Influence of high density lipoprotein cholesterol levels on circulating monocytic angiogenic cells functions in individuals with type 2 diabetes mellitus.

BACKGROUND: High-density lipoproteins (HDLs) can exert anti-atherogenic effects. On top of removing excess cholesterol through reverse cholesterol transport, HDLs play beneficial actions on endothelial function and integrity. In particular, HDLs are strong determinant of endothelial progenitor cells (EPCs) number and function. To gain further insights into such an effect we characterized in vitro functionality of circulating "early" EPCs obtained from 60 type 2 diabetes individuals with low HDL-cholesterol (HDL-C) and 59 with high HDL-C levels. METHODS: After an overnight fast, venous blood was drawn in EDTA tubes and processed within 2-h from sampling. Peripheral blood mononuclear cells were isolated and plated on fibronectin coated culture dishes; after 3 days culture, adherent cells positive for Dil-ac-LDL/Lectin dual fluorescent staining were identified as monocytic angiogenic cells (MACs). After 5-7 days culture in EBM-2 medium, adherent cells were evaluated for viability/proliferation (MTT assay), senescence (beta-galactosidase activity detection), migration (modified Boyden chamber using VEGF as chemoattractant), adhesion capacity (on fibronectin-coated culture dishes) and ROS production (ROS-sensitive fluorescent probe CM-H2DCFDA). RESULTS: MACs obtained from diabetic individuals with high HDL-C had 23% higher viability compared to low HDL-C (111.6 ± 32.7% vs. 90.5 ± 28.6% optical density; p = 0.002). H2O2 exposure impaired MACs viability to a similar extent in both groups (109.2 ± 31.7% vs. 74.5 ± 40.8% in high HDL-C, p < 0.0001; 88.3 ± 25.5% vs. 72.3 ± 22.5% in low-HDL, p = 0.004). MACs senescence was comparable in the two groups (102.7 ± 29.8% vs. 99.2 ± 27.8%; p = 0.703) and was only slightly modified by exposure to H2O2. There was no difference in the MACs migration capacity between the two groups (91.3 ± 34.2% vs. 108.7 ± 39.5%; p = 0.111), as well as in MACs adhesion capacity (105.2 ± 32.7% vs. 94.1 ± 26.1%; p = 0.223). Finally, ROS production was slightly thought not significantly higher in MACs from type 2 diabetes individuals with low- than high-HDL. After stratification of HDL-C levels into quartiles, viability (p < 0.0001) and adhesion (p = 0.044) were higher in Q4 than in Q1-Q3. In logistic regression analysis, HDL-C was correlated to MACs viability and adhesion independently of HbA1c or BMI, respectively. CONCLUSIONS: Our data suggest that in type 2 diabetes subjects, HDL-cholesterol is an independent determinant of circulating MACs functional capacities-mainly viability, to a lesser extent adhesion-likely contributing also through this mechanism to cardiovascular protection even in type 2 diabetes.

Metabolic regulation of GLP-1 and PC1/3 in pancreatic α-cell line.

BACKGROUND AND AIMS: An intra-islet incretin system has been recently suggested to operate through modulation of the expression and activity of proconvertase 1/3 and 2 (PC1/3, PC2) in pancreatic alpha-cell accounting for local release of GLP-1. Little is known, whether this alpha-cell activity can be affected by the metabolic alterations occurring in type 2 diabetes, such as hyperglycemia, hyperlipidemia or hyperglucagonemia. MATERIALS AND METHODS: AlphaTC1/6 cells from a mice pancreatic cell line were incubated in the presence of two glucose (G) concentration (5.5 and 16.7 mM) for 16 h with or without free fatty acid, IL6 or glucagon. GLP-1 secretion was measured by ELISA and expression of PC1/3 and PC2 by RT-PCR and western blot; cell viability was determined by MTT method, Reactive Oxygen Species generation (ROS) by H2DCFDA fluorescence and apoptosis by Annexin staining and Propidium Iodine (PI) fluorescence. RESULTS: Upon 16.7G incubation, GLP-1 secretion (total and active) was significantly increased in parallel with a significant increment in PC1/3 expression, a slight increase in cell viability and ROS generation and by a decrement in PC2 expression with no change in cell apoptosis. When cells were incubated at 5.5mM glucose with FFA, also an increment in GLP-1 secretion and PC1/3 expression was observed together an increment in ROS generation, a decrement in cell viability, and a modest increment in apoptosis. When incubated with 16.7mM glucose with FFA, the increment in GLP-1 secretion was reduced to basal, accompanied by an increment in apoptosis and ROS generation. This was also observed with IL-6, but in this case, no modification in ROS generation or apoptosis was observed when compared to 16.7mM glucose. The presence of glucagon did not modify any of the parameters studied. CONCLUSION: These data suggest that under hyperglycemic, hyperlipidemia or inflammatory conditions, alpha cells can increase expression PC1/3 and activate GLP-1 secretion, which may contribute protecting both alpha and beta-cells from glucose and lipotoxicity, while this effect seems to be lost in the presence of both pathophysiological conditions.

