Biomedical subjects

P L Brubaker

Publications and source records attributed to P L Brubaker.

At least 19 recordsLinked to original sources

PubMed2006

From cradle to grave: pancreatic beta-cell mass and glucagon-like peptide-1.

Type 2 diabetes mellitus and its clinical correlates, including impaired fasting blood glucose, obesity and insulin resistance, represent a significant public health issue worldwide, with the prevalence of these metabolic conditions increasing exponentially. Given the staggering financial costs and human suffering incurred by diabetes and its co-morbid conditions, any safe new therapeutic interventions that prove to have a beneficial effect in reducing the incidence of diabetes in susceptible individuals or in preventing progression of the disease would have major public health benefits. Studies on the regulation of beta-cell mass have demonstrated a remarkable plasticity, from fetal through adult life, as well as in response to a variety of stresses. These findings are considered in this review in the context of newer studies on the intestinal hormone, glucagon-like peptide-1, which not only enhances beta-cell function, but also stimulates beta-cell growth, neogenesis and survival.

Adult↗

PubMed2005

Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B.

AIMS/HYPOTHESIS: The gut hormone glucagon-like peptide-1 (GLP-1) decreases beta cell apoptosis in a protein kinase B (PKB)-dependent fashion, and increases islet cell mass and function in vivo. In contrast, cytokines induce beta cell apoptosis, leading to decreased islet mass and type 1 diabetes. In the present study we used rat INS-1E beta cells and primary rat islet cells to examine the potential role of PKB as a mediator of the effect of GLP-1 on cytokine-induced apoptosis. METHODS: Cell viability was determined by MTT assay, and apoptosis and necrosis by Hoechst 33342-propidium iodide staining. Immunoblot analysis was used to detect changes in protein expression, including active (phosphorylated) and total PKB, phosphorylated and total glycogen synthase kinase-3beta, activated caspase-3 and inducible nitric oxide synthase. Reactive oxygen species were determined by 1,7-dichlorofluorescein (DCF) analysis, and mutant forms of PKB were introduced into cells using adenoviral vectors. RESULTS: Incubation of INS-1E cells with cytokines (IL-1beta, TNF-alpha and interferon-gamma; 10-50 ng/ml) for 18 h significantly decreased cell viability (by 44%, p<0.001), cell proliferation (by 80%, p<0.001), and activation of PKB (by 67%, p<0.001). Pre-treatment with exendin-4 (10(-7) mol/l), a long-acting GLP-1 receptor agonist, partially protected the cells against cytokine-induced toxicity (p<0.01) in association with a reduction in cytokine-induced inhibition of PKB phosphorylation (p<0.05). Exendin-4 pre-treatment did not change cell proliferation. Cytokine treatment increased apoptosis (by 156%, p<0.05) and necrosis (from undetectable to 2.6% of cells). These increases were both reduced by pre-treatment with exendin-4 (p<0.05-0.01). Furthermore, cytokine-induced apoptosis and necrosis were significantly increased in cells infected with kinase-dead PKB (p<0.05), and the protective effect of exendin-4 on both parameters was fully abolished in these cells. Similar changes were observed in primary islet cells. In parallel with these changes, exendin-4 decreased the cytokine-induced activation of caspase-3 (by 46%, p<0.05), and decreased levels of inducible nitric oxide synthase (by 71%, p<0.05) and reactive oxygen species (by 27%, p<0.05). CONCLUSIONS/INTERPRETATION: The results of our study indicate that GLP-1 plays a protective role against cytokine-induced apoptosis and necrosis in beta cells through a PKB-dependent signalling pathway.

Animals↗

PubMed2004

Extrahypothalamic expression of the glucagon-like peptide-2 receptor is coupled to reduction of glutamate-induced cell death in cultured hippocampal cells.

