. 2025 Jan 9;188(1):175-186.e20.

doi: 10.1016/j.cell.2024.10.032. Epub 2024 Nov 12.

A β-hydroxybutyrate shunt pathway generates anti-obesity ketone metabolites

Maria Dolores Moya-Garzon¹, Mengjie Wang², Veronica L Li³, Xuchao Lyu¹, Wei Wei⁴, Alan Sheng-Hwa Tung⁴, Steffen H Raun⁵, Meng Zhao⁶, Laetitia Coassolo⁶, Hashim Islam⁷, Barbara Oliveira⁷, Yuqin Dai⁸, Jan Spaas⁹, Antonio Delgado-Gonzalez¹⁰, Kenyi Donoso¹¹, Aurora Alvarez-Buylla¹², Francisco Franco-Montalban¹³, Anudari Letian¹⁴, Catherine P Ward¹⁵, Lichao Liu¹⁶, Katrin J Svensson⁶, Emily L Goldberg¹⁴, Christopher D Gardner¹⁵, Jonathan P Little⁷, Steven M Banik¹⁷, Yong Xu¹⁸, Jonathan Z Long¹⁹

Affiliations

¹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA; Wu Tsai Human Performance Alliance, Stanford University, Stanford, CA, USA.
² USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.
³ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Department of Chemistry, Stanford University, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA; Wu Tsai Human Performance Alliance, Stanford University, Stanford, CA, USA.
⁴ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Department of Biology, Stanford University, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
⁵ Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
⁶ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Stanford Diabetes Research Center, Stanford University, Stanford, CA, USA; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
⁷ School of Health and Exercise Sciences, University of British Columbia, Kelowna, BC, Canada.
⁸ Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
⁹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
¹⁰ Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA.
¹¹ Department of Urology, Stanford University School of Medicine, Stanford, CA, USA.
¹² Department of Biology, Stanford University, Stanford, CA, USA.
¹³ Departamento de Química Farmacéutica y Orgánica, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain.
¹⁴ Department of Physiology, University of California, San Francisco, San Francisco, CA, USA.
¹⁵ Stanford Prevention Research Center, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
¹⁶ Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
¹⁷ Department of Chemistry, Stanford University, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
¹⁸ USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA. Electronic address: yongx@bcm.edu.
¹⁹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA; Stanford Diabetes Research Center, Stanford University, Stanford, CA, USA; Wu Tsai Human Performance Alliance, Stanford University, Stanford, CA, USA; The Phil & Penny Knight Initiative for Brain Resilience at the Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA. Electronic address: jzlong@stanford.edu.

PMID: 39536746
PMCID: PMC11724754
DOI: 10.1016/j.cell.2024.10.032

A β-hydroxybutyrate shunt pathway generates anti-obesity ketone metabolites

Maria Dolores Moya-Garzon et al. Cell. 2025.

. 2025 Jan 9;188(1):175-186.e20.

doi: 10.1016/j.cell.2024.10.032. Epub 2024 Nov 12.

Authors

Affiliations

¹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA; Wu Tsai Human Performance Alliance, Stanford University, Stanford, CA, USA.
² USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.
³ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Department of Chemistry, Stanford University, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA; Wu Tsai Human Performance Alliance, Stanford University, Stanford, CA, USA.
⁴ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Department of Biology, Stanford University, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
⁵ Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
⁶ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Stanford Diabetes Research Center, Stanford University, Stanford, CA, USA; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
⁷ School of Health and Exercise Sciences, University of British Columbia, Kelowna, BC, Canada.
⁸ Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
⁹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
¹⁰ Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA.
¹¹ Department of Urology, Stanford University School of Medicine, Stanford, CA, USA.
¹² Department of Biology, Stanford University, Stanford, CA, USA.
¹³ Departamento de Química Farmacéutica y Orgánica, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain.
¹⁴ Department of Physiology, University of California, San Francisco, San Francisco, CA, USA.
¹⁵ Stanford Prevention Research Center, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
¹⁶ Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
¹⁷ Department of Chemistry, Stanford University, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
¹⁸ USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA. Electronic address: yongx@bcm.edu.
¹⁹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Sarafan ChEM-H, Stanford University, Stanford, CA, USA; Stanford Diabetes Research Center, Stanford University, Stanford, CA, USA; Wu Tsai Human Performance Alliance, Stanford University, Stanford, CA, USA; The Phil & Penny Knight Initiative for Brain Resilience at the Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA. Electronic address: jzlong@stanford.edu.

