Flux profile and modularity analysis of time-dependent metabolic changes of de novo adipocyte formation

2007 ◽  
Vol 292 (6) ◽  
pp. E1637-E1646 ◽  
Author(s):  
Yaguang Si ◽  
Jeongah Yoon ◽  
Kyongbum Lee
Keyword(s):  
Metabolic Flux ◽  
Type I Collagen ◽  
De Novo ◽  
Cell Number ◽  
Pentose Phosphate ◽  
Gene Profiling ◽  
Flux Analysis ◽  
Type I ◽  
White Adipocyte ◽  
Modularity Analysis

White adipose tissue (WAT) mass is the main determinant of obesity and associated health risks. WAT expansion results from increases in white adipocyte cell number and size, which in turn reflect a series of shifts in the cellular metabolic state. To quantitatively profile the metabolic alterations occurring during de novo adipocyte formation, metabolic flux analysis (MFA) was used in conjunction with a novel modularity analysis algorithm on differentiating 3T3-L1 preadipocytes. Use of a type I collagen gel as an effective long-term culture substrate was also assessed. The calculated flux distributions predicted the sequential activation of several intracellular cross-compartmental pathways, including lipogenesis, the pentose phosphate pathway, and the malate cycle, in good agreement with earlier isotopic tracer experiments and gene profiling studies. Partition of the adipocyte metabolic network into highly interacting reaction subgroups suggested a functional reorganization of the major pathways consistent with the lipid-loading phenotype of the adipocyte. Flux and modularity analysis results together point to the flux distribution around pyruvate as a key indicator of adipocyte lipid accumulation.

scholarly journals Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

68 Flux analysis of aerobic glycolysis in bovine blastocysts and CT1 cells

2019 ◽  
Vol 31 (1) ◽  
pp. 159
Author(s):  
J. Chung ◽  
R. Clifford ◽  
G. Sriram ◽  
C. Keefer
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Metabolic Flux ◽  
Aerobic Glycolysis ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Flux Analysis ◽  
Metabolic Modelling ◽  
Chromatography Mass Spectrometry

Embryo quality and maternal recognition are crucial for successful initiation of bovine pregnancy. Previous studies have proposed that better quality embryos use aerobic glycolysis to meet a high demand for biomass components. While hexoses are the principal carbon sources that provide energy to glycolysis, little is known about partitioning of hexoses into metabolic pathways or alteration of partitioning when different hexoses are simultaneously available. Specific metabolic utilisation of 13C-labelled substrates can be quantified by gas chromatography-mass spectrometry, an excellent noninvasive approach for studying cellular metabolism. To assess hexose flux through central metabolism, bovine blastocysts and CT1 cells (a bovine trophectoderm cell line) were cultured in SOF-based media supplemented with combinations of 50% uniformly labelled (U) and 50% naturally abundant (NA) glucose (Glc) or fructose (Fru) (U−13C Glc+NA Glc, U−13C Fru+NA Fru, U−13C Glc+NA Fru, and U−13C Fru+NA Glc), such that total hexose concentration was 1.5mM. Metabolites in spent media from 24-h cultures of single or 5 blastocysts (40-μL drops; 5% CO2, 5% O2, 90% N2) and 1-, 2-, 3-, 6-, 8-, and 24-h incubations of CT1 cells (150 μL; ~3×104 cells per well; 5% CO2, 95% air) were extracted with a MeOH-CHCl3 reagent, derivatized, and analysed by gas chromatography-mass spectrometry. Measurement of mass isotopomer distributions of metabolites, chiefly pyruvate, lactate, and amino acids, followed by correction for natural abundances and metabolic modelling, revealed several insights. For instance, five Day 7 or Day 8 blastocysts (Day 0=fertilization) supplied with U−13C Glc+NA Fru displayed 13C enrichments of 80.3%±1.4% for pyruvate and 71.6%±2.8% for lactate, whereas when supplied with U−13C Fru+NA Glc, they displayed lower 13C enrichments of 5.7%±2.4% for pyruvate and 2.8%±0.4% lactate (mean±standard deviation, n=3 to 4). Metabolic modelling revealed that when Glc and Fru are simultaneously available, the blastocysts used 2.5±0.2 moles of Fru per 100 moles of Glc used. Furthermore, 13C enrichment of pyruvate was 42.0±0.6% when U−13C Glc+NA Glc was supplied and 37.8±2.7% when U−13C Fru+NA Fru was supplied. Lactate enrichments followed a similar trend. This indicates that, individually, Glc and Fru were utilised majorly through aerobic glycolysis with some involvement of the pentose phosphate pathway. Alanine was negligibly labelled in all of the experiments, suggesting either a low TCA flux or that alanine is diluted by extra- or intracellular amino or fatty acids. Single blastocysts and CT1 cells showed a similar labelling pattern when hexoses were available. Following Glc depletion at 8h in CT1 cultures, the 13C enrichments of alanine and citrate in the media increased, suggesting a sharp alteration of metabolic state. These findings demonstrate that metabolic flux can be comprehensively analysed for single bovine blastocysts and CT1 cell metabolism models that of the blastocyst. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2015-67015-23237 from the USDA National Institute of Food and Agriculture.

