Abstract
N-Lactoyl-phenylalanine (Lac-Phe) is a lactate-derived circulating metabolite that reduces feeding and obesity, but the molecular mechanisms that underlie the metabolic benefits of Lac-Phe remain unknown. Here we show that Lac-Phe directly inhibits hypothalamic neurons that express Agouti-related protein (AgRP), resulting in an indirect activation of anorexigenic neurons in the paraventricular nucleus of the hypothalamus (PVH). Both AgRP inhibition and PVH activation are required to mediate Lac-Phe-induced hypophagia. Lac-Phe-mediated inhibition of AgRP neurons occurs through activation of the ATP-sensitive potassium (KATP) channel, whereas inhibition of the KATP channel blunts the effects of Lac-Phe to suppress feeding. Together, these results reveal the molecular and neurobiological mechanisms by which Lac-Phe mediates metabolic improvements and suggest this exercise-induced metabolite might have therapeutic benefits in various human diseases.
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Acknowledgements
The investigators were supported by grants from the USDA/CRIS (51000-064-01S to Y.X., 3092-51000-062-04(B)S to C.W.), American Heart Association (23POST1030352 to Hailan Liu), National Institutes of Health (F32DK134121 to K.M.C.; R01DK136479 to Y.X., R01DK136526 to J.Z.L., T32GM13854 to V.L.L.), a Bio-X SIGF Graduate Student Fellowship to V.L.L. and Texas Children’s Research Scholar funds to Y.H.
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Hailan Liu, V.L.L., Q.L., Y.H., J.Z.L. and Y.X. conceived of the project and experimental design and wrote the paper. Hailan Liu, V.L.L., Q.L. and Y.H. performed the procedures, data acquisition and analyses. Y. Liu, S.C. and Hueyxian Wong assisted with feeding studies. N.Y. and Yongjie Yang assisted in molecular and cellular experiments. Hesong Liu, X.F., K.M.M., Hueyzhong Wong, M.Y., L.T., J.C.B., Y. Li, M.W., Y.D., Y.S., O.Z.G., Yuxue Yang, J.H., M.E.B. and S.V.J. contributed to the generation of study mice and data discussion. C.W., B.R.A. and D.K. were involved in study design and data discussion.
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V.L.L., Y.H., J.Z.L. and Y.X. are listed as inventors on a patent covering lactoyl amino acids for the treatment of metabolic diseases (E-160-2023-0-PC-01, N-lactoyl-phenylalanine [Lac-Phe] compound derivatives, Stanford University and Baylor College of Medicine). The other authors declare no conflict of interest.
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Extended data
Extended Data Fig. 1 Lac-Phe does not induce adverse effects.
(a) Three-hour food intake in chow-fed female WT mice (2 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). p = 0.016. (b) Three-hour food intake in HFD-fed female WT mice (3 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). p = 0.0047. (c) Three-hour kaolin intake in male WT mice (3 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). (d) Flavor preference ratio in male WT mice (3 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). (e) Travel distance, velocity, number of entries into the center and time spent in the center during OFT in male WT mice (3 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). (f) Travel distance, velocity, number of entries into the open arms and time spent in the open arms during OFT in male WT mice (3 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). (g) Number of entries into the closed arms and time spent in the closed arms during OFT in male WT mice (3 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). (h) Time spent in immobility in FST in male WT mice (3 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). (i) Sucrose preference ratio in SPT in male WT mice (3 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). N = 8 mice in each group for a-i.Data are mean ± SEM with individual data points in (a-i). Two-sided unpaired t-test was used in (a-i). *, p < 0.05; **, p < 0.01.
Extended Data Fig. 2 Lac-Phe activates NTS and PVH neurons.
(a-b) Representative fluorescent images showing c-Fos (a) and quantification (b) in the NTS in HFD-fed female WT mice (4 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). N = 3 mice in each group. p = 0.00014. (c-d) Representative fluorescent images showing c-Fos (c) and quantification (d) in the PVH in HFD-fed female WT mice. N = 3 mice in each group. p < 0.0001. (e-f) Representative microscopic images showing mCherry signals in the NTS of control and NTSTRAP-Di mice (e) or in the PVH of control and PVHTRAP-Di mice (f). N = 3 biological replicates in each group. (g) A representative current-clamp trace showing effects of CNO (10 μM, 5 s puff) on a mCherry+ PVH neuron in PVHTRAP-Di mice. (h-i) Resting membrane potential (h) and firing frequency (i) of mCherry+ PVH neurons during the baseline and 10 μM CNO treatment. N = 5 neurons from 3 different mice. p = 0.0017 for h; p = 0.0007 for i. Data are mean ± SEM with individual data points in (b, d) and individual data points in (h-i). Two-sided unpaired t-test was used in (b, d); Two-sided paired t-tests were used in (h-i). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Extended Data Fig. 3 Rabies tracing from Lac-Phe-activated PVH neurons.
