Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2

Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates intestinal growth and up-regulates villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia resulting from total parenteral nutrition. However, the mechanism underlying these actions has remained unclear. Here we report the cloning and characterization of cDNAs encoding rat and human GLP-2 receptors (GLP-2R), a G protein-coupled receptor superfamily member expressed in the gut and closely related to the glucagon and GLP-1 receptors. The human GLP-2R gene maps to chromosome 17p13.3. Cells expressing the GLP-2R responded to GLP-2, but not GLP-1 or related peptides, with increased cAMP production (EC50 = 0.58 nM) and displayed saturable high-affinity radioligand binding (Kd = 0.57 nM), which could be displaced by synthetic rat GLP-2 (Ki = 0.06 nM). GLP-2 analogs that activated GLP-2R signal transduction in vitro displayed intestinotrophic activity in vivo. These results strongly suggest that GLP-2, like glucagon and GLP-1, exerts its actions through a distinct and specific novel receptor expressed in its principal target tissue, the gastrointestinal tract.     

Histopathological variations among cases of Wilms'' tumor in Bangladesh and its relationship with prognosis

Both glucagon and glucagon-like peptide-1 (GLP-1) play an important role in the regulation of nutrient homeostasis. In this study, the tissue distributions of the expression of receptor genes for glucagon and GLP-1 were examined. Expression of glucagon receptor gene was detected in liver, kidney, ileum and pancreatic islets but not in brain. In contrast, expression of GLP-1 receptor gene was detected in brain, pancreas and pancreatic islets but not in liver, kidney, or ileum. To investigate the existence and characteristics of glucagon and GLP-1 receptors on pancreatic beta cells, expression of the receptor genes and translational regulation of the expression of the receptor genes by glucose were analyzed in a mouse pancreatic beta cell line, MIN6 cells. In the cDNA pool of MIN6 cells, both glucagon and GLP-1 receptor genes were identified and showed higher expression level in MIN6 cells cultured under high glucose condition than in those cultured under low glucose condition. These results suggest that glucagon and GLP-1 receptor genes are expressed in pancreatic beta cells and their expression is upregulated by glucose.     

The Xenopus proglucagon gene encodes novel GLP-1-like peptides with insulinotropic properties

The proglucagon gene encodes several hormones that have key roles in the regulation of metabolism. In particular, glucagon-like peptide (GLP-1), a potent stimulus of insulin secretion, is being developed as a therapy for the treatment of non-insulin-dependent diabetes mellitus. To define structural moieties of the molecule that convey its insulinotropic activity, we have cloned and characterized the proglucagon gene from the amphibian, Xenopus laevis. Unexpectedly, these cDNAs were found to encode three unique glucagon-like-1 peptides, termed xenGLP-1A, xenGLP-1B, and xenGLP-1C in addition to the typical proglucagon-derived hormones glucagon and GLP-2. xenGLP-1A, -1B, and -1C were synthesized and tested for their ability to bind and activate the human GLP-1 receptor (hGLP-1R), and to stimulate insulin release from rat pancreas. All three Xenopus GLP-1-like peptides bind effectively to the hGLP-1R and stimulate cAMP production. Surprisingly, xenGLP-1B(1-30) demonstrated higher affinity for the hGLP-1R than hGLP-1 (IC50 of 1.1 +/- 0.4 nM vs. 4.4 +/- 1.0 nM, respectively, P < 0.02) and was equipotent to hGLP-1 in stimulating cAMP production (EC50 of 0.17 +/- 0.02 nM vs. 0.6 +/- 0. 2 nM, respectively, P > 0.05). Further studies demonstrated that hGLP-1, xenGLP-1A, -1B, and -1C stimulate comparable insulin release from the pancreas. These results demonstrate that despite an average of nine amino acid differences between the predicted Xenopus GLPs and hGLP-1, all act as hGLP-1R agonists.     

Cholecystokinin-B/gastrin receptor: a novel molecular probe for human small cell lung cancer

The brain-gut hormones, gastrin and cholecystokinin, have a trophic effect on the gastrointestinal mucosa in vivo and promote the growth of several neoplastic cell lines. In this study, cholecystokinin-B/gastrin receptor has been demonstrated to provide a novel molecular marker for the diagnosis of small cell lung cancer by using biopsy specimens. Physiological expression of the receptor mRNA is detectable in particular areas of the human brain, stomach, and pancreas but not in the lung. The receptor mRNA was detected selectively in all small cell lung cancer (10 cases) with a RT-PCR assay. By contrast, it was detectable in only 1 of 13 squamous cell carcinomas or 21 adenocarcinomas of the lung. Thus, the cholecystokinin-B/gastrin receptor could be an attractive therapeutic target for small cell lung cancer.     

