University of Kentucky
Characterization of GLP-1 Receptor in Flox & Knockout Mice
Grade Level at Time of Presentation
Junior
Major
Biology
Minor
Spanish
KY House District #
48
KY Senate District #
26
Faculty Advisor/ Mentor
Dr. Zhenheng Guo, PhD; Dr. Ming Gong, PhD
Department
Pharmacology & Nutritional Sciences
Abstract
Type 2 Diabetes mellitus (T2DM) is one of the most prevalent and costly diseases concerning modern society, characterized by impaired insulin action, inducing chronic hyperglycemia. Glucagon-like peptide-1 (GLP-1) receptor agonists (RAs) are an emerging class of drugs proven extremely effective in treating T2DM and obesity. GLP-1 is a peptide secreted by enteroendocrine L cells along the digestive tract. Secreted GLP-1 acts on the pancreas to stimulate insulin, inhibit glucagon secretion, and suppress appetite. Challenges in GLP-1R research are antibody specificity and inconsistent effects of knocking out GLP-1R globally. This study aims to examine the efficacy of tamoxifen-induced knockout of GLP1-R on protein, mRNA, and DNA levels.
Breeding of Glp-1r loxP-flanked (flox) mice with tamoxifen-inducible ubiquitous Cre (UBC-Cre-ERT2) mice on a C57Bl/6 genetic background results in Cre-mediated deletion of Glp-1r in widespread tissues. The flox Glp-1r sequence is greater than 6000 bp, but after cleavage is 387 bp. The 30 second annealing step in the PCR protocol allows only for amplification of sequences less than 500 bp. Results reflected successful cleavage of Glp-1r. Western blotting identified that, among the three GLP-1R antibodies tested, the ProteinTech antibody most efficaciously detects GLP-1R, showing significant difference between GLP-1R flox and KO mice in duodenum (p
Results support effectiveness of tamoxifen-induced GLP-1R knockout in duodenum and hypothalamus. GLP-1R KO in the liver demonstrates low success, potentially attributable to uneven Cre expression. In the future, we hope this novel mouse model may be used for GLP-1R experiments examining its relationship to comorbidities of T2D.
Characterization of GLP-1 Receptor in Flox & Knockout Mice
Type 2 Diabetes mellitus (T2DM) is one of the most prevalent and costly diseases concerning modern society, characterized by impaired insulin action, inducing chronic hyperglycemia. Glucagon-like peptide-1 (GLP-1) receptor agonists (RAs) are an emerging class of drugs proven extremely effective in treating T2DM and obesity. GLP-1 is a peptide secreted by enteroendocrine L cells along the digestive tract. Secreted GLP-1 acts on the pancreas to stimulate insulin, inhibit glucagon secretion, and suppress appetite. Challenges in GLP-1R research are antibody specificity and inconsistent effects of knocking out GLP-1R globally. This study aims to examine the efficacy of tamoxifen-induced knockout of GLP1-R on protein, mRNA, and DNA levels.
Breeding of Glp-1r loxP-flanked (flox) mice with tamoxifen-inducible ubiquitous Cre (UBC-Cre-ERT2) mice on a C57Bl/6 genetic background results in Cre-mediated deletion of Glp-1r in widespread tissues. The flox Glp-1r sequence is greater than 6000 bp, but after cleavage is 387 bp. The 30 second annealing step in the PCR protocol allows only for amplification of sequences less than 500 bp. Results reflected successful cleavage of Glp-1r. Western blotting identified that, among the three GLP-1R antibodies tested, the ProteinTech antibody most efficaciously detects GLP-1R, showing significant difference between GLP-1R flox and KO mice in duodenum (p
Results support effectiveness of tamoxifen-induced GLP-1R knockout in duodenum and hypothalamus. GLP-1R KO in the liver demonstrates low success, potentially attributable to uneven Cre expression. In the future, we hope this novel mouse model may be used for GLP-1R experiments examining its relationship to comorbidities of T2D.