We analyzed previously generated stable monocyte-derived cell line carrying mutation JAK2 V617F. Evaluation of the expression of pro- and antifibrotic factors revealed changes in the production of MMPs and their inhibitors, growth factors, galectin-3, and pentraxin 3 in cells carrying mutation JAK2 in comparison with control non-modified cells.
Obesity and type 2 diabetes mellitus (T2DM) are often combined and pathologically affect many tissues due to changes in circulating bioactive molecules. In this work, we evaluated the effect of blood plasma from obese (OB) patients or from obese patients comorbid with diabetes (OBD) on skeletal muscle function and metabolic state. We employed the mouse myoblasts C2C12 differentiation model to test the regulatory effect of plasma exposure at several levels: (1) cell morphology; (2) functional activity of mitochondria; (3) expression levels of several mitochondria regulators, i.e., Atgl, Pgc1b, and miR-378a-3p. Existing databases were used to computationally predict and analyze mir-378a-3p potential targets. We show that short-term exposure to OB or OBD patients’ plasma is sufficient to affect C2C12 properties. In fact, the expression of genes that regulate skeletal muscle differentiation and growth was downregulated in both OB- and OBD-treated cells, maximal mitochondrial respiration rate was downregulated in the OBD group, while in the OB group, a metabolic switch to glycolysis was detected. These alterations correlated with a decrease in ATGL and Pgc1b expression in the OB group and with an increase of miR-378a-3p levels in the OBD group.
Assessment of cellular rates of amino acid consumption and release in vitro allows the study of cell culture in a time-course experiment without any cell damage. Determination of the release of amino acid metabolites that initially were not present in the media provides more reliable information about the processes of growth and differentiation in comparison with determination of amino acid consumption rates. Homoarginine (hArg), a derivative of arginine, is generated as the minor product in the reaction catalyzed by L-Arginine: glycine amidinotransferase, where L-lysine serves as an acceptor for amidine group instead of glycine. Ornithine is another product generated in this reaction from arginine. Thus, the goal of the present study was to evaluate the rate of hArg and ornithine accumulation in comparison to the rate of consumption of other amino acids in the course of C2C12 myoblast differentiation. The release time profiles were similar for hArg and ornithine, with the maximum corresponding to the second day of differentiation. The shift for hArg at this time point was detected with greater reliability (p < 0.002) than for ornithine and other amino acids. We suggest that hArg and ornithine could serve as markers to monitor the processes of myoblasts growth and differentiation.
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