Fructose-1,6-bisphosphate couples glycolytic flux to activation of Ras

Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1Δ) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency. © 2017 The Author(s)

The creatine kinase system and pleiotropic effects of creatine

The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure–function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans

Regulation of Ras by Glycolytic Flux: Screen for interfering compounds and physiological relevance

In this work, we further develop our hypothesis, which poses that high-rate glycolytic metabolism stimulates Ras activation, via the highly controlled glycolytic intermediate fructose-1,6-bisphosphate (Fru1,6bisP). It is well established that oncogenic Ras activity stimulates the glycolytic flux. Hence, Fru1,6bisP-induced Ras activation might close a reciprocal stimulatory/feedback loop that locks cancer metabolism in a state that favors lactate fermentation and rapid cell proliferation. In order to study the physiological relevance of Fru1,6bisP-induced Ras activation, we first studied possible correlations between Fru1,6bisP levels, Ras activation levels and growth rate in yeast cells. Next, using the human H-RAS protein and its activator SOS1, we further elaborate on the molecular mechanism of this interaction, and show that Fru1,6bisP interacts with SOS1 to increase the dissociation rate of the H-RAS/SOS1 complex, within physiologically relevant concentrations of Fru1,6bisP. Further highlighting the evolutionary conservation of this mechanism, we show that glucose triggers Ras activation in human embryonic kidney cells and cancer cells, just like in yeast cells. Anticipating the clinical relevance of this mechanism, we identified small molecules that might interfere with Fru1,6bisP-induced Ras activation, and could thus be used for the development of anti-cancer drugs. Finally, we use a specific yeast mutant (i.e. tps1∆) to study why sugar phosphates upstream of the glycolytic enzyme GAPDH, especially Fru1,6bisP, continue to hyperaccumulate when glucose triggers an apoptotic response in this mutant. We show that inadequate pH homeostasis in the tps1∆ strain is closely correlated with the steep pH dependency of GAPDH, and that preventing intracellular acidification is sufficient to lower the extent of Fru1,6bisP accumulation. Finally, we consolidate the results obtained in this dissertation with the existing literature, and elaborate on how we view the relevance of our hypothesis for the study and targeting of cancer metabolism. In addition, we revisit the assumption that intracellular phosphate depletion is the cause of the glycolytic deregulation in the tps1∆ glucose growth defect, and instead discuss a causal role of defective cytosolic pH homeostasis.status: publishe

The biomechanic origin of sprint performance enhancement after one-week creatine supplementation.

In order to test whether an improvement of maximal sprinting speed after creatine (Cr) supplementation was due to the increase of stride frequency (SF), stride length (SL) or both, 7 subjects ran 4 consecutive sprints after 1 week of placebo or Cr supplementation. SF and SL were assessed by a triaxial accelerometer. Compared to the placebo, Cr induced an increase of running speed (+1.4% p < 0.05) and SF (+1.5%, p < 0.01), but not of SL. The drop in performance following repeated sprints was partially prevented by Cr. In conclusion, exogenous Cr enhanced sprinting performance by increasing SF. This result may be related to the recent findings of shortening in muscular relaxation time after Cr supplementation

Efeitos da suplementação de creatina na força máxima e na amplitude do eletromiograma de mulheres fisicamente ativas Effect of creatine supplementation in maximal strength and electromyogram amplitude of physically active women

A suplementação de creatina apresenta ação ergogênica na força muscular. Entretanto, não há consenso deste efeito na força isométrica máxima e na amplitude do eletromiograma (EMG). Assim, o objetivo deste estudo foi analisar os efeitos da suplementação de creatina na força isométrica máxima e na amplitude do EMG em mulheres fisicamente ativas. Vinte e sete mulheres (idade 23,04 ± 1,82 anos, massa corporal 58,37 ± 6,10kg, estatura 1,63 ± 0,05m e índice de massa corporal 21,93 ± 2,02kg/m²) foram designadas aleatoriamente para os grupos creatina (GCr) (n = 13) e placebo (GPL) (n = 14), os quais ingeriram diariamente, durante seis dias, 20g de creatina mono-hidratada e 20g de maltodextrina, respectivamente. Antes e depois da suplementação, a força foi medida em um dinamômetro isométrico durante contração isométrica voluntária máxima (CIVM) de extensão unilateral do joelho (três séries de 6s intervaladas por 180s), com captação simultânea dos valores root mean square (RMS) do EMG obtido no músculo vasto lateral. A ANOVA de dois critérios de classificação (dois momentos x dois grupos) e o teste de Wilcoxon foram utilizados na análise estatística dos dados paramétricos e não paramétricos (p < 0,05). Após a suplementação, o GCr aumentou significativamente a força, com incrementos de 7,85% (p = 0,002), 7,31% (p = 0,001) e 5,52% (p = 0,001) para a primeira, segunda e terceira séries, respectivamente. Para este mesmo grupo, os valores RMS aumentaram significativamente na terceira série (p = 0,026). O GPL não apresentou alterações significativas. Os resultados sugerem que a suplementação de creatina aumenta a força isométrica máxima e que a amplitude do EMG pode ser utilizada como indicador dessas alterações de desempenho.<br>Creatine supplementation has shown to enhance muscular strength. However, there is not a consensus on this effect on maximal isometric strength neither on electromyogram (EMG) amplitude. Thus, the aim of this study was to analyze the creatine supplementation effects on maximal isometric strength and EMG amplitude in physically active women. 27 women (age 23.04 ± 1.82 years, body mass 58.37 ± 6.10kg, height 1.63±0.05m and body mass index 21.93 ± 2.02kg/m²) were randomly assigned in creatine (CrG) (n = 13) or placebo group (PLG) (n = 14). The CrG and PLG ingested 20g/day of creatine and 20g/day of maltodextrin during six days, respectively. The strength was measured before and after supplementation using a isometric dynamometer during a maximal isometric voluntary contraction (MIVC) of a unilateral knee extension (3 sets of 6s with 180s rest period), and the EMG was acquired from the vastus lateralis muscle and its amplitude quantified using the root mean square (RMS) values. Two-way ANOVA (2 groups x 2 moments) was used for parametric data and Wilcoxon test for non-parametric analysis (p < 0.05). After supplementation, CrG significantly enhanced strength, with increase of 7.85% (p = 0.002); 7.31% (p = 0.001) and 5.52% (p = 0.000) for the first, second and third trials, respectively. In CrG, the RMS values significantly increased on the third trial (p = 0.026). No changes were found in GPL. Results suggest that creatine supplementation enhance maximal isometric strength and EMG amplitude can be useful to identify these performance modifications