Regulation of Glucagon-Like Peptide-1 Synthesis and Secretion in the GLUTag Enteroendocrine Cell Line¹

Abstract

Glucagon-like peptide-1 (GLP-1) released from the intestine is a potent stimulator of glucose-dependent insulin secretion. To elucidate the factors regulating GLP-1 secretion, we have studied the enteroendocrine GLUTag cell line. GLP-1 secretion was stimulated in a dosedependent fashion by activation of protein kinase A or C with forskolin or phorbol 12,13-dibutyrate, respectively (by 2.3 Ϯ 0.5-fold at 100 M and 4.3 Ϯ 0.6-fold at 0.3 M, respectively; P Ͻ 0.01-0.001). Of the regulatory peptides tested, only glucose-dependent insulinotropic peptide stimulated the release of GLP-1 (by 2.3 Ϯ 0.2-fold at 0.1 M; P Ͻ 0.001); glucagon was without effect, and paradoxically, the inhibitory neuropeptide somatostatin-14 increased secretion slightly (by 1.6 Ϯ 0.3-fold at 0.01 M; P Ͻ 0.05). In tests of several neurotransmitters, only the cholinergic agonists carbachol and bethanechol stimulated peptide secretion in a dose-dependent fashion (by 2.3 Ϯ 0.5-and 1.7 Ϯ 0.3-fold at 1000 M; P Ͻ 0.05-0.001); the ␤-adrenergic agonist isoproterenol and the chloride channel inhibitor ␥-aminobutyric acid did not affect release of GLP-1. Long chain monounsaturated fatty acids (18:1), but not saturated fatty acids (16:0), also stimulated the release of GLP-1 (by 1.7 Ϯ 0.1-fold at 150 M; P Ͻ 0.001). Consistent with the presence of a cAMP response element in the proglucagon gene, activation of the protein kinase A-dependent pathway with forskolin increased proglucagon messenger RNA transcript levels by 2-fold (P Ͻ 0.05); glucose-dependent insulinotropic peptide and phorbol 12,13-dibutyrate were without effect. Therefore, by comparison with results obtained using primary L cell cultures or in vivo models, GLUTag cells appear to respond appropriately to the regulatory mechanisms controlling intestinal GLP-1 secretion. (Endocrinology 139: 4108 -4114, 1998) G LUCAGON-LIKE peptide-1 (GLP-1) is a potent stimulator of glucose-dependent insulin secretion. Administration of GLP-1 to patients with type II diabetes normalizes both fasting and postprandial glycemia (1-13), not only through stimulation of insulin release, but also through concomitant inhibition of glucagon secretion (5, 9, 11, 13) and gastric motility (5, 14) and, possibly, enhancement of insulin sensitivity (8,15,16). GLP-1 is normally synthesized and secreted by the intestinal L cell (17-21); thus, stimulation of endogenous secretion represents an alternative approach to increasing levels of GLP-1 in type II diabetes. It is therefore essential that the factors regulating GLP-1 release from the L cell be elucidated.A number of in vitro cell culture systems have been developed as models of the intestinal L cell, each of which has both advantages and drawbacks. For example, fetal rat intestinal cell (FRIC) cultures are heterogeneous in their cell population, although they have proven to be an excellent model of the rat L cell otherwise, releasing GLP-1 in response to a wide variety of different signal transduction pathways and extracellular mediators (18, 20 -24). FRIC cells hav...

Transcriptional Activation of the Proglucagon Gene by Lithium and β-Catenin in Intestinal Endocrine L Cells

“…We hypothesized that if PKA activates proglucagon gene transcription via inactivating GSK-3␤, lithium may also activate proglucagon mRNA expression. We examined this hypothesis in the mouse large intestinal GLUTag (13,(45)(46)(47) and the mouse small intestinal STC-1 (14) cell lines, as well as in the primary (FRIC) rat intestinal cultures. After incubation with 10 mM LiCl (48) for 4 h, both cell lines demonstrated ϳ3-fold increased proglucagon mRNA expression ( Fig.…”

TCF-4 Mediates Cell Type-specific Regulation of Proglucagon Gene Expression by β-Catenin and Glycogen Synthase Kinase-3β

“…A challenging question that remains is whether the cultivated cell lines used in these studies are good surrogates of the primary glu producing cells. Our previous studies have demonstrated that the GLUTag cell line responds appropriately to the regulatory factors known to control glu gene expression and GLP-1 synthesis and secretion in primary gut endocrine cells, including cAMP/protein kinase A, glucose-dependent insulinotropic peptide, and bethanechol (26,40,50,51). Likewise, the ␣-TC-1 and InR1-G9 cell lines have been routinely used as ␣ cell models to study glu gene expression and glucagon synthesis and secretion (19,20,30,(52)(53)(54)(55)(56).…”

Flavone Hispidulin Stimulates Glucagon‐Like Peptide‐1 Secretion and Ameliorates Hyperglycemia in Streptozotocin‐Induced Diabetic Mice

“…Mouse GluTag L‐cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5.5 m m glucose, 10% fetal bovine serum (FBS), and 1% penicillin/ streptomycin . For GLP‐1 secretion experiments, cells were seeded into poly‐ d ‐lysine coated 12‐well plate (10 5 cells per well).…”

“…Cell Culture: Mouse GluTag L-cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5.5 mm glucose, 10% fetal bovine serum (FBS), and 1% penicillin/ streptomycin. [74] For GLP-1 secretion experiments, cells were seeded into poly-d-lysine coated 12well plate (10 5 cells per well). After 24 h, cells were washed with Krebs Ringer Buffer (KRB, 129 mm NaCl, 2.5 mm CaCl 2 , 1.2 mm MgSO 4 , 4.8 mm KCl, 1.2 mm KH 2 PO 4 , 10 mm HEPES, 5 mm NaHCO 3 , pH 7.4) supplemented with 0.2% bovine serum albumin (BSA), and treated with various concentrations of hispidulin or with various flavonoids as indicated for 1 h. Active GLP-1 secreted in supernatants was measured using an ELISA kit.…”