Proglucagon-derived glucagon-like peptide-2 (GLP-2) is liberated in enteroendocrine cells and neurons. GLP-2 regulates energy absorption and epithelial integrity in the gastrointestinal tract, whereas GLP-2 action in the central nervous system remains poorly defined. We identified proglucagon and GLP-2 receptor (GLP-2R) mRNA transcripts by RT-PCR in multiple regions of the developing and adult rat central nervous system. GLP-2R mRNA transcripts were localized by in situ hybridization to the hippocampus, hypothalamus, nucleus of the solitary tract, parabrachial nucleus, supramammillary nucleus, and substantia nigra. The bioactive form of GLP-2, GLP-2-(1-33) was detected by RIA and HPLC analysis in the fetal and adult brainstem and hypothalamus. GLP-2 stimulated increases in cAMP accumulation in postnatal d 8, but not embryonic d 14, dispersed neonatal rat brainstem tissues. The actions of GLP-2 were independent of the GLP-1R antagonist exendin-(9-39), and GLP-2 stimulated cAMP accumulation in hippocampal cell cultures from both wild-type and GLP-1R(-/-) mice. GLP-2 significantly reduced glutamate-induced excitotoxic injury in hippocampal cells via a protein kinase A-dependent pathway, but had no effect on the rate of cell proliferation. These findings establish the presence of a functional GLP-2-GLP-2R axis in the developing rodent brain and demonstrate that GLP-2 exerts cytoprotective actions in cells derived from the central nervous system.

Animals↗

PubMed2004

Minireview: Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system.

Gut peptides exert diverse effects regulating satiety, gastrointestinal motility and acid secretion, epithelial integrity, and both nutrient absorption and disposal. These actions are initiated by activation of specific G protein-coupled receptors and may be mediated by direct or indirect effects on target cells. More recent evidence demonstrates that gut peptides, exemplified by glucagon-like peptides-1 and 2 (GLP-1 and GLP-2), directly regulate signaling pathways coupled to cell proliferation and apoptosis. GLP-1 receptor activation enhances beta-cell proliferation and promotes islet neogenesis via activation of pdx-1 expression. The proliferative effects of GLP-1 appear to involve multiple intracellular pathways, including stimulation of Akt, activation of protein kinase Czeta, and transactivation of the epidermal growth factor receptor through the c-src kinase. GLP-1 receptor activation also promotes cell survival in beta-cells and neurons via increased levels of cAMP leading to cAMP response element binding protein activation, enhanced insulin receptor substrate-2 activity and, ultimately, activation of Akt. These actions of GLP-1 are reflected by expansion of beta-cell mass and enhanced resistance to beta-cell injury in experimental models of diabetes in vivo. GLP-2 also promotes intestinal cell proliferation and confers resistance to cellular injury in a variety of cell types. Administration of GLP-2 to animals with experimental intestinal injury promotes regeneration of the gastrointestinal epithelial mucosa and confers resistance to apoptosis in an indirect manner via yet-to-be identified GLP-2 receptor-dependent regulators of mucosal growth and cell survival. These proliferative and antiapoptotic actions of GLP-1 and GLP-2 may contribute to protective and regenerative actions of these peptides in human subjects with diabetes and intestinal disorders, respectively.

Animals↗

PubMed2004

Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells.

AIMS/HYPOTHESIS: The incretin hormone glucagon-like peptide-1 augments islet cell mass in vivo by increasing proliferation and decreasing apoptosis of the beta cells. However, the signalling pathways that mediate these effects are mostly unknown. Using a clonal rat pancreatic beta cell line (INS-1), we examined the role of protein kinase B in mediating beta-cell growth and survival stimulated by glucagon-like peptide-1. METHODS: Immunoblot analysis was used to detect active (phospho-) and total protein kinase B. Proliferation was assessed using (3)H-thymidine incorporation, while apoptosis was quantitated using 4'-6-diamidino-2-phenylindole staining and APO percentage apoptosis assay. Kinase-dead and wild-type protein kinase B was introduced into cells using adenoviral vectors. RESULTS: Glucagon-like peptide-1 rapidly activated protein kinase B in INS-1 cells (by 2.7+/-0.7-fold, p<0.05). This effect was completely abrogated by inhibition, with wortmannin, of the upstream activator of protein kinase B, phosphatidylinositol-3-kinase. Glucagon-like peptide-1 also stimulated INS-1 cell proliferation in a dose-dependent manner (by 1.8+/-0.5-fold at 10(-7) mol/l, p<0.01), and inhibited staurosporine-induced apoptosis (by 69+/-12%, p<0.05). Both of these effects were also prevented by wortmannin treatment. Ablation of protein kinase B by adenovirus-mediated overexpression of the kinase-dead form of protein kinase Balpha prevented protein kinase B phosphorylation and completely abrogated both cellular proliferation ( p<0.05) and protection from drug-induced cellular death ( p<0.01) induced by glucagon-like peptide-1. CONCLUSIONS/INTERPRETATION: These results identify protein kinase B as an essential mediator linking the glucagon-like peptide-1 signal to the intracellular machinery that modulates beta-cell growth and survival.

