RESUMEN
BACKGROUND: Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis. METHODS: Various nanobodies against the mouse transferrin receptor were fused to neurotensin and injected peripherally in mice. Neurotensin is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery. Continuous body temperature measurements were used as a readout for brain penetration of nanobody-neurotensin fusions after its peripheral administration. Full temperature curves were analyzed using two-way ANOVA with Dunnett multiple comparisons tests. RESULTS: One anti-transferrin receptor nanobody coupled to neurotensin elicited a drop in body temperature following intravenous injection. Epitope binning indicated that this nanobody bound a distinct transferrin receptor epitope compared to the non-crossing nanobodies. This brain-penetrating nanobody was used to characterize the in vivo hypothermia model. The hypothermic effect caused by neurotensin is dose-dependent and could be used to directly compare peripheral administration routes and various nanobodies in terms of brain exposure. CONCLUSION: This method led to the discovery of an anti-transferrin receptor nanobody that can reach the brain via receptor-mediated transcytosis after peripheral administration. This method could be used to assess novel proteins for brain-penetrating capabilities using a target-engaging readout.
Asunto(s)
Temperatura Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Neurotensina/farmacología , Receptores de Transferrina/inmunología , Anticuerpos de Dominio Único/farmacología , Transcitosis/fisiología , Animales , Camélidos del Nuevo Mundo , Femenino , Masculino , Ratones , Neurotensina/administración & dosificación , Receptores de Neurotensina/efectos de los fármacos , Anticuerpos de Dominio Único/administración & dosificaciónRESUMEN
Neurotensin (NTS)-polyplex is a multicomponent nonviral vector that enables gene delivery via internalization of the neurotensin type 1 receptor (NTSR1) to dopaminergic neurons and cancer cells. An approach to improving its therapeutic safety is replacing the viral karyophilic component (peptide KPSV40; MAPTKRKGSCPGAAPNKPK), which performs the nuclear import activity, by a shorter synthetic peptide (KPRa; KMAPKKRK). We explored this issue and the mechanism of plasmid DNA translocation through the expression of the green fluorescent protein or red fluorescent protein fused with KPRa and internalization assays and whole-cell patch-clamp configuration experiments in a single cell together with importin α/ß pathway blockers. We showed that KPRa electrostatically bound to plasmid DNA increased the transgene expression compared with KPSV40 and enabled nuclear translocation of KPRa-fused red fluorescent proteins and plasmid DNA. Such translocation was blocked with ivermectin or mifepristone, suggesting importin α/ß pathway mediation. KPRa also enabled NTS-polyplex-mediated expression of reporter or physiological genes such as human mesencephalic-derived neurotrophic factor (hMANF) in dopaminergic neurons in vivo. KPRa is a synthetic monopartite peptide that showed nuclear import activity in NTS-polyplex vector-mediated gene delivery. KPRa could also improve the transfection of other nonviral vectors used in gene therapy.
Asunto(s)
Portadores de Fármacos/síntesis química , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Neurotensina/administración & dosificación , Fragmentos de Péptidos/síntesis química , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Núcleo Celular/metabolismo , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Masculino , Ratones , Modelos Animales , Nanopartículas/química , Neurotensina/genética , Neurotensina/farmacocinética , Técnicas de Placa-Clamp , Plásmidos/genética , Ratas , Receptores de Neurotensina/metabolismo , Análisis de la Célula Individual , Técnicas EstereotáxicasRESUMEN
The current study has determined the ability of exendin-4 to augment the antidiabetic benefits of the recently characterised GIP/xenin hybrid, (DAla2)GIP/xenin-8-Gln. As such, combined activation of metabolic pathways linked to various gut derived hormones has been shown to exert complementary beneficial metabolic effects in diabetes. (DAla2)GIP/xenin-8-Gln and exendin-4 were administered twice daily to high fat fed (HFF) or db/db mice for 28 days and antidiabetic benefits assessed. Persistence of beneficial metabolic effects in HFF mice was also examined. Twice-daily injection of (DAla2)GIP/xenin-8-Gln for 28 days in HFF mice significantly reduced energy intake, body weight, circulating glucose, HbA1c and improved glucose tolerance and insulin sensitivity. Overall pancreatic islet, alpha- and beta-cell areas were reduced, with concurrent reduction in alpha- and beta-cell proliferation that was more apparent in the combined treatment group. Addition of exendin-4 to (DAla2)GIP/xenin-8-Gln therapy did not significantly improve metabolic control. Remarkably, beneficial effects were still evident 14 days following complete cessation of peptide administration. Thus, circulating glucose and insulin, HbA1c concentrations and glucose tolerance were still significantly improved when compared to control HFF mice on day 42, with minimal changes to pancreatic islet architecture. In contrast to HFF mice, combined treatment of db/db mice with (DAla2)GIP/xenin-8-Gln plus exendin-4 was required to induce beneficial effects on key metabolic parameters, which were not observed with either treatment alone. This included improvements in glucose tolerance and insulin sensitivity, but no effect on pancreatic architecture. These studies highlight the clear, and persistent, metabolic advantages of sustained activation of GLP-1 receptors, alongside concurrent activation of related GIP and xenin cell signalling pathways, in diabetes.