Download PDF
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
Further Information

Publication History

Received: March 3, 2004 First decision: May 13, 2004

Accepted: September 9, 2004

Publication Date:
23 March 2005 (online)

Also available at
    Permissions and Reprints

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 (Kd = 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

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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 3 Berridge M. Inositol trisphosphate and calcium signaling.  Nature. 1993;  361 315-325
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 9 Exton J. Cell signaling through guanine nucleotide binding regulatory proteins (G proteins) and phospholipases.  Eur J Biochem. 1997;  243 10-20
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 32 Munson P, Rodbard D. Ligand: A versatile computerized approach for characterization of ligand binding systems.  Analytical Biochemistry. 1980;  107 220-239
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 34 Nichols C, Lederer W. The mechanism of kATP channel inhibition by ATP.  J General Physiol. 1991;  94 1095-1098
    PubMedSearch in Google Scholar
  • 35 Offermanns S, Lida-Klein A, Segre G, Mi S. Gαq family members couple parathyroid hormone/PTH related peptide and calcitonin receptors to phospholipase C in COS-7 cells.  Mol Endocrinol. 1996;  10 566-574
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 46 Tucker J D, Dhanvantari S, Brubaker P L. Proglucagon processing in islet and intestinal cell lines.  Regulatory Peptides. 1996;  62 29-35
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar
  • 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
    PubMedSearch in Google Scholar
  • 55 Zar J. Biostatistical Analysis. New Jersey; Prentice-Hall 1984
    Search in Google Scholar
  • 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
    CrossrefPubMedSearch in Google Scholar

Joseph Dillon

Division of Endocrinology
Room 3E10 VAMC
University of Iowa

Iowa City

IA 52246 · USA

Phone: + 3193380581 ext. 7640

Fax: + 31 93 39 70 25

Email: joseph-dillon@uiowa.edu