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Microbial metabolite delta-valerobetaine is a diet-dependent obesogen

Nature Metabolism volume 3, pages 1694–1705 (2021)Cite this article

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Abstract

Obesity and obesity-related metabolic disorders are linked to the intestinal microbiome. However, the causality of changes in the microbiome–host interaction affecting energy metabolism remains controversial. Here, we show the microbiome-derived metabolite δ-valerobetaine (VB) is a diet-dependent obesogen that is increased with phenotypic obesity and is correlated with visceral adipose tissue mass in humans. VB is absent in germ-free mice and their mitochondria but present in ex-germ-free conventionalized mice and their mitochondria. Mechanistic studies in vivo and in vitro show VB is produced by diverse bacterial species and inhibits mitochondrial fatty acid oxidation through decreasing cellular carnitine and mitochondrial long-chain acyl-coenzyme As. VB administration to germ-free and conventional mice increases visceral fat mass and exacerbates hepatic steatosis with a western diet but not control diet. Thus, VB provides a molecular target to understand and potentially manage microbiome–host symbiosis or dysbiosis in diet-dependent obesity.

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Fig. 1: δ-VB is a microbiome-derived mitochondrial metabolite.
Fig. 2: δ-VB is a microbiome-derived metabolite.
Fig. 3: VB decreases fatty acid oxidation.
Fig. 4: VB increases lipid accumulation in host tissues.
Fig. 5: Long-term VB treatment in GF and conventional mice.
Fig. 6: VB participates in microbiome–mitochondria communication to reprogramme host lipid metabolism.
Fig. 7: Clinical associations of plasma VB with microbiome manipulation and obesity-related phenotypes in people.

Data availability

RNA-seq data are available at the NCBI Gene Expression Omnibus (GEO) GSE185525 and GSE145012. Source data are provided with this paper. Other data that support the findings of this study are available on request from the corresponding authors.

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Acknowledgements

This work was supported by grant nos. T32 GM008602 (K.H.L.), P30 ES019776 (D.P.J.), R01 ES023485 (D.P.J., Y.M.G.), U2C ES030163 (D.P.J.), R2C DK118619 (D.P.J.), S10 OD018006 (D.P.J.), K01 DK102851 (J.A.A.), R03 DK117246 (J.A.A.), K24 DK096574 (T.R.Z., J.A.A.), R21HD089056 (M.B.V.), R00AA021803 (S.M.Y.), R01AA026086 (S.M.Y.) and R01AI064462 (A.S.N.). Additionally, we acknowledge support from the Georgia Clinical and Translational Science Alliance grant no. UL1 TR002378 and the Emory-Georgia Tech Predictive Health Institute and Center for Health Discovery and Well-Being. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

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Author notes

  1. These authors contributed equally Ken H. Liu, Joshua A. Owens, Bejan Saeedi, Andrew S. Neish, Dean P. Jones.

Authors and Affiliations

  1. Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA

    Ken H. Liu, Michael Orr, Xin Hu, Jolyn Fernandes, Chunyu Ma, Samantha M. Yeligar, Karan Uppal, Young-Mi Go & Dean P. Jones

  2. Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA

    Joshua A. Owens, Catherine E. Cohen, Crystal Naudin, Trevor Darby, Miriam B. Vos & Rheinallt M. Jones

  3. Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA

    Bejan Saeedi, Mary Catherine Camacho, Sarah Hunter-Chang, David VanInsberghe, Colleen S. Kraft & Andrew S. Neish

  4. Division of Endocrinology, Metabolism, and Lipids, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA

    Moriah P. Bellissimo, Jessica A. Alvarez & Thomas R. Ziegler

  5. Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA

    Samuel Druzak, Kristal Maner-Smith & Eric Ortlund

  6. Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA, USA

    Thota Ganesh

  7. Atlanta Veterans Affairs Health Care System, Decatur, GA, USA

    Samantha M. Yeligar

  8. Division of Infectious Disease, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA

    Michael H. Woodworth & Colleen S. Kraft

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Contributions

K.H.L., J.A.O., B.S., D.P.J. and A.S.N. conceived the work. K.H.L., B.S., D.P.J., A.S.N., J.A.O., C.N., Y.-M.G., M.H.W., C.S.K., S.M.Y. and E.O. designed experiments. K.H.L., B.S., J.A.O., C.E.C., M.P.B., C.N., T.D., S.D., K.M.-S., M.O., X.H., J.F., M.C.C., S.H.-C., T.G., T.R.Z., M.B.V., J.A.A., D.v.I., M.H.W., C.S.K., C.M., R.M.J. and K.U. performed experiments, acquired or analysed data for this study. K.H.L., B.S., J.A.O., Y.-M.G., D.P.J. and A.S.N. prepared the paper.

