RESUMO
OBJECTIVE: A plant-based dietary pattern, the Portfolio Diet, has been shown to lower LDL cholesterol and other cardiovascular disease risk factors. However, no study has evaluated the association of this diet with incident type 2 diabetes. RESEARCH DESIGN AND METHODS: This analysis included 145,299 postmenopausal women free of diabetes at baseline in the Women's Health Initiative (WHI) Clinical Trials and Observational Study from 1993 to 2021. Adherence to the diet was assessed with a score based on six components (high in plant protein [soy and pulses], nuts, viscous fiber, plant sterols, and monounsaturated fat and low in saturated fat and cholesterol) determined from a validated food-frequency questionnaire. We used Cox proportional hazards models to estimate hazard ratios (HRs) and 95% CIs of the association of the Portfolio Diet, alongside the Dietary Approaches to Stop Hypertension (DASH) and Mediterranean diets, with incident type 2 diabetes, with adjustment for potential confounders. RESULTS: Over a mean follow-up of 16.0 years, 13,943 cases of incident type 2 diabetes were identified. In comparisons of the highest with the lowest quintiles of adherence, the HRs for risk of incident type 2 diabetes were 0.77 (95% CI 0.72, 0.82) for the Portfolio Diet, 0.69 (0.64, 0.73) for the DASH diet, and 0.78 (0.74, 0.83) for the Mediterranean diet. These findings were attenuated by 10% after additional adjustment for BMI. CONCLUSIONS: Greater adherence to the plant-predominant Portfolio, DASH, and Mediterranean diets was prospectively associated with lower risk of type 2 diabetes in postmenopausal women.
Assuntos
Diabetes Mellitus Tipo 2 , Dieta Mediterrânea , Feminino , Humanos , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/prevenção & controle , Estudos Prospectivos , Fatores de Risco , Dieta , Saúde da MulherRESUMO
PURPOSE: The objectives of the ongoing, Phase 3, open-label extension trial enliGHten are to assess the long-term safety and efficacy of weekly administered long-acting growth hormone lonapegsomatropin in children with growth hormone deficiency. METHODS: Eligible subjects completing a prior Phase 3 lonapegsomatropin parent trial (heiGHt or fliGHt) were invited to participate. All subjects were treated with lonapegsomatropin. Subjects in the United States switched to the TransCon hGH Auto-Injector when available. Endpoints were long-term safety, annualized height velocity, pharmacodynamics [insulin-like growth factor-1 SD score (SDS) values], and patient- and caregiver-reported assessments of convenience and tolerability. RESULTS: Lonapegsomatropin treatment during enliGHten was associated with continued improvements in height SDS through week 104 in treatment-naïve subjects from the heiGHt trial (-2.89 to -1.37 for the lonapegsomatropin group; -3.0 to -1.52 for the daily somatropin group). Height SDS also continued to improve among switch subjects from the fliGHt trial (-1.42 at fliGHt baseline to -0.69 at week 78). After 104 weeks, the average bone age/chronological age ratio for each treatment group was 0.8 (0.1), showing only minimal advancement of bone age relative to chronological age with continued lonapegsomatropin treatment among heiGHt subjects. Fewer local tolerability reactions were reported with the TransCon hGH Auto-Injector compared with syringe/needle. CONCLUSIONS: Treatment with lonapegsomatropin continued to be safe and well-tolerated, with no new safety signals identified. Children treated with once-weekly lonapegsomatropin showed continued improvement of height SDS through the second year of therapy without excess advancement of bone age.
