PMC

Knockdown of PGC-1α augmented high glucose-induced cytochrome c release and caspases activation. HUVECs were treated with Ad-shPGC-1α for 24 h in prior incubation with high glucose for another 48 h. (A) Western blot analysis of cytochrome c (Cyto-c) protein expression in the mitochondrial and cytosol. (B and C) Densitometric analysis of cytochrome c expression in the mitochondrial (B) and cytosol (C). (D) Densitometric analysis of cytochrome c release from mitochondria to cytoplasm. (E and F) Protein expression of caspase-3 (E) and -9 (F) were measured by western blot. **P < 0.01 vs. control, ##P < 0.01 vs. high glucose, n = 6.

High glucose-induced cell injury was associated with decreased PGC-1α expression. A. HUVECs were incubated with different concentrations of high glucose (HG) for 48 h. Cell viability was analyzed by cell counting kit-8 (CCK-8). B. The cells were treated with high glucose (15 mmol/L) for the indicated times. CCK-8 analysis was performed. C. Western blot showed that glucose inhibited PGC-1α expression dose-dependently. D. After treatment mentioned in B, PGC-1α protein level was determined by western blot. All data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. control, n = 6.

PGC-1α deficiency exacerbated high glucose-induced mitochondrial membrane dysfunction. HUVECs were infected with Ad-Scr or Ad-shp PGC-1α for 24 h, and then were cultured in high glucose medium for another 48 h. A. After treatment, cells were loaded with calcein-AM with Co²⁺, representative photographs of inner mitochondrial membrane permeabilization was captured by confocal microscopy. B. Quantitative measurements of calcein fluorescence intensity assessed by microplate reader. C. Loss of mitochondrial membrane potential was measured using JC-1 staining by confocal microscopy. Representative images of JC-1 derived fluorescence in HUVECs. D. Quantitative analysis of the ratio of red/green fluorescence. **P < 0.01 vs. control, ##P < 0.01 vs. high glucose alone, n = 6.

PGC-1α deficiency exaggerated ECs apoptosis through activation of VDAC1. HUVECs were pre-treated with Ad-Scr or Ad-shPGC-1α for 24 h following incubation with high glucose in the absence or presence of DIDS. A. Bcl-2 and Bax protein levels were examined by western blot. B. Representative images for cells loaded with H₂DCF-DA (10 μmol/L) was captured with a fluorescence microscopy. C. Quantitative analysis of DCF fluorescence intensity assessed by microplate reader. D. Western blot showed that Ad-shPGC-1α inhibited high glucose-induced Bip/GRP78 expression. E. VDAC isoforms (VDAC1, VDAC2 and VDAC3) expression levels were determined by western blot using β-actin as an internal control. F. cell apoptosis was determined by Annexin V/PI staining. G. Quantitative analysis of the percentage of apoptotic cells. **P < 0.01 vs. control, ##P < 0.01 vs. high glucose, &&P < 0.01 vs. high glucose+Ad-shPGC-1α, n = 6.

Lack of PGC-1α promoted high glucose-induced cell injury and apoptosis in HUVECs. (A and B) Western blot (A) and qPCR (B) showed that infection with adenovirus containing PGC-1α small hairpin RNA (Ad-PGC-1α) at multiplicity of infection (MOI) of 10, 20 and 40 for 24 h significantly decreased PGC-1α expression, but transfection with negative shRNA (Ad-Scr) did not change endogenous PGC-1α expression. (C) HUVECs were infected with Ad-Scr or Ad-shPGC-1α in the absence or presence of high glucose. Cell viability was determined by CCK-8. (D) After treatment as described in (C), HUVECs apoptosis was determined by Annexin V/PI staining followed by flow cytometry. (E). Quantitative analysis of the percentage of apoptotic cells. *P < 0.05, **P < 0.01 vs. control, ##P < 0.01 vs. high glucose alone, n = 6.