A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism:lessons from class B crystal structuress

The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiologic significance of this is yet to be elucidated. Previous work has implicated R2.60190 , N3.43240 , Q7.49394 , and H6.52363 as key residues involved in peptide-mediated biased agonism, with R2.60190 , N3.43240 , and Q7.49394 predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict additional key molecular interactions with these amino acids. We have introduced E6.53364 A, N3.43240 Q, Q7.49493N, and N3.43240 Q/Q7.49 Q/Q7.49493N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium, or extracellular signal-regulated kinase (ERK) signaling. A polar interaction between E6.53364 and R2.60190 was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49394 , but not R2.60190 /E6.53364 was critical for calcium mobilization for all three peptides. Mutation of N3.43240 and Q7.49394 had differential effects on individual peptides, providing evidence for molecular differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.

Control of the endocrine pancreas by gastrointestinal peptides

This thesis concerns the mechanism through which enteral delivery of glucose results in a larger insulin response than an equivalent parenteral glucose load. Preliminary studies in which mice received a glucose solution either intragastrically or intraperitoneally confirmed this phenomenon. An important regulatory system in this respect is the entero-insular axis, through which insulin secretion is influenced by neural and endocrine communication between the gastrointestinal tract and the pancreatic islets of Langerhans. Using an in vitro system involving static incubation of isolated (by collagenase digestion) islets of Langerhans, the effect of a variety of gastrointestinal peptides on the secretion of the four main islet hormones, namely insulin, glucagon, somatostatin and pancreatic polypeptide, was studied. The gastrointestinal peptides investigated in this study were the secretin family, comprising secretin, glucagon, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and growth hormone releasing factor (GRF). Gastrin releasing peptide (GRP) was also studied. The results showed that insulin release was stimulated by all peptides studied except PHI, glucagon release was stimulated by all peptides tested, except GRF which suppressed glucagon release, somatostatin release was stimulated by GIP and GRF but suppressed by VIP, PHI, glucagon and secretin, and PP release was stimulated by GIP and GRF, but suppressed by PHI. The insulinotropic effect of GRP was investigated further. A perifusion system was used to examine the time-course of insulin release from isolated islets after stimulation with GRP. GRP was shown to be insulinotropic only in the presence of physiologically elevated glucose concentrations and both first and second phases of insulin release were augmented. There was no effect at substimulatory or very high glucose concentrations. Studies using a cultured insulin-secreting islet cell line, the RINm5F cell line, were undertaken to elucidate the intracellular mechanism of action of GRP. This peptide did not enhance insulin release via an augmentation of glucose metabolism, or via the adenylate cyclase/cyclic AMP secondary messenger system. The pattern of changes of cytosolic free calcium in response to GRP, which involved both mobilization of intracellular stores and an influx of extracellular calcium, suggested the involvement of phosphatidylinositol bisphosphate breakdown as a mediator of the effect of GRP on insulin secretion.

Receptor Activity Modifying Proteins (RAMPs) interact with the VPAC2 Receptor and CRF1 receptors and modulate their function

Background and Purpose Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-Activating peptide 2 receptor (VPAC) and the type 1 corticotrophin releasing factor receptor (CRF) has been examined. Experimental Approach GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by elisa. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca mobilization and GTPγS binding to G, G, G and G were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2 mice was assessed. Key Results The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC enhanced the cell surface expression of all three RAMPs. CRF enhanced the cell surface expression of RAMP2; the cell surface expression of CRF was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF: RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2 mice, there was a loss of responsiveness to CRF. Conclusions and Implications The VPAC and CRF receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF, coupling to RAMP2 may be of physiological significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

High-strength resorbable brushite bone cement with controlled drug-releasing capabilities

Brushite cements differ from apatite-forming compositions by consuming a lot of water in their setting reaction whereas apatite-forming cements consume little or no water at all. Only such cement systems that consume water during setting can theoretically produce near-zero porosity ceramics. This study aimed to produce such a brushite ceramic and investigated whether near elimination of porosity would prevent a burst release profile of incorporated antibiotics that is common to prior calcium phosphate cement delivery matrices. Through adjustment of the powder technological properties of the powder reactants, that is particle size and particle size distribution, and by adjusting citric acid concentration of the liquid phase to 800 mM, a relative porosity of as low as 11% of the brushite cement matrix could be achieved (a 60% reduction compared to previous studies), resulting in a wet unprecompacted compressive strength of 52 MPa (representing a more than 100% increase to previously reported results) with a workable setting time of 4.5 min of the cement paste. Up to 2 wt.% of vancomycin and ciprofloxacin could be incorporated into the cement system without loss of wet compressive strength. It was found that drug release rates could be controlled by the adjustable relative porosity of the cement system and burst release could be minimized and an almost linear release achieved, but the solubility of the antibiotic (vancomycin > ciprofloxacin) appeared also to be a crucial factor.