scholarly journals Endothelial cell crosstalk improves browning but hinders white adipocyte maturation in 3D engineered adipose tissue

2020 ◽  
Vol 12 (4) ◽  
pp. 81-89
Author(s):  
Jennifer H Hammel ◽  
Evangelia Bellas
Keyword(s):  
Endothelial Cells ◽  
Adipose Tissue ◽  
Type I Collagen ◽  
Network Formation ◽  
Uncoupling Protein ◽  
Collagen Gel ◽  
Cell Types ◽  
Vascular Network ◽  
Type I ◽  
White Adipocyte

Abstract Central to the development of adipose tissue (AT) engineered models is the supporting vasculature. It is a key part of AT function and long-term maintenance, but the crosstalk between adipocytes and endothelial cells is not well understood. Here, we directly co-culture the two cell types at varying ratios in a 3D Type I collagen gel. Constructs were evaluated for adipocyte maturation and function and vascular network organization. Further, these constructs were treated with forskolin, a beta-adrenergic agonist, to stimulate lipolysis and browning. Adipocytes in co-cultures were found to be less mature than an adipocyte-only control, shown by smaller lipid droplets and downregulation of key adipocyte-related genes. The most extensive vascular network formation was found in the 1:1 co-culture, supported by vascular endothelial growth factor (VEGF) upregulation. After forskolin treatment, the presence of endothelial cells was shown to upregulate PPAR coactivator 1 alpha (PGC-1α) and leptin, but not uncoupling protein 1 (UCP1), suggesting a specific crosstalk that enhances early stages of browning.

scholarly journals Human platelet-rich plasma improves the nesting and differentiation of human chondrocytes cultured in stabilized porous chitosan scaffolds

2017 ◽  
Vol 8 ◽  
pp. 204173141769754 ◽  
Author(s):  
Maria Sancho-Tello ◽  
Sara Martorell ◽  
Manuel Mata Roig ◽  
Lara Milián ◽  
MA Gámiz-González ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Articular Chondrocytes ◽  
Platelet Rich Plasma ◽  
Cartilage Regeneration ◽  
Cell Number ◽  
Type I ◽  
Type Ii ◽  
Cell Counts ◽  
Porous Chitosan ◽  
Chitosan Scaffolds

The clinical management of large-size cartilage lesions is difficult due to the limited regenerative ability of the cartilage. Different biomaterials have been used to develop tissue engineering substitutes for cartilage repair, including chitosan alone or in combination with growth factors to improve its chondrogenic properties. The main objective of this investigation was to evaluate the benefits of combining activated platelet-rich plasma with a stabilized porous chitosan scaffold for cartilage regeneration. To achieve this purpose, stabilized porous chitosan scaffolds were prepared using freeze gelation and combined with activated platelet-rich plasma. Human primary articular chondrocytes were isolated and cultured in stabilized porous chitosan scaffolds with and without combination to activated platelet-rich plasma. Scanning electron microscopy was used for the morphological characterization of the resulting scaffolds. Cell counts were performed in hematoxylin and eosin–stained sections, and type I and II collagen expression was evaluated using immunohistochemistry. Significant increase in cell number in activated platelet-rich plasma/stabilized porous chitosan was found compared with stabilized porous chitosan scaffolds. Chondrocytes grown on stabilized porous chitosan expressed high levels of type I collagen but type II was not detectable, whereas cells grown on activated platelet rich plasma/stabilized porous chitosan scaffolds expressed high levels of type II collagen and type I was almost undetectable. In summary, activated platelet-rich plasma increases nesting and induces the differentiation of chondrocytes cultured on stabilized porous chitosan scaffolds.

scholarly journals Identification and characterization of a fibroblast marker: FSP1.

