Adipocytes play a central role in whole-body energy homoeostasis. Complex regulatory transcriptional networks control adipogensis, with ligand-dependent activation of PPARγ (peroxisome proliferator-activated receptor γ) being a decisive factor. Yet the identity of endogenous ligands promoting adipocyte differentiation has not been established. Here we present a critical evaluation of the role of LOXs (lipoxygenases) during adipocyte differentiation of 3T3-L1 cells. We show that adipocyte differentiation of 3T3-L1 preadipocytes is inhibited by the general LOX inhibitor NDGA (nordihydroguaiaretic acid) and the 12/15-LOX selective inhibitor baicalein. Baicalein-mediated inhibition of adipocyte differentiation was rescued by administration of rosiglitazone. Treatment with baicalein during the first 4 days of the differentiation process prevented adipocyte differentiation; supplementation with rosiglitazone during the same period was sufficient to rescue adipogenesis. Accordingly, we demonstrate that adipogenic conversion of 3T3-L1 cells requires PPARγ ligands only during the first 4 days of the differentiation process. We show that the baicalein-sensitive synthesis of endogenous PPARγ ligand(s) increases rapidly upon induction of differentiation and reaches a maximum on days 3–4 of the adipocyte differentiation programme. The conventional platelet- and leucocyte-type 12(S)-LOXs and the novel eLOX-3 (epidermis-type LOX-3) are expressed in white and brown adipose tissue, whereas only eLOX-3 is clearly expressed in 3T3-L1 cells. We suggest that endogenous PPARγ ligand(s) promoting adipocyte differentiation are generated via a baicalein-sensitive pathway involving the novel eLOX-3.
Abbreviations used: ACC-1, acetyl-CoA carboxylase; ADD-1, adipocyte determination and differentiation-dependent factor; aP2, adipocyte lipid-binding protein; BAT, brown adipose tissue; C/EBPα, CCAAT/enhancer-binding protein; COX, cyclo-oxygenase; CMV, cytomegalovirus; DBD, DNA-binding domain; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular-signal-regulated kinase; FAS, fatty acid synthase; FBS, fetal bovine serum; GST, glutathione S-transferase; LBD, ligand-binding domain; LPL, lipoprotein lipase; LOX, lipoxygenase; eLOX-3, epidermis-type LOX-3; l12(S)-LOX, leucocyte-type 12(S)-LOX; p12(S)-LOX, platelet-type 12(S)-LOX; MAPK, mitogen-activated protein kinase; MDI, isobutylmethylxanthine (‘methylisobutylxanthine’), dexamethasone and insulin; NDGA, nordihydroguaiaretic acid; PPAR, peroxisome proliferator-activated receptor; RT-PCR, reverse transcription–PCR; TBP, TATA box-binding protein; UAS, upstream activator sequence; WAT, white adipose tissue.
These authors contributed equally to this work.