased risk of obesity. We found that 84 identifable biochemicals that were signifcantly altered due to the VDR status. When challenged with a HFD, 530 metabolites showed discrete changes. Tese changes were observed due to variations in carbohydrate, protein, lipid, and xenobiotic pathways. Te 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 infuenced female mice more than the males. At the protein level, we found that FXR expression was increased afer 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 profles 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 microbiota actively participates in host metabolism by regulating metabolites generated from bile acid metabolism via VDR-FXR signaling. VDR defciency alters the metabolite profle and FXR expression in the host. Our data suggest the tissue specifcity and gender diferences 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 deletion indicating changes in glycogen metabolism as well as lipid and amino acid super pathways, whereas RF analysis using data derived from HFD fed VDRloxP 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. Te amino acid metabolite taurine is known to be protective against infammation, apoptosis, and oxidative stress29. Te kynurenine pathway is associated with infammatory neurological disorders30.

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A Threonylphenylalanine Lysylleucine Lanosterol 1-methinicotinamide Hepatodecasphingosine Indolepropionate Ceramide Valylleucine N-acetylputrescine Alanylleucine Palmitoylcarnitine 7-methylguanine Margaroycarnitine Oleoyl ethanolamide Sphingosine 3-ureidopropionate Oleoylcarnitine Putrescine Docosadienoate Eicosenoate N-Stearoyl-sphingosine

Increasing Importance to group Separation 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

B 20 ]

0 Comp.2 [13.82%

-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 classifcation 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. Diferent colors indicate diferent biochemicals like, light blue = amino acid, purple = cofactors &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

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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 MFM FMM F F M F M FMF M F M F M F M FMF M F M Chow HFFD Chow HFD Chow HFD ChowCh HFDDCChChow HFD ChowChCh HFFDFD LoxP/LoxP ΔIEC LoxP/LoxP ΔIEC LoxP/LoxP ΔIEC VDR VDR VDR VDR VDR VDR

Cis-Uroconate TTrransans-Uroconat-Uroconate B 6.0 5.0 6.0 4.0 3.0 4.0 2.0 2.0 1.0 0.0 F M F M FMM F 0.0 Chow HFD Chow HFD F M F M FFMM F M LoxP ΔIEC ChowChoow HFDDCChhowhoow HHFD LoxPL ΔIEC VDR VDR VDR VDR

Alpha-tocopherol Gamma-tocopherol/beta-tocopherol C 1.4 2.0

1.2 1.6 1.0 1.2 0.8

0.6 0.8

0.4 0.4 F M F F F M M M F M F M F M F M Chow HFDHChow FD Chow HFD Chow HFD LoxP ΔIEC VDR VDR LoxP ΔIEC VDR VDR

N1,N12-diacetylspermine D 8.0

6.0

4.0

2.0

0.0 F M F M F M F M Chow HFDChow HFD ΔIEC VDRVLoxP DR

Figure 7. HFD altered the metabolite profle: Intestinal specifc 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 afer HFD feeding. Tese 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). Signifcance is established at adjusted 0.05 < P < 0.1.

and can afect 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 profle 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 signifcant factors in shaping the microbiome of diet-induced obese mice35. Te 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 infammation. Our study demonstrates that long-chain fatty acids (LCFAs) and acylcarnitines are signifcantly elevated in VDR defcient 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 subsequently, infammatory cells in close proximity to the colon can produce reactive oxygen species36. As a result, VDR defciency may cause lower levels of tocopherols, which may be indicative of an increased risk for colon cancer.

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Taurolithocholate 3-sulfate Taurocholenate sulfate* A 7.0 12.0

