title: Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay.
abstract: BACKGROUND
  G-protein-coupled bile acid receptor 1, also known as TGR5 is known to be involved in glucose homeostasis. In animal models, treatment with a TGR5 agonist induces incretin secretion to reduce hyperglycemia. Betulinic acid, a triterpenoid present in the leaves of white birch, has been introduced as a selective TGR5 agonist. However, direct activation of TGR5 by betulinic acid has not yet been reported.
  
  
  METHODS
  Transfection of TGR5 into cultured Chinese hamster ovary (CHO-K1) cells was performed to establish the presence of TGR5. Additionally, TGR5-specific small interfering RNA was employed to silence TGR5 in cells (NCI-H716 cells) that secreted incretins. Uptake of glucose by CHO-K1 cells was evaluated using a fluorescent indicator. Amounts of cyclic adenosine monophosphate and glucagon-like peptide were quantified using enzyme-linked immunosorbent assay kits.
  
  
  RESULTS
  Betulinic acid dose-dependently increases glucose uptake by CHO-K1 cells transfected with TGR5 only, which can be considered an alternative method instead of radioligand binding assay. Additionally, signals coupled to TGR5 activation are also increased by betulinic acid in cells transfected with TGR5. In NCI-H716 cells, which endogenously express TGR5, betulinic acid induces glucagon-like peptide secretion via increasing calcium levels. However, the actions of betulinic acid were markedly reduced in NCI-H716 cells that received TGR5-silencing treatment. Therefore, the present study demonstrates the activation of TGR5 by betulinic acid for the first time.
  
  
  CONCLUSIONS
  Similar to the positive control lithocholic acid, which is the established agonist of TGR5, betulinic acid has been characterized as a useful agonist of TGR5 and can be used to activate TGR5 in the future.
authors: Division of Cardiology, Department of Internal Medicine, Zhongxing Branch of Taipei City Hospital; Department of History and Geography, University of Taipei, Taipei, Taiwan.
authors: Shih-Hsiang
authors: SH
authors: Lo
authors: Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
authors: Kai-Chung
authors: KC
authors: Cheng
authors: Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; Department of Medical Research.
authors: Ying-Xiao
authors: YX
authors: Li
authors: Department of Medical Research; Department of Neurosurgery, Chi-Mei Medical Center, Yong Kang.
authors: Chin-Hong
authors: CH
authors: Chang
authors: Department of Medical Research; Institute of Medical Science, College of Health Science, Chang Jung Christian University, Tainan.
authors: Juei-Tang
authors: JT
authors: Cheng
authors: Department of Surgery, Pingtung Hospital; Division of Neurosurgery, Department of Surgery, Kaohsiung Medical University Chung-Ho Memorial Hospital, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors: Kung-Shing
authors: KS
authors: Lee
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D006801:Humans; D053978:Pentacyclic Triterpenes; D043562:Receptors, G-Protein-Coupled / Q000819:agonists*; D013329:Structure-Activity Relationship; D014315:Triterpenes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D000094062:Betulinic Acid
chemical_list: C473772:GPBAR1 protein, human; D053978:Pentacyclic Triterpenes; D043562:Receptors, G-Protein-Coupled; D014315:Triterpenes; D000094062:Betulinic Acid
keywords: CHO-K1 cell; NCI-H716 cell; lithocholic acid; siRNA; transfection