The Recombinant Rat Glucagon-Like Peptide-1 Receptor, Expressed in an α-Cell Line, Is Coupled to Adenylyl Cyclase Activation and Intracellular Calcium Release

J. S. Dillon; M. Lu; S. Bowen; L. L. Homan

doi:10.1055/s-2005-837526

Experimental and Clinical Endocrinology & Diabetes, Table of Contents

Exp Clin Endocrinol Diabetes 2005; 113(3): 182-189
DOI: 10.1055/s-2005-837526

Article

J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York

The Recombinant Rat Glucagon-Like Peptide-1 Receptor, Expressed in an α-Cell Line, Is Coupled to Adenylyl Cyclase Activation and Intracellular Calcium Release

J. S. Dillon¹ , M. Lu² , S. Bowen¹ , L. L. Homan¹

¹Division of Endocrinology, Department of Internal Medicine, University of Iowa, Iowa City, IA, USA
²Department of Ophthalmology, Children's Hospital, Harvard Medical School, Boston, MA, USA

Abstract

The glucagon-like peptide-1 (GLP-1) receptor is expressed on α-cells, though its functional significance is unknown. The endogenous β-cell GLP-1 receptor is coupled to adenylyl cyclase, cell depolarization, activation of voltage-dependent Ca²⁺ channels (VDCC) and extracellular Ca²⁺ influx ([Lu et al. 1993 b]). In contrast, the signaling pathways of the GLP-1 receptor in α-cells are poorly understood. To determine the signaling mechanisms of the α-cell GLP-1 receptor, we established a stable pancreatic islet α-cell line expressing the recombinant rat GLP-1 receptor (INR1-SF2), using INRl-G9 cells. These INRl-G9 cells do not express endogenous GLP-1 receptor. In INR1-SF2 cells, GLP-1 bound to the recombinant receptor (K_d = 0.9 nM) and increased cAMP (ED₅₀ = 0.6 nM). GLP-1 increased the free cytosolic Ca²⁺ ([Ca²⁺]_i) (ED₅₀ = 50 nM) by release from intracellular stores, but did not affect INR1-SF2 cell phosphoinositol turnover. Despite expressing VDCC, the INR1-SF2 cells were not depolarized by GLP-1, even in the presence of glucose. This contrasts with the depolarizing action of GLP-1 in β-cells in the presence of glucose ([Lu et al., 1993 b]).

This study establishes that a single GLP-1 receptor species can mediate the effects of GLP-1 through multiple signaling pathways, including the adenylyl cyclase system and intracellular Ca²⁺ release, in an α-cell type. Furthermore, since GLP-1 is unable to cause cellular depolarization or activate VDCC in INR1-SF2 cells, these data suggest that glucose-induced membrane depolarization may be crucial for GLP-1 to further activate VDCC and potentiate glucose-stimulated insulin release in β-cells. Finally this study describes a cell line that can be used as a model system for evaluation of GLP-1 signaling in α-cells.