Primary diffuse large B cell lymphoma of the uterine cervix successfully treated by combined chemotherapy alone: A case report.

RATIONALE: Primary lymphomas of the uterine cervix are a rare disease. They are often misdiagnosed because of their rarity and because they can be easily confused with a squamous cell carcinoma of the cervix, as they are usually presented as exophytic mass with vaginal bleeding as their most common symptoms. Nevertheless, considering that both the prognosis and the treatment are completely different between them, differential diagnosis should be taken into account. PATIENT CONCERNS: A case of a 51-year-old woman with a primary diffuse large B-cell lymphoma of the cervix is presented. DIAGNOSES: Diagnosis of this tumor was a challenge for pathologists and clinicians, as four biopsies were needed to achieve a final diagnosis. INTERVENTIONS: Patient was successfully treated with combined Rituximab and chemotherapy (R-CHOP) alone. OUTCOMES: Complete remission, confirmed through biopsy, was reached after six courses of chemotherapy. At 2-years follow up, patient is alive and free of disease. LESSONS: Considering that the prognosis and treatment of primary malignant lymphoma of the cervix are completely different than that of the squamous cell carcinoma, awareness of this disease should be considered in the differential diagnosis.

Gastrointestinal hormones stimulate growth of Foregut Neuroendocrine Tumors by transactivating the EGF receptor.

Foregut neuroendocrine tumors [NETs] usually pursuit a benign course, but some show aggressive behavior. The treatment of patients with advanced NETs is marginally effective and new approaches are needed. In other tumors, transactivation of the EGF receptor (EGFR) by growth factors, gastrointestinal (GI) hormones and lipids can stimulate growth, which has led to new treatments. Recent studies show a direct correlation between NET malignancy and EGFR expression, EGFR inhibition decreases basal NET growth and an autocrine growth effect exerted by GI hormones, for some NETs. To determine if GI hormones can stimulate NET growth by inducing transactivation of EGFR, we examined the ability of EGF, TGFα and various GI hormones to stimulate growth of the human foregut carcinoid,BON, the somatostatinoma QGP-1 and the rat islet tumor,Rin-14B-cell lines. The EGFR tyrosine-kinase inhibitor, AG1478 strongly inhibited EGF and the GI hormones stimulated cell growth, both in BON and QGP-1 cells. In all the three neuroendocrine cell lines studied, we found EGF, TGFα and the other growth-stimulating GI hormones increased Tyr(1068) EGFR phosphorylation. In BON cells, both the GI hormones neurotensin and a bombesin analogue caused a time- and dose-dependent increase in EGFR phosphorylation, which was strongly inhibited by AG1478. Moreover, we found this stimulated phosphorylation was dependent on Src kinases, PKCs, matrix metalloproteinase activation and the generation of reactive oxygen species. These results raise the possibility that disruption of this signaling cascade by either EGFR inhibition alone or combined with receptor antagonists may be a novel therapeutic approach for treatment of foregut NETs/PETs.

The Src kinase Yes is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters, but not pancreatic growth factors, which stimulate its association with numerous other signaling molecules.

For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK(1)-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130(Cas), PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects.

Normalizing action of exendin-4 and GLP-1 in the glucose metabolism of extrapancreatic tissues in insulin-resistant and type 2 diabetic states.