Animals↗

PubMed2002

Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 ameliorates the symptoms of diabetes through stimulation of insulin secretion and enhancement of beta-cell mass. We have therefore investigated the effects of glucagon-like peptide-1 on the development of diabetes, using db/db mice as a model of Type II diabetes. METHODS: The potent glucagon-like peptide-1 analogue Exendin-4 or vehicle (control) was administered (i.p.; 1 nmol/kg) to obese 6-week old db/db mice daily for 14 days ( n=10). RESULTS: By 8 weeks of age, control db/db mice developed hyperglycaemia (fasting: 10.4+/-0.5 mmol/l), hyperinsulinaemia and impaired glucose tolerance. However, Exendin-4 treatment prevented hyperglycaemia (fasting: 6.1+/-1.0 mmol/l, p<0.01), with reduced plasma insulin concentrations ( p<0.001) and improved glucose tolerance ( p<0.05). Peripheral insulin sensitivity was not affected. However, insulin release in vivo and in vitro from the perfused pancreas was improved by Exendin-4, as were pancreatic insulin concentrations (0.54+/-0.02 vs 0.32+/-0.01 micro g/mg protein, p<0.05). These changes occurred in conjunction with increased beta-cell mass (3.01+/-0.31 vs 2.22+/-0.22 mg, p<0.05) and proliferation (BrdU(+) beta-cells: 1.08+/-0.20 vs 0.47+/-0.11%, p<0.05), as well as decreased apoptosis (Tunel (+) beta-cells: 0.37+/-0.06 vs 1.20+/-0.21%). Western blot demonstrated increased expression of Akt1 (by fivefold, p<0.01) and p44 MAP kinase (by sixfold, p<0.01), and decreased activation of caspase-3 (by 30%, p<0.05). CONCLUSION/INTERPRETATION: Our results suggest that Ex4 treatment delays the onset of diabetes in 6-8 week old db/db mice, through a mechanism involving Akt1 and expansion of the functional beta-cell mass.

Animals↗

PubMed2002

Cellular specificity of proexendin-4 processing in mammalian cells in vitro and in vivo.

Glucagon-like peptide-1 (GLP-1) is a potent stimulator of glucose-dependent insulin secretion. Exendin-4(1-39) (Ex-4), isolated from Gila monster venom, is a highly specific GLP-1 receptor agonist that exhibits a prolonged duration of action in vivo. Although the processing mechanisms underlying liberation of GLP-1 from its prohormone have been elucidated, those for Ex-4 remain unknown. To examine the requirements for proEx-4 processing in mammalian cells, BHK fibroblasts, InR1-G9 islet A cells, and AtT-20 corticotropes, which express different prohormone convertases (furin, prohormone convertase 2, and prohormone convertase 1, respectively) were transfected with full-length lizard proEx-4, and the processing of proexendin was examined by HPLC and RIA (n = 3). All of the transfected cell lines exhibited Ex-4-like immunoreactivity in the media, and Ex-4-like immunoreactivity was detected in extracts of InR1-G9 and AtT-20 cells. However, only media and extracts from AtT-20 cells (not InR1-G9 and BHK cells) contained a single peak by HPLC corresponding to synthetic Ex-4. To establish whether proEx-4 can be processed to Ex-4 in nonimmortalized mammalian cells in vivo, the molecular forms of exendin-4 were examined in mice expressing a metallothionein-proEx-4 transgene (n = 3-6 for both males and females). ProEx4 mRNA transcripts were detected by RT-PCR in a broad range of both endocrine and nonendocrine tissues. Ex-4-like immunoreactivity was detected in pituitary, fat, adrenals, and testes; however HPLC analyses demonstrated that processed Ex-4 was found only in adrenals and testes. These results indicate that lizard proEx-4 is processed to mature bioactive Ex-4 in both rodent endocrine and nonendocrine mammalian cell types in vitro and in murine tissues in vivo. These findings may be useful for engineering cells that express a lizard pro-Ex4 transgene for the treatment of type 2 diabetes.

Adrenocorticotropic Hormone↗

PubMed2002

Structure-function of the glucagon receptor family of G protein-coupled receptors: the glucagon, GIP, GLP-1, and GLP-2 receptors.