Key words

GLP-1 receptor - alpha cells - INR1-G9 cells - intracellular Ca²⁺ release

Full Text

References

1 Abou-Samra A-B, Juppner H, Force T, Freeman M, Kong X, Schipani E, Urena P, Richards J, Bonventre J, Potts J, Kronenberg H, Segre G. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: A single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. Proc National Acad Sciences USA. 1992; 89 2732-2736
2 Amatruda T, Gerard N, Gerard C, MI S. Specific interactions of chemoattractant factor receptors with G-proteins. J Biol Chem. 1993; 14 10139-10144
3 Berridge M. Inositol trisphosphate and calcium signaling. Nature. 1993; 361 315-325
4 Canonico P, Cronin M, Thorner M, Macleod R. Human pancreatic GRF stimulates phosphatidylinositol-labeling in cultured anterior pituitary cells. Am J Physiol. 1983; 245 E587-590
5 Dillon J S, Tanizawa Y, Wheeler M B, Leng X H, Ligon B B, Rabin D U, Yoo-Warren H, Permutt M A, Boyd A E. Cloning and functional expression of the human glucagon-like peptide-1 (GLP-1) receptor. Endocrinology. 1993; 133 1907-1910
6 Ding W G, Renstrom E, Rorsman P, Buschard K, Gromada J. Glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide stimulate calcium-induced secretion in rat alpha-cells by a protein kinase A-mediated mechanism. Diabetes. 1997; 46 792-800
7 Drucker D J, Philippe J, Mojsov S. Proglucagon gene expression and posttranslational processing in a hamster islet cell line. Endocrinology. 1988; 123 1861-1867
8 Eddlestone G, Oldham S, Lipson L, Premdas F, Biegelman P. Electrical activity, cAMP concentration, and insulin release in mouse pancreatic islets of Langerhans. Am J Physiol. 1985; 248 C145-153
9 Exton J. Cell signaling through guanine nucleotide binding regulatory proteins (G proteins) and phospholipases. Eur J Biochem. 1997; 243 10-20
10 Fehmann H C, Habener J F. Homologous desensitization of the insulinotropic glucagon-like peptide-1 (7 - 37) receptor on insulinoma (HIT-T15) cells. Endocrinology. 1991; 128 2880-2888
11 Goke R, Conlon J. Receptors for glucagon-like peptide-1(7 - 36) amide on rat insulinoma-derived cells. J Endocrinol. 1988; 116 357-362
12 Gopel S O, Kanno T, Barg S, Weng X G, Gromada J, Rorsman P. Regulation of glucagon release in mouse α-cells by kATP channels and inactivation of TTX-sensitive Na⁺ channels. J Physiol. 2000; 528 509-520
13 Graber M, Bockenstedt L, Weiss A. Signaling via the inositol phospholipid pathway by T cell antigen receptor is limited by receptor number. Journal of Immunology. 1991; 146 2935-2943
14 Gromada J, Dissing S, Bokvist K, Renstrom E, Frokjaer-Jensen J, Wulff B S, Rorsman P. Glucagon-like peptide 1 increases cytoplasmic calcium in insulin-secreting beta tc3-cells by enhancement of intracellular calcium mobilization. Diabetes. 1995; 44 767-774
15 Gutniak M, Orskov C, Holst J J, Ahren B, Efendic S. Antidiabetogenic effect of glucagon-like peptide-1(7 - 36) amide in normal subjects and patients with diabetes mellitus. New Engl J Med. 1992; 326 1316-1322
16 Gutniak M K, Juntti-Berggren L, Hellstrom P M, Guenifi A, Holst J J, Efendic S. Glucagon-like peptide 1 enhances the insulinotropic effect of glibenclamide in NIDDM patients and in the perfused rat pancreas. Diabetes Care. 1996; 19 857-863
17 Heller R S, Aponte G W. Intra-islet regulation of hormone secretion by glucagon-like peptide-1-(7 - 36) amide. Am J Physiol. 1995; 269 G852-860
18 Heller R S, Kieffer T J, Habener J F. Insulinotropic glucagon-like peptide 1 receptor expression in glucagon-producing alpha-cells of the rat endocrine pancreas. Diabetes. 1997; 46 785-791
