RESUMEN
Diabetic retinopathy (DR) is one of the most prevalent secondary complications associated with diabetes. Specifically, Type 1 Diabetes Mellitus (T1D) has an immune component that may determine the evolution of DR by compromising the immune response of the retina, which is mediated by microglia. In the early stages of DR, the permeabilization of the blood-retinal barrier allows immune cells from the peripheral system to interact with the retinal immune system. The use of new bioactive molecules, such as 3-(2,4-dihydroxyphenyl)phthalide (M9), with powerful anti-inflammatory activity, might represent an advance in the treatment of diseases like DR by targeting the immune systems responsible for its onset and progression. Our research aimed to investigate the molecular mechanisms involved in the interaction of specific cells of the innate immune system during the progression of DR and the reduction in inflammatory processes contributing to the pathology. In vitro studies were conducted exposing Bv.2 microglial and Raw264.7 macrophage cells to proinflammatory stimuli for 24 h, in the presence or absence of M9. Ex vivo and in vivo approaches were performed in BB rats, an animal model for T1D. Retinal explants from BB rats were cultured with M9. Retinas from BB rats treated for 15 days with M9 via intraperitoneal injection were analyzed to determine survival, cellular signaling, and inflammatory markers using qPCR, Western blot, or immunofluorescence approaches. Retinal structure images were acquired via Spectral-Domain-Optical Coherence Tomography (SD-OCT). Our results show that the treatment with M9 significantly reduces inflammatory processes in in vitro, ex vivo, and in vivo models of DR. M9 works by inhibiting the proinflammatory responses during DR progression mainly affecting immune cell responses. It also induces an anti-inflammatory response, primarily mediated by microglial cells, leading to the synthesis of Arginase-1 and Hemeoxygenase-1(HO-1). Ultimately, in vivo administration of M9 preserves the retinal integrity from the degeneration associated with DR progression. Our findings demonstrate a specific interaction between both retinal and systemic immune cells in the progression of DR, with a differential response to treatment, mainly driven by microglia in the anti-inflammatory action. In vivo treatment with M9 induces a switch in immune cell phenotypes and functions that contributes to delaying the DR progression, positioning microglial cells as a new and specific therapeutic target in DR.
Asunto(s)
Diabetes Mellitus Tipo 1 , Retinopatía Diabética , Modelos Animales de Enfermedad , Microglía , Animales , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/patología , Retinopatía Diabética/inmunología , Ratas , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Retina/efectos de los fármacos , Retina/patología , Retina/metabolismo , Células RAW 264.7 , Masculino , Benzofuranos/farmacología , Benzofuranos/uso terapéutico , Inmunomodulación/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/patología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/complicaciones , Ratas Endogámicas BBRESUMEN
The depleting Vß13a T cell receptor monoclonal antibody (mAb) 17D5 prevents both induced and spontaneous autoimmune diabetes in BB rats. Here it was tested in congenic DRLyp/Lyp rats, all of which spontaneously developed diabetes. Starting at 40 days of age, rats were injected once weekly with either saline, His42 Vß16 mAb, or 17D5 mAb and monitored for hyperglycemia. Diabetes occurred in 100% (n = 5/5) of saline-treated rats (median age, 66 days; range 55-73), and in 100% (n = 6/6) of His42-treated rats (median age, 69 days; range 59-69). Diabetes occurred in fewer (n = 8/11, 73%) 17D5-treated rats at a later age (median 76 days, range 60-92). Three (27%) of the 17D5-treated rats were killed at 101-103 days of age without diabetes (17D5 no-diabetes rats). Survival analysis demonstrated that 17D5 mAb delayed diabetes onset. Saline- and His42-treated rats had severely distorted islets with substantial loss of insulin-positive cells. These rats exhibited prominent hyaluronan (HA) staining, with the intra-islet HA+ accumulations measuring 5,000 ± 2,400 µm2 and occupying 36 ± 12% of islet area, and severe (grade 4) insulitis with abundant infiltration by CD68+, CD3+, and CD8+ cells. The 17D5 mAb-treated rats with delayed diabetes onset exhibited less severe insulitis (predominantly grade 3). In contrast, the 17D5 no-diabetes rats had mostly normal islets, with insulin+ cells representing 76 ± 3% of islet cells. In these rats, the islet HA deposits were significantly smaller than in the diabetic rats; the intra-islet HA+ areas were 1,200 ± 300 µm2 and accounted for 8 ± 1% of islet area. Also, islet-associated CD68+ and CD3+ cells occurred less frequently (on average in 60 and 3% of the islets, respectively) than in the diabetes rats (present in >95% of the islets). No CD8+ cells were detected in islets in all 17D5 no-diabetes rats. We conclude that mAb 17D5 delayed diabetes in DRLyp/Lyp rats and markedly reduced expression of HA and concomitant infiltration of CD68+, CD3+, and CD8+ cells. Our findings underscore the importance of refining immune suppression in prevention or intervention clinical trials to use mAb reagents that are directed against specific T cell receptors.