(a) Representative fluorescent images showing dsRed (red) and DAPI (blue) expression in the PVH, LS, NAcSh, POA, PVT, SON, cHypo, and MVe of male TRAP2 mice with Lac-Phe-activated PVH neurons infected by EnvA-ΔG-Rabies-dsRed. (b) RNAscope images showing tdTomato, Mc4r and Npy1r expression in the PVH of male TRAP2/Rosa26-LSL-tdTomato mice following Lac-Phe TRAP. (c-e) Quantification of the percentage of tdTomato+ cells expressing Mc4r (c), or Npy1r (d), or Mc4r and Npy1r (e). N = 3 mice.
Extended Data Fig. 4 i.p. injection of Lac-Phe increases its concentration in the ARH.
(a-c) Body weight (a), body composition (b), and blood glucose (c) changes in chow- and HFD-fed male mice. N = 19 mice in each group. p < 0.0001 for b; p = 0.0086 for c. (d-e) Lac-Phe concentration in the serum (d) and ARH (e) of chow-fed male mice at 0, 5, 10, 30, 60 and 120 min after an i.p. injection of 50 mg/kg Lac-Phe. N = 4 mice for 0 and 5 min, N = 3 for 10, 30, 60, and 120 min. p = 0.0022 for 0 vs 5; p = 0.0005 for 0 vs 10; p = 0.015 for 0 vs 30 min in d; p = 0.0003 for 0 vs 5; p = 0.0011 for 0 vs 10 min in e. (f-g) Lac-Phe concentration in the serum (f) and ARH (g) of HFD-fed male mice at 0, 5, 10, 30, 60 and 120 min after an i.p. injection of 50 mg/kg Lac-Phe. N = 4 mice for 0, 5 and 10 min, N = 3 for 30, 60, and 120 min. p = 0.0085 for 0 vs 5; p = 0.0035 for 0 vs 10; p = 0.0059 for 0 vs 30, p = 0.0425 for 0 vs 60 min in f; p = 0.0009 for 0 vs 5; p = 0.0184 for 0 vs 10; p = 0.0293 for 0 vs 30, p = 0.0372 for 0 vs 60 min in g. (h-i) Serum (h) and ARH (i) Lac-Phe concentrations in chow and HFD-fed male mice at 0, 5, 10, 30, 60 and 120 min after an i.p. injection of 50 mg/kg Lac-Phe. Note that panels h-i were plotted using the same data from d-g. Data are mean ± SEM with individual data points in (b, d-g) and mean ± SEM in (a, c, h-i). One-way ANOVA analysis was used in (d-g); two-way ANOVA analysis was used in (a-c, h-i). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Extended Data Fig. 5 Lac-Phe activates AgRP neurons during fasting conditions.
(a-b) Temporal changes in the resting membrane potential (a) and firing frequency (b) of AgRP neurons in the ARH in response to 2.5 μM (puff 5 s) lactate or phenylalanine. N = 7 neurons from 3 different mice. (c) Representative current-clamp traces showing effects of vehicle or Lac-Phe (5 μM) on AgRP neurons in overnight fasted male mice. (d-e) Temporal changes in the resting membrane potential (d) and firing frequency (e) of AgRP neurons in male mice following vehicle or Lac-Phe application. N = 6 neurons from 3 different mice for each treatment. p < 0.0001 for d and e. (f) Representative current-clamp traces showing effects of vehicle or Lac-Phe (5 μM) on AgRP neurons in overnight fasted female mice. (g-h) Temporal changes in the resting membrane potential (g) and firing frequency (h) of AgRP neurons in female mice following vehicle or Lac-Phe application. N = 6 neurons from 3 different mice for each treatment. p = 00001 for g, p < 0.0001 for h. Data are mean ± SEM in (a-b, d-e, g-h). Two-way ANOVA analysis followed by post hoc Sidak tests was used in (a-b, d-e, g-h). ***, p < 0.001; ****, p < 0.0001.
Extended Data Fig. 6 Lac-Phe inhibits AgRP neurons.
