Glucagon-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.
METHODS: Using 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.
RESULTS: Rodent 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.
CONCLUSIONS: These 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.