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Anticuerpos Monoclonales/farmacología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Complejo CD3/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Experimental/inmunología , Ácido Hialurónico/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/prevención & control , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Polimorfismo de Nucleótido Simple/genética , Ratas Endogámicas BBRESUMEN
Previous studies demonstrated that an enriched environment (EE) exposure improves cognitive functions, synaptic plasticity, neurogenesis, and induction of brain-derived neurotrophic factor (BDNF) in multiple brain regions of laboratory animal models. Also, studies on the sex-dependent effects of exposure to EE during adolescence on adult cognitive functions are less. Therefore, the present experiment was aimed to assess the effects of EE during adolescence on passive avoidance learning and memory, nociception, and prefrontal cortex (PFC) BDNF mRNA levels in the adult male and female rats. Our results indicated that housing in the EE during adolescence improves passive avoidance memory and increases nociceptive response against thermal stimulus in both sexes. Findings of our study also showed an increased BDNF level in the PFC of female animals. As a result, sex differences can affect the expression of BDNF mRNA in the PFC. Further research concerning the precise mechanisms underlying sex hormone-dependent production of BDNF in PFC is critical.
Asunto(s)
Reacción de Prevención , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Memoria , Nocicepción , Animales , Femenino , Vivienda para Animales , Masculino , Corteza Prefrontal/metabolismo , Ratas , Ratas Endogámicas BBRESUMEN
Retinal homeostasis is under both diurnal and circadian regulation. We sought to investigate the diurnal expression of autophagy proteins in normal rodent retina and to determine if this is impaired in diabetic retinopathy. C57BL/6J mice and Bio-Breeding Zucker (BBZ) rats were maintained under a 12h/12h light/dark cycle and eyes, enucleated over a 24 h period. Eyes were also collected from diabetic mice with two or nine-months duration of type 1 diabetes (T1D) and Bio-Breeding Zucker diabetic rat (BBZDR/wor rats with 4-months duration of type 2 diabetes (T2D). Immunohistochemistry was performed for the autophagy proteins Atg7, Atg9, LC3 and Beclin1. These autophagy proteins (Atgs) were abundantly expressed in neural retina and endothelial cells in both mice and rats. A differential staining pattern was observed across the retinas which demonstrated a distinctive diurnal rhythmicity. All Atgs showed localization to retinal blood vessels with Atg7 being the most highly expressed. Analysis of the immunostaining demonstrated distinctive diurnal rhythmicity, of which Atg9 and LC3 shared a biphasic expression cycle with the highest level at 8:15 am and 8:15 pm. In contrast, Beclin1 revealed a 24-h cycle with the highest level observed at midnight. Atg7 was also on a 24-h cycle with peak expression at 8:15am, coinciding with the first peak expression of Atg9 and LC3. In diabetic animals, there was a dramatic reduction in all four Atgs and the distinctive diurnal rhythmicity of these autophagy proteins was significantly impaired and phase shifted in both T1D and T2D animals. Restoration of diurnal rhythmicity and facilitation of autophagy protein expression may provide new treatment strategies for diabetic retinopathy.
Asunto(s)
Autofagia/genética , Trastornos Cronobiológicos/complicaciones , Ritmo Circadiano/genética , Complicaciones de la Diabetes/genética , Retinopatía Diabética/genética , Retina/patología , Animales , Femenino , Humanos , Masculino , Ratones , Ratas , Ratas Endogámicas BBRESUMEN
Studies performed in humans and animal models have implicated the environment in the etiology of type 1 diabetes (T1D), but the nature and timing of the interactions triggering ß cell autoimmunity are poorly understood. Virus infections have been postulated to be involved in disease mechanisms, but the underlying mechanisms are not known. It is exceedingly difficult to establish a cause-and-effect relationship between viral infection and diabetes in humans. Thus, we have used the BioBreeding Diabetes-Resistant (BBDR) and the LEW1.WR1 rat models of virus-induced disease to elucidate how virus infection leads to T1D. The immunophenotype of these strains is normal, and spontaneous diabetes does not occur in a specific pathogen-free environment. However, ß cell inflammation and diabetes with many similarities to the human disease are induced by infection with the parvovirus Kilham rat virus (KRV). KRV-induced diabetes in the BBDR and LEW1.WR1 rat models is limited to young animals and can be induced in both male and female rats. Thus, these animals provide a powerful experimental tool to identify mechanisms underlying virus-induced T1D development.
