Endocrine Abstracts (2003) 5 P76

Development of a bioassay for leptin agonists and antagonists

M Fazeli, H Zarkesh-Esfahani, M Maamra, S Justice & RJM Ross

Divison of Clinical Sciences North, University of Sheffield, Sheffield, UK.

Introduction: Identifying leptin agonists and antagonists requires a reliable bioassay responsive to physiological levels of leptin. The leptin receptor exists in multiple isoforms but Ob-Rb, the full length receptor, mediates the biological actions of leptin and its predominant signalling pathway is through STAT3. We report the development of a bioassay based on a STAT3 firefly luciferase reporter. Materials and Methods: HEK 293 cells were transfected with varying amounts of mammalian expression vectors for Ob-Rb, SIE-luciferase reporter, STAT3, and the internal control of transfection Renilla luciferase using calcium phosphate transfection. Sixteen hours after transfection, cells were transferred into serum free medium supplemented with leptin (0-100 ng/ml) and incubated for either 2 or 6 hours. Luciferase activity in cell lysates was determined, corrected for transfection efficiency and results reported as fold induction over cells not exposed to leptin. Results: The maximal fold induction in cells transfected with Ob-Rb in the absence of a STAT3 expression vector was 1.7±0.24 at 100 ng/ml leptin. The co-transfection STAT3 augmented fold induction to 4 plus/minus 0.37, although at high levels of STAT3 expression there was increased background and reduced sensitivity. The transfection of Ob-Rb 2ug, STAT3 1ug, SIE-luciferase reporter 2ug, and Renilla 0.03ug per 12-well plate gave optimal results with maximal fold induction of 7±0.16 and the minimal concentration of leptin required for significant activation of STAT3 was 3.1 ng/ml. Six hours exposure to leptin gave the best standard curve, although 2 hours also gave reproducible results and these protocols may be of value for detecting antagonist activity when a short incubation period is required. In conclusion: we describe a robust leptin bioassay based on the detection of STAT3 activation.

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