GLP 1 Regulated Intestinal Cell’s Insulin Expression and Selfadaptation before the Onset of Type 2 Diabetes (original) (raw)
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Glucagon-Like Peptide-1 Promotes Islet Cell Growth and Inhibits Apoptosis in Zucker Diabetic Rats
constant remodeling of islet cell mass mediated by proliferative and apoptotic stimuli ensures a dynamic response to a changing demand for insulin. In this study, we investigated the effect of glucagon-like peptide-1 (GLP-1) in Zucker diabetic rats, an animal model in which the onset of diabetes occurs when the proliferative potential and the rate of -cell apoptosis no longer compensate for the increased demand for insulin. We subjected diabetic rats to a 2-d infusion of GLP-1 and tested their response to an ip glucose tolerance test. GLP-1 produced a significant increase of insulin secretion, which was paralleled by a decrease in plasma glucose levels (P < 0.001 and P < 0.01, respectively). Four days after the removal of the infusion pumps, rats were killed and the pancreas harvested to study the mechanism by which GLP-1 ameliorated glucose tolerance. Ex vivo immunostaining with the marker of cell proliferation, Ki-67, showed that the metabolic changes observed in rats treated with GLP-1 were associated with an increase in cell proliferation of the endocrine and exocrine component of the pancreas. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, a marker of cellular apoptosis, indicated a reduction of apoptotic cells within the islet as well in the exocrine pancreas in GLP-1-treated rats. Double immunostaining for the apoptotic marker caspase-3 and for insulin showed a significant reduction of caspase-3 expression and an increase in insulin content in GLP-1-treated animals. Finally, staining of pancreatic sections with the nuclear dye 4,6- Diaminidino-2-phenyl-dihydrochloride demonstrated a marked reduction of fragmented nuclei in the islet cells of rats treated with GLP-1. Our findings provide evidence that the beneficial effects of GLP-1 in Zucker diabetic rats is mediated by an increase in islet cell proliferation and a decrease of cellular apoptosis. (Endocrinology 143: 4397–4408, 2002)
Insulin Regulates Glucagon-Like Peptide-1 Secretion from the Enteroendocrine L Cell
Endocrinology, 2009
Insulin resistance and type 2 diabetes mellitus are associated with impaired postprandial secretion of glucagon-like peptide-1 (GLP-1), a potent insulinotropic hormone. The direct effects of insulin and insulin resistance on the L cell are unknown. We therefore hypothesized that the L cell is responsive to insulin and that insulin resistance impairs GLP-1 secretion. The effects of insulin and insulin resistance were examined in well-characterized L cell models: murine GLUTag, human NCI-H716, and fetal rat intestinal cells. MKR mice, a model of chronic hyperinsulinemia, were used to assess the function of the L cell in vivo. In all cells, insulin activated the phosphatidylinositol 3 kinase-Akt and MAPK kinase (MEK)-ERK1/2 pathways and stimulated GLP-1 secretion by up to 275 ± 58%. Insulin resistance was induced by 24 h pretreatment with 10−7m insulin, causing a marked reduction in activation of Akt and ERK1/2. Furthermore, both insulin-induced GLP-1 release and secretion in response ...