Asunto(s)
Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/virología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/virología , Infecciones por Parvoviridae/complicaciones , Parvovirus/inmunología , Animales , Glucemia/análisis , Técnicas de Cultivo de Célula , Línea Celular , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/etiología , Femenino , Glucosuria , Inflamación/inmunología , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/virología , Masculino , Ratas , Ratas Endogámicas BB , Ratas Endogámicas Lew , Ratas Endogámicas WFRESUMEN
OBJECTIVE: The need of today's research is to develop successful and reliable diabetic animal models for understanding the disease susceptibility and pathogenesis. Enormous success of animal models had already been acclaimed for identifying key genetic and environmental factors like Idd loci and effects of microorganisms including the gut microbiota. Furthermore, animal models had also helped in identifying many therapeutic targets and strategies for immune-intervention. In spite of a quite success, we have acknowledged that many of the discovered immunotherapies are working on animals and did not have a significant impact on human. Number of animal models were developed in the past to accelerate drug discovery pipeline. However, due to poor initial screening and assessment on inequivalent animal models, the percentage of drug candidates who succeeded during clinical trials was very low. Therefore, it is essential to bridge this gap between pre-clinical research and clinical trial by validating the existing animal models for consistency. RESULTS AND CONCLUSION: In this review, we have discussed and evaluated the significance of animal models on behalf of published data on PUBMED. Amongst the most popular diabetic animal models, we have selected six animal models (e.g. BioBreeding rat, "LEW IDDM rat", "Nonobese Diabetic (NOD) mouse", "STZ RAT", "LEPR Mouse" and "Zucker Diabetic Fatty (ZDF) rat" and ranked them as per their published literature on PUBMED. Moreover, the vision and brief imagination for developing an advanced and robust diabetic model of 21st century was discussed with the theme of one miceone human concept including organs-on-chips.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Evaluación Preclínica de Medicamentos/tendencias , Hipoglucemiantes/farmacología , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Tipo 2/inducido químicamente , Diabetes Mellitus Tipo 2/genética , Predicción , Humanos , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Ratas Endogámicas BB , Ratas Endogámicas Lew , Ratas Zucker , Especificidad de la Especie , EstreptozocinaRESUMEN
Abstract Norepinephrine in the basolateral amygdala (BLA) plays a pivotal role in mediating the effects of stress on memory functions in the hippocampus, however, the functional contribution of β1-adrenergic receptors on the BLA inputs to the CA1 region of hippocampus and memory function are not well understood. In the present study the role of β1-adrenoreceptor in the BLA on memory, neuronal arborization and long-term potentiation (LTP) in the CA1 region of hippocampus was examined by infusion the β1-adrenoreceptor agonist (Dobutamine; 0.5µl/side) or antagonist (Atenolol; 0.25µL/side) bilaterally into the BLA before foot-shock stress. Passive avoidance test results showed that Step-through latency time was significantly decreased in the stress group rats one, four and seven days after the stress, which intra-BLA injection of Atenolol or Dobutamine before stress couldn't attenuate this reduction. Barnes-maze results revealed that infusion of Dobutamine and Atenolol significantly reduced spatial memory indicators such as increased latency time, the number of errors and the distance traveling to achieve the target hole in the stress group. These learning impairments in stress rats correlated with a reduction of LTP in hippocampal CA1 synapses in-vivo, which infusion of Dobutamine and Atenolol couldn't attenuate the population spike amplitude and mean-field excitatory postsynaptic potentials (fEPSP) slope reduction induced by stress. Also, the Golgi-Cox staining demonstrated that infusion of Atenolol attenuated stress decreased CA1 region dendritic and axonal arborization. These results suggest that β1-adrenergic receptors activation or block seem to exacerbate stress-induced hippocampal memory deficits and this effect is independent of CA1 LTP modulation.