Enhanced glucose-dependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor -/- mice

Incretins are gastrointestinal hormones that act on the pancreas to potentiate glucose-stimulated insulin secretion. Despite the physiological importance of the enteroinsular axis, disruption of glucagon-like peptide (GLP)-1 action is associated with only modest glucose intolerance in GLP-1 receptor -/- (GLP-1R -/-) mice. We show here that GLP-1R -/- mice exhibit compensatory changes in the enteroinsular axis via increased glucose-dependent insulinotropic polypeptide (GIP) secretion and enhanced GIP action. Serum GIP levels in GLP-1R -/- mice were significantly elevated versus those in +/+ control mice after an oral glucose tolerance test (369 +/- 40 vs. 236 +/- 28 pmol/l; P < or = 0.02). Furthermore, GIP perfusion of mice pancreas and isolated islets in the presence of elevated glucose concentrations elicited a significantly greater insulin response in GLP-1R -/- than in +/+ mice (P < or = 0.02-0.05). In contrast, no significant perturbation in the insulin response to perfused glucagon was detected under conditions of low (4.4 mmol/l) or high (16.6 mmol/l) glucose in GLP-1R -/- mice. Total pancreatic insulin but not glucagon content was significantly reduced in GLP-1R -/- compared with in +/+ mice (77 +/- 9 vs. 121 +/- 10 pmol/mg protein; P < or = 0.005). These observations suggest that upregulation of the GIP component of the enteroinsular axis, at the levels of GIP secretion and action, modifies the phenotype resulting from interruption of the insulinotropic activity of GLP-1 in vivo.     

Glucagonlike peptide 1: a newly discovered gastrointestinal hormone

Glucagonlike peptide (GLP) 1, a peptide of 30 amino acids with 50% sequence homology to glucagon, results from expression of the glucagon gene in the L cells of the distal intestinal mucosa. It is secreted early in response to mixed meals by mechanisms involving the presence of unabsorbed nutrients in the gut lumen or the absorptive process itself, but other mechanisms may also be involved. GLP-1 has two important actions. First, it stimulates insulin secretion and inhibits glucagon secretion and thereby inhibits hepatic glucose production and lowers blood glucose levels. It may have effects on glucose clearance independent of its pancreatic effects. It acts on recently cloned G protein-coupled specific receptors and seems to increase insulin secretion via cyclic adenosine monophosphate-dependent increases in intracellular calcium. It has been suggested that activation of the beta cells by GLP-1 is a prerequisite for glucose-induced insulin secretion. Second, it also potently inhibits gastrointestinal secretion and motility and is likely to act as an "ileal brake," possibly after activation of cerebral receptors. Therefore, GLP-1 physiologically seems to signal nutritional abundancy and enhance deposition of nutrients. Because of these effects, however, the peptide can completely normalize blood glucose levels in type 2 diabetics and is therefore of considerable pharmaceutical interest.     

Distribution of fibroblast growth factor (FGF)-2 and FGF receptor-1 messenger RNA expression and protein presence in the mid-trimester human fetus

Fibroblast growth factors (FGF) are known to have key roles in embryonic growth and morphogenesis, but their presence and contributions to fetal development are unclear. In particular, little information exists as to the relevance of FGF and their specific receptors to human fetal development. We studied the anatomical distribution of messenger RNA encoding FGF-2 and one of its high affinity receptors, FGFR1, using in situ hybridization in a variety of human fetal tissues in early second trimester. Corresponding protein distributions were determined by immunohistochemistry. Both FGF-2 and FGFR1 mRNA and proteins were found to be present in every organ and tissue examined, but with defined cellular localizations. In skeletal muscle, both FGF-2 and FGFR1 mRNA and peptides were present in differentiated fibers, and both co-localized to proliferating chondrocytes of the epiphyseal growth plate. FGF-2 and FGFR1 mRNA and peptides were also present within cardiac or gastrointestinal smooth muscle. Within the gastrointestinal tract FGF-2 mRNA and peptide were located in the submucosal tissue, whereas FGFR1 was expressed within the overlying mucosa. Similarly, in skin, FGF-2 was expressed within the dermis whereas FGFR1 mRNA and peptide were most apparent in the stratum germinativum of the epidermis. In kidney and lung, FGFR1 mRNA was located in the tubular and alveolar epithelia respectively, whereas FGF-2 was expressed in both epithelial and mesenchymal cell populations. Both growth factor and receptor were widespread in both neuroblasts and glioblasts in the cerebral cortex of the brain. Immunoreactivity for FGF-2 and FGFR1 was seen in all vascular endothelial cells of major vessels and capillaries. Within the skin, kidney, lung, and intestine FGF-2 immunoreactivity was found in basement membranes underlying epithelia, and was associated with the extracellular matrix and plasma membranes of many cell types. The results show that FGF-2 and one of its receptors are widely expressed anatomically in the mid-trimester human fetus.     

Cloning and characterization of the human galanin GALR2 receptor

We present the molecular cloning and characterization of the human galanin receptor, hGALR2. hGALR2 shares 85%, 39%, and 57% amino acid identities to rGALR2, hGALR1, and hGALR3, respectively. hGALR2, along with rGALR2, can be distinguished from the other cloned galanin receptors by a tolerance for both N-terminal extension and C-terminal deletion of galanin, as well as by a primary signaling mechanism involving phosphatidyl inositol hydrolysis and calcium mobilization. By RT-PCR, GALR2 mRNA was abundant in human hippocampus, hypothalamus, heart, kidney, liver, and small intestine. A weak GALR2 mRNA signal was detected in human retina, and no signal was detected in cerebral cortex, lung, spleen, stomach, or pituitary.     