Exendin-4 (Ex-4) mimics glucagon-like peptide-1 (GLP-1 or GCG as listed in the HUGO database), being anti-diabetic and anorectic, in stimulating glucose and lipid metabolism in extrapancreatic tissues. We studied the characteristics of Ex-4 and GLP-1 action, during prolonged treatment, on GLUTs expression (mRNA and protein), glycogen content (GC), glucose transport (GT), glycogen synthase a (GSa), and kinase (PI3K and MAPKs) activity, in liver, muscle, and fat of insulin-resistant (IR, by fructose) and type 2 diabetic (T2D, streptozotocin at birth) rats compared with normal rats. In both IR and T2D, the three tissues studied presented alterations in all measured parameters. In liver, GLP-1 and also Ex-4 normalized the lower than normal Glut2 (Slc2a2) expression and showed a trend to normalize the reduced GC in IR, and GLP-1, like Ex-4, also in T2D, effects mediated by PI3K and MAPKs. In skeletal muscle, neither GLP-1 nor Ex-4 modified Glut4 (Slc2a4) expression in either experimental model but showed normalization of reduced GT and GSa, in parallel with the normalization of reduced PI3K activity in T2D and MAPKs in both models. In adipose tissue, the altered GLUT4 expression in IR and T2D, along with reduced GT in IR and increased GT in T2D, and with hyperactivated PI3K in both, became normal after GLP-1 and Ex-4 treatment; yet, MAPKs, that were also higher, became normal only after Ex-4 treatment. The data shows that Ex-4, as well as GLP-1, exerts a normalizing effect on IR and T2D states through a distinct post-receptor mechanism, the liver being the main target for Ex-4 and GLP-1 to control glucose homeostasis.

PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades.

The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal (GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin (CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA was present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCK(A)-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125(FAK) and IKKα/ß, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ's direct association with AKT, RafA, RafC and Lyn. These results show for the first time the PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth).

Pharmacology and selectivity of various natural and synthetic bombesin related peptide agonists for human and rat bombesin receptors differs.

The mammalian bombesin (Bn)-receptor family [gastrin-releasing peptide-receptor (GRPR-receptor), neuromedin B-receptor (NMB receptor)], their natural ligands, GRP/NMB, as well as the related orphan receptor, BRS-3, are widely distributed, and frequently overexpressed by tumors. There is increased interest in agonists for this receptor family to explore their roles in physiological/pathophysiological processes, and for receptor-imaging/cytotoxicity in tumors. However, there is minimal data on human pharmacology of Bn receptor agonists and most results are based on nonhuman receptor studies, particular rodent-receptors, which with other receptors frequently differ from human-receptors. To address this issue we compared hNMB-/GRP-receptor affinities and potencies/efficacies of cell activation (assessing phospholipase C activity) for 24 putative Bn-agonists (12 natural, 12 synthetic) in four different cells with these receptors, containing native receptors or receptors expressed at physiological densities, and compared the results to native rat GRP-receptor containing cells (AR42J-cells) or rat NMB receptor cells (C6-glioblastoma cells). There were close correlations (r=0.92-99, p<0.0001) between their affinities/potencies for the two hGRP- or hNMB-receptor cells. Twelve analogs had high affinities (≤ 1 nM) for hGRP receptor with 15 selective for it (greatest=GRP, NMC), eight had high affinity/potencies for hNMB receptors and four were selective for it. Only synthetic Bn analogs containing ß-alanine(11) had high affinity for hBRS-3, but also had high affinities/potencies for all GRP-/hNMB-receptor cells. There was no correlation between affinities for human GRP receptors and rat GRP receptors (r=0.131, p=0.54), but hNMB receptor results correlated with rat NMB receptor (r=0.71, p<0.0001). These results elucidate the human and rat GRP-receptor pharmacophore for agonists differs markedly, whereas they do not for NMB receptors, therefore potential GRP-receptor agonists for human studies (such as Bn receptor-imaging/cytotoxicity) must be assessed on human Bn receptors. The current study provides affinities/potencies on a large number of potential agonists that might be useful for human studies.

Bombesin receptor subtype-3 agonists stimulate the growth of lung cancer cells and increase EGF receptor tyrosine phosphorylation.