The glucagon-like peptides include glucagon, GLP-1, and GLP-2, and exert diverse actions on nutrient intake, gastrointestinal motility, islet hormone secretion, cell proliferation and apoptosis, nutrient absorption, and nutrient assimilation. GIP, a related member of the glucagon peptide superfamily, also regulates nutrient disposal via stimulation of insulin secretion. The actions of these peptides are mediated by distinct members of the glucagon receptor superfamily of G protein-coupled receptors. These receptors exhibit unique patterns of tissue-specific expression, exhibit considerable amino acid sequence identity, and share similar structural and functional properties with respect to ligand binding and signal transduction. This article provides an overview of the biology of these receptors with an emphasis on understanding the unique actions of glucagon-related peptides through studies of the biology of their cognate receptors.

Alternative Splicing↗

PubMed2001

Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo.

Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala(2), its clinical application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala(2)]GLP-1 and [Gly(2)]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [D-Ala(2)]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His(1)-GLP-1, glucagon((1-5)-, glucagon((1-10))-, PACAP(1-5)-, VIP(1-5)-, and secretin((1-10))-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala(8/11/12/16)] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the alpha-amino group of His(1) and replacement of Ala(2) result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.

Amino Acid Sequence↗

PubMed2001

A glucagon-like peptide-1 receptor agonist and an antagonist modify macronutrient selection by rats.

The hypothesis that peripheral glucagon-like peptide-1 (GLP-1) is a regulator of both food intake and macronutrient selection in rats was tested by administration of its antagonist, exendin 9-39, and its agonist, exendin 4. The effect of exendin 9-39 given intraperitoneally (i.p.) on food intake was measured after carbohydrate, protein or fat preloads, and on choice between a protein-free, high carbohydrate (CHO) diet and a high protein, low carbohydrate (PRO) diet. The effect of exendin 4 on selection between the CHO and PRO diets was also investigated. Exendin 9-39 significantly enhanced food intake suppression occurring after glucose, but not after corn oil or albumin preloads. In diet selection studies, exendin 9-39 selectively decreased intake of only the CHO diet. In contrast, exendin 4 decreased intake of only the PRO diet. Thus, we suggest that peripheral GLP-1 plays a role in the regulation of macronutrient selection as well as food intake in rats.

Animals↗

PubMed2001

Coregulation of glucagon-like peptide-1 synthesis with proglucagon and prohormone convertase 1 gene expression in enteroendocrine GLUTag cells.

The insulinotropic hormone glucagon-like peptide-1 (GLP-1) is synthesized in the intestinal L cell by prohormone convertase 1 (PC1)-mediated posttranslational processing of proglucagon. Previous studies have demonstrated that proglucagon gene transcription in the L cell is stimulated by the protein kinase A (PKA) pathway through a cAMP response element (CRE). Because the PC1 gene contains two functional CREs, the present studies were conducted to investigate whether the PC1 and proglucagon genes are coregulated by PKA, and to elucidate the temporal relationship(s) of PC1 and proglucagon gene expression with production of GLP-1, in the intestinal cell. The GLUTag enteroendocrine cell line, which is known to express the proglucagon gene and to synthesize and secrete GLP-1, was used as a model. Proglucagon and PC1 messenger RNA transcript levels were both increased after 12 h (but not 24 h) of treatment of GLUTag cells with forskolin/isobutylmethylxanthine (IBMX), by 2.7 +/- 0.3- and 2.4 +/- 0.3-fold, respectively, compared with controls (P < 0.01-0.001). Activation of PKA resulted in a 2.1 +/- 0.1-fold increase in PC1 reporter construct expression (P < 0.001) at 12 h, which was dependent on the presence of the CRE, and a 13- to 24-fold increment in PC1 protein levels (P < 0.01) at 12 and 24 h. Similarly, forskolin/IBMX increased secretion of GLP-1, by 1.8 +/- 0.2- and 2.2 +/- 0.6-fold at 12 and 24 h, respectively (P < 0.05-0.01). Although the cell content of GLP-1 was diminished after 12 h of treatment (P < 0.001), GLP-1 levels increased back to control values after 24 h of forskolin/IBMX treatment (P < 0.01 vs. 12-h levels). Thus, PKA-induced secretion of GLP-1 from the L cell is followed by restoration of the cellular peptide levels through a PKA-mediated, CRE-dependent up-regulation of proglucagon and PC1 gene expression.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Monounsaturated fatty acid diets improve glycemic tolerance through increased secretion of glucagon-like peptide-1.