Asunto(s)
Animales , Masculino , Ratas , Estrés Fisiológico/efectos de los fármacos , Norepinefrina/metabolismo , Dobutamina/farmacología , Región CA1 Hipocampal/efectos de los fármacos , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Complejo Nuclear Basolateral/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Ratas Endogámicas BB , Hipocampo/efectos de los fármacosRESUMEN
Purpose: To appraise the curative effect of ginsenoside Rb1 and trigonelline in diabetic nephropathy and to analyze the expression analysis of microRNAs and their target genes during experimental diabetic renal lesions in rats. Methods: Wistar rats were made diabetic by intraperitoneal injection of 55 mg/kg streptozotocin. According to their fasting blood glucose values and initial body weight, diabetic rats were assigned to specific groups and treated as follows: DN group (tap water, n = 10), A group (ginsenoside Rb1, 40 mg/kg, n = 10), B group (trigonelline, 20 mg/kg, n = 10) and the C group (ginsenoside Rb1 and trigonelline, 60 mg/kg, m(ginsenoside Rb1) : m (trigonelline) = 2:1, n = 10). The control group was treated with tap water (n = 10). All rats were gavaged with drugs or tap water once daily for 12 weeks. Results: Renal dysfunction, oxidative stress, and pathological alteration were significantly alleviated by a combination of ginsenoside Rb1 and trigonellin (C group). Some miRNAs were expressed differentially in Con, DN, A and C groups. Results of immunohistochemistry and western blotting showed that Wnt and ß-catenin were expressed differentially in Con, DN, and C groups. Conclusion: Ginsenoside Rb1 and trigonelline could prevent the development of diabetic renal lesions by regulating the expression of miR-3550 and further associating with the Wnt/ß-catenin signaling.
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Alcaloides/farmacología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Ginsenósidos/farmacología , MicroARNs/biosíntesis , Animales , Diabetes Mellitus Experimental , Nefropatías Diabéticas/tratamiento farmacológico , Riñón/metabolismo , Riñón/patología , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Endogámicas BB , EstreptozocinaRESUMEN
BACKGROUND: A complex interplay between a failing intestinal barrier and low-grade inflammation leading to sensorimotor disturbances is an often-cited mechanism in the pathogenesis of functional gastrointestinal disorders (FGID). However, the cause-consequence relationship between these features has not been clearly established. We previously described jejunal alterations in the normoglycemic BB-rat (BBDP-N) model proposing this model as a suitable animal model to study FGID pathophysiology. The current study explores colonic permeability, inflammation, and sensitivity of the BB-rat. METHODS: Colonic tissue of BBDP-N and control (BBDR) rats at 50, 90, 110, 160, and 220 days (n ≥ 7 per group) was used to assess intestinal permeability in Ussing chambers and inflammation, including infiltration by eosinophils, mast cells, and eosinophil peroxidase (EPO) activity. Anxiety-like symptoms were evaluated at 50, 90, and 220 days and colonic sensitivity at 160 and 220 days by measuring the visceromotor response (VMR) to isobaric colorectal distensions. KEYS RESULTS: Lamina propria eosinophil and mast cell infiltration and increased EPO activity were demonstrated from 90 days onward. Increased permeability and myenteric ganglionitis were observed in the oldest BBDP-N rats. At 220 days, the VMR was significantly increased suggesting colonic hypersensitivity. At the same age, increased anxiety-like behavior was observed. CONCLUSION AND INFERENCES: We demonstrated a lamina propria eosinophil and mast cell infiltration preceding visceral hypersensitivity in the colon of the BBDP-N rat, reminiscent of patients with FGID. These findings help elucidating pathogenetic pathways in FGID and further validate the BBDP-N rat as an attractive model to study pathophysiology and therapy of FGID.
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Colon/patología , Enfermedades Gastrointestinales/patología , Hiperalgesia/patología , Inflamación/patología , Mucosa Intestinal/patología , Animales , Permeabilidad Capilar/fisiología , Modelos Animales de Enfermedad , Enfermedades Gastrointestinales/complicaciones , Hiperalgesia/etiología , Masculino , Ratas , Ratas Endogámicas BBRESUMEN
Type 1 diabetes can have deleterious effects on skeletal muscle and its microvasculature. Our laboratory has recently identified murine double minute-2 as a master regulator of muscle microvasculature by controlling expression levels of two key molecular actors of the angio-adaptive process: the pro-angiogenic vascular endothelial growth factor-A and the anti-angiogenic thrombospondin-1. Here, we show for the first time that in the soleus and plantaris muscles of the diabetes-prone BioBreeding rats, a rodent model of autoimmune type 1 diabetes, murine double minute-2 protein levels are significantly decreased, coinciding with elevated protein levels of thrombospondin-1 and its transcription factor forkhead box O1. Significant capillary regression was observed to similar extent in soleus and plantaris muscles of type 1 diabetic rats. Elevated blood glucose levels were correlated with the loss of capillaries, the reduction in murine double minute-2 expression and with the elevations in thrombospondin-1. Vascular endothelial growth factor-A protein levels were unaltered or even increased in diabetic animals, yet type 1 diabetic animals had less vascular endothelial growth factor receptor-2 abundance. The vascular endothelial growth factor-A/thrombospondin-1 ratio, a good indicator of skeletal muscle angio-adaptive environment, was decreased in type 1 diabetic muscle. Our results suggest that the murine double minute-2-forkhead box O1-thrombospondin-1 pathway plays an important role in angio-regulation of the skeletal muscle in the pathophysiological context of type 1 diabetes.