The effects of bombesin receptor subtype-3 (BRS-3) agonists were investigated on lung cancer cells. The BRS-3 agonist (DTyr(6), (Ala(11), Phe(13), Nle(14)) bombesin(6-14) (BA1), but not gastrin releasing peptide (GRP) or neuromedin B (NMB) increased significantly the clonal growth of NCI-H1299 cells stably transfected with BRS-3 (NCI-H1299-BRS-3). Also, BA1 addition to NCI-H727 or NCI-H1299-BRS-3 cells caused Tyr(1068) phosphorylation of the epidermal growth factor receptor (EGFR). Similarly, (DTyr(6), R-Apa(11), Phe(13), Nle(14)) bombesin(6-14) (BA2) and (DTyr(6), R-Apa(11), 4-Cl,Phe(13), Nle(14)) bombesin(6-14) (BA3) but not gastrin releasing peptide (GRP) or neuromedin B (NMB) caused EGFR transactivation in NCI-H1299-BRS-3 cells. BA1-induced EGFR or ERK tyrosine phosphorylation was not inhibited by addition of BW2258U89 (BB(2)R antagonist) or PD168368 (BB(1)R antagonist) but was blocked by (DNal-Cys-Tyr-DTrp-Lys-Val-Cys-Nal)NH(2) (BRS-3 ant.). The BRS-3 ant. reduced clonal growth of NCI-H1299-BRS-3 cells. BA1, BA2, BA3 and BRS-3 ant. inhibit specific (125)I-BA1 binding to NCI-H1299-BRS-3 cells with an IC(50) values of 1.1, 21, 15 and 750nM, respectively. The ability of BRS-3 to regulate EGFR transactivation in NCI-H1299-BRS-3 cells was reduced by AG1478 or gefitinib (EGFR tyrosine kinase inhibitors), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant), Tiron (superoxide scavenger) and DPI (NADPH oxidase inhibitor). These results demonstrate that BRS-3 agonists may stimulate lung cancer growth as a result of EGFR transactivation and that the transactivation is regulated by BRS-3 in a Src-, reactive oxygen and matrix metalloprotease-dependent manner.

Characteristic of GLP-1 effects on glucose metabolism in human skeletal muscle from obese patients.

Direct effects of GLP-1, kinase-mediated, on glucose and lipid metabolism in rat and human extrapancreatic tissues, are amply documented and also changes in type-2 diabetic (T2D) patients. Here, we explored the characteristics of the GLP-1 action and those of its analogs Ex-4 and Ex-9, on muscle glucose transport (GT) and metabolism in human morbid obesity (OB), as compared with normal and T2D subjects. In primary cultured myocytes from OB, GT and glycogen synthase a (GSa) activity values were lower than normal, and comparable to those reported in T2D patients; GT was increased by either GLP-1 or Ex-9 in a more efficient manner than in normal or T2D, up to normal levels; the Ex-4 increasing effect on GSa activity was two times that in normal cells, while Ex-9 failed to modify the enzyme activity. In OB, the control value of all kinases analyzed - PI3K, PKB, MAPKs, and p70s6K - although lower than that in normal or T2D subjects, the cells maintained their response capability to GLP-1, Ex-4, Ex-9 and insulin, with some exceptions. GLP-1 and exendins showed a direct normalizing action in the altered glucose uptake and metabolism in the muscle of obese subjects, which in the case of GLP-1 could account, at least in part, for the reported restoration of the metabolic conditions of these patients after restrictive surgery.

Bombesin receptor-mediated imaging and cytotoxicity: review and current status.

The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered.

Pharmacology of putative selective hBRS-3 receptor agonists for human bombesin receptors (BnR): affinities, potencies and selectivity in multiple native and BnR transfected cells.

The orphan receptor, bombesin receptor subtype-3(BRS-3) is a G-protein-coupled receptor classified in the bombesin (Bn) receptor family because of its high homology (47-51%) with other members of this family [gastrin-releasing peptide receptor [GRPR] and neuromedin B receptor [NMBR]]. There is increasing interest in BRS-3, because primarily from receptor knockout studies, it seems important in energy metabolism, glucose control, insulin secretion, motility and tumor growth. Pharmacological tools to study the role of BRS-3 in physiology/pathophysiology are limited because the natural ligand is unknown and BRS-3 has low affinity for all naturally occurring Bn-related peptides. However, a few years ago a synthetic high-affinity agonist [dTyr(6),betaAla(11),Phe(13),Nle(14)]Bn-(6-14) was described but was nonselective for BRS-3 over other Bn receptors. Based on this peptide, in various studies a number of putative selective, high-potency hBRS-3 agonists were described, however the results on their selectivity are conflicting in a number of cases. The purpose of the present study was to thoroughly study the pharmacology of four of the most select/potent putative hBRS-3 agonists (#2-4, 16a). Each was studied in multiple well-characterized Bn receptor-transfected cells and native Bn receptor bearing cells, using binding studies, alterations in cellular signaling (PLC, PKD) and changes in cellular function(growth). Two peptides (#2, #3) had nM affinities/potencies for hBRS-3, peptide #4 had low affinity/potency, and peptide #16a very low (>3000 nM). Peptide#3 had the highest selectivity for hBRS-3 (100-fold), whereas #2, 4 had lower selectivity. Peptide #16a's selectivity could not be determined because of its low affinity/potencies for all hBn receptors. These results show that peptide #3 is the preferred hBRS-3 agonist for studies at present, although its selectivity of only 100-fold may limit its utility in some cases. This study underscores the importance of full pharmacological characterization of newly reported selective agonists.

Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells.

Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr(1068) of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transected with NMB receptors. The transactivation of the EGF receptor or the tyrosine phosphorylation of ERK caused by NMB-like peptides was inhibited by AG1478 or gefitinib (tyrosine kinase inhibitors) and NMB receptor antagonist PD168368 but not the GRP receptor antagonist, BW2258U89. The transactivation of the EGF receptor caused by NMB-like peptides was inhibited by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), or transforming growth factor (TGF)alpha antibody. The transactivation of the EGF receptor and the increase in reactive oxygen species caused by NMB-like peptides was inhibited by N-acetylcysteine (NAC) or Tiron. Gefitinib inhibited the proliferation of NCI-H1299 cells and its sensitivity was increased by the addition of PD168368. The results indicate that the NMB receptor regulates EGF receptor transactivation by a mechanism dependent on Src as well as metalloprotease activation and generation of reactive oxygen species.

Molecular basis for the selectivity of the mammalian bombesin peptide, neuromedin B, for its receptor.

The mammalian bombesin (Bn) peptides, neuromedin B (NMB) and gastrin-releasing peptide (GRP), have widespread actions in many tissues, and their effects are mediated by two closely related G-protein-coupled receptors, the NMBR and GRPR. Little is known about the structural determinants of NMBR selectivity for NMB, in contrast to GRP selectivity for the GRPR, which has been extensively studied. To provide insight, chimeric NMBR-GRPR loss-of-affinity and gain-of-affinity mutants were made, as well as NH(2)-terminally truncated NMBR and point mutants using site-directed mutagenesis. Receptors were expressed in Balb-3T3-cells or CHOP cells, and affinities were determined. NMB had 115-fold greater affinity for NMBR than GRPR. Receptor-chimeric studies showed that NMBR selectivity for NMB was primarily determined by differences in the third extracellular (EC3) regions of GRPR-NMBR and adjacent upper-transmembrane-5 (TM5) region. In this region, 24 NMB gain-of-affinity GRPR mutants or NMBR loss-of-affinity point/combination mutants were made. Three gain-of-affinity mutant GRPRs [[A198I] (EC3), [H202Q] (EC3), [S215I] (upper TM5)] had increased NMB affinity (2.4-21-fold), and these results were confirmed with NMBR loss-of-affinity mutants [I199A,Q203H,I215S-NMBR]. The combination mutant [A198I,S215]GRPR had the greatest effect causing a complete NMB gain-of-affinity. The importance of differences at position 199NMBR or 203NMBR was studied by substituting amino acids with various properties. Our results show that NMBR selectivity for NMB is due to differences in the EC3 of NMBR-GRPR and the adjacent upper-TM5 region. Within these regions, isoleucines in NMBR [position 199 (EC3)] (instead of A198GRPR) and in 215NMBR (TM5) (instead of S214GRPR), as well as Q203NMBR (instead of H202GRPR) are responsible for high NMB-affinity/selectivity of NMBR. The effect at position 199 is primarily due to differences in hydrophobicity of the substitution, whereas steric factors and charge of the substitution at position 203 were important determinants of NMB selectivity.

Characterization of putative GRP- and NMB-receptor antagonist's interaction with human receptors.

