Endotoxin-induced activation of hypoxia-inducible factor 1α in cultured human hepatocytes and monocytes: impact on cellular and mitochondrial respiration

Introduction

Hypoxia-inducible factor 1α (HIF1α) is a transcriptional factor activated by hypoxia. HIF1α coordinates cell adaptation to hypoxia and modulates cellular metabolism and respiration. Recent data suggest that HIF1α may also be activated via proinflammatory mediators and Toll-like receptors under normoxic conditions. The aim of this study was to evaluate whether lipopolysaccharide (LPS) could increase HIF1α expression in a time-dependent and dose-dependent manner in cultured human hepatocytes and monocytes, and to determine a possible role in the modulation of cellular respiration.

Methods

Cultured human hepatocytes (HepG2) and monocytes (MM6) were exposed to cobalt chloride, hypoxia (1.5% oxygen) and different concentrations of LPS. The time-course expression of HIF1α was determined by western blotting. Mitochondrial respiration was assessed after cell permeabilization with a protocol of stimulation-inhibition of each mitochondrial complex using the Oxygraph 2 K (Oroboros Instruments, Innsbruck, Austria) and Datlab 4.2 software for data acquisition.

Results

Hypoxia, cobalt chloride and LPS induced accumulation of HIF1α in both cell lines in comparison with controls. In monocytes, HIF1α was detected after 4 hours of normoxic LPS incubation at a concentration of 1 mg/ml. In cultured hepatocytes, HIF1α was detected after 2 hours of normoxic LPS incubation at a concentration of 1 mg/ml. Cellular respiration of permeabilized cultured hepatocytes was not affected after 6 hours (complex I and II respiratory control ratio (RCR)-dependent respiration: controls: 1.7 ± 0.4 vs LPS: 2.2 ± 0.4 and controls: 2.4 ± 1 vs LPS: 3.4 ± 0.5, respectively, P > 0.05, n = 5) or after 24 hours (complex I and II RCR-dependent respiration: controls: 2 ± 0.4 vs LPS: 1.9 ± 0.5 and controls: 3.9 ± 1.7 vs LPS: 4.5 ± 2.6, respectively, P > 0.05, n = 6) of normoxic LPS 1 mg/ml incubation (P > 0.05 for both).

Conclusion

LPS induces the expression of HIF1α in human monocytes and hepatocytes under normoxic conditions. Exposing hepatocytes to LPS (1 mg/ml) for 6 and 24 hours does not impair their cellular respiration.