Ca2+-induced changes in energy metabolism and viability of melanoma cells

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. We show here that a rise in intracellular-free calcium ion (Ca2+), induced by Ca2+-ionophore A23187, exerted a deleterious effect on glycolysis and viability of B16 melanoma cells. Ca2+-ionophore caused a dose-dependent detachment of phosphofructokinase (EC 2.7.1.11), one of the key enzymes of glycolysis, from cytoskeleton. It also induced a decrease in the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis. All these changes occurred at lower concentrations of the drug than those required to induce a reduction in viability of melanoma cells. We also found that low concentrations of Ca2+-ionophore induced an increase in adenosine 5′-triphosphate (ATP), which most probably resulted from the increase in mitochondrial-bound hexokinase, which reflects a defence mechanism. This mechanism can no longer operate at high concentrations of the Ca2+-ionophore, which causes a decrease in mitochondrial and cytosolic hexokinase, leading to a drastic fall in ATP and melanoma cell death. The present results suggest that drugs which are capable of inducing accumulation of intracellular-free Ca2+ in melanoma cells would cause a reduction in energy-producing systems, leading to melanoma cell death. © 1999 Cancer Research Campaig

Effects of calorie restriction on life span of microorganisms

Calorie restriction (CR) in microorganisms such as budding and fission yeasts has a robust and well-documented impact on longevity. In order to efficiently utilize the limited energy during CR, these organisms shift from primarily fermentative metabolism to mitochondrial respiration. Respiration activates certain conserved longevity factors such as sirtuins and is associated with widespread physiological changes that contribute to increased survival. However, the importance of respiration during CR-mediated longevity has remained controversial. The emergence of several novel metabolically distinct microbial models for longevity has enabled CR to be studied from new perspectives. The majority of CR and life span studies have been conducted in the primarily fermentative Crabtree-positive yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, but studies in primarily respiratory Crabtree-negative yeast and obligate aerobes can offer complementary insight into the more complex mammalian response to CR. Not only are microorganisms helping characterize a conserved cellular mechanism for CR-mediated longevity, but they can also directly impact mammalian metabolism as part of the natural gut flora. Here, we discuss the contributions of microorganisms to our knowledge of CR and longevity at the level of both the cell and the organism

Measurement of [18F]-fluorodeoxyglucose incorporation into human osteoblast–An experimental method

An evaluation of human osteoblast metabolism usually involves measurements of the by-products of bone matrix elaboration. The assessment of glycolytic activity of osteoblasts is not a standard tool in most of the reports, but might be of value by providing a direct indicator of cellular metabolism. Measurement of the incorporation of [18F]-fluorodeoxyglucose, which is not further degradable following its conversion into glycose-6-phosphate during glycolysis and is trapped in this form within the cells, can be used as an effective research tool for estimation of osteoblast metabolism. In order to estimate the [18F]-fluorodeoxyglucose incorporation we used cultured human osteoblast-like cells. Following incubation of the culture samples in a glucose free medium with 5 μ Ci [18F]-fluorodeoxyglucose we measured the radioactivity of the cell fraction, as a percent from the initial dose, and compared to the incorporation values in cells treated by protoporphyrine IX (10−5 M), an endogenous pro-apoptotic agent. To compare the response of [18F]-fluorodeoxyglucose incorporation studies, following treatment of cells with the protoporphyrine IX, to other experimental cell metabolism evaluation methods, we performed a parallel comparison of alkaline phospatase activity, which is a standard measurement tool of osteoblast metabolism, in the control and treatment groups. A narrow range of 0.22–1.36% of [18F]-fluorodeoxyglucose incorporation per million cells was found. Additionally in the protoporphyrine IX treated cells a significant 62% decrease of [18F]-fluorodeoxyglucose incorporation was observed (p < .05). A parallel significant decrease in alkaline phosphatase activity (p < .001) was found in the cells treated by the protoporphyrine IX. Therefore we suggest that the presented method of [18F]-fluorodeoxyglucose incorporation measurement can be utilized as an effective research tool for estimation of the cellular glycolitic activity in human osteoblast-like cells in vitro

Microtubule assembly in meiotic extract requires glycogen

We identified a clarified extract containing the soluble factors for microtubule assembly. We found that microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly

Bcl-xl regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FoATP synthase

Anti-apoptotic Bcl2 family proteins such as Bcl-xL protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-xL enhances the efficiency of energy metabolism. Our evidence indicates that Bcl-xLinteracts directly with the ?-subunit of the F1FO ATP synthase, decreasing an ion leak within the F1FO ATPase complex and thereby increasing net transport of H+ by F1FO during F1FO ATPase activity. By patch clamping submitochondrial vesicles enriched in F1FO ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-xL activity increases the membrane leak conductance. In addition, recombinant Bcl-xL protein directly increases the level of ATPase activity of purified synthase complexes, and inhibition of endogenous Bcl-xL decreases the level of F1FO enzymatic activity. Our findings indicate that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-xL-expressing neurons

Systems Biology approach to metabolomics in cancer studies

The astonishing development of high-throughput techniques in the last decades has fostered a renewed, dynamical comprehension of cell and tissue metabolism, giving unexpected insights into the ‘systemic aspects’ of cancer, namely pointing out that metabolism should be considered a truly “systems property. Both internal and microenvironmental cues tightly cooperate in shaping tissue metabolomic fingerprint. tumour metabolome hardly could be mechanistically linked to the linear dynamics of few gene regulatory networks; otherwise it is likely to be the complex end point of several interacting non-linear pathways, involving both cells and their microenvironment. As such, tumour metabolism might be considered an emerging, “systems property”, arising at the integrated scale of the whole system and behaving like an “attractor” in a specific space phase defined by thermodynamic constraints . Therefore, metabolomics ‘strategies’ are settled in order to understand complex biological systems from an integrated (‘holistic’) point of view. Metabolomics measurements are hence correlated with the time-dependent changes in concentrations of other components (proteins, gene-expression data), in order to obtain an integrated model of the gene-protein-metabolite interactions. Such framework represents a meaningful discontinuity with respect to the reductionist and qualitative molecular biology, and discloses new perspective to scientific researc