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High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy

Nature Metabolism volume 2, pages 110–125 (2020)Cite this article

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Abstract

High-protein diets are commonly utilized for weight loss, yet they have been reported to raise cardiovascular risk. The mechanisms underlying this risk are unknown. Here, we show that dietary protein drives atherosclerosis and lesion complexity. Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mammalian target of rapamycin (mTOR) signalling. This is causal in plaque progression, because the effects of dietary protein are abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis induced by atherogenic lipids, a process that involves mammalian target of rapamycin complex 1 (mTORC1)-dependent inhibition of mitochondrial autophagy (mitophagy), accumulation of dysfunctional mitochondria and mitochondrial apoptosis. Using macrophage-specific mTORC1- and autophagy-deficient mice, we confirm this amino acid–mTORC1–autophagy signalling axis in vivo. Our data provide insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies is important to define the vascular effects of protein-based weight loss regimens.

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Fig. 1: High-protein diets increase atherosclerotic plaque formation and plaque complexity.
Fig. 2: High-protein diets elevate amino acids levels in vivo and activate mTOR signalling in plaque macrophages.
Fig. 3: High-protein diets accelerate atherogenesis through macrophage mTORC1 signalling.
Fig. 4: Leucine-mediated activation of mTORC1 synergizes with atherogenic lipids to induce mitochondria-dependent apoptosis of macrophages.
Fig. 5: Leucine-mediated activation of mTORC1 leads to accumulation of dysfunctional mitochondria and ROS production in macrophages.
Fig. 6: Leucine-mediated activation of mTORC1 inhibits mitophagy in macrophages.
Fig. 7: Autophagy deficiency reverses the atheroprotective effect of Raptor silencing in macrophages.
Fig. 8: Leucine activates mTORC1 signalling and regulates downstream targets in a dosage-dependent manner.
Fig. 9: Graphical summary of the progression of events from ingestion of a protein meal to the deleterious effects on atherosclerotic plaque complexity.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data for Figs. 4 and 8 and Extended Data Figs. 3, 4 and 6 are provided with the paper.

Change history

  • 09 September 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

This work was supported by National Institutes of Health grant no. R01 HL125838, VA MERIT I01 BX003415, American Diabetes Association grant no. 1-18-IBS-029, Washington University Diabetic Cardiovascular Disease Center and Diabetes Research Center grant no. P30 DK020579, Washington University Mass Spectrometry core grant nos. P41GM103422 and P30DK056341, a grant from the Longer Life Foundation and the Foundation for Barnes-Jewish Hospital.

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Authors and Affiliations

  1. Department of Medicine, Cardiovascular Division, Washington University School of Medicine, St Louis, MO, USA

    Xiangyu Zhang, Ismail Sergin, Trent D. Evans, Se-Jin Jeong, Astrid Rodriguez-Velez, Divya Kapoor, Sunny Chen, Eric Song, Karyn B. Holloway, Abhinav Diwan, Nathan O. Stitziel, Joel D. Schilling & Babak Razani

  2. John Cochran VA Medical Center, St Louis, MO, USA

    Xiangyu Zhang, Se-Jin Jeong, Divya Kapoor, Karyn B. Holloway & Abhinav Diwan

  3. Department of Medicine, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, St Louis, MO, USA

    Jan R. Crowley & Irfan J. Lodhi

  4. University Health Network, Peter Munk Cardiac Center, University of Toronto, Toronto, Ontario, Canada

    Slava Epelman

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

    Conrad C. Weihl

  6. Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC, USA

    Daping Fan

  7. Department of Nutrition, Washington University School of Medicine, St Louis, MO, USA

    Bettina Mittendorfer & Babak Razani

  8. Department of Pathology & Immunology, Washington University School of Medicine, St Louis, MO, USA

    Joel D. Schilling & Babak Razani

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Contributions

X.Z. and B.R. designed the studies and wrote the manuscript. X.Z., I.S., T.D.E., S.J., A.R., D.K., S.C., E.S., K.B.H. and J.R.C. performed and analysed the experiments. X.Z. and I.S. prepared the figures. D.K., S.E., C.C.W., A.D., D.F., B.M., N.O.S., J.D.S., I.J.L. and B.R. provided the reagents, advised on the experimental design and performed critical reading of the manuscript.

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Correspondence to Babak Razani.

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The authors declare no competing interests.