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 M M FMFMF MFM F F M F MMF Chow HFDChow HFD Chow HFD Chow HFD LoxP ΔIEC VDR LoxP VDR ΔIEC VDR 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 FFFM F M F M FFFFFM 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-specifc 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. Diferences 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). Signifcance 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 infammation 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 shif in the metabolic capacitance of the microbiota is also associated with the severity of nonalcoholic 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 signifcantly down, and Eggerthella were increased. Intestinal specifc deletion of VDR leads to dysbiosis due to increased E. coli and Bacteroides and decreased butyrate-producing bacteria7. Tis 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 profle. Accordingly, the percentage of abundance of Parabacteroides distasonis (PD) signifcantly 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-infammatory efect on LPS-induced infammation. 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-infammatory 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 defciency 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 benefcial efects depending on the VDR status51. Gut microbiota controls neurobehavior via modulating brain insulin sensitivity and metabolism of tryptophan, the precursor of serotonin52. Increased infux of tryptophan into the brain by HFD could be related to increased blood insulin levels. VDRΔlyz group displayed signifcant downregulation of quinolinate and tocopherol pathway derived metabolites and increase in nicotinamide. Quinolinic acid acts as a neurotoxin, proinfammatory mediator, and prooxidant molecule53. Loss of intestinal VDR also increased kynurenine, a pathway associated with infammatory neurological disorder30. Tese changes indicate the role of VDR in neurophysiology. Te role of VDR/vitamin D in the gut-brain axis needs further investigation in future research. We have demonstrated gender diferences in metabolites that may be regulated by VDR status. Female mice were shown to be more afected by VDR deletion than male mice. Higher circulating bile acids were observed in obese and type-2 diabetes54. Tus, signifcant elevation of secondary bile acids in VDR defcient females indicates