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético/efectos de los fármacos , Exenatida/administración & dosificación , Polipéptido Inhibidor Gástrico/administración & dosificación , Neurotensina/administración & dosificación , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 2/prevención & control , Dieta Alta en Grasa/efectos adversos , Encefalinas/administración & dosificación , Hormonas Gastrointestinales/administración & dosificación , Hemoglobina Glucada/metabolismo , Hipoglucemiantes/administración & dosificación , Insulina/sangre , Ratones , Resultado del TratamientoRESUMEN
Neurotensin (NT) demonstrates ambiguous activity on inflammatory processes. The present study was undertaken to test the potential anti-inflammatory activity of NT in a murine model of non-atopic asthma and to establish the contribution of NTR1 receptors. Asthma was induced in BALB/c mice by skin sensitization with dinitrofluorobenzene followed by intratracheal hapten provocation. The mice were treated intraperitoneally with NT, SR 142948 (NTR1 receptor antagonist) + NT or NaCl. Twenty-four hours after the challenge, airway responsiveness to nebulized methacholine was measured. Bronchoalveolar lavage fluid (BALF) and lungs were collected for biochemical and immunohistological analysis. NT alleviated airway hyperreactivity and reduced the number of inflammatory cells in BALF. These beneficial effects were inhibited by pretreatment with the NTR1 antagonist. Additionally, NT reduced levels of IL-13 and TNF-α in BALF and IL-17A, IL12p40, RANTES, mouse mast cell protease and malondialdehyde in lung homogenates. SR 142948 reverted only a post-NT TNF-α decrease. NT exhibited anti-inflammatory activity in the hapten-induced asthma. Reduced leukocyte accumulation and airway hyperresponsiveness indicate that this beneficial NT action is mediated through NTR1 receptors. A lack of effect by the NTR1 blockade on mast cell activation, oxidative stress marker and pro-inflammatory cytokine production suggests that other pathways can be involved, which requires further research.
Asunto(s)
Antiinflamatorios/administración & dosificación , Asma/tratamiento farmacológico , Haptenos/efectos adversos , Neurotensina/administración & dosificación , Animales , Antiinflamatorios/farmacología , Asma/inducido químicamente , Asma/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inyecciones Intraperitoneales , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Cloruro de Metacolina/administración & dosificación , Cloruro de Metacolina/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Neurotensina/farmacología , Pirazoles/administración & dosificación , Pirazoles/farmacología , Quinolinas/administración & dosificación , Quinolinas/farmacologíaRESUMEN
Opioid and neurotensin (NT) receptors are expressed in both central and peripheral nervous systems where they modulate nociceptive responses. Nowadays, opioid analgesics like morphine remain the most prescribed drugs for the treatment of moderate to severe pain. However, despite their daily used, opioids can produce life-threatening side effects, such as constipation or respiratory depression. Besides, NT analogs exert strong opioid-independent analgesia. Here, we thus hypothesized that the combined use of opioid and NT agonists would require lower doses to produce significant analgesic effects, hence decreasing opioid-induced adverse effects. We used isobologram analyses to determine if the combination of a NT brain-penetrant analog, An2-NT(8-13) with morphine results in an inhibitory, synergistic or additive analgesic response. We found that intravenous administration of An2-NT(8-13) reduced by 90% the nocifensive behaviors induced by formalin injection, at the dose of 0.018â¯mg/kg. Likewise, subcutaneous morphine reduced pain by 90% at 1.8â¯mg/kg. Importantly, isobologram analyses revealed that the co-injection of An2-NT(8-13) with morphine induced an additive analgesic response. We finally assessed the effects of morphine and An2-NT(8-13) on the gastrointestinal tract motility using the charcoal meal test. As opposed to morphine which significantly reduced the intestinal motility at the analgesic effective dose of 1.8â¯mg/kg, An2-NT(8-13) did not affect the charcoal meal intestinal transit at 0.018â¯mg/kg. Interestingly, at the dose providing 90% pain relief, the co-administration of morphine with An2-NT(8-13) had a reduced effect on constipation. Altogether, these results suggest that combining NT agonists with morphine may improve its analgesic/adverse effect ratio.