19 Henquin J, Meissner H. The ionic, electrical, and secretory effects of endogenous cyclic adenosine monophosphate in mouse pancreatic β-cells: Studies with forskolin. Endocrinology. 1984; 115 1125-1134
20 Holz G G, Leech C A, Heller R S, Castonguay M, Habener J F. cAMP-dependent mobilization of intracellular calcium stores by activation of ryanodine receptors in pancreatic beta-cells. A calcium signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7 - 37). J Biol Chem. 1999; 274 14147-14156
21 Holz G G, Kuhtreiber W M, Habener J F. Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7 - 37). Nature. 1993; 361 362-365
22 Holz G H, Kuhtreiber W M, Habener J F. Induction of glucose competence in pancreatic beta cells by glucagon-like peptide-1(7 - 37). Transactions of the Association of American Physicians. 1992; 105 260-267
23 Kanse S M, Kreymann B, Ghatei M A, Bloom S R. Identification and characterization of glucagon-like peptide-1 7 - 36 amide-binding sites in the rat brain and lung. FEBS Letters. 1988; 241 209-212
24 Komatsu R, Matsuyama T, Namba M, Watanabe N, Itoh H, Kono N, Tarui S. Glucagonostatic and insulinotropic action of glucagon like peptide 1-(7 - 36)-amide. Diabetes. 1989; 38 902-905
25 Ling Z, Wu D, Zambre Y, Flamez D, Drucker D J, Pipeleers D G, Schuit F C. Glucagon-like peptide 1 receptor signaling influences topography of islet cells in mice. Virchows Archiv. 2001; 438 382-387
26 Login I, Judd A, Macleod R. Association of ⁴⁵Ca²⁺ mobilization with stimulation of growth hormone release by GH-releasing factor in dispersed normal male pituitary cells. Endocrinology. 1986; 118 239-243
27 Lu M, Soltoff S, Yaney G, Boyd A E. The mechanisms underlying the glucose dependence of arginine vasopressin induced insulin secretion in β cells. Endocrinology. 1993 a; 132 2141-2148
28 Lu M, Wheeler M B, Leng X H, Boyd A E. The role of the free cytosolic calcium level in beta-cell signal transduction by gastric inhibitory polypeptide and glucagon-like peptide 1 (7 - 37). Endocrinology. 1993 b; 132 94-100
29 Marie J C, Rosselin G, Skoglund G. Pancreatic beta-cell receptors and g proteins coupled to adenylyl cyclase. Ann New York Acad Sciences. 1996; 805 122-132
30 Moens K, Heimberg H, Flamez D, Huypens P, Quartier E, Ling Z, Pipeleers D, Gremlich S, Thorens B, Schuit F. Expression and functional activity of glucagon, glucagon-like peptide 1, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells. Diabetes. 1996; 45 257-261
31 Montrose-Rafizadeh C, Avdonin P, Garant M J, Rodgers B D, Kole S, Yang H, Levine M A, Schwindinger W, Bernier M. Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells. Endocrinology. 1999; 140 1132-1140
32 Munson P, Rodbard D. Ligand: A versatile computerized approach for characterization of ligand binding systems. Analytical Biochemistry. 1980; 107 220-239
33 Nelson T, Gaines K, Rajan A, Berg M, Boyd A E. Increased cytosolic calcium: A signal for sulfonylurea-stimulated insulin release from beta cells. J Biol Chem. 1987; 262 2608-2612
34 Nichols C, Lederer W. The mechanism of kATP channel inhibition by ATP. J General Physiol. 1991; 94 1095-1098
35 Offermanns S, Lida-Klein A, Segre G, Mi S. G_αq family members couple parathyroid hormone/PTH related peptide and calcitonin receptors to phospholipase C in COS-7 cells. Mol Endocrinol. 1996; 10 566-574
36 Orskov C, Poulsen S S. Glucagon like peptide-1-(7 - 36)-amide receptors only in islets of Langerhans. Autoradiographic survey of extracerebral tissues in rats. Diabetes. 1991; 40 1292-1296
37 Rajan A, Hill R, Boyd A E. Effect of rise in cAMP levels on Ca²⁺ influx through voltage dependent Ca²⁺ channels in HIT cells. Diabetes. 1989; 38 874-880