Assuntos
Nanismo Hipofisário , Hormônio do Crescimento Humano , Estatura , Criança , Transtornos do Crescimento/tratamento farmacológico , Hormônio do Crescimento , Hormônio do Crescimento Humano/efeitos adversos , HumanosRESUMO
INTRODUCTION: The phase 3 fliGHt Trial evaluated the safety and tolerability of once-weekly lonapegsomatropin, a long-acting prodrug, in children with growth hormone deficiency (GHD) who switched from daily somatropin therapy to lonapegsomatropin. METHODS: This multicenter, open-label, 26-week phase 3 trial took place at 28 sites across 4 countries (Australia, Canada, New Zealand, and the USA). The trial enrolled 146 children with GHD, 143 of which were previously treated with daily somatropin. All subjects received once-weekly lonapegsomatropin 0.24 mg human growth hormone/kg/week. The primary outcome measure was safety and tolerability of lonapegsomatropin over 26 weeks. Secondary outcome measures assessed annualized height velocity (AHV), height standard deviation score (SDS), and IGF-1 SDS at 26 weeks. RESULTS: Subjects had a mean prior daily somatropin dose of 0.29 mg/kg/week. Treatment-emergent adverse events (AEs) reported were similar to the published AE profile of daily somatropin therapies. After switching to lonapegsomatropin, the least-squares mean (LSM) AHV was 8.7 cm/year (95% CI: 8.2, 9.2) at Week 26 and LSM height SDS changed from baseline to Week 26 of +0.25 (95% CI: 0.21, 0.29). Among switch subjects, the LSM for average IGF-1 SDS was sustained at Weeks 13 and 26, representing an approximate 0.7 increase from baseline (prior to switching from daily somatropin therapy). Patient-reported outcomes indicated a preference for weekly lonapegsomatropin among both children and their parents. CONCLUSIONS: Lonapegsomatropin treatment outcomes were as expected across a range of ages and treatment experiences. Switching to lonapegsomatropin resulted in a similar AE profile to daily somatropin therapy.
Assuntos
Substituição de Medicamentos , Nanismo Hipofisário , Hormônio do Crescimento , Hormônio do Crescimento Humano , Estatura , Criança , Nanismo Hipofisário/tratamento farmacológico , Transtornos do Crescimento/tratamento farmacológico , Hormônio do Crescimento/uso terapêutico , Hormônio do Crescimento Humano/uso terapêutico , Humanos , Fator de Crescimento Insulin-Like I/uso terapêuticoRESUMO
Pancreatic ß-cell membranes and presynaptic areas of neurons contain analogous protein complexes that control the secretion of bioactive molecules. These complexes include the neuroligins (NLs) and their binding partners, the neurexins (NXs). It has been recently reported that both insulin secretion and the proliferation rates of ß-cells increase when cells are co-cultured with full-length NL-2 clusters. The pharmacological use of full-length protein is always problematic due to its unfavorable pharmacokinetic properties. Thus, NL-2-derived short peptide was conjugated to the surface of polyamidoamine-based (PAMAM) dendrimers. This nanoscale composite improved ß-cell functions in terms of the rate of proliferation, glucose-stimulated insulin secretion (GSIS), and functional maturation. This functionalized dendrimer also protected ß-cells under cellular stress conditions. In addition, various novel peptidomimetic scaffolds of NL-2-derived peptide were designed, synthesized, and conjugated to the surface of PAMAM in order to increase the biostability of the conjugates. However, after being covered by peptidomimetics, PAMAM dendrimers were inactive. Thus, the original peptide-based PAMAM dendrimer is a leading compound for continued research that might provide a unique starting point for designing an innovative class of antidiabetic therapeutics that possess a unique mode of action.