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tauroursodeoxycholicstearoylthioproline sphingomyelin acid (d18:1/18:0) sulfate (2) -5 5 1-stearoyl-2-arachidonoyl-GPE1-(1-enyl-palmitoyl)-2-arachidonoyl-G...N-acetyl-beta-glucosaminylaminegamma-glutamyl-epsilon-lysinphenylalanylalaninecysteinetrans-urocanatevalylglutaminetyrosylglycincis-urocanateasparagineagmatinearginine s-sulfate e e (18:0/2... 7-alpha-hydroxy-3-oxo-4-cholestenoate...N-(2-hydroxypalmitoyl)-sphingosine5-methylthioadenosineN-acetylmethioninealpha-ketobutyrateN-acetylarginineisoleucylglycinedeoxycarnitinphenylacetatindolin-2-oncitrullineornithinelysine eee (MTA) (d... lactosyl-N-palmitoyl-sphingosine(163beta-hydroxy-5-cholestenoatlinolenoylcarnitinehydroxystearate3-dehydrodeoxycholatstearoylcarnitine3-hydroxybehenatedehydrolithocholateeicosenoate12-ketolithocholate6-oxolithocholaterucate or 17)-methylstearate (22:1n9) (20:1)e sulfat(C18) (C18:3)* e (a19:0* e or(d18... i... Group 18-methylnonadecanoate10-nonadecenoate2-palmitoylglycerolnonadecanoate2-hydroxyarachidate*2-hydroxypalmitat2-hydroxystearatearachidatemargaratestearate (18:0) (17:0)(20:0) (19:0)e (16:0)(19:1n9) (i20:0) LoxP dihomo-linolenoylcarnitine1-(1-enyl-palmitoyl)-2-palmitoyl-GPC1-(1-enyl-palmitoyl)-2-oleoyl-GPC1-(1-enyl-palmitoyl)-2-arachidonoyl-G...1-(1-enyl-palmitoyl)-2-linoleoyl-GPC1-(1-enyl-palmitoyl)-GPCglycerophosphorylcholine1-(1-enyl-oleoyl)-GPEarachidonoylcarnitinedihomo-linoleatedocosadienoatepalmitoleatepalmitate (16:0) (16:1n7) (22:2n6) (20:2n6) (C20:4(P-18:1) (P-16:0)(GPC (C20:3n3) *) *(P-...... o...... ceramideN-stearoyl-sphingosineN-stearoyl-sphinganineN-oleoyl-sphingosineeicosanoylsphingosinehexadecasphingosineheptadecasphingosine4-cholesten-3-ondesmosterolsphinganinsphingosine (d18:1/20:0,(d18:1/17:0,(d18:1/14:0,e e (d18:1/18:1) (d16:1)*d16:1/22:0,d17:1/18:0)d16:1/16:0) (d17:1(d20:1)(d18:1/18:0)(d18:0/18:0))* *d20...* VDR Chow N-acetyl-isoputreanine*N-acetyl-beta-alaninemevalonolactonphytosphingosinemannitol/sorbitolpyroglutamine*arabitol/xylitolpantoatebetainribitol e e LoxP N1-Methyl-4-pyridone-3-carboxamideN1-Methyl-2-pyridone-5-carboxamide1-(3-aminopropyl)-2-pyrrolidon5-hydroxyindoleacetate3-amino-2-piperidon1-methylhistaminN-acetylglutamineN-acetylhistidinexanthurenatehypotaurinecarnitinecreatinurate e e e e VDR Chow N,N,N-trimethyl-alanylprolinesuccinylcarnitine2,8-quinolinediol2-hydroxyphenylacetate4-hydroxymandelatglyciteinsulfate* sulfate (2 sulfat(C4-DC) e e ) betaine... ΔIEC 3-(3-hydroxyphenyl)propionateribulonate/xylulonate/lyxonateN-formylanthranilicaconitatehydroquinone2,4-dihydroxybutyratedaidzein4-hydroxybenzoathomostachydrine*equolanthranilatvanillactatcitrate sulfat sulfate [ciseee orsulfat (1trans]e) acide * sulfate trigonelline3-hydroxy-2-ethylpropionateethylN2,N5-diacetylornithineN-glycolylneuraminatep-cresolN-carbamoylalanine2-hydroxyoctanoatpropionylglycine3-methyladipatethylmalonatstachydrinequinat alpha-glucopyranosidee glucuronide* (N'-methylnicotinate)e e e VDR Chow 3-hydroxyphenylacetatebeta-guanidinopropanoathydantoin-5-propionatmethylmalonate1-methylnicotinamid2'-O-methylcytidinN-formylmethionineO-sulfo-L-tyrosinebiopterin (MMA)e e e sulfate e ΔIEC