PMID: 39536746
PMCID: PMC11724754
DOI: 10.1016/j.cell.2024.10.032

Abstract

β-Hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve the interconversion of BHB and primary energy intermediates. Here, we identify a previously undescribed BHB secondary metabolic pathway via CNDP2-dependent enzymatic conjugation of BHB and free amino acids. This BHB shunt pathway generates a family of anti-obesity ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. The most abundant BHB-amino acid, BHB-Phe, is a ketosis-inducible congener of Lac-Phe that activates hypothalamic and brainstem neurons and suppresses feeding. Conversely, CNDP2-KO mice exhibit increased food intake and body weight following exogenous ketone ester supplementation or a ketogenic diet. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, enzymatic amino acid BHB-ylation defines a ketone shunt pathway and bioactive ketone metabolites linked to energy balance.

Keywords: BHB; enzyme; ketone; metabolite; metabolomics; obesity.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests A provisional patent application has been filed by Stanford University on BHB-amino acids for the treatment of cardiometabolic disease.

Figures

**Fig. 1.. CNDP2 catalyzes amino acid BHB-ylation in vitro.**
(A) Schematic of CNDP2-dependent Lac-Phe synthesis and the proposed CNDP2-dependent BHB-Phe synthesis reaction. (B) BHB-Phe synthesis activity of cell lysates transfected with GFP or mouse CNDP2-flag. (C,D) Synthesis activity of cell lysates transfected with GFP or mouse CNDP2-flag and incubated with the indicated monocarboxylate with Phe (C) or the indicated amino acid with BHB (D). (E,F) Michaelis-Menten kinetics of recombinant purified mouse CNDP2-flag protein with BHB (E) or lactate (F) as the organic acid donor. (G) Molecular docking of BHB-Phe into the mouse CNDP2 active site. (H,I) BHB-Phe synthesis activity (H) or Lac-Phe synthesis activity (I) of HEK293T cells transfected with the indicated mouse WT or mutant CNDP2 plasmid. For enzyme assays, organic acids and amino acids were incubated at a concentration of 20 mM at 37°C for 1 hour. For B-F and H,I, N=3–4/group. Data for B-F and H,I are shown as means ± SEM. P-values were calculated by Student’s two-sided t-test.

**Fig. 2.. CNDP2 is the principal BHB-amino acid synthetase in mouse tissues.**
(A) Western blot of the indicated mouse tissues using an anti-CNDP2 (top) or anti-tubulin (bottom) antibody. (B-F) Enzyme activities of tissues from WT or CNDP2-KO mice when provided with BHB and Phe (B), BHB and Leu (C), BHB and Val (D), lactate and Phe (E), or carnosine (F) as substrates. For enzyme assays, organic acids and amino acids were incubated at a concentration of 20 mM at 37°C for 1 hour. For B-F, N=3–4/group. Data for B-F are shown as means ± SEM. P-values were calculated by Student’s two-sided t-test.

**Fig. 3.. Detection and ketosis-inducibility of BHB-amino acids in mouse plasma.**
(A-D) Tandem mass spectrometry fragmentation of the authentic standard (left) and co-elution of the standard and the endogenous peak from mouse plasma (right) using the indicated multiple reaction monitoring transition for BHB-Phe (A), BHB-Val (B), BHB-Leu (C), and BHB-Met (D). (E-H) BHB-amino acid quantitation in 8–9 week old male C57BL/6J mouse plasma at baseline, after 1 week on ketogenic diet (Research Diets D21021803), after a 24 h fast or 30 min post ketone monoester drink administration by oral gavage (3 g KE/kg of body weight). For E-H, N=5/group, with the baseline N=15 (pooled from each of the three groups). Data for E-H are shown as box-and-whisker plots. P-values were calculated by Student’s two-sided t-test.

**Fig. 4.. Genetic regulation of BHB-amino acids by CNDP2 and HMGCL.**
(A) Schematic of ketone biochemical pathways and the genetic mouse models used. (B-D) BHB-amino acid quantitation in plasma from 4–10-week-old male WT and CNDP2-KO mice at 60 min post ketone monoester drink administration by oral gavage (3 g KE/kg of body weight) (B), from 7–16-week-old female WT and CNDP2-KO mice after 1 week on ketogenic diet (Research Diets D06040601, C), or from Hmgcl (fl/fl) vs Alb-Hmgcl(−/−) mice after a 24 h fast (D). For B, N = 7 for WT and 4 for KO. For C, N = 8 per group. For D, N = 5 for Hmgcl (fl/fl), N = 4 for Alb-Hmgcl(−/−). Data for B-D are shown as box-and-whisker plots. P-values were calculated by Student’s two-sided t-test.

