Glycolytic flux-signaling controls mouse embryo mesoderm development
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PROVIDER: PRJEB55095 | ENA |
REPOSITORIES: ENA
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Project description:How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here, we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). Overexpression of cytoPFKFB3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPFKFB3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.
| S-EPMC9771359 | biostudies-literature
Project description:How metabolism is rewired during embryonic development is still largely unknown, as it remains a major technical challenge to resolve metabolic activities or metabolite levels with spatiotemporal resolution. Here, we investigated metabolic changes during development of organogenesis-stage mouse embryos, focusing on the presomitic mesoderm (PSM). We measured glycolytic labeling kinetics from 13C-glucose tracing experiments and detected elevated glycolysis in the posterior, more undifferentiated PSM. We found evidence that the spatial metabolic differences are functionally relevant during PSM development. To enable real-time quantification of a glycolytic metabolite with spatiotemporal resolution, we generated a pyruvate FRET-sensor reporter mouse line. We revealed dynamic changes in cytosolic pyruvate levels as cells transit toward a more anterior PSM state. Combined, our approach identifies a gradient of glycolytic activity across the PSM, and we provide evidence that these spatiotemporal metabolic changes are intrinsically linked to PSM development and differentiation.
| S-EPMC5337618 | biostudies-literature
Project description:Metabolic heterogeneity modulates productivity, antibiotic resistance and cancer aggressiveness. Since metabolic fluxes represent the functional output of metabolism, with glycolytic flux correlating with highly-productive phenotypes and cancer, such flux map will be indicative of the cellular metabolic state. Therefore, the quantification of metabolic fluxes is vital to identify the existence of metabolic subpopulations and to understand the process of their emergence at the single-cell level. However, so far inference of metabolic fluxes in individual cells is not possible as no method is available. Here, we developed a biosensor for glycolytic flux measurements in single yeast cells drawing on the robust correlation between fructose-1,6-bisphosphate (FBP) and flux levels in yeast, and using the B. subtilis FBP-binding transcription factor CggR. We followed a systematic engineering approach starting from promoter design, computational protein design and protein engineering, accompanied by strict characterization of the biosensor using different biochemical methods, proteomics, metabolomics and physiological analyses. As proof of principle, we applied the biosensor in vivo in the search for metabolic subpopulations in yeast cultures and, using fluorescence microscopy, we demonstrated that quiescent yeast cells have low glycolytic fluxes in comparison to coexisting dividing cells. We anticipate that our biosensor will contribute with unprecedented resolution for the study of metabolic subpopulations, to understand how and why metabolic subpopulations emerge and, very importantly, give clues on how to counteract the undesirable effects of such.
2019-12-17 | PXD012964 | Pride
Project description:Following UV irradiation of skin, dendritic cells (DCs) differentiating from the bone marrow (BM) of mice have a reduced ability to prime new immune responses; their reduced immunogenicity is maintained for at least 16 weeks in UV-chimeric mice. We hypothesized that different metabolic states underpin changes in DC function. Compared with DCs from the BM of non-irradiated mice, DCs from the BM of UV-irradiated mice produced more lactate and utilized greater amounts of glucose, a profile that was supported by greater glycolytic flux when incubated in low-serum-containing medium. Responses to a mitochondrial stress test were similar suggesting that the DCs from the BM of UV-irradiated mice had not switched from a profile of oxidative phosphorylation, but were imprinted for greater glycolytic responses. After microarray profiling, RT-qPCR confirmation and Ingenuity pathway analysis, greater expression of the enzyme, 3-hydroxyanthranilate 3,4-dioxygenase, was identified as a potential contributor to increased glycolysis by BM-differentiated DCs. This enzyme provides the final step of the biosynthetic pathway from tryptophan to quinolinate, the universal de novo precursor to the pyridine ring of nicotinamide adenine dinucleotide (NAD), and may provide a mechanism to ensure sufficient NAD is available to support enhanced glycolysis. Increased lactate production was also measured for DCs from the BM of 16-week engrafted UV-chimeric mice and suggests long-lasting imprinting of progenitor cells for altered immunometabolism in their progeny cells. This study provides evidence of changes to metabolic states that associate with altered DC function.