N-acetyl-2-aminooctanoateN-acetyl-3-methylhistidine*3-hydroxypyridine6-hydroxyindoleC-glycosyltryptophancaffeicequolN-acetylpyrralineisobutyrylglycine7-methylguanintigloylglycin glucuronide acid esulfate sulfate sulfate e * VDR Chow 1,2,3-benzenetriolN-acetyl-1-methylhistidine*2-acetamidophenol4-methylhexanoylglycine4-methylcatechol2-amino-p-cresoladipoylcarnitinephenylpropionylglycineindolepropionylglycinphenol2-butenoylglycinp-cresolerythritol glucuronid sulfate (C6-DC)e sulfate sulfate sulfate e e (2) glycinep-hydroxybenzyl-glucosinolat1-ribosyl-imidazoleacetate*3-methylcrotonylglycinnicotinamide5-methylthioribose*hexanoylglycineallantoicurea conjugate acid N-oxide of* C10H14O2e e (1)* Δlyz S-(3-hydroxypropyl)mercapturic3,4-dihydroxyphenylacetate4-hydroxycinnamatedihydrocaffeatedihydroferulic4-vinylphenolphenylacetylglycinisocaproylglycindihydrobiopterinphenolhippuratallantoinorotidin sulfatee e sulfatacid esulfate esulfate sulfat (2e)e sulfat acide (... VDR Chow dimethylguanidino2-hydroxyhippuratetrimethylamine2-aminophenolferulicN-(2-furoyl)glycincinnamoylglycinguanidinoacetat3-indoxylferulylglycinecatecholisocitriccreatinin acid lactone esulfat sulfat 4-sulfat (1e eN-oxidsulfat)ee valerice (salicylurate)ee acid (DMGV)* docosahexaenoateeicosapentaenoate3,4-methylenearachidonate4-hydroxyhippuratindoleacetylglycine2'-deoxyguanosineisovalerylglycin2'-deoxycytidinepicolinoylglycinevalerylglycinebutyrylglycinegulonate* (20:4n6) heptanoylglycinee e (DHA;(EPA; 20:5n322:6n3)) Gender docosapentaenoatedihomo-linoleoylcarnitineeicosenoylcarnitinelignoceroylcarnitinemargaroylcarnitinearachidoylcarnitinebehenoylcarnitinepalmitoylcarnitine2-stearoyl-GPEoleoylcarnitine1-stearoyl-GPIN-acetylputrescinputrescine (C18:1(18:0) (18:0)e (C16)(C22) (C17)* (C20)* (C20:1)(C24)(n6)* *DPA; (C20:2)** * 22:5n6) 1-palmitoyl-2-stearoyl-GPC1-(1-enyl-palmitoyl)-GPE1-(1-enyl-stearoyl)-GPEpalmitoleoylcarnitinemyristoleoylcarnitineglycerophosphoethanolaminelinoleoylcarnitinemyristoylcarnitine1-palmitoyl-GPC1-stearoyl-GPE1-stearoyl-GPC3-hydroxysuberatecholate sulfate (18:0) (16:0)(C18:2)* (C14) (C14:1)*(C16:1)* (P-18:0) (P-16:0) (16:0/18:0** ) Female octadecenedioylcarnitineN-palmitoyl-sphinganineoctadecanedioylcarnitinepalmitoleoylmargaroyl7,12-diketolithocholatestearoyl3-dehydrocholatebilirubincholesterolbiliverdincholate (E,E)*(Z,Z)ethanolamide ethanolamide* ethanolamide (d18:0/16:0) (C18:1-DC)(C18-DC)* * * sphingomyelinhydroxypalmitoylpalmitoylN-palmitoyl-sphingosineceramide(3'-5')-guanylyladenosine*(3'-5')-adenylylguanosine*(3'-5')-guanylylcytidin(3'-5')-guanylyluridin5-aminovaleratcadaverin dihydrosphingomyelinsphingomyeline(d18:2/24:1, e(d17:1/16:0,(d18:1/24:1, sphingomyelinee d18:1/24:2) (d18:1/16:0)(d18:1/16:0 d18:1/15:0...d18:2/24:0)* (d18:0..(d18:1...