Diets enriched in monounsaturated fatty acids (MUFA)s have been shown to benefit glycemic control. Furthermore, MUFAs specifically stimulate secretion of the antidiabetic hormone, Glucagon-like peptide-1 (GLP-1) in vitro. To determine whether the MUFA-induced benefit in glycemic tolerance in vivo is due to increased GLP-1 release, lean Zucker rats were pair-fed a synthetic diet containing 5% fat derived from either olive oil (OO; 74% MUFA) or coconut oil (CO; 87% saturated fatty acids; SFA) for 2 weeks. Food intake and body weight gain were similar for both groups over the feeding period. The OO group had improved glycemic tolerance compared with the CO group in both oral and duodenal glucose tolerance tests [area under curve (AUC) 121 +/- 61 vs. 290 +/- 24 mM.120 min, P < 0.05; and 112 +/- 28 vs. 266 +/- 65 mM.120 min, P < 0.05, respectively]. This was accompanied by increased secretion of gut glucagon-like immunoreactivity (gGLI; an index of GLP-1 levels) in the OO rats compared with the CO rats (402 +/- 96 vs. 229 +/- 33 pg/ml at t = 10 min, P < 0.05). Tissue levels of GLP-1 and plasma insulin and glucagon levels were not different between the two groups. To determine the total contribution of GLP-1 to the enhanced glycemic tolerance in OO rats, the GLP-1 receptor antagonist exendin(9-39) (Ex(9-39)) was infused 3 min before a duodenal glucose tolerance test. Ex(9-39) abolished the benefit in glycemic tolerance conferred by OO feeding (OO+Ex(9-39) vs. CO+Ex(9-39), P = NS), and resulted in a deterioration of glycemic tolerance in the OO+Ex(9-39) group when compared with the OO controls (AUC 331 +/- 21 vs. 112 +/- 28 mM.120 min, P < 0.05). To probe the mechanism by which the OO diet enhanced GLP-1 secretion, a GLP-1-secreting L cell line was incubated for 24 h with either 100 microM oleic acid (MUFA) or 100 microM palmitic acid (SFA) and subsequently challenged with GIP, a known stimulator of the L cell. Preexposure to oleic acid but not to palmitic acid significantly increased GIP-induced GLP-1 secretion when compared with controls (55 +/- 12% vs. 34 +/- 9%, P < 0.01). These results demonstrate that the benefit in glycemic tolerance obtained with MUFA diets occurs in association with increased GLP-1 secretion, through a mechanism of enhanced L cell sensitivity. These results suggest that diet therapy with MUFAs may be useful for the treatment of patients with impaired glucose tolerance and/or type 2 diabetes through increased GLP-1 secretion.

Administration, Oral↗

PubMed2001

Therapeutic potential of the intestinotropic hormone, glucagon-like peptide-2.

Glucagon-like peptide-2 (GLP-2) is a newly discovered growth factor that has been demonstrated to enhance intestinal growth and function in normal rodents and to prevent damage and facilitate intestinal repair in various animal models of intestinal insufficiency. A recent study has demonstrated that GLP-2 also acts as an intestinotropin in humans with short-bowel syndrome. The high degree of specificity of GLP-2 for induction of intestinal growth, without affecting growth of other peripheral tissues, is determined by the highly localized expression of the GLP-2 receptor in the intestinal epithelium. In this article, we review the regulation of GLP-2 in physiology, from synthesis to metabolism, with a particular emphasis on potential targets in this pathway for therapeutic manipulation of GLP-2 actions. We also discuss the various animal models of intestinal insufficiency that have been used to demonstrate the therapeutic potential of this intestinotropic hormone, including short bowel, intestinal atrophy, enteritis and colitis. The results of these studies indicate that GLP-2 is a promising therapeutic agent for the treatment of various forms of intestinal insufficiency in humans.

Animals↗

PubMed2000

Sustained expression of exendin-4 does not perturb glucose homeostasis, beta-cell mass, or food intake in metallothionein-preproexendin transgenic mice.