**Fig. 5.. BHB-Phe suppresses food intake and body weight.**
(A-E) Food intake (A), ambulatory movement (B), oxygen consumption (VO₂) (C), carbon dioxide production (VCO₂) (D), and respiratory exchange ratio (RER) (E) of singly housed 29-week-old male DIO mice following a single injection of vehicle or BHB-Phe (50 mg/kg, IP) over a 10 h period in metabolic chambers. (F,G) Change in body weight (F) and cumulative food intake (G) of singly housed 28-week-old male DIO mice treated with vehicle or BHB-Phe (50 mg/kg/day, IP). Starting body weights were vehicle: 46.4 ± 1.4 g, BHB-Phe 45.7 ± 1.0 g (mean ± SEM). (H) Change in body weight (left) and daily food intake (right) of group housed 15-week-old male DIO mice after 6 days of treatment with vehicle, BHB-Phe (50 mg/kg/day, IP) or vehicle-treated pair-fed mice. Starting body weights were vehicle: 39.4 ± 2.3 g, BHB-Phe: 38.5 ± 0.8 g, and pair-fed: 37.8 ± 0.6 g (mean ± SEM). (I) Change in body weight (left) and food intake (right) of group housed 13-week-old male DIO mice after 9 days of treatment with vehicle, BHB-Phe, BHB, or phenylalanine (50 mg/kg/day, IP). Starting body weights were vehicle: 36.2 ± 0.7 g, BHB-Phe: 37.9 ± 2.1 g, BHB: 36.8 ± 1.1 g, Phe: 34.5 ± 0.5 g (mean ± SEM). (J,K) Change in body weight (J) and food intake (K) of group housed 14–16-week-old male DIO mice after 9 days of treatment with Phe-Phe, BHB-Lys, Leu-Leu, BHB-Phe (50 mg/kg/day, IP) or vehicle. Starting body weights were vehicle: 41.2 ± 1.3 g, BHB-Phe: 39.1 ± 1.7 g, BHB-Lys: 40.1 ± 2.2 g, Phe-Phe: 40.8 ± 1.4 g, Leu-Leu: 41.2 ± 1.3 g (mean ± SEM). (L,M) Change in body weight (L) and food intake (M) of singly housed 19–32-week-old male WT and CNDP2-KO mice that had been rendered obese by high fat diet feeding for 11–19 weeks receiving ketone esters (3 g/kg/day, PO). Starting body weights were WT: 46.8 ± 1.4 g, CNDP2-KO: 47.1 ± 1.3, P > 0.05 (mean ± SEM). For A-E, N = 7 for vehicle, N = 8 for BHB-Phe. For F,G, N = 10 per group. For H-K, N = 5 per group. For L,M, N = 9 per group. Data in A-G, J, L, and M are shown as the mean ± SEM. Data in H, I, and K are shown as box-and-whisker plots. P-values were calculated by Student’s two-sided t-test or by two-way ANOVA.

**Fig. 6.. TRAP/c-Fos mapping of BHB-Phe- and Lac-Phe-activated neurons in the brain.**
(A) A schematic diagram of the experimental design for mapping BHB-Phe- and Lac-Phe-activated neurons. TRAP, targeted recombination in active populations. (B) Heat map showing the number of Veh^TRAP, BHB-Phe^TRAP, Veh^c-Fos and Lac-Phe^c-Fos labeled neurons in various brain regions. ARH, arcuate nucleus of the hypothalamus; DMH, dorsomedial hypothalamus; LH, lateral hypothalamus; LPBN, lateral parabrachial nucleus; NTS, nucleus of the solitary tract; PVH, paraventricular hypothalamus; SCN, superchiasmatic nucleus; VMH, ventromedial hypothalamus. (C,D) Quantification (C) and representative sections (D) of TRAP+/c-Fos+ neurons in the indicated brain regions. For B and C, N = 3 per group. Data in C are shown as mean ± SEM. Scale bars, 100 μm.

**Fig. 7.. Human CNDP2 activity and BHB-amino acids in human plasma.**
(A,B) BHB-Phe synthetase activity of recombinant human CNDP2 provided with the indicated substrates (A) and Michaelis-Menten kinetics of recombinant human CNDP2 protein with increasing concentrations of BHB substrate (B). (C-E) Top: BHB-Phe synthesis activity of cell lysates from WT or CNDP2-KO human cell lines U937 (C), Caco-2 (D) or PANC-1 (E). Bottom: Western blot using an anti-CNDP2 (upper) or anti-tubulin (lower) antibody for WT and CNDP2-KO U937 (C), Caco-2 (D), and PANC-1 (E) cells. (F,G) Levels of BHB-amino acids (F) or the indicated metabolite (G) in human plasma at baseline or 60 minutes post ketone ester drink administration (0.3 g/kg ketone ester). For A and C-E, reactions were performed with 20 mM substrates at 37°C for 1 hour. For A-E, N=3–5/group. For F and G, N=7/group. For A-E, data are shown as mean ± SEM. For F and G, data are shown as box-and-whisker pots. P-values were calculated by Student’s two-sided t-test.

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A β-hydroxybutyrate shunt pathway generates anti-obesity ketone metabolites

Affiliations

A β-hydroxybutyrate shunt pathway generates anti-obesity ketone metabolites

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases

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