All-trans-retinoic acid represses obesity and insulin resistance by activating both peroxisome proliferation-activated receptor beta/delta and retinoic acid receptor

Abstract

Many biological activities of all-trans-retinoic acid (RA) are mediated by the ligand-activated transcription factors termed retinoic acid receptors (RARs), but this hormone can also activate the nuclear receptor peroxisome proliferation-activated receptor beta/delta (PPARbeta/delta). We show here that adipocyte differentiation is accompanied by a shift in RA signaling which, in mature adipocytes, allows RA to activate both RARs and PPARbeta/delta, thereby enhancing lipolysis and depleting lipid stores. In vivo studies using a dietary-induced mouse model of obesity indicated that onset of obesity is accompanied by downregulation of adipose PPARbeta/delta expression and activity. RA treatment of obese mice induced expression of PPARbeta/delta and RAR target genes involved in regulation of lipid homeostasis, leading to weight loss and improved insulin responsiveness. RA treatment also restored adipose PPARbeta/delta expression. The data indicate that suppression of obesity and insulin resistance by RA is largely mediated by PPARbeta/delta and is further enhanced by activation of RARs. By targeting two nuclear receptors, RA may be a uniquely efficacious agent in the therapy and prevention of the metabolic syndrome.

FIG. 1.

Expression of RA binding proteins and receptors during adipocyte differentiation. (a) Immunoblots of RARα, RARβ, and RARγ in 3T3-L1 cell prior to (pre) and after (adipocyte) differentiation. (b) Immunoblot of CRABP-II in preadipocytes (lane 0) and at indicated times after differentiation induction. (c) Levels of CRABP-II mRNA in preadipocytes and differentiated adipocytes. (d and e) Levels of mRNA of Cyp26a in preadipocytes (d) and in differentiated adipocytes (e) treated with denoted ligands (0.1 μM, 6 h). (f) Level of PPARβ/δ mRNA in preadipocytes and differentiated adipocytes treated with the denoted ligands (0.1 μM, 6 h). (g) Immunoblot of PPARβ/δ in preadipocytes and differentiated adipocytes. (h) Immunoblot of FABP5 expression in preadipoctyes (lane 0) and at the indicated times after differentiation induction. (i) Level of FABP5 mRNA in preadipocytes and differentiated adipocytes. *, P < 0.01; **, P < 0.05 (versus preadipocytes).

FIG. 2.

PPARβ/δ target genes are induced upon adipocyte differentiation and in response to RA. (a to g) Levels of denoted PPARβ/δ target genes in preadipocytes (Pre) and in differentiated (Adipocyte) adipocytes treated with RA, the PPARβ/δ-selective ligand GW0742, or the RAR-selective ligands TTNPB or 9cRA (0.1 μM, 4 h) (*, P = 0.05; and **, P = 0.01 [versus preadipocytes]; #, P = 0.01 [versus untreated control]). (h and i) FABP5 is involved in adipocyte differentiation. Preadipocytes were infected with a lentivirus harboring FABP5shRNA or an empty virus (E.V) and then induced to differentiate. (h) Triglyceride assays were carried out upon completion of differentiation indicating that the decreased FABP5 levels resulted in a lower triglyceride content (***, P < 0.001 [versus empty virus]). (i) Cells were observed under normal phase microscopy (×10 magnification). Adipocytes that differentiated under low FABP5 levels displayed fewer lipid droplets.

FIG. 3.

sIn adipocytes, RA induces PPARβ/δ target genes in an FABP5- and PPARβ/δ-dependent manner, activates Akt signaling, and enhances lipolysis. (a and b) PDK1 mRNA in the absence or presence of FABP5 shRNA or PPAR-β/δ shRNA (*, P < 0.01 [versus empty virus controls]). (c) Immunoblots of total and phosphorylated Akt1 in differentiated adipocytes treated with insulin (5 nM, 20 min) or the denoted ligands (0.1 μM, 12 h). Experiments were repeated three times with similar results. (d) The top panel shows representative immunoblots of total and phosphorylated GSK3β in differentiated adipocytes infected with a control lentivirus or a lentivirus harboring FABP5shRNA and treated with insulin (5 nM, 30 min) or the denoted ligands (0.1 μM, 16 h). The bottom panel shows the quantitation of effects of insulin and ligands on GSK3β phosphorylation (mean ± standard deviation, n = 3). *, P < 0.05 (versus corresponding empty virus controls). (e) Differentiated adipocytes were infected with a control lentivirus or lentivirus harboring FABP5 shRNA or PPARβ/δ shRNA, treated with the denoted ligands (0.1 μM, 24 h), and lipolysis assays were carried out. **, P < 0.05 (versus untreated control). (f) Triglyceride levels in mature adipocytes treated with denoted ligands as in panel e. **, P < 0.05 (versus untreated control). (g) Levels of mRNA of HSL in adipocytes infected with empty lentivirus or virus harboring PPARβ/δ shRNA for 5 days and then treated with the denoted ligands (0.1 μM, 4 h.). **, P < 0.05 (versus untreated empty virus). ***, P = 0.05 (versus GW0742-treated empty virus).