RESUMO
A 29-year-old woman was referred for new-onset diabetes mellitus after her glycosylated haemoglobin (HbA1c) was found to be 10.2%. Three years earlier, the patient's HbA1c-measured by the same clinical laboratory-had been 5.5%. The newer HbA1c was discordant with fasting glucose levels and a lack of diabetes-associated symptoms. The laboratory reported that their assay methodology remained unchanged and also that no haemoglobin variants were detected. Further investigation, however, revealed, first, that the patient carried a haemoglobin alpha chain mutation (Hb Wayne) that can sometimes cause assay interference and, second, that although the laboratory's assay methodology had not changed, their assay instrument had. Depending on assay methodology, haemoglobin variants can cause HbA1c assay interference and the presence of these variants may not be detected by the performing laboratory. Interference may not only be dependent on assay methodology but also on the assay instrument used.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Erros de Diagnóstico , Hemoglobinas Glicadas/genética , Testes Hematológicos/instrumentação , Hemoglobinas Anormais/genética , Adulto , Portador Sadio , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Hemoglobinas Glicadas/análise , Testes Hematológicos/estatística & dados numéricos , Humanos , Avaliação de Resultados da Assistência ao PacienteRESUMO
Both pancreatic ß-cell membranes and presynaptic active zones of neurons include in their structures similar protein complexes, which are responsible for mediating the secretion of bioactive molecules. In addition, these membrane-anchored proteins regulate interactions between neurons and guide the formation and maturation of synapses. These proteins include the neuroligins (e.g., NL-2) and their binding partners, the neurexins. The insulin secretion and maturation of ß-cells is known to depend on their 3-dimensional (3D) arrangement. It was also reported that both insulin secretion and the proliferation rates of ß-cells increase when cells are cocultured with clusters of NL-2. Use of full-length NL-2 or even its exocellular domain as potential ß-cell functional enhancers is limited by the biostability and bioavailability issues common to all protein-based therapeutics. Thus, based on molecular modeling approaches, a short peptide with the potential ability to bind neurexins was derived from the NL-2 sequence. Here, we show that the NL-2-derived peptide conjugates onto innovative functional maghemite (γ-Fe2O3)-based nanoscale composite particles enhance ß-cell functions in terms of glucose-stimulated insulin secretion and protect them under stress conditions. Recruiting the ß-cells' "neuron-like" secretory machinery as a target for diabetes treatment use has never been reported before. Such nanoscale composites might therefore provide a unique starting point for designing a novel class of antidiabetic therapeutic agents that possess a unique mechanism of action.
Assuntos
Nanopartículas , Animais , Moléculas de Adesão Celular Neuronais , Compostos Férricos , Hipoglicemiantes , Insulina , Camundongos , Proteínas do Tecido NervosoRESUMO
Contact between ß-cells is necessary for their normal function. Identification of the proteins mediating the effects of ß-cell-to-ß-cell contact is a necessary step toward gaining a full understanding of the determinants of ß-cell function and insulin secretion. The secretory machinery of the ß-cells is nearly identical to that of central nervous system (CNS) synapses, and we hypothesize that the transcellular protein interactions that drive maturation of the two secretory machineries upon contact of one cell (or neural process) with another are also highly similar. Two such transcellular interactions, important for both synaptic and ß-cell function, have been identified: EphA/ephrin-A and neuroligin/neurexin. Here, we tested the role of another synaptic cleft protein, CADM1, in insulinoma cells and in rat and human islet ß-cells. We found that CADM1 is a predominant CADM isoform in ß-cells. In INS-1 cells and primary ß-cells, CADM1 constrains insulin secretion, and its expression decreases after prolonged glucose stimulation. Using a coculture model, we found that CADM1 also influences insulin secretion in a transcellular manner. We asked whether extracellular CADM1 interactions exert their influence via the same mechanisms by which they influence neurotransmitter exocytosis. Our results suggest that, as in the CNS, CADM1 interactions drive exocytic site assembly and promote actin network formation. These results support the broader hypothesis that the effects of cell-cell contact on ß-cell maturation and function are mediated by the same extracellular protein interactions that drive the formation of the presynaptic exocytic machinery. These interactions may be therapeutic targets for reversing ß-cell dysfunction in diabetes.
Assuntos
Moléculas de Adesão Celular/metabolismo , Exocitose/fisiologia , Imunoglobulinas/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Sintaxina 1/metabolismo , Animais , Molécula 1 de Adesão Celular , Comunicação Celular/fisiologia , Linhagem Celular , Líquido Extracelular/metabolismo , Humanos , Secreção de Insulina , Ratos , Especificidade da EspécieRESUMO
The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/ß-catenin signaling. This TNKS activity uses NAD+ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD+ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD+ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD+ since it is mirrored by the NAD+ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD+ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD+ production to expand NAD+ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD+ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD+ production by ATP suggests that nutritional energy may enhance the functions of other NAD+-driven enzymes including sirtuins.
