Klepinin 2017 MiP2017
Adenylate kinase (AK) is a ubiquitous enzyme critical in intracellular energy transfer processes, the maintenance of energy homeostasis, and a major player in AMP metabolic signaling circuits in some highly differentiated cells. However, the role of the AK network in aging, during cancerogenesis is poorly understood. The main limitation in research of the AK network is that cytosolic and mitochondrial AK metabolic flux cannot be separated due to the absence of isoform specific inhibitor.
For this purpose, a simple oxygraphic method was developed that enables directly and semi-quantitatively estimate the distribution between cytosolic AK1 and mitochondrial AK2 localized in the intermembrane space, both in isolated cells and tissue samples (biopsy material). Experiments were performed on isolated rat mitochondria or permeabilized material, including undifferentiated and differentiated neuroblastoma Neuro-2a cells, pluripotent human embryonic stem cells, relatively null potent embryonal carcinoma cells (2102Ep cells line), HL-1 cells, isolates rat heart cardiomyocytes (CM) as well as on human colorectal and breast cancer postoperative samples.
Intracellular distribution of AK1 and AK2 can be detected by high-resolution respirometry due to their functional coupling with ATP synthesis in mitochondria. Extracellular ADP which is not related to AK flux was eliminated by pyruvate kinase and its substrate phosphoenolpyruvate to ensure specific AK2 functional coupling with mitochondria. Distribution of AK1 and AK2 is expressed by specific AK index. Our study showed that among 1 year old rats total AK activity had already decreased in CM. Nevertheless, in all studied age groups no significant changes in AK1 and AK2 distribution were noticed. In ageing rat CMs it seems, that drastic alteration in CK network can be compensated by both AK1 and AK2. Furthermore, in different cancer cell lines in contrast to the creatine kinase pathway, the AK phosphotransfer pathway is up-regulated and the expression of AK2 is higher than AK1 in these malignant cells. The study of biopsy material showed that instead of mitochondrial creatine kinase the AK network was the main energy transfer pathway in colorectal and breast cancer. The respirometric method also revealed a considerable difference in mitochondrial affinity for AMP between nontransformed cells and tumor cells.
In summary, we developed a simple and rapid oxygraphic method to estimate semi-quantitatively distribution of AK1 and AK2 in mammalian tissues and cultured cells. Our study showed that although, in aging rat CMs, the AK network slightly decreases, the AK1 and AK2 distribution remain unchanged. This method was tested also in cancer postoperative material and revealed that it was sensitive enough to perform on tumor biopsies. The data show that AK2 highly expressed predominantly in poorly differentiated cells with a high proliferative index, and that strong differences exist between highly-differentiated and tumor cells in the affinity of their mitochondrial respiration for exogenous AMP. Further studies will be required to identify the exact role of AK pathway in aging heart muscle and cancerogenesis.
Labels: MiParea: Respiration, Comparative MiP;environmental MiP Pathology: Aging;senescence, Cancer
Organism: Human, Rat Tissue;cell: Heart, Stem cells Preparation: Permeabilized tissue, Isolated mitochondria
- Klepinin A(1), Ounpuu L(1), Guzun R(2,3), Chekulayev V(1), Timohhina N(1), Tepp K(1), Shevchuk I(1), Schlattner U(2,3), Kaambre T(1)
- Lab Bioenergetics, Nat Inst Chem Phys Biophys, Akadeemia tee 23, Tallinn, Estonia
- Lab Fundamental Applied Bioenergetics, Univ Grenoble Alpes, France
- Inserm, Grenoble, France. – [email protected]