* ) . Male N-acetylglucosaminylasparagineglycerophosphoserine*cysteinephenylacetyltaurinN-linoleoyltaurine*N-stearoyltaurineN-oleoyltaurinN-acetyltaurinN-acetylglycinsuccinimidekynurenineguanintaurinee sulfinice acide taurohyodeoxycholictaurochenodeoxycholattauroursodeoxycholat3-hydroxyanthranilattauro-beta-muricholatglycocholatetaurodeoxycholatprotoporphyrinN-acetylprolineprolylglycinetaurocholatsarcosine e sulfat IXee e acide e Diet 1-palmitoyl-galactosylglycerolN1,N12-diacetylspermine1-palmitoyl-GPG1-stearoyl-GPG1-oleoyl-GPGisovalerateheptanoatecaprylatecaproatevalerateI-urobilinogenicotinamidbenzoate (5:0 (6:0(8:0 e(i5:0(7:0n))) (18:1) ))(18:0) (16:0)* * (16:0)* 1-pentadecanoylglycerol1-methyl-5-imidazoleacetatgamma-glutamylisoleucine*5-methyl-2'-deoxycytidin4-hydroxyphenylacetategamma-glutamylglycingamma-glutamylvalineN-formylphenylalanine2'-deoxyuridine2'-deoxyinosinN6-formyllysinethymidinetyrosol e e e (15:0)e Chow uridine-2',3'-cyclichexadecenedioate3-(4-hydroxyphenyl)propionate3-(3-hydroxyphenyl)propionate1,5-anhydroglucitol2,3-dihydroxyisovalerateN6-succinyladenosin3-hydroxydecanoate2'-deoxyadenosineindolelactatspermidineadenincytidinee e monophosphat (C16:1-DC) (1,5-AGe ) * e pyridoxine1-linoleoyl-GPE1-linoleoyl-GPG1-linoleoyl-GPI1-oleoyl-GPEN-acetyl-cadaverine1-oleoyl-GPIN-acetylthreonineN-acetylcitrullinimidazole5-methylcytosineN-acetylserine lactat (Vitamin (18:1) (18:1) e(18:2) (18:2) B6***) HFD 1,2-dilinoleoyl-galactosylglycerol1,2-dilinolenoyl-galactosylglyceroltartronate1-palmitoyl-GPIN,N-dimethylalanindeoxymugineic1-oleoyl-GPCdimethylglycineN-methyl-GABAcystathioninekynurenatnaringeninglycitein e(hydroxymalonate (18:1 acid(16:0)e) ) (1... (... gamma-tocopherol/beta-tocopherol1-lignoceroyl-GPCpalmitoyldaidzeingenisteinlinoleoylcholinepalmitoylcholinesoyasaponinformononetioleoylcholineafromosin sulfateethanolamidesulfate*n III I* (2) (24:0) oleoyl-linolenoyl-glycerollinoleoyl-linolenoyl-glycerololeoyl-linoleoyl-glycerollinolenoyl-linolenoyl-glycerollinoleoyl-linoleoyl-glycerollinolenoyllinoleoylgamma-tocotrienooleoylalpha-tocotrieno ethanolamide ethanolamid ethanolamidel l e (18:1/18:2)... (18:1/18:3... (18:2/18... (18:2/1... (18:3/... Super Pathway 1-palmitoyl-2-linoleoyl-GPCpalmitoyl-linoleoyl-glycerol1-oleoyl-2-linoleoyl-GPC1,2-dilinoleoyl-GPCglycerolcholineribulose/xylulosegalacturonat12,13-DiHOMED-urobilinarabinosglucosxylosee phosphat 3-phosphate e e e(18:2/18:2 (18:1/18:2)* (16:0/18... (16:0/18:2) ) 1-linoleoyl-2-linolenoyl-GPC3-phenylpropionate1,2-dilinoleoyl-GPEN-acetylmethionine(3'-5')-adenylyladenosine*(3'-5')-adenylylcytidin(3'-5')-adenylyluridin4-hydroxycinnamatferuloylputrescinN-acetylhistamineN-acetylmuramateenterolactonhistamine e e e (hydrocinnamatesulfoxide(18:2/18:2)*ee (18:2/18...) Amino Acid 1-palmitoyl-2-docosahexaenoyl-GPC1-palmitoyl-2-arachidonoyl-GPE1-palmitoyl-2-palmitoleoyl-GPC1-stearoyl-2-linoleoyl-GPC1-oleoyl-2-linoleoyl-GPE1-palmitoyl-2-oleoyl-GPC1,2-dipalmitoyl-GPCN-acetylneuraminatstigmastadienonadenosinglyceratecholinefucose e e e (16:0/16:0 (18:1/18:2)* (16:0/18:1 (18:0/18:2)*) (16:0/.. (16:0/..) (16..... hexadecatrienoate3-hydroxydodecanedioate2-linoleoylglycerol1-linoleoylglycerol2-oleoylglycerol1-oleoylglycerol3-hydroxysebacatealpha-tocopherolnicotianaminestigmasteromevalonateergosterollanosterol l (18:1) (18:2)(16:3n3)* Carbohydrate linolenate5-(2-Hydroxyethyl)-4-methylthiazoletridecenedioatedehydrophytosphingosine*hydroxymethylpyrimidin13-HODEretinollinoleatepheophorbidedelta-tocopherolbeta-sitosterolcampesterolfucosterol (Vitamin (18:2n6) [alpha+ 9-HODE A A)(C13:1-DC)* or gamma;e (18:3n3 