FIG. 4.

Effects of RA treatment on obese mice fed a high-fat/high-carbohydrate diet. (a) Whole-body weight of obese mice (n = 16) and obese mice treated with RA (n = 14; *, P < 0.001). (b) Dorsal view of obese mice and obese mice treated with RA for 5 weeks. (c) Tissue weights of epididymal/abdominal WAT, brown adipose tissue (BAT), gastronemius and soleus skeletal muscle, and livers of obese mice (n = 9) and mice treated with RA for 5 weeks (n = 7; *, P < 0.001 [versus untreated mice]). (d and e) Hematoxylin and eosin staining of cross-sections of WATs (d) and livers (e) of untreated and RA-treated mice. Tissues were preserved in 10% formalin. Samples were embedded in paraffin, sectioned, and stained at the Pathology Core Facility of Case Western Reserve University. Bar, 100 mm. (f) Food intake of untreated and RA-treated obese mice (*, P < 0.001; **, P = 0.05 [versus untreated animals]). (g) The mitochondrial marker SDH in gastronemius muscle after a 5-week RA visualized by immunoblots (top) and by immunofluorescence microscopy (bottom). Photographs were taken at a magnification of ×20. (h) Glucose tolerance test in obese mice (n = 7), RA-treated obese mice (n = 7), and lean control mice (n = 4). **, P = 0.05; ***, P = 0.005 (versus untreated obese mice).

FIG. 5.

Expression of RARs, PPARβ/δ, FABP5, and PPARβ/δ target genes in lean, obese, RA- and GW0742-treated mice. (a and b) Levels of mRNA for denoted RARs (a) and PPARβ/δ and FABP5 (b) in the adipose tissues of lean, obese, and RA-treated obese mice (n = 3 per group; *, P < 0.005; **, P = 0.04 [versus lean mice]). (c) The top panel shows immunoblots of FABP5 in the adipose tissues of lean, obese, and RA-treated mice. Each lane represents an individual mouse. The bottom panel shows the quantitation of the immunoblots in the top panels (‡, P = 0.07 [versus untreated obese mice]). (d) Levels of mRNA for the denoted genes in WAT and skeletal muscles of lean, obese, and obese mice treated with RA for 5 weeks (n = 3; #, P = 0.05 [versus untreated obese mice]). (e) The top panel shows immunoblots of total and phospho-Akt1 in WAT of lean, obese, and mice treated with RA for 5 weeks. The bottom panel shows the quantitation of the immunoblots in the top panels (*, P < 0.005 [versus obese mice]). (f) Levels of mRNA for the denoted PPARβ/δ target genes in adipose tissues and skeletal muscles of untreated mice and mice treated with RA for 2 days. #, P = 0.05 (versus untreated obese mice).

FIG. 6.

Effects of RA, GW0742, and TTNPB on gene expression in obese mice and in HepG2 cells. (a and b) Levels of mRNA of PPARβ/δ target genes (a) and of PPARβ/δ and FABP5 (b) in untreated, RA-treated, and GW0742-treated mice after 3 weeks of treatment (0.16 mg/day). *, P ≤ 0.05 (versus obese untreated mice). (c) Levels of adipose HSL and UCP1 and hepatic apo A1 in obese mice fed sesame oil (obese), RA, GW0742, or TTNPB for 2 days (0.16 mg/day). WATs and livers were harvested, RNA was isolated, and mRNA levels were examined by Q-PCR. *, P ≤ 0.05; **, P = 0.09; ***, P = 0.06 (versus obese untreated mice). (d) Levels of apo A1 mRNA in HepG2 cells treated with the denoted ligands (0.1 μM, 4 h). *, P ≤ 0.05 (versus untreated mice).