Asunto(s)
Analgésicos Opioides/administración & dosificación , Neurotensina/administración & dosificación , Dimensión del Dolor/efectos de los fármacos , Fragmentos de Péptidos/administración & dosificación , Péptidos/administración & dosificación , Receptores de Neurotensina/agonistas , Receptores Opioides mu/agonistas , Animales , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Masculino , Morfina/administración & dosificación , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-Dawley , Receptores de Neurotensina/metabolismo , Receptores Opioides mu/metabolismoRESUMEN
Several studies have suggested that neurotensin receptors (NTRs) and neurotensin (NT) greatly affect the growth and survival of pancreatic ductal adenocarcinoma (PDAC). Developing NTR-targeted PET probes could therefore be important for the management of a pancreatic cancer patient by providing key information on the NTR expression profile noninvasively. Despite the initial success on the synthesis of 18F-labeled NT PET probes, the labeling procedure generally requires lengthy steps including azeotropic drying of 18F. Using a straightforward chelation method, here we report the simple preparation of aluminum-18F-NOTA-NT starting from aqueous 18F. The cell binding test demonstrated that [19F]AlF-NOTA-NT maintained high receptor-binding affinity to NTR1. This probe was then further evaluated in NTR1 positive pancreatic tumor models (AsPC-1 and PANC-1). After the administration of [18F]AlF-NOTA-NT, small animal PET studies showed a high contrast between tumor and background in both models at 1 and 4 h time points. A blocking experiment was performed to demonstrate the receptor specificity: the tumor uptake in AsPC1 without and with blocking agent was 1.0 ± 0.2 and 0.1 ± 0.0%ID/g, respectively, at 4 h post injection. In summary, a NTR specific PET agent, [18F]AlF-NOTA-NT, was prepared through the simple chelation method. This NTR-targeted PET probe may not only be used to detect NTR1 positive pancreatic tumors (diagnosis), but also it may be fully integrated to NTR target therapy leading to personalized medicine (theranostic).
Asunto(s)
Carcinoma Ductal Pancreático/diagnóstico por imagen , Neoplasias Pancreáticas/diagnóstico , Radiofármacos/administración & dosificación , Receptores de Neurotensina/metabolismo , Aluminio/administración & dosificación , Aluminio/química , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Flúor/administración & dosificación , Flúor/química , Radioisótopos de Flúor/administración & dosificación , Radioisótopos de Flúor/química , Compuestos Heterocíclicos con 1 Anillo/administración & dosificación , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Ratones , Ratones Desnudos , Imagen Molecular/métodos , Terapia Molecular Dirigida/métodos , Neurotensina/administración & dosificación , Neurotensina/química , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Tomografía de Emisión de Positrones/métodos , Radiofármacos/química , Receptores de Neurotensina/antagonistas & inhibidores , Nanomedicina Teranóstica/métodos , Microtomografía por Rayos X/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Silk fibroin (SF) films containing a peptide, neurotensin (NT), stimulated by iontophoresis were developed aiming to modulate the inflammatory process and prevent the growth of microorganisms typical of wounds. NT-loaded SF films composition shows predominance of ß-sheet structures that conferred adequate mechanical properties, transparency, moderate roughness and low swelling index to fibroin films. Infrared spectroscopy and thermal analysis suggested the presence of non-covalent interactions between NT and fibroin. Using the MALDI imaging technique, it was possible to visualize the homogeneous NT distribution throughout the film surface, in addition to its prolonged release for up to 72â¯h. In vitro studies in E. coli liposaccharide-stimulated macrophages showed a significant reduction of interleukins production after NT-loaded film application, whereas NT solution did not reduce them. Bi-laminated NT-loaded fibroin films containing silver electrodes provided a burst release of NT when anodic iontophoresis was applied, enabling a rapid onset of drug action. In addition, anodic iontophoresis presented a bacteriostatic effect against gram-positive microorganisms. Different iontophoresis densities, from 0.2 to 0.6â¯mA/cm2, did not significantly reduce fibroblast viability after 30â¯min of application. In conclusion, iontophoretic-stimulated peptide-loaded fibroin films could be a promising platform for the treatment of wounds.
Asunto(s)
Antibacterianos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Fibroínas/química , Neurotensina/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Animales , Antibacterianos/química , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Bombyx/química , Supervivencia Celular , Fibroblastos , Interleucinas/metabolismo , Iontoforesis/métodos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Pruebas de Sensibilidad Microbiana , Neurotensina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Tridecapeptide Neurotensin (NT) is widely distributed in the central nervous system where it acts as a neurotransmitter and neuromodulator. The central nucleus of amygdala (CeA), part of the limbic system, plays an important role in learning, memory, anxiety and reinforcing mechanisms. Our previous data showed that NT microinjected into the CeA has positive reinforcing properties. We supposed that these effects might be due to modulations of the mesolimbic dopamine system. The aim of our study was to examine in the CeA the possible effects of NT and dopamine interaction on reinforcement by conditioned place preference test. Male Wistar rats were microinjected bilaterally with 100â¯ng NT or 2⯵g D1 dopamine receptor antagonist alone, or D1 dopamine antagonist 15â¯min before 100â¯ng NT treatment or vehicle solution into the CeA. Other animals received 4⯵g D2 dopamine receptor antagonist Sulpiride alone, or administration of D2 dopamine receptor antagonist 15â¯min before 100â¯ng NT treatment or vehicle solution into the CeA. Rats that received 100â¯ng NT spent significantly more time in the treatment quadrant during the test session. Pre-treatment with the D1 dopamine antagonist, blocked the effects of NT. D2 dopamine receptor antagonist pretreatment could prevent the positive reinforcing effects of NT as well. Antagonists themselves did not influence the place preference. Our results show that the rewarding effect of NT can be due to the modulation of DA system, since its effects could be blocked by either D1 dopamine or D2 dopamine antagonist preteatment.
