Fig 2: PC was upregulated in macrophages of humans and mice with atherosclerosis. A) Heatmap of differential genes from the publicly available RNA sequencing data (GSE203250) of RAW 264.7 macrophages treated with VLDL‐sized emulsion particles or vehicle (n = 3). B) PC mRNA levels in HMDMs and BMDMs treated with oxidized low‐density lipoproteins (oxLDL, 80 µg mL−1) for 24 h (n = 6). C,D) Cells were treated with oxLDL (80 µg mL−1) for 24 h (n = 5). Representative immunofluorescence images of PC and CD68 in C) HMDMs and D) BMDMs. Scale bars: 40 µm. E) PC mRNA levels in the PBMCs of healthy participants (non‐CAD) (n = 27; male vs female: 15:12) and patients with CAD (CAD) (n = 53; male vs female: 35:18). F) Representative immunofluorescence images of PC and CD68 in the plaques of aortic sinuses from ApoE−/− male mice fed with chow or HFD (n = 5). The fluorescence intensity of PC was quantified. Scale bars: 100 µm. Data are presented as means ± SD. B–D,F) Unpaired two‐tailed t‐test was used. E) Two‐way analysis of variance with Tukey's correction was used. VLDL, very low‐density lipoprotein; HMDM, human monocyte‐derived macrophage; BMDM, bone marrow‐derived macrophage; CAD, coronary artery diseases; PBMC, peripheral blood mononuclear cell; HFD, high‐fat diet; PC, pyruvate carboxylase.

Fig 3: PC upregulated HIF‐1α by preventing its proteasome degradation in macrophages. A,B) BMDMs were isolated from male PCMKO mice and treated with cycloheximide (CHX, 50 µg mL−1), along with MG132 (10 µmol L−1) or chloroquine (CQ, 20 µmol L−1) at different timepoints (n = 6). Representative western blotting A) and quantification B) of HIF‐1α protein in BMDMs with different treatments. C,D) Mouse primary BMDMs were transfected with HA‐HIF‐1α plasmids and Ad‐LacZ or Ad‐PC, followed by CHX treatment at the indicated time (n = 5). HIF‐1α expression C) and quantification D) in BMDMs at different timepoints are presented. E,F) BMDMs were isolated from male PCfl/fl and PCMKO mice and treated with DMSO or MG132 (10 µmol L−1) (n = 6). Representative western blotting E) and quantification F) of HIF‐1α protein in BMDMs with different treatments. G,H) Mouse primary BMDMs were treated with MG132 (10 µmol L−1) and transfected with Ad‐LacZ or Ad‐PC, followed by LPS (100 ng mL−1) stimulation for 12 h (n = 6). Lysates were immunoprecipitated using the HIF‐1α antibody and immunoblotted with antibodies against the indicated proteins. Representative western blotting G) and quantification H) of indicated proteins in BMDMs with different treatments. I,J) BMDMs were isolated from PCfl/fl and PCMKO mice fed with HFD (n = 8). Selected proteins expression I) and quantification J) in BMDMs are presented. K,L) BMDMs were isolated from male PCfl/fl and PCMKO mice and treated with lactate (5 mm) for 12 h (n = 6). Representative western blotting K) and quantification L) of HIF‐1α protein in BMDMs with different treatments. M,N) BMDMs from male ApoE−/‐ mice fed with a chow diet or HFD were collected and transfected with Ad‐LacZ or Ad‐PC (n = 6). HIF‐1α expression M) and quantification N) in BMDMs are presented. O,P) BMDMs from male PCfl/fl and PCMKO mice administrated with AAV‐PCSK9DY and fed with a chow diet or HFD were collected (n = 8). HIF‐1α expression O) and quantification P) in BMDMs are presented. Data are presented as means ± SD. B) One‐way analysis of variance (ANOVA) with Tukey's correction was used. D) Two‐way ANOVA with Bonferroni's multiple comparisons test was used. F,H,L,N,P) Two‐way ANOVA with Tukey's correction was used. J) Unpaired two‐tailed t‐test was used. DMSO, dimethyl sulfoxide; CHX, cycloheximide; CQ chloroquine; HFD, high‐fat diet; BMDM, bone marrow‐derived macrophage; HIF‐1, hypoxia‐inducible factor 1; PC, pyruvate carboxylase.

Fig 4: Isoginkgetin attenuates atherosclerosis in male and female Apoe-/- mice. (A) Scheme of study design. Male and female Apoe-/- mice were induced by high cholesterol diet for 7 weeks. Animals then received ISOGK treatment (20 mg/kg/day) for another 8 weeks. (B) The body weights were shown after ISOGK treatment for 8 weeks (n = 10 or 11). Two-tailed Student's t test. (C) Serum ALT, AST levels of Apoe-/- mice treated with or without ISOGK (n = 10 or 11). Two-tailed Student's t test. (D) Representative images of Oil Red O staining of en face aortas from male and female Apoe-/- mice fed high cholesterol diet. (E) Quantification of en face plaque areas as a percentage of the area in D (n = 10 or 11). Two-tailed Student's t test. (F) The representative images of Oil Red O (upper) staining and H&E (lower) staining of the aortic sinus from male and female Apoe-/- mice fed high cholesterol diet. Arrow indicates necrotic core area. (G) Quantitative analysis of F (n = 6 or 7). Two-tailed Student's t test. The data are means ± SEM, n.s., not significant, **P < 0.01, ***P < 0.001.

Fig 5: Characteristics of ApoE−/− mice after CL316,243 treatment.(A) Body weight (n = 10). (B) Blood glucose levels (n = 9). (C) Serum triglycerides (n = 10). (D) Serum cholesterol (n = 10). Student’s t test was used to determine statistical difference.