Induction of glutathione biosynthesis by glycine-based treatment mitigates atherosclerosis

Abstract

Lower circulating levels of glycine are consistently reported in association with cardiovascular disease (CVD), but the causative role and therapeutic potential of glycine in atherosclerosis, the underlying cause of most CVDs, remain to be established. Here, following the identification of reduced circulating glycine in patients with significant coronary artery disease (sCAD), we investigated a causative role of glycine in atherosclerosis by modulating glycine availability in atheroprone mice. We further evaluated the atheroprotective potential of DT-109, a recently identified glycine-based compound with dual lipid/glucose-lowering properties. Glycine deficiency enhanced, while glycine supplementation attenuated, atherosclerosis development in apolipoprotein E-deficient (Apoe^-/-) mice. DT-109 treatment showed the most significant atheroprotective effects and lowered atherosclerosis in the whole aortic tree and aortic sinus concomitant with reduced superoxide. In Apoe^-/- mice with established atherosclerosis, DT-109 treatment significantly reduced atherosclerosis and aortic superoxide independent of lipid-lowering effects. Targeted metabolomics and kinetics studies revealed that DT-109 induces glutathione formation in mononuclear cells. In bone marrow-derived macrophages (BMDMs), glycine and DT-109 attenuated superoxide formation induced by glycine deficiency. This was abolished in BMDMs from glutamate-cysteine ligase modifier subunit-deficient (Gclm^-/-) mice in which glutathione biosynthesis is impaired. Metabolic flux and carbon tracing experiments revealed that glycine deficiency inhibits glutathione formation in BMDMs while glycine-based treatment induces de novo glutathione biosynthesis. Through a combination of studies in patients with CAD, in vivo studies using atherosclerotic mice and in vitro studies using macrophages, we demonstrated a causative role of glycine in atherosclerosis and identified glycine-based treatment as an approach to mitigate atherosclerosis through antioxidant effects mediated by induction of glutathione biosynthesis.

Keywords: Amino acids; Atherosclerosis; Glutathione; Glycine; Macrophages.

Graphical abstract

Fig. 1

Circulating glycine is decreased in patients with significant coronary artery disease and glycine deprivation enhances atherosclerosis in Apoe^−/−mice. (A) Patients with acute chest pain suspected to be of cardiac origin underwent coronary computed tomography angiography (CCTA) to assess atherosclerosis in the coronary arteries (n = 95). Twenty-four patients were found to have significant coronary artery disease (sCAD) and 71 patients were found to have no sCAD. Representative CCTA with curved multiplanner reconstruction and 3D volume rendering from patients with and without sCAD are shown. Significant coronary artery stenosis is indicated by yellow arrows. Serum glycine concentrations were analyzed by LC-MS/MS (B) in all patients, and (C) in 24 age-, sex-, and blood pressure-matched patients with or without sCAD. (D–E)Apoe^−/− mice were fed an amino acid-defined Western diet with glycine (WD_AA + Gly) or without glycine (WD_AA -Gly) for 10 weeks. Atherosclerotic plaque area in the aortic sinus was analyzed (n = 6–7, scale bar: 200 μm). Data are presented as violin plots or as mean ± SEM showing all points. Statistical differences were compared using unpaired t-test or Mann-Whitney U t-test.

