Miméticos do “glucagon-like peptide-1” (GLP-1) e o seu potencial farmacêutico no controlo da diabetes tipo 2 e da obesidade

A diabetes mellitus do tipo 2 é caracterizada pela resistência à insulina e pela disfunção das células β do pâncreas. Os péptidos gastrintestinais, “gastric inhibitory polypeptide” (GIP) e “glucagon-like peptide-1” (GLP-1), são hormonas incretinas que estimulam, maioritariamente, a produção de insulina pós-prandial. Formulações contendo GLP-1 possuem um grande potencial no tratamento desta doença. Porém, o GLP-1 é eficaz apenas quando administrado por via parentérica. Para o tratamento da diabetes mellitus tipo 2 são usados análogos do GLP‑ 1 ou miméticos da incretina os quais são eficazes por via subcutânea. The pathogenesis of diabetes mellitus type 2 includes insulin resistance and progressive β-cell dysfunction. The gastrointestinal peptides, gastric inhibitory polypeptide (GIP) and glucagon‑like peptide-1 (GLP-1), are incretin hormones which are responsible for the major part of postprandial insulin secretion. Formulations containing GLP-1 have a great potential in the treatment of diabetes mellitus type 2. Nonetheless, GLP-1 is only efficient by continuous parenteral administration. GLP-1 analogues or incretin mimetics, exendine-4, are active after subcutaneous injection and can be used in the treatment of diabetes mellitus of type 2.

The role of the IL-1 type 1 receptor in the life, death and differentiation of adult hippocampal neural precursor cells

Neurogenesis occurs in two distinct regions of the adult brain; the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, and the subventricular zone (SVZ) lining the lateral ventricles. It is now well-known that adult hippocampal neurogenesis can be modulated by a number of intrinsic and extrinsic factors e.g. local signalling molecules, exercise, environmental enrichment and learning. Moreover, levels of adult hippocampal neurogenesis decrease with age, at least in rodents, and alterations in hippocampal neurogenesis have been reported in animal models and human studies of neuropsychiatric and neurodegenerative conditions. Neuroinflammation is a common pathological feature of these conditions and is also a potent modulator of adult hippocampal neurogenesis. Recently, the orphan nuclear receptor TLX has been identified as an important regulator of adult hippocampal neurogenesis as its expression is necessary to maintain the neural precursor cell (NPC) pool in the adult DG. Likewise, exposure of animals to voluntary exercise has been consistently demonstrated to promote adult hippocampal neurogenesis. Lentivirus (LV)- mediated gene transfer is a useful tool to elucidate gene function and to explore potential therapeutic candidates across an array of conditions as it facilitates sustained gene expression in both dividing and post-mitotic cell populations. Both intrinsic and extrinsic factors are important regulators of adult hippocampal neurogenesis. Examining how these factors are affected by an inflammatory stimulus, and the subsequent effects on adult hippocampal neurogenesis provides important information for the development of novel treatment strategies for neuropsychiatric and neurodegenerative conditions in which adult hippocampal neurogenesis is impaired. The aims of the series of experiments presented in this thesis were to examine the effect of the pro-inflammatory cytokine interleukin-1β (IL-1β) on adult hippocampal NPCs both in vitro and in vivo. In vitro, we have shown that IL-1β reduces proliferation of adult hippocampal NPCs in a dose and time-dependent manner. In addition, we have demonstrated that TLX expression is reduced by IL-1β. Blockade of IL-1β signalling prevented both the IL-1β-induced reduction in cell proliferation and TLX expression. In vivo, we examined the effect of short term and long term exposure to LV-IL-1β in sedentary mice and in mice exposed to voluntary running. We demonstrated that impaired hippocampal neurogenesis is only evident after long term exposure to IL-1β. In mice exposed to voluntary running, hippocampal neurogenesis is significantly increased following short-term but not long-term exposure to running. Moreover, short-term running effectively prevents any IL-1β-induced effects on hippocampal neurogenesis; however, no such effects are seen following long-term exposure to running.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.

Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Comparative effects of GLP-1 and GIP on cAMP production, insulin secretion, and in vivo antidiabetic actions following substitution of Ala8/Ala2 with 2-aminobutyric acid

The two major incretin hormones, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP), are currently being considered as prospective drug candidates for treatment of type 2 diabetes. Interest in these gut hormones was initially spurred by their potent insulinotropic activities, but a number of other antihyperglycaemic actions are now established. One of the foremost barriers in progressing GLP-1 and GIP to the clinic concerns their rapid degradation and inactivation by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV). Here, we compare the DPP IV resistance and biological properties of Abu(8)/ Abu(2) (2-aminobutyric acid) substituted analogues of GLP-1 and GIP engineered to impart DPP IV resistance. Whereas (Abu(8))GLP-1 was completely stable to human plasma (half-life > 12h), GLP-1, GIP, and (Abu(2))GIP were rapidly degraded (half-lives: 6.2, 6.0, and 7.1 h, respectively). Native GIP, GLP-1, and particularly (Abu(8))GLP-1 elicited significant adenylate cyclase and insulinotropic activity, while (Abu(2))GIP was less effective. Similarly, in obese diabetic (ob/ob) mice, GIP, GLP-1, and (Abu(8))GLP-1 displayed substantial glucose-lowering and insulin -releasing activities, whereas (Abu(2))GIP was only weakly active. These studies illustrate divergent effects of penultimate amino acid Ala(8)/Ala(2) substitution with Abu on the biological properties of GLP-1 and GIP, suggesting that (Abu(8))GLP-1 represents a potential candidate for future therapeutic development. (C) 2004 Elsevier Inc. All rights reserved.