38 Ribalet B, Ciani S, Eddlestone G. ATP mediates both activation and inhibition of kATP channel activity via cAMP dependent protein kinase in insulin secreting cell lines. J General Physiol. 1989; 94 693-796
39 Richter G, Goke R, Goke B, Schmidt H, Arnold R. Characterization of glucagon-like peptide-1(7 - 36)amide receptors of rat lung membranes by covalent cross-linking. FEBS Letters. 1991; 280 247-250
40 Sato M, Kataoka R, Dingus J, Wilcox M, Hildebrandt J, Lanier S. Factors determining specificity of signal transduction by G-protein coupled receptors. J Biol Chem. 1995; 270 15269-15276
41 Satoh F, Beak S A, Small C J, Falzon M, Ghatei M A, Bloom S R, Smith D M. Characterization of human and rat glucagon-like peptide-1 receptors in the neurointermediate lobe: Lack of coupling to either stimulation or inhibition of adenylyl cyclase. Endocrinology. 2000; 141 1301-1309
42 Schirra J, Sturm K, Leicht P, Arnold R, Goke B, Katschinski M. Exendin(9 - 39)amide is an antagonist of glucagon-like peptide-1(7 - 36)amide in humans. J Clin Invest. 1998; 101 1421-1430
43 Sculptoreanu A, Scheuer T, Catterall W. Voltage-dependent potentiation of L-type Ca²⁺ channels due to phosphorylation by cAMP-dependent protein kinase. Nature. 1993; 364 240-243
44 Takaki R, Ono J, Nakamura M, Yokogawa Y, Kumae S, Hiraoka T, Yamaguchi K, Hamaguchi K, Uchida S. Isolation of glucagon-secreting cell lines by cloning insulinoma cells. In Vitro Cellular and Developmental Biology. 1986; 22 120-126
45 Takhar S, Gyomorey S, Su R C, Mathi S K, Li X, Wheeler M B. The third cytoplasmic domain of the GLP-1(7 - 36 amide) receptor is required for coupling to the adenylyl cyclase system. Endocrinology. 1996; 137 2175-2178
46 Tucker J D, Dhanvantari S, Brubaker P L. Proglucagon processing in islet and intestinal cell lines. Regulatory Peptides. 1996; 62 29-35
47 Uttenthal L O, Blazquez E. Characterization of high-affinity receptors for truncated glucagon-like peptide-1 in rat gastric glands. FEBS Letters. 1990; 262 139-141
48 Wang X, Cahill C M, Pineyro M A, Zhou J, Doyle M E, Egan J M. Glucagon-like peptide-1 regulates the beta cell transcription factor, PDX-1, in insulinoma cells. Endocrinology. 1999; 140 4904-4907
49 Wheeler M, Nishitani J, Buchan A, Kopin A, Chey W, Chang T-M, Leiter A. Identification of a transcriptional enhancer important for enteroendocrine and pancreatic islet cell-specific expression of the secretin gene. Mol Cell Biol. 1992; 12 3531-3539
50 Wheeler M B, Lu M, Dillon J S, Leng X H, Chen C, Boyd A E. Functional expression of the rat glucagon-like peptide-1 receptor, evidence for coupling to both adenylyl cyclase and phospholipase-C. Endocrinology. 1993; 133 57-62
51 Widmann C, Burki E, Dolci W, Thorens B. Signal transduction by the cloned glucagon-like peptide-1 receptor: Comparison with signaling by the endogenous receptors of beta cell lines. Mol Pharmacol. 1994; 45 1029-1035
52 Winicov I, Gershengorn M. Receptor density determines secretory response patterns mediated by inositol lipid derived second messengers. J Biol Chem. 1989; 264 9438-9443
53 Xu G, Stoffers D A, Habener J F, Bonner-Weir S. Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes. 1999; 48 2270-2276
54 Yang H, Egan J M, Wang Y, Moyes C D, Roth J, Montrose M H, Montrose-Rafizadeh C. GLP-1 action in L6 myotubes is via a receptor different from the pancreatic GLP-1 receptor. Am J Physiol. 1998; 275 C675-683
55 Zar J. Biostatistical Analysis. New Jersey; Prentice-Hall 1984
56 Zhou J, Wang X, Pineyro M A, Egan J M. Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes. 1999; 48 2358-2366

Joseph Dillon

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