Activation of glucagon-like peptide (GLP)-1 receptor signaling promotes glucose lowering via multiple mechanisms, including regulation of food intake, glucose-dependent insulin secretion, and stimulation of beta-cell mass. As GLP-1 exhibits a short t(12) in vivo, the biological consequences of prolonged GLP-1 receptor signaling remains unclear. To address this question, we have now generated metallothionein promoter-preproexendin (MT-Ex) transgenic mice. MT-Ex mice process preproexendin correctly, as is made evident by detection of circulating plasma exendin-4 immunoreactivity using high pressure liquid chromatography and an exendin-4-specific radioimmunoassay. Despite elevated levels of exendin-4, fasting plasma glucose and glucose clearance following oral and intraperitoneal glucose tolerance tests are normal in MT-Ex mice. Induction of transgene expression significantly reduced glycemic excursion during both oral and intraperitoneal glucose tolerance tests (p < 0.05) and increased levels of glucose-stimulated insulin following oral glucose administration (p < 0.05). Despite evidence that exendin-4 may induce beta-cell proliferation, beta-cell mass and islet histology were normal in MT-Ex mice. MT-Ex mice exhibited no differences in basal food intake or body weight; however, induction of exendin-4 expression was associated with reduced short term food ingestion (p < 0.05). In contrast, short term water intake was significantly reduced in the absence of zinc in fluid-restricted MT-Ex mice (p < 0.05). These findings illustrate that sustained elevation of circulating exendin-4 is not invariably associated with changes in glucose homeostasis, increased beta-cell mass, or reduction in food intake in mice in vivo.

Animals↗

PubMed2000

Structural determinants for activity of glucagon-like peptide-2.

Glucagon-like peptide-2 (GLP-2) is a 33 amino acid gastrointestinal hormone that regulates epithelial growth in the intestine. Dipeptidylpeptidase IV cleaves GLP-2 at the position 2 alanine, resulting in the inactivation of peptide activity. To understand the structural basis for GLP-2 action, we studied receptor binding and activation for 56 GLP-2 analogues with either position 2 substitutions or alanine replacements along the length of the peptide. The majority of position 2 substitutions exhibited normal to enhanced GLP-2 receptor (GLP-2R) binding; in contrast, position 2 substitutions were less well tolerated in studies of receptor activation as only Gly, Ile, Pro, alpha-aminobutyric acid, D-Ala, or nor-Val substitutions exhibited enhanced GLP-2R activation. In contrast, alanine replacement at positions 5,6,17, 20, 22, 23, 25, 26, 30, and 31 led to diminished GLP-2R binding. Position 2 substitutions containing Asp, Leu, Lys, Met, Phe, Trp, and Tyr, and Ala substitutions at positions 12 and 21 exhibited normal to enhanced GLP-2R binding but greater than 75% reduction in receptor activation. D-Ala(2), Pro(2) and Gly(2), Ala(16) exhibited significantly lower EC(50)s for receptor activation than the parent peptide (p < 0.01-0.001). Circular dichroism analysis indicated that the enhanced activity of these GLP-2 analogues was independent of the alpha-helical content of the peptide. These results indicate that single amino acid substitutions within GLP-2 can confer structural changes to the ligand-receptor interface, allowing the identification of residues important for GLP-2R binding and receptor activation.

Amino Acid Sequence↗

PubMed2000

Oral delivery of glucagon-like peptide-1 in a modified polymer preparation normalizes basal glycaemia in diabetic db/db mice.

