Bernardo Yusta

Human [Gly2]GLP-2 reduces the severity of colonic injury in a murine model of experimental colitis

The pathology of Crohns disease and ulcerative colitis is characterized by chronic inflammation and destruction of the gastrointestinal epithelium. Although suppression of inflammatory mediators remains the principle component of current disease therapeutics, strategies for enhancing repair and regeneration of the compromised intestinal epithelium have not been widely explored. The demonstration that a peptide hormone secreted by the intestinal epithelium, glucagon-like peptide-2 (GLP-2), is a potent endogenous stimulator of intestinal epithelial proliferation in the small bowel prompted studies of the therapeutic efficacy of GLP-2 in CD1 and BALB/c mice with dextran sulfate (DS)-induced colitis. We report here that a human GLP-2 analog (h[Gly2]GLP-2) significantly reverses weight loss, reduces interleukin-1 expression, and increases colon length, crypt depth, and both mucosal area and integrity in the colon of mice with acute DS colitis. The effects of h[Gly2]GLP-2 in the colon are mediated in part via enhanced stimulation of mucosal epithelial cell proliferation. These observations suggest that exploitation of the normal mechanisms used to regulate intestinal proliferation may be a useful adjunct for healing mucosal epithelium in the presence of active intestinal inflammation.The pathology of Crohns disease and ulcerative colitis is characterized by chronic inflammation and destruction of the gastrointestinal epithelium. Although suppression of inflammatory mediators remains the principle component of current disease therapeutics, strategies for enhancing repair and regeneration of the compromised intestinal epithelium have not been widely explored. The demonstration that a peptide hormone secreted by the intestinal epithelium, glucagon-like peptide-2 (GLP-2), is a potent endogenous stimulator of intestinal epithelial proliferation in the small bowel prompted studies of the therapeutic efficacy of GLP-2 in CD1 and BALB/c mice with dextran sulfate (DS)-induced colitis. We report here that a human GLP-2 analog (h[Gly2]GLP-2) significantly reverses weight loss, reduces interleukin-1 expression, and increases colon length, crypt depth, and both mucosal area and integrity in the colon of mice with acute DS colitis. The effects of h[Gly2]GLP-2 in the colon are mediated in part via enhanced stimulation of mucosal epithelial cell proliferation. These observations suggest that exploitation of the normal mechanisms used to regulate intestinal proliferation may be a useful adjunct for healing mucosal epithelium in the presence of active intestinal inflammation.

Glucagon-like peptide 2 decreases mortality and reduces the severity of indomethacin-induced murine enteritis.

Glucagon-like peptides (GLPs) are secreted from enteroendocrine cells in the gastrointestinal tract. GLP-1 actions regulate blood glucose, whereas GLP-2 exerts trophic effects on intestinal mucosal epithelium. Although GLP-1 actions are preserved in diseases such as diabetes, GLP-2 action has not been extensively studied in the setting of intestinal disease. We have now evaluated the biological effects of a human GLP-2 analog in the setting of experimental murine nonsteroidal antiinflammatory drug-induced enteritis. Human (h)[Gly2]GLP-2 significantly improved survival whether administered before, concomitant with, or after indomethacin. h[Gly2]GLP-2-treated mice exhibited reduced histological evidence of disease activity, fewer intestinal ulcerations, and decreased myeloperoxidase activity in the small bowel ( P < 0.05, h[Gly2]GLP-2- vs. saline-treated controls). h[Gly2]GLP-2 significantly reduced cytokine induction, bacteremia, and the percentage of positive splenic and hepatic bacterial cultures ( P < 0.05). h[Gly2]GLP-2 enhanced epithelial proliferation ( P < 0.05 for increased crypt cell proliferation in h[Gly2]GLP-2- vs. saline-treated mice after indomethacin) and reduced apoptosis in the crypt compartment ( P < 0.02). These observations demonstrate that a human GLP-2 analog exerts multiple complementary actions that serve to preserve the integrity of the mucosal epithelium in experimental gastrointestinal injury in vivo.

