RESUMO
AIM: To investigate the effects of the novel glucose-dependent insulinotropic polypeptide (GIP) analogue, ZP4165, on body weight and glycaemic control in rodents, and to investigate if ZP4165 modulates the anti-obesity and anti-hyperglycaemic effects of a glucagon-like peptide-1 (GLP-1) agonist (liraglutide). METHODS: The acute insulinotropic effect of ZP4165 was investigated in rats during an oral glucose tolerance test. The long-term effects of ZP4165 on body weight and glycaemic control, either alone or in combination with liraglutide, were assessed in diet-induced obese mice and diabetic db/db mice. RESULTS: ZP4165 showed insulinotropic action in rats. The GIP analogue did not alter the body weight of obese mice but enhanced GLP-1-induced weight loss. In diabetic mice, 4 weeks' dosing with ZP4165 reduced glycated haemoglobin levels vs vehicle by an extent similar to the GLP-1 agonist. CONCLUSIONS: ZP4165 potentiated the anti-obesity effect of a GLP-1 agonist in obese mice and improved glycaemic control in diabetic mice. These studies support further investigation of dual-incretin therapy as a more effective treatment option than mono GLP-1 medication for type 2 diabetes mellitus and obesity.
Assuntos
Fármacos Antiobesidade/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Polipeptídeo Inibidor Gástrico/análogos & derivados , Polipeptídeo Inibidor Gástrico/uso terapêutico , Hiperglicemia/prevenção & controle , Hipoglicemiantes/uso terapêutico , Obesidade/tratamento farmacológico , Receptores dos Hormônios Gastrointestinais/agonistas , Animais , Fármacos Antiobesidade/sangue , Fármacos Antiobesidade/farmacocinética , Fármacos Antiobesidade/farmacologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Desenho de Fármacos , Quimioterapia Combinada , Polipeptídeo Inibidor Gástrico/farmacocinética , Polipeptídeo Inibidor Gástrico/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células HEK293 , Meia-Vida , Humanos , Hipoglicemiantes/sangue , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Incretinas/farmacologia , Incretinas/uso terapêutico , Liraglutida/farmacologia , Liraglutida/uso terapêutico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Obesidade/sangue , Obesidade/metabolismo , Ratos Sprague-Dawley , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Redução de Peso/efeitos dos fármacosRESUMO
microRNAs are small regulatory RNAs that are currently emerging as new biomarkers for cancer and other diseases. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood serum or plasma. These types of samples represent a challenge in terms of microRNA quantification. A newly developed method for microRNA qPCR using Locked Nucleic Acid (LNA)-enhanced primers enables accurate and reproducible quantification of microRNAs in scarce clinical samples. Here we show that LNA-based microRNA qPCR enables biomarker screening using very low amounts of total RNA from FFPE samples and the results are compared to microarray analysis data. We also present evidence that the addition of a small carrier RNA prior to total RNA extraction, improves microRNA quantification in blood plasma and laser capture microdissected (LCM) sections of FFPE samples.
Assuntos
MicroRNAs/análise , Reação em Cadeia da Polimerase/métodos , Fixadores , Formaldeído , Humanos , Lasers , MicroRNAs/sangue , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Inclusão em ParafinaRESUMO
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.
Assuntos
Anfetamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Sintaxina 1/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Corpo Estriado/citologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Glutationa Transferase/metabolismo , Humanos , Rim/citologia , Mesencéfalo/citologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sinaptossomos/metabolismo , TransfecçãoRESUMO
To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of approximately 3.6 x 10(-9) cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-beta2AR (beta2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the Vmax and KM values for [3H]dopamine uptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Microdomínios da Membrana/metabolismo , Animais , Linhagem Celular , Toxina da Cólera/metabolismo , Colesterol/deficiência , Citoesqueleto/metabolismo , Difusão , Humanos , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de FluorescênciaRESUMO
Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux.
Assuntos
Anfetaminas/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Neurônios/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Benzilaminas/farmacologia , Transporte Biológico/efeitos dos fármacos , Western Blotting/métodos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Inibidores Enzimáticos/farmacologia , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mesencéfalo/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Sulfonamidas/farmacologia , Transfecção/métodosRESUMO
ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Dissulfetos/metabolismo , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Antagonistas Adrenérgicos alfa/farmacocinética , Sequência de Aminoácidos , Antineoplásicos/farmacocinética , Transporte Biológico , Biotinilação , Western Blotting , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Dissulfetos/química , Resistência a Múltiplos Medicamentos , Humanos , Técnicas Imunoenzimáticas , Rim/metabolismo , Mitoxantrona/farmacocinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/química , Prazosina/farmacocinéticaRESUMO
The human dopamine transporter (hDAT) contains a C-terminal type 2 PDZ (postsynaptic density 95/Discs large/zona occludens 1) domain-binding motif (LKV) known to interact with PDZ domain proteins such as PICK1 (protein interacting with C-kinase 1). As reported previously, we found that, after deletion of this motif, hDAT was retained in the endoplasmic reticulum (ER) of human embryonic kidney (HEK) 293 and Neuro2A cells, suggesting that PDZ domain interactions might be critical for hDAT targeting. Nonetheless, substitution of LKV with SLL, the type 1 PDZ-binding sequence from the beta2-adrenergic receptor, did not disrupt plasma membrane targeting. Moreover, the addition of an alanine to the hDAT C terminus (+Ala), resulting in an LKVA termination sequence, or substitution of LKV with alanines (3xAla_618-620) prevented neither plasma membrane targeting nor targeting into sprouting neurites of differentiated N2A cells. The inability of +Ala and 3xAla_618-620 to bind PDZ domains was confirmed by lack of colocalization with PICK1 in cotransfected HEK293 cells and by the inability of corresponding C-terminal fusion proteins to pull down purified PICK1. Thus, although residues in the hDAT C terminus are indispensable for proper targeting, PDZ domain interactions are not required. By progressive substitutions with beta2-adrenergic receptor sequence, and by triple-alanine substitutions in the hDAT C terminus, we examined the importance of epitopes preceding the LKV motif. Substitution of RHW(615-617) with alanines caused retention of the transporter in the ER despite preserved ability of this mutant to bind PICK1. We propose dual roles of the hDAT C terminus: a role independent of PDZ interactions for ER export and surface targeting, and a not fully clarified role involving PDZ interactions with proteins such as PICK1.