Fig 2: DSHT therapy promotes cholesterol metabolism in serum and enhances the expression of ABCA1, ABCG1, and LDLR in the aorta. Serum (A) ApoA1 and (B) ApoB levels, and (C) their ratio. (D) Relative mRNA expression levels of cholesterol metabolism-related ABCA1, ABCG1, and LDLR genes. Data are presented as mean ± standard error of mean of the samples from mice in the vehicle, AT, DSHT, and DSHT + AT groups (n = 5 for all). * p < 0.05; ** p < 0.01; *** p < 0.001, compared with the vehicle group (Mann–Whitney U test).

Fig 3: Metabolic enhancer and exercise independently attenuate diet-induced pro-atherogenic serum profile. (A–C) Serum content of circulating PCSK9, ApoA1, and ApoB (n = 8–11). (D) Quantitative real-time PCR analysis of hepatic mRNA abundance of indicated genes (n = 5–6). (E) Immunofluorescent staining of LDLR in the livers of mice fed NCD or HFD supplemented with either ME or exercise. Scale bars, 200 µm. All data are shown as mean ± SD. *, denotes p < 0.05 by one-way ANOVA with Tukey multiple comparison testing using Prism 6 (GraphPad).

Fig 4: Inhibition of HIF-1α attenuates hypoxia-mediated GM130 degradation and lipid accumulation in the small intestine of HFD-fed mice.a Oil Red O staining of small intestinal epithelial cells. Eight-week-old mice were fed either a NCD or HFD for 16 weeks. PX-478 (10 mg/kg) was administered via oral gavage three times a week starting at 6 weeks of HFD feeding. Scale bar, 50 μm. b The protein expression of HIF-1α, GM130, PLIN2 and β-actin was measured by immunoblotting. The protein expression was normalized to that of β-actin and was measured via statistical analysis. The data are presented as the mean ± standard deviation (s.d.) (n = 3 independent experiments), *P < 0.05. c Bnip3 mRNA expression was quantified by qRT‒PCR. The data are presented as the mean ± s.d. (n = 5 independent experiments), *P < 0.05 and **P < 0.01. d The protein expression of GM130 and PLN2 was observed by immunofluorescence staining. Scale bar, 50 μm. A statistical histogram of the fluorescence intensity is shown. The data are presented as the mean ± s.d. (n = 5 independent experiments), *P < 0.05. e TEM images of the Golgi apparatus in intestinal epithelial cells. Scale bar, 2 μm (top), 500 nm (bottom). f The ApoA1 concentration in the intestinal jejunum tissue was measured using an ELISA. The data are presented as the mean ± s.d. (n = 5 independent experiments), *P < 0.05. g The ApoB48 concentration in the serum was measured using an ELISA after treatment with 10 μl/g (body weight) lard oil. The data are presented as the means ± s.d. (n = 5 independent experiments), *P < 0.05. h The serum LDL and TG levels were determined using ELISAs. The data are presented as the mean ± s.d. (n = 5 independent experiments), *P < 0.05 and **P < 0.01.

Fig 5: GM130 degradation increases Golgi condensation and lipid accumulation via NEDD4 under hypoxic conditions.a Immunofluorescence staining for GM130 and BODIPY was performed to visualize the Golgi apparatus and lipids, respectively. ApoA1 was used as a marker for lipoproteins (chylomicron/HDL), the ER tracker was used as a marker for the ER and DAPI was used as a marker for the nuclei. Scale bar, 5 μm. Statistical histograms of the BODIPY intensity (b), Mender’s colocalization coefficient of BODIPY-APOA1 (c) and Mender’s colocalization coefficient of the ER tracker APOA1 (d) are shown. The data are presented as the means ± standard deviation (s.d.) (n = 5 independent experiments), *P < 0.05. For a–d, the Fhs74Int cells were transfected with either siNEDD4 or siHIF-1α before treatment with oleic acid (OA) and CoCl2. e The protein expression of HIF-1α, DGAT-1, ACOX1, I-FABP and β-actin was determined by immunoblotting. The protein expression was normalized to that of β-actin and was measured via statistical analysis. The data are presented as the mean ± s.d. (n = 3 independent experiments), *P < 0.05. f Fhs74Int cells were transfected with the indicated plasmids and treated with CoCl2. Immunofluorescence staining for HA and BODIPY was performed to visualize the transfected cells and lipids. Scale bar, 10 μm. The intensity of BODIPY in the transfected cells was statistically analyzed. The data are presented as the mean ± s.d. (n = 5 independent experiments), *P < 0.05. g–i Fhs74int cells were transfected with either siCon or siGolga2 (g) before treatment with OA and CoCl2 (h and i). g Immunofluorescence staining for GALANT2 and GM130 was performed to visualized the Golgi apparatus. A statistical histogram of the cis-Golgi cisternae length is shown. The data are presented as the mean ± s.d. (n = 100), ***P < 0.001. h Immunofluorescence staining for GALNT2 and BODIPY was performed to visualize the Golgi apparatus and lipids, respectively. A statistical histogram of the cis-Golgi cisternae length is shown. The data are presented as the mean ± s.d. (n = 100), ***P < 0.001. i A statistical histogram of the BODIPY intensity. The data are presented as the mean ± s.d. (n = 5 independent experiments), *P < 0.05. j The concentration of free fatty acids was measured via GC‒MS. The data are presented as the mean ± s.d. (n = 5 independent experiments), *P < 0.05 and **P < 0.01. k A statistical histogram of the cis-Golgi cisternae length. The data are presented as the mean ± s.d. (n = 100), ***P < 0.001. l Transfected cells treated with OA. A statistical histogram of the ORO intensity in transfected cells is shown. The data are presented as the mean ± s.d. (n = 5 independent experiments), *P < 0.05. Fhs74int cells were transfected with either siCon or siGolga2 before being transfected with either Myc or Myc_Golga2. Immunofluorescence staining for GALNT2 and BODIPY was performed to visualize the Golgi apparatus and lipids, respectively. Green Fluorescent Protein (GFP) indicates plasmid-transfected cells. Scale bar, 20 μm.