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Ketone body β -hydroxybutyrate is an autophagy-dependent vasodilator

McCarthy, Cameron G., Chakraborty, Saroj, Singh, Gagandeep, Yeoh, Beng San, Schreckenberger, Zachary J., Singh, Avinash, Mell, Blair, Bearss, Nicole R., Yang, Tao, Cheng, Xi, Vijay-Kumar, Matam, Wenceslau, Camilla F., Joe, Bina·2021·JCI Insight
DOI 10.1172/jci.insight.149037PMC PMC8564907
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Abstract

Autophagy has long been associated with longevity, and it is well established that autophagy reverts and prevents vascular deterioration associated with aging and cardiovascular diseases. Currently, our understanding of how autophagy benefits the vasculature is centered on the premise that reduced autophagy leads to the accumulation of cellular debris, resulting in inflammation and oxidative stress, which are then reversed by reconstitution or upregulation of autophagic activity. Evolutionarily, autophagy also functions to mobilize endogenous nutrients in response to starvation. Therefore, we hypothesized that the biosynthesis of the most physiologically abundant ketone body, β-hydroxybutyrate (βHB), would be autophagy dependent and exert vasodilatory effects via its canonical receptor, Gpr109a. To the best of our knowledge, we have revealed for the first time that the biosynthesis of βHB can be impaired by preventing autophagy. Subsequently, βHB caused potent vasodilation via potassium channels but not Gpr109a. Finally, we observed that chronic consumption of a high-salt diet negatively regulates both βHB biosynthesis and hepatic autophagy and that reconstitution of βHB bioavailability prevents high-salt diet–induced endothelial dysfunction. In summary, this work offers an alternative mechanism to the antiinflammatory and antioxidative stress hypothesis of autophagy-dependent vasculoprotection. Furthermore, it reveals a direct mechanism by which ketogenic interventions (e.g., intermittent fasting) improve vascular health.