Endocrinology, 2003
The peptide hormone, glucagon-like peptide 1 (GLP-1), has been shown to increase glucose-dependent insulin secretion, enhance insulin gene transcription, expand islet cell mass, and inhibit -cell apoptosis in animal models of diabetes. The aim of the present study was to evaluate whether GLP-1 could improve function and inhibit apoptosis in freshly isolated human islets. Human islets were cultured for 5 d in the presence, or absence, of GLP-1 (10 nM, added every 12 h) and studied for viability and expression of proapoptotic (caspase-3) and antiapoptotic factors (bcl-2) as well as glucose-dependent insulin production. We observed better-preserved three-dimensional islet morphology in the GLP-1-treated islets, compared with controls. Nuclear condensation, a feature of cell apoptosis, was inhibited by GLP-1. The reduction in the number of apoptotic cells in GLP-1-treated islets was particularly evi-dent at d 3 (6.1% apoptotic nuclei in treated cultures vs. 15.5% in controls; P < 0.01) and at d 5 (8.9 vs. 18.9%; P < 0.01). The antiapoptotic effect of GLP-1 was associated with the downregulation of active caspase-3 (P < 0.001) and the up-regulation of bcl-2 (P < 0.01). The effect of GLP-1 on the intracellular levels of bcl-2 and caspase-3 was observed at the mRNA and protein levels. Intracellular insulin content was markedly enhanced in islets cultured with GLP-1 vs. control (P < 0.001, at d 5), and there was a parallel GLP-1-dependent potentiation of glucose-dependent insulin secretion (P < 0.01 at d 3; P < 0.05 at d 5). Our findings provide evidence that GLP-1 added to freshly isolated human islets preserves morphology and function and inhibits cell apoptosis. (Endocrinology 144:
Regulatory Peptides, 2002
Supraphysiological doses of glucagon-like peptide-2 (GLP-2) have been shown to induce intestinal growth by increasing villus height and crypt depth and by decreasing apoptosis, but a physiological effect of GLP-2 has not yet been demonstrated. Earlier, we found elevated levels of endogenous GLP-2 in untreated streptozotocin diabetic rats associated with marked intestinal growth. In the present study, we investigated the role of endogenous GLP-2 for this adaptive response. We included four groups of six rats: (1) diabetic rats treated with saline, (2) diabetic rats treated with non-specific antibodies, (3) diabetic rats treated with polyclonal GLP-2 antibodies and (4) non-diabetic control rats treated with saline. All animals were treated with once daily intraperitoneal injections for 13 days and killed on day 14. Diabetic rats treated with saline or non-specific antibodies had a significantly ( P < 0.01) increased area of mucosa (13.00 F 0.64 and 13.37 F 0.60 mm 2 , respectively) in the proximal part of the small intestine compared with non-diabetic controls (7.97 F 0.70 mm 2 ). In contrast, diabetic rats treated with GLP-2 antibodies had a significantly ( P < 0.01) smaller increase in area of mucosa in the proximal part of the small intestine (10.84 F 0.44 mm 2 ). Antibody treatment had no effect on body weight, blood glucose concentrations and food intake. Thus, blocking of endogenous GLP-2 in a model of adaptive intestinal growth reduces the growth response, providing strong evidence for a physiological growth factor function of GLP-2. D
Endocrine, 2002
Glucagon-like peptide 1 (GLP-1) content of the intestinal tract was recently found to be lower in diabetesprone BioBreeding (BBdp) rats than in the corresponding control animals (BBc rats), a finding compatible with the idea that an inflammatory intestinal state precedes insulitis in these diabetes-prone animals. This study aimed at measuring GLP-1 content of the intestinal tract both in another animal model of type 1 diabetes and in an animal model of type 2 diabetes. GLP-1 content of the jejunum, ileum, colon, and cecum was measured in male and female adult control rats and animals injected with streptozotocin (STZ) either during the neonatal period or 7 d before sacrifice. GLP-1 content of the intestinal tract was higher in type 1 diabetic rats than in control animals. Such was not the case in the type 2 diabetic rats. The findings recorded in the rats injected with STZ either during the neonatal period or later in life indicate that hyperglycemia and/or insulin deficiency do not cause a decrease in GLP-1 content of the intestinal tract. On the contrary, such a content may increase when the glucose intolerance and hypoinsulinemia are sufficiently pronounced, as was the case in the type 1 diabetic rats. These findings are thus compatible with the view that the decreased GLP-1 content of the intestinal tract in BBdp rats may result from intestinal inflammation.