1995 ◽  
Vol 130 (2) ◽  
pp. 393-405 ◽  
Author(s):  
F Strutz ◽  
H Okada ◽  
C W Lo ◽  
T Danoff ◽  
R L Carone ◽  
...  
Keyword(s):  
Cell Fate ◽  
Calcium Binding ◽  
Type I Collagen ◽  
De Novo ◽  
Mesangial Cells ◽  
In Vivo Studies ◽  
Type I ◽  
Tubular Epithelium ◽  
Collagen Gels

We performed subtractive and differential hybridization for transcript comparison between murine fibroblasts and isogenic epithelium, and observed only a few novel intracellular genes which were relatively specific for fibroblasts. One such gene encodes a filament-associated, calcium-binding protein, fibroblast-specific protein 1 (FSP1). The promoter/enhancer region driving this gene is active in fibroblasts but not in epithelium, mesangial cells or embryonic endoderm. During development, FSP1 is first detected by in situ hybridization after day 8.5 as a postgastrulation event, and is associated with cells of mesenchymal origin or of fibroblastic phenotype. Polyclonal antiserum raised to recombinant FSP1 protein stained the cytoplasm of fibroblasts, but not epithelium. Only occasional cells stain with specific anti-FSP1 antibodies in normal parenchymal tissue. However, in kidneys fibrosing from persistent inflammation, many fibroblasts could be identified in interstitial sites of collagen deposition and also in tubular epithelium adjacent to the inflammatory process. This pattern of anti-FSP1 staining during tissue fibrosis suggests, as a hypothesis, that fibroblasts in some cases arise, as needed, from the local conversion of epithelium. Consistent with this notion that FSP1 may be involved in the transition from epithelium to fibroblasts are experiments in which the in vitro overexpression of FSP1 cDNA in tubular epithelium is accompanied by conversion to a mesenchymal phenotype, as characterized by a more stellate and elongated fibroblast-like appearance, a reduction in cytokeratin, and new expression of vimentin. Similarly, tubular epithelium submerged in type I collagen gels exhibited the conversion to a fibroblast phenotype which includes de novo expression of FSP1 and vimentin. Use of the FSP1 marker, therefore, should further facilitate both the in vivo studies of fibrogenesis and the mapping of cell fate among fibroblasts.

scholarly journals Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

2013 ◽  
Vol 79 (7) ◽  
pp. 2336-2348 ◽  
Author(s):  
Tanja Hanke ◽  
Katharina Nöh ◽  
Stephan Noack ◽  
Tino Polen ◽  
Stephanie Bringer ◽  
...  
Keyword(s):  
Phase Ii ◽  
Growth Phase ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Glucose Dehydrogenase ◽  
Gluconobacter Oxydans ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Content Type ◽  
Activity Measurements

ABSTRACTIn this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes inGluconobacter oxydans621H with glucose were studied by13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For13C-MFA, cells were cultivated with specifically13C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. SinceG. oxydanslacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP).13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.

scholarly journals Extracellular Matrix Proteins Modulate Antimigratory and Apoptotic Effects of Doxorubicin

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Georges Said ◽  
Marie Guilbert ◽  
Hamid Morjani ◽  
Roselyne Garnotel ◽  
Pierre Jeannesson ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tumor Cells ◽  
Topoisomerase I ◽  
Type I Collagen ◽  
De Novo ◽  
Acquired Resistance ◽  
Chemotherapeutic Agents ◽  
Type I ◽  
Induced Apoptosis ◽  
Apoptotic Effects

Anticancer drug resistance is a multifactorial process that includes acquired and de novo drug resistances. Acquired resistance develops during treatment, while de novo resistance is the primary way for tumor cells to escape chemotherapy. Tumor microenvironment has been recently shown to be one of the important factors contributing to de novo resistance and called environment-mediated drug resistance (EMDR). Two forms of EMDR have been described: soluble factor-mediated drug resistance (SFM-DR) and cell adhesion-mediated drug resistance (CAM-DR). Anthracyclines, among the most potent chemotherapeutic agents, are widely used in clinics against hematopoietic and solid tumors. Their main mechanism of action relies on the inhibition of topoisomerase I and/or II and the induction of apoptosis. Beyond this well-known antitumor activity, it has been recently demonstrated that anthracyclines may display potent anti-invasive effects when used at subtoxic concentrations. In this paper, we will describe two particular modes of EMDR by which microenvironment may influence tumor-cell response to one of these anthracyclines, doxorubicin. The first one considers the influence of type I collagen on the antimigratory effect of doxorubicin (CAM-DR). The second considers the protection of tumor cells by thrombospondin-I against doxorubicin-induced apoptosis (SFM-DR).

