N-acetylcysteine inhibits lipid accumulation in mouse embryonic adipocytes

A Pieralisi, C Martini, D Soto, M C Vila, J C Calvo, L N Guerra, A Pieralisi, C Martini, D Soto, M C Vila, J C Calvo, L N Guerra

Abstract

Oxidative stress plays critical roles in the pathogenesis of diabetes, hypertension, and atherosclerosis; some authors reported that fat accumulation correlates to systemic oxidative stress in human and mice, but cellular redox environment effect on lipid accumulation is still unclear. In our laboratory we used mouse embryonic fibroblasts (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC) and accumulate lipids, as obesity model. Here we analyzed the role of the well-known antioxidant and glutathione precursor N-acetylcysteine (NAC) in cellular MAPK modulation and lipid accumulation. We evaluated the effect of NAC on the adipogenic differentiation pathway using different doses: 0.01, 0.1, 1 and 5mM; no toxic doses in these cells. A dose of 5mM NAC [DCN-5] provoked a significant decrease in triglyceride accumulation (72±10 [DCN-5] vs 169±15 [DC], p<0.01), as well in Oil Red O stained neutral lipid content (120±2 [DCN-5] vs 139±12 [DC], p<0.01). Molecular mechanisms responsible for adipogenic differentiation involve increase of the expression of phosphoERK½ and phosphoJNK, 5mM NAC treatment inhibited both pERK½ and pJNK protein levels. We also evaluated the mitotic clonal expansion (MCE) which takes place during adipogenesis and observed an increase in DC at a rate of 1.5 cells number compared to CC at day 2, whereas the highest doses of NAC significantly inhibited MCE. Our results suggest that NAC inhibits lipid accumulation and the MAPK phosphorylation in mouse embryonic fibroblasts during adipogenic differentiation and further contribute to probe the importance of cellular redox environment in adipogenesis.

Keywords: Adipogenesis; Antioxidants; Kinases; Lipids; MEF; N-acetylcysteine.

Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Fig. 1
Fig. 1
MEF at day 10 of differentiation. (A) Control cells (CC); (B) MDI-treated cells (DC). Lipid droplets are shown in black. Representative results from one of three experiments with similar results are shown.
Fig. 2
Fig. 2
NAC effect on triglyceride (Tg) accumulation in MEF at day 10 of differentiation. MDI cells were treated with 0.01 mM NAC (DCN-0.01), 0.1 mM NAC (DCN-0.1), 1 mM NAC (DCN-1) or 5 mM NAC (DCN-5). NAC was added at day 0 and replaced every day for 10 days. Control cells (CC), MDI-treated cells (DC) and MDI–NAC-treated cells were harvested at day 10 and, Tg determinations were performed. Cellular Tg content was normalized by cellular protein content. Tg content of control cells (CC) was set to 100, data represent the percentage of CC. The results are the average of four independent experiments (mean±SD). *p<0.01: control cells (CC) vs MDI-treated cells (DC); *p<0.01: MDI – 5 mM NAC-treated cells (DCN-5) vs MDI-treated cells (DC).
Fig. 3
Fig. 3
NAC effect on neutral lipid accumulation visualized with Oil Red O in MEF at day 10 of differentiation. MDI cells were treated with 0.01 mM NAC (DCN-0.01), 0.1 mM NAC (DCN-0.1), 1 mM NAC (DCN-1) or 5 mM NAC (DCN-5). NAC was added at day 0 and replaced every day for 10 days. Control cells (CC), MDI-treated cells (DC) and MDI–NAC-treated cells were harvested at day 10 and Oil Red O dye was extracted and determined. Cellular lipid content was normalized by cell number. Lipid content of control cells (CC) was set to 100, data represent the percentage of CC. The results are the average of four independent experiments (mean±SD). *p<0.01: control cells (CC) vs MDI-treated cells (DC); *p<0.01: MDI – 5 mM NAC treated cells (DCN-5) vs MDI-treated cells (DC).
Fig. 4
Fig. 4
NAC effect on Oil Red O stained neutral lipid in MEF at day 10 of differentiation. Cells were stained with Oil Red O and photographed: (A) Control cells (CC); (B) MDI-treated cells (DC); (C) MDI – 1 mM NAC-treated cells (DCN-1); (D) MDI – 5 mM NAC-treated cells (DCN-5). Stained lipid droplets are shown. Representative results from one of four experiments with similar results are shown.
Fig. 5
Fig. 5
Concentration-dependent effect of NAC on MEF viability. MDI cells were treated with 0.01 mM NAC (DCN-0.01), 0.1 mM NAC (DCN-0.1), 1 mM NAC (DCN-1) or 5 mM NAC (DCN-5). NAC was added at day 0 and replaced every day for 10 days. Control cells (CC), MDI-treated cells (DC) and MDI–NAC-treated cells were harvested at day 10 and, MTT assays were performed. The absorbance of MDI-treated cells (DC) was set to 100% of cell viability, data represent the percentage of CC. The results shown are the averages of four independent experiments (mean±SD).
Fig. 6
Fig. 6
Kinases phosphorylation in response to NAC treatment. (A) pJNK expression was evaluated; the results were normalized to JNK expression. (B) pERK½ expression was evaluated; the results were normalized to ERK½ expression. Comparison of control cells (CC); MDI cells (DC) and MDI + NAC treated cells (DCN-0.01: 0.01 mM NAC; DCN-0.05: 0.05 mM NAC; DCN-1: 1 mM NAC; DCN-5: 5 mM NAC). The results are expressed as arbitrary units, arbitrary units of control cells (CC) was set to 1. The values represent the fold increase in protein expression compared to control cells (CC). The bars show the average of two different experiments (means±SD). Representative results from one of two independent western blot experiments with similar results are shown.
Fig. 7
Fig. 7
NAC effect on MEF during mitotic clonal expansion (MCE). Comparison of control cells (CC); MDI cells (DC); MDI + NAC treated cells (DCN). Cells were harvested at day 1 (DC 24 h, DCN 24 h) or at day 2 (DC 48 h, DCN 48 h) of the differentiation protocol. The concentration of NAC is shown. Cells were counted and viable cells were evaluated by trypan blue stain exclusion. Cell number of control cells (CC) was set to 1, data represent the percentage of CC. Results are the average of four different experiments (mean±SD). *p<0.01: MDI - 1 mM NAC-treated cells vs MDI-treated cells (DC); *p<0.01: MDI – 5 mM NAC-treated cells vs MDI-treated cells (DC).

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