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6′- dibehenate) - A novel adjuvant inducing both strong CMI and antibody responses:a novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6′-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4°C and 25°C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-γ cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-γ was identified as CD4 T cells.

Carbon monoxide inhibits sprouting angiogenesis and vascular endothelial growth factor receptor-2 phosphorylation

Carbon monoxide (CO) is a gaseous autacoid known to positively regulate vascular tone; however, its role in angiogenesis is unknown. The aim of this study was to investigate the effect of CO on angiogenesis and vascular endothelial growth factor (VEGF) receptor-2 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were cultured on growth factor- reduced Matrigel and treated with a CO-releasing molecule (CORM-2) or exposed to CO gas (250 ppm). Here, we report the surprising finding that exposure to CO inhibits vascular endothelial growth factor (VEGF)-induced endothelial cell actin reorganisation, cell proliferation, migration and capillary-like tube formation. Similarly, CO suppressed VEGF-mediated phosphorylation of VEGFR-2 at tyrosine residue 1175 and 1214 and basic fibroblast growth factor- (FGF-2) and VEGF-mediated Akt phosphorylation. Consistent with these data, mice exposed to 250 ppm CO (1h/day for 14 days) exhibited a marked decrease in FGF-2-induced Matrigel plug angiogenesis (p<0.05). These data establish a new biological function for CO in angiogenesis and point to a potential therapeutic use for CO as an anti-angiogenic agent in tumour suppression.

Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in placenta growth factor (PlGF) deficiency

Placenta growth factor (PlGF) deficient mice are fertile at a Mendelian ratio. Interestingly, low maternal plasma levels of PlGF are strongly associated with early onset of preeclampsia, a pregnancy hypertensive disorder characterised by high blood pressure, proteinuria and fetal growth restriction. PlGF is increasingly being recognised as an early diagnostic biomarker, but the physiological importance of PlGF in the pathogenesis of preeclampsia is unknown. We investigated whether the decreased levels of PlGF in pregnancy exacerbate the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1 and the potential of hydrogen sulphide to ameliorate these effects. Pregnant PlGF−/− mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at 1 × 109 pfu/ml at E10.5 and mean arterial blood pressure (MAP), biochemical and histological analysis of maternal kidney, placenta and embryos were assessed at the end of pregnancy. Ad-sFlt-1 significantly increased MAP and induced severe glomerular endotheliosis in PlGF−/− mice compared to wild-type animals. Soluble Flt-1 also significantly elevated albumin–creatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury. Furthermore, sFlt-1 over expression increased fetal resorption rate in the PlGF−/− mice and promoted abnormal placental vascularisation. To determine whether placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/− placentas and embryos in dams and exposed to high sFlt-1 environment. These mothers showed reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF−/− mice. Furthermore, treatment with hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria observed in Ad-sFlt-1 treated pregnant PlGF−/− mice. Our study shows that placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 associated with preeclampsia and activation of the hydrogen sulphide pathway may rescue preeclampsia phenotypes even under low PlGF environment.

Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in Placenta Growth Factor (PlGF) deficiency

INTRODUCTION: Low circulating levels of placenta growth factor (PlGF) is strongly associated with the onset of preeclampsia, a maternal hypertensive disorder characterized by high blood pressure and proteinuria after 20 weeks of gestation. Although, PlGF-deficient mice are born healthy and fertile at a Mendelian ratio, the physiological importance of PlGF in the pathogenesis of preeclampsia is unclear. We hypothesised that decreased levels of PlGF in pregnancy exacerbates the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1. METHODS: Pregnant PlGF-/- mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at high (i) 1.5x109 pfu/ml and low (ii) 0.5x109 pfu/ml doses. Mean arterial blood pressure (MBP), biochemical and histological assessments of maternal kidney, placenta and embryos were performed. RESULTS: Ad-sFlt-1 significantly increased MBP and induced severe glomerular endotheliosis in PlGF-/- mice at E10.5 gestation compared to wild-type animals. High sFlt-1 also significantly elevated albumincreatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury.At a high dose of sFlt-1, there was complete fetal resorption in the pregnant PlGF-/- mice, and even the lower dose of sFlt-1 induced severe fetal resorption and abnormal placental vascularization. Hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria in Ad-sFlt-1 treated pregnant PlGF-/- mice. To determine if placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/- placentas and embryos were generated in wild-time dams and exposed to high sFlt-1 environment. This resulted in reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF-/- mice. CONCLUSIONS: Placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 in preeclampsia and the hydrogen sulphide pathway may rescue preeclampsia phenotypes.