Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

Changes in availability and utilisation of 02 and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O-2. Upon 2-4 h moderate hypoxia, HIF-alpha protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1 alpha dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2 alpha levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2 alpha levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O-2 and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2 alpha decreased after 24 h glucose deprivation. This effect, associated with increased AMPK alpha phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2 alpha accumulation, which became mainly glucose-dependent. Overall, the availability of O-2 and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-alpha. (C) 2014 Elsevier Inc. All rights reserved.