Metabolic Plasticity Enables Circadian Adaptation to Acute Hypoxia in Zebrafish Cells.

BACKGROUND/AIMS: Reduced oxygen availability, hypoxia, is frequently encountered by organisms, tissues and cells, in aquatic environments as well as in high altitude or under pathological conditions such as infarct, stroke or cancer. The hypoxic signaling pathway was found to be mutually intertwined with circadian timekeeping in vertebrates and, as reported recently, also in mammals. However, the impact of hypoxia on intracellular metabolic oscillations is still unknown. METHODS: For determination of metabolites we used Multilabel Reader based fluorescence and luminescence assays, circadian levels of Hypoxia Inducible Factor 1 alpha and oxidized peroxiredoxins were semi quantified by Western blotting and ratiometric quantification of cytosolic and mitochondrial H2O2 was achieved with stable transfections of a redox sensitive green fluorescent protein sensor into zebrafish fibroblasts. Circadian oscillations of core clock gene mRNA´s were assessed using realtime qPCR with subsequent cosine wave fit analysis. RESULTS: Here we show that under normoxia primary metabolic activity of cells predominately occurs during day time and that after acute hypoxia of two hours, administrated immediately before each sampling point, steady state concentrations of glycolytic key metabolites such as glucose and lactate reveal to be highly rhythmic, following a circadian pattern with highest levels during the night periods and reflecting the circadian variation of the cellular response to hypoxia. Remarkably, rhythms in glycolysis are transferred to cellular energy states under normoxic conditions, so that ADP/ATP ratios oscillate as well, which is the first evidence for cycling ADP/ATP pools in a metazoan cell line to our knowledge. Furthermore, the hypoxia induced alterations in rhythms of glycolysis lead to the alignment of three major cellular redox systems, namely the circadian oscillations of NAD+/NADH and NADP+/NADPH ratios and of increased nocturnal levels of oxidized peroxiredoxins, resulting in a highly oxidized nocturnal cellular environment. Of note, circadian rhythms of cytosolic H2O2 remain unaltered, while the transcriptional clock is already attenuated, as it is known to occur also under chronic hypoxia. CONCLUSION: We therefor propose that the realignment of metabolic redox oscillations might initiate the observed hypoxia induced attenuation of the transcriptional clock, based on the reduced binding affinity of the CLOCK/BMAL complex to the DNA in an oxidized environment.

Follicular growth after xenotransplantation of cryopreserved/thawed human ovarian tissue in SCID mice: dynamics and molecular aspects.

PURPOSE: To study the influence of xenotransplantation on follicular recruitment and growth in cryopreserved/thawed human ovarian tissue. METHOD: Two 3-mm pieces of cryopreserved/thawed human ovarian tissue obtained from female cancer patients (n = 11) were xenotransplanted into a subcutaneous neck pouch of 6-week-old ovarectomized SCID mice (n = 33) for 4 (n = 18) and 12 (n = 15) weeks. RESULT: Thirty-two out of 33 mice survived the entire observation periods. Graft recovery rate was 95.58 % (65 of 68 grafts). The percentages of primordial follicles after 4 weeks (P < 0.001) and 12 weeks (P = 0.009) of grafting were significantly lower in comparison to pregraft controls. The percentage of secondary follicle was significantly higher after 4 weeks of grafting (P = 0.018) and after 12 weeks (P = 0.001) of grafting in comparison to pregraft controls. Ki67 immunohistochemistry showed that proliferative follicles were significantly higher after 4 and 12 weeks of grafting compared to pregraft controls (P < 0.001). All follicles analyzed by TUNEL staining appeared healthy after xenotransplantation. The expression level of PTEN was reduced by 2.47-fold after 4 weeks of xenotransplantation, and this result was significant when 2-ΔCt were analyzed (P = 0.042). CONCLUSION: The higher proportion of growing follicles compared to resting follicles observed after xenotransplantation is most likely due to downregulation of PTEN gene expression followed by acceleration of follicular recruitment.

Chronodisruption increases cardiovascular risk in zebrafish via reduced clearance of senescent erythrocytes.

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in pathogenesis. The bi-directionality of the interaction between both pathways has been shown on physiological and only recently also on molecular level. But the consequences of a disturbed circadian rhythm for the hypoxic response and the cardiovascular system have never been addressed in any organism. Here we show that the hypoxic response of animals subjected to chronodisruption is reduced by approximately 30%, as reflected by decreased expression levels of hypoxia inducible factor 1 and its down-stream target genes erythropoietin, responsible for the generation of red blood cells (RBC) and vascular endothelial growth factor, which is essential for proper vascularization. Beside malformations of their vascular beds, chronodisrupted animals surprisingly revealed elevated numbers of senescent erythrocytes under normoxic conditions, due to a reduced clearance rate via apoptosis. Over-aged erythrocytes in turn are characterized by decreased oxygen transport capacities and an increased tendency for aggregation, explaining the higher mortality of chronodisrupted animals observed in our study. The present study shows for the first time that chronodisruption strongly interferes with the hypoxic signalling cascade, increasing the cardiovascular risk in zebrafish due to elevated proportions of senescent erythrocytes. The results might shed new light on the etiology of the increased cardiovascular risk observed among shiftworkers.