Asunto(s)
Núcleo Amigdalino Central/metabolismo , Condicionamiento Clásico/fisiología , Dopamina/metabolismo , Neurotensina/metabolismo , Conducta Espacial/fisiología , Animales , Benzazepinas/farmacología , Núcleo Amigdalino Central/efectos de los fármacos , Condicionamiento Clásico/efectos de los fármacos , Masculino , Microinyecciones , Neurotensina/administración & dosificación , Neurotransmisores/farmacología , Ratas Wistar , Receptores de Dopamina D1/antagonistas & inhibidores , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Conducta Espacial/efectos de los fármacos , Sulpirida/farmacologíaRESUMEN
We previously demonstrated that infusion of an intestinal peptide called xenin-25 (Xen) amplifies the effects of glucose-dependent insulinotropic polypeptide (GIP) on insulin secretion rates (ISRs) and plasma glucagon levels in humans. However, these effects of Xen, but not GIP, were blunted in humans with type 2 diabetes. Thus, Xen rather than GIP signaling to islets fails early during development of type 2 diabetes. The current crossover study determines if cholinergic signaling relays the effects of Xen on insulin and glucagon release in humans as in mice. Fasted subjects with impaired glucose tolerance were studied. On eight separate occasions, each person underwent a single graded glucose infusion- two each with infusion of albumin, Xen, GIP, and GIP plus Xen. Each infusate was administered ± atropine. Heart rate and plasma glucose, insulin, C-peptide, glucagon, and pancreatic polypeptide (PP) levels were measured. ISRs were calculated from C-peptide levels. All peptides profoundly increased PP responses. From 0 to 40 min, peptide(s) infusions had little effect on plasma glucose concentrations. However, GIP, but not Xen, rapidly and transiently increased ISRs and glucagon levels. Both responses were further amplified when Xen was co-administered with GIP. From 40 to 240 min, glucose levels and ISRs continually increased while glucagon concentrations declined, regardless of infusate. Atropine increased resting heart rate and blocked all PP responses but did not affect ISRs or plasma glucagon levels during any of the peptide infusions. Thus, cholinergic signaling mediates the effects of Xen on insulin and glucagon release in mice but not humans.
Asunto(s)
Glucagón/metabolismo , Intolerancia a la Glucosa/sangre , Insulina/metabolismo , Neurotensina/farmacología , Polipéptido Pancreático/metabolismo , Receptores Colinérgicos/metabolismo , Transducción de Señal , Adulto , Atropina/administración & dosificación , Atropina/farmacología , Glucemia/metabolismo , Estudios Cruzados , Femenino , Polipéptido Inhibidor Gástrico/administración & dosificación , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Secreción de Insulina , Masculino , Persona de Mediana Edad , Neurotensina/administración & dosificaciónRESUMEN
Combined modulation of peptide hormone receptors including, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and xenin, have established benefits for the treatment of diabetes. The present study has assessed the biological actions and therapeutic efficacy of a novel exendin-4/xenin-8-Gln hybrid peptide, both alone and in combination with the GIP receptor agonist (DAla2)GIP. Exendin-4/xenin-8-Gln was enzymatically stable and exhibited enhanced insulin secretory actions when compared to its parent peptides. Exendin-4/xenin-8-Gln also possessed ability to potentiate the in vitro actions of GIP. Acute administration of exendin-4/xenin-8-Gln in mice induced appetite suppressive effects, as well as significant and protracted glucose-lowering and insulin secretory actions. Twice daily administration of exendin-4/xenin-8-Gln, alone or in combination with (DAla2)GIP, for 21-days significantly reduced non-fasting glucose and increased circulating insulin levels in high fat fed mice. In addition, all exendin-4/xenin-8-Gln treated mice displayed improved glucose tolerance, insulin sensitivity and metabolic responses to GIP. Combination therapy with (DAla2)GIP did not result in any obvious further benefits. Metabolic improvements in all treatment groups were accompanied by reduced pancreatic beta-cell area and insulin content, suggesting reduced insulin demand. Interestingly, body weight, food intake, circulating glucagon, metabolic rate and amylase activity were unaltered by the treatment regimens. However, all treatment groups, barring (DAla2)GIP alone, exhibited marked reductions in total- and LDL-cholesterol. Furthermore, exendin-4 therapy also reduced circulating triacylglycerol. This study highlights the positive antidiabetic effects of exendin-4/xenin-8-Gln, and suggests that combined modulation of GLP-1 and xenin related signalling pathways represents an exciting treatment option for type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Polipéptido Inhibidor Gástrico/administración & dosificación , Péptido 1 Similar al Glucagón/administración & dosificación , Hipoglucemiantes/administración & dosificación , Animales , Glucemia/efectos de los fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Combinación de Medicamentos , Exenatida/administración & dosificación , Exenatida/química , Polipéptido Inhibidor Gástrico/química , Glucagón/química , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/química , Glucosa/metabolismo , Humanos , Hipoglucemiantes/química , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Neurotensina/administración & dosificación , Neurotensina/químicaRESUMEN