Corresponding authors

Correspondence to Andrew S. Neish or Dean P. Jones.

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The authors declare no competing interests.

Additional information

Peer review information Nature Metabolism thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Ashley Castellanos-Jankiewicz and Pooja Jha.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Additional mass spectrometry analysis of VB.

A Mass spectrometric analysis of VB in a) lung and brain of conventional (C) and germ-free (GF) mice and b) Ion dissociation spectra (MS/MS) analysis of 160.1332 m/z (hcd 30) in caecal contents and mouse diet. VB was not present in GF cecal contents, or the control chow (Teklad) diet used for GF mouse experiments. The fragmentation of 160.1332 m/z in GF cecal contents and GF control diet is consistent with valine betaine (Supplementary Information). c) Ion fragmentation spectra of 160.1332 m/z in GF control diet and extracted ion chromatograms of 160.1332 m/z in all diets used in this study. There was no peak for 160.1332 m/z observed in the Western diet. c) Extracted ion chromatograms of 160.1332 m/z from mouse diets used for this study. VB is not present in the sterilized chow (Teklad) used as the control diet for GF and conventionalization experiments. The conventional chow (Labdiet), which was not autoclavable, was used as the control for conventional mouse experiments. This diet contained a minor peak for VB (red brackets), but the later eluting major peak (0.8m) had a fragmentation pattern observed for 160.1332 m/z (valine betaine) in the GF diet. The small amount of VB present in the Labdiet chow is not the major source of VB in conventional mice since serum VB was equivalent between WD-fed (which does not contain VB) and Labdiet chow-fed conventional mice (Extended data 6c). d) (Nε, Nε, Nε)-trimethyllysine (TML) is a precursor to δ-valerobetaine (VB). (Nε, Nε, Nε)-trimethyllysine (100 mg/kg) or (13C Nε, 13C Nε, 13C Nε)-trimethyllysine (25 mg/kg) was gavaged into conventional mice for 3 days. The formation of unlabelled VB (160.1332 m/z) from TML and labeled VB (163.1431 m/z) from isotopically labeled TML is shown here along with accurate mass MS1 and ion fragmentation spectra consistent with VB and labeled VB.

Extended Data Fig. 2 VB inhibits mitochondrial fatty acid oxidation.

a) Tracing the oxidation of 13C16 palmitic acid in HepG2 cells to examine the effect of VB treatment on cellular mitochondrial fatty acid oxidation. 12 hour pretreatment with VB (green) decreased (p = 0.0015 (AUC - Area under the curve), two-tailed student’s t-test) the formation of labeled acetyl-CoA (bottom middle) by approximately 75% compared to vehicle (blue). Addition of carnitine back to cells pretreated with VB for 12 hours (purple) restored the carnitine-dependent formation of mitochondrial acetyl-CoA (p = 0.49 (AUC), two-tailed student’s t-test). Co-treatment of VB with the addition of stable isotope-labeled palmitate (red) decreased the formation of labeled acetyl-CoA by approximately 25% compared to vehicle (p = 0.04 (AUC), two-tailed student’s t-test). Data for VB, carnitine, and other metabolites are shown to illustrate VB treatment does not affect uptake of labeled 13C16 palmitate (middle left), or the conjugation of labeled palmitate to CoA (middle middle). VB treatment decreased carnitine approximately 20% after one hour (top middle) and these changes drive decreased formation of labeled palmitoylcarnitine (middle right), labeled acetyl-CoA (bottom middle), labeled acetylcarnitine (bottom left), and labeled citrate (bottom right). Each data point represents the average of 3 biological replicates ± standard deviation. b) The effects of VB on mitochondrial respiration is dependent on the availability of fuel substrates. In the presence of glucose and glutamine, VB does not decrease basal respiration (ANOVA (F = 6.239, p = 0.0014) with post-hoc test for linear trend (R2 = 0.0078, p = 0.50)) or maximum respiratory capacity after addition of FCCP (ANOVA (F = 2.223, p = 0.10). VB does not influence basal respiration with culture conditions with palmitate but without glucose and glutamine (ANOVA (F = 9.729, p = 0.001) with post-hoc test for linear trend (R2 = 0.075, p < 0.096). Each data point represents at least 8 biological replicates.