Assuntos
Insulinoma/patologia , NAD/química , Tanquirases/química , Células 3T3 , Acrilamidas/química , Trifosfato de Adenosina/química , Animais , Catálise , Metabolismo Energético/genética , Glucose/química , Células HEK293 , Humanos , Camundongos , Nicotinamida Fosforribosiltransferase/química , Piperidinas/química , Complexo de Endopeptidases do Proteassoma/química , Processamento de Proteína Pós-Traducional , Ratos , Ubiquitina/químicaRESUMO
Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function. Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions. Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.
Assuntos
Comunicação Celular/fisiologia , Técnicas de Cocultura/métodos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Células HEK293 , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Mapas de Interação de ProteínasRESUMO
Neuroligin-2 is a transmembrane, cell-surface protein originally identified as an inhibitory synapse-associated protein in the central nervous system. Neuroligin-2 is also present on the pancreatic beta-cell surface, and there it engages in transcellular interactions that drive functional maturation of the insulin secretory machinery; these are necessary for normal insulin secretion. The effects of neuroligin-2 deficiency on brain and neuronal function and morphology and on behavior and coordination have been extensively characterized using neuroligin-2 knockout mice. The effects of absent neuroligin-2 expression on islet development and function, however, are unknown. Here, to help test whether neuroligin-2 is necessary for normal islet development, we characterized islet morphology in mice lacking neuroligin-2. To test whether-as predicted by our earlier co-culture studies-absence of neuroligin-2 impairs beta cell function, we compared glucose-stimulated insulin secretion by islets from mutant and wild-type mice. Our results show that while islets from neuroligin-2-deficient mice do not to appear to differ architecturally from wild-type islets, they are smaller, fewer in number, and contain beta cells with lower insulin content. Evaluation of transcript levels suggests that upregulation of neuroligin-1 helps compensate for loss of neuroligin-2. Surprisingly, under both basal and stimulating glucose levels, isolated islets from the knockout mice secreted more of their intracellular insulin content. Rat islets with shRNA-mediated neuroligin-2 knockdown also exhibited increased insulin secretion. Neurexin transcript levels were lower in the knockout mice and, consistent with our prior finding that neurexin is a key constituent of the insulin granule docking machinery, insulin granule docking was reduced. These results indicate that neuroligin-2 is not necessary for the formation of pancreatic islets but that neuroligin-2 influences islet size and number. Neuroligin-2-perhaps through its effects on the expression and/or activity of its binding partner neurexin-promotes insulin granule docking, a known constraint on insulin secretion.
Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologia , Proteínas do Tecido Nervoso/deficiência , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RatosRESUMO
Normal glucose-stimulated insulin secretion is dependent on interactions between neighboring ß cells. Elucidation of the reasons why this cell-to-cell contact is essential will probably yield critical insights into ß cell maturation and function. In the central nervous system, transcellular protein interactions (i.e. interactions between proteins on the surfaces of different cells) involving neuroligins are key mediators of synaptic functional development. We previously demonstrated that ß cells express neuroligin-2 and that insulin secretion is affected by changes in neuroligin-2 expression. Here we show that the effect of neuroligin-2 on insulin secretion is mediated by transcellular interactions. Neuroligin-2 binds with nanomolar affinity to a partner on the ß cell surface and contributes to the increased insulin secretion brought about by ß cell-to-ß cell contact. It does so in a manner seemingly independent of interactions with neurexin, a known binding partner. As in the synapse, transcellular neuroligin-2 interactions enhance the functioning of the submembrane exocytic machinery. Also, as in the synapse, neuroligin-2 clustering is important. Neuroligin-2 in soluble form, rather than presented on a cell surface, decreases insulin secretion by rat islets and MIN-6 cells, most likely by interfering with endogenous neuroligin interactions. Prolonged contact with neuroligin-2-expressing cells increases INS-1 ß cell proliferation and insulin content. These results extend the known parallels between the synaptic and ß cell secretory machineries to extracellular interactions. Neuroligin-2 interactions are one of the few transcellular protein interactions thus far identified that directly enhance insulin secretion. Together, these results indicate a significant role for transcellular neuroligin-2 interactions in the establishment of ß cell function.