Methamphetamine (METH) is a psychostimulant that exhibits significant abuse potential. Although METH addiction is a major health and societal concern, no drug is currently approved for its therapeutic management. METH activates the central dopaminergic "reward" circuitry, and with repeated use increases levels of the neuromodulatory peptide neurotensin in the nucleus accumbens and ventral tegmental area. Previous studies in rats suggest that neurotensin agonism decreases METH self-administration, but these studies did not examine the effect of neurotensin agonism on the pattern of self-administration or open field locomotion. In our studies, we established intravenous METH self-administration in male, DBA/2J mice (fixed ratio 3, 2 hr sessions) and examined the effect of pretreatment with the NTS1 receptor agonist PD149163 on METH self-administration behavior. Locomotion following PD149163 was also measured up to 2 hours after injection on a rotarod and in an open field. Pretreatment with PD149163 (0.05 and 0.10 mg/kg, s.c.) significantly decreased METH self-administration. The pattern of responding suggested that PD149163 decreased motivation to self-administer METH initially in the session with more normal intake in the second hour of access. Voluntary movement in the open-field was significantly decreased by both 0.05 and 0.10 mg/kg (s.c.) PD149163 from 10-120 minutes after injection, but rotarod performance suggested that PD149163 did not cause frank sedation. These results suggest that a systemically delivered NTS1 receptor agonist decreases METH self-administration in mice. The pattern of self-administration suggests that PD149163 may acutely decrease motivation to self-administer METH before the drug is experienced, but cannot rule out that depression of voluntary movement plays a role in the decreased self-administration.
Asunto(s)
Metanfetamina/toxicidad , Neurotensina/análogos & derivados , Neurotensina/metabolismo , Autoadministración/métodos , Trastornos Relacionados con Sustancias/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Dopamina/metabolismo , Humanos , Ratones , Ratones Endogámicos DBA , Neurotensina/administración & dosificación , Receptores de Neurotensina/metabolismo , Trastornos Relacionados con Sustancias/fisiopatologíaRESUMEN
Long-term exposure to antipsychotics like haloperidol can increase sensitivity to dopamine agonist stimulation. This could contribute to treatment failure and increase relapse to psychosis. Chronic antipsychotic treatment elevates neurotensin levels in the nucleus accumbens (NAc), where the neuropeptide modulates dopamine function by signalling through NTS1 receptors. We hypothesized that increasing neurotensin activity in the NAc attenuates the expression of antipsychotic-induced dopamine supersensitivity, which is indicated by a potentiated psychomotor response to amphetamine. Rats received either continuous (CONT-HAL; achieved via subcutaneous osmotic minipump) or intermittent (INT-HAL; achieved via daily subcutaneous injection) haloperidol treatment for 16-17 days. Three to 5 days later, we injected neurotensin into the NAc and measured amphetamine-induced locomotion. Only CONT-HAL rats showed potentiated amphetamine-induced locomotion, indicating dopamine supersensitivity. Compared to intra-NAc saline, intra-NAc neurotensin suppressed amphetamine-induced locomotion in CONT-HAL rats, but not in INT-HAL or control rats. In a new cohort of CONT-HAL and INT-HAL rats, we measured striatal levels of proneurotensin mRNA and NTS1 receptors. The two treatments led to overlapping but also distinct neurochemical profiles. Neither treatment altered NTS1 receptor levels in the NAc, but both increased proneurotensin mRNA levels in the NAc core. In the caudate-putamen, only INT-HAL increased NTS1 receptor levels, while only CONT-HAL increased proneurotensin mRNA expression. Thus, antipsychotic-induced dopamine supersensitivity enhances the ability of neurotensin in the NAc to regulate dopamine-mediated behaviours, and this likely does not involve changes in local levels of NTS1 receptors or proneurotensin mRNA. We conclude that increasing neurotensin activity could be considered to attenuate antipsychotic-induced dopamine supersensitivity.
