RESUMO
Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic alpha cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr(-/-)). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr(-/-) mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to alpha cell, and to a lesser extent, delta cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr(-/-) mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and alpha and delta cell numbers. Furthermore, the lean phenotype of Gcgr(-/-) mice suggests glucagon action may be involved in the regulation of whole body composition.
Assuntos
Glicemia/metabolismo , Glucagon/sangue , Ilhotas Pancreáticas/patologia , Receptores de Glucagon/genética , Receptores de Glucagon/fisiologia , Animais , Peso Corporal , Calorimetria , Divisão Celular , AMP Cíclico/metabolismo , Epididimo/metabolismo , Epinefrina/farmacologia , Glucose/metabolismo , Hormônios/metabolismo , Hiperplasia , Imuno-Histoquímica , Insulina/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fenótipo , Fatores de TempoRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.
Assuntos
Receptores dos Hormônios Gastrointestinais/química , Animais , Células CHO , Células COS , Cricetinae , AMP Cíclico/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Ligação Proteica , Ratos , Receptores dos Hormônios Gastrointestinais/genética , Deleção de Sequência , Transdução de Sinais , TransfecçãoRESUMO
The receptors for the two structurally related insulinotropic hormones Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-Like Peptide-1 (GLP-1) share approximately 40% sequence identity and demonstrate complete specificity for their endogenous ligands, while utilizing similar second messenger pathways. In the current study chimeric GIP-GLP-1 receptors were prepared, and the effect of domain-exchange on ligand binding and adenylyl cyclase activation examined. A chimera (CH-2) consisting of the first 132 amino acids of the external N-terminal (NT) domain bound 125I-GIP with high affinity (27.77 +/- 11.85 nM). However, for receptor coupling to cAMP production it was necessary to extend the NT into the first transmembrane (TM-1) region (CH-3: IC50 = 9.04 +/- 1.07 nM; EC50 = 17.1 +/- 3.5 nM). A chimera which included part of TM-3 (CH-4) demonstrated binding and signalling (IC50 = 8.33 +/- 0.14 nM; EC50 = 467.5 +/- 173.6 pM) similar to the wild type receptor (IC50 = 1.33 +/- 0.19 nM; EC50 = 497.9 +/- 211.7 pM). Surprisingly constructs CH-2 and CH-3, while devoid of detectable 125I-GLP-1 binding, were capable of eliciting GLP-1-specific cAMP production (EC50s CH-2 = 81.4 +/- 19.6 nM; CH-3 = 5.99 +/- 0.68 nM) suggesting that receptor activation is not completely dependent on high affinity receptor binding. These data clearly demonstrate that the NT domain of the GIP receptor acts as the ligand-specific binding domain and that the first transmembrane domain is important for receptor activation.
Assuntos
Polipeptídeo Inibidor Gástrico/farmacologia , Glucagon/farmacologia , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas/farmacologia , Receptores dos Hormônios Gastrointestinais/química , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Glucagon/química , Receptores de Glucagon/metabolismo , Adenilil Ciclases/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Células CHO , Células COS , Clonagem Molecular , Cricetinae , AMP Cíclico/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Cinética , Modelos Estruturais , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Estrutura Secundária de Proteína , Ratos , Receptores dos Hormônios Gastrointestinais/biossíntese , Receptores de Glucagon/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
GIP (Glucose-dependent Insulinotropic Polypeptide) is an important regulator of insulin secretion. The effects of truncated forms of the peptide, GIP(10-30), GIP(6-30amide) and GIP(7-30), on binding of 125I-GIP(1-42) to GIP receptors in transfected CHO-KI cells, and on cyclic AMP responses to GIP(1-42), have been studied with a view to defining further the receptor binding region of GIP, and to establish whether such truncated peptides exhibit agonist or antagonist activity. All three peptides were found to be receptor antagonists, however GIP(6-30amide) exhibited receptor binding affinity equivalent to that of GIP(1-42) in competitive binding studies (IC50 = 3.08+/-0.57 nM). GIP(6-30amide) inhibited GIP(1-42)-induced cAMP production by 58% at a concentration of 100 nM, whereas GIP(10-30) and GIP(7-30), inhibited only in the microM range. GIP(6-30amide) therefore contains the high affinity binding region of GIP and is a potent inhibitor of GIP(1-42) action in vitro.