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Comunicação Celular/fisiologia , Proliferação de Células , Regulação da Expressão Gênica/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Células HEK293 , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-DawleyRESUMO
Neurexins are a family of transmembrane, synaptic adhesion molecules. In neurons, neurexins bind to both sub-plasma membrane and synaptic vesicle-associated constituents of the secretory machinery, play a key role in the organization and stabilization of the presynaptic active zone, and help mediate docking of synaptic vesicles. We have previously shown that neurexins, like many other protein constituents of the neurotransmitter exocytotic machinery, are expressed in pancreatic ß cells. We hypothesized that the role of neurexins in ß cells parallels their role in neurons, with ß-cell neurexins helping to mediate insulin granule docking and secretion. Here we demonstrate that ß cells express a more restricted pattern of neurexin transcripts than neurons, with a clear predominance of neurexin-1α expressed in isolated islets. Using INS-1E ß cells, we found that neurexin-1α interacts with membrane-bound components of the secretory granule-docking machinery and with the granule-associated protein granuphilin. Decreased expression of neurexin-1α, like decreased expression of granuphilin, reduces granule docking at the ß-cell membrane and improves insulin secretion. Perifusion of neurexin-1α KO mouse islets revealed a significant increase in second-phase insulin secretion with a trend toward increased first-phase secretion. Upon glucose stimulation, neurexin-1α protein levels decrease. This glucose-induced down-regulation may enhance glucose-stimulated insulin secretion. We conclude that neurexin-1α is a component of the ß-cell secretory machinery and contributes to secretory granule docking, most likely through interactions with granuphilin. Neurexin-1α is the only transmembrane component of the docking machinery identified thus far. Our findings provide new insights into the mechanisms of insulin granule docking and exocytosis.
Assuntos
Exocitose/fisiologia , Glicoproteínas/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Neuropeptídeos/metabolismo , Vesículas Secretórias/metabolismo , Animais , Células Cultivadas , Expressão Gênica/fisiologia , Glucose/farmacologia , Glicoproteínas/genética , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Células Secretoras de Insulina/fisiologia , Vesículas Sinápticas/fisiologia , Fatores de Transcrição/fisiologia , Animais , Western Blotting , Brefeldina A/farmacologia , Proteínas de Ligação a DNA/genética , Imuno-Histoquímica , Insulina/fisiologia , Microscopia Confocal , Células PC12 , Inibidores da Síntese de Proteínas/farmacologia , RNA/química , RNA/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/fisiologiaRESUMO
The composition of the beta-cell exocytic machinery is very similar to that of neuronal synapses, and the developmental pathway of beta-cells and neurons substantially overlap. beta-Cells secrete gamma-aminobutyric acid and express proteins that, in the brain, are specific markers of inhibitory synapses. Recently, neuronal coculture experiments have identified three families of synaptic cell-surface molecules (neurexins, neuroligins, and SynCAM) that drive synapse formation in vitro and that control the differentiation of nascent synapses into either excitatory or inhibitory fully mature nerve terminals. The inhibitory synapse-like character of the beta-cells led us to hypothesize that members of these families of synapse-inducing adhesion molecules would be expressed in beta-cells and that the pattern of expression would resemble that associated with neuronal inhibitory synaptogenesis. Here, we describe beta-cell expression of the neuroligins, neurexins, and SynCAM, and show that neuroligin expression affects insulin secretion in INS-1 beta-cells and rat islet cells. Our findings demonstrate that neuroligins and neurexins are expressed outside the central nervous system and help confer an inhibitory synaptic-like phenotype onto the beta-cell surface. Analogous to their role in synaptic neurotransmission, neurexin-neuroligin interactions may play a role in the formation of the submembrane insulin secretory apparatus.