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Figure 1 β-Hydroxybutyrate biosynthesis is autophagy dependent. β-Hydroxybutyrate (βHB) was measured in serum samples from nonfasted (free access to food) and fasted (24 hours) Dahl S (A) and Dahl R (B) rats (both low-salt diet fed), pretreated with and without lysosome acidification inhibitor chloroquine (2 × 50 mg/kg) or AMPKα inhibitor dorsomorphin (25 mg/kg). Mean ± SEM. n = 5–10. Two-way ANOVA: *P < 0.05, ****P < 0.0001.
Figure 1 β-Hydroxybutyrate biosynthesis is autophagy dependent. β-Hydroxybutyrate (βHB) was measured in serum samples from nonfasted (free access to food) and fasted (24 hours) Dahl S (A) and Dahl R (B) rats (both low-salt diet fed), pretreated with and without lysosome acidification inhibitor chloroquine (2 × 50 mg/kg) or AMPKα inhibitor dorsomorphin (25 mg/kg). Mean ± SEM. n = 5–10. Two-way ANOVA: *P < 0.05, ****P < 0.0001.CC BY · PubMed Central
Figure 2 β-Hydroxybutyrate causes vasodilation via potassium channels and not nitric oxide biosynthesis or cyclooxygenase-derived products. Concentration-response curves to β-hydroxybutyrate (βHB) in mesenteric resistance arteries from low-salt diet–fed Dahl S and Dahl R rats (A). Some arteries from Dahl S rats were endothelium denuded (E-) (B); incubated with tetraethylammonium (TEA, 10 mmol/L) (C); contracted in response to potassium chloride (KCl, 120 mmol/L), as opposed to phenylephrine (10 μmol/L) (D); incubated with Nω-Nitro-L-arginine methyl ester (L-NAME, 100 μmol/L) (E); or incubated with indomethacin (10 μmol/L) (F). pH responses to βHB in Krebs buffer (G). Concentration-response curves to βHB in arteries from Dahl S rats were also incubated with UCL 1684 (100 nmol/L) and TRAM-34 (10 μmol/L) combined, iberiotoxin (100 nmol/L), barium chloride (100 μmol/L), glybenclamide (1 μmol/L), or ouabain (100 μmol/L) (H). The same control data are presented in multiple panels. n = 4–21. Mean ± SEM. Two-way ANOVA: *P < 0.05 (B–D and F). One-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; t test: *P < 0.05 (H).
Figure 2 β-Hydroxybutyrate causes vasodilation via potassium channels and not nitric oxide biosynthesis or cyclooxygenase-derived products. Concentration-response curves to β-hydroxybutyrate (βHB) in mesenteric resistance arteries from low-salt diet–fed Dahl S and Dahl R rats (A). Some arteries from Dahl S rats were endothelium denuded (E-) (B); incubated with tetraethylammonium (TEA, 10 mmol/L) (C); contracted in response to potassium chloride (KCl, 120 mmol/L), as opposed to phenylephrine (10 μmol/L) (D); incubated with Nω-Nitro-L-arginine methyl ester (L-NAME, 100 μmol/L) (E); or incubated with indomethacin (10 μmol/L) (F). pH responses to βHB in Krebs buffer (G). Concentration-response curves to βHB in arteries from Dahl S rats were also incubated with UCL 1684 (100 nmol/L) and TRAM-34 (10 μmol/L) combined, iberiotoxin (100 nmol/L), barium chloride (100 μmol/L), glybenclamide (1 μmol/L), or ouabain (100 μmol/L) (H). The same control data are presented in multiple panels. n = 4–21. Mean ± SEM. Two-way ANOVA: *P < 0.05 (B–D and F). One-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; t test: *P < 0.05 (H).CC BY · PubMed Central
Figure 3 β-Hydroxybutyrate–induced vasodilation is not mediated via Gpr109a or Gpr41. β-Hydroxybutyrate (βHB) concentration-response curves in mesenteric resistance arteries from WT and Gpr109a–/– (A) and WT and Gpr41–/– (B) mice. Mean ± SEM. n = 3–9.
Figure 3 β-Hydroxybutyrate–induced vasodilation is not mediated via Gpr109a or Gpr41. β-Hydroxybutyrate (βHB) concentration-response curves in mesenteric resistance arteries from WT and Gpr109a–/– (A) and WT and Gpr41–/– (B) mice. Mean ± SEM. n = 3–9.CC BY · PubMed Central
Figure 4 High-salt diet negatively regulates β-hydroxybutyrate synthesis and autophagic activity. β-Hydroxybutyrate (βHB) was measured in sera from nonfasted (free access to food) and fasted (24 hours) Dahl S and Dahl R rats that had consumed a low- or high-salt diet for 8 weeks (A). Protein expression analysis was performed for LC3B-II normalized to LC3B-I in liver biopsies from fasted and nonfasted low-salt diet– and high-salt diet–fed Dahl S and Dahl R rats (B). Representative images of immunoblots and densitometric analysis. Histological analysis was performed for fibrosis (C) and lipid droplets (D) in liver biopsies from nonfasted low-salt diet– and high-salt diet–fed Dahl S and Dahl R rats. Scale bar: 50 μm. Circulating liver enzymes (E and F), total cholesterol (G), triglycerides (H), and glucose (I) were measured in sera from nonfasted Dahl S and Dahl R rats that had consumed a low- or high-salt diet. Mean ± SEM. n = 4–10. Two-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (A and B); t test: *P < 0.05 (E–I).