Asunto(s)
Antipsicóticos/administración & dosificación , Haloperidol/administración & dosificación , Neurotensina/metabolismo , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Anfetamina/farmacología , Animales , Núcleo Caudado/efectos de los fármacos , Núcleo Caudado/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , Dopamina/metabolismo , Masculino , Movimiento/efectos de los fármacos , Movimiento/fisiología , Neurotensina/administración & dosificación , Putamen/efectos de los fármacos , Putamen/metabolismo , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Receptores de Neurotensina/metabolismoRESUMEN
We examined the impact of crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling pathways on the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in the context of human pancreatic ductal adenocarcinoma (PDAC). Stimulation of PANC-1 or MiaPaCa-2 cells with insulin and neurotensin, a potent mitogenic combination of agonists for these cells, promoted striking YAP nuclear localization and decreased YAP phosphorylation at Ser127 and Ser397 Challenging PDAC cells with either insulin or neurotensin alone modestly induced the expression of YAP/TEAD-regulated genes, including connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), and CXCL5, whereas the combination of neurotensin and insulin induced a marked increase in the level of expression of these genes. In addition, siRNA-mediated knockdown of YAP/TAZ prevented the increase in the expression of these genes. A small-molecule inhibitor (A66), selective for the p110α subunit of PI3K, abrogated the increase in phosphatidylinositol 3,4,5-trisphosphate production and the expression of CTGF, CYR61, and CXCL5 induced by neurotensin and insulin. Furthermore, treatment of PDAC cells with protein kinase D (PKD) family inhibitors (CRT0066101 or kb NB 142-70) or with siRNAs targeting the PKD family prevented the increase of CTGF, CYR61, and CXCL5 mRNA levels in response to insulin and neurotensin stimulation. Thus, PI3K and PKD mediate YAP activation in response to insulin and neurotensin in pancreatic cancer cells.Implications: Inhibitors of PI3K or PKD disrupt crosstalk between insulin receptor and GPCR signaling systems by blocking YAP/TEAD-regulated gene expression in pancreatic cancer cells. Mol Cancer Res; 15(7); 929-41. ©2017 AACR.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma Ductal Pancreático/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Neoplasias Pancreáticas/genética , Fosfoproteínas/genética , Proteína Quinasa C/genética , Antígenos CD/genética , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Quimiocina CXCL5/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Proteína 61 Rica en Cisteína/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/administración & dosificación , Insulina/metabolismo , Neurotensina/administración & dosificación , Neurotensina/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Pirimidinas/administración & dosificación , Receptor de Insulina/genética , Receptores Acoplados a Proteínas G/genética , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Characterization of in vitro and in vivo catabolism of therapeutic proteins has increasingly become an integral part of discovery and development process for novel proteins. Unambiguous and efficient identification of catabolites can not only facilitate accurate understanding of pharmacokinetic profiles of drug candidates, but also enables follow up protein engineering to generate more catabolically stable molecules with improved properties (pharmacokinetics and pharmacodynamics). Immunoaffinity capture (IC) followed by top-down intact protein analysis using either matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry analysis have been the primary methods of choice for catabolite identification. However, the sensitivity and efficiency of these methods is not always sufficient for characterization of novel proteins from complex biomatrices such as plasma or serum. In this study a novel bottom-up targeted protein workflow was optimized for analysis of proteolytic degradation of therapeutic proteins. Selective and sensitive tagging of the alpha-amine at the N-terminus of proteins of interest was performed by immunoaffinity capture of therapeutic protein and its catabolites followed by on-bead succinimidyloxycarbonylmethyl tri-(2,4,6-trimethoxyphenyl N-terminus (TMPP-NTT) tagging. The positively charged hydrophobic TMPP tag facilitates unambiguous sequence identification of all N-terminus peptides from complex tryptic digestion samples via data dependent liquid chromatgraphy-tandem mass spectroscopy. Utility of the workflow was illustrated by definitive analysis of in vitro catabolic profile of neurotensin human Fc (NTs-huFc) protein in mouse serum. The results from this study demonstrated that the IC-TMPP-NTT workflow is a simple and efficient method for catabolite formation in therapeutic proteins.
Asunto(s)
Neurotensina/sangre , Compuestos Onio/química , Compuestos Organofosforados/química , Fragmentos de Péptidos/sangre , Coloración y Etiquetado/métodos , Secuencia de Aminoácidos , Animales , Biotransformación , Cromatografía Liquida , Humanos , Técnicas de Inmunoadsorción , Ratones , Neurotensina/administración & dosificación , Fragmentos de Péptidos/química , Electricidad Estática , Espectrometría de Masas en Tándem , Tripsina/químicaRESUMEN
Previous studies have shown that activation of ventral midbrain NMDA receptors is required to initiate sensitization by amphetamine. In view of the recent evidence that neurotensin modulates ventral midbrain glutamate neurotransmission, we tested the hypothesis that neurotensin is acting upstream to glutamate to initiate sensitization to the behavioral and neurochemical effects of amphetamine. During a first testing phase, adult male rats implanted with bilateral ventral midbrain cannulae were injected every second day for three days with D-[Tyr11]neurotensin (1.5 nmol/side), the preferred NMDA GluN2A/B antagonist, CPP (40 or 120 pmol/side), the selective GluN2B antagonist, Ro04-5595 (200 or 1200 pmol/side), CPP (40 or 120 pmol/side) + D-[Tyr11]neurotensin (1.5 nmol/side) or Ro04-5595 (200 or 1200 pmol/side) + D-[Tyr11]neurotensin (1.5 nmol/side) and locomotor activity was measured immediately after the injection. Five days after the last central injection, the locomotor response or the expression of phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) in neurons of different limbic nuclei was measured following a systemic injection of amphetamine sulfate (0.75 mg/kg, i.p.). Results show that amphetamine induced significantly stronger locomotor activity and pERK1/2 expression in the nucleus accumbens shell and infralimbic cortex in neurotensin pre-exposed animals than in controls (vehicle pre-exposed). These sensitization effects initiated by neurotensin were prevented by CPP, but not Ro04-5595. These results support the hypothesis that neurotensin is stimulating glutamate neurotransmission to initiate neural changes that sub-serve amphetamine sensitization and that glutamate is acting on NMDA receptors that are mostly likely composed of GluN2A, but not GluN2B, subunits. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.