o... adenosinediosmin(R)-3-hydroxybutyrylcarnitin2-oxindole-3-acetatebeta-cryptoxanthincarotenepheophytincoumestrogenisteindaidzeinequol (diosmetin diol l5'-monophosphate A (3(2(1) 7-rutinoside)e (AMP) Co-factors &Vitamins glycosyl-N-palmitoyl-sphingosine2'-deoxyadenosine2'-deoxycytidineoctadecadienedioatelignoceroyluridine(S)-3-hydroxybutyrylcarnitinethymidinealpha-tocopherolsphingadieninemyo-inositolquinolinatebiotin 5'-monophosphate 5'-monophosphat ethanolamide 5'-monophosphate acetat 5'-monophosphat (C18:2-DC)e (24:0) (UMP)e * *(d18...e 1-palmitoyl-digalactosylglycerolarachidoyloctadecanedioatenicotinateN-alpha-acetylornithineN-delta-acetylornithinediacetylspermidine3-ureidoisobutyrate10-hydroxystearat3-ureidopropionatN-methylalanin ribonucleosideethanolamidee e e(C18-DC)* (20:0)* (16:0)* Energy 1-palmitoyl-2-oleoyl-GPE1,2-dipalmitoyl-GPEbehenoyl(3'-5')-cytidylylcytidine*(3'-5')-cytidylyluridine*(3'-5')-uridylylcytidine*3,4-dihydroxybutyrate(3'-5')-uridylyluridinindole-3-carboxylat2,8-quinolinediol9,10-DiHOMmannosfructosee ethanolamideE e (16:0/16:0)* (22:0) (16:0/18:1* ) gamma-glutamylphenylalaninegamma-glutamyl-alpha-lysinegamma-glutamylmethioninegamma-glutamylthreoninegamma-glutamylglutamingamma-glutamyltyrosinediaminopimelatN6-methyllysinesedoheptulosepyridoxaminpantetheinpantethineanserine e e e e 3-methylglutarate/2-methylglutarat6-oxopiperidine-2-carboxylate2-methylcitrate/homocitrateN6-carboxyethyllysine2,3-dimethylsuccinatimidazole5-hydroxyhexanoate2-hydroxyglutarateglutarateindolepropionatedihydroferulatindoleacetatchiro-inositol (C5-DC propionatee ) ee e Lipid 1-palmitoyl-2-linoleoyl-galactosylgly...undecanedioatedodecanedioateglycerophosphoglycero16-hydroxypalmitate3-aminoisobutyratesebacategalactitol3-sulfo-L-alanincysteinylglycinehomoserinevanillatecysteine (dulcitol)(C10-DC)e (C11-DC)(C12-DC)l 1-palmitoyl-2-linoleoyl-digalactosylg...beta-hydroxyisovalerat1-palmitoyl-GPE2-hydroxyheptanoatemaltotrioseenterodiolfumaratepyrralinepyridoxalgentisatemaltosribonatmalateee (16:0)* e Nucleotide 1-palmitoyl-2-linoleoyl-GPEcytidineoxalatethiamin3-dehydroshikimateN-methylpipecolatcarboxyethyl-GABAsuberateazelateN-methylprolinepyridoxatferulateglycero l (ethanedioate)(Vitamin(C9-DC)5'-monophosphate e(C8-DC) B1e) (5'-CMP)(16:0/18:2) 2,6-dihydroxybenzoicN6-carboxymethyllysinglycerophosphoinositol*dimethylmalonic1-linoleoyl-GPC3-methylglutaconatepimelate2-hydroxyadipatsyringicglucuronatdiosmetimalonatsinapate eacidn (C7-DC)e e (18:2)acid acide Partially Charecterized 3-hydroxy-3-methylglutaratN-acetylaspartate3-hydroxyphenylacetateN-acetylphenylalaninetrans-4-hydroxyproline3-hydroxyhexanoateN-acetyltryptophan2-isopropylmalatmethylsuccinatN-acetylvalinepicolinatesalicylatsuccinatee e e (NAA) e 2-hydroxybutyrate/2-hydroxyisobutyrateN,N,N-trimethyl-5-aminovaleratN,N-dimethyl-5-aminovalerat2-hydroxy-3-methylvalerat3-(4-hydroxyphenyl)lactatealpha-hydroxyisovalerat2,4,6-trihydroxybenzoateN-carbamoylaspartatphenyllactatephenethylaminelysylleucinegluconatlactate e (PLA) e e e e e Peptides dimethylarginine1-methyl-4-imidazoleacetatalpha-hydroxyisocaproateN6,N6,N6-trimethyllysine3-hydroxyadipate*1-methyladenin1-methylguaninN-acetylcysteine2-aminoadipate3-formylindoltryptamineserotonintyramine e e (SDMA + ADMA)e