Fig. 2

Glycine-based treatment reduces atherosclerotic plaque size and superoxide. (A)Apoe^−/− mice were fed a Western diet (WD) for 12 weeks and orally administered water supplemented with DT-109 (1 mg/g body weight/day) or equivalent levels of leucine (Leu, 0.33 mg/g body weight/day), glycine (Gly, 0.66 mg/g body weight/day) or water control (H₂O). (B–C) Atherosclerotic plaques in the aortic sinus were analyzed for plaque area using H&E (n = 8, scale bar: 200 μm). (D–E) Whole aortas were stained en face with oil red O (ORO) and plaque area was expressed as a percentage of the total aorta area (n = 6–8). (F) Livers were collected at endpoint and analyzed for glutathione metabolic genes. Expression of Gss (glutathione synthetase), Gclm/c (glutamate-cysteine ligase modifier and catalytic subunits, respectively), Gsr (glutathione disulfide reductase), Gpx3/6 (glutathione peroxidase 3 and 6, respectively), and Gstt2 (glutathione S-transferase theta 2) was normalized to Gapdh and expressed as fold change from WD with water (H₂O) treatment (n = 8). (G–H) Aortic sinuses were analyzed for superoxide using dihydroethidieum (DHE, red) with a nuclear counterstain (DAPI, blue). DHE fluorescence for each treatment was expressed as fold change from WD with H₂O treatment (n = 8, scale bar: 200 μm). Data are presented as mean ± SEM showing all points. Statistical differences were compared using one-way ANOVA with Tukey's post hoc analysis or Kruskal-Wallis test followed by Dunn's post-hoc analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Glycine-based intervention reduces atherosclerosis independent of lipid-lowering effects. (A)Apoe^−/- mice were fed a Western diet for 12 weeks to develop atherosclerosis (baseline), then placed onto standard diet (SD) for additional 8 weeks with oral administration of water supplemented with DT-109 (0.5 mg/g body weight/day) or equivalent levels of leucine (Leu, 0.17 mg/g body weight/day), glycine (Gly, 0.33 mg/g body weight/day) or water control (H₂O). (B) Plasma total cholesterol (TC), and (C) plasma triglycerides (TG) at endpoint (n = 8). (D–E) Atherosclerotic plaques in the aortic sinus were analyzed for plaque area using H&E (n = 8, scale bar: 200 μm). (F–G) Whole aortas were stained en face with oil red O (ORO) and plaque area was expressed as a percentage of the total aorta area (n = 8). Data are presented as mean ± SEM showing all points. Statistical differences were compared using one-way ANOVA with Tukey's post hoc analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Glycine-based intervention reduces plaque superoxide and enhances GSH formation in mononuclear cells.Apoe^−/- mice were fed a Western diet for 12 weeks to develop atherosclerosis (baseline), then placed onto standard diet (SD) for additional 8 weeks with oral administration of water supplemented with DT-109 (0.5 mg/g body weight/day) or equivalent levels of leucine (Leu, 0.17 mg/g body weight/day), glycine (Gly, 0.33 mg/g body weight/day) or water control (H₂O). (A) Livers were collected at endpoint and analyzed for glutathione metabolic genes. Expression of Gss (glutathione synthetase), Gclm/c (glutamate-cysteine ligase modifier and catalytic subunits, respectively), Gsr (glutathione disulfide reductase), Gpx6 (glutathione peroxidase 6), and Gstt2/3 (glutathione S-transferase theta 2 and 3, respectively) was normalized to Gapdh and expressed as fold change from SD with water (H₂O) treatment (n = 8). (B–C) Aortic sinuses were analyzed for superoxide using dihydroethidieum (DHE, red) with a nuclear counterstain (DAPI, blue). DHE fluorescence for each treatment was expressed as fold change from WD with H₂O treatment (n = 8, scale bar: 200 μm). (D–F) C57BL/6J mice were orally administered DT-109 (0.5 mg/g body weight). At baseline (0 h), 2, 6, and 24 h, blood was collected and mononuclear cells were isolated. GSH and GSSG in mononuclear cells were analyzed using LC-MS/MS and expressed as concentrations (mmol/L) or GSH:GSSG ratio relative to baseline (n = 5). Data are presented as mean ± SEM. Statistical differences were compared using one-way ANOVA with Tukey's post hoc analysis or Kruskal-Wallis test followed by Dunn's post-hoc analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Glycine-based treatment decreases superoxide generation in macrophages through GSH formation. Bone marrow-derived macrophages (BMDMs) were treated with glycine-depleted metabolic media supplemented with water control, glycine (1 mM) or DT-109 (1 mM) for 18 h. (A) Following treatment, BMDMs were labeled with dihydroethidium (DHE, red) for assessment of superoxide and nuclei were stained using Hoechst (blue) (scale bar: 100 μm). (B) DHE fluorescence was measured as mean fluorescence intensity normalized to the number of nuclei per high powered field and expressed as fold change from water control (n = 3). (C) Schematic diagram illustrating the production of GSH by glutamate-cysteine ligase, with the addition of inhibitors (Gclm KO, glycine depletion) to enhance superoxide production (γ-GC, γ-glutamylcysteine). (D) BMDMs were isolated from Gclm^+/+ and Gclm^−/- mice, and the absence of GCLM was confirmed by Western blot using GAPDH as loading control (n = 3). (E) Following 18 h treatment with glycine-depleted media supplemented with water control, glycine (1 mM) or DT-109 (1 mM), superoxide was analyzed in BMDMs from Gclm^−/- mice using DHE (scale bar: 100 μm). (F) DHE fluorescence was measured as mean fluorescence intensity normalized to the number of nuclei per high powered field and expressed as fold change from water control (n = 4). Data are presented as mean ± SEM showing all points. Statistical differences were compared using one-way ANOVA with Tukey's post hoc analysis or Kruskal-Wallis test followed by Dunn's post-hoc analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Glycine-based treatment induces de novo GSH synthesis in macrophages. (A) Schematic representation of labeling de novo synthesized GSH from ¹³C₅-labeled glutamine in BMDMs. BMDMs were treated with glycine-depleted media for 18 h, then incubated for 5 h with glycine-depleted media supplemented with ¹³C₅-labeled glutamine (1 mM) and water control (cont.), glycine (Gly, 1 mM), or DT-109 (1 mM). Isotopologue distribution of indicated cellular metabolites as determined by LC-MS/MS (n = 5–6): (B) glutamine, (C) glutamate, (D) γ-glutamylcysteine, (E) M+5 isotopologue of γ-glutamylcysteine, (F) GSH, and (G) M+5 isotopologue of GSH. Metabolite peak area was normalized to total measurable ions in the sample. Data are presented as mean ± SEM. Statistical differences were compared using one-way ANOVA with Tukey's post hoc analysis.

Fig. 7

Proposed model of glycine-based treatment for atherosclerosis through antioxidant effects mediated by induction of GSH biosynthesis.