RESUMO
In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.
Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Inflamação , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/inervação , Neurônios Aferentes/fisiologia , Canais de Cátion TRPV/fisiologia , Animais , Autoimunidade , Capsaicina/farmacologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Resistência à Insulina , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiopatologia , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Neurônios Aferentes/efeitos dos fármacos , Substância P/farmacologia , Linfócitos T/imunologia , Canais de Cátion TRPV/genéticaRESUMO
The venom of the spitting cobra, Naja naja sputatrix contains highly potent alpha-neurotoxins (NTXs) in addition to phospholipase A2 (PLA2) and cardiotoxin (CTX). In this study, we report the complete characterization of three genes that are responsible for the synthesis of three isoforms of alpha-NTX in the venom of a single spitting cobra. DNA amplification by long-distance polymerase chain reaction (LD-PCR) and genome walking have provided information on the gene structure including their promoter and 5' and 3' UTRs. Each NTX isoform is approximately 4 kb in size and contains three exons and two introns. The sequence homology among these isoforms was found to be 99%. Two possible transcription sites were identified by primer extension analysis and they corresponded to the adenine (A) nucleotide at positions +1 and -45. The promoter also contains two TATA boxes and a CCAAT box. Putative binding sites for transcriptional factors AP-2 and GATA are also present. The high percentage of similarity observed among the NTX gene isoforms of N. n. sputatrix as well as with the alpha-NTX and kappa-NTX genes from other land snakes suggests that the NTX gene has probably evolved from a common ancestral gene.
Assuntos
Mapeamento Cromossômico/métodos , Neurotoxinas/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Proteínas Cardiotóxicas de Elapídeos/genética , Códon de Iniciação/genética , Elapidae/metabolismo , Éxons , Íntrons , Dados de Sequência Molecular , Fosfolipases A/genética , Fosfolipases A2 , Filogenia , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Sinapses/metabolismoRESUMO
cDNAs encoding 4 short chain alpha-neurotoxins from Malayan spitting cobra (Naja naja sputatrix) venom were cloned and expressed in Escherichia coli. The recombinant toxins possessed identical amino acid sequences to alpha-neurotoxins. This is the first report on cloning and expression of isoforms of neurotoxins from a species of spitting cobra. Two of these isoforms were also identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), capillary electrophoresis followed by mass spectrometry and characterized by protein sequencing. Based on the variable amino acid residues, the neurotoxins in N. n. sputatrix could be assigned to 2 major groups, 10E11T and 10Q11A, which could be further subdivided into 10E11T28S: 10E11T28G and 10Q11A28S; 10Q11A28G respectively. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. Phylogenetic analysis based on molecular properties of the toxins provided further support for the classification of N. n. sputatrix neurotoxin into 2 fundamental groups.
