HK3 Regulation and Protection Figure 4. Overexpression of HKIII protects against oxidant induced cell death. HEK cells overexpressing HKIII or GFP were treated with 500 mM of H2O2, and assayed for cell death using propidium iodide (PI) staining as an indicator of compromised membrane integrity and Annexin V as a marker of apoptosis. (A) Representative flow output demonstrating a reduction in the number of PI positive cells after treatment with H2O2 (500 mM, 6 h) in samples overexpressing HKIII v GFP. (B) Cell viability was significantly increased in cells overexpressing HKIII compared to GFP after exposure to 500 mM H2O2 at the 6 and 12 h time points, as determined by propidium iodide (PI) exclusion. (C) Representative flow output demonstrating a reduction in the number of cells positive for the apoptotic marker Annexin V after treatment with H2O2 (500 mM, 6 h) in samples overexpressing HKIII v GFP. (D) HKIII overexpression resulted in decrease in apoptotic cell death after exposure to 500 mM H2O2 at the 6 and 12 h time points, as determined by AnnexinV. Cell counts were normalized to counts obtained without H2O2 treatment. *P,0.05 vs GFP. Data are presented as mean6SEM. N = 3–6. doi:10.1371/journal.pone.0013823.g004 HKIII overexpression leads to increased mitochondrial membrane potential and mitochondrial biogenesis The increased energy reserve and reduced ROS levels observed in stressed cells overexpressing HKIII suggest that HKIII may have an impact on the mitochondria. Previous studies have shown that HKI and II attenuated the loss of mitochondrial membrane potential in cells exposed to oxidative stress [27]. We next studied the effects of HKIII overexpression on membrane potential using TMRE as previously described [27]. Overexpression of HKIII did not impact overall TMRE fluorescence at baseline; however TMRE fluorescence was significantly higher in cells overexpressing HKIII-GFP after exposure to oxidative stress compared to GFP controls (Figures 6A-E, S4A and B). Studies have also shown that elevated glucose metabolism is associated with increased mitochondrial biogenesis [36,37], so we compared the expression of genes known to play a role in mitochondrial biogenesis and the overall mitochondrial DNA content in HEK293 cells transfected with HKIII-GFP or GFP. Overexpression of HKIII increased mRNA levels of PGC-1a and the nuclear regulatory factors (NRF) 1 and 2a (Figure 7A), which regulate the expression of nuclear genes involved in mitochondrial biogenesis. HKIII overexpression also induced expression of several genes involved in the TCA cycle, fatty acid transport, and b-oxidation (Figure 7B). Total mitochondrial DNA content was also higher in cells that overexpressed HKIII-GFP than in GFP-overexpressing cells (Figure 7C). Thus, the overexpression of HKIII appears to increase mitochondrial biogenesis. This process could also contribute to the cytoprotective effects of HKIII. PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e13823