Asunto(s)
Anfetamina/administración & dosificación , Mesencéfalo/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Actividad Motora/efectos de los fármacos , Neurotensina/administración & dosificación , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/fisiología , Neuronas/metabolismo , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Ratas , Ratas Long-Evans , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
Delivery efficiency with gene transfection is a pivotal point in achieving maximized therapeutic efficacy and has been an important challenge with central nervous system (CNS) diseases. In this study, neurotensin (NT, a neuro-specific peptide)-conjugated polyethylenimine (PEI)-modified reduced graphene oxide (rGO) nanoparticles with precisely controlled two-stage near-infrared (NIR)-laser photothermal treatment to enhance the ability to target neurons and achieve high gene transfection in neurons. First-stage NIR laser irradiation on the cells with nanoparticles attached on the surface can increase the permeability of the cell membrane, resulting in an apparent increase in cellular uptake compared to untreated cells. In addition, second-stage NIR laser irradiation on the cells with nanoparticles inside can further induce endo/lysosomal cavitation, which not only helps nanoparticles escape from endo/lysosomes but also prevents plasmid DNA (pDNA) from being digested by DNase I. At least double pDNA amount can be released from rGO-PEI-NT/pDNA under NIR laser trigger release compared to natural release. Moreover, in vitro differentiated PC-12 cell and in vivo mice (C57BL/6) brain transfection experiments have demonstrated the highest transfection efficiency occurring when NT modification is combined with external multi-stage stimuli-responsive NIR laser treatment. The combination of neuro-specific targeting peptide and external NIR-laser-triggered aid provides a nanoplatform for gene therapy in CNS diseases.
Asunto(s)
Grafito/administración & dosificación , Enfermedades Neurodegenerativas/tratamiento farmacológico , Neuronas/efectos de los fármacos , Neurotensina/administración & dosificación , Óxidos/administración & dosificación , Animales , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Grafito/química , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Nanopartículas/química , Neurotensina/química , Óxidos/química , Plásmidos/metabolismo , Polietileneimina/química , Ratas , Espectroscopía Infrarroja Corta/métodos , Transfección/métodosRESUMEN
Neurotensin (NT) is a peptide expressed in the brain and in the gastrointestinal tract. Brain NT inhibits food intake, but the effects of peripheral NT are less investigated. In this study, peripheral NT decreased food intake in both mice and rats, which was abolished by a NT antagonist. Using c-Fos immunohistochemistry, we found that peripheral NT activated brainstem and hypothalamic regions. The anorexigenic effect of NT was preserved in vagotomized mice but lasted shorter than in sham-operated mice. This in combination with a strong increase in c-Fos activation in area postrema after ip administration indicates that NT acts both through the blood circulation and the vagus. To improve the pharmacokinetics of NT, we developed a pegylated NT peptide, which presumably prolonged the half-life, and thus, the effect on feeding was extended compared with native NT. On a molecular level, the pegylated NT peptide increased proopiomelanocortin mRNA in the arcuate nucleus. We also investigated the importance of NT for the decreased food intake after gastric bypass surgery in a rat model of Roux-en-Y gastric bypass (RYGB). NT was increased in plasma and in the gastrointestinal tract in RYGB rats, and pharmacological antagonism of NT increased food intake transiently in RYGB rats. Taken together, our data suggest that NT is a metabolically active hormone, which contributes to the regulation of food intake.
Asunto(s)
Regulación del Apetito/efectos de los fármacos , Derivación Gástrica , Neurotensina/administración & dosificación , Animales , Ingestión de Alimentos/efectos de los fármacos , Femenino , Masculino , Ratones Endogámicos C57BL , Neurotensina/antagonistas & inhibidores , Neurotensina/sangre , Ratas Sprague-Dawley , Sacarosa , VagotomíaRESUMEN
Xenin is a gut hormone that reduces food intake by partly acting through the hypothalamus via neurotensin receptor 1 (Ntsr1). However, specific signaling pathways that mediate xenin-induced feeding suppression are not fully understood. Activation of Ntsr1 leads to the activation of the extracellular signal-regulated kinase (ERK). Hypothalamic ERK participates in the regulation of food intake by mediating the effect of hormonal signals. Therefore, we hypothesized that the anorectic effect of xenin is mediated by hypothalamic ERK signaling. To address this hypothesis, we compared levels of phosphorylation of ERK1/2 (pERK1/2) in the hypothalamus of both control and xenin-treated mice. The effect of xenin on ERK1/2 phosphorylation was also examined in mouse hypothalamic neuronal cell lines with or without Ntsr1. We also examined the effect of the blockade of central ERK signaling on xenin-induced feeding suppression in mice. The intraperitoneal (i.p.) injection of xenin caused a significant increase in the number of pERK1/2-immunoreactive cells in the hypothalamic arcuate nucleus. The majority of pERK1/2-positive cells expressed neuronal nuclei (NeuN), a marker for neurons. Xenin treatment increased pERK1/2 levels in one cell line expressing Ntsr1 but not another line without Ntsr1 expression. Both i.p. and intracerebroventricular (i.c.v.) injections of xenin reduced food intake in mice. The i.c.v. pre-treatment with U0126, a selective inhibitor of ERK1/2 upstream kinases, did not affect xenin-induced reduction in food intake. These findings suggest that although xenin activates ERK signaling in subpopulations of hypothalamic neurons, xenin does not require the activation of hypothalamic ERK signaling pathway to elicit feeding suppression.
