title: Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A.
abstract: OBJECTIVE
  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.
authors.affiliation: Kovler Diabetes Center, The University of Chicago, USA ; Department of Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, USA.
authors.forename: Sindhu
authors.initials: S
authors.lastname: Rajan
authors.affiliation: Kovler Diabetes Center, The University of Chicago, USA ; Department of Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, USA.
authors.forename: Lorna M
authors.initials: LM
authors.lastname: Dickson
authors.affiliation: Kovler Diabetes Center, The University of Chicago, USA ; Department of Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mathew
authors.affiliation: Kovler Diabetes Center, The University of Chicago, USA ; Department of Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, USA ; Committee on Molecular Metabolism and Nutrition, The University of Chicago, USA.
authors.forename: Caitlin M O
authors.initials: CM
authors.lastname: Orr
authors.affiliation: Kovler Diabetes Center, The University of Chicago, USA ; Department of Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, USA.
authors.forename: Johanne H
authors.initials: JH
authors.lastname: Ellenbroek
authors.affiliation: Kovler Diabetes Center, The University of Chicago, USA ; Department of Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, USA ; Committee on Molecular Metabolism and Nutrition, The University of Chicago, USA.
authors.forename: Louis H
authors.initials: LH
authors.lastname: Philipson
authors.affiliation: Kovler Diabetes Center, The University of Chicago, USA ; Department of Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, USA ; Committee on Molecular Metabolism and Nutrition, The University of Chicago, USA.
authors.forename: Barton
authors.initials: B
authors.lastname: Wicksteed
keywords: GLP-1 receptor; Hyperglycemia; Protein kinase A; Small ubiquitin-related modifier
references.citation: Anticancer Res. 2008 Nov-Dec;28(6A):3749-56
references.pmid: 19189660
references.citation: J Clin Endocrinol Metab. 2004 Jul;89(7):3469-73
references.pmid: 15240633
references.citation: J Pharm Pharmacol. 2004 Dec;56(12):1477-92
references.pmid: 15563754
references.citation: Pharmacol Ther. 2007 Mar;113(3):546-93
references.pmid: 17306374
references.citation: Diabetologia. 2009 Feb;52(2):199-207
references.pmid: 19037628
references.citation: J Biol Chem. 2005 Sep 2;280(35):31294-302
references.pmid: 15987680
references.citation: J Biol Chem. 1989 Aug 5;264(22):13321-8
references.pmid: 2546952
references.citation: Mol Endocrinol. 1996 Jan;10 (1):62-75
references.pmid: 8838146
references.citation: Clin Biochem. 1981 Oct;14(5):230-6
references.pmid: 7037227
references.citation: Diabetes. 2003 Feb;52(2):380-6
references.pmid: 12540611
references.citation: Diabetes. 2013 Oct;62(10):3316-23
references.pmid: 23818527
references.citation: Semin Cell Dev Biol. 2000 Aug;11(4):235-42
references.pmid: 10966857
references.citation: Cell Metab. 2011 Mar 2;13(3):308-19
references.pmid: 21356520
references.citation: Biochem J. 2010 Apr 28;428(1):55-65
references.pmid: 20196770
references.citation: Am J Physiol Endocrinol Metab. 2012 Mar 15;302(6):E714-23
references.pmid: 22234371
references.citation: Nat Rev Mol Cell Biol. 2004 Dec;5(12):959-70
references.pmid: 15573134
references.citation: Mol Cell Endocrinol. 2014 Feb 15;382(2):938-49
references.pmid: 24275181
references.citation: Diabetologia. 2003 Oct;46(10):1383-93
references.pmid: 13680124
references.citation: Diabetes. 2004 Dec;53 Suppl 3:S205-14
references.pmid: 15561912
references.citation: Biochem Soc Trans. 2008 Oct;36(Pt 5):916-9
references.pmid: 18793161
references.citation: Diabetologia. 2008 Apr;51(4):632-40
references.pmid: 18292985
references.citation: J Biol Chem. 2012 Jun 8;287(24):20483-9
references.pmid: 22549777
references.citation: Lancet. 2002 Mar 9;359(9309):824-30
references.pmid: 11897280
references.citation: Peptides. 2014 Feb;52:134-42
references.pmid: 24406898
references.citation: Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4445-50
references.pmid: 12672969
references.citation: Pancreas. 1996 Oct;13(3):273-82
references.pmid: 8884849
references.citation: J Mol Biol. 2000 Apr 28;298(2):329-39
references.pmid: 10764601
references.citation: Diabetologia. 2015 Feb;58(2):324-33
references.pmid: 25381556
references.citation: J Biol Chem. 2011 Feb 11;286(6):4718-26
references.pmid: 21123177
references.citation: PLoS One. 2010 Aug 11;5(8):e11996
references.pmid: 20711444
references.citation: Br J Pharmacol. 1996 Jun;118(3):769-75
references.pmid: 8762106
references.citation: J Clin Endocrinol Metab. 2012 Apr;97(4):1363-70
references.pmid: 22319034
references.citation: Am J Physiol Endocrinol Metab. 2010 Mar;298(3):E622-33
references.pmid: 20009023
references.citation: Lancet. 2012 Jun 16;379(9833):2270-8
references.pmid: 22683137
references.citation: Annu Rev Biochem. 1995;64:689-719
references.pmid: 7574498
references.citation: J Biol Chem. 1993 Apr 5;268(10):6843-6
references.pmid: 8463209
references.citation: J Biol Chem. 1987 Sep 25;262(27):13111-9
references.pmid: 2820963
references.citation: J Biol Chem. 2013 Nov 15;288(46):33283-91
references.pmid: 24100033
references.citation: Lancet. 2006 Nov 11;368(9548):1696-705
references.pmid: 17098089
references.citation: J Biol Chem. 2001 Jun 15;276(24):21110-20
references.pmid: 11274216
references.citation: Diabetes. 2007 Jun;56(6):1551-8
references.pmid: 17360984
references.citation: Diabetes. 2008 Mar;57(3):584-93
references.pmid: 18025410
references.citation: Diabetes. 2010 Jul;59(7):1572-4
references.pmid: 20587800
references.citation: Diabetes. 2011 May;60(5):1599-607
references.pmid: 21441444
references.citation: Diabetes. 2007 Aug;56(8):1951-9
references.pmid: 17513701
references.citation: Diabetes Care. 2001 Aug;24(8):1416-21
references.pmid: 11473079
references.citation: Diabetes Care. 2009 May;32(5):762-8
references.pmid: 19196887
references.citation: J Gen Physiol. 2008 Sep;132(3):329-38
references.pmid: 18695009
references.citation: FASEB J. 2003 Jan;17(1):91-3
references.pmid: 12475913
references.citation: Peptides. 1994;15(3):453-6
references.pmid: 7937318
references.citation: J Biol Chem. 1996 Aug 16;271(33):19957-63
references.pmid: 8702711
references.citation: Diabetes. 2001 Nov;50(11):2505-13
references.pmid: 11679428
references.citation: Diabetologia. 2006 Dec;49(12):2920-9
references.pmid: 17053883
references.citation: Diabetes Metab. 2013 May;39(3):195-201
references.pmid: 23643349
references.citation: Diabetes. 2002 Feb;51 Suppl 1:S122-9
references.pmid: 11815470
references.citation: Diabetologia. 2000 Jul;43(7):852-8
references.pmid: 10952457