Physiology and Pharmacology of the Enteroendocrine Hormone Glucagon-Like Peptide-2

Glucagon-like peptide-2 (GLP-2) is a 33-amino-acid proglucagon-derived peptide secreted from enteroendocrine L cells. GLP-2 circulates at low basal levels in the fasting period, and plasma levels rise rapidly after food ingestion. Renal clearance and enzymatic inactivation control the elimination of bioactive GLP-2. GLP-2 increases mesenteric blood flow and activates proabsorptive pathways in the gut, facilitating nutrient absorption. GLP-2 also enhances gut barrier function and induces proliferative and cytoprotective pathways in the small bowel. The actions of GLP-2 are transduced via a single G protein-coupled receptor (GLP-2R), expressed predominantly within the gastrointestinal tract. Disruption of GLP-2R signaling increases susceptibility to gut injury and impairs the adaptive mucosal response to refeeding. Sustained augmentation of GLP-2R signaling reduces the requirement for parenteral nutrition in human subjects with short-bowel syndrome. Hence GLP-2 integrates nutrient-derived signals to optimize mucosal integrity and energy absorption.

The Glucagon-like Peptide-2 Receptor Mediates Direct Inhibition of Cellular Apoptosis via a cAMP-dependent Protein Kinase-independent Pathway

Glucagon and the glucagon-like peptides regulate metabolic functions via signaling through a glucagon receptor subfamily of G protein-coupled receptors. Activation of glucagon-like peptide-2 receptor (GLP-2R) signaling maintains the integrity of the intestinal epithelial mucosa via regulation of crypt cell proliferation. Because GLP-2 decreases mortality and reduces intestinal apoptosis in rodents after experimental injury, we examined whether GLP-2R signaling directly modifies the cellular response to external injury. We show here that activation of GLP-2R signaling inhibits cycloheximide-induced apoptosis in baby hamster kidney fibroblasts expressing a transfected GLP-2 receptor. GLP-2 reduced DNA fragmentation and improved cell survival, in association with reduced activation of caspase-3 and decreased poly(ADP-ribose) polymerase cleavage and reduced caspase-8 and caspase-9-like activities. Both GLP-2 and forskolin reduced mitochondrial cytochrome crelease and decreased the cycloheximide-induced cleavage of caspase-3 in the presence or absence of the PKA inhibitor H-89. Similarly, GLP-2 increased cell survival following cycloheximide in the presence of the kinase inhibitors PD98054 and LY294002. These findings provide evidence that signaling through G protein-coupled receptors of the glucagon superfamily is directly linked to regulation of apoptosis and suggest the existence of a cAMP-dependent protein kinase-, phosphatidylinositol 3-kinase-, and mitogen-activated protein kinase-independent pathway coupling GLP-2R signaling to caspase inhibition and cell survival.

Identification of Glucagon-like Peptide-2 (GLP-2)-activated Signaling Pathways in Baby Hamster Kidney Fibroblasts Expressing the Rat GLP-2 Receptor

Glucagon-like peptide-2 (GLP-2) promotes the expansion of the intestinal epithelium through stimulation of the GLP-2 receptor, a recently identified member of the glucagon-secretin G protein-coupled receptor superfamily. Although activation of G protein-coupled receptors may lead to stimulation of cell growth, the mechanisms transducing the GLP-2 signal to mitogenic proliferation remain unknown. We now report studies of GLP-2R signaling in baby hamster kidney (BHK) cells expressing a transfected rat GLP-2 receptor (BHK-GLP-2R cells). GLP-2, but not glucagon or GLP-1, increased the levels of cAMP and activated both cAMP-response element- and AP-1-dependent transcriptional activity in a dose-dependent manner. The activation of AP-1-luciferase activity was protein kinase A (PKA) -dependent and markedly diminished in the presence of a dominant negative inhibitor of PKA. Although GLP-2 stimulated the expression of c-fos, c-jun, junB, and zif268, and transiently increased p70 S6 kinase in quiescent BHK-GLP-2R cells, GLP-2 also inhibited extracellular signal-regulated kinase 1/2 and reduced serum-stimulated Elk-1 activity. Furthermore, no rise in intracellular calcium was observed following GLP-2 exposure in BHK-GLP-2R cells. Although GLP-2 stimulated both cAMP accumulation and cell proliferation, 8-bromo-cyclic AMP alone did not promote cell proliferation. These findings suggest that the GLP-2R may be coupled to activation of mitogenic signaling in heterologous cell types independent of PKA via as yet unidentified downstream mediators of GLP-2 action in vivo.