(a) The experimental design for recording AgRP neuronal activity 1 h after vehicle or Lac-Phe injection. (b) Representative current-clamp traces of AgRP neurons recorded 1 h after vehicle or Lac-Phe injection in male AgRP-IRES-Cre/Rosa26-LSL-tdTomato mice. (c-d) Resting membrane potential (c) and firing frequency (d) of AgRP neurons in male mice following vehicle or Lac-Phe administration. N = 17 (vehicle) or 18 (Lac-Phe) neurons from 3 different mice in each group. p < 0.0001 for c, p = 0.0077 for d. (e) Representative current-clamp traces of AgRP neurons recorded 1 h after vehicle or Lac-Phe injection in female AgRP-IRES-Cre/Rosa26-LSL-tdTomato mice. (f-g) Resting membrane potential (f) and firing frequency (g) of AgRP neurons in female mice following vehicle or Lac-Phe administration. N = 14 (vehicle) or 16 (Lac-Phe) neurons from 3 different mice in each group. p = 0.0011 for f, p = 0.0298 for g. (h) Representative current-clamp traces of AgRP neurons recorded 3 h after vehicle or Lac-Phe injection in female AgRP-IRES-Cre/Rosa26-LSL-tdTomato mice. (i-j) Resting membrane potential (i) and firing frequency (j) of AgRP neurons in female mice following vehicle or Lac-Phe administration. N = 14 neurons from 3 different mice in each group. p = 0.010 for I, p = 0.012 for j. Data are mean ± SEM with individual data points in (c-d, f-g, i-j). Two-sided unpaired t-tests were used in (c-d, f-g, i-j). *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
Extended Data Fig. 7 AgRP neurons mediate exercise-induced hypophagia.
(a) A representative microscopic image showing expression of GCaMP6m in AgRP neurons in the ARH with the fiber photometry probe track. N = 6 biological replicates. (b) Representative microscopic images showing pPDH immunofluorescence (green) in tdTomato-labelled AgRP neurons (red) in male AgRP-IRES-Cre/Rosa26-LSL-tdTomato mice (4 months of age) receiving i.p. injections of vehicle or Lac-Phe (50 mg/kg). (c) Quantifications of pPDH+/tdTomato+ cells. N = 3 mice in each group. p < 0.0001. (d) Quantification for the number of Agrp+ cells in the ARH of control and AgRPDTA mice. N = 9 mice for control, N = 11 mice for AgRPDTA. p < 0.0001. (e) Body weight gain in control and AgRPDTA mice after HFD feeding. N = 9 mice for control, N = 11 mice for AgRPDTA. P = 0.0104. (f) A representative current-clamp trace showing effects of CNO (10 μM, 10 s puff) on an AgRP neurons in AgRPDq mice. (g-h) Resting membrane potential (g) and firing frequency (h) of AgRP neurons during the baseline and 10 μM CNO treatment. N = 7 neurons from 3 different mice. p = 0.0002 for g, p = 0.0017 for h. (i) Representative microscopic images showing c-Fos immunofluorescence (green) in tdTomato-labelled AgRP neurons (red) in male AgRP-IRES-Cre/Rosa26-LSL-tdTomato mice (4 months of age) that were infected with AAV8-FLEX-hM3Dq-mCherry, followed by i.p. injections of saline or CNO (1 mg/kg). (j) Quantifications of c-Fos+/tdTomato+ cells. N = 3 mice in each group. p < 0.0001. Data are mean ± SEM with individual data points in (c-d, j) and mean ± SEM in (e) or individual data points in (g-h). Two-sided unpaired t-tests were used in (c-d, j); two-sided paired t-tests were used in (g-h); two-way ANOVA analysis was used in (e). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Extended Data Fig. 8 Lac-Phe does not activate POMC neurons.
(a) A representative current-clamp trace showing effects of Lac-Phe (5 μM, 5 s puff) on a POMC neuron in POMC-CreERT2/Rosa26-LSL-tdTomato mice. (b-c) Temporal changes in the resting membrane potential (b) and firing frequency (c) of POMC neurons in the ARH in response to vehicle or 5 μM Lac-Phe (puff 5 s). (d) Representative microscopic images showing β-endorphin immunofluorescence (green) in tdTomato-labelled ARH neurons (red) in male TRAP2/Rosa26-LSL-tdTomato mice (3 months of age) following Lac-Phe TRAP. N = 3 mice. Data are mean ± SEM in (b, c). Two-way ANOVA analysis followed by post hoc Sidak tests was used in (b, c).
