Circulating metabolite homeostasis achieved through mass action

ABSTRACT Homeostasis maintains serum metabolites within physiological ranges. For glucose, this requires insulin, which suppresses glucose production while accelerating its consumption. For

other circulating metabolites, a comparable master regulator has yet to be discovered. Here we show that, in mice, many circulating metabolites are cleared via the tricarboxylic acid cycle

(TCA) cycle in linear proportionality to their circulating concentration. Abundant circulating metabolites (essential amino acids, serine, alanine, citrate, 3-hydroxybutyrate) were

administered intravenously in perturbative amounts and their fluxes were measured using isotope labelling. The increased circulating concentrations induced by the perturbative infusions

hardly altered production fluxes while linearly enhancing consumption fluxes and TCA contributions. The same mass action relationship between concentration and consumption flux largely held

across feeding, fasting and high- and low-protein diets, with amino acid homeostasis during fasting further supported by enhanced endogenous protein catabolism. Thus, despite the copious

regulatory machinery in mammals, circulating metabolite homeostasis is achieved substantially through mass action-driven oxidation. Access through your institution Buy or subscribe This is a

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ANALYSIS OF INTACT HUMAN LIVER TISSUE EX VIVO Article Open access 29 August 2024 RESTRICTION OF ESSENTIAL AMINO ACIDS DICTATES THE SYSTEMIC METABOLIC RESPONSE TO DIETARY PROTEIN DILUTION

Article Open access 09 June 2020 ISLET HORMONES AT THE INTERSECTION OF GLUCOSE AND AMINO ACID METABOLISM Article 07 March 2025 DATA AVAILABILITY All data and materials will be provided on

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S.H. was supported by a National Institutes of Health (NIH) grant (no. 4R00DK117066). T.G.A. was supported by NIH grant no. DK109714 and a U.S. Department of Agriculture National Institute

of Food and Agriculture grant no. NC1184-NJ14240. C.J. was supported by NIH grant no. 1R01AA02912. This work was supported by the NIH Pioneer (no. 1DP1DK113643) and Paul G. Allen Family

Foundation grants (no. 0034665) and Ludwig Cancer Research. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Molecular Biology, Princeton University, Princeton, NJ, USA Xiaoxuan

Li & Joshua D. Rabinowitz * Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA Sheng Hui, Won Dong Lee, Cholsoon Jang & Joshua D. Rabinowitz *

Department of Molecular Metabolism, Harvard T. H. Chan School of Public Health, Boston, MA, USA Sheng Hui * Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ, USA

Emily T. Mirek, William O. Jonsson & Tracy G. Anthony * Department of Chemistry, Princeton University, Princeton, NJ, USA Won Dong Lee, Xianfeng Zeng & Joshua D. Rabinowitz *

Department of Biological Chemistry, University of California Irvine, Irvine, CA, USA Cholsoon Jang * Ludwig Institute for Cancer Research, Princeton Branch, Princeton, NJ, USA Joshua D.

Rabinowitz Authors * Xiaoxuan Li View author publications You can also search for this author inPubMed Google Scholar * Sheng Hui View author publications You can also search for this author

inPubMed Google Scholar * Emily T. Mirek View author publications You can also search for this author inPubMed Google Scholar * William O. Jonsson View author publications You can also

search for this author inPubMed Google Scholar * Tracy G. Anthony View author publications You can also search for this author inPubMed Google Scholar * Won Dong Lee View author publications

You can also search for this author inPubMed Google Scholar * Xianfeng Zeng View author publications You can also search for this author inPubMed Google Scholar * Cholsoon Jang View author

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CONTRIBUTIONS X.L., S.H., C.J. and J.D.R. designed the study. X.L. performed most of the experiments and data analysis. E.T.M. and T.G.A. contributed to the breeding and BCAA infusions on

BCKDK knockout mice. W.O.J. performed the comprehensive laboratory animal monitoring system study on the BCKDK knockout mice. W.D.L. contributed to the hyperinsulinaemic–euglycaemic clamp

study. X.Z. provided the portal vein data for the amino acids. X.L., C.J. and J.D.R. wrote the manuscript. All authors discussed and commented on the manuscript. CORRESPONDING AUTHORS

