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

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 ¹⁸O 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

¹⁸O-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 ¹⁸O labeled water and found that ¹⁸O 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. ¹⁸O 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. ¹⁸O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of ¹⁸O isotope labeling in combination with metabolomics to uncover changes in cellular metabolism that are not detectable by time-resolved metabolomics.</p></div

The CDP-choline cycle is active during 3T3-L1 preadipocyte differentiation.

<p>(A) Choline metabolism and the potential for ¹⁸O incorporation from H₂¹⁸O. 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 ¹⁸O 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 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

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

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% H₂O or H₂¹⁸O. Shown are means ± SE, n = 3 biological replicates. ND, not detected.</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

Image_1_Photoprotective Properties of Isothiocyanate and Nitrile Glucosinolate Derivatives From Meadowfoam (Limnanthes alba) Against UVB Irradiation in Human Skin Equivalent.TIF

<p>Exposure to ultraviolet B (UVB) irradiation of the skin leads to numerous dermatological concerns including skin cancer and accelerated aging. Natural product glucosinolate derivatives, like sulforaphane, have been shown to exhibit chemopreventive and photoprotective properties. In this study, we examined meadowfoam (Limnanthes alba) glucosinolate derivatives, 3-methoxybenzyl isothiocyanate (MBITC) and 3-methoxyphenyl acetonitrile (MPACN), for their activity in protecting against the consequences of UV exposure. To that end, we have exposed human primary epidermal keratinocytes (HPEKs) and 3D human skin reconstructed in vitro (EpiDerm^TM FT-400) to UVB insult and investigated whether MBITC and MPACN treatment ameliorated the harmful effects of UVB damage. Activity was determined by the compounds’ efficacy in counteracting UVB-induced DNA damage, matrix-metalloproteinase (MMP) expression, and proliferation. We found that in monolayer cultures of HPEK, MBITC and MPACN did not protect against a UVB-induced loss in proliferation and MBITC itself inhibited cell proliferation. However, in human reconstructed skin-equivalents, MBITC and MPACN decrease epidermal cyclobutane pyrimidine dimers (CPDs) and significantly reduce total phosphorylated γH2A.X levels. Both MBITC and MPACN inhibit UVB-induced MMP-1 and MMP-3 expression indicating their role to prevent photoaging. Both compounds, and MPACN in particular, showed activity against UVB-induced proliferation as indicated by fewer epidermal PCNA+ cells and prevented UVB-induced hyperplasia as determined by a reduction in reconstructed skin epidermal thickness (ET). These data demonstrate that MBITC and MPACN exhibit promising anti-photocarcinogenic and anti-photoaging properties in the skin microenvironment and could be used for therapeutic interventions.</p