Extended Data Fig. 9 Lac-Phe enhances KATP channels in AgRP neurons of female mice.
(a) Representative voltage-clamp traces for KATP currents in AgRP neurons of female mice at the baseline (black) or in response to 5 μM Lac-Phe (red) in the absence or presence of tolbutamide. (b) Quantifications of KATP currents in AgRP neurons in response to various treatments as described in (a). N = 6 or 8 neurons from 3 different mice. p < 0.0001 for vehicle vs Lac-Phe and Lac-Phe vs Lac-Phe+Tol. (c) Representative current-clamp traces showing effects of vehicle, Lac-Phe (5 μM), or Lac-Phe (5 μM) plus tolbutamide (100 μM) on AgRP neurons in the ARH of female mice. (d-e) Temporal changes in the resting membrane potential (d) and firing frequency (e) of AgRP neurons in the ARH in response to various treatments as described in (c). N = 6 neurons from 3 different female mice. p < 0.0001 for vehicle vs Lac-Phe and Lac-Phe vs Lac-Phe+Tol in both d and e. Data are mean ± SEM with individual data points in (b) and mean ± SEM in (d, e). Two-way ANOVA analysis followed by post hoc Sidak tests was used in (b, d, e). *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
Extended Data Fig. 10 Validation of AAV-sgKcnj11.
(a) A schematic illustration for stereotaxic injections of AAV-spCas9 and AAV-sgKcnj11-FLEX-mCherry (ARHΔKcnj11), or AAV-GFP (control) into the ARH of male WT mice. (b) Three-hour food intake in HFD-fed male control (N = 8) or ARHΔKcnj11 (N = 9) mice (4 months of age) receiving i.p. injection of vehicle or Lac-Phe (50 mg/kg, i.p.). p < 0.0001 for vehicle vs Lac-Phe in control mice, p = 0.0039 for vehicle vs Lac-Phe in ARHΔKcnj11 mice, #### represents p < 0.0001 for Lac-Phe treated control vs ARHΔKcnj11 mice. (c) A representative microscopic image showing mCherry in the ARH of AgRP-IRES-Cre/Cas9fl/+ mice following AAV-sgKcnj11-FLEX-mCherry injection. N = 8 biological replicates. (d) Body weight gain in control and AgRPΔKcnj11 mice after HFD feeding. N = 8 mice in each group. p = 0.0069. (e) A representative current-clamp trace showing effects of diazoxide on AgRP neurons in the ARH of control mice. (f-g) Changes in the resting membrane potential (f) and firing frequency (g) of AgRP neurons in control mice following diazoxide treatment. N = 5 neurons. p = 0.0133 for f, p = 0.0138 for g. (h) A representative current-clamp trace showing effects of diazoxide on AgRP neurons in the ARH of AgRPΔKcnj11 mice. (i-j) Changes in the resting membrane potential (i) and firing frequency (j) of AgRP neurons in AgRPΔKcnj11 mice following diazoxide treatment. N = 6 neurons. (k) Representative voltage-clamp traces for KATP currents in AgRP neurons of control and AgRPΔKcnj11 mice in response to diazoxide. (l) Quantifications of KATP currents in AgRP neurons of control (N = 5) and AgRPΔKcnj11 (N = 6) mice. p = 0.00045. (m) Three-hour food intake in HFD-fed male control or ARHΔKcnj11 mice (4 months of age) receiving i.p. injection of vehicle or lorcaserin (9 mg/kg, i.p.). N = 5 mice in each group. p = 0.0136 and 0.0015 for vehicle vs lorcaserin in control and ARHΔKcnj11 mice, respectively. (n) Three-hour food intake in HFD-fed male control or AgRPΔKcnj11 mice (4 months of age) receiving i.p. injection of vehicle or lorcaserin (9 mg/kg, i.p.). N = 8 mice in each group. p < 0.0001 for vehicle vs lorcaserin in control and AgRPΔKcnj11 mice. Data are mean ± SEM with individual data points in (b, l-n) and mean ± SEM in (d) or individual data points in (f-g, i-j). Two-sided unpaired t-tests were used in (l); two-sided paired t-tests were used in (f-g, i-j); two-way ANOVA analysis was used in (b, d, m-n). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
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Liu, H., Li, V.L., Liu, Q. et al. Lac-Phe induces hypophagia by inhibiting AgRP neurons in mice. Nat Metab 7, 2004–2017 (2025). https://doi.org/10.1038/s42255-025-01377-9
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DOI: https://doi.org/10.1038/s42255-025-01377-9
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