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Branched-chain amino acid catabolism in muscle affects systemic BCAA levels but not insulin resistance

Nature Metabolism volume 5, pages 589–606 (2023)Cite this article

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Abstract

Elevated levels of plasma branched-chain amino acids (BCAAs) have been associated with insulin resistance and type 2 diabetes since the 1960s. Pharmacological activation of branched-chain α-ketoacid dehydrogenase (BCKDH), the rate-limiting enzyme of BCAA oxidation, lowers plasma BCAAs and improves insulin sensitivity. Here we show that modulation of BCKDH in skeletal muscle, but not liver, affects fasting plasma BCAAs in male mice. However, despite lowering BCAAs, increased BCAA oxidation in skeletal muscle does not improve insulin sensitivity. Our data indicate that skeletal muscle controls plasma BCAAs, that lowering fasting plasma BCAAs is insufficient to improve insulin sensitivity and that neither skeletal muscle nor liver account for the improved insulin sensitivity seen with pharmacological activation of BCKDH. These findings suggest potential concerted contributions of multiple tissues in the modulation of BCAA metabolism to alter insulin sensitivity.

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Fig. 1: Systemic BCAA oxidation improves insulin sensitivity.
Fig. 2: Increased SM BCAA oxidation does not affect insulin sensitivity in mice fed normal chow.
Fig. 3: Increased SM BCAA oxidation does not affect insulin sensitivity in mice fed a variety of HFDs.
Fig. 4: Decreased SM BCAA oxidation does not affect insulin sensitivity in mice fed normal chow.
Fig. 5: Decreased SM BCAA oxidation does not affect insulin sensitivity in mice fed HFD.
Fig. 6: Increased liver BCAA oxidation does not affect insulin sensitivity in mice fed HFD.
Fig. 7: Decreased liver BCAA oxidation does not affect insulin sensitivity in mice fed normal chow or HFD.
Fig. 8: Double knockout of BCKDK in both SM and liver has no effect on insulin sensitivity.

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Data availability

All data generated or analysed during this study are included in the published article and its supplementary information files. The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank R. Miller and K. Filipski for their feedback. M.C.B. was supported by a National Institutes of Health (NIH) grant no. F31DK132839. Z.A. was supported by NIH grant nos. DK114103 and CA248315. This work was also supported in part by Pfizer. We also thank the Penn Vector Core at the University of Pennsylvania for producing the custom AAV8 vectors, the Comparative Pathology Core at the University of Pennsylvania School of Veterinary Medicine for preparing and staining the mouse muscle slides, and J. Davis at the Rodent Metabolic Phenotyping Core, supported in part by Penn Diabetes Research Center grant nos. P30-DK19525 and S10-OD025098, for the lean mass body weight measurements. Metabolic studies were in part supported by NIH Diabetes Research Center grant no. P30 DK019525.

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Authors and Affiliations

  1. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Megan C. Blair, Qingwei Chu, Jae Woo Jung, Jessie Axsom, Marc R. Bornstein, Chelsea Thorsheim, Kristina Li & Zoltan Arany

  2. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Michael D. Neinast & Joshua D. Rabinowitz

  3. Department of Biological Chemistry, University of California Irvine, Irvine, CA, USA

    Cholsoon Jang

  4. Kyoto Prefectural University of Medicine, Kyoto, Japan

    Atsushi Hoshino

  5. Washington University School of Medicine, St Louis, MO, USA

    Steven Yang

  6. Pfizer Inc., Cambridge, MA, USA

    Rachel J. Roth Flach & Bei B. Zhang

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Contributions

M.C.B. and Z.A. designed the project. M.C.B. generated most of the data, analysed all the data, created the figures and wrote the text. M.D.N. generated most of the LC–MS data, assisted in the generation of data for some of the figure panels and contributed substantially to the early stages of project design. C.J. generated some of the LC–MS data. Q.C. performed all catheterization surgeries. J.W.J. designed, optimized and assisted in performing the BCKDH activity assays. J.A., M.R.B., C.T. and K.L. each assisted with data collection. A.H. generated the BckdkloxP/loxP animals. S.Y. generated the DbtloxP/loxP animals. R.J.R.F. and B.B.Z. gave support and feedback throughout the data generation process. J.D.R. assisted, in collaboration with M.D.N. and C.J., with the use of the LC–MS instruments. Z.A. contributed substantial support to the project with regular feedback and idea generation, as well as editing the figures and text.

