Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling

• Published in: Differentiation (London) 2014-06, Vol.87 (5), p.220-229
• Main Authors: Skop, Vojtech, Cahova, Monika, Dankova, Helena, Papackova, Zuzana, Palenickova, Eliska, Svoboda, Petr, Zidkova, Jarmila, Kazdova, Ludmila
• Format: Article
• Language: English
• Subjects: 3T3-L1 Cells; Adipocytes; Adipocytes - cytology; Adipocytes - drug effects; Adipogenesis - drug effects; Adipogenesis - genetics; Animals; Asparagine - pharmacology; Autophagy; Autophagy - drug effects; Autophagy - genetics; Cell Differentiation - drug effects; Cell Differentiation - genetics; Differentiation; Gene Expression Regulation, Developmental - drug effects; Gene Expression Regulation, Developmental - genetics; Mice; Mitochondria; Mitochondria - drug effects; Mitochondria - metabolism; Preadipocytes
• ISSN: 0301-4681; 1432-0436
• DOI: 10.1016/j.diff.2014.06.002

Autophagy is essential for successful white adipocyte differentiation but the data regarding the timing and relevance of autophagy action during different phases of adipogenesis are limited. We subjected 3T3-L1 preadipocytes to a standard differentiation protocol and inhibited the autophagy within time-limited periods (days 0–2; 2–4; 4–6; 6–8) by asparagine or 3-methyladenine. In the normal course of events, both autophagy flux and the mRNA expression of autophagy related genes (Atg5, Atg12, Atg16, beclin 1) is most intensive at the beginning of differentiation (days 0–4) and then declines. The initiation of differentiation is associated with a 50% reduction of the mitochondrial copy number on day 2 followed by rapid mitochondrial biogenesis. Preadipocytes and differentiated adipocytes differ in the mRNA expression of genes involved in electron transport (Nufsd1, Sdhb, Uqcrc1); ATP synthesis (ATP5b); fatty acid metabolism (CPT1b, Acadl); mitochondrial transporters (Hspa9, Slc25A1) and the TCA cycle (Pcx, Mdh2) as well as citrate synthase activity. Autophagy inhibition during the first two days of differentiation blocked both phenotype changes (lipid accumulation) and the gene expression pattern, while having no or only a marginal effect over any other time period. Similarly, autophagy inhibition between days 0–2 inhibited mitotic clonal expansion as well as mitochondrial network remodeling. In conclusion, we found that autophagy is essential and most active during an initial stage of adipocyte differentiation but it is dispensable during its later stages. We propose that the degradation of preadipocyte cytoplasmic structures, predominantly mitochondria, is an important function of autophagy during this phase and its absence prevents remodeling of the mitochondrial gene expression pattern and mitochondrial network organization. •Autophagy is most intensive during early phase (day 0–2) of 3T3-L1 differentiation.•Autophagy inhibition at early phase, but not later, prevents 3T3-L1 differentiation.•Mitochondria copy number is reduced by 50% at the early phase of differentiation.•Pre- and differentiated 3T3-L1 differ in gene expression pattern and enzyme equipment.•Early-phase autophagy inhibition forestalls mitochondrial network remodeling.