2015-10-30 | GSE74468 | GEO
Project description:Following UV irradiation of skin, dendritic cells (DCs) differentiating from the bone marrow (BM) of mice have a reduced ability to prime new immune responses; their reduced immunogenicity is maintained for at least 16 weeks in UV-chimeric mice. We hypothesized that different metabolic states underpin changes in DC function. Compared with DCs from the BM of non-irradiated mice, DCs from the BM of UV-irradiated mice produced more lactate and utilized greater amounts of glucose, a profile that was supported by greater glycolytic flux when incubated in low-serum-containing medium. Responses to a mitochondrial stress test were similar suggesting that the DCs from the BM of UV-irradiated mice had not switched from a profile of oxidative phosphorylation, but were imprinted for greater glycolytic responses. After microarray profiling, RT-qPCR confirmation and Ingenuity pathway analysis, greater expression of the enzyme, 3-hydroxyanthranilate 3,4-dioxygenase, was identified as a potential contributor to increased glycolysis by BM-differentiated DCs. This enzyme provides the final step of the biosynthetic pathway from tryptophan to quinolinate, the universal de novo precursor to the pyridine ring of nicotinamide adenine dinucleotide (NAD), and may provide a mechanism to ensure sufficient NAD is available to support enhanced glycolysis. Increased lactate production was also measured for DCs from the BM of 16-week engrafted UV-chimeric mice and suggests long-lasting imprinting of progenitor cells for altered immunometabolism in their progeny cells. This study provides evidence of changes to metabolic states that associate with altered DC function. Female C57BL/6J (CD45.2 alloantigen) and B6.SJL-Ptprca (CD45.1 alloantigen) mice were obtained from the Animal Resources Centre (Murdoch, Western Australia). A bank of TL40W/12RS lamps (Philips, Amsterdam, The Netherlands) emitting broadband UVR with 65% UVB (280-320 nm) and peak emission at 313 nm was used. Twenty-four h prior to irradiation, a uniform area of dorsal skin of mice was shaved (8 cm2). To administer UVR, mice were held in perspex compartments which were covered with 0.2 mm polyvinyl chloride plastic to eliminate wavelengths <290 nm. The compartments were placed 20 cm beneath the UV lamps and up to 8 kJ/m2 UVR was delivered. Eight kJ/m2 UVR is equivalent to 3-4 minimal erythemal doses . The UVB output by the lamps was measured using a UVX radiometer (Ultraviolet Products Inc., Upland, CA). Mice were 6-10 wk old at the time of irradiation unless otherwise stated. Mice were subcutaneously implanted with two PGE2 pellets, each containing 0.1 mg with a constant release of 4.76 µg/day over 21 days (total 9.52 µg/day, Innovative Research of America, Sarasota, FL), three days prior to isolation of BM cells. The pellets were inserted into the loose skin at the top of the back. Freshly-isolated BM cells were cultured for 5 days in RPMI-1640 (HyClone, GE Health Care Life Sciences, Logan, UT) supplemented with 10% FCS (RPMI-10), 10 ng/ml GM-CSF and 10 ng/ml IL-4 (Peprotech Inc, Rocky Hill, NJ) at a density of 8 x 105 cells/ml in 24 well plates to promote CD11c+ cell differentiation (medium replaced on days 2 and 4). Non-adherent cells were enriched to >95% CD11c+ cells (confirmed by flow cytometry) using anti-CD11c magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and autoMACS-Pro (Miltenyi Biotec) separation. Total RNA was extracted from triplicate preparations of BM-differentiated CD11c+ cells. Two sample groups; BM-differentiated DC sample from C57BL/6J mice injected with 2 x 10^6 BM cells from a naïve congenic B6.SJL-Ptprca mouse BM-differentiated DC sample from C57BL/6J mice injected with 2 x 10^6 BM cells from a UV irradiated congenic B6.SJL-Ptprca mouse
2015-10-30 | E-GEOD-74468 | biostudies-arrayexpress
Project description:Adrenal Abcg1 controls cholesterol flux and steroidogenesis
| PRJNA1011305 | ENA
