Regulation of Adipogenesis by Natural and Synthetic REV-ERB Ligands

Abstract

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptor's activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.

Structure of REV-ERBβ Ligand-binding Domain Bound to a Porphyrin Antagonist

“…Interestingly, even though heme acts as an agonist in cells causing REV-ERB to recruit NCoR and suppression of target gene transcription, in biochemical assays such as Luminex (10) or TR-FRET (25) the natural porphyrin agonist, heme, causes a decrease in corepressor peptide recruitment. This appears to be a unique feature of heme as synthetic agonists that induce NCoR recruitment in a cellular context cause an increase in corepressor peptide binding in biochemical assays in contrast to heme (13). Thus, based on the effects of CoPP in this experiment and its structural similarity to heme, we could not predict whether this compound would function as a REV-ERB agonist or antagonist.…”

“…The proton carrier frequency was set coincident with the water resonance for all experiments. Backbone assignments for the apo-REV-ERB␤ or bound to zinc(II) protoporphyrin IX (ZnPP, 1:2 ratio) were obtained using three-dimensional NMR experiments available in Bruker Topspin 3.0, including TROSY versions of the HNCO, HNCA, HN(CO)CA, HN(CA)CB, HN(COCA)CB, as well as 15 N-NOESY-HSQC, using 2 H, 13 C, 15 N-labeled REV-ERB␤-LBD (1.2 mM). Data were processed with Topspin and analyzed by NMRview (23).…”

“…We further characterized GSK4112 as a dual REV-ERB␣/REV-ERB␤ agonist that activated REV-ERB activity in cell-based models of adipogenesis (13). We also described the first REV-ERB antagonist, SR8278, which was developed based on the GSK4112 scaffold (14).…”

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor

“…Heme levels increase throughout adipogenesis at a rate closely matching that of Rev-erb␣ degradation. Similarly, decreasing cellular heme levels with 3-amino-1,2,4-triazole increases the steadystate levels of Rev-erb␣ in adipocytes (86). However, heme did not affect degradation of Rev-erb␣ in cells treated with the translation inhibitor cycloheximide (94).…”

Biological aging alters circadian mechanisms in murine adipose tissue depots

“…Together, this core circadian regulatory complex constitutes a self-contained feedback loop whose rate-limiting protein levels oscillate in a ∼24-h manner. It is noteworthy that BMAL1, nocturnin, and Rev-erb α have all been implicated as transcriptional regulators of adipogenesis in pre-adipocyte cell models (Shimba et al 2004(Shimba et al , 2005Kawai et al 2010;Kumar et al 2010).…”