Assuntos
Polipeptídeo Inibidor Gástrico/antagonistas & inibidores , Polipeptídeo Inibidor Gástrico/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Animais , Sítios de Ligação , Ligação Competitiva , Células CHO , Linhagem Celular , Cricetinae , AMP Cíclico/biossíntese , Polipeptídeo Inibidor Gástrico/química , Técnicas In Vitro , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ratos , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , TransfecçãoRESUMO
A 537 base pair cDNA clone for murine GIP has been isolated. The elucidated sequence encodes an open reading frame of 432 base pairs which codes for a 144 amino acid precursor. Murine GIP is predicted to differ from the human hormone by three amino acid substitutions: arginine for histidine at position 18, arginine for lysine at position 30 and serine for lysine at position 34. GIP mRNA levels in STC6-14 cells incubated in the presence of varying glucose concentrations was investigated using a competitive-PCR method. In the presence of a 5-mM glucose stimulus, 1 x 10(5) GIP cells were found to contain 3.9 +/- 0.59 amol of GIP mRNA while the same number of cells contained 11.6 +/- 1.4 amol when subjected to a high (25 mM) glucose stimulus.
Assuntos
Polipeptídeo Inibidor Gástrico/biossíntese , Regulação da Expressão Gênica , Glucose/farmacologia , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Relação Dose-Resposta a Droga , Polipeptídeo Inibidor Gástrico/genética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Neoplásico/análise , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Incretins are endogenous peptides released from the gastrointestinal tract into the circulation during a meal that potentiate glucose-stimulated insulin secretion. At present, there are two established incretins: glucose-dependent insulinotropic polypeptide (GIP) and the truncated glucagon-like peptides (tGLPs), which are now being investigated for use in the treatment of diabetes mellitus. In the present study we cloned a rat islet GIP receptor complementary DNA (GIP-R1) to answer several important questions regarding the ligand-binding and intracellular signaling properties of the GP receptor. GIP-R1, when expressed transiently in monkey kidney (COS-7) or stably in Chinese hamster ovary (CHO-K1) cells, demonstrated comparable high affinity binding for either synthetic porcine (sp) GIP or synthetic human (sh) GIP. The IC50 values for displacement of [125I]spGIP in CHO-K1 cells were 2.6 +/- 0.8 and 3.1 +/- 0.9 nM for two different preparations of shGIP, and 3.7 +/- 1.5 and 3.6 +/- 0.4 nM for two preparations of spGIP. Saturation isotherms obtained with both intact cells and membranes gave monophasic binding curves with apparent Kd values of 204 +/- 17 and 334 +/- 94 pM, respectively. Cells expressed 12-15 x 10(3) receptors/cell. In COS-7 cells, spGIP and shGIP also exhibited similar IC50 values (7.6 +/- 1.2 and 8.9 +/- 1.8 nM, respectively). The receptor in CHO-K1 cells bound GIP-(1-30) with lower affinity (IC50 = 39 +/- 17 nM), whereas the fragments GIP-(19-30), GIP-(18-28), and GIP-(21-26) showed no apparent binding. The specificity of the receptor was further examined using several structurally related peptides. Surprisingly, exendin-(9-39) [Ex-(9-39)], a GLP-1 receptor antagonist, and Ex-4-(1-39), a GLP-1 receptor agonist, demonstrated some affinity for the GIP receptor, with 39% and 21% displacement of [125I]spGIP, respectively, at 1 microM. Other members of the secretin/vasoactive intestinal peptide family of peptides tested showed no interaction. GIP-R1 receptor binding correlated with activation of the adenylyl cyclase system, whereby spGIP and shGIP evoked concentration-dependent increases in cAMP accumulation with EC50 values of 8.7 +/- 1.5 x 10(-10)M and 8.1 +/- 1.6 x 10(-10)M for spGIP and shGIP, respectively. Increases in cAMP in the presence of 10 nM spGIP were not dependent on the ambient glucose concentration, with 22- and 18-fold increases in cAMP accumulation at 0.1 and 5.5 mM glucose, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)