Regulation of Aerobic Glycolysis by Na/K‐ATPase

Mammals express Na/K‐ATPase isoforms in a tissue‐specific manner. Besides the canonical ion‐pumping function of the enzyme complex, we and others have demonstrated the importance of α1 Na/K‐ATPase/Src interaction in cellular signal transduction. Interestingly, α2 Na/K‐ATPase lacks Src interaction. Using cell lines expressing wild‐type or mutant forms of rat α1 and α2 in α1‐knockdown pig kidney cells, the aim of this study was to test whether the difference in Src interaction confers an isoform‐specific phenotype to cellular metabolism.Compared to α1‐expressing cells, α2 cells exhibited a 30% increase in aerobic glycolysis measured by Seahorse Extracellular Flux Analyzer. Consistently, direct measurement of lactic acid content in the medium indicated that both short term (4h) and long term (3 days) release were significantly increased in cell cultures. Consistently, ATP generation of α2‐expressing cells was more dependent on glucose. Indeed, α1‐expressing cells exhibited about 25% drop in cellular ATP content after 45 minutes in 50 mM 2‐DG, while a larger drop of over 50% was observed in α2 cells in the same condition. In α2‐expressing cells, failure to activate glycolysis in response to inhibition of oxidative respiration indicated that the glycolytic reserve was drastically reduced compared to α1‐expressing cells, suggesting that they lacked metabolic plasticity and were more prone to metabolic stress. Taken together, these data suggested that Na/K‐ATPase regulates aerobic glycolysis in a α‐isoform‐specific manner in mammalian cells, with α2 resulting in an increase of glycolysis known as a Warburg Effect. In keeping with well‐known characteristics of cells exhibiting a Warburg Effect, α2 cells were highly dependent on glucose for their growth and survival with significant reduction in number upon glucose‐deprivation that was not observed in α1 cells. Consistent with a critical role of Na/K‐ATPase/Src interaction in this isoform‐specific function, cells expressing mutant forms of α1 which were unable to interact with Src, showed increased glycolysis, decreased metabolic plasticity and high reliance on glucose comparable to those of α2 cells. Conversely, cells expressing mutant form of α2 which was able to interact with Src, showed a α1‐like aerobic glycolysis phenotype. Physiologically, it is known that α2 isoform is highly expressed in tissues/cells such as glycolytic skeletal muscle fibers, astrocytes and adipocytes where glucose is utilized as a major fuel for ATP generation or for providing building materials as well as metabolic intermediates. Thus, our new findings could be highly relevant to Na/K‐ATPase α2‐specific cell physiology. We speculate that co‐expression of α1 in α2‐expressing cells could allow a dynamic regulation of bioenergetics that meet the need of muscle, astrocytes and adipocytes.Support or Funding InformationMarshall Institute for Interdisciplinary Research and NIH Grant HL‐109015