Pyruvate metabolism and its importance in determining cell fate
A seminal discovery in our understanding cell metabolism was the identification of the mitochondrial pyruvate carrier (MPC) by our laboratory in 2012. The existence of this carrier had been predicted for more than 40 years, and its molecular identification has now opened fresh avenues for investigating its importance using diverse molecular biological, biochemical, microscopy and genetic approaches.
Pyruvate is the end product of glycolysis in the cytosol and it can be either imported into the mitochondria where it forms the principal substrate for the TCA cycle, leading ultimately to the reduction of molecular oxygen and production of high levels of ATP; or it can be retained in the cytosol and converted to lactate by lactate dehydrogenase (LDH), a reaction which also refuels the glycolytic pathway. This latter cycle is important in times of oxidative stress, for example in muscle cells during intense physical exercise, while under normoxic conditions, aerobic glycolysis is characteristic of highly proliferating cells, since many of the biochemical intermediates of the glycolytic pathway provide raw materials for rapid generation of biomass.
Aeobic glycolysis is also exploited by rapidly growing cancer cells and in this context is often referred to as the Warburg effect. Import of pyruvate into the mitochondria by the MPC is thus a critical event in determining cell fate. We have shown that loss of MPC in mice has profound effects on metabolic homeostasis and also perturbs neurotransmitter balance. We are interested in exploring the relevance of these changes in major diseases including cancer, type II diabetes and neurological diseases such as epilepsy.
Figure 2a: Schematic representation of the bioluminescence resonance energy transfer (BRET) assay used to monitor activity of the MPC in real time. The MPC complex is located in the inner mitochondrial membrane and the two subunits MPC2 and MPC1 are tagged with an energy donor (Rluc8), or a fluorescence emitter (Venus) respectively. Transport of pyruvate through the channel leads to a conformational change within the complex, which brings the two tags into closer proximity leading to a measurable change in the fluorescence spectra.
The goals of our current research are,
- to understand how the MPC is regulated. We have recently developed a real-time biosensor of MPC activity based on bioluminescence resonance energy transfer (BRET), with which we can monitor pyruvate import into mitochondria in different types of primary cells and tumour cells (Figure 2). This will be helpful in understanding the factors that regulate the MPC. Using the same assay we are also looking for small molecule modulators of MPC activity for potential therapeutic applications;
- to identify novel carriers for importing critical metabolites into mitochondria;
- to explore the role of the MPC in controlling synaptic function in vitro and in vivo.
Figure 2b: BRET kinetics in either rat pancreatic β cells (INS-1E) which rely extensively on glucose derived pyruvate for mitochondrial production of ATP by oxidative phosphorylation, or HEK293 cells in which cytosolic glycolysis provides the main source of ATP. Both cell types respond to direct stimulation by pyruvate (red lines) however in the presence of glucose, only the pancreatic cells transport pyruvate derived through glycolysis (blue lines). Basal activity of the MPC in both cell lines is measured in the presence of PBS alone (black lines).