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An exercise-inducible metabolite that suppresses feeding and obesity

Abstract

Exercise confers protection against obesity, type 2 diabetes and other cardiometabolic diseases1,2,3,4,5. However, the molecular and cellular mechanisms that mediate the metabolic benefits of physical activity remain unclear6. Here we show that exercise stimulates the production of N-lactoyl-phenylalanine (Lac-Phe), a blood-borne signalling metabolite that suppresses feeding and obesity. The biosynthesis of Lac-Phe from lactate and phenylalanine occurs in CNDP2+ cells, including macrophages, monocytes and other immune and epithelial cells localized to diverse organs. In diet-induced obese mice, pharmacological-mediated increases in Lac-Phe reduces food intake without affecting movement or energy expenditure. Chronic administration of Lac-Phe decreases adiposity and body weight and improves glucose homeostasis. Conversely, genetic ablation of Lac-Phe biosynthesis in mice increases food intake and obesity following exercise training. Last, large activity-inducible increases in circulating Lac-Phe are also observed in humans and racehorses, establishing this metabolite as a molecular effector associated with physical activity across multiple activity modalities and mammalian species. These data define a conserved exercise-inducible metabolite that controls food intake and influences systemic energy balance.

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Fig. 1: Lac-Phe is strongly induced in blood plasma after a single bout of running in mice and racehorses.
Fig. 2: CNDP2-dependent and lactate-dependent production and secretion of Lac-Phe in vitro.
Fig. 3: Lac-Phe suppresses food intake and obesity and improves glucose homeostasis.
Fig. 4: Increased food intake and obesity in mice genetically deficient in Lac-Phe production.
Fig. 5: Sustained increases in Lac-Phe following human exercise.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files. Source data are provided with this paper.

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Acknowledgements

We thank members of the Long, Xu, Snyder, Richter and Svensson laboratories, and L. Sylow for helpful discussions. This work was supported by the NIH (DK124265 and DK130541 to J.Z.L.; DK113954, DK115761, DK117281 and DK120858 to Y.X.; GM113854 to V.L.L.; and AR072695 to K.v.d.W), the Ono Pharma Foundation (research grant to J.Z.L.), BASF (research grant to J.Z.L.), the USDA (51000-064-01S to Y.X.), the American Heart Association (20POST35120600 to Y.H.), the Novo Nordisk Foundation (NNF17OC0027274 and NNF18OC00334072 to E.A.R.) and PXE International (research grant to K.v.d.W.).

Author information

Authors and Affiliations

Authors

Contributions

V.L.L. performed the exercise studies, gain and loss of function studies, in vitro studies and MS analyses. Y.H. performed the acute feeding experiments (with the help of H.L.). K.C. re-analysed the MS data from the previously published human exercise experiment. J.T.K., A.L.W., J.T.T. and A.S.-H.T. assisted in tissue collection and exercising mice for the chronic running experiments. X.L. assisted in preparing plasma samples for MS analyses. P.-J.H.Z. performed the metabolic chamber studies. R.S.J. performed studies using ABCC5-KO mice. B.M. assisted in MS analyses. K.Y.L. and A.C.Y. assisted in collecting brain tissue. C.S.C., C.T.V., B.K. and E.A.R. performed the human exercise studies. W.W. and S.M.T. assisted in the mouse running experiments. B.C.M. and R.M.A. assisted in the horse metabolomics studies. S.M.B. synthesized Lac-Phe. J.Z.L., Y.X., G.A.W., A.S., B.K., E.A.R., M.P.S. and K.v.d.W. supervised the work. V.L.L., J.Z.L., Y.H. and Y.X. conceived the experiments and wrote the manuscript with contribution from the other authors.

Corresponding authors

Correspondence to Yong Xu or Jonathan Z. Long.

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Competing interests

Stanford University is in the process of applying for a patent application (US2022027261) covering lactoyl amino acids for the treatment of metabolic diseases that lists J.Z.L., V.L.L., S.M.B., Y.X. and Y.H. as inventors.

Peer review

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Nature thanks Tamas Horvath and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Additional metabolomic characterization of Lac-Phe and lactoyl amino acid dynamics in mouse and thoroughbred racehorse exercise.

(a) Schematic of speed and incline in the acute running protocol for mice. (b) Extracted ion chromatograms the endogenous m/z = 236.0928 peak in mouse plasma in comparison with a synthetic Lac-Phe standard. (c, d) Fragmentation spectra (c) extracted ion chromatogram (d) of the the endogenous m/z = 236.0928 peak in horse plasma in comparison with a synthetic Lac-Phe standard. (e) Quantitation of Lac-Phe levels in the indicated tissue from either sedentary mice (blue) or after a single bout of exhaustive running (red). (f, g) Quantitation of the indicated lactoyl amino acid level in blood plasma from mouse (f) or racehorse (g). For (e) and (f), N = 5/group; for (g), N = 10/group. Data are shown as mean ± SEM. All experiments were performed once. P-values were calculated by Student’s two-sided t-test

Source data

Extended Data Fig. 2 Additional characterization and validation of CNDP2 protein in cells and in mice.

(a) Schematic of the chemical reaction catalyzed by CNDP2 to produce Lac-Phe. (b) Cndp2 mRNA expression across tissues from Tabula Muris. (c-e) Anti-CNDP2 (top) and anti-tubulin (bottom) Western blots in WT and CNDP2-KO RAW264.7 (c), RT4 (d), or TKPTS (e) cells. (f-h) Anti-CNDP2 (top) and anti-actin (bottom) Western blots of liver (f), kidney (g), or quadraceps (h) harvested from either sedentary mice (left three lanes) or mice after a single bout of exhaustive exercise (right three lanes). For gel source data, see Supplementary Fig. 1. For (c), experiments were performed twice. For (d-h), experiments were performed once.

