RESUMO
Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is characterized by intrahepatic triglyceride accumulation and can progress to metabolic dysfunction-associated steatohepatitis (MASH) and liver fibrosis. Hepatic de novo lipogenesis (DNL), activated by glucose and insulin, is a central pathway contributing to early-stage development of MASLD. The emerging global prevalence of MASLD highlights the urgent need for pharmaceutical intervention to combat this health threat. However, the identification of novel drugs that could inhibit hepatic DNL is hampered by a lack of reliable, insulin-sensitive, human, in vitro, hepatic models. Here, we report human skin stem cell-derived hepatic cells (hSKP-HPC) as a unique in vitro model to study insulin-driven DNL (iDNL), evidenced by both gene expression and lipid accumulation readouts. Insulin-sensitive hSKP-HPC showed increased sterol regulatory element-binding protein 1c (SREBP-1c) expression, a key transcription factor for DNL. Furthermore, this physiologically relevant in vitro human steatosis model allowed both inhibition and activation of the iDNL pathway using reference inhibitors and activators, respectively. Optimisation of the lipid accumulation assay to a high-throughput, 384-well format enabled the screening of a library of annotated compounds, delivering new insights on key players in the iDNL pathway and MASLD pathophysiology. Together, these results establish the value of the hSKP-HPC model in preclinical development of antisteatotic drugs to combat MASLD.
Assuntos
Insulina , Hepatopatia Gordurosa não Alcoólica , Humanos , Insulina/metabolismo , Lipogênese/genética , Fígado/metabolismo , Hepatócitos/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Triglicerídeos/metabolismo , Células-Tronco/metabolismoRESUMO
Myelinated fibers are essential for the rapid and efficient propagation of nerve information throughout the body. These fibers result from an intimate crosstalk between myelinating glia and the myelinated axons and, because it is difficult to fully reproduce these interactions in vitro, the basic molecular mechanisms that regulate myelination, demyelination, and remyelination remain unclear. Schwann cells produce myelin in the peripheral nervous system (PNS) and remain associated with the axons of peripheral neurons throughout axonal migration to the target. In order to investigate more closely the biology of myelinated fibers, we developed a local transgenesis approach based on the injection of engineered viral vectors in the sciatic nerve of mice to locally transduce peripheral nerve cells. This approach represents an alternative to germline modifications as it facilitates and speed up the investigation of peripheral nerve biology in vivo. Indeed the protocol we describe here requires just 3 weeks to complete. The injection of engineered viral vectors in the sciatic nerve of mice is a reproducible and straightforward method for introducing exogenous factors into myelinating Schwann cells and myelinated axons in vivo in order to investigate specific molecular mechanisms.
Assuntos
Vetores Genéticos/genética , Células de Schwann/metabolismo , Células de Schwann/virologia , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Transdução Genética , Transgenes , Animais , Expressão Gênica , Camundongos , Regiões Promotoras GenéticasRESUMO
Fast nerve conduction relies on successive myelin segments that electrically isolate axons. Segment geometry-diameter and length-is critical for the optimization of nerve conduction and the molecular mechanisms allowing this optimized geometry are partially known. We show here that peripheral myelin elongation is dynamically regulated by stimulation of YAP (Yes-associated protein) transcription cofactor activity during axonal elongation and limited by inhibition of YAP activity via the Hippo pathway. YAP promotes myelin and non-myelin genes transcription while the polarity protein Crb3, localized at the tips of the myelin sheath, activates the Hippo pathway to temper YAP activity, therefore allowing for optimal myelin growth. Dystrophic Dy(2j/2j) mice mimicking human peripheral neuropathy with reduced internodal lengths have decreased nuclear YAP which, when corrected, leads to longer internodes. These data show a novel mechanism controlling myelin growth and nerve conduction, and provide a molecular ground for disease with short myelin segments.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Axônios/metabolismo , Proteínas de Membrana/metabolismo , Bainha de Mielina/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Axônios/ultraestrutura , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Células HEK293 , Via de Sinalização Hippo , Humanos , Glicoproteínas de Membrana , Camundongos , Fenótipo , Fosforilação , Ratos , Células de Schwann/metabolismo , Transcrição Gênica , Proteínas de Sinalização YAPRESUMO
SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.