Corresponding author

Correspondence to Zoltan Arany.

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R.J.R.F. and B.B.Z. are employees of Pfizer. This work was in part supported by Pfizer. The other authors declare no competing interests.

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Nature Metabolism thanks Yibin Wang, Owen McGuinness and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Isabella Samuelson, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 BT2 treatment, acutely or chronically, does not affect body weight.

a–c, Western quantification of the ratio p-BCKDH/BCKDH in fasted tissues; n = 6 vehicle- and n = 6 BT2-treated. d, Plasma BCAA concentration in HFD-fed mice fasted for 5-hr or re-fed for 2-hr after an overnight fast; n = 10 vehicle- and n = 10 BT2-treated. Mice were gavaged with 50 mg/kg/day of either vehicle or BT2 for 4 weeks prior to plasma collection. e, Body weights of mice used for acute BT2 experiment; n = 10 vehicle- and n = 10 BT2-treated. f, Body weights of mice used for chronic BT2 experiment; n = 10 vehicle- and n = 10 BT2-treated. g, Body weights of mice used for chronic BT2 GTT (at treatment day 28); n = 10 vehicle- and n = 10 BT2-treated. Mice used for these experiments were male C57BL/6 J diet-induced obesity (DIO) mice ordered from the Jackson Laboratory. They were age 10–12 weeks and on HFD for 4–6 weeks. Data are presented as means ± SEM. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: ***p < 0.001. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures. Exact p-values: a, p = 0.000097. c, p = 0.000556. d, valine p = 0.000002, leucine p = 0.000014, isoleucine p = 0.000557.

Extended Data Fig. 2 BCKDK SM knockout is specific and does not affect body weight or muscle physiology on chow diet.

a–d, Gene expression data via qPCR of Bckdk, Bckdk exon 6 & 7 (deleted region), Bckdha, and Bckdhb, respectively; n = 3 Bckdkl/l (WT) and n = 5 Bckdkl/l-HSA-CreER (Cre). e, Body weights; n = 8 WT and n = 8 Cre. f-j, Western quantification of BCKDK in fasted tissues normalized to loading control; n = 3 WT and n = 3 Cre. k-o, Western quantification of the ratio pBCKDH/BCKDH in fasted tissues; n = 3 WT and n = 3 Cre. p, Western blotting for p-ACLY and ACLY in fasted livers; n = 6 WT and n = 6 Cre. 14-3-3 is the loading control. q, Western quantification of the ratio p-ACLY/ACLY from the blot in p. r, H&E staining of tibialis anterior (TA) muscle. Representative images from n = 4 WT and n = 3 Cre mice. s, Ratio of 3-HIB/valine, and t, Concentration of 3-HIB in the plasma from mice fasted for 5-hr (n = 10 WT and n = 8 Cre) or re-fed for 2-hr after an overnight fast (n = 10 WT and n = 8 Cre). Mice used for these experiments were male, aged 8–10 weeks and fed normal chow. Data are presented as means ± SEM. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: *p < 0.05, **p < 0.01, ***p < 0.001. Exact p-values: a, p = 0.000060. b, p = 0.000020. g, p = 0.008871. h, p = 0.031431. l, p = 0.039537. m, p = 0.015917. s, fasted p = 0.002253, re-fed p = 0.029746. t, re-fed p = 0.023789.

Source data

Extended Data Fig. 3 Increased SM BCAA oxidation is specific in a fasted state and does not affect body weight or glucose tolerance on Western diet.