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Peer review information Primary Handling Editor: Pooja Jha.

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Extended data

Extended Data Fig. 1 High Protein diets increase atherosclerotic plaque formation and plaque complexity without altering serum metabolites.

(a) Average daily food intake over 1 week for ApoE-KO mice fed a standard Western diet (n = 5) or high protein Western diet (n = 5) (b–d) Cohorts of ApoE-KO mice were placed on a standard Western diet (Std. WD) or high protein Western diet (HP WD) and after 8 weeks, (b) Body composition (fat and lean weights) (Std. WD: n = 4; HP WD: n = 5), (c) glucose tolerance test (GTT) and glucose AUC (Std. WD: n = 7; HP WD: n = 9), and (d) serum cholesterol, glucose, triglycerides, and free fatty acids (Std. WD: n = 11; HP WD: n = 14) were measured. (e) Quantification of atherosclerotic plaque burden using Oil Red O-stained aortic root sections from mice fed standard or high protein Western diets for 16 weeks; representative roots shown on right (Std. WD: n = 12; HP WD: n = 11). (f) Measurements of serum cholesterol in cohorts of ApoE-KO mice after 16 weeks of standard or high protein Western diets (Std WD: n = 6; HP WD: n = 8). (g-i) Plaque composition quantified by immunofluorescence staining of aortic root sections for (g) macrophage (MOMA-2+) (Std. WD: n = 12; HP WD: n = 13), (h) apoptosis (TUNEL+) (Std. WD: n = 13; HP WD: n = 13), (i) and necrotic core (acellular) (Std. WD: n = 13; HP WD: n = 13). For all graphs, data are presented as mean ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired t-test.

Extended Data Fig. 2 Levels of select L-amino acids in serum, aorta, and splenic macrophages after high protein challenge.

(a) Serum levels of 8 L-amino acids by mass spectrometry from mice fed standard or high protein Western diets for 8 weeks (Std. WD: n = 8; HP WD: n = 8). (b–d) Time course measurement of the levels of 8 L-amino acids in serum (n = 3) (b), splenic macrophages (n = 2) (c), and atherosclerotic aortas (n = 2) (d) by mass spectrometry after high protein gavage. For all graphs, data are presented as mean ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired t-test for a and one-way ANOVA with Dunnett’s test for b–d.

Extended Data Fig. 3 Leucine is amongst the best amino acid inducers of mTOR signaling in cultured primary macrophages.

(a, b) Immunoblot analysis of thioglycollate-elicited peritoneal macrophages (PMACs) (a) and bone marrow derived macrophages (BMDM) (b) after 30min of incubation with amino acid-free or regular/amino acid-sufficient medium to assess mTORC1 signaling using phospho- and total S6K and S6 levels as readouts. (c) Representative immunofluorescence images of BMDMs after 15min of incubation with amino acid-free or regular/amino acid-sufficient medium to assess co-localization between mTOR and Lamp2. (d, e) Immunoblot analysis of macrophages after 30min of incubation with regular medium or amino acid-free medium with and without 20 different L-amino acids to assess mTORC1 signaling using phospho- and total S6 levels as readouts. Representative blots (d) and quantification of pS6/total S6 ratio for five independent experiments (e). The best three mTOR inducers and three non-inducers are listed at right. (f, g) Immunofluorescence imaging of macrophages after 15min of incubation with regular medium or amino acid-free medium with and without the best three mTOR inducers and non-inducers to assess co-localization between mTOR and Lamp2. Representative images (f) and quantification of the mTOR/Lamp2 co-localization (+aa: n = 42; -aa: n = 74; Leu: n = 26; Arg: n = 38; Glu: n = 12; Gln: n = 34; Phe: n = 37; Thr: n = 43 cells) (g). For all graphs, data are presented as mean ±SEM.

Source data

Extended Data Fig. 4 Body weight and common serum metabolites of control and mϕRaptor KO mice fed a standard or LCHP Western diet.

