All-trans retinoic acid induces lipophagy through the activation of the AMPK-Beclin1 signaling pathway and reduces Rubicon expression in adipocytes.

Lipophagy is defined as a lipolysis pathway that degrades lipid droplet (LD) via autophagy. All-trans retinoic acid (atRA), a metabolite of vitamin A, stimulates lipolysis through hormone-sensitive lipase and ß-oxidation. However, the regulation of lipolysis by atRA-induced autophagy in adipocytes remains unclear. In this study, we investigated the effect of atRA on autophagy in epididymal fat of mice and the molecular mechanisms of autophagy in 3T3-L1 adipocytes. Western blotting showed that atRA decreased the expression of p62, a cargo receptor for autophagic degradation, and increased the expression of the lipidated LC3B (LC3B-II), an autophagy marker, in epididymal fat. Next, we confirmed that atRA increased autophagic flux in differentiated 3T3-L1 cells using the GFP-LC3-RFP-LC3ΔG probe. Immunofluorescent staining revealed that the colocalization of LC3B with perilipin increased in differentiated 3T3-L1 cells treated with atRA. The knockdown of Atg5, an essential gene in autophagy induction, partly suppressed the atRA-induced release of non-esterified fatty acid (NEFA) from LDs in differentiated 3T3-L1 cells. atRA time-dependently elicited the phosphorylation of AMPK and Beclin1, autophagy-inducing factors, in mature 3T3-L1 adipocytes. Inversely, atRA decreased the protein expression of Rubicon, an autophagy repressor, in differentiated 3T3-L1 cells and epididymal fat. Interestingly, the expression of ALDH1A1, atRA-synthesizing enzymes, increased in epididymal fat with decreased protein expression of Rubicon in aged mice. These results suggest that atRA may partially induce lipolysis through lipophagy by activating the AMPK-Beclin1 signaling pathway in the adipocytes and increased atRA levels may contribute to decreased Rubicon expression in the epididymal fat of aged mice. (248/250 words).

Sulforaphane induces lipophagy through the activation of AMPK-mTOR-ULK1 pathway signaling in adipocytes.

Lipophagy, a form of selective autophagy, degrades lipid droplet (LD) in adipose tissue and the liver. The chemotherapeutic isothiocyanate sulforaphane (SFN) contributes to lipolysis through the activation of hormone-sensitive lipase and the browning of white adipocytes. However, the details concerning the regulation of lipolysis in adipocytes by SFN-mediated autophagy remain unclear. In this study, we investigated the effects of SFN on autophagy in the epididymal fat of mice fed a high-fat diet (HFD) or control-fat diet and on the molecular mechanisms of autophagy in differentiated 3T3-L1 cells. Western blotting revealed that the protein expression of lipidated LC3 (LC3-II), an autophagic substrate, was induced after 3T3-L1 adipocytes treatment with SFN. In addition, SFN increased the LC3-II protein expression in the epididymal fat of mice fed an HFD. Immunofluorescence showed that the SFN-induced LC3 expression was co-localized with LDs in 3T3-L1 adipocytes and with perilipin, the most abundant adipocyte-specific protein, in adipocytes of mice fed an HFD. Next, we confirmed that SFN activates autophagy flux in differentiated 3T3-L1 cells using the mCherry-EGFP-LC3 and GFP-LC3-RFP-LC3ΔG probe. Furthermore, we examined the induction mechanisms of autophagy by SFN in 3T3-L1 adipocytes using western blotting. ATG5 knockdown partially blocked the SFN-induced release of fatty acids from LDs in mature 3T3-L1 adipocytes. SFN time-dependently elicited the phosphorylation of AMPK, the dephosphorylation of mTOR, and the phosphorylation of ULK1 in differentiated 3T3-L1 cells. Taken together, these results suggest that SFN may provoke lipophagy through AMPK-mTOR-ULK1 pathway signaling, resulting in partial lipolysis of adipocytes.