Assuntos
Alternativas aos Testes com Animais/métodos , Testes de Toxicidade/métodos , Alternativas aos Testes com Animais/legislação & jurisprudência , Alternativas aos Testes com Animais/organização & administração , Animais , Biomarcadores/análise , Células Cultivadas , Qualidade de Produtos para o Consumidor , União Europeia , Regulamentação Governamental , Ensaios de Triagem em Larga Escala , Humanos , Técnicas In VitroRESUMO
Schwann cells produce myelin sheath around peripheral nerve axons. Myelination is critical for rapid propagation of action potentials, as illustrated by the large number of acquired and hereditary peripheral neuropathies, such as diabetic neuropathy or Charcot-Marie-Tooth diseases, that are commonly associated with a process of demyelination. However, the early molecular events that trigger the demyelination program in these diseases remain unknown. Here, we used virally delivered fluorescent probes and in vivo time-lapse imaging in a mouse model of demyelination to investigate the underlying mechanisms of the demyelination process. We demonstrated that mitochondrial calcium released by voltage-dependent anion channel 1 (VDAC1) after sciatic nerve injury triggers Schwann cell demyelination via ERK1/2, p38, JNK, and c-JUN activation. In diabetic mice, VDAC1 activity was altered, resulting in a mitochondrial calcium leak in Schwann cell cytoplasm, thereby priming the cell for demyelination. Moreover, reduction of mitochondrial calcium release, either by shRNA-mediated VDAC1 silencing or pharmacological inhibition, prevented demyelination, leading to nerve conduction and neuromuscular performance recovery in rodent models of diabetic neuropathy and Charcot-Marie-Tooth diseases. Therefore, this study identifies mitochondria as the early key factor in the molecular mechanism of peripheral demyelination and opens a potential opportunity for the treatment of demyelinating peripheral neuropathies.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio , Colestenonas/farmacologia , Doenças Desmielinizantes/tratamento farmacológico , Mitocôndrias/metabolismo , Células de Schwann/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Doença de Charcot-Marie-Tooth/tratamento farmacológico , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Obesos , Camundongos SCID , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/patologia , Ratos , Células de Schwann/efeitos dos fármacos , Canal de Ânion 1 Dependente de Voltagem/antagonistas & inibidores , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
The myelin sheath that covers a large amount of neurons is critical for their homeostasis, and myelinating glia mitochondria have recently been shown to be essential for neuron survival. However morphological and physiological properties of these organelles remain elusive. Here we report a method to analyze mitochondrial dynamics and morphology in myelinating Schwann cells of living mice using viral transduction and time-lapse multiphoton microscopy. We describe the distribution, shape, size and dynamics of mitochondria in live cells. We also report mitochondrial alterations in Opa1(delTTAG) mutant mice cells at presymptomatic stages, suggesting that mitochondrial defects in myelin contribute to OPA1 related neuropathy and represent a biomarker for the disease.
Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Bainha de Mielina/fisiologia , Bainha de Mielina/ultraestrutura , Células de Schwann/fisiologia , Células de Schwann/ultraestrutura , Animais , GTP Fosfo-Hidrolases/deficiência , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Imagem com Lapso de TempoRESUMO
The myelin sheath is essential for the rapid and efficient propagation of action potentials. However, our understanding of the basic molecular mechanisms that regulate myelination, demyelination and remyelination is limited. Schwann cells produce myelin in the peripheral nervous system and remain associated with the axons of peripheral neurons throughout axonal migration to the target. Owing to the intimate relationship between these cell types it is difficult to fully reproduce their function in vitro. For this reason, we developed an approach based on the injection of an engineered virus into the sciatic nerve of mice to locally transduce peripheral nerve cells. This approach can be used as an alternative to germline transgenesis to facilitate the investigation of peripheral nerve biology in vivo. The detailed protocol, described here, requires 3 weeks to complete. In comparison with genetic modification strategies, this protocol is a fast, reproducible and straightforward method for introducing exogenous factors into myelinating Schwann cells and myelinated axons in vivo to investigate specific molecular mechanisms.
