Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala(8)-substituted GLP-1 analogues

The hormone glucagonlike peptide-1(736)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucosedependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the epsilon-amino group in the side chain of the Lys(26) residue and to combine this modification with substitutions of the Ala(8) residue, namely Val or aminobutyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val(8),Lys(pal)(26)]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal beta-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val(8),Lys(pal)(26)]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val8,Lys(pal)26]GLP-1 did not demonstrate acute glucoselowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability.

Enhanced cAMP generation and insulin-releasing potency of two novel Tyr(1)-modified enzyme-resistant forms of glucose-dependent insulinotropic polypeptide is associated with significant antihyperglycaemic activity in spontaneous obesity-diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin hormone, which potentiates glucose-induced insulin secretion. Antihyperglycaemic actions of GIP provide significant potential in Type 11 diabetes therapy. However, inactivation of GIP by the enzyme dipeptidyl peptidase IV (DPP IV) and its consequent short circulating half-life limit its therapeutic use. Therefore two novel Tyr(1)-Modified analogues of GIP, N-Fmoc-GIP (where Fmoc is 9-fluorenylmethoxycarbonyl) and N-palmitate-GIP, were synthesized and tested for metabolic stability and biological activity. Both GIP analogues were resistant to degradation by DPP IV and human plasma. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, both analogues exhibited a 2-fold increase in cAMP-generating potency compared with native GIP (EC50 values of 9.4, 10.0 and 18.2 nM respectively). Using clonal BRIN-BD11 cells, both analogues demonstrated strong insulinotropic activity compared with native GIP (P <0.01 to P <0.001). In obese diabetic (ob/ob) mice, administration of N-Fmoc-GIP or N-palmitate-GIP (25 nmol/kg) together with glucose (18 mmol/kg) significantly reduced the peak 15 min glucose excursion (1.4- and 1.5-fold respectively; P <0.05 to P <0.01) compared with glucose alone. The area under the curve (AUC) for glucose was significantly lower after administration of either analogue compared with glucose administered alone or in combination with native GIP (1.5-fold; P <0.05). This was associated with a significantly greater AUC for insulin (2.1-fold; P <0.001) for both analogues compared with native GIP. A similar pattern of in vivo responsiveness was evident in lean control mice. These data indicate that novel N-terminal Tyr(1) modification of GIP with an Fmoc or palmitate group confers resistance to degradation by DPP IV in plasma, which is reflected by increased in vitro potency and greater insulinotropic and antihyperglycaemic activities in an animal model of Type 11 diabetes mellitus.

Improved stability, insulin-releasing activity and antidiabetic potential of two novel N-terminal analogues of gastric inhibitory polypeptide: N-acetyl-GIP and pGlu-GIP

Aims/hypothesis: This study examined the plasma stability, biological activity and antidiabetic potential of two novel N-terminally modified analogues of gastric inhibitory polypeptide (GIP).

Methods: Degradation studies were carried out on GIP, N-acetyl-GIP (Ac-GIP) and N-pyroglutamyl-GIP (pGlu-GIP) in vitro following incubation with either dipeptidylpeptidase IV or human plasma. Cyclic adenosine 3'5' monophosphate (cAMP) production was assessed in Chinese hamster lung fibroblast cells transfected with the human GIP receptor. Insulin-releasing ability was assessed in vitro in BRIN-BD11 cells and in obese diabetic (ob/ob) mice.

Results: GIP was rapidly degraded by dipeptidylpeptidase IV and plasma (t1/2 2.3 and 6.2 h, respectively) whereas Ac-GIP and pGlu-GIP remained intact even after 24 h. Both Ac-GIP and pGlu-GIP were extremely potent (p<0.001) at stimulating cAMP production (EC50 values 1.9 and 2.7 nmol/l, respectively), almost a tenfold increase compared to native GIP (18.2 nmol/l). Both Ac-GIP and pGlu-GIP (10–13–10–8 mmol/l) were more potent at stimulating insulin release compared to the native GIP (p<0.001), with 1.3-fold and 1.2-fold increases observed at 10–8 mol/l, respectively. Administration of GIP analogues (25 nmol/kg body weight, i.p.) together with glucose (18 mmol/kg) in (ob/ob) mice lowered (p<0.001) individual glucose values at 60 min together with the areas under the curve for glucose compared to native GIP. This antihyperglycaemic effect was coupled to a raised (p<0.001) and more prolonged insulin response after administration of Ac-GIP and pGlu-GIP (AUC, 644±54 and 576±51 ng·ml–1·min, respectively) compared with native GIP (AUC, 257±29 ng·ml–1·min).

Conclusion/interpretation: Ac-GIP and pGlu-GIP, show resistance to plasma dipeptidylpeptidase IV degradation, resulting in enhanced biological activity and improved antidiabetic potential in vivo, raising the possibility of their use in therapy of Type II (non-insulin-dependent) diabetes mellitus.

Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells. (C) 2008 by Radiation Research Society.