Asunto(s)
Ingestión de Alimentos , Hipotálamo/metabolismo , Sistema de Señalización de MAP Quinasas , Neurotensina/metabolismo , Animales , Línea Celular , Ingestión de Alimentos/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotensina/administración & dosificación , FosforilaciónRESUMEN
Xenin-25 (Xen) is a neurotensin-related peptide secreted by a subset of enteroendocrine cells located in the proximal small intestine. Many effects of Xen are mediated by neurotensin receptor-1 on neurons. In healthy humans with normal glucose tolerance (NGT), Xen administration causes diarrhea and inhibits postprandial glucagon-like peptide-1 (GLP-1) release but not insulin secretion. This study determines (i) if Xen has similar effects in humans with Roux-en-Y gastric bypass (RYGB) and (ii) whether neural pathways potentially mediate effects of Xen on glucose homeostasis. Eight females with RYGB and no history of type 2 diabetes received infusions with 0, 4 or 12pmol Xen/kg/min with liquid meals on separate occasions. Plasma glucose and gastrointestinal hormone levels were measured and insulin secretion rates calculated. Pancreatic polypeptide and neuropeptide Y levels were surrogate markers for parasympathetic input to islets and sympathetic tone, respectively. Responses were compared to those in well-matched non-surgical participants with NGT from our earlier study. Xen similarly increased pancreatic polypeptide and neuropeptide Y responses in patients with and without RYGB. In contrast, the ability of Xen to inhibit GLP-1 release and cause diarrhea was severely blunted in patients with RYGB. With RYGB, Xen had no statistically significant effect on glucose, insulin secretory, GLP-1, glucose-dependent insulinotropic peptide, and glucagon responses. However, insulin and glucose-dependent insulinotropic peptide secretion preceded GLP-1 release suggesting circulating GLP-1 does not mediate exaggerated insulin release after RYGB. Thus, Xen has unmasked neural circuits to the distal gut that inhibit GLP-1 secretion, cause diarrhea, and are altered by RYGB.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diarrea/metabolismo , Insulina/metabolismo , Neurotensina/administración & dosificación , Adolescente , Adulto , Anciano , Glucemia , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/patología , Diarrea/inducido químicamente , Diarrea/fisiopatología , Femenino , Derivación Gástrica/métodos , Polipéptido Inhibidor Gástrico/metabolismo , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Humanos , Secreción de Insulina , Masculino , Persona de Mediana Edad , Neuropéptido Y/metabolismo , Neurotensina/efectos adversos , Neurotensina/metabolismo , Polipéptido Pancreático/metabolismoRESUMEN
UNLABELLED: Peripheral muscarinic acetylcholine receptors regulate insulin and glucagon release in rodents but their importance for similar roles in humans is unclear. Bethanechol, an acetylcholine analogue that does not cross the blood-brain barrier, was used to examine the role of peripheral muscarinic signaling on glucose homeostasis in humans with normal glucose tolerance (NGT; n = 10), impaired glucose tolerance (IGT; n = 11), and type 2 diabetes mellitus (T2DM; n = 9). Subjects received four liquid meal tolerance tests, each with a different dose of oral bethanechol (0, 50, 100, or 150 mg) given 60 min before a meal containing acetaminophen. Plasma pancreatic polypeptide (PP), glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), glucose, glucagon, C-peptide, and acetaminophen concentrations were measured. Insulin secretion rates (ISRs) were calculated from C-peptide levels. Acetaminophen and PP concentrations were surrogate markers for gastric emptying and cholinergic input to islets. The 150 mg dose of bethanechol increased the PP response 2-fold only in the IGT group, amplified GLP-1 release in the IGT and T2DM groups, and augmented the GIP response only in the NGT group. However, bethanechol did not alter ISRs or plasma glucose, glucagon, or acetaminophen concentrations in any group. Prior studies showed infusion of xenin-25, an intestinal peptide, delays gastric emptying and reduces GLP-1 release but not ISRs when normalized to plasma glucose levels. Analysis of archived plasma samples from this study showed xenin-25 amplified postprandial PP responses ~4-fold in subjects with NGT, IGT, and T2DM. Thus, increasing postprandial cholinergic input to islets augments insulin secretion in mice but not humans. TRIAL REGISTRATION: ClinicalTrials.gov NCT01434901.