Source data

Extended Data Fig. 3 VB alters carnitine metabolism in mice.

VB treatment increased a) circulating and b) hepatic VB in conventional mice [Kruskal-Wallis analysis to determine group-wise differences with Dunn’s multiple comparisons test (serum VB males (n = 3 per treatment) – KW statistic 7.2, p = 0.0036; serum VB females (Vehicle n = 5, 10 mg/kg n = 6, 100 mg/kg n = 6) – KW statistic 10.84, p = 0.0008; liver VB males – KW statistic 6.489, p = 0.0107; liver VB females – KW statistic 12.94, p < 0.0001)]. c, d) Pathway enrichment analysis of serum metabolites correlated with VB treatment shows VB alters carnitine shuttle metabolism in male and female mice (p < 0.05, mummichog p-value based on permutation analysis). e) VB decreases circulating carnitines in mice. Correlation heatmaps are based on Spearman’s correlation values with red corresponding to Spearman’s rho = 1 and blue Spearmen’s rho = -1. f) VB treatment increases urinary carnitine in mice (n = 3, time 0 p = 0.126, time 2h p = 0.001, time 6h p = 5.86e-005, two-tailed student’s t-test). g) Fasting does not change circulating (n = 6, p = 0.95, two-tailed student’s t-test) or hepatic (n = 5, p = 0.24, two-tailed student’s t-test) VB compared to fed mice.

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Extended Data Fig. 4 VB decreases circulating and hepatic beta-hydroxybutyrate.

VB decreases circulating and hepatic beta-hydroxybutyrate, produced from mitochondrial fatty acid oxidation during fasting. Kruskal-Wallis with Dunn’s multiple comparisons test was used for fasted serum and liver analyses (n = 8 vehicle, n = 3 10 mg/kg, n = 5 100 mg/kg for male and female). Male serum KW statistic 7.864, p = 0.0085; female serum KW statistic 10.46, p = 0.0006. Male liver KW statistic 7.864, p = 0.0085; female liver KW statistic 11.73, p = 0.0001. Control vs. 100 mg/kg was significantly different for all comparisons in fasted mice. For fed mice, (n = 3 vehicle, n = 3 10 mg/kg, n = 3 100 mg/kg for male and female), one-way ANOVA with Dunnett’s multiple comparison tests was used. Male serum (ANOVA F = 0.9472, p = 0.4390); Female serum (ANOVA F = 6.8, p = 0.028, vehicle vs. 100 mg/kg significant); Male liver (ANOVA F = 0.9032, p = 0.454); Female liver (ANOVA F = 4.295, p = 0.0695).

Extended Data Fig. 5 Untargeted lipidomics of VB treated mice.

VB alters neutral lipid profiles liver, heart, and brain of male and female mice. Neutral lipids from untargeted lipidomic profiling with average fold-change greater than 2 in 100 mg/kg VB-treated mice (n = 5 male, n = 5 female) versus control (n = 5 male, n = 5 female).

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Extended Data Fig. 6 Baseline data for long-term mouse experiments with VB.

a) Comparison of VB in GF mouse serum and liver after treatment with 25 mg/kg VB with conventional mice on respective control diets. Samples were analyzed after 6 week treatment with VB and normal conventional mice. GF mice control diet was Teklad 2019S chow and conventional mice control diet was LabDiets 5001 chow. Serum GF+VB (n = 5) and conventional (n = 5) comparison (p = 0.01, two-tailed t-test with conventional mice having approximately 1.7x circulating VB compared to GF mice treated with VB. Liver GF+VB (n = 5) and conventional (n = 5) comparison (p = 0.0004, two-tailed t-test with conventional mice having approximately 0.74x liver VB compared to GF mice treated with VB. b) Comparison of carnitine in mouse serum and liver after treatment with 25 mg/kg in GF mice with conventional mouse (control diet) data for comparison. Carnitine is decreased in conventional mice compared to GF mice. VB treatment to GF mice led to serum and liver carnitine concentrations equivalent to conventional mice. (Two-tailed unpaired student t-tests: Serum GFCD vs. GFCD+VB p = 0.0001, GFCD vs. Conventional CD p < 0.0001, GFCD+VB vs. Conventional CD p = 0.395; Liver GFCD vs. GFCD+VB p < 0.0001, GFCD vs. Conventional CD p < 0.0001, GFCD+VB vs. Conventional CD p = 0.6559) c) Comparison of circulating VB in conventional mice (male M, female F) between Western diet (WD) and control chow (Two-tailed unpaired student t-tests: CD male vs. WD male p = 0.4982; CD female vs. WD female p = 0.1653).