Enhanced sensitivity of protein kinase B/Akt to insulin in hypoxia is independent of HIF1 alpha and promotes cell viability

Maintenance of oxygen homeostasis is a key requirement to ensure normal mammalian cell growth and differentiation. Hypoxia arises when oxygen demand exceeds supply, and is a feature of multiple human diseases including stroke, cancer and renal fibrosis. We have investigated the effect of hypoxia on kidney cells, and observed that insulin-induced cell viability is increased in hypoxia. We have characterized the role of protein kinase B (PKB/ Akt) in these cells as a potential mediator of this effect. PKB/Akt activity was increased by low oxygen concentrations in kidney cells, and insulin-stimulated activation of PKB/Akt was stronger, more rapid and more sustained in hypoxia. Reduction of HIF1 alpha levels using antimycin-A or siRNA targeting HlF1 alpha did not affect PKB/Akt activation in hypoxia. Pharmacologic stabilization of HIF1 alpha independent of hypoxia did not increase insulin-stimulated PKB/Akt activation. Although increased insulin-stimulated cell viability was observed in hypoxia, no differences in the degree of insulin-stimulated glucose uptake were observed in L6 muscle cells in hypoxia compared to normoxia. Thus, PKB/Akt may regulate specific cellular responses to growth factors such as insulin under adverse conditions such as hypoxia. alpha 2007 Elsevier GmbH. All rights reserved.

Anti-apoptotic effects of Z alpha-1 antitrypsin in human bronchial epithelial cells.

α1-antitrypsin (α1-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of α1-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z α1-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved.

Control, M variant α1-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-κB activation and induced expression of a selection of pro- and anti-apoptotic genes.

Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-κB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-κB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies.

The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Partial agonism, neutral antagonism, and inverse agonism at the human wild-type and constitutively active cholecystokinin-2 receptors

Cholecystokinin receptor-2 (CCK2R) is a G protein receptor that regulates a number of physiological functions. Activation of CCK2R and/or expression of a constitutively active CCK2R variant may contribute to human diseases, including digestive cancers. Search for antagonists of the CCK2R has been an important challenge during the last few years, leading to discovery of a set of chemically distinct compounds. However, several early-discovered antagonists turned out to be partial agonists. In this context, we carried out pharmacological characterization of six CCK2R antagonists using COS-7 cells expressing the human CCK2R or a CCK2R mutant having a robust constitutive activity on inositol phosphates production, and we investigated the molecular mechanisms which, at a CCK2R binding site, account for these features. Results indicated that three compounds, 3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3- yl)-N'-(3-methylphenyl)urea (L365,260), 4-{[2-[[3-(lH-indol-3-yl)-2- methyl-1-oxo-2-[[[1.7.7-trimethyl-bicyclo[2.2.1]hept-2-yl)-oxy]carbonyl]amino] propyl]amino]-1-phenylethyl]amino-4-oxo-[lS-la.2[S*(S*)]4a]} -butanoate N-methyl-D-glucamine (PD135, 158), and (R)-1-naphthalenepropanoic acid, b-[2-[[2-(8-azaspiro-[4.5]dec-8-ylcarbonyl)-4,6-dimethylphenyl]amino]-2- oxoethyl] (CR2945), were partial agonists; one molecule, 1-[(R)-2,3-dihydro-1- (2,3-dihydro-1-(2-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl] -3-(3-methylphenyl)urea (YM022), was a neutral antagonist; and two compounds, N-(+)-[1-(adamant-1-ylmethyl)-2,4-dioxo-5-phenyl2,3,4,5-tetrahydro-1H-1, 5-benzodiazepin-3-yl]-N'-phenylurea (GV150,013X) and ([(N-[methoxy-3 phenyl] N-[N-methyl N-phenyl carbamoylmethyl], carbomoylmethyl)-3 ureido]-3-phenyl)2-propionic acid (RPR101,048), were inverse agonists. Furthermore, target- and pharmacophore-based docking of ligands followed by molecular dynamic simulation experiments resulted in consistent motion of aromatic residues belonging to a network presumably important for activation, thus providing the first structural explanations for the different pharmacological profiles of tested compounds. This study confirms that several referenced so-called antagonists are in fact partial agonists, and because of this undesired activity, we suggest that newly generated molecules should be preferred to efficiently block CCK2R-related physiological effects. Furthermore, data on the structural basis for the different pharmacological features of CCK2R ligands will serve to further clarify CCK2R mechanism of activation. Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics.

SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr(346/356/366)) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1-12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr(346/356/366) in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na+ conductance (G (Na)) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr(346/356/366) phosphorylation. Although G (Na) is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 mu M) acutely activated SGK1 and the peak of this response (2-3 h) coincided with the induction of G (Na), and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G (Na), transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G (Na) but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na+ absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G (Na), its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.

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.

Use of synthetic peptides to locate novel integrin alpha(2)beta(1)-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triple-helical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

Klebsiella pneumoniae increases the levels of Toll-like receptors 2 and 4 in human airway epithelial cells

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IkappaBalpha-dependent NF-kappaBeta pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.

Insulin receptor substrate-2 is expressed in kidney epithelium and up-regulated in diabetic nephropathy

Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-ß1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase-A computational perspective on severe epimerase-deficiency galactosemia

UDP-galactose 4'-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD(+) cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Background: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown.

Methods: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses.

Results: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1).

Conclusions: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.