Correspondence to Cholsoon Jang or Joshua D. Rabinowitz. ETHICS DECLARATIONS COMPETING INTERESTS J.D.R. is a cofounder and stockholder in Toran and Serien Therapeutics and advisor to and

stockholder in Agios Pharmaceuticals, Kadmon, Bantam Pharmaceutical, Colorado Research Partners, Rafael Pharmaceuticals, Barer Institute and L.E.A.F. Pharmaceuticals. The other authors

declare no competing interests. PEER REVIEW INFORMATION _Nature Metabolism_ thanks Adam Rose and the other, anonymous, reviewers for their contribution to the peer review of this work.

Alfredo Gimenez-Cassina and George Caputa were the primary handling editors. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 PERTURBATIVE INFUSION OUTCOMES FOR DIFFERENT REGULATORY MECHANISMS. Green arrows reflect labeled metabolite

fluxes including the experimenter-controlled influx from infusion. Blue arrows reflect unlabeled metabolite fluxes, including endogenous production. Green and blue circles are labeled and

unlabeled metabolites, respectively. Red circles represent red blood cells. (A) Mass action. Labeled metabolites accumulate linearly with infusion rate, with unlabeled metabolite

concentrations and fluxes not altered. (B) Active consumption induction. Labeled metabolites accumulate less than linearly with infusion rate, with unlabeled metabolite levels decreased but

fluxes unaltered. (C) Consumption saturation. Labeled metabolites accumulate more than linearly with infusion rate, with unlabeled metabolites levels increased but fluxes unaltered. (D)

Feedback inhibition of production. Unlabeled metabolite levels and fluxes are decreased. EXTENDED DATA FIG. 2 CLEARANCE OF METABOLITE BOLUSES IS CONSISTENT WITH MASS ACTION KINETICS. (A)

Mice were fasted from 9 AM to 5 PM (8 h fasting). At 5 PM, mice were injected with an intravenous bolus of the indicated [U-13C] metabolite at a low, medium, or high dose (as specified in

the methods). Blood was taken 5, 15, 30, and 60 min after the injection, and the concentration of labeled metabolite in serum was measured by LC-MS. For experiments involving branched-chain

amino acids, all three were given together, with only the indicated amino acid in labeled form. Labeled metabolite concentration was plotted against time post bolus. Lines are exponential

decay curves fitted with mean value of each group. (B) Pseudo-first-order consumption rate constants from bolus and perturbative infusions. The slopes (α) calculated from the infusion

experiments were plotted against the elimination constants (γ) calculated from the bolus experiments for each circulating metabolite. Line is linear regression fit. Source data EXTENDED DATA

FIG. 3 ELEVATED PORTAL VEIN ALANINE IN FED MICE. (A) Circulating metabolite concentrations in the fasted and refed state. Fasting and feeding schedules were the same as Fig. 4a. Blood was

taken at 5 PM for the fasted group and 11 PM for the refed group. Mean±SD. n = 4 mice. (B, C) Insulin does not alter concentrations or consumption fluxes of valine, lysine, and alanine. (b)

Serum metabolite levels from hyperinsulinemic-euglycemic clamp (2.5 mU/kg/min insulin) and control (saline) experiments. Mice were fasted from 10 AM to 5 PM. The clamp was performed from 3

PM to 5 PM and blood was collected at 5 PM. Mean±s.d. n = 4 mice. (c) Consumption fluxes in the above clamp condition based on non-perturbative infusion of a mixture of 13C-valine,

13C-lysine, and 13C-alanine. The 13C-infusion was initiated 2.5 h prior to starting insulin to induce the hyperinsulinemic clamp and continued throughout the clamp experiment. Blood samples

were taken immediately prior to or 120-min after initiation of the clamp. Mean±s.d. n = 4 mice. (D) Metabolite concentration ratios between the portal vein and tail vein of fasted (7.5 h

fast starting at 9:30 AM, with sampling at 5 PM) or ad lib fed mice (with sampling at 11 PM). Mean±s.d. n = 4 mice. (E) Calculation of alanine consumption flux-concentration relationship

