All-trans retinoic acid induces oxidative phosphorylation and mitochondria biogenesis in adipocytes

Abstract

A positive effect of all-trans retinoic acid (ATRA) on white adipose tissue (WAT) oxidative and thermogenic capacity has been described and linked to an in vivo fat-lowering effect of ATRA in mice. However, little is known about the effects of ATRA on mitochondria in white fat. Our objective has been to characterize the effect of ATRA on mitochondria biogenesis and oxidative phosphorylation (OXPHOS) capacity in mature white adipocytes. Transcriptome analysis, oxygraphy, analysis of mitochondrial DNA (mtDNA), and flow cytometry-based analysis of mitochondria density were performed in mature 3T3-L1 adipocytes after 24 h incubation with ATRA (2 µM) or vehicle. Selected genes linked to mitochondria biogenesis and function and mitochondria immunostaining were analyzed in WAT tissues of ATRA-treated as compared with vehicle-treated mice. ATRA upregulated the expression of a large set of genes linked to mtDNA replication and transcription, mitochondrial biogenesis, and OXPHOS in adipocytes, as indicated by transcriptome analysis. Oxygen consumption rate, mtDNA content, and staining of mitochondria were increased in the ATRA-treated adipocytes. Similar results were obtained in WAT depots of ATRA-treated mice. We conclude that ATRA impacts mitochondria in adipocytes, leading to increased OXPHOS capacity and mitochondrial content in these cells.

Keywords: adipose tissue; browning; vitamin A.

Fig. 1.

Oxygen consumption in 3T3-L1 adipocytes exposed to 2 µM of ATRA was measured as described in Materials and Methods using Clarke’s electrode. Control refers to control cells, which received the vehicle (DMSO). Data are the mean ± SEM of three independent cultures per treatment condition.

Fig. 2.

mtDNA to nuclear DNA ratio in 3T3-L1 adipocytes exposed to the indicated ATRA doses for 24 h (A) and 48 h (B). Control refers to control cells, which received the vehicle (DMSO). Data are the mean ± SEM of three independent cultures per treatment condition. mtDNA to nuclear DNA ratio in 3T3-L1 adipocytes preincubated with RAR antagonist (AGN 193109; 10 µM) or PPARδ antagonist (GSK0660; 10 µM) for 1 h and exposed to 2 µM of ATRA for 24 h (C). Control refers to control cells, which received the vehicles (ethanol and DMSO). Data are the mean ± SEM of two independent cultures per treatment condition.

Fig. 3.

MitoTracker® Green FM staining of 3T3-L1 adipocytes after treatment for 24 h with vehicle (control) or 2 µM ATRA. Incubation with the dye and flow cytometry sorting of the stained cells were conducted as described in Materials and Methods.

Fig. 4.

Representative light microscopy micrographs of CoxIV immunostained sections of retroperitoneal (A–D) and inguinal (E, F) white adipose depots of control (A, C, E) and ATRA-treated (B, D, F) mice. Cells from control animals present sparse COXIV positivity as compared with those of ATRA-treated mice. Dotted line-encircled areas in A and B are shown magnified in C and D, respectively, to illustrate COXIV positivity in the cytoplasm of unilocular adipocytes. COXIV positive multilocular adipocytes as found in the inguinal depot of ATRA-treated mice are illustrated in F. Scale bars indicate 50 µm (A, B), 10 µm (C, D), and 20 µm (E, F).