Project description:Metabolic fluxes may be regulated "hierarchically," e.g., by changes of gene expression that adjust enzyme capacities (V(max)) and/or "metabolically" by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the approximately 10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50-20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75-100% of the regulation of protein levels. Keywords: Condition comparison
2007-10-17 | GSE9232 | GEO
Project description:Metabolism is tightly coupled with the process of aging, and tumorigenesis. However, the mechanisms regulating metabolic properties in different contexts remain unclear. Cellular senescence is widely recognized as an important tumor suppressor function and accompanies metabolic remodeling characterized by increased mitochondrial oxidative phosphorylation (OXPHOS). Here we showed retinoblastoma (RB) is required for the increased OXPHOS in oncogene-induced senescent (OIS) cells. Combined metabolic and gene expression profiling revealed that RB mediated activation of the glycolytic pathway in OIS cells, causing upregulation of several glycolytic genes and concomitant increases in the levels of associated metabolites in the glycolytic pathway. Knockdown of these genes by small interfering RNAs (siRNAs) resulted in decreased mitochondrial respiration, suggesting that RB-mediated glycolytic gene activation promotes metabolic flux into the OXPHOS pathway. These results suggest that coordinate transcriptional activation of metabolic genes by RB enables OIS cells to maintain metabolically bivalent states that both glycolysis and OXPHOS are highly active. Collectively, our findings demonstrated a previously unrecognized function of RB in OIS cells. To understand the role of RB, we investigated the effect of RB1-knockdown in the transcription profile of oncogene-induced senescent (OIS) cells. IMR90 ER:Ras cells were treated with 100 nM 4-OHT for 6 days to induce senescence. RNA was isolated 6 days after OHT treatment and hybridized to Affymetrix microarrays. SiRNA transfection (control siRNA or siRB1) was performed 4 days before RNA isolation.
2015-05-26 | E-GEOD-60652 | biostudies-arrayexpress
Project description:Circadian clock and Smad2/3/4-mediated Nodal signaling regulate multiple physiological and pathological processes. However, it remains unknown whether Clock directly cross-talks with Nodal signaling and how this would regulate embryonic development. Here we show that Clock1a coordinated mesoderm development and primitive hematopoiesis in zebrafish embryos by directly up-regulating Nodal-Smad3 signaling. We found that Clock1a is expressed both maternally and zygotically throughout early zebrafish development. We also noted that Clock1a alterations produce embryonic defects with shortened body length, lack of the ventral tail fin, or partial defect of the eyes. Clock1a regulates the expression of the mesodermal markers ntl, gsc, and eve1 and of the hematopoietic markers scl, lmo2, and fli1a Biochemical analyses revealed that Clock1a stimulates Nodal signaling by increasing expression of Smad2/3/4. Mechanistically, Clock1a activates the smad3a promoter via its E-box1 element (CAGATG). Taken together, these findings provide mechanistic insight into the role of Clock1a in the regulation of mesoderm development and primitive hematopoiesis via modulation of Nodal-Smad3 signaling and indicate that Smad3a is directly controlled by the circadian clock in zebrafish.
| S-EPMC5572899 | biostudies-other
Project description:Metabolic fluxes may be regulated "hierarchically," e.g., by changes of gene expression that adjust enzyme capacities (V(max)) and/or "metabolically" by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the approximately 10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50-20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75-100% of the regulation of protein levels. Experiment Overall Design: To quantify the regulation of the Vmax values and the fluxes at the different levels of gene expression, we measured how the fluxes through the glycolytic enzymes, the Vmax values, and the concentrations of these enzymes and their corresponding mRNA concentrations change when yeast is exposed to aerobic and anaerobic (with and without challenges.
2008-06-16 | E-GEOD-9232 | biostudies-arrayexpress