Assuntos
Marcação de Genes/métodos , Células de Schwann/metabolismo , Nervo Isquiático/citologia , Transdução Genética/métodos , Adenoviridae , Animais , Vetores Genéticos/genética , Lentivirus , Camundongos , RNA Interferente Pequeno/genética , Células de Schwann/citologia , Transgenes/genéticaRESUMO
The spatial distributions of lipids, amyloid-beta deposits, markers of neurons and glial cells were imaged, at submicrometer lateral resolution, in brain structures of a mouse model of Alzheimer's disease using a new methodology that combines time-of-flight secondary ion mass spectrometry (ToF-SIMS) and confocal fluorescence microscopy. The technology, which enabled us to simultaneously image the lipid and glial cell distributions in Tg2576 mouse brain structures, revealed micrometer-sized cholesterol accumulations in hippocampal regions undergoing amyloid-beta deposition. Such cholesterol granules were either associated with individual amyloid deposits or spread over entire regions undergoing amyloidogenesis. Subsequent immunohistochemical analysis of the same brain regions showed increased microglial and astrocytic immunoreactivity associated with the amyloid deposits, as expected from previous studies, but did not reveal any particular astrocytic or microglial feature correlated with cholesterol granulation. However, dystrophic neurites as well as presynaptic vesicles presented a distribution similar to that of cholesterol granules in regions undergoing amyloid-beta accumulation, thus indicating that these neuronal endpoints may retain cholesterol in areas with lesions. In conclusion, the present study provides evidence for an altered cholesterol distribution near amyloid deposits that would have been missed by several other lipid analysis methods, and opens for the possibility to study in detail the putative liaison between lipid environment and protein structure and function in Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Colesterol/metabolismo , Neuroglia/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Imunofluorescência/métodos , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Espectrometria de Massa de Íon Secundário/métodosRESUMO
System x(c)- exchanges intracellular glutamate for extracellular cystine, giving it a potential role in intracellular glutathione synthesis and nonvesicular glutamate release. We report that mice lacking the specific xCT subunit of system x(c)- (xCT(-/-)) do not have a lower hippocampal glutathione content, increased oxidative stress or brain atrophy, nor exacerbated spatial reference memory deficits with aging. Together these results indicate that loss of system x(c)- does not induce oxidative stress in vivo. Young xCT(-/-) mice did however display a spatial working memory deficit. Interestingly, we observed significantly lower extracellular hippocampal glutamate concentrations in xCT(-/-) mice compared to wild-type littermates. Moreover, intrahippocampal perfusion with system x(c)- inhibitors lowered extracellular glutamate, whereas the system x(c)- activator N-acetylcysteine elevated extracellular glutamate in the rat hippocampus. This indicates that system x(c)- may be an interesting target for pathologies associated with excessive extracellular glutamate release in the hippocampus. Correspondingly, xCT deletion in mice elevated the threshold for limbic seizures and abolished the proconvulsive effects of N-acetylcysteine. These novel findings sustain that system x(c)-) is an important source of extracellular glutamate in the hippocampus. System x(c)(-) is required for optimal spatial working memory, but its inactivation is clearly beneficial to decrease susceptibility for limbic epileptic seizures.
Assuntos
Sistema y+ de Transporte de Aminoácidos/fisiologia , Espaço Extracelular/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/fisiologia , Sistema Límbico/fisiologia , Memória de Curto Prazo/fisiologia , Estresse Oxidativo/fisiologia , Convulsões/fisiopatologia , Percepção Espacial/fisiologia , Animais , Western Blotting , Encéfalo/anatomia & histologia , DNA/genética , Eletroencefalografia , Genótipo , Glutationa/metabolismo , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdiálise , Atividade Motora/fisiologia , Neuroglia/fisiologia , Desempenho Psicomotor/fisiologia , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/induzido quimicamente , Convulsões/genéticaRESUMO
Two structurally distinct peptides, angiotensin IV and LVV-haemorphin 7, both competitive high-affinity inhibitors of insulin-regulated aminopeptidase (IRAP), were found to enhance aversion-associated and spatial memory in normal rats and to improve performance in a number of memory tasks in rat deficits models. These findings provide compelling support for the development of specific, high-affinity inhibitors of the enzyme as new cognitive enhancing agents. Different classes of IRAP inhibitors have been developed including peptidomimetics and small molecular weight compounds identified through in silico screening with a homology model of the catalytic domain of IRAP. The proof of principal that inhibition of IRAP activity results in facilitation of memory has been obtained by the demonstration that the small-molecule IRAP inhibitors also exhibit memory-enhancing properties.
Assuntos
Cognição/efeitos dos fármacos , Cistinil Aminopeptidase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nootrópicos/farmacologia , Animais , Cistinil Aminopeptidase/metabolismo , Inibidores Enzimáticos/química , Humanos , Nootrópicos/químicaRESUMO
Adult neural stem cell proliferation is dynamic and has the potential for massive self-renewal yet undergoes limited cell division in vivo. Here, we report an epigenetic mechanism regulating proliferation and self-renewal. The recruitment of the PI3K-related kinase signaling pathway and histone H2AX phosphorylation following GABA(A) receptor activation limits subventricular zone proliferation. As a result, NSC self-renewal and niche size is dynamic and can be directly modulated in both directions pharmacologically or by genetically targeting H2AX activation. Surprisingly, changes in proliferation have long-lasting consequences on stem cell numbers, niche size, and neuronal output. These results establish a mechanism that continuously limits proliferation and demonstrates its impact on adult neurogenesis. Such homeostatic suppression of NSC proliferation may contribute to the limited self-repair capacity of the damaged brain.