Figure 4 High-salt diet negatively regulates β-hydroxybutyrate synthesis and autophagic activity. β-Hydroxybutyrate (βHB) was measured in sera from nonfasted (free access to food) and fasted (24 hours) Dahl S and Dahl R rats that had consumed a low- or high-salt diet for 8 weeks (A). Protein expression analysis was performed for LC3B-II normalized to LC3B-I in liver biopsies from fasted and nonfasted low-salt diet– and high-salt diet–fed Dahl S and Dahl R rats (B). Representative images of immunoblots and densitometric analysis. Histological analysis was performed for fibrosis (C) and lipid droplets (D) in liver biopsies from nonfasted low-salt diet– and high-salt diet–fed Dahl S and Dahl R rats. Scale bar: 50 μm. Circulating liver enzymes (E and F), total cholesterol (G), triglycerides (H), and glucose (I) were measured in sera from nonfasted Dahl S and Dahl R rats that had consumed a low- or high-salt diet. Mean ± SEM. n = 4–10. Two-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (A and B); t test: *P < 0.05 (E–I).CC BY · PubMed Central
Figure 5 Reconstitution of β-hydroxybutyrate bioavailability prevents high-salt diet–induced endothelial dysfunction in Dahl S and Dahl R rats. Acetylcholine (ACh) concentration-response curves in mesenteric resistance arteries from low-salt diet–fed (LS-fed) Dahl S rats (A), high-salt diet–fed (HS-fed) Dahl S rats (B), and HS-fed animals Dahl S rats with 1,3-butanediol (1,3-BD; 20% v/v) (C) after incubation with Nω-Nitro-L-arginine methyl ester (L-NAME, 100 μmol/L). ACh concentration-response curves in L-NAME–incubated arteries from HS-fed Dahl S rats that were either coincubated with tetraethylammonium (TEA, 10 mmol/L) (D) or contracted in response to potassium chloride (KCl, 120 mmol/L), as opposed to phenylephrine (10 μmol/L) (E). ACh concentration-response curves in L-NAME–incubated arteries from HS-fed Dahl S rats that were also coincubated with UCL 1684 (100 nmol/L) and TRAM-34 (10 μmol/L) combined, iberiotoxin (100 nmol/L), barium chloride (100 μmol/L), or glybenclamide (1 μmol/L) (F). ACh concentration-response curves in arteries from LS-fed Dahl R rats (G), HS-fed Dahl R rats (H), and HS-fed Dahl R rats treated with 1,3-BD (I) after incubation with indomethacin (10 μmol/L). Mean ± SEM. n = 4–11. Two-way ANOVA: *P < 0.05 (A–E); 1-way ANOVA: **P < 0.01 (F); nonlinear regression (logEC50): *P < 0.05 (H).
Figure 5 Reconstitution of β-hydroxybutyrate bioavailability prevents high-salt diet–induced endothelial dysfunction in Dahl S and Dahl R rats. Acetylcholine (ACh) concentration-response curves in mesenteric resistance arteries from low-salt diet–fed (LS-fed) Dahl S rats (A), high-salt diet–fed (HS-fed) Dahl S rats (B), and HS-fed animals Dahl S rats with 1,3-butanediol (1,3-BD; 20% v/v) (C) after incubation with Nω-Nitro-L-arginine methyl ester (L-NAME, 100 μmol/L). ACh concentration-response curves in L-NAME–incubated arteries from HS-fed Dahl S rats that were either coincubated with tetraethylammonium (TEA, 10 mmol/L) (D) or contracted in response to potassium chloride (KCl, 120 mmol/L), as opposed to phenylephrine (10 μmol/L) (E). ACh concentration-response curves in L-NAME–incubated arteries from HS-fed Dahl S rats that were also coincubated with UCL 1684 (100 nmol/L) and TRAM-34 (10 μmol/L) combined, iberiotoxin (100 nmol/L), barium chloride (100 μmol/L), or glybenclamide (1 μmol/L) (F). ACh concentration-response curves in arteries from LS-fed Dahl R rats (G), HS-fed Dahl R rats (H), and HS-fed Dahl R rats treated with 1,3-BD (I) after incubation with indomethacin (10 μmol/L). Mean ± SEM. n = 4–11. Two-way ANOVA: *P < 0.05 (A–E); 1-way ANOVA: **P < 0.01 (F); nonlinear regression (logEC50): *P < 0.05 (H).CC BY · PubMed Central

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Metadata

Authors
McCarthy, C. G., Chakraborty, S., Singh, G., Yeoh, B. S., Schreckenberger, Z. J., Singh, A., Mell, B., Bearss, N. R., Yang, T., Cheng, X., Vijay-Kumar, M., Wenceslau, C. F., & Joe, B.
ORCID
Venue
JCI Insight
Publisher
American Society for Clinical Investigation
Published
2021-10-22
Volume
6
Issue
20
ISSN
2379-3708
DOI
10.1172/jci.insight.149037
PMC
PMC8564907
Cited by
74
References
5
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • American Heart Association (18POST34060003)
  • National Institutes of Health (K99HL151889,R01CA219144,R00GM118885,R01HL149762,R01HL143082)

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