using tail or portal vein data. Portal vein concentrations were calculated by multiplying alanine data from Fig. 4a by the portal/tail vein TIC ratio from (d). Lines are linear fits to each

dataset. Source data EXTENDED DATA FIG. 4 BCKDK WHOLE-BODY KO MICE ARE LETHARGIC WHEN FED LOW PROTEIN DIET. (A) Valine consumption flux versus circulating concentration in whole-body BCKDK

knockout (KO) mice and littermate control mice (WT). Both groups were infused with [U-13C]valine as in Fig. 2. Lines are linear fits to each dataset. (B) KO and WT mice were fed either 20%

or 5% protein diet for 7 days and blood was taken at 5 PM on the last day. Mean±SD. n = 5 WT and 4 KO mice. (C) Activity of KO and WT mice fed 5% protein diet as measured using metabolic

chambers. Mean±SD. n = 6 mice. Source data EXTENDED DATA FIG. 5 TCA OXIDATION MEDIATES MASS ACTION-DRIVEN CONSUMPTION. Tissue TCA labeling-concentration relationship for fasted perturbative

infusions. Data are as in Fig. 5, except for (A) Use of succinate rather than malate to read out tissue TCA labeling or (B) Measurement of malate labeling across additional organs. Lines are

linear fits to the data with intercept set to zero. Source data EXTENDED DATA FIG. 6 PROTEIN SYNTHESIS RATES ARE INSENSITIVE TO BRANCHED-CHAIN AMINO ACID INFUSION. (A) Calculation of

protein synthesis rate. Mice were infused with [U-13C]valine as described in Fig. 6a (fasted group). Tissues were harvested and valine labeling fraction in hydrolyzed tissue proteins were

plotted against time (left). Protein synthesis rate was then calculated based on the slopes (right) using the equation shown (below). Lines are linear fitting with mean values of each

tissue. n = 2 mice per time point for each condition. (B) Tissue protein synthesis rates do not increase with infusion rate. n = 2 mice per time point for each condition. (C) Tissue protein

synthesis rate does not change in response to perturbative valine infusion. Line is mean of the data as the slope is not significant. (D) Tissue protein labeling was consistent from

different BCAAs. Mice were infused with [U-13C]valine, [U-13C]leucine, or [U-13C]isoleucine as in (a). n = 2 mice per time point for each condition. Source data EXTENDED DATA FIG. 7 PROTEIN

DEGRADATION RATE DOES NOT CHANGE IN RESPONSE TO PERTURBATIVE VALINE INFUSION. Data were from the experiments in Fig. 2 (valine panel). Line is mean of the data as the slope is not

significant. Source data EXTENDED DATA FIG. 8 RAW DATA SUPPORTING THE DETERMINATION OF PROTEIN SYNTHESIS RATES AFTER FEEDING. Mice were infused with [U-13C]valine at the same condition as

described in Fig. 6a (refed group). Lines are linear fitting with mean values of each tissue. n = 2 mice per timepoint. Source data EXTENDED DATA FIG. 9 OVER 2 WEEKS, DIETARY PROTEIN

FRACTION HAS LITTLE EFFECT ON BODY WEIGHT OR FOOD INTAKE. Mice were fed high- (HP), normal- (NP), and low-protein (LP) diets ad lib for 2 weeks. (A) Body weight gain. (B) Food intake. Mean ±

SD, n = 6 mice. Source data EXTENDED DATA FIG. 10 TCA OXIDATION MEDIATES MASS ACTION-DRIVEN VALINE CONSUMPTION UNDER HIGH-, MEDIUM-, AND LOW-PROTEIN DIET. Tissue malate labeling relative to

serum valine labeling from non-perturbative infusion of [U-13C]valine, as in Fig. 7c. Lines are linear fits to the data with intercept set to zero. Source data SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Tables 1–7. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 3

Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA FIG. 6 Statistical source data. SOURCE DATA FIG. 7 Statistical

source data. SOURCE DATA EXTENDED DATA FIG. 2 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data.

SOURCE DATA EXTENDED DATA FIG. 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 7 Statistical source data. SOURCE DATA

EXTENDED DATA FIG. 8 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 9 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 10 Statistical source data. RIGHTS AND PERMISSIONS

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