a, Body weights of mice; n = 8 Bckdkl/l (WT) and n = 9 Bckdkl/l-HSA-CreER (Cre). b, Plasma BCAA levels, c, Ratio of 3-HIB/valine, and d, Relative amount of 3-HIB in mice after a 5-hr fast (fasted) and in a fed state collected at 10 pm (fed); n = 12 WT and n = 17 Cre. e, Fasting plasma pool size of various amino acids, and f, Steady-state plasma labeling of BCAAs by U13C-BCAAs (fraction) during steady-state infusion of U13C-BCAAs before (basal, 5-hr fasted) and during (clamp, 7-hr fasted) HIEC in n = 6 WT and n = 6 Cre mice. g, Normalized labeling of TCA cycle intermediates by U13C-BCAAs in liver, h, iBAT, and i, gWAT at the end of the infusion and HIEC (7-hr fasted); n = 6 WT and n = 6 Cre. j, BCKDH complex activity measured in fasted quad samples fed HFD for 4 weeks; n = 4 WT and n = 3 Cre. k, 2 g/kg glucose IP-GTT in 5-hr fasted mice fed Western diet for 5 weeks; n = 8 WT and n = 9 Cre. l, Area-under-the-curve (AUC) for the GTT in k. Mice used in these experiments were male, aged 18–24 weeks and fed Western diet for 5–12 weeks. Data are presented as means ± SEM. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: *p < 0.05, **p < 0.01, ***p < 0.001. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures. Exact p-values: b, valine fasted p < 0.000001, leucine fasted p < 0.000001, isoleucine fasted p < 0.000001, isoleucine fed p = 0.029124. c, fasted p = 0.013891. f, valine basal p = 0.030476, isoleucine basal p = 0.046680. g, glutamate p = 0.004926. h, malate p = 0.034456. I, succinate p = 0.039056.

Extended Data Fig. 4 Increased SM BCAA oxidation is specific in a fed state and does not affect body weight or glucose tolerance on HFD.

a, Ratio of 3-HIB/valine, and b, Concentration of 3-HIB in mice fasted for 5-hr or re-fed for 2-hr after an overnight fast; n = 10 Bckdkl/l (WT) and n = 8 Bckdkl/l-HSA-CreER (Cre). c, 1.5 g/kg IP-GTT in 5-hr fasted mice; n = 11 WT and n = 14 Cre. d, Area-under-the-curve (AUC) for GTT in c. e, Insulin concentration at t = 0 and t = 20 min (n = 11 WT and n = 14 Cre) during GTT from c. f, Weights; n = 10 WT and n = 8 Cre. g, Lean mass; n = 7 WT and n = 9 Cre. h, Weights; n = 11 WT and n = 14 Cre. i-k, Quantification of BCKDK in re-fed tissues normalized to loading control. l-n Quantification of the ratio pBCKDH/BCKDH in re-fed tissues. o, Western blotting for p-ACLY&ACLY in re-fed livers. 14-3-3 is the loading control. p, Quantification of the ratio p-ACLY/ACLY. n = 6 WT and n = 6 Cre for i-p. q, Labeling fraction of BCAAs in plasma before (t = 0) and 10 min after gavage (t = 10) of U13C-BCAAs in mice re-fed for 2-hr, and labeling fraction of TCA cycle intermediates 10 min after gavage of U13C-BCAAs in re-fed r, liver, s, iBAT, and t, gWAT; n = 8 WT and n = 8 Cre. u, BCKDH activity in re-fed quads; n = 4 WT and n = 3 Cre. v, Plasma BCKAs in Bckdkl/l-HSA-CreER mice treated with 100 mg/kg of vehicle (Veh; n = 6) or BT2 (n = 7) the night before and morning of collection. w, Weights of Bckdkl/l-HSA-CreER mice treated with Veh (n = 6) or BT2 (n = 7). Mice in a, b, & f were male, aged 14–24 weeks and fed HFD for 4–5 weeks. Mice in c-e, & h were male, aged 20–26 weeks and fed HFD for 10 weeks. Mice in g & l-u were male, aged 16–17 weeks and fed HFD for 4 weeks. Mice in v & w were male, aged 12–20 and fed HFD for 4 weeks. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: *p < 0.05, **p < 0.01, ***p < 0.001. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures. Exact p-values: a, fasted p = 0.024668. j, p = 0.000013. m, p = 0.000017. v, KIC/KMV p = 0.008925.

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Extended Data Fig. 5 SM DBT knockout is specific and does not affect body weight, muscle physiology, or glucose tolerance on chow diet.

a-d, Gene expression data via qPCR of Dbt, Dbt exon 6 (deleted region), Bckdha, and Bckdhb, respectively; n = 3 Dbtl/l (WT) and n = 5 Dbtl/l-HSA-CreER (Cre). e-g, Western quantification of DBT in fasted tissues normalized to loading control; n = 3 WT and n = 3 Cre. h, H&E staining of tibialis anterior (TA) muscle. Representative images from n = 3 WT and n = 3 Cre. I, Ratio of 3-HIB/valine, and j, Concentration of 3-HIB from mice fasted for 5-hr (n = 10 WT and n = 8 Cre) or re-fed for 2-hr after an overnight fast (n = 11 WT and n = 9 Cre). k, Body weights; n = 10 WT and n = 8 Cre. l, 2 g/kg glucose IP-GTT in 5-hr fasted mice; n = 10 WT and n = 10 Cre. m, Area-under-the-curve (AUC) for the GTT in l. n, Insulin concentration at t = 0 and t = 20 min (n = 10 WT and n = 7 Cre) during GTT from l. Mice used in these experiments were male, aged 10–19 weeks fed normal chow. Data are presented as means ± SEM. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: *p < 0.05 and ***p < 0.001. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures. Exact p-values: a, p = 0.039254. b, p = 0.043912. f, p = 0.000749. g, p = 0.011125.