ErbB Signaling Is Required for the Proliferative Actions of GLP-2 in the Murine Gut

BACKGROUND & AIMS Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide hormone secreted by enteroendocrine cells in response to nutrient ingestion. GLP-2 stimulates crypt cell proliferation leading to expansion of the mucosal epithelium; however, the mechanisms transducing the trophic effects of GLP-2 are incompletely understood. METHODS We examined the gene expression profiles and growth-promoting actions of GLP-2 in normal mice in the presence or absence of an inhibitor of ErbB receptor signaling, in Glp2r(-/-) mice and in Egfr(wa2) mice harboring a hypomorphic point mutation in the epidermal growth factor receptor. RESULTS Exogenous GLP-2 administration rapidly induced the expression of a subset of ErbB ligands including amphiregulin, epiregulin, and heparin binding (HB)-epidermal growth factor, in association with induction of immediate early gene expression in the small and large bowel. These actions of GLP-2 required a functional GLP-2 receptor because they were eliminated in Glp2r(-/-) mice. In contrast, insulin-like growth factor-I and keratinocyte growth factor, previously identified mediators of GLP-2 action, had no effect on the expression of these ErbB ligands. The GLP-2-mediated induction of ErbB ligand expression was not metalloproteinase inhibitor sensitive but was significantly diminished in Egfr(wa2) mice and completed abrogated in wild-type mice treated with the pan-ErbB inhibitor CI-1033. Furthermore, the stimulatory actions of GLP-2 on crypt cell proliferation and bowel growth were eliminated in the presence of CI-1033. CONCLUSIONS These findings identify the ErbB signaling network as a target for GLP-2 action leading to stimulation of growth factor-dependent signal transduction and bowel growth in vivo.

ErbB Activity Links the Glucagon-Like Peptide-2 Receptor to Refeeding-Induced Adaptation in the Murine Small Bowel

BACKGROUND & AIMS The small bowel mucosa is sensitive to nutrients and undergoes rapid adaptation to nutrient deprivation and refeeding through changes in apoptosis and cell proliferation, respectively. Although glucagon-like peptide-2 (GLP-2) exerts trophic effects on the gut and levels increase with refeeding, mechanisms linking GLP-2 to mucosal adaptation to refeeding remain unclear. METHODS Fasting and refeeding were studied in wild-type (WT) and Glp2r(-/-) mice and in WT mice treated with the pan ErbB inhibitor CI-1033. Experimental end points included intestinal weights, histomorphometry, gene and protein expression, and crypt cell proliferation. RESULTS Fasting was associated with significant reductions in small bowel mass, decreased crypt plus villus height, and reduced crypt cell proliferation. Refeeding increased plasma levels of GLP-2, reversed small bowel atrophy, increased villus height and cell number, and stimulated jejunal crypt cell proliferation. In contrast, refeeding failed to increase small bowel weight, crypt cell proliferation, or villus cell number in Glp2r(-/-) mice. Levels of mRNA transcripts for egf, kgf, and igfr were lower in fasted Glp2r(-/-) mice. Epidermal growth factor but not insulin-like growth factor-1 restored the intestinal adaptive response to refeeding in Glp2r(-/-) mice. Furthermore, CI-1033 prevented adaptive crypt cell proliferation, Akt activation, and induction of ErbB ligand gene expression after refeeding. Up-regulation of ErbB ligand expression and intestinal Akt phosphorylation were significantly diminished in refed Glp2r(-/-) mice. CONCLUSIONS These findings identify Glp2r and ErbB pathways as essential components of the signaling network regulating the adaptive mucosal response to refeeding in the mouse intestine.