The mammalian bombesin (Bn) peptides neuromedin B (NMB) and gastrin-releasing peptide (GRP) actions are mediated by two receptors (NMB-receptor, GRP-receptor) which are widely distributed in the GI tract and CNS. From primarily animal studies NMB/GRP-receptor activation has physiological/pathophysiological effects in the CNS and GI tract including stimulating of growth of cancers and normal tissues. Whereas these Bn-receptors' effects have been extensively studied in nonhuman cells and animals, little is known of the physiological/pathological role(s) in humans, largely due to lack of potent antagonists. To address this issue we compared NMB/GRP-receptor affinity/potency of 10 chemical classes of putative antagonists (35 compounds) for human Bn-receptors by performing binding studies or assessing abilities to activate hGRP/hNMB-receptor [assessing phospholipase C activation] in four different cells containing native Bn-receptors or transfected receptors. From binding studies 23 were GRP-receptor-preferring, 4 were NMB-receptor, and 8 nonselective. For the hGRP-receptor-preferring analogues none showed hGRP-receptor agonist activity, but 13 were full or partial hNMB-receptor agonists at hNMB-receptors. For hNMB-receptor-preferring analogues none were agonists. Analogue #24 ([(3-Ph-Pr(6)), His(7), d-Ala(11), d-Pro(13), Psi(13-14), Phe(14)]Bn(6-14)NH2) and analogue #7 [d-Phe(6), Leu(13), Psi(CH(2)NH), Cpa(14)]Bn(6-14) were the most potent (0.2-1.4nM) and selective (>10,000-fold) for the hGRP-receptor with analogue #7.5 [d-Tpi(6), Leu(13), Psi(CH2NH), Leu(14)]Bn(6-14)[RC-3095] (0.2-1.4nM) slightly less selective. Analogue #34 (PD168368) had the highest affinity for hNMB-receptor (1.32-1.58nM) and the greatest selectivity (2298-6952-fold) for the hNMB-receptor. These results demonstrate numerous putative hGRP/hNMB-receptor antagonists identified in nonhuman cells and/or animals have agonist activity at the hNMB-receptor, limiting their potential usefulness. However, a number were identified which were potent/selective for human Bn-receptors and should be useful for investigating their roles in human physiological/pathophysiological conditions.

Gastrointestinal growth factors and hormones have divergent effects on Akt activation.

Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigated in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters [CCK, bombesin, carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations (pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations (nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory effects can be seen, which are mediated by different mechanisms.

Characteristics of GLP-1 and exendins action upon glucose transport and metabolism in type 2 diabetic rat skeletal muscle.

Exendin-4, a peptide 53% structurally homologous with glucagon-like peptide 1 (GLP-1), is insulinotropic and has an antidiabetic effect even more prolonged than that of GLP-1. Exendin-9 is an antagonist of GLP-1 receptor and action in several cell systems, but shows GLP-1- and exendin-4-agonistic characteristics in human muscle cells and tissue. The action of GLP-1 upon glucose transport and metabolism in muscle is mediated by specific receptors. In this study we investigated the effect of both exendin-4 and -9, relative to that of GLP-1, upon glucose transport and metabolism in the skeletal muscle from a streptozotocin-induced type 2 diabetic rat model, compared to normal. In normal rats, exendin-4, like GLP-1 and insulin, enhanced glucose uptake. This effect, which is mediated to a certain extent by some kinases (PI3K/ PKB, p70s6k and MAPKs), may be caused by the peptide acting, at least in part, through the muscle GLP-1 receptors. Exendin-9 also stimulated the same kinases, except for PKB, but failed to modify basal glucose uptake. Type 2 diabetic rats showed lower than normal basal muscle glucose transport and oxidation value, and higher glycogen synthase alpha activity and pyruvate release; however, no modification of glucose uptake by GLP-1 or exendin-4 was detected, at variance with insulin, and basal activity of PI3K/PKB was lower than normal, while that of p70s6k and MAPKs was higher. GLP-1 failed to affect the activity of any of the kinases, while exendin-4 increased the activity of PI3K, p70s6k and MAPKs, but not PKB, suggesting that this enzyme plays a major role in exendin-4 effect upon glucose transport in muscle.

Progress in developing cholecystokinin (CCK)/gastrin receptor ligands that have therapeutic potential.