Extended Data Fig. 6 Decreased SM BCAA oxidation is specific and does not affect body weight or glucose tolerance on HFD.

a, Plasma labeling of BCAAs after U13C-BCAAs gavage (fraction) in 5-hr fasted mice; n = 8 Dbtl/l (WT) and n = 8 Dbtl/l-HSA-CreER (Cre). b, BCKDH complex activity measured in fasted quads from n = 4 WT and n = 4 Cre mice. Labeling fraction of TCA cycle intermediates 10 min after gavage of U13C-BCAAs in c, liver d, iBAT, & e, gWAT in 5-hr fasted mice; n = 8 WT and n = 8 Cre. f, Body weights; n = 10 WT and n = 14 Cre. g, Lean mass; n = 7 WT and n = 13 Cre. Fasting plasma pool size of various amino acids during steady-state infusion of U13C-BCAAs before (basal, 5-hr fasted) and during (clamp, 7-hr fasted) HIEC in n = 6 WT and n = 6 Cre mice. h, Plasma BCAA concentration in 5-hr fasted or 2-hr re-fed after an overnight fast in mice; n = 8 WT and n = 10 Cre. i, Steady-state plasma labeling of BCAAs by infusion of U13C-BCAAs before (basal) and during (clamp) HIEC; n = 6 WT and n = 6 Cre mice fed. j, 1.5 g/kg glucose IP-GTT in 5-hr fasted mice fed HFD for 4 weeks; n = 8 WT and n = 9 Cre. k, Area-under-the-curve (AUC) for the GTT in j. l, Insulin concentration at t = 0 and t = 20 min (n = 8 WT and n = 9 Cre) during the GTT from j. m, Plasma BCAA concentration in 5-hr fasted mice before (t = 0), n = 8 WT and n = 9 Cre, and 20 min after (t = 20), n = 6 WT and n = 7 Cre, a gavage of BCAAs. Mice used in these experiments were male, aged 10–20 weeks fed HFD for 4–8 weeks. Data are presented as means ± SEM. Comparisons of two groups use two-tailed Student’s t-test. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures.

Extended Data Fig. 7 Liver BCKDK knockout does not affect body weight or glucose tolerance at various time points on HFD or after a BCAA challenge.

a-c, Western quantification of BCKDK in tissues normalized to loading control; n = 3 Bckdkl/l-AAV8:GFP (GFP) and n = 3 Bckdkl/l-AAV8:Cre (Cre). d-f, Western quantification of the ratio pBCKDH/BCKDH; n = 3 GFP and n = 3 Cre. g, Body weights of n = 9 GFP and n = 10 Cre mice on HFD. h, Plasma labeling of BCAAs after U13C-BCAAs gavage (fraction) 5-hr fasted mice; n = 4 GFP and n = 5 Cre. Labeling fraction of TCA cycle intermediates 10 min post U13C-BCAA gavage in i, quad j, iBAT, & k, gWAT in 5-hr fasted mice; n = 4 GFP and n = 5 Cre. l, BCKDH complex activity assay measured in fasted livers from n = 4 GFP and n = 3 Cre mice. m, 1.5 g/kg glucose IP-GTT in 5-hr fasted mice fed HFD after a BCAA gavage given at t = 0; n = 8 GFP and n = 10 Cre. n, Area-under-the-curve (AUC) for the GTT in m. o, Plasma BCAA concentration of 5-hr fasted plasma before (t = 0) (n = 8 GFP and n = 9 Cre) and 20 min after (t = 20) (n = 8 GFP and n = 10 Cre) a BCAA gavage from GTT + BCAA gavage in m. Mice used in these experiments were male, aged 8–26 weeks and on HFD for 2–12 weeks. Data are presented as means ± SEM. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: *p < 0.05 and **p < 0.01. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures. Exact p-values: a, p = 0.029151. d, p = 0.005367.

