Citation

  • Authors: Zhu Y. et al.
  • Year: 2022
  • Journal: Phytomedicine 100 154085
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: C2C12
      Description: Murine myoblasts
    2. Name: HEK-293
      Description: Human embryonic kidney Fibroblast
      Known as: HEK293, 293

Method

HEK mBRS-3 stable cells were transiently transfected with β-arrestin2 tagged with an enhanced green fluorescent protein (β-arrestin2-EGFP) (2 μg) by using jetOPTIMUS Reagent (Polyplus, France), following the manufacturer's instruction. Cells were plated into poly-D-lysine–coated cell culture dish (35 mm, glass-bottom) at approximately 0.2 × 106 cells/dish. Twenty four hours after transfected, the culture medium was replaced with serum-free DMEM for 30 min prior to each experiment. Cells were stimulated with MK-5046 (1 µM) or ODN (1 µM) for 5 min and stained with 10 μg/mL DAPI for 2 min. Images were captured with a Leica TCS SP8 fluorescence microscopy system. C2C12 cells were transiently transfected with pmCherry-GLUT4-myc expression vector DNA (2 μg) using jetOPTIMUS Reagent by following the manufacturer's instructions. Cells were then plated into poly-D-lysine–coated cell culture dish (35 mm, glass-bottom) at approximately 1 × 105 cells / dish overnight. After transfected for 24 h, the cells were starved for 2 h in glucose and serum-free DMEM, and then treated with ODN (1 μM) or insulin (0.1 μM) for 1 h in high glucose DMEM containing 10% FBS. The Bantag-1 (0.1 μM) was added 10 min before the agonist. The fluorescence of pmCherry was visualized under a Leica LP8 confocal microscope.

Abstract

Background: Bombesin Receptor Subtype-3 (BRS-3, Bombesin-like receptor, BB3) is an orphan G-protein coupled receptor (GPCR). Recent studies have shown that BRS-3 played a vital role in glucose regulation, insulin secretion, and energy homeostasis. Therefore, discovering more novel exogenous ligands with diverse structures for BRS-3 will be of great importance for target validation and drug development. Purpose: In this study, we aim to discover new agonists of BRS-3 from our natural compound libraries, providing a new probe to study the function of BRS-3. Study design: Multiple cell-based assays and in vivo experiments were performed to identify the new ligand. Methods: BRS-3 overexpression cells were coupled with FLIPR assay, homogeneous time-resolved fluorescence (HTRF) IP-ONE assay, dynamic mass redistribution (DMR) assay, β-arrestin2 recruitment assay, and western blot to determine receptor activation and downstream signaling events. To further validate the target of BRS-3, a series of in vitro and in vivo experiences were conducted, including glucose uptake, glucose transporter type 4 (GLUT4) transportation in C2C12, and oral glucose tolerance test (OGTT) in mice. Results: We discovered and identified oridonin as a novel small molecule agonist of BRS-3, with a moderate affinity (EC50 of 2.236 × 10-7 M in calcium mobilization assay), specificity, and subtype selectivity. Further in vitro and in vivo tests demonstrated that oridonin exerted beneficial effects in glucose homeostasis through activating BRS-3. Conclusions: Oridonin, as the discovered new ligand of BRS-3, provides a valuable tool compound to investigate BRS-3's function, especially for target validation in type 2 diabetes and obesity. Oridonin is promising as a lead compound in the treatment of metabolic disorders. Compared to the known agonists of BRS-3, we can take advantage of the multiple reported pharmacological activities of ODN as a natural product and assess whether these pharmacological activities are regulated by BRS-3. This may facilitate the discovery of novel functions of BRS-3.

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