Effect of GLP-1 Treatment on GLUT2 and GLUT4 Expression in Type 1 and Type 2 Rat Diabetic Models
Endocrine, 2001
Glucagon-like peptide-1 (GLP-1) is an incretin with glucose-dependent insulinotropic and insulin-independent antidiabetic properties that exerts insulin-like effects on glucose metabolism in rat liver, skeletal muscle, and fat. This study aimed to search for the effect of a prolonged treatment, 3 ds, with GLP-1 on glucotransporter GLUT2 expression in liver, and on that of GLUT4 in skeletal muscle and fat, in rats. Normal rats and streptozotocin-induced type 1 and type 2 diabetic models were used; diabetic rats were also treated with insulin for comparison. In normal rats, GLP-1 treatment reduced in the three tissues the corresponding glucotransporter protein level, without modifying their mRNA. In the type 2 diabetic model, GLP-1, like insulin, stimulated in liver and fat only the glucotransporter translational process, while in the muscle an effect at the GLUT4 transcriptional level was also observed. In the type 1 diabetic model, GLP-1 apparently exerted in the liver only a posttranslational effect on GLUT2 expression; in muscle and fat, while insulin was shown to have an action on GLUT4 at both transcriptional and translational levels, the effect of GLP-1 was restricted to glucotransporter translation. In normal and diabetic rats, exogenous GLP-1 controlled the glucotransporter expression in extrapancreatic tissues participating in the overall glucose homeostasis-liver, muscle, and fatwhere the effect of the peptide seems to be exerted only at the translational and/or posttranslational level; in muscle and fat, the presence of insulin seems to be required for GLP-1 to activate the transcriptional process. The stimulating action of GLP-1 on GLUT2 and GLUT4 expression, mRNA or protein, could be a mechanism by which, at least in part, the peptide exerts its lowering effect on blood glucose.
Endocrinology, 2003
The activation of the glucagon-like peptide-1 (GLP-1) receptor has been shown to have an important role in the functional activity of islet -cells and in the expansion of the islet cell mass. Constant remodeling of islet cell mass is mediated in vivo by proliferative and apoptotic stimuli to ensure a dynamic response to a changing demand for insulin. The present study was undertaken to investigate the biological activity of GLP-1 when cells were challenged by a proapoptotic stimulus. We have shown that activation of the GLP-1 receptor inhibits H 2 O 2-induced apoptosis in a cultured mouse insulinoma cell line, termed MIN6. GLP-1 reduced DNA fragmentation and improved cell survival. This was mediated by an increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. GLP-1 also prevented the H 2 O 2-dependent cleavage of poly-(ADP-ribose)-polymerase. Inhibition of the GLP-1-dependent increase of cAMP by Rp-cAMP blocked the antiapoptotic action of GLP-1, as determined by DNA fragmentation and poly-(ADP-ribose)-polymerase assays and by detection of Bcl-2 and Bcl-xL protein levels. Investigation of the role of the protein kinases, PI-3 kinase (PI3K) and MAPK, by use of the inhibitors PD098059 and LY294002 demonstrated that the activation of PI3K, but not MAPK, was required to prevent proapoptotic events in cells exposed to H 2 O 2. The present study provides evidence that GLP-1 has an antiapoptotic action mediated by a cAMP-and PI3K-dependent signaling pathway. (Endocrinology 144: 1444-1455, 2003) A POPTOSIS, OR PROGRAMMED cell death, is a physiological mode of remodeling tissues during organogenesis and adulthood. The process is characterized by morphological changes, including condensation of the nuclear chromatin, DNA fragmentation, cellular shrinkage, and the formation of apoptotic bodies, which are membrane-bound cellular constituents (1). Apoptotic cell death is an energyrequiring process that involves de novo synthesis of proteins. Various molecules have been demonstrated to regulate, by promoting or inhibiting, the cellular changes that lead to apoptosis. These include the Bcl-2 protein family (2), the caspase family (3), caspase-activated deoxyribonuclease, and inhibitor of caspaseactivated deoxyribonuclease (4). Numerous studies have demonstrated that cell apoptosis plays an important role in both the physiological remodeling of the pancreas after birth and pathological pancreatic damage in diabetes (5, 6). Lally et al. (7) and Augstein et al. (8) demonstrated that a great increase of islet cell apoptosis in animal models of type I diabetes correlated with the progression of -cell, leading to the onset of hyperglycemia. Recent findings that free fatty acids, glucose, sulfonylurea, and amylin cause -cell apoptosis in vitro suggest that apoptosis may also be involved in the pathogenesis of type II diabetes (9, 