Extended Data Fig. 8 Liver DBT knockout does not affect body weight or glucose tolerance on multiple diets or after a BCAA challenge.

a-c, Western quantification of DBT in tissues normalized to loading control; n = 3 Dbtl/l-AAV8:GFP (GFP) and n = 3 Dbtl/l-AAV8:Cre (Cre). d, Body weights of n = 10 GFP and n = 10 Cre mice on chow and HFD. e, Plasma labeling of BCAAs after U13C-BCAA gavage (fraction); n = 4 GFP and n = 5 Cre. Labeling fraction of TCA cycle intermediates 10 min post U13C-BCAA gavage in f, quad g, iBAT, & h, gWAT in 5-hr fasted mice; n = 4 GFP and n = 5 Cre. i, BCKDH complex activity measured in fasted livers from n = 4 GFP and n = 4 Cre mice. j, 1.5 g/kg glucose IP-GTT in 5-hr fasted mice fed HFD for 4 weeks after a BCAA gavage given at t = 0; n = 8 GFP and n = 9 Cre. k, Area-under-the-curve (AUC) for the GTT in j. l, Insulin concentration at t = 0 and t = 20 min (n = 8 GFP and n = 9 Cre) during GTT from j. m, Plasma BCAA concentration in 5-hr fasted mice before (t = 0) and 20 min after (t = 20) a gavage of BCAAs from GTT + BCAA gavage in j; n = 8 GFP and n = 9 Cre. Mice used in these experiments were male, aged 16–24 weeks and fed normal chow for 4–5 weeks, followed by HFD for 4–6 weeks. Data are presented as means ± SEM. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: *p < 0.05. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures. Exact p-values: b, p = 0.025494. m, valine t = 0 p = 0.031669, leucine t = 0 p = 0.033539.

Extended Data Fig. 9 Double knockout of BCKDK in both SM and liver is specific in fasted and re-fed states.

a–d, Western quantification of BCKDK in fasted tissues normalized to loading control collected from n = 3 WT and n = 3 BCKDK double KO (Double KO) mice. e-h, Western quantification of the ratio pBCKDH/BCKDH in fasted tissues; n = 3 WT and n = 3 Double KO. i, Western blotting for p-ACLY and ACLY in fasted livers. 14-3-3 is the loading control; n = 6 WT and n = 6 Double KO. j, Western quantification of the ratio p-ACLY/ACLY from the blot in i. k, Body weights of n = 10 WT and n = 10 Double KO mice on HFD. l, Lean mass of n = 8 WT and n = 8 Double KO mice on HFD. m, Ratio of 3-HIB/valine, and n, Concentration of 3-HIB in plasma in mice fasted for 5-hr or re-fed for 2-hr after an overnight fast; n = 10 WT and n = 10 Double KO mice fed HFD. o-r, Western quantification of BCKDK in re-fed tissues normalized to loading control; n = 3 WT and n = 3 Double KO. s-v, Western quantification of the ratio pBCKDH/BCKDH in re-fed tissues; n = 3 WT and n = 3 Double KO. w, Western blotting for p-ACLY and ACLY in re-fed livers. 14-3-3 is the loading control; n = 6 WT and n = 6 Double KO. x, Western quantification of the ratio p-ACLY/ACLY from the blot in w. y, Body weights of n = 8 BCKDK double KO + vehicle (Veh)-treated and n = 8 BCKDK double KO + BT2-treated mice fed HFD. Mice used for these experiments were aged 13–23 weeks and fed HFD for 6–7 weeks. Data are presented as means ± SEM. Comparisons of two groups use two-tailed Student’s t-test with significance defined as: *p < 0.05 and ***p < 0.001. Experiments with multiple comparisons at different time-points use two-way ANOVA with repeated measures. Exact p-values: a, p = 0.023348. b, p = 0.047025. e, p = 0.044708. f, p = 0.000955. m, fasted p = 0.018524. o, p = 0.000230. s, p = 0.044860. u, p = 0.031191.

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Blair, M.C., Neinast, M.D., Jang, C. et al. Branched-chain amino acid catabolism in muscle affects systemic BCAA levels but not insulin resistance. Nat Metab 5, 589–606 (2023). https://doi.org/10.1038/s42255-023-00794-y

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