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. 2014 May;21(5):836-44.
doi: 10.1038/cdd.2014.24. Epub 2014 Feb 28.

Low-dose radiation exposure induces a HIF-1-mediated adaptive and protective metabolic response

R Lall  1 , S Ganapathy  2 , M Yang  2 , S Xiao  1 , T Xu  1 , H Su  1 , M Shadfan  1 , J M Asara  3 , C S Ha  1 , I Ben-Sahra  2 , B D Manning  2 , J B Little  2 , Z-M Yuan  2
Affiliations

Low-dose radiation exposure induces a HIF-1-mediated adaptive and protective metabolic response

R Lall et al. Cell Death Differ. 2014 May.

Abstract

Because of insufficient understanding of the molecular effects of low levels of radiation exposure, there is a great uncertainty regarding its health risks. We report here that treatment of normal human cells with low-dose radiation induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis resulting in increased radiation resistance. This metabolic change is highlighted by upregulation of genes encoding glucose transporters and enzymes of glycolysis and the oxidative pentose phosphate pathway, concomitant with downregulation of mitochondrial genes, with corresponding changes in metabolic flux through these pathways. Mechanistically, the metabolic reprogramming depends on HIF1α, which is induced specifically by low-dose irradiation linking the metabolic pathway with cellular radiation dose response. Increased glucose flux and radiation resistance from low-dose irradiation are also observed systemically in mice. This highly sensitive metabolic response to low-dose radiation has important implications in understanding and assessing the health risks of radiation exposure.