Distribution of prostaglandin E receptors in the rat gastrointestinal tract

AIMS: In order to study the role of prostaglandin in the regulation of the gastrointestinal functions, gene expression of prostaglandin receptors along the rat gastrointestinal tracts were investigated. METHODS: Rats were used for the study. The combination of counterflow elutriation separation of mucosal cells and Northern blot analysis was used to detect the gene expression of prostaglandin receptors in gastrointestinal tracts. RESULTS: In small intestine and colon, prostaglandin E2 EP1 and EP3 receptor mRNAs were mainly localized in the deeper intestinal wall containing muscle layers. EP4 receptor gene expression, on the other hand, was detected in the intestinal mucosal layer. In the stomach, EP1 mRNA was detected in gastric muscle layers, whereas EP3 and EP4 receptor gene expression was mainly present in the gastric mucosal layer containing epithelial cells. In gastric epithelial cells, parietal cells were found to have both EP3 and EP4 receptors. At lower concentrations, prostaglandin E2 inhibited gastric acid secretion by parietal cells probably through EP4 receptors. At higher concentrations, however, it stimulated it. On the other hand, mucous cells possessed only EP4 receptor mRNA. CONCLUSIONS: Thus, it is suggested that prostaglandin E2 modulates gastrointestinal functions through at least three different prostaglandin receptors (EP1, EP3, and EP4), each of which has a distinct contribution in the gastrointestinal tract.     

Glucagon-like peptide-1--a new hormone and a new drug

Glucagon-like peptide-1 (GLP-1 is an insulinotropic hormone, which is secreted from endocrine cells of the intestinal mucosa in relation to meal ingestion. It plays an important role as an incretin hormone; thus, mice with a null-muation in the gene encoding the GLP-1 receptor are glucose intolerant. In addition, GLP-1 inhibits gastrointestinal secretion and motility and is thought to act as one of the hormones of the "ileal brake". The insulinotropic effect of GLP-1 is preserved in patients with non insulin-dependent diabetes mellitus (NIDDM) and, because GLP-1 also inhibits glucagon secretion, it effectively lowers blood glucose in such, and given as an intravenous infusion it may completely normalise blood glucose. Furthermore, because its actions on insulin and glucagon secretion are dependent on the blood glucose levels it will not cause hypoglycemia. Efforts are therefore currently being made to employ GLP-1 or analogues thereof in clinical diabetes treatment, not least because recent investigations have shown that GLP-1, perhaps due to its gastrointestinal actions, is capable of reducing food intake in humans.     

Antibody against synthetic rat PTH peptide (1-34) blocks PTH-mediated cAMP formation and phosphate transport in opossum kidney cells

The expression of the NMDA subtype of glutamate receptors was investigated by Western blot analysis and RT-PCR in cultured chick Bergmann and Müller glial cells. Using subunit-specific antibodies directed to the carboxy terminus of the rat NMDAR2A/B we detected the expression of the NMDAR2 subunit in both kinds of culture. The functional subunit of the NMDA receptor, NMDAR1, was detected by means of RT-PCR. These results, together with our previous functional characterization of NMDA receptors in radial glia, provide conclusive evidence for the expression of functional NMDA receptor/channels in Bergmann and Muller glia cells. Our findings strengthen the notion of a modulatory role of glial cells in synaptic transmission.     

Infection control systems in the United States: its history and problems

To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, alpha TC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 +/- 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 +/- 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.     

Co-amplification of a novel cyclophilin-like gene (PPIE) with L-myc in small cell lung cancer cell lines

Specific genetic alterations affecting proto-oncogenes of the myc gene family are frequently detected in human lung cancer. Among 11 SCLC cell lines with L-myc gene amplification, four were found to have alteration of the RLF gene by Southern blot and RT-PCR analyses. One cell line, NCI-H378, contained aberrantly-sized L-myc-hybridizing bands by Southern and Northern blot hybridization but had no alteration of RLF. Some L-myc-hybridizing cDNAs from NCI-H378 contained a novel sequence with close homology to the cyclophilins joined to antisense L-myc exon 2 sequence. Full length cDNAs isolated from human skeletal muscle containing only the novel sequence identify open reading frames of 301 and 296 amino acids and differ in the C-terminal region by 22 and 17 amino acids. This gene, tentatively named PPIE (peptidyl-prolyl cis-trans isomerase E), has 83% amino acid identity with the central conserved region of cyclophilin A, is evolutionarily conserved by Southern blot, and exhibits differential tissue expression with highest levels found in muscle and brain. Co-amplification of PPIE was observed in seven of eleven L-myc amplified cell lines. Analysis of radiation hybrids suggests that the gene order is RLF-PPIE-L-myc on chromosome 1p and pulse-field gel electrophoresis localizes all three genes to an 800 megabase Mlu I fragment. The prognostic and functional consequences of PPIE gene amplification in SCLC can now be determined.     

Hydration of polyethylene glycol-grafted liposomes

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.