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento Alternativo , Animais , Western Blotting , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular Tumoral , Glicoproteínas/genética , Glicoproteínas/metabolismo , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Humanos , Secreção de Insulina , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The enzyme glutamate decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes. The mechanism whereby autoreactivity to GAD65, an intracellular protein, is triggered is unknown, and it is possible that immunoreactive GAD65 is released by injured pancreatic islet beta-cells. There is a great need for methods by which to detect and monitor ongoing islet injury. If GAD65 were released and, furthermore, were able to reach the circulation, it could function as a marker of beta-cell injury. Here, a novel GAD65 plasma immunoassay is used to test the hypotheses that beta-cell injury induces GAD65 discharge in vivo and that discharged GAD65 reaches the bloodstream. Plasma GAD65 levels were determined in rats treated with alloxan, and with diabetogenic and low, subdiabetogenic doses of streptozotocin. beta-Cell injury resulted in GAD65 release into the circulation in a dose-dependent manner, and low-dose streptozotocin resulted in a more gradual increase in plasma GAD65 levels than did diabetogenic doses. Plasma GAD65 levels were reduced in rats that had undergone partial pancreatectomy and remained undetectable in mice. Together, these data demonstrate that GAD65 can be released into the circulation by injured beta-cells. Autoantigen shedding may contribute to the pathogenesis of islet autoimmunity in the multiple low-dose streptozocin model and perhaps, more generally, in other forms of autoimmune diabetes. These results demonstrate that, as is true with other tissues, islet injury, at least in some circumstances, can be monitored by use of discharged, circulating proteins. GAD65 is the first such confirmed protein marker of islet injury.
Assuntos
Aloxano/farmacologia , Glutamato Descarboxilase/sangue , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , Estreptozocina/farmacologia , Aloxano/administração & dosagem , Animais , Biomarcadores/sangue , Peptídeo C/sangue , Morte Celular , Relação Dose-Resposta a Droga , Meia-Vida , Injeções Intraperitoneais , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pancreatectomia , Ratos , Ratos Wistar , Estreptozocina/administração & dosagemRESUMO
BACKGROUND: Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells. GAD65 may also be released by transplanted islets undergoing immunological rejection. To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum or plasma will be necessary. Ideally, this assay would also be resistant to interference by anti-GAD65 autoantibodies. METHODS: A novel, magnetic bead-based assay was developed based on GAD65 capture by a monoclonal antibody directed to the only region of the protein known not to be significantly targeted by autoantibodies. A subsequent denaturation step allows sensitive immunodetection to proceed using anti-GAD65 polyclonal antibodies that would otherwise potentially be blocked by bound autoantibodies. RESULTS: The GAD65 assay worked equally well with serum and plasma as with a solution of bovine serum albumin (BSA). The limit of blank was 31 pg/mL and did not differ significantly in the BSA solution (27 pg/mL). Mean recovery of GAD65 from the plasma of control subjects and GAD65 autoantibody-positive and -negative subjects with type 1 diabetes was 101 +/- 4.6%, 88 +/- 7.8%, and 99 +/- 7.0% (+/- SEM), respectively. The assay was used to quantify both recombinant GAD65 and the GAD65 content of human and rodent islets and other tissue extracts that were added to human plasma samples. CONCLUSIONS: A sensitive, autoantibody-resistant GAD65 assay has been developed that is compatible with detection in serum and plasma and therefore will likely also work with a variety of other biologic fluids. This assay may enable the use of circulating GAD65 as a biomarker of islet damage or transplant rejection and will facilitate in vivo studies of the pathogenesis of anti-GAD65 autoreactivity.
