Comparative bioenergetic assessment of transformed cells using a cell energy budget platform.

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.

Immunonutrition and surgical practice.

Immunonutrition generally refers to the effect of the provision of specific nutrients on the immune system. These nutrients typically have immunoenhancing properties, and recent advances in nutrition support involve studies designed to exploit the desirable biological properties of these nutrients. The term immunonutrition strictly implies that we are focusing on the effect of certain nutrients on aspects of the immune system. However, in reality immunonutrition also refers to studies that not only examine the function of lymphocytes and leucocytes, but which also study the influence of key nutrients on the acute-phase response, the inflammatory response and on gastrointestinal structure and function. The interest, therefore, is on the impact of immunonutrition on all aspects of host defence mechanisms in response to a catabolic stress. Major surgery evokes an acute-phase response, a transient immunosuppression and alterations in gastrointestinal function. Normal function is usually restored after a few days; however, in a subgroup of patients homeostasis may be lost and development of the systemic inflammatory response syndrome (SIRS) ensues. Results of recent clinical trials suggest that provision of immunomodulatory nutrients, including glutamine, arginine, n-3 polyunsaturated fatty acids and dietary nucleotides, may promote restoration of normal tissue function post-operatively and prevent the occurrence of SIRS.

Host defense--a role for the amino acid taurine?

Taurine (2-aminoethane sulphonic acid), a ubiquitous beta-amino acid is conditionally essential in man. It is not utilized in protein synthesis but found free or in some simple peptides. Derived from methionine and cysteine metabolism, taurine is known to play a pivotal role in numerous physiological functions. Some of the roles with which taurine has been associated include osmoregulation, antioxidation, detoxification and stimulation of glycolysis and glycogenesis. Intracellular taurine is maintained at high concentrations in a variety of cell types and alteration of cell taurine levels is difficult. The role of taurine within the cell appears to be determined by the cell type. Recent research has determined a regulatory role for taurinechloramine, the product formed by the reaction between taurine and neutrophil derived hypochlorous acid on macrophage function. Plasma taurine levels are also high, although decreases are observed in response to surgical injury and numerous pathological conditions including cancer and sepsis. Supplementary taurine replenishes decreased plasma taurine. Although commonly used as a dietary supplement in the Far East, the potential advantages of dietary taurine supplementation have not as yet been fully recognized in the Western World; this is an area which could prove to be beneficial in the clinical arena.

Dexamethasone and lipopolysaccharide regulation of taurine transport in Caco-2 cells.

Intracellular enterocytic levels of the immunomodulator taurine decrease significantly in response to trauma and surgical insult. The effect of physiological stress on enterocyte taurine uptake is unknown. The aim of this study was to compare taurine transport under basal and stressed conditions using the human intestinal Caco-2 cell line in vitro. Caco-2 cells were incubated with 10 nM [1,2-3H]taurine at 37 degrees C and 5% CO2 and taurine uptake was examined over the range of 0.1-10 microM to determine kinetic parameters of the transporter. The culture medium was then supplemented with dexamethasone and/or lipopolysaccharide (LPS) and taurine uptake was calculated as picomoles per milligram protein per hour. Statistics were by unpaired Student's t test. Taurine uptake was hyperbolically related to taurine concentration and obeyed Michaelis-Menten kinetics with a K(m) of 5.27 +/- 0.95 microM and Vmax of 1125.43 +/- 130.9 pmole/mg protein/ hour. Dexamethasone (1-1000 microM) significantly reduced taurine uptake by up to 66.15%. LPS (1 microgram/ml) impaired transport of taurine by 15.7%, and in combination with dexamethasone (100 microM) by 42.4%. All results are mean of at least three experiments and P < 0.05. We have established that taurine uptake by enterocytes is downregulated by dexamethasone. This may relate to the decreased intestinal levels of taurine observed in trauma and surgery patients. Further study may elucidate mechanisms whereby homeostasis of enterocyte taurine might be maintained during sepsis.