Extended Data Fig. 3 Additional characterization of the effects of Lac-Phe administration to diet-induced obese mice.

(a, b) Phenotype associations of the single nucleotide polymorphisms rs373836366 (a) and rs780772968 (b) from the Type 2 Diabetes Knowledge Portal. (c, d) Plasma Lac-Phe levels (c) and blood lactate levels (d) in plasma of mice following a single injection of Lac-Phe (50 mg/kg, IP). (e-g) 12 h oxygen consumption VO2 (e), carbon dioxide production VCO2 (f), and respiratory exchange ratio RER (g) of 22-week old DIO mice following a single injection of vehicle or Lac-Phe (50 mg/kg, IP). (h-j) Food intake (h), kaolin intake (i), and water intake (j) in 21-week old DIO mice following a single injection of vehicle or Lac-Phe (50 mg/kg, IP). (k, l) Plasma leptin (k) and acyl-ghrelin (l) levels in 17-week old DIO mice 30 min after a single injection of vehicle or Lac-Phe (50 mg/kg, IP). For (c), (d), (k), and (l), N = 3/group. For (e-j), N = 7/group. Data are shown as means ± SEM. All experiments were performed once. P-values were calculated by Student’s two-sided t-test

Source data

Extended Data Fig. 4 Metabolic effects of Lac-Phe administration to chow-fed, lean mice.

(a) Lac-Phe levels in plasma of male lean mice (22–27 g) following a single injection of Lac-Phe (50 mg/kg, IP). (b-f) 12 h food consumption (b), ambulatory activity (c), oxygen consumption VO2 (d), carbon dioxide production VCO2 (e), and respiratory exchange ratio RER (f) of chow fed lean mice following a single injection of Lac-Phe (50 mg/kg, IP). (g) 24 h food intake in lean mice after a single injection of Lac-Phe at the indicated dose. For (a), N = 3/group. For (b-f), N = 8/group. For (g), N = 5/group. Data are shown as means ± SEM. For (a-f), experiments were performed once. For (g), experiments were performed two times. P-values were calculated by Student’s two-sided t-test

Source data

Extended Data Fig. 5 Metabolic effects of oral Lac-Phe administration to diet-induced obese mice.

(a, b) Change in body weight (a) and daily food intake (b) of 16-week diet-induced obese mice treated with Lac-Phe (50 mg/kg/day, PO). N = 5/group. Data are shown as means ± SEM. Experiments were performed once

Source data

Extended Data Fig. 6 Additional characterization of CNDP2-KO mice.

(a) Treadmill time until exhaustion for WT and CNDP2-KO (“KO”) mice. (b-d) Plasma levels of the indicated lactoyl amino acid in WT or CNDP2-KO mice in the sedentary state or after a single bout of acute exhaustive running (“exercise”). (e) Plasma levels of carnosine in sedentary WT or CNDP2-KO mice. (f, g) Body weight (f) and cumulative daily food intake (g) of WT (blue) or CNDP2-KO (red) mice under high fat diet, sedentary conditions. For (a), N = 6 for WT and N = 8 for CNDP2-KO; for (b-d), N = 6/group; for (e), N = 6 for WT and N = 5 for CNDP2-KO; for (f,g), N = 8 for WT and N = 11 for CNDP2-KO. Data are shown as means ± SEM. For (a-g), experiments were performed once. P-values for (a-e) were calculated by Student’s two-sided t-test

Source data

Extended Data Fig. 7 Plasma Lac-Phe levels in WT and ABCC5-KO mice sedentary mice or after a single bout of treadmill running to exhaustion.

N = 3/group. Data are shown as means ± SEM. Experiments were performed once. P-values were calculated by Student’s two-sided t-test

Source data

Extended Data Fig. 8 Additional characterization of plasma Lac-Phe levels in humans.

(a, b) Tandem MS fragmentation (a) and co-elution (b) of an authentic Lac-Phe standard and the endogenous m/z = 236.0928 mass from human plasma run on the Snyder laboratory untargeted metabolomics platform (see Methods). (c) Time course of phenylalanine levels in blood before and after a single acute bout of treadmill running from the human acute treadmill exercise study (Cohort 1, N = 36). (d) Time course of lactate levels before and after sprint (red), resistance (blue), and endurance (light blue) exercise from the human crossover acute exercise study (Cohort 2, N = 8). For (c, d), data are shown as mean ± SEM, **p < 0.01, ***p < 0.001. Experiments were performed once. P-values were calculated by two-way ANOVA with repeated measures

Source data

Supplementary information

Supplementary Fig. 1

Original source images for all data obtained by electrophoretic separation.

Reporting Summary

Supplementary Table 1

Excel file with additional metabolomics data. Tab 1: targeted metabolomics from mouse plasma after an acute bout of treadmill running versus sedentary condition. Tab 2: XCMS analysis of untargeted metabolomics from mouse plasma after an acute bout of treadmill running versus sedentary condition. Tab 3: targeted metabolomics of racehorse plasma before versus after the race. Tab 4: untargeted metabolomics of racehorse plasma before versus after the race. Tab 5: MS parameters for targeted metabolomics analyses of mouse and racehorse plasma.

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Li, V.L., He, Y., Contrepois, K. et al. An exercise-inducible metabolite that suppresses feeding and obesity. Nature 606, 785–790 (2022). https://doi.org/10.1038/s41586-022-04828-5

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