Edited by James Murray, PhD.
Thousands of researchers around the world are studying the connections between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlights how Abcam's MitoSciences range of research tools has contributed to this effort. The full list of 23 original research papers published this month using the MitoSciences range of products can be found here.
Past issues are available for review in the archives.
Table of Contents
I. Autophagy, senescence and tumor growth
II. Ectopic OXPHOS
III. PDH regulation by glucose
I. Autophagy, senescence and tumor growth
Several reports support the theory that senescent fibroblasts can stimulate neighboring tumor growth in aged animals. However, the mechanism by which this occurs remains unclear. This month, Capparelli et al. expand upon their theory explaining this phenomenon, termed ‘two compartment tumor metabolism'. Here tumor cells release oxidants, inducing oxidative stress in neighboring fibroblasts. The oxidative stress in fibroblasts drives autophagy, mitophagy (mitochondrial dysfunction), increased aerobic glycolysis and senescence. This heightened catabolic state increases cellular nutrients and metabolic fuels such as lactate, ketone bodies, glutamine and free fatty acids, which are transferred to the neoplastic epithelial cells in a paracrine fashion stimulating anabolic processes necessary for tumorigenesis. As a result, cancer cells increase their OXPHOS and oxidative capacity. In this recently published work, the researchers address these points, showing that cell lines over-expressing various autophagy-related genes do undergo significant autophagy and mitophagy as measured by methods including Western blotting of OXPHOS complexes. These mitochondria-deficient, autophagic, fibroblast ‘feeder' cells become more glycolytic and produce more extracellular lactate and ketone bodies. Co-cultured neighboring cancer cells show an increase in mitochondrial staining in response. This effect is compartment specific. Expression of autophagy genes in cancer cells themselves inhibited their growth. Finally a similar effect is observed in vivo where injection of autophagic fibroblasts enhanced the metastatic capacity of epithelial breast cancer cells in mice.
Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis via glycolysis and ketone production. Cell Cycle 2012. Capparelli C, Guido C, Whitaker-Menezes D, Bonuccelli G, Balliet R, Pestell TG, Goldberg AF, Pestell RG, Howell A, Sneddon S, Birbe R, Tsirigos A, Martinez-Outschoorn U, Sotgia F, Lisanti MP.
II. Ectopic OXPHOS – ATP synthase and respiratory chain on the cell surface
The ATP synthase enzyme is often considered a specific marker for mitochondria and exclusively located within the inner membrane of the mitochondrion. However a number of studies, dating back to Das et al. 1999
The presence of this enzyme at the cell surface has been shown in a number of cell types including endothelial cells, hepatocytes, keratinocytes and adipocytes, principally by immunofluorescent localization or GFP tagging of enzyme subunits. Its function, whether generating extracellular ATP or acting as a proton pump or channel, is unclear. Also, the enzyme may act as a receptor, for example to angiostatin, inhibitor protein or HDL, and appears to be sensitive to ATP synthase inhibitors. For more information see MitoNews Dec 2005.
This month Chang et al showed by immunofluorescent confocal microscopy, flow cytometry and Western blotting the presence of the ATP synthase subunit beta (ATPB) and also a representative subunit from each of the other OXPHOS complexes (NDUFB4, SDHA, UQCRC2, and COX5A )
The ectopic ATP synthase in lung cancer cells was sensitive to the specific inhibitor citreoviridin, which reduced extracellular pH and ATP concentration, induced cell cycle arrest, inhibited proliferation and anchorage-dependant growth. Citreoviridin did not affect membrane potential within mitochondria, indicating its action was at the cell surface.
The authors went on to examine the effects of citreoviridin at the proteomic level and identified that the most altered pathway was protein folding. Up regulation or activation of a number of ER stress and oxidative stress response proteins were identified. Pretreatment of cells with N-acetyl cysteine reduced oxidative stress and increase cell viability. Finally, the authors postulate that this response may be signaled by p53 and showed that a p53 null cell line is significantly less sensitive to citreoviridin.
The connection between ectopic OXPHOS activity and ER stress is unclear. However the authors propose that changes in extracellular ATP, proton pumping/acidification, ROS generation by the respiratory chain or calcium homeostasis may be involved.
Ectopic ATP synthase blockade suppresses lung adenocarcinoma growth by activating the unfolded protein responseCancer Res. 2012. Chang HY, Huang HC, Huang TC, Yang PC, Wang YC, Juan HF.
Also this month:
Fluorescence imaging of mitochondria in cultured skin fibroblasts: a useful method for detection of oxidative phosphorylation defects. Pediatr Res 2012 De Paepe B, Smet J, Vanlander A, Seneca S, Lissens W, De Meirleir L, Vandewoestyne M, Deforce D, Rodenburg RJ, Van Coster R.
Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J Physiol. 2012. Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, Schroder HD, Boushel R, Helge JW, Dela F, Hey-Mogensen M.
III. PDH regulation by glucose
Pyruvate dehydrogenase is central to the regulation of glucose metabolism. The enzyme activity is regulated by key metabolic intermediates, products, and substrates. In addition the enzyme is inactivated at three different phosphorylation sites in subunit E1alpha by a set of four kinases (PDK1-4), which are expressed in differently in various tissues and the action of two phosphatases (PDP1-2) lead to dephosphorylation and activation.
This month, Akhmedov, et al. published work from their studies of PDH regulation in pancreatic beta cells (store and release insulin). They showed that basal levels of glucose (2 mM) reduced PDH activity to 91% compared to the activity of a PDP1 treated, fully dephosphorylated, form of the enzyme. The activity was decreased to 78% when cells were stimulated higher concentrations of glucose (16.7 mM). These PDH activity data agreed well with PDH activity measurements obtained by following cellular 14CO2 production from supplied 14C-pyruvate. PDH E1alpha phosphorylation levels also agreed with these findings. However, despite this phosphorylation induced decrease in PDH activity, the flux of pyruvate through the oxidative pathway was unaffected. This may be due to an excess of PDH in beta cells or a compensatory increase in metabolic substrates into the mitochondrion such as pyruvate, as has been observed and proposed in previous cell studies of Liu, et al.
Pyruvate dehydrogenase E1α phosphorylation is induced by glucose but does not control metabolism-secretion coupling in INS-1E clonal β-cells.BBA 2012. Akhmedov D, De Marchi U, Wollheim CB, Wiederkehr A.
Proteins and Lysates
- Calyculin treated HeLa cell lysate – matched control samples for phosphorylation in WB (ab135395)