UC Davis

The increasing prevalence of type 2 diabetes (T2D) and obesity poses substantial concerns and economic challenges to the healthcare system. Type 2 Diabetes is characterized by disruption of glucose homeostasis. Insulin is the primary driver of glucose homeostasis and released by pancreatic -cells in response to nutritional and endocrine signals. Glucose stimulated insulin secretion is augmented by the actions of incretin hormones including Glucagon-Like peptide 1 (GLP-1), which activates the GLP-1 receptor (GLP-1R) in islets producing the “incretin effect”. Because of their insulinotropic properties, incretin drugs, such as GLP-1 receptor agonists are used as therapeutic agents to maintain glucose homeostasis in type-2 diabetes. However, concerns regarding the long-term effects of these drugs and development of tolerance to GLP-1R agonist persist. In this study, we investigate the role of G protein-coupled receptor-associated sorting protein 1 (GASP1), a critical regulator involved in post-endocytic trafficking of GLP-1R, on development of tolerance to GLP-1R agonists. By combining CRISPR-Cas9 technique, Homogenous Time Resolved Fluorescence (HTRF) biochemical assay, transgenic mice and in vivo and ex vivo animal studies, we found the following:

• GASP1 is expressed in both human embryonic kidney (HEK 293) and rat insulinoma derived insulin producing INS-1 cells.

• Following acute treatment with the GLP-1R agonist Exendin-4 (Ex-4), both HEK 293 and INS-1 cells demonstrate a dose-dependent increase in intracellular cyclic adenosine monophosphate (cAMP) levels.

• Prolonged Ex-4 pretreatment of HEK 293 and INS-1 cells with Ex-4 results in loss of responsiveness of the receptor (i.e “Tolerance”).

•CRISPR-Cas9-mediated removal of GASP1 in both HEK 293 and INS-1 cells did not impact acute GLP-1R signaling. However, GASP1 knockout prevented the development of tolerance in response to prolonged Ex-4 pretreatment, indicating that GASP1 plays a role in regulating the post-endocytic trafficking of GLP-1R.

• Similarly, in INS-1 cells, deletion of GASP1 has no effect on incretin-mediated insulin secretion in response to Ex-4. However, upon prolonged Ex-4 pretreatment wild-type (WT) INS-1 cells show reduced incretin response, whereas GASP1 knockout (KO) INS-1 cells retained the incretin effect.

• Furthermore, in a longitudinal mouse islet insulin secretion assay, both GASP1 wild-type and beta-cell specific GASP1 deleted (β-GASP1-KO) islets show robust incretin effect and increase in insulin secretion when stimulated with Ex-4 acute treatment.

• Interestingly, after an Ex-4 pretreatment, GASP1-WT islets show reduced insulin secretion indicating development of tolerance while β-GASP1-KO islets maintained their incretin effect.

• Importantly, both GASP1-WT and β-GASP1-KO islets displayed a substantial incretin effect after a 24-hour recovery period, suggesting that the observed tolerance effect is not due to any inherent unhealthiness of the islets.

• In WT mice which are chronically treated with Ex-4 for six weeks show development of tolerance to glucose-stimulated insulin secretion effect of Ex-4.

• Furthermore, in WT mice treated chronically with Ex-4, there is not only the development of tolerance to exogenous Ex-4 treatment but also to their endogenous incretins.

• In mice with selective disruption of GASP1 in pancreatic beta cells (β-GASP1-KO mice), the acute treatment of oral glucose and Ex-4 are indistinguishable from WT mice.

• However, chronic Ex-4 treatment of β-GASP1-KO mice with Ex-4 does not develop tolerance to either exogenous Ex-4 or endogenous incretins.

This study highlights the pivotal role of GASP1 in regulating the post-endocytic trafficking of GLP-1R in pancreatic β-cells and its impact on receptor function during prolonged drug administration. These findings also emphasize the critical role of GASP1-mediated GLP-1R trafficking in the development of tolerance to incretin drugs and offers potential novel strategies for improving therapeutic efficacy. Therefore, gaining a deeper understanding of the molecular mechanisms governing GASP1-mediated GLP-1R trafficking could help in the development of improved therapies utilizing GLP-1R agonists to effectively combat type 2 diabetes and obesity.