scholarly journals Development and Validation of Fluorescently Labeled, Functional Type I Collagen Molecules

2021 ◽  
Author(s):  
Seyed Mohammad Siadat ◽  
Monica E Susilo ◽  
Jeffrey A Paten ◽  
Alexandra A Silverman ◽  
Charles A DiMarzio ◽  
...  
Keyword(s):  
Type I Collagen ◽  
De Novo ◽  
Type I ◽  
Growth And Remodeling ◽  
Fibril Structure ◽  
Collagen Molecules ◽  
Vitro Model ◽  
Development And Validation ◽  
Network Morphology

While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are quantified for the first time. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ~1.2 labels preserves them. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labelled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development, is endocytosed rapidly by cells and is translocated into synthesized matrix networks within 24 hours.

scholarly journals Biomechanical assessment of remote and postinfarction scar remodeling following myocardial infarction

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mihaela Rusu ◽  
Katrin Hilse ◽  
Alexander Schuh ◽  
Lukas Martin ◽  
Ioana Slabu ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Heart Failure ◽  
Type I Collagen ◽  
De Novo ◽  
Heart Function ◽  
Type Iii Collagen ◽  
Type I ◽  
Compensatory Mechanisms ◽  
Biomechanical Assessment ◽  
Type V

AbstractThe importance of collagen remodeling following myocardial infarction (MI) is extensively investigated, but little is known on the biomechanical impact of fibrillar collagen on left ventricle post-MI. We aim to identify the significant effects of the biomechanics of types I, III, and V collagen on physio-pathological changes of murine hearts leading to heart failure. Immediately post-MI, heart reduces its function (EF = 40.94 ± 2.12%) while sarcomeres’ dimensions are unchanged. Strikingly, as determined by immunohistochemistry staining, type V collagen fraction significantly grows in remote and scar for sustaining de novo-types I and III collagen fibers’ assembly while hindering their enzymatic degradation. Thereafter, the compensatory heart function (EF = 63.04 ± 3.16%) associates with steady development of types I and III collagen in a stiff remote (12.79 ± 1.09 MPa) and scar (22.40 ± 1.08 MPa). In remote, the soft de novo-type III collagen uncoils preventing further expansion of elongated sarcomeres (2.7 ± 0.3 mm). Once the compensatory mechanisms are surpassed, the increased turnover of stiff type I collagen (>50%) lead to a pseudo-stable biomechanical regime of the heart (≅9 MPa) with reduced EF (50.55 ± 3.25%). These end-characteristics represent the common scenario evidenced in patients suffering from heart failure after MI. Our pre-clinical data advances the understanding of the cause of heart failure induced in patients with extended MI.

Cell-matrix and cell-cell interactions modulate apoptosis of bronchial epithelial cells

1997 ◽  
Vol 272 (1) ◽  
pp. L28-L37 ◽  
Author(s):  
K. Aoshiba ◽  
S. I. Rennard ◽  
J. R. Spurzem
Keyword(s):  
Epithelial Cells ◽  
Type I Collagen ◽  
Bronchial Epithelium ◽  
Cell Aggregation ◽  
Cell Number ◽  
Bronchial Epithelial Cells ◽  
Cell Interactions ◽  
Type I ◽  
Bronchial Epithelial ◽  
Cell Cell

Apoptosis is an important process maintaining cell number and tissue structure. To determine whether cell-extracellular matrix (ECM) and cell-cell interactions modulate apoptosis in bronchial epithelium, we cultured human bronchial epithelial cells in different conditions and evaluated the cells for apoptosis. We found that plating cells in conditions that prevent cell-ECM adhesion induced apoptosis. Plating cells on type I collagen, fibronectin, and biosynthesized matrix prevented apoptosis, due at least in part to integrin-mediated adhesion. When cells were cultured at high density but under conditions preventing cell-substratum adhesion, aggregation occurred. Apoptosis was inversely correlated with aggregation. Cell-cell adhesion in these conditions was mediated at least partly by integrins containing alpha v. Cell aggregation was not associated with activation of a signaling pathway that is usually activated by cell-ECM adhesion, phosphorylation of focal adhesion kinase, but was associated with Bcl-2 protein expression, consistent with the concept that Bcl-2 protects against apoptosis. We conclude that both cell-ECM and cell-cell interactions, likely mediated in part by integrins, modulate apoptosis in bronchial epithelium.

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