(a) Immunoblot analysis of control and Raptor KO macrophages after 30min of incubation with regular medium or amino acid-free medium with and without leucine to assess mTORC1 activity using phospho- and total S6K and S6 as readouts. (b) Immunofluorescence imaging of macrophages after 15min of incubation with regular medium or amino acid-free medium with and without leucine to assess co-localization between mTOR and Lamp2. Representative images (left) and quantification of the mTOR/Lamp2 co-localization (right) (Control: +aa: n = 42; -aa: n = 52; Leu: n = 44; ATG5 KO: +aa: n = 27; -aa: n = 40; Leu: n = 43 cells). (c) Total body weights of control (n = 22) and mϕRaptor-KO mice (n = 18) (all on ApoE-KO background) fed a standard Western diet for 8 weeks. (d) Measurements of serum cholesterol, glucose, triglycerides, and free fatty acids in control and mϕRaptor-KO mice after 8 weeks of Western diet feeding (n = 15–25 per group). (e) Measurements of serum cholesterol, glucose, triglycerides, and free fatty acids in cohorts of mϕRaptor-KO mice (on ApoE-null background) fed a standard Western diet or high protein Western diet for 8 weeks (n = 15 per group). For all graphs, data are presented as mean ±SEM. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey’s test for b, two-tailed unpaired t-test for c.

Source data

Extended Data Fig. 5 Leucine-mediated activation of mTORC1 synergizes with atherogenic lipids to induce mitochondrial-dependent apoptosis in macrophages.

(a, b) Control and Raptor-KO macrophages were treated with vehicle, 500µg/ml cholesterol crystals (CC) with or without leucine and apoptosis assessed by Caspase-3/7 immunofluorescence staining. Shown are (a) representative images and (b) quantification of >103 cells from acquired images (Control: -aa: n = 11; Leu: n = 9; cc: n = 11; cc+Leu: n = 11; Raptor-KO: n = 11 image fields). For all graphs, data are presented as mean ±SEM. *P < 0.05, ***P < 0.001, one-way ANOVA with Tukey’s test.

Extended Data Fig. 6 Leucine-mediated activation of mTORC1 inhibits autophagy in macrophages.

(a) Immunoblot analysis of phospho- and total ULK1 levels in macrophages incubated with amino acid-free medium with and without leucine for the indicated times. (b, c) The autophagy marker LC3 was evaluated in macrophages incubated with regular medium or amino acid-free medium with and without leucine for 30 minutes using (b) immunoblot analysis or (c) quantification of LC3 intensity (+aa: n = 52; -aa: n = 52; Leu: n = 52 cells) and number of puncta (+aa: n = 97; -aa: n = 87; Leu: n = 97 cells) by immunofluorescence microscopy. (d) Phospho- and total ULK1 levels were determined in Control and Raptor KO macrophages incubated with regular medium or amino acid-free medium with and without leucine for 30 minutes. (e) LC3 levels in control and Raptor KO macrophages incubated with vehicle or 100nM Bafilomycin for 2 hours. (f) LC3 intensity (Control: +aa: n = 39; -aa: n = 27; Leu: n = 30; Raptor-KO: +aa: n = 31; -aa: n = 30; Leu: n = 30 cells) and number of puncta (n = 52 cells/group) were analyzed by immunofluorescence microscopy in control and Raptor KO macrophages incubated with regular medium or amino acid-free medium with and without leucine for 30 minutes. (g) Quantification of LC3 intensity by immunofluorescence microscopy in control and ATG5-KO macrophages incubated with regular medium or amino acid-free medium with and without leucine for 30 minutes (Control: +aa: n = 52; -aa: n = 50; Leu: n = 51; ATG5 KO: +aa: n = 36; -aa: n = 35; Leu: n = 47 cells). (h) ATG5-KO macrophages were co-incubated with vehicle or CC ± leucine and percent of caspase 3/7-positive cells were quantified in >103 cells from acquired images (-aa: n = 13; -aa+cc: n = 11; Leu: n = 10; Leu+ cc.: n = 12 image fields). For all graphs, data presented as mean ±SEM. *P < 0.05, one-way ANOVA with Tukey’s test.

Source Data

Extended Data Fig. 7 Common serum metabolites of mϕRaptor-KO and dual mϕRaptor/mϕATG5-KO (DKO) mice fed a standard Western diet.

Measurements of serum cholesterol, glucose, triglycerides, and free fatty acids in cohorts of mϕRaptor-KO (n = 18) and mϕRaptor/ mϕATG5-KO (DKO) mice (n = 14) (all on ApoE-null background) fed a standard Western diet for 8 weeks.

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Source Data Extended Data Fig. 4

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Zhang, X., Sergin, I., Evans, T.D. et al. High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy. Nat Metab 2, 110–125 (2020). https://doi.org/10.1038/s42255-019-0162-4

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