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Figures

Figure 1
Figure 1
Cellular radioadaptive response is very sensitive to environmental oxygen concentration. (a) Human fibroblasts were cultured under conventional condition (20–21% O2). For assessing the effect of 0.1Gy on 4.0 Gy-induced DNA damage, cells were pretreated with a dose of 0.1 Gy or sham treated, after 12 h, followed by a 0 or 4.0 Gy treatment. The cells were harvested 1 h after the 4 Gy treatment and subjected to immunostaining with γH2AX (red) and DAPI (blue). (b) Quantification of γH2AX-positive cells shown in a. Bars represent mean±S.D. of (∼100 cells per sample) three independent experiments. *<0.05, **<0.01. (c). Human fibroblasts were maintained at 5% O2 for at least 12 h before being irradiated and analyzed as described in a. (d) Quantitative analysis of γH2AX-positive cells shown in c was carried out as in b. (e) Human fibroblasts were pretreated with or without 10 mM NAC for 1 h. The cells were either sham or 0.1 Gy irradiated and harvested 1 h after for immunostaining with carboxy-H2DCFDA (#C400, Invitrogen/Molecular probes, green) and DAPI (blue). (f) Fibroblasts were treated with 10 mM NAC (+NAC) for 1 h and then subjected to the treatment and analysis as in a. (g) Quantitative analysis of γH2AX-positive cells shown in f was carried out as in b
Figure 2
Figure 2
Low-dose radiation induces a metabolic shift. Human fibroblasts were either sham treated (S) or irradiated at 0.1 Gy and 12 h after the treatment; (a) culture media from an equal number of fibroblasts (1 × 106) were collected for lactate measurement. The numbers are mean±S.D. from three independent experiments. *<0.05, **<0.01. (b) An aliquot of cells were harvested and mRNA were isolated for qRT-PCR analysis of MCT expression with 18 s as an internal standard. The numbers are fold change of MCT mRNA levels in 0.1 Gy relative to sham-treated cells as mean±S.D. from three independent experiments. (c) Glucose consumption was determined in cells as in a and the numbers are mean±S.D. from three independent experiments. (d) Fibroblasts were treated as in a and subjected to analysis using the XF Analyzer according to the manufacture's protocol (Seahorse Bioscience). The numbers are mean±S.D. from three independent experiments. (e–g). Fibroblasts treated as in a and the cells were incubated with [1,2-13C]-glucose for 15 min prior to metabolite extraction and targeted LC-MS/MS analysis. The ratio of 13C labeled to unlabeled (12C) metabolites was measured by LC-MS/MS are presented as mean±S.D. over three independent samples, *<0.05, **<0.01. Metabolites with P-values for pair-wise comparisons <0.05 are shown. (h) mRNAs as in b were analyzed with qRT-PCR for the expression of the indicated genes. The numbers are mean±S.D. from three independent experiments. (i) Cell lysates were analyzed by western blot with the indicated antibody. Human fibroblasts treated as in a were harvested 12 h after the treatment and analyzed by either qRT-PCR (j) or immunostaining (k) of GLUT-1 or 3
Figure 3
Figure 3
The glucose metabolism is necessary for low-dose IR-induced resistance. Human fibroblasts cultured in normal media (a) low glucose (2 mM) media (b) were pretreated with a dose of 0.1 Gy or sham treated, followed after 12 h by a 0 or 4.0 Gy treatment. The cells were fixed 1 h post 4 Gy treatment and co-immunostained with γH2AX (red) and DAPI (blue). (c) Fibroblasts were treated with 2-DG (5 mM) 2 h before 4 Gy treatment and subject to the treatment and analysis as in a. (d) Human fibroblasts were transfected with either siLDHα or siG6PD (e). The knockdown efficiency was determined by qRT-PCR analysis of mRNA levels (Supplementary Figure 2D). The cells were subjected to the treatment at 48 h post transfection, treated and analyzed as in a. The fibroblasts along with siControl (siGL2, a siRNA sequence targeting Luciferase gene) expressing cells (f) were also subjected to colony survival assays (g and h). The numbers are mean±S.D. from three independent experiments, *<0.05, **<0.01
Figure 4
Figure 4
Low-dose IR-induced metabolic changes and radiation resistance are mediated by HIF-1α. Human fibroblasts were treated with a dose of 0.1 Gy or sham treated. The cells were harvested 12 h later and subjected to HIF-1α (red) and DAPI (blue) immunostaining (a), western blot (b) or mRNA measurement by qRT-PCR (c). (d) Fibroblasts were treated with DMSO (-NAC) or 10 mM NAC (+NAC) for 1 h and then subjected to the treatment as in a. The cells were immunostained with HIF-1α. Fibroblasts were transfected with control (siGL2) or siHIF-1α. The HIF-1α knockdown efficiency is shown in Supplementary Figure 4B. The cells were treated as in a and mRNAs were extracted for qRT-PCR for the expression of the indicated metabolic genes (e) or GLUT1 and 3 (f). The numbers are fold change of the mRNA levels in 0.1 Gy-treated cells relative to sham-treated cells as mean±S.D. from three independent experiments. (g) siGL2 or siHIF-1α expressing cells were treated and subjected to the colony survival assay as described in Figure 3f. The numbers are mean±S.D. from three independent experiments, *<0.05, **<0.01
Figure 5
Figure 5
Distinct dose response of p53 and HIF-1α to irradiation. (a). Fibroblasts were irradiated with the indicated dose and harvested 12 h later. GLUT-3 mRNA was measured. (b) Fibroblasts were irradiated with the indicated dose and harvested 3 h later. Cellular mRNA was extracted for qRT-PCR for the expression of p21. The numbers are fold change of the mRNA levels in irradiated cells relative to sham-treated cells as mean±S.D. from three independent experiments. (c). Fibroblasts treated as in a or b were analyzed by immunostaining of p21 or GLUT-3 and counterstaining with DAPI
Figure 6
Figure 6
Low-dose irradiation induces glucose flux and radiation resistance in vivo. (a) BALB/C mice (4–6 weeks) were either sham-treated or 0.1 Gy irradiated. The expression of HIF1α or GLUT-3 in the small intestine was examined 12 h post treatment by immunohistochemical staining. (b) Mice were treated with a 12-h interval between a 0.1 Gy low-dose and 2 Gy high dose. Live animal imaging was performed 1 h after the 2 Gy irradiation using a procedure described in Materials and Methods to monitor the update of labeled glucose. The representative optical images are shown. (c) The quantitative data were acquired, analyzed using the manufacturer's Living Image 3.2 software and presented as means ±S.D. (d). Mice (6 mice per group) were irradiated as described in b and treated with 100 μl saline or 2-DG (200mg/kg body weight) for 2 h prior to the 2 Gy radiation treatment. The animals were harvested 12 h post-2 Gy irradiation. The spleen and small intestines were harvested and subjected to TUNEL assays. The representative images are shown. (e) The numbers of TUNEL positive cells were quantified using the J program and presented as mean ±S.D. of six mice. P<0.005. For the quantification in the GI track, the TUNEL positive cells from 5 villas permouse were counted
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