Assuntos
Glutamato Descarboxilase/sangue , Fragmentos de Peptídeos/sangue , Autoanticorpos , Autoantígenos/sangue , Diabetes Mellitus/sangue , Diabetes Mellitus/imunologia , Glutamato Descarboxilase/imunologia , Humanos , Técnicas de Imunoadsorção , Células Secretoras de Insulina/metabolismo , Fragmentos de Peptídeos/imunologia , Plasma/química , Proteínas Recombinantes/sangue , Soro/química , Extratos de Tecidos/químicaAssuntos
Educação Médica/economia , Financiamento Governamental , Pesquisadores/economia , Apoio ao Desenvolvimento de Recursos Humanos/economia , Bolsas de Estudo/economia , Financiamento Governamental/legislação & jurisprudência , Médicos/economia , Pesquisa/economia , Pesquisa/educação , Apoio à Pesquisa como Assunto , Apoio ao Desenvolvimento de Recursos Humanos/legislação & jurisprudência , Estados UnidosRESUMO
gamma-Aminobutyric acid (GABA) is stored in microvesicles in pancreatic islet cells. Because GAD65 and GAD67, which catalyze the formation of GABA, are cytoplasmic, the existence of an islet vesicular GABA transporter has been postulated. Here, we test the hypothesis that the putative transporter is the vesicular inhibitory amino acid transporter (VIAAT), a neuronal transmembrane transporter of GABA and glycine. We sequenced the human VIAAT gene and determined that the human and rat proteins share over 98% sequence identity. In vitro expression of VIAAT and immunoblotting of brain and islet lysates revealed two forms of the protein: an approximately 52-kDa and an approximately 57-kDa form. By immunoblotting and immunohistochemistry, we detected VIAAT in rat but not human islets. Immunohistochemical staining showed that in rat islets, the distribution of VIAAT expression parallels that of GAD67, with increased expression in the mantle. GABA, too, was found to be present in islet non-beta-cells. We conclude that VIAAT is expressed in rat islets and is more abundant in the mantle and that expression in human islets is very low or nil. The rat islet mantle differs from rat and human beta-cells in that it contains only GAD67 and relatively increased levels of VIAAT. Cells that express only GAD67 may require higher levels of VIAAT expression.
Assuntos
Sistemas de Transporte de Aminoácidos , Proteínas de Transporte/genética , Expressão Gênica , Ilhotas Pancreáticas/química , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Química Encefálica , Proteínas de Transporte/análise , Proteínas de Transporte/química , Glutamato Descarboxilase/análise , Glicina/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Isoenzimas/análise , Camundongos , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos BB , Alinhamento de Sequência , Análise de Sequência , Distribuição Tecidual , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/metabolismoRESUMO
While both isoforms of glutamic acid decarboxylase (GAD) function as important autoantigens in autoimmune diabetes mellitus-GAD65 in humans and GAD67 in the NOD mouse-GAD67 is not synthesized in human pancreatic islets and is thought not to be an autoantigen in human diabetes. We have recently shown, however, that human islets contain a GAD67 splice variant: GAD25. Given the evidence that GAD67 could be a key diabetogenic autoantigen in the NOD mouse and the high prevalence of GAD65 autoantibodies in human type 1 diabetes, it became important to ask whether there is also immune reactivity to GAD25 in type 1 diabetes-possibly implicating it in the pathogenesis of the disease-and whether GAD25 reactivity could, like GAD65 reactivity, function as a clinically useful marker for the disease. We also hypothesized that the presence of autoantibodies to the smaller splice variant could be a cause of the up to 30% prevalence of GAD67 autoreactivity associated with type 1 diabetes. We therefore analyzed GAD25 reactivity in 105 newly-diagnosed children with type 1 diabetes and 74 control subjects. While 14 (13%) of the diabetic subjects were positive for GAD67 autoantibodies, only 3 (3%) were positive for GAD25 reactivity, none of which were GAD67 antibody-positive. Analysis of reactivity to a GAD67 chimera was consistent with GAD67 binding activity being due to cross-reactive GAD65 antibodies. Immunostaining confirmed the presence of GAD25 in human islets, revealing GAD25-positive cells to be sparse. Our results indicate that autoreactivity to GAD25 is rare in newly diagnosed type 1 diabetes and does not underlie GAD67 reactivity.