The effect of a dietary nitrate supplementation in the form of a single shot of beetroot juice on static and dynamic apnoea performance

Introduction: The purpose of the present study was to assess the effects of acute nitrate (NO3-)-rich beetroot juice supplementation on peripheral oxygen saturation (SpO2), heart rate (HR), and pulmonary gas exchange during submaximal static and dynamic apnoea. Methods: Nine (six male, three female) trained apneists (age: 39.6 ± 8.2 years, stature: 170.4 ± 11.5cm, body mass: 72.0 ± 11.5 kg) performed three submaximal static apnoeas at 60%, 70% and 80% of the participant’s current reported personal best time, followed by three submaximal (~ 75% or personal best distance) dynamic apnoeas following the consumption of either a 140 ml concentrated NO3--rich beetroot juice (BRJ; 7.7 mmol NO3-) or a NO3--depleted placebo (PLA; 0.1 mmol NO3-) in double-blind randomised manner. HR and SpO2 were measured via fingertip pulse oximetry at the nadir, and online gas analysis was used to assess pulmonary oxygen uptake (V̇O2) during recovery following breath-holds. Results: There were no differences (P <0.05) between conditions for HR (PLA = 59 ± 11 bpm and BRJ = 61 ± 12 bpm), SpO2 (PLA = 83 ± 14% and BRJ = 84 ± 9%) or V̇O2 (PLA = 1.00 ± 0.22 L.min-1 and BRJ = 0.97 ± 0.27 L.min-1). Conclusion: The consumption of 7.7mmol of beetroot juice supplementation prior to a series of submaximal static and dynamic apnoeas did not induce a significant change in SpO2, HR and V̇O2, when compared to placebo. Therefore there is no apparent physiological response that may benefit free-divers as a result of the supplementation

Cell biology of stress: Cytoplasmic rearrangements and signaling events

Recent reports show that formation of COPII coated vesicles and ER exit are regulated by kinases and that more generally, many components of the secretory pathway have been found phosphorylated. However the conditions under which these events occur are so far poorly understood. As the main aim of this thesis, we asked how the function and organization of the early secretory pathway respond to nutrient stress. In particular, we examine the behavior the components of the early secretory pathway under nutrient restriction, their contribution to other processes that are also regulated by the same stress (such as protein translation arrest and stress granule formation), and the nature of the signaling events regulating these responses. We follow up on the results of a kinase screen designed to identify regulators of the early secretory pathway and find that the extracellularly regulated kinase 7 (Erk7, also known as MAPK15) mediates the response of the ERES to serum starvation. These findings establish that the early secretory pathway is sensitive to nutrient signals, that pathways sensing nutrient abundance regulate its functional organization and that the key ERES component, Sec16 is the platform that integrates these signals. These results fuelled the further experiments to examine the behavior of the ERES components upon amino-acid starvation. We find that this response is even more dramatic than the one observed with serum deprivation. Indeed the ERES are remodeled into the Sec bodies, a novel, non-membrane bound, reversible structure that does not support protein transport and acts as a reservoir for ERES components. We find that the formation of Sec bodies is critical to cell survival and re-adaptation to normal growth conditions after the stress is relieved. Our quest to understand the nature of this novel structure reveals that it behaves like a liquid droplet, linking it to the cytoplasmic reorganization that occurs during stress, a well-documented manifestation of which is the assembly of stress granules. The similarities we observed between the Sec bodies and the stress granules prompted us to investigate the hypothesis that the formation of two structures is somehow linked. Indeed, we find that even though Sec bodies and stress granules are distinct both morphologically and functionally the specific ERES components that are required for Sec body assembly are also necessary for stress granule formation. These observations reveal a so far unexplored link between ER exit and mRNA sorting and turnover. Furthermore we study the stress granule assembly under a different type of stress, heat exposure, which does not affect the early secretory pathway. We find that TORC2 signaling is required for their formation. Having characterized the heat sensitivity phenotype of Rictor mutant flies we set out to understand the molecular mechanisms of this phenotype. Using S2 cells initially and then confirming our observations in Drosophila tissues we show that during heat stress TORC2 mediates the assembly of stress granules, possibly via its effector, Akt

Phospho-Rasputin Stabilization by Sec16 Is Required for Stress Granule Formation upon Amino Acid Starvation

Most cellular stresses induce protein translation inhibition and stress granule formation. Here, using Drosophila S2 cells, we investigate the role of G3BP/Rasputin in this process. In contrast to arsenite treatment, where dephosphorylated Ser142 Rasputin is recruited to stress granules, we find that, upon amino acid starvation, only the phosphorylated Ser142 form is recruited. Furthermore, we identify Sec16, a component of the endoplasmic reticulum exit site, as a Rasputin interactor and stabilizer. Sec16 depletion results in Rasputin degradation and inhibition of stress granule formation. However, in the absence of Sec16, pharmacological stabilization of Rasputin is not enough to rescue the assembly of stress granules. This is because Sec16 specifically interacts with phosphorylated Ser142 Rasputin, the form required for stress granule formation upon amino acid starvation. Taken together, these results demonstrate that stress granule formation is fine-tuned by specific signaling cues that are unique to each stress. These results also expand the role of Sec16 as a stress response protein

TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules

The kinase TORis found in two complexes, TORC1,which is involved in growth control, and TORC2, whose roles are less well defined. Here, we asked whether TORC2 has a role in sustaining cellular stress. We show that TORC2 inhibition in Drosophila melanogaster leads to a reduced tolerance to heat stress, whereas sensitivity to other stresses is not affected. Accordingly, we show that upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for maintaining the level of its known target, Akt1 (also known as PKB). We show that the phosphorylation of the stress-activated protein kinases is not modulated by TORC2 nor is the heat-induced upregulation of heat-shock proteins. Instead, we show, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears to be less efficient in the absence of TORC2 function.We propose that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules

Phospho-rasputin stabilization by Sec16 is required for stress granule formation upon amino acid starvation

Most cellular stresses induce protein translation inhibition and stress granule formation. Here, using Drosophila S2 cells, we investigate the role of G3BP/Rasputin in this process. In contrast to arsenite treatment, where dephosphorylated Ser142 Rasputin is recruited to stress granules, we find that, upon amino acid starvation, only the phosphorylated Ser142 form is recruited. Furthermore, we identify Sec16, a component of the endoplasmic reticulum exit site, as a Rasputin interactor and stabilizer. Sec16 depletion results in Rasputin degradation and inhibition of stress granule formation. However, in the absence of Sec16, pharmacological stabilization of Rasputin is not enough to rescue the assembly of stress granules. This is because Sec16 specifically interacts with phosphorylated Ser142 Rasputin, the form required for stress granule formation upon amino acid starvation. Taken together, these results demonstrate that stress granule formation is fine-tuned by specific signaling cues that are unique to each stress. These results also expand the role of Sec16 as a stress response protein

Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae

Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes