18 research outputs found
Proteome-Driven Elucidation of Adaptive Responses to Combined Vitamin E and C Deficiency in Zebrafish
The purpose of this study was to determine the system-wide consequences
of deficiencies in two essential micronutrients, vitamins E and C,
on the proteome using zebrafish (<i>Danio rerio</i>) as
one of the few vertebrate models that similar to humans cannot synthesize
vitamin C. We describe a label-free proteomics workflow to detect
changes in protein abundance estimates dependent on vitamin regimes.
We used ion-mobility-enhanced data-independent tandem mass spectrometry
to determine differential regulation of proteins in response to low
dietary levels of vitamin C with or without vitamin E. The detection
limit of the method was as low as 20 amol, and the dynamic range was
five orders of magnitude for the protein-level estimates. On the basis
of the quantitative changes obtained, we built a network of protein
interactions that reflect the whole organism’s response to
vitamin C deficiency. The proteomics-driven study revealed that in
vitamin-E-deficient fish, vitamin C deficiency is associated with
induction of stress response, astrogliosis, and a shift from glycolysis
to glutaminolysis as an alternative mechanism to satisfy cellular
energy requirements
The CDP-choline cycle is active during 3T3-L1 preadipocyte differentiation.
<p>(A) Choline metabolism and the potential for <sup>18</sup>O incorporation from H<sub>2</sub><sup>18</sup>O. Adapted from Fagone and Jackowski [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157118#pone.0157118.ref041" target="_blank">41</a>]. (B-F) Peak area ratios (M+H+2 peak over the M+H peak, a measure of <sup>18</sup>O incorporation) of choline metabolites. (G-M) Temporal changes in choline metabolites during 3T3-L1 preadipocyte differentiation. Shown are means ± SE, n = 3 biological replicates except time 0 h, where n = 6. *** p < 0.0001 and ** p < 0.001 from a two-tailed t-test. ns, not significant where p > 0.05.</p
Short-chain peptide levels are reflective of proteasome activity.
<p>(A-D) Relative levels of short-chain peptides in the presence of various concentrations of inhibitors of proteasome activity (epoxomicin; Epox), autophagosome-lysosome fusion (bafilomycin A1; Baf), and aminopeptidase activity (bestatin; Best). 0.1% DMSO served as a vehicle control. Shown are means ± SE, n = 3 biological replicates. ND, not detected.</p
<sup>18</sup>O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes
<div><p>The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with <sup>18</sup>O labeled water and found that <sup>18</sup>O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. <sup>18</sup>O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. <sup>18</sup>O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of <sup>18</sup>O isotope labeling in combination with metabolomics to uncover changes in cellular metabolism that are not detectable by time-resolved metabolomics.</p></div
Glutaminolysis is active during 3T3-L1 preadipocyte differentiation.
<p>(A) Peak area ratio (M+H+2 peak over the M+H peak, a measure of <sup>18</sup>O incorporation) of glutamate. (B) Scheme of glutamine hydrolysis for production of the anaplerotic amino acid glutamate. Shown are means ± SE, n = 3 biological replicates.</p
Time course metabolomics of differentiating adipocytes.
<p>PCA-DA scores plot representing metabolomic analysis (positive ion) of differentiating and control (0.1% DMSO) 3T3-L1 fibroblasts during the early phase of differentiation. The times refer to the amount of time passed after the addition of the differentiation cocktail or DMSO. n = 3 biological replicates for all time points except 0 h, where n = 6. Shown are means ± SE.</p
Temporal changes in polyamine, glutathione, and amino acid metabolism during 3T3-L1 preadipocyte differentiation.
<p>Time course profiles of metabolites involved in glutathione metabolism (A-C), polyamine metabolism (D-G), and amino acid metabolism (H) over the first 48 h of adipocyte differentiation. Control cells had 0.1% DMSO. (I) Hypothetical scheme linking proline degradation to reactive oxygen species (ROS) formation and polyamine biosynthesis in differentiating 3T3-L1 preadipocytes. n = 3 biological replicates for all time points except time 0 h, where n = 6. Shown are means ± SE.</p
Short-chain peptide levels are reflective of matrix metalloproteinase activity.
<p>(A-C) Relative levels of short chain peptides in the presence of batimastat (10 μM), a broad spectrum matrix metalloproteinase inhibitor. 0.1% DMSO served as a vehicle control. Shown are means ± SE, n = 3 biological replicates. *** p < 0.0001 and ** p < 0.001 and * p > 0.05 from a two-tailed t-test.</p
Short-chain peptides are products of peptide hydrolysis.
<p>Representative total ion chromatograms (A, B) and peak areas of peptide M+H+2 peaks (C) of differentiating (for 24 h) 3T3-L1 preadipocytes treated with 6% H<sub>2</sub>O or H<sub>2</sub><sup>18</sup>O. Shown are means ± SE, n = 3 biological replicates. ND, not detected.</p
Temporal changes in products of protein degradation during adipocyte differentiation.
<p>Time course profiles of short-chain peptides (A-C), methylhistidine (D), and bilirubin (E) over the first 48 h of adipocyte differentiation. Control cells had only 0.1% DMSO. n = 3 biological replicates for all time points except time 0 h, where n = 6. Shown are means ± SE.</p