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First published online 30 September 2008
doi: 10.1242/jcs.034660

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Mitochondrial dysfunction and HIF1 stabilization in inflammation

The Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK

View larger version (36K): [in this window] [in a new window]   Fig. 1. Effect of activation of J774.A1 M on their consumption of O2 and generation of NO. Activation of M with 10 U ml-1 IFN and 10 ng ml-1 LPS progressively inhibited mitochondrial O2 consumption and concomitantly increased the release of NO into the extracellular fluid (A), and decreased the spare respiratory capacity (B). The different components of cellular O2 consumption by untreated and SEITU-treated control M did not change throughout the experimental period. Administration of 500 µM SEITU at the time of activation prevented the decrease in cellular (A) and mitochondrial O2 consumption (not shown) and spare respiratory capacity (B). (C) The non-mitochondrial uptake of O2 was greatly enhanced after 12 hours of activation; this could be reduced by the administration of 10 µM oxyhaemoglobin or abolished by treatment with SEITU. *, Significantly different from values in non-activated cells; , Significantly different from IFN+LPS-treated cells (P<0.05, one way ANOVA, Tukey's post hoc test). Results are mean±s.d., n=5.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Effect of activation of M on their glycolytic metabolism. The rate of glucose uptake (A), rate of lactate release (B) and LDH activity in cell homogenates (C) were determined in untreated cells or in those activated with IFN and LPS. Co-administration of SEITU partly prevented the increase in all three parameters of glycolytic metabolism that was observed in activated cells. Mean±s.d., n=3-5. There were significant differences (P<0.05, one way ANOVA, Tukey's post hoc test) in the glucose uptake rates, lactate release rates and LDH activities between the IFN + LPS + SEITU-treated group and all the other groups at each sampling time, except at time zero.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Contribution of oxidative phosphorylation and glycolysis to ATP production in activated M. Total cellular [ATP] and ATP synthesis rates (glycolytic and mitochondrial) of M treated with IFN + LPS (A) and IFN + LPS + SEITU (B) are shown. Treatment with SEITU prevented the drop in mitochondrial synthesis of ATP and reduced the enhanced glycolytic production of ATP in activated M. It also prevented the drop in total cellular [ATP]. Cellular [ATP] was determined by the luciferase assay, the rate of synthesis of ATP by glycolysis was computed from the lactate production rate, and the rate of synthesis of ATP by mitochondria was computed from the oligomycin-sensitive mitochondrial oxygen consumption rate, assuming a P:O ratio of 1:2.4. *, Total cellular ATP significantly different from values in non-activated cells (0 hour). Results are mean±s.d., n=5.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Effect of activation of M on their proliferation. Changes in cell density relative to the initial density are shown in A, and BrdU incorporation into DNA after 12 hours activation and 4 hours incubation with BrdU are shown in B. Activation led to complete arrest of cell proliferation that could be partially reversed by treatment with SEITU. *, Significantly different from values in non-activated cells; , Significantly different from IFN + LPS-treated cells (P<0.05, ANOVA, Tukey's test). Results are mean±s.d., n=5.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Effect of activation of M on the stabilization of HIF1. (A) The time course of HIF1 stabilization following activation and (B) the corresponding densitometry values normalized with -tubulin and relative to non-activated controls. Cobalt chloride (CoCl2) was used as a positive control for HIF1 in A. (C) Cells were transfected with HIF1 siRNA or scrambled siRNA as a control. After 24 hours they were treated with the agents indicated and incubated for 12 hours. Treatment with IFN + LPS resulted in stabilization of HIF1 under normoxic conditions. This was reduced in the cells in which HIF1 had been silenced, and in those treated with SEITU. Densitometry values, normalized with -tubulin and relative to that of control transfected M (in the case of HIF1) and control transfected and activated M (in the case of iNOS) are shown below each lane. (D) Silencing HIF1 reduced the generation of NO and (E) partially preserved mitochondrial oxygen consumption in activated M. (F) The upregulation of glycolysis in activated M was attenuated by silencing HIF1 and by treatment with SEITU; it was completely abolished by a combination of the two treatments. (G) The cellular ATP content was reduced in M in which HIF1 had been silenced and was reduced further by activation, an effect that was prevented by SEITU. *, Significantly different from control transfected values. Results are mean±s.d., n=3-5, and representative western blots are shown.

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