AIMS/HYPOTHESIS: The insulinotropic hormone, glucagon-like peptide-1 has been proposed for the treatment of patients with Type II (non-insulin-dependent) diabetes mellitus. As glucagon-like peptide-1 is rapidly cleaved at L-ala2 by dipeptidylpeptidase IV, D-ala2-glucagon-like peptide-1 was synthesized and shown to have dipeptidylpeptidase IV resistance in vitro and enhanced bioactivity in mice during an oral glucose challenge. The actions of D-ala2-glucagon-like peptide-1 were, however, lost within 4 h of injection, thus necessitating frequent and invasive treatment if it is to be used therapeutically. To circumvent this problem, a microsphere of D-ala2-glucagon-like peptide-1 that could be given orally was developed. METHODS: We encapsulated D-ala2-glucagon-like peptide-1 in poly(lactide-co-glycolide)-COOH with olive oil as a filler, using phase inversion. The microspheres were tested in vivo by oral gavage in mice at t = 0 h followed by repeated oral glucose tolerance tests at t = 0, 4 and 8 h. RESULTS: The D-ala2-glucagon-like peptide-1-microspheres lowered the glycaemic response to the 4 h oral glucose challenge in both normal CD1 and diabetic db/db mice, by 41 +/- 12% (p <0.001) and 27 +/- 5% (p < 0.001), respectively and by 19 +/- 11% (p < 0.05) and 28 +/- 4% (p < 0.001), respectively during the 8-h test. At 4 h after the oral gavage, basal glycaemia in the diabetic mice was reduced from 13 +/- 1 mmol/l to 10 +/- 1 mmol/l and was reduced further 8h after treatment from 12 +/- 1 mmol/l to 8 +/- 1 mmol/l (p < 0.05). Giving D-ala2-glucagon-like peptide-1 alone orally had no effect on glycaemia. CONCLUSION/INTERPRETATION: The data presented here suggest that a similar microsphere preparation could be useful in the delivery of glucagon-like peptide-1 to patients with Type II diabetes.

3-O-Methylglucose↗

PubMed2000

Enzymatic- and renal-dependent catabolism of the intestinotropic hormone glucagon-like peptide-2 in rats.

The intestinotropic hormone glucagon-like peptide (GLP)-2-(1-33) is cleaved in vitro to GLP-2-(3-33) by dipeptidyl peptidase IV (DP IV). To determine the importance of DP IV versus renal clearance in the regulation of circulating GLP-2-(1-33) levels in vivo, GLP-2-(1-33) or the DP IV-resistant analog [Gly(2)]GLP-2 was injected in normal or DP IV-negative rats and assayed by HPLC and RIA. Normal rats showed a steady degradation of GLP-2-(1-33) to GLP-2-(3-33) over time, whereas little or no conversion was detected for GLP-2-(1-33) in DP IV-negative rats and for [Gly(2)]GLP-2 in normal rats. To determine the role of the kidney in clearance of GLP-2-(1-33) from the circulation, normal rats were bilaterally nephrectomized, and plasma immunoreactive GLP-2 levels were measured. The slope of the disappearance curves for both GLP-2-(1-33) and [Gly(2)]GLP-2 were significantly reduced in nephrectomized compared with non-nephrectomized rats (P < 0.01). In contrast to both GLP-2-(1-33) and [Gly(2)]GLP-2, GLP-2-(3-33) did not stimulate intestinal growth in a murine assay in vivo. Thus the intestinotropic actions of GLP-2-(1-33) are determined both by the actions of DP IV and by the kidney in vivo in the rat.

Amino Acid Sequence↗

PubMed2000

Circulating levels of glucagon-like peptide-2 in human subjects with inflammatory bowel disease.

Glucagon-like peptide-2 (GLP-2) is a recently characterized intestine-derived peptide that exerts trophic activity in the small and large intestine. Whether circulating levels of GLP-2 are perturbed in the setting of human inflammatory bowel disease (IBD) remains unknown. The circulating levels of bioactive GLP-2-(1-33) compared with its degradation product GLP-2-(3-33) were assessed using a combination of RIA and HPLC in normal and immunocompromised control human subjects and patients hospitalized for IBD. The activity of the enzyme dipeptidyl peptidase IV (DP IV), a key determinant of GLP-2-(1-33) degradation was also assessed in the plasma of normal controls and subjects with IBD. The circulating levels of bioactive GLP-2-(1-33) were increased in patients with either ulcerative colitis (UC) or Crohn's Disease (CD; to 229 +/- 65 and 317 +/- 89%, P < 0.05, of normal, respectively). Furthermore, the proportion of total immunoreactivity represented by intact GLP-2-(1-33), compared with GLP-2-(3-33), was increased from 43 +/- 3% in normal healthy controls to 61 +/- 6% (P < 0.01) and 59 +/- 2% (P < 0.01) in patients with UC and CD, respectively. The relative activity of plasma DP IV was significantly reduced in subjects with IBD compared with normal subjects (1.4 +/- 0.3 vs. 5.0 +/- 1.1 mU/ml, respectively; P < 0.05). These results suggest that patients with active IBD may undergo an adaptive response to intestinal injury by increasing the circulating levels of bioactive GLP-2-(1-33), facilitating enhanced repair of the intestinal mucosal epithelium in vivo.

Adaptation, Physiological↗