alpha-ketoglutaramate*4-imidazoleacetate5,6-dihydrouridineadipate2-oxoarginine*pseudouridinpantothenatemannonategalactonatargininate*pipecolateectoinorotatee (C6-DCe* e ) Xenobiotics 5-methyluridine2S,3R-dihydroxybutyrate2R,3R-dihydroxybutyrate4-guanidinobutanoatN2,N6-diacetyllysinN1-methyladenosinearabonate/xylonatefructosyllysinerythronate*guanosinthreonateinosineuridine e e (ribothymidine)ee N-acetylglucosamine/N-acetylgalactosa...1-palmitoyl-2-dihomo-linolenoyl-GPC1-stearoyl-2-arachidonoyl-GPC1-palmitoyl-2-arachidonoyl-GPC1-(1-enyl-palmitoyl)-2-oleoyl-GPE1-myristoyl-2-palmitoyl-GPC1-stearoyl-2-oleoyl-GPE1-stearoyl-2-oleoyl-GPCS-1-pyrroline-5-carboxylat7-ketodeoxycholat3-ketosphinganinehyocholatexanthosine e (18:0/18:1e (14:0/16:0 (18:0/2... (16:0/.. (P-...) (...). N-palmitoyl-phytosphingosine(12hexadecasphinganinehexadecadienoate6-beta-hydroxylithocholatetetradecadienoate3-hydroxypalmitatalpha-muricholat3-hydroxyoleate*3-hydroxylauratebeta-muricholatdeoxycholatlithocholat or 13)-methylmyristatee e eee (14:2)(16:2n6) (d16:0)** (a15:0 (t18:0/1... or ... docosapentaenoatedihomo-linolenate10-heptadecenoate3b-hydroxy-5-cholenoictaurolithocholatedeoxycholicoleate/vaccenatetaurocholenatepentadecanoateglutaminylleucine3-hydroxystearateadrenatemyristate (22:4n6(14:0) acid sulfate* (15:0) 3-sulfat)(18:1) (20:3n3 (17:1n7)(n3 acidDPA;e or n6 22:5n3)) gamma-glutamylleucinethreonylphenylalaninemethionineglycylisoleucineleucylalanineleucylglycinalanylleucineglycylleucinvalylglycineglycylvalinevalylleucinaspartatealanine e esulfoxide phenylalanylglycintryptophylglycinleucylglutamine*methionineisoleucinethreoninglutaminhistidineleucineglycinprolineserinvalinee ee e e 2-hydroxylignocerate*2-hydroxynervonate2-hydroxydecanoate2-hydroxybehenatemethioninediacetylchitobiosmethylphosphatephenylalaninphosphato-TyrosinL-urobilintryptophatyrosine en sulfone ee * flavinglutamate,hexadecanedioatethiaminnicotinamideformiminoglutamate2-hydroxyquinolineN6-acetyllysinhomocitrullinhypoxanthin4-thiouracilpterin adeninemononucleotide monophosphat gamma-methyle eriboside edinucleotide (C16-DC) (FMNe este(FAD) r ) octadecenedioateeicosenedioate3-hydroxybutyrateisohyodeoxycholatN-acetyltyrosinebeta-alanineursocholatnicotinatglutamatxanthincytosinuracilribose eee e (C20:1-DC) (C18:1-DC)e(BHBA) * * 4-methylthio-2-oxobutanoat4-hydroxyphenylpyruvateN('1)-acetylspermidineriboflavinN-acetylasparagine2'-O-methyluridinN-acetylglutamatN-acetylalanineN2-acetyllysinergothioneineallo-threonin5-oxoprolinethymine (Vitamine e e B2) e Group 4-methyl-2-oxopentanoate3-methyl-2-oxovalerate3-methyl-2-oxobutyratealpha-ketoglutaratN-acetylisoleucinN-acetylleucinphenylpyruvatepyruvate e ee GENDER UICH-00155UICH-00135UICH-00147UICH-00137UICH-00133UICH-00138UICH-00162UICH-00163UICH-00170UICH-00169UICH-00171UICH-00173UICH-00175UICH-00174UICH-00176UICH-00177UICH-00172UICH-00178UICH-00168UICH-00166UICH-00167UICH-00129UICH-00153UICH-00157UICH-00123UICH-00127UICH-00158UICH-0015UICH-00131UICH-00148UICH-00139UICH-00142UICH-00124UICH-00125UICH-00126UICH-001459UICH-00132UICH-00144UICH-00146UICH-00150UICH-00143UICH-00130UICH-00152UICH-00136UICH-00149UICH-00156UICH-00128UICH-00160UICH-001UICH-001UICH-0UICH-0UICHUIUICUIC60H-C ARAM_DIET