Gastrin and cholecystokinin (CCK) are two of the oldest hormones and within the past 15 years there has been an exponential increase in knowledge of their pharmacology, cell biology, receptors (CCK1R and CCK2R), and roles in physiology and pathological conditions. Despite these advances there is no approved disease indication for CCK receptor antagonists and only a minor use of agonists. In this review, the important factors determining this slow therapeutic development are reviewed. To assess this it is necessary to briefly review what is known about the roles of CCK receptors (CCK1R and CCK2R) in normal human physiology, their role in pathologic conditions, the selectivity of available potent CCKR agonists/antagonists as well as to review their use in human conditions to date and the results. Despite extensive studies in animals and in humans, recent studies suggest that monotherapy with CCK1R agonists will not be effective in obesity, nor CCK2R antagonists in panic disorders or CCK2R antagonists to inhibit growth of pancreatic cancer. Areas that require more study include the use of CCK2R agonists for imaging tumors and radiotherapy, CCK2R antagonists in hypergastrinemic states especially with long-term PPI use and for potentiation of analgesia as well as use of CCK1R antagonists for a number of gastrointestinal disorders [motility disorders (irritable bowel syndrome, dyspepsia, and constipation) and pancreatitis (acute and chronic)].

The action of GLP-1 and exendins upon glucose transport in normal human adipocytes, and on kinase activity as compared to morbidly obese patients.

A role of GLP-1 (glucagon-like peptide-1) in the recovery of the metabolic conditions of morbidly obese patients after bariatric surgery has been proposed. Exendin 4 (Ex-4) and exendin 9 (Ex-9) both have GLP-1-like effects upon glucose metabolism in human myocytes. We investigated in normal human adipocytes the effect of GLP-1, Ex-4 and Ex-9, compared with insulin upon the activity of PI3K, PKB, MAPKs and p70s6 kinases, and the participation of these enzymes in their action upon 2-deoxy-D-glucose transport by using potential inhibitors. The study was extended to morbidly obese patients. In normal subjects, GLP-1, Ex-4 and insulin, but not Ex-9, increased glucose uptake. In addition, GLP-1 and Ex-4 stimulated PI3K and MAPKs, similar to insulin, but not PKB. Ex-9 only enhanced PI3K, while none affected p70s6k. Inhibition of both PI3K and MAPKs blocked the stimulatory action of GLP-1, Ex-4 and insulin upon glucose transport. In obese patients, basal PI3K, PKB and MAPK activity was, as a rule, lower than that in normal subjects, while cells maintained their normal incremental response to GLP-1, Ex-4 or insulin; Ex-9 induced a clear stimulation of p42 MAPK. In summary, in normal human adipocytes, GLP-1 and Ex-4 have a protein kinase-dependent increasing effect upon glucose transport, which is impaired in obese patients. The participation of GLP-1 in the normalization of the metabolic conditions of the obese may occur through its effects on lipid metabolism or through effects upon glucose transport and/or metabolism in the liver and muscle, which in human obesity remain to be investigated.

Effect of GLP-1 on D-glucose transport, lipolysis and lipogenesis in adipocytes of obese subjects.

GLP-1 has anorectic properties and regulates fuel homeostasis through both its insulinotropic and insulinotrophic actions and effects in extrapancreatic tissue. This study is aimed at characterizing the response to GLP-1 of adipocytes from obese patients, in terms of D-glucose transport and lipid metabolism, in comparison with data from normal subjects. Adipocytes were obtained by enzymatic digestion from the abdominal fat tissue of 25 morbidly obese patients and 8 normal subjects undergoing bariatric or inguinal hernia surgery, respectively. Basal GLUT4 expression, D-glucose transport, glycerol release and lipogenesis were measured in cells treated, when required, with 10(-12)-10(-9) M GLP-1, insulin, glucagon and the GLP-1 structurally related peptides, exendin-4 and exendin-9. In obese patients, versus normal subjects, a trend towards lower values was found in GLUT4 protein or mRNA, although the differences were not statistically significant; insulin-stimulated glucose uptake was higher and cells did not respond to GLP-1, while both exendins (10(-10) and 10(-9) M) exerted an inhibitory action; basal lipolysis was higher and so was the effect of GLP-1 and glucagon, whereas insulin abolished the lipolytic action of all peptides; both basal lipogenesis and the response to insulin were higher while GLP-1 and exendin-4 were ineffective. These results document the analogies and dissimilarities between the response to GLP-1, exendin-4 and exendin-9, as well as to insulin and glucagon, relative to glucose transport and lipid metabolism of fat tissue from obese patients versus normal subjects, the reduced lipogenic effect and enhanced lipolytic action of GLP-1 being, perhaps, adequate for its therapeutic use in obesity.