N-terminal His(7)-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid in-activation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His(7)-modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1 (9-36),amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC50 values 32(.)9 and 6(.)7 nM, respectively) compared with native GLP-1 (IC50 0(.)37 nM). Similarly, both analogues stimulated cAMP production with EC50 values of 16(.)3 and 27 nM respectively compared with GLP-1 (EC50 4(.)7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5(.)6 mM glucose (P

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.

Neuropeptide Y Y(1) receptor regulates protein turnover and constitutive gene expression in hypertrophying cardiomyocytes.

Increased levels of neuropeptide Y correlate with severity of left ventricular hypertrophy in vivo. At cardiomyocyte level, hypertrophy is characterised by increased mass and altered phenotype. The aims were to determine the contributions of increased synthesis and reduced degradation of protein to neuropeptide Y-mediated increase in mass, assess effects on gene expression, and characterise neuropeptide Y Y receptor subtype involvement. Neuropeptide Y (10 nM) increased protein mass of adult rat ventricular cardiomyocytes maintained in culture (24 h) (16%>basal) and de novo protein synthesis (incorporation of [14C]phenylalanine) (18%>basal). Neuropeptide Y (100 nM) prevented degradation of existing protein at 8 h. Actinomycin D (5 µM) attenuated increases in protein mass to neuropeptide Y (=1 nM) but not to neuropeptide Y (10 nM). [Leu31, Pro34]neuropeptide Y (10 nM), an agonist at neuropeptide Y Y1 receptors, increased protein mass (25%>basal) but did not stimulate protein synthesis. Neuropeptide Y-(3–36) (10 nM), an agonist at neuropeptide Y Y2 receptors, increased protein mass (29%>basal) and increased protein synthesis (13%>basal), respectively. Actinomycin D (5 µM) abolished the increase in protein mass elicited by neuropeptide Y-(3–36) but not that by [Leu31, Pro34]neuropeptide Y. BIBP3226 [(R)-N2-(diphenylacetyl)-N-(4-hydroxyphenylmethyl)-d-arginine amide] (1 µM), a neuropeptide Y Y1 receptor subtype-selective antagonist, and T4 [neuropeptide Y-(33–36)]4, a neuropeptide Y Y2 receptor subtype-selective antagonist, attenuated the increase in protein mass to 100 nM neuropeptide Y by 68% and 59%, respectively. Neuropeptide Y increased expression of the constitutive gene, myosin light chain-2 (MLC-2), maximally at 12 h (4.7-fold>basal) but did not induce (t=36 h) expression of foetal genes (atrial natriuretic peptide (ANP), skeletal-a-actin and myosin heavy chain-ß). This increase was attenuated by 86% and 51%, respectively, by BIBP3226 (1 µM) and T4 [neuropeptide Y-(33–36)]4 (100 nM). [Leu31, Pro34]neuropeptide Y (100 nM) (2.4-fold>basal) and peptide YY-(3–36) (100 nM) (2.3 fold>basal) increased expression of MLC-2 mRNA at 12 h. In conclusion, initiation of cardiomyocyte hypertrophy by neuropeptide Y requires activation of both neuropeptide Y Y1 and neuropeptide Y Y2 receptors and is associated with enhanced synthesis and attenuated degradation of protein together with increased expression of constitutive genes but not reinduction of foetal genes.

Effects of sub-chronic exposure to naturally occurring N-terminally truncated metabolites of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), GIP(3-42) and GLP-1 (9-36)amide, on insulin secretion and glucose homeostasis in ob/ob mice

Glucose-dependent insulinotrophic polypepticle (GIP) and glucagon-like peptide-1 (GLP-1) are important enteroendocrine hormones that are rapidly degraded by an ubiquitous enzyme dipeptidyl peptidase IV to yield truncated metabolites GIP(3-42) and GLP-1 (9-36)amide. In this study, we investigated the effects of sub-chronic exposure to these major circulating forms of GIP and GLP-1 on blood glucose control and endocrine pancreatic function in obese diabetic (ob/ob) mice. A once daily injection of either peptide for 14 days had no effect on body weight, food intake or pancreatic insulin content or islet morphology. GLP-1(9-36)amide also had no effect on plasma glucose homeostasis or insulin secretion. Mice receiving GIP(3-42) exhibited small but significant improvements in non-fasting plasma glucose, glucose tolerance and glycaemic response to feeding. Accordingly, plasma insulin responses were unchanged suggesting that the observed enhancement of insulin sensitivity was responsible for the improvement in glycaemic control. These data indicate that sub-chronic exposure to GIP and GLP-1 metabolites does not result in physiological impairment of insulin secretion or blood glucose control. GIP(3-42) might exert an overall beneficial effect by improving insulin sensitivity through extrapancreatic action.

Lys(9) for Glu(9) substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His(7) and Ala(8), can greatly improve resistance to this enzyme. Little research has assessed what effect Glu(9)-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu(9) of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue, (Lys(9))GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys(9))GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor. (C) 2004 Elsevier Inc. All rights reserved.