Prevotella dentalis Escherichia coli

Escherichia coli C ) 30

B ) 1.0 40 20 ) 30 0.8 10 20 0.6 10 0.4 0 Percentage of 0 0.2 abundance (%

Percentage of -10 abundance (% VDR LoxP Δlyz Percentage of -10 0.0 LoxP ΔIEC VDR abundance (% LoxP ΔIEC VDR VDR VDR VDR

Parabacteroides sp. CT06 Akkermansia muciniphila Parabacteroides sp. CT06 0.5 0.08 ) ) 0.5 ) * 0.4 0.4 0.06 0.3 0.3 0.04 0.2 0.2 0.1 0.02 0.1 Percentage of Percentage of Percentage of abundance (% abundance (% abundance (% 0.0 0.0 0.00 LoxP Δlyz VDR LoxP VDR ΔIEC VDR LoxP VDR ΔIEC VDR VDR

Parabacteroides distasonis Parabacteroides distasonis 0.20 * ) 0.20 ) 0.15 0.15 0.10 0.10 0.05 0.05 Percentage of Percentage of abundance (%

abundance (% 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 defcient animals to the control group as well as that were kept on HFD or a chow diet. Diferent 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-specifc VDRΔlyz mice. Each dot indicates percentage of abundance in each mice sample. Signifcance is established at adjusted P < 0.05.

that VDR defciency plays a critical role in bile acid accumulation. Te same increase was not observed in males, suggesting that sex hormones might also play a role in bile acid accumulation. Tis might be the reason why Vitamin D defciency 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.

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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. Te absence of VDR leads to altered metabolites, which contribute to the disease state. Signifcance was established at adjusted P < 0.05.

Te increase in polyamines was more signifcant in male VDRΔIEC mice, indicating the gender diferences in metabolite-VDR interactions. One of the limitations of our study is that it does not provide information on urolithin and that certain biochemicals did not reach levels of signifcance. However, we did clearly demonstrate that in the absence of VDR,

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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 specifcally 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 deficiency not only drives derangements in gut-microbiome but also signifcantly alters the metabolite profle in a tissue- and gender-specifc 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. Tese fndings 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. Te 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 specifc 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. Tis was a three-way (Genotype, Diet Treatment, and Gender) study design. To further evaluate the diet efect 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. Te 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 identifcation 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 frst 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. Te resulting extract was divided into fve 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 briefy on a TurboVap (Zymark) to remove the organic solvent. Te 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 bufer (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 fnally visualized using an ECL kit. Te relative abundance of protein was determined using Image-J (NIH) sofware. Te gels/blots used in fgures are checked their compliance with the digital image and integrity policies in Nature publisher.

Immunohistochemical staining (IHC). Immunohistochemistry (IHC) was performed using sections of parafn-embedded liver tissue as previously described6. Sections were incubated in primary antibody FXR (Santacruz, CA, USA) 1:50 diluted for in blocking bufer) at 4 °C overnight. Ten 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 fuorescence 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 amplifcation (to increase the total amount of DNA but without selecting for any specifc sequences) and for the initiation of the sequencing reaction, again without selection for any specifc sequence from the source genomic DNA. Millions to hundreds of millions of short sequences (generally 150 bases, in pairs) are generated using Illumina sequencing platforms. Tese data were analyzed by (a) searching for lineage-specifc 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

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identify diferences in the relative abundance of microbial features (i.e., taxa, genes, and pathways) between different groups of samples. We used the UIC genomic facility for our studies.

Statistical analysis. Raw data were extracted, peak-identifed and QC processed using Metabolon’s hard- ware and sofware. Compounds were identifed by comparison to library entries of purifed standards or recur- rent unknown entities. Furthermore, biochemical identifcations 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 quantifed and data were normalized as necessary. Te analysis data were presented as a fold change ratio of treatment vs. control. All the tests were two-sided. Te numbers of biochemicals were summarized as statistical signifcance 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 difered signifcantly between genotypes and treatment groups. Tree-way ANOVA was further conducted to identify biochemicals exhibiting signifcant interaction and main efects 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. Te matched pairs t-test is used to test whether two unknown means are diferent 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 hierarchical 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 diferences.

Ethics approval and consent to participate. No human study. All animal studies were performed following ACC guidelines at the University of Illinois at Chicago (UIC), IL, USA. Data availability Data and material will be available by request.

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