Much of the cellular metabolic network relies on normal physiological oxygen tensions for normal function. When environmental oxygen tensions change, such as occurs during exercise, cells respond by altering their metabolism to maintain function and viability. However, this metabolic adaptation may have a dark side, as inadequate oxygenation of cells plays a pathogenic role in a number of diseases, including cancer, cardiovascular disease, obstructive sleep apnoea and diabetes.
The Tennant Research Group is focused on improving our understanding of how cells adapt their metabolism in response to changes in oxygen tension. By doing so, we will improve our knowledge of when and how this adaptation goes wrong, and as a consequence design new therapies to improve our long-term health.
An example trace performed on an Oroboros high resolution respirometry instrument. Oxygen consumption by permeabilised cells was recorded in response to respiratory complex I substrates (malate-glutamate) or complex II substrate (succinate).
U87, glioblastoma cell line, stained for lipid droplets (green). Left hand image shows cells cultured in normoxia oxygen, and image on right shows cells after culturing for 24 hours in very low (0.3%) oxygen. Note the significant increase in lipid droplets in low oxygen conditions.
A trace of the peaks detected by our mass spectrometer after gas chromatographic separation of the polar metabolites produced by cells. To see which metabolites were being made from the amino acid glutamine by these cells, we gave the cells glutamine enriched in the stable isotope, 13C, and the mass spectrum was investigated for incorporation of those atoms into other polar metabolites within the cells. This type of analysis allows us to compare how cells use nutrients when their environment changes - such as when they are exposed to low oxygen.
We use a number of techniques to study the effect of changes in environmental oxygen tension on cellular function, and in particular their metabolism. These include direct measurements of their use of oxygen in response to different nutrients using high resolution respirometry (A). With this technology, we can investigate how changes in genotype alter the cellular use of oxygen at its simplest, or how well the mitochondrial metabolism of nutrients such as pyruvate or glutamate are coupled to ATP generation. Alongside this, we can also image some of the metabolic constituents of the cell using confocal microscopy. Using this method, we can qualitatively assess how changes in oxygen tension alter the use or storage of various nutrients - such as lipids as shown in B.
Our metabolic investigations are underpinned by the use of two complementary technologies - mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. After incubating cells with one or more nutrients enriched in a stable isotope, such as 13C, intracellular and extracellular metabolites are extracted and the resulting mixtures analysed using both platforms. The complexity of the samples are resolved through either gas chromatography (mass spectroscopy) or through the use of 2D or pseudo-2D NMR experiments.
Example of a spin system (L) and cross section through this (R) of the C4 of glutamate as analysed with a 1H-13C-HSQC experiment, after 24 hours of incubation of cells with 13C-[U]-glutamine. The characteristic 'splitting' of the central peak (not observed here due to high 13C incorporation) into two major doublets - one due to the neighbouring C5 also being 13C, and one due to the other neighbour - the C3 being 13C. By using the non-model isotopomer simulation software programme, Metabolab, and combining these data with the GC-MS data (see C) within the analysis, we can accurately the often complex mix of isobaric and non-isobaric isotopomers of a metabolite and thereby infer metabolic pathway use by a cell population.
Our main research focus is on understanding the role of hypoxia in tumour formation and progression. Hypoxic regions, which arise in most tumour types, are therapy-resistant and often represent the source of the cells with the most malignant phenotype. However, hypoxia also enforces metabolic adaptation on tumour cells that is not otherwise found in a healthy individual. Improving our knowledge of the precise nature of these adaptations will allow us to specifically target hypoxic cancer cells, thereby improving patient outcomes.
Although hypoxic regions are observed in most tumour types, we investigate some of those in which hypoxia is known to play a major negative role, including gliomas. In addition, we study tumours that contain genetic mutations that lead to them acquiring hypoxic phenotypes even when oxygen is not limiting. Mutations in one such enzyme complex, succinate dehydrogenase (SDH), leads to paragangliomas and pheochromocytomas, which we are currently studying in collaboration with Dr. Judith Favier (Paris). In addition, we are investigating the role hypoxia in the transformation of stromal phenotype and metabolism during tumour formation (multiple myeloma). To find out more about each area, click on the boxes to the right.
The resulting datasets are analysed simultaneously using Metabolab (http://metabolab.uk/), bespoke software written by Dr. Christan Ludwig (a PI at the same Institute as the Tennant group), which provides high resolution datasets that describe the use of stable isotope-enriched nutrients provided by cells, thereby allowing interpretation of their metabolic phenotype.