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Extended Data Fig. 7 Effects of 8-week VB treatment on weight gain and adipose tissue mass in control or Western diet in conventional female mice.

Effects of 8-week VB treatment on weight gain and adipose tissue mass in control or Western diet in conventional female mice (n = 5 per treatment). The combination of Western Diet with VB led to approximately a 3-6% increase in body weight compared to Western Diet alone in female mice, however these results were not statistically significant at a p-value threshold of 0.05 (p = 0.14, one-tailed t-test). In control-diet fed female conventional mice (n = 5 per treatment), VB did not increase weight gain (p-value for increase in weight = 0.9803 (1 – 0.0197), one-tailed t-test). VB treatment increased perigonadal visceral adipose tissue (VAT) and posterior subcutaneous adipose (SubQ) tissue mass in conventional female mice fed a Western diet. VB treatment increased interscapular brown adipose tissue (BAT) mass on a control diet but did not increase BAT mass with the Western diet in conventional female mice. One-tailed t-tests with p < 0.05 used to test for an increase in adipose tissue mass following VB treatment.

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Extended Data Fig. 8 Cytokine panel following long-term VB treatment to male and female conventional mice.

a) Plasma biomarkers of glucose tolerance/insulin resistance (insulin, glucagon, resistin) in conventional mice after 8 weeks of VB treatment (n = 5 per treatment, outliers removed by Robust regression and Outlier removal (ROUT) in Prism 6.0). (One-way ANOVA with Sidak’s multiple comparisons: Insulin male F = 1.648, p = 0.2205; Insulin female F = 1.034, p = 0.4059; Glucagon male F = 0.5165, p = 0.6772; Glucagon female F = 1.723, p = 0.2025; Resistin male F = 4.747, p = 0.0149 [CD vs. CDVB not significant, WD vs. WDVB not significant]; Resistin female F = 2.687, p = 0.0814) b) Plasma biomarkers of inflammation (IL-6, TNF-alpha, MCP-1) in conventional mice after 8 weeks of VB treatment. (One-way ANOVA with Sidak’s multiple comparisons: IL-6 male F = 0.9316, p = 0.4497; IL-6 female F = 1.646, p = 0.2185; TNF-alpha male F = 1.645, p = 0.2186; TNF-alpha female F = 3.241, p = 0.0499 [CD vs. CDVB not significant, WD vs. WDVB not significant]; MCP-1 male F = 2.467, p = 0.1021; MCP-1 female F = 2.048, p = 0.1477).

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Extended Data Fig. 9 Transcription factor analysis for Western Diet-fed GF mice.

Transcription factor enrichment analysis shows that Ppara target genes are upregulated by a) microbiome acquisition and VB treatment in control diet fed GF mice. Ppara target genes are downregulated by VB treatment in Western diet (WD) fed GF mice. b) VB treatment in WD fed mice downregulates genes linked to Ppara which function in mitochondria and lipid processing pathways (p-value < 0.005, FC < 0.8).

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Extended Data Fig. 10 Urinary carnitine correlations with urinary VB.

Urinary carnitine is correlated with urinary VB in humans (n = 143, Pearson’s R = 0.754, p < 0.00001).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

Supplementary Table 1

Diet composition for diets used in this study.

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Liu, K.H., Owens, J.A., Saeedi, B. et al. Microbial metabolite delta-valerobetaine is a diet-dependent obesogen. Nat Metab 3, 1694–1705 (2021). https://doi.org/10.1038/s42255-021-00502-8

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