A Shifty Target: Tumor-Initiating Cells and Their Metabolism.

Tumor-initiating cells (TICs), or cancer stem cells, constitute highly chemoresistant, asymmetrically dividing, and tumor-initiating populations in cancer and are thought to play a key role in metastatic and chemoresistant disease. Tumor-initiating cells are isolated from cell lines and clinical samples based on features such as sphere formation in stem cell medium and expression of TIC markers, typically a set of outer membrane proteins and certain transcription factors. Although both bulk tumor cells and TICs show an adaptive metabolic plasticity, TIC metabolism is thought to differ and likely in a tumor-specific and growth condition-dependent pattern. In the context of some common solid tumor diseases, we here review reports on how TIC isolation methods and markers associate with metabolic features, with some focus on oxidative metabolism, including fatty acid and lipid metabolism. These have emerged as significant factors in TIC phenotypes, and in tumor biology as a whole. Other sections address mitochondrial biogenesis and dynamics in TICs, and the influence of the tumor microenvironment. Further elucidation of the complex biology of TICs and their metabolism will require advanced methodologies.

Comparison of Mitochondrial Incubation Media for Measurement of Respiration and Hydrogen Peroxide Production.

High-Resolution FluoRespirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function. For harmonization of results in a common database, we compared respiration and H2O2 production of permeabilized HEK 293T cells measured in MiR05 (sucrose and K-lactobionate), Buffer Z (K-MES and KCl), MiR07 (combination of MiR05 and Buffer Z), and MiRK03 (KCl). Respiration in a simple substrate-uncoupler-inhibitor titration protocol was identical in MiR05, Buffer Z, and MiR07, whereas oxygen fluxes detected with MiRK03 were consistently lower in all coupling and electron transfer-pathway states. H2O2 production rates were comparable in all four media, while assay sensitivity was comparatively low with MiR05 and MiR07 and higher but declining over time in the other two media. Stability of assay sensitivity over experimental time was highest in MiR05 but slightly less in MiR07. Taken together, MiR05 and Buffer Z yield comparable results on respiration and H2O2 production. Despite the lower sensitivity, MiR05 was selected as the medium of choice for FluoRespirometry due to the highest stability of the sensitivity or calibration constant observed in experiments over periods of up to 2 h.

Measurement of ß-oxidation capacity of biological samples by respirometry: a review of principles and substrates.

Oxidation of fatty acids is a major source of energy in the heart, liver, and skeletal muscle. It can be measured accurately using respirometry in isolated mitochondria, intact cells, and permeabilized cells or tissues. This technique directly measures the rate of oxygen consumption or flux at various respiratory states when appropriate substrates, uncouplers, and inhibitors are used. Acylcarnitines such as palmitoylcarnitine or octanoylcarnitine are the commonly used substrates. The ß-oxidation pathway is prone to feedforward inhibition resulting from accumulation of short-chain acyl-CoA and depletion of CoA, but inclusion of malate or carnitine prevents accumulation of these intermediaries and CoA depletion.

Early cardiovascular changes occurring in diet-induced, obese insulin-resistant rats.

The metabolic syndrome is recognized as a cluster of disturbances associated with obesity, type 2 diabetes and hypertension. Over the past two decades, the number of people with the metabolic syndrome has increased at an alarming rate. This increase is associated with the global epidemic of both obesity and diabetes. Cardiovascular mortality is increased among diabetics and obesity-related insulin-resistant patients, and obesity is currently recognized as independent risk factor for cardiovascular disease. We aimed to establish the effects of a short period of an altered diet on the heart using a rat model of hyperphagia-induced obesity (diet supplemented with sucrose and condensed milk for 8 weeks = DIO) compared to age-matched controls. Isolated, perfused hearts were subjected to global or regional ischaemia/reperfusion. Function on reperfusion was recorded and infarct size determined. A plasma lipid profile was established via HPLC-based methods and proteins involved in metabolic signalling determined either by western blotting or RT-PCR. 8 weeks of diet resulted in whole-body but not myocardial insulin resistance, increased plasma triglyceride and phospholipid levels as well as increased lipid peroxidation. Despite the similar baseline function, hearts from DIO animals showed significantly poorer postischaemic recovery than controls (41.9 % RPP recovery vs 57.9 %, P < 0.05, n = 7-11/group) but surprisingly, smaller infarct size (24.95 ± 1.97 vs 47.26 ± 4.05 % of the area at risk, P < 0.005, n = 8/group). Basal phosphorylation of PKB/Akt was elevated but IRS-1 and SERCA-2 expression severely downregulated. In conclusion, after only 8 weeks of a slight change in diet, the rat heart shows signs of metabolic remodelling. Some of these changes may be protective but others may be detrimental and eventually lead to maladaptation.