Glucagon-like Peptide-2 Does Not Modify the Growth or Survival of Murine or Human Intestinal Tumor Cells

Glucagon-like peptide-2 (GLP-2) secreted from enteroendocrine cells exerts proabsorptive, regenerative, and cytoprotective actions in the normal and injured gut epithelium. Hence, sustained GLP-2 receptor (GLP-2R) activation represents a strategy under investigation for the prevention and treatment of chemotherapy-induced mucositis. Nevertheless, the consequences of increased GLP-2R signaling for the growth and survival of intestinal tumor cells remain poorly understood. We studied the proliferative and cytoprotective actions of GLP-2 in human colon cancer cells stably transfected with the GLP-2R and in nude mice harboring GLP-2R(+) human colon cancer cells. The importance of the GLP-2R for tumor growth was also examined in Apc(Min/+) mice chronically treated with exogenous GLP-2 and in Apc(Min/+):Glp2r(-/-) mice. GLP-2 increased cyclic AMP accumulation and produced cell-specific activation of growth and survival pathways in DLD-1, SW480, and HT29 cells. However, GLP-2 did not stimulate cell growth or attenuate cycloheximide-, LY294002-, indomethacin-, or chemotherapy-induced cytotoxicity in vitro. Moreover, chronic GLP-2 administration had no effect on the growth of human colon cancer cell xenografts in nude mice in vivo. Daily GLP-2 treatment for 7 weeks increased growth of normal gut mucosa but did not increase the number or size of polyps in Apc(Min/+) mice, and genetic disruption of the Glp2r gene in Apc(Min/+) mice did not modify polyp size or number. Taken together, although GLP-2R activation engages signaling pathways promoting cell proliferation and cytoprotection in the normal gut epithelium, sustained direct or indirect modulation of GLP-2R signaling does not modify intestinal tumor cell growth or survival.

Intestinotrophic glucagon-like peptide-2 (GLP-2) activates intestinal gene expression and growth factor-dependent pathways independent of the vasoactive intestinal peptide gene in mice.

The enteroendocrine and enteric nervous systems convey signals through an overlapping network of regulatory peptides that act either as circulating hormones or as localized neurotransmitters within the gastrointestinal tract. Because recent studies invoke an important role for vasoactive intestinal peptide (VIP) as a downstream mediator of glucagon-like peptide-2 (GLP-2) action in the gut, we examined the importance of the VIP-GLP-2 interaction through analysis of Vip(-/-) mice. Unexpectedly, we detected abnormal villous architecture, expansion of the crypt compartment, increased crypt cell proliferation, enhanced Igf1 and Kgf gene expression, and reduced expression of Paneth cell products in the Vip(-/-) small bowel. These abnormalities were not reproduced by antagonizing VIP action in wild-type mice, and VIP administration did not reverse the intestinal phenotype of Vip(-/-) mice. Exogenous administration of GLP-2 induced the expression of ErbB ligands and immediate-early genes to similar levels in Vip(+/+) vs. Vip(-/-) mice. Moreover, GLP-2 significantly increased crypt cell proliferation and small bowel growth to comparable levels in Vip(+/+) vs. Vip(-/-) mice. Unexpectedly, exogenous GLP-2 administration had no therapeutic effect in mice with dextran sulfate-induced colitis; the severity of colonic injury and weight loss was modestly reduced in female but not male Vip(-/-) mice. Taken together, these findings extend our understanding of the complex intestinal phenotype arising from loss of the Vip gene. Furthermore, although VIP action may be important for the antiinflammatory actions of GLP-2, the Vip gene is not required for induction of a gene expression program linked to small bowel growth after enhancement of GLP-2 receptor signaling.

Triiodothyronine (T3) induces neurite formation and increases synthesis of a protein related to MAP 1B in cultured cells of neuronal origin.

Neuroblastoma (N2A) cells were found to develop axon-like neurite extensions when grown in the presence of triiodothyronine (T3), while C6 cells (of glial origin) did not. Analysis of radiolabelled protein synthesis showed that, in N2A only, T3 increased the synthesis of a polypeptide corresponding in electrophoretic mobility to the microtubule-associated protein MAP 1B. Immunoblotting of total cell proteins with a monoclonal antibody confirmed that this polypeptide was immunologically related to MAP 1B. Further studies using indirect immunofluorescence with monoclonal antibodies against both tubulin and MAP 1B showed that both antigens were present in neurites. Taken together, these results suggest that T3 may control maturation of neural tissue via effects on the microtubule-associated proteins in cells of neuronal origin.