10). Bonner-Weir and colleagues (11) have shown that, in a Zucker diabetic fatty rat model, the onset of diabetes is caused by an excessive rate of -cell death, not by an inefficient replication capacity (11). In a recent study, we demonstrated that treatment with glucagon-like peptide-1 (GLP-1) drastically reduced the number of apoptotic cells in the pancreas of Zucker diabetic rats. This was indicated by a decrease of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive-cells, a down-regulation of caspase-3 expression, and an increased expression of Bcl-2 and insulin (12). The present study was undertaken to investigate whether GLP-1 had a direct antiapoptotic effect on insulin-secreting cells, independently from the amelioration of insulin secretion and the acquired glucose control that follow its administration in vivo. We also investigated some of the early events characterizing the signaling pathway that mediates the antiapoptotic action of GLP-1. Materials and Methods Mouse insulinoma cell line, MIN6, was a kind gift from Dr. Donald F. Steiner (Howard Hughes Medical Institute, The University of Chicago, IL); Fetal bovine serum (FBS), PBS, penicillin-streptomycin, and cell culture media were obtained from Life Technologies, Inc. (Rockville, MD). The anti-Bcl-xL, anti-Bcl-2, and anti--actin antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Electrochemiluminescence Western blotting detection reagents, Hybond-C nitrocellulose membrane, and cAMP RIA kit were purchased from Amersham Pharmacia Biotech (Denver, CO); X-Omat AR autoradiographic films were from Eastman Kodak Co. (New Haven, CT). H 2 O 2 , dimethyl sulfoxide, ethanol, LY294002, PD098059, and Hoechst 33342 were obtained from Sigma (St. Louis, MO); GLP-1, exendin-4, and exendin(9-39) were purchased from American Peptide Co. (Sunnyvale, CA); Rp-cAMP was purchased from Calbiochem (La Jolla, CA); Annexin-V-FLUOS staining kit was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Comassie dye assay was purchased from Bio-Rad Laboratories, Inc.
Endocrinology, 1998
Glucagon-like peptide-1(7-36)amide (GLP-1) is a potent insulinotropic peptide released from the small intestine. To investigate the regulation of GLP-1 secretion, we established a GLP-1 release assay based on primary canine intestinal L-cells. The ileal mucosa was digested with collagenase/EDTA to a single cell suspension and enriched for L-cells by counterstream centrifugal elutriation. We performed release assays on the cultured cells after 36 h, and GLP-1 in the supernatant was determined by enzyme-linked immunoabsorbent assay (ELISA). Glucose-dependent insulinotropic peptide (GIP) dose dependently stimulated the release of GLP-1 and resulted in a 2-fold increase at 100 nM GIP. This effect was fully inhibited by 10 nM somatostatin. However, neither basal or GIP stimulated GLP-1 secretion were affected by ambient glucose concentrations from 5-25 mM. The receptorindependent secretagogues  phorbol myristate acetate and forskolin dose dependently increased the secretion of GLP-1; effects inhibited by staurosporine and H8 respectively. Costimulation with GIP and phorbol ester, but not forskolin, resulted in an additive response. Furthermore, the effect of GIP could be inhibited by H8 but not by staurosporine. These results indicate that glucose does not directly stimulate canine L-cells. It is more probable that glucose releases GIP from the upper intestine that in turn stimulates GLP-1 secretion. The ability of GIP to stimulate GLP-1 secretion is probably mediated through activation of protein kinase A.
Journal of Clinical Investigation, 1987
Insulin secretion is controlled by a complex set of factors that include not only glucose but amino acids, catecholamines, and intestinal hormones. We report that a novel glucagon-like peptide, co-encoded with glucagon in the glucagon gene is a potent insulinotropic factor. The glucagon gene encodes a proglucagon that contains in its sequence glucagon and additional glucagonlike peptides (GLPs). These GLPs are liberated from proglucagon in both the pancreas and intestines. GLP-I exists in at least two forms: 37 amino acids GLP-I(1-37), and 31 amino acids, GLP-I(7-37). We studied the effects of synthetic GLP-Is on insulin secretion in the isolated perfused rat pancreas. In the presence of 6.6 mM glucose, GLP-I(7-37) is a potent stimulator of insulin secretion at concentrations as low as 5 X 10-11 M (3to 10-fold increases over basal). GLP-I(1-37) had no effect on insulin secretion even at concentrations as high as 5 X 10-7 M. The earlier demonstration of specific liberation of GLP-I(7-37) in the intestine and pancreas, and the magnitude of the insulinotropic effect at such low concentrations, suggest that GLP-I(7-37) participates in the physiological regulation of insulin secretion.