Cell experiment:

HEK293 cells are maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37℃ under 5% CO2. HepG2 cells are maintained and routinely propagated in minimum essential medium supplemented with 10% fetal bovine serum at 37℃ under 5% CO2. 24 h prior to transfection, cells are plated in 96-well plates at a density of 15×103 cells/well. Transfections are performed using LipofectamineTM 2000. 16 h post-transfection, the cells are treated with vehicle or SR3335. 24 h post-treatment, the luciferase activity is measured using the Dual-GloTM luciferase assay system. The values indicated represent the means±S.E. from four independently transfected wells. The experiments are repeated at least three times[1].

Animal experiment:

Mice[1] 30 week old Diet induced obese (DIO) C57BL/6 male mice are purchased from Jackson Laboratories that are maintained on a 65% Kcal high-fat diet from weaning. DIO mice are treated twice per day (07:00h and 18:00h) with 15 mg/kg SR3335 or vehicle for 6 days i.p. Pyruvate tolerance test is conducted on day 6 of the treatment. Food is removed from mice in the morning after SR3335 injection, fasted for 6 hours and the pyruvate tolerance test is conducted at 13:00h. Time 0 blood glucose is measured taken from the tail nip and the pyruvate challenge is initiated by injection of 2g/kg of pyruvate i.p. followed by measuring blood glucose at 15, 30 and 60 min following the injection. Blood glucose is measured by one touch ultra glucose-meter.

SR3335 is a synthetic selective inverse agonist of RORα with Ki value of 220 nM [1][2].
The retinoic acid receptor-related orphan receptor α (RORα) is an orphan receptor that plays an important role in the regulation of metabolism.
SR3335 prevented the expression of endogenous RORα in HepG2. And it may suppress hepatic glucose production in type 2 diabetics. SR3335 was able to repress the expression of G6Pase. In ChIP trial, ML-176 reduced the amount of SRC2 at the G6Pase promoter [3]. In a GAL4-RORR ligand binding domain cotransfection assay, SR3335 inhibited the constitutive activity of RORα and the Ki was calculated as 220 nM [1].
When treat diet-induced obese mice with SR3335 (15 mg/kg), after injected with pyruvate, the plasma glucose levels were significantly lower at each time point compared with vehicle-treated animals. The results indicated SR3335 suppressed hepatic gluconeogenesis [1].
References:
[1]. Naresh Kumar, Douglas J. Kojetin, et al. Identification of SR3335 (ML176): a Synthetic RORα Selective Inverse Agonist. ACS Chem Biol. 2011 March 18; 6(3): 218-222.
[2]. Laura A. Solt, Thomas P. Burris. Action of RORs and their ligands in (patho)physiology. Trends in Endocrinology & Metabolism, 2012, 23(12): 619-627.
[3]. Kumar N, Nuhant P, Solt LA, et al. Identification of a novel selective inverse agonist probe and analogs for the Retinoic acid receptor-related Orphan Receptor Alpha (RORα). Probe Reports from the NIH Molecular Libraries Program [Internet], Bethesda (MD): National Center for Biotechnology Information (US),2010-.2010.