Metabolic and structural properties of human obestatin {1-23} and two fragment peptides

Obestatin is a peptide produced in the oxyntic mucosa of the stomach and co-localizes with ghrelin on the periphery of pancreatic islets. Several studies demonstrate that obestatin reduces food and water intake, decreases body weight gain, inhibits gastrointestinal motility, and modulates glucose-induced insulin secretion. In this study we evaluated the acute metabolic effects of human obestatin {1-23} and fragment peptides {1-10} or {11-23} in high-fat fed mice, and then investigated their solution structure by NMR spectroscopy and molecular modelling. Obestatins {1-23} and {11-23} significantly reduced food intake (86% and 90% respectively) and lowered glucose responses to feeding, whilst leaving insulin responses unchanged. No metabolic changes could be detected following the administration of obestatin (1-10). In aqueous solution none of the obestatin peptides possessed secondary structural features. However, in a 2,2,2-trifluoroethanol (TFE-d(3))-H2O solvent mixture, the structure of obestatin {1-23} was characterized by an a-helix followed by a single turn helix conformation between residues Pro(4) and Gln(15) and His(19) and Ala(22) respectively. Obestatin {1-10} showed no structural components whereas {11-23} contained an a-helix between residues Val(14) and Ser(20) in a mixed solvent. These studies are the first to elucidate the structure of human obestatin and provide clear evidence that the observed a-helical structures are critical for in vivo activity. Future structure/function studies may facilitate the design of novel therapeutic agents based on the obestatin peptide structure. (C) 2010 Elsevier Inc. All rights reserved.

Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P

Kinetic analysis of the cleavage of human protease-activated receptor-1/2/3 and 4 using quenched-fluorescent peptide substrates

Protease-activated receptors [PARs] are a family of G-protein-coupled seven-transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten-residue peptide spanning the receptor activation cleavage site and using progress-curve kinetics, k(cat)/K-m values were determined.

Immunocytochemical Localization of Substance P Receptors (NK-1 Receptors) in Human Gingival Tissue

Substance P (SP) is a member of the structurally related family of neuropeptides known as the tachykinins. In addition to neurotransmitter roles, the tachykinins are also known to modulate local inflammation which depends on signalling between the neuropeptide molecules and target cells and tissues. SP mediates its effects through a specific receptor, known as the substance P receptor or the neurokinin 1 (NK-1) receptor. The NK-1 receptor is a G-protein associated integral membrane protein and although it has been studied in a wide range of tissues, to date there has been no published data on the localisation of the NK-1 receptor in human gingival tissue. Objective: The aim of this study was to examine the distribution of the NK-1 receptor in human gingival tissue using immunocytochemistry. Method: Gingival tissue was obtained from patients undergoing periodontal surgery. Tissue was fixed in paraformaldehyde and embedded in wax for sectioning. Sections were dewaxed in xylene and then rehydrated in alcohols and phosphate buffered saline. Rehydrated sections were probed with rabbit polyclonal antibody to human NK-1 receptor which was subsequently detected using anti-rabbit horseradish peroxidase conjugate and diaminobenzidine as substrate. Results: Immunocytochemistry revealed that the NK-1 receptor was distributed along nerve fibres and blood vessel endothelial cells, suggesting these areas are main targets for the actions of SP via the NK-1 receptor. Conclusion: This is the first immunocytochemical report of NK-1 receptors in human gingival tissue and provides evidence for possible NK-1 mediated biological effects of SP in human gingival tissue from periodontitis patients.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8) GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]iodophenyl]2-ethoxy propanoic acid], which is specific for the ? isoform of the peroxisomal proliferator activated receptor (PPAR?), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPAR?1 and to a GST (glutathione S-transferase) fusion protein contg. the ligand binding domain of human PPAR?1 (KD = 70 nM). Using this ligand, the authors characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition expts., rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, resp.). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPAR?1 were comparable with those detd. in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an ?-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPAR?1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPAR? and that the pharmacol. of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.

The role of human demographic history in determining the distribution and frequency of transferase-deficient galactosaemia mutations

Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa. Heredity (2010) 104, 148-154; doi:10.1038/hdy.2009.84; published online 29 July 2009

P2Y(1) antagonists: Combining receptor-based modeling and QSAR for a quantitative prediction of the biological activity based on consensus scoring

P2Y(1) is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y(1) homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.

Achaete-scute homolog-1 linked to remodeling and preneoplasia of pulmonary epithelium

The basic helix-loop-helix protein achaete-scute homolog-1 (ASH1) is involved in lung neuroendocrine (NE) differentiation and tumor promotion in SV40 transgenic mice. Constitutive expression of human ASH-1 (hASH1) in mouse lung results in hyperplasia and remodeling that mimics bronchiolization of alveoli (BOA), a potentially premalignant lesion of human lung carcinomas. We now show that this is due to sustained cellular proliferation in terminal bronchioles and resistance to apoptosis. Throughout the airway epithelium the expression of anti-apoptotic Bcl-2 and c-Myb was increased and Akt/mTOR pathway activated. Moreover, the expression of matrix metalloproteases (MMPs) including MMP7 was specifically enhanced at the bronchiolo-alveolar duct junction and BOA suggesting that MMPs play a key role in this microenvironment during remodeling. We also detected MMP7 in 70% of human BOA lesions. Knockdown of hASH1 gene in human lung cancer cells in vitro suppressed growth by increasing apoptosis. We also show that forced expression of hASH1 in immortalized human bronchial epithelial cells decreases apoptosis. We conclude that the impact of hASH1 is not limited to cells with NE phenotype. Rather, constitutive expression of hASH1 in lung epithelium promotes remodeling through multiple pathways that are commonly activated during lung carcinogenesis. The collective results suggest a novel model of BOA formation via hASH1-induced suppression of the apoptotic pathway. Our study yields a promising new preclinical tool for chemoprevention of peripheral lung carcinomas. © 2007 USCAP, Inc All rights reserved.