Assuntos
Células-Tronco Adultas/fisiologia , Ciclo Celular/fisiologia , Proliferação de Células , Reparo do DNA/fisiologia , Epigênese Genética/fisiologia , Histonas/metabolismo , Células-Tronco Neurais/fisiologia , Transdução de Sinais/fisiologia , Animais , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Estatísticas não ParamétricasRESUMO
The physiological importance of the insulin responsive glucose transporter GLUT4 in adipocytes and muscle in maintaining glucose homeostasis is well established. A key protein associated with this process is the aminopeptidase IRAP which co-localizes with GLUT4 in specialized vesicles, where it plays a tethering role. In this study, we investigated the distribution of both GLUT4 and IRAP in the kidney to gain insights into the potential roles of these proteins in this organ. Both IRAP and GLUT4 immunostaining was observed in the epithelial cells of the proximal and distal tubules and thick ascending limbs in the cortex, but very little overlap between GLUT4 and IRAP immunoreactivity was observed. GLUT4 staining was consistent with a vesicular localization, whereas IRAP staining was predominantly on the luminal surface. In the principal cells of the inner medulla collecting duct (IMCD), IRAP immunoreactivity was detected throughout the cell, with limited overlap with the vasopressin responsive water channel aquaporin-2 (AQP-2). AQP-2 levels were observed to be two-fold higher in IRAP knockout mice. Based on our results, we propose that GLUT4 plays a role in shunting glucose across epithelial cells. In the kidney cortex, IRAP, in concert with other peptidases, may be important in the generation of free amino acids for uptake, whereas in the principal cells of the inner medulla IRAP may play a localized role in the regulation of vasopressin bioactivity.
Assuntos
Cistinil Aminopeptidase/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Rim/metabolismo , Animais , Aquaporina 2/metabolismo , Córtex Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Sprague-DawleyRESUMO
Malfunctioning of system x(c)(-), responsible for exchanging intracellular glutamate for extracellular cystine, can cause oxidative stress and excitotoxicity, both important phenomena in the pathogenesis of Parkinson's disease (PD). We used mice lacking xCT (xCT(-/-) mice), the specific subunit of system x(c)(-), to investigate the involvement of this antiporter in PD. Although cystine that is imported via system x(c)(-) is reduced to cysteine, the rate-limiting substrate in the synthesis of glutathione, deletion of xCT did not result in decreased glutathione levels in striatum. Accordingly, no signs of increased oxidative stress could be observed in striatum or substantia nigra of xCT(-/-) mice. In sharp contrast to expectations, xCT(-/-) mice were less susceptible to 6-hydroxydopamine (6-OHDA)-induced neurodegeneration in the substantia nigra pars compacta compared to their age-matched wild-type littermates. This reduced sensitivity to a PD-inducing toxin might be related to the decrease of 70% in striatal extracellular glutamate levels that was observed in mice lacking xCT. The current data point toward system x(c)(-) as a possible target for the development of new pharmacotherapies for the treatment of PD and emphasize the need to continue the search for specific ligands for system x(c)(-).
Assuntos
Sistema y+ de Transporte de Aminoácidos/deficiência , Dopamina/fisiologia , Neurônios/efeitos dos fármacos , Oxidopamina/toxicidade , Envelhecimento/fisiologia , Sistema y+ de Transporte de Aminoácidos/fisiologia , Animais , Corpo Estriado/metabolismo , Cistina/metabolismo , Glioma/metabolismo , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Camundongos , Camundongos Knockout , Doenças Neurodegenerativas/prevenção & controle , Neurônios/metabolismo , Substância Negra/metabolismo , Células Tumorais CultivadasRESUMO
The AT(4) ligands, angiotensin IV and LVV-hemorphin 7, elicit robust effects on facilitating memory by binding to a specific site in the brain historically termed the angiotensin AT(4) receptor. The identification of the AT(4) receptor as insulin-regulated aminopeptidase (IRAP) is controversial, with other proteins speculated to be the target(s) of these peptides. In this study we have utilized IRAP knockout mice to investigate IRAP in the brain. We demonstrate that the high-affinity binding site for angiotensin IV is absent in IRAP knockout mice brain sections in parallel with the loss of IRAP immunostaining, providing irrefutable proof that IRAP is the specific high-affinity binding site for AT(4) ligands. However, our characterization of the behavioural phenotype of the IRAP knockout mice revealed a totally unexpected finding. In contrast to the acute effects of IRAP inhibitors in enhancing memory, deletion of the IRAP gene resulted in mice with an accelerated, age-related decline in spatial memory that was only detected in the Y maze paradigm. Moreover, no alterations in behaviour of the IRAP knockout mice were observed that could assist in elucidating the endogenous substrate(s). Our results highlight the importance of analysing the behavioural phenotype of knockout mice across different ages and in distinct memory paradigms.
