Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A

Abstract

ObjectiveGlucagon-like peptide 1 (GLP-1) enhances insulin secretion and protects β-cell mass. Diabetes therapies targeting the GLP-1 receptor (GLP-1R), expressed in numerous tissues, have diminished dose-response in patients with type 2 diabetes compared with healthy human controls. The aim of this study was to determine the mechanistic causes underlying the reduced efficacy of GLP-1R ligands.MethodsUsing primary mouse islets and the β-cell line MIN6, outcomes downstream of the GLP-1R were analyzed: Insulin secretion; phosphorylation of the cAMP-response element binding protein (CREB); cAMP responses. Signaling systems were studied by immunoblotting and qRT-PCR, and PKA activity was assayed. Cell surface localization of the GLP-1R was studied by confocal microscopy using a fluorescein-tagged exendin-4 and GFP-tagged GLP-1R.ResultsRodent β-cells chronically exposed to high glucose had diminished responses to GLP-1R agonists including: diminished insulin secretory response; reduced phosphorylation of (CREB); impaired cAMP response, attributable to chronically increased cAMP levels. GLP-1R signaling systems were affected by hyperglycemia with increased expression of mRNAs encoding the inducible cAMP early repressor (ICER) and adenylyl cyclase 8, reduced PKA activity due to increased expression of the PKA-RIα subunit, reduced GLP-1R mRNA expression and loss of GLP-1R from the cell surface. To specifically examine the loss of GLP-1R from the plasma membrane a GLP-1R-GFP fusion protein was employed to visualize subcellular localization. Under low glucose conditions or when PKA activity was inhibited, GLP-1R-GFP was found at the plasma membrane. Conversely high glucose, expression of a constitutively active PKA subunit, or exposure to exendin-4 or forskolin led to GLP-1R-GFP internalization. Mutation of serine residue 301 of the GLP-1R abolished the glucose-dependent loss of the receptor from the plasma membrane. This was associated with a loss of an interaction between the receptor and the small ubiquitin-related modifier (SUMO), an interaction that was found to be necessary for internalization of the receptor.ConclusionsThese data show that glucose acting, at least in part, via PKA leads to the loss of the GLP-1R from the cell surface and an impairment of GLP-1R signaling, which may underlie the reduced clinical efficacy of GLP-1R based therapies in individuals with poorly controlled hyperglycemia.

Enhanced AMPA Receptor Trafficking Mediates the Anorexigenic Effect of Endogenous Glucagon-like Peptide-1 in the Paraventricular Hypothalamus

“…We took advantage of Fluorescein-Trp 25 – Exendin-4 (F-Exn4), which has been shown to have a similar anorexic effect as Exn4 at the same dose (Rajan et al, 2015; Reiner et al, 2016), and is a high affinity ligand of GLP-1R. We incubated live hypothalamic brain slices for 15 min in artificial cerebrospinal fluid (ACSF) with 100 nM F-Exn4 before fixing the tissue and performing immunohistochemistry (IHC).…”

The complexity of signalling mediated by the glucagon-like peptide-1 receptor

“…The receptor contains a classical caveolin-1 binding motif within the second intracellular loop of the receptor and has been shown to interact and co-localize with the protein intracellularly [28]. GLP-1R induced internalization has been proposed to occur via Gaq activation of PKC and ERK1/2 phosphorylation, although conditions of chronic hyperglycaemia have been suggested to stimulate endocytosis via PKA phosphorylation and SUMOylation of the receptor [27,29].…”

Is it safe to acutely discontinue insulin therapy in patients with chronic hyperglycaemia starting GLP-1R agonists?

“…They exert their effects by binding to GLP-1R on pancreatic beta-cells but studies have shown that in chronic hyperglycaemia there is downregulation of GLP-1R 9 10. Rajan et al found that there was internalisation of GLP-1R when rodent beta-cells were subjected to chronic hyperglycaemia 9. They also showed that when liraglutide was administered to young db/db (diabetic) mice exposed to moderate hyperglycaemia, the GLP-1R agonist was much more potent compared with older db/db mice subjected to more severe hyperglycaemic states and proposed that intensive insulin therapy to normalise glucose levels before induction of GLP-1R mimetics may improve their efficacy in individuals with type 2 diabetes 9.…”

“…Rajan et al found that there was internalisation of GLP-1R when rodent beta-cells were subjected to chronic hyperglycaemia 9. They also showed that when liraglutide was administered to young db/db (diabetic) mice exposed to moderate hyperglycaemia, the GLP-1R agonist was much more potent compared with older db/db mice subjected to more severe hyperglycaemic states and proposed that intensive insulin therapy to normalise glucose levels before induction of GLP-1R mimetics may improve their efficacy in individuals with type 2 diabetes 9. However, there was no suggestion of whether age influenced the poorer liraglutide efficacy in the older group of mice or whether the main factor was their more severe hyperglycaemia.…”