Assuntos
Envelhecimento/metabolismo , Angiotensina II/análogos & derivados , Encéfalo/metabolismo , Cistinil Aminopeptidase/metabolismo , Transtornos da Memória/metabolismo , Percepção Espacial/fisiologia , Angiotensina II/metabolismo , Animais , Cistinil Aminopeptidase/genética , Transportador de Glucose Tipo 4/metabolismo , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , Testes Neuropsicológicos , Fenótipo , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Reconhecimento Psicológico/fisiologiaRESUMO
Approximately one-quarter of people over the age of 65 are estimated to suffer some form of cognitive impairment, underscoring the need for effective cognitive-enhancing agents. Insulin-regulated aminopeptidase (IRAP) is potentially an innovative target for the development of cognitive enhancers, as its peptide inhibitors exhibit memory-enhancing effects in both normal and memory-impaired rodents. Using a homology model of the catalytic domain of IRAP and virtual screening, we have identified a class of nonpeptide, small-molecule inhibitors of IRAP. Structure-based computational development of an initial "hit" resulted in the identification of two divergent families of compounds. Subsequent medicinal chemistry performed on the highest affinity compound produced inhibitors with nanomolar affinities (K(i) 20-700 nM) for IRAP. In vivo efficacy of one of these inhibitors was demonstrated in rats with an acute dose (1 nmol in 1 microl) administered into the lateral ventricles, improving performance in both spatial working and recognition memory paradigms. We have identified a family of specific IRAP inhibitors that is biologically active which will be useful both in understanding the physiological role of IRAP and potentially in the development of clinically useful cognitive enhancers. Notably, this study also provides unequivocal proof of principal that inhibition of IRAP results in memory enhancement.
Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Memória/efeitos dos fármacos , Nootrópicos/farmacologia , Animais , Bioensaio , Domínio Catalítico , Desenho de Fármacos , Masculino , Modelos Moleculares , Ratos , Ratos Sprague-Dawley , Reconhecimento Psicológico/efeitos dos fármacosRESUMO
It is proposed that insulin-regulated aminopeptidase (IRAP) is the site of action of two peptides, angiotensin IV and LVV-hemorphin 7, which have facilitatory effects on learning and memory. In fat and muscles, IRAP codistributes with the insulin-responsive glucose transporter GLUT4 in specialised vesicles, where it plays a role in the tethering and/or trafficking of these vesicles. This study investigated whether an analogous system exists in two functionally distinct regions of the brain, the hippocampus and the cerebellum. In the hippocampus, IRAP was found in the pyramidal neurones where it exhibited a high degree of colocalisation with GLUT4. Consistent with the role of GLUT4 in insulin-responsive tissues, the glucose transporter was thought to be responsible for facilitating glucose uptake into these pyramidal neurones in response to potassium-induced depolarisation or cAMP activation as the glucose influx was sensitive to indinavir treatment. Angiotensin IV and LVV-hemorphin 7 enhanced this activity-dependent glucose uptake in hippocampal slices. In contrast, in the cerebellum, where the distribution of IRAP was dissociated from GLUT4, the effect of the peptides on glucose uptake was absent. We propose that the modulation of glucose uptake by angiotensin IV and LVV-hemorphin 7 is region-specific and is critically dependent on a high degree of colocalisation between IRAP and GLUT4. These findings also confirm a role for IRAP and GLUT4 in activity-dependent glucose uptake in hippocampal neurones.
Assuntos
Cistinil Aminopeptidase/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Desoxiglucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Hemoglobinas/metabolismo , Hipocampo/citologia , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Fragmentos de Peptídeos/metabolismoRESUMO
Insulin-regulated aminopeptidase, IRAP, is an abundant protein that was initially cloned from a rat epididymal fat pad cDNA library as a marker protein for specialized vesicles containing the insulin-responsive glucose transporter GLUT4, wherein it is thought to participate in the tethering and trafficking of GLUT4 vesicles. The same protein was independently cloned from human placental cDNA library as oxytocinase and is proposed to have a primary role in the regulation of circulating oxytocin (OXY) during the later stages of pregnancy. More recently, IRAP was identified as the specific binding site for angiotensin IV, and we propose that it mediates the memory-enhancing effects of the peptide. This protein appears to have multiple physiological roles that are tissue- and domain-specific; thus the protein can be specifically targeted for treating different clinical conditions.