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Glucemia/metabolismo , Capilares/metabolismo , Diabetes Mellitus Tipo 1/sangre , Angiopatías Diabéticas/sangre , Músculo Esquelético/irrigación sanguínea , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Trombospondina 1/metabolismo , Animales , Biomarcadores/sangre , Capilares/patología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/patología , Modelos Animales de Enfermedad , Ratas Endogámicas BB , Ratas Endogámicas , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin ß1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling.
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Axones/metabolismo , Ganglios Espinales/lesiones , Biosíntesis de Proteínas , Nervio Ciático/lesiones , Serina-Treonina Quinasas TOR/biosíntesis , Regiones no Traducidas 3' , Animales , Tamaño de la Célula , Ratones , Ratones Endogámicos , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Endogámicas BB , Ratas Sprague-Dawley , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , NucleolinaRESUMEN
AIMS/HYPOTHESIS: Genetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available. METHODS: We used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells. RESULTS: DRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5-7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet-1 h-1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 109 ± 9.5 × 107 vs 3.8 × 109 ± 5.8 × 107 µm3; p = 0.044) and small sized islets (1.6 × 109 ± 5.1 × 107 vs 1.4 × 109 ± 4.5 × 107 µm3; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 µl min-1 [g pancreas]-1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats. CONCLUSIONS/INTERPRETATION: The present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.
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Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Células Secretoras de Insulina/citología , Insulina/metabolismo , Adenosina Difosfato/química , Adenosina Trifosfato/química , Animales , Glucemia/metabolismo , Femenino , Genotipo , Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , Células de Langerhans/metabolismo , Masculino , Páncreas/metabolismo , Perfusión , Ratas , Ratas Endogámicas BB , Ratas WistarRESUMEN
In spite of the fact that dissolution time profiles of 250mg ursodeoxycholic acid (UCDA) capsules developed by Sponsor and 250mg hard capsules produced by Ursofalk®, Dr. Falk Pharma GmbH, indicated similarity (f2=60.6), a bioavailability study indicated unexpected differences in the formulations. To find an explanation of the in vivo performance of the compared formulations, the dissolution profiles were analyzed using a novel dissolution theory considering: The dissolution model was applied to the measured data using SADAPT. Despite Cmax and AUC values showing higher values after administration of the test product, a reduction of UDCA particle size for the test formulation was suggested for reformulation. The decision was based on the strongly pH-dependent UDCA solubility, formation of insoluble crystals at low pH condition and the known high pH fluctuations ranging from pH1 to 8 in empty stomach. The performed reformulation led to increased dissolution rate of the test product and to a positive bioequivalence study which compared the reformulated test generic formulation with two reference products purchased from two highly regulated markets.
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Liberación de Fármacos , Ácido Ursodesoxicólico/farmacocinética , Administración Oral , Adulto , Animales , Área Bajo la Curva , Cápsulas , Estudios Cruzados , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Modelos Biológicos , Modelos Químicos , Tamaño de la Partícula , Ratas Endogámicas BB , Ácido Ursodesoxicólico/administración & dosificación , Ácido Ursodesoxicólico/químicaRESUMEN
Microtubules can regulate GPCR (G protein-coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the ß-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance of this pathway in vascular smooth muscle cells, we investigated the role of microtubule stability on ß-adrenoceptor signaling in rat renal and mesenteric arteries. In isometric tension experiments, incubation with the microtubule inhibitors colchicine and nocodazole enhanced isoprenaline-mediated relaxations of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated the effect of colchicine on isoprenaline relaxations, whereas iberiotoxin-a BKCa channel blocker-had no effect. In addition, colchicine improved the relaxations to the Kv7.2 to 7.5 activator, S-1, in both renal and mesenteric artery segments compared with dimethyl sulfoxide incubation. We determined that increased mesenteric artery myocytes treated with colchicine showed increased Kv7.4 membrane expression, but Western blot analysis showed no change in total Kv7.4 protein. This study is the first to show microtubule disruption improves the ß-adrenoceptor-mediated relaxations of mesenteric and renal arteries and determine this enhancement to be because of increased membrane expression of the Kv7 voltage-gated potassium channels.
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Canales de Potasio KCNQ/metabolismo , Microtúbulos/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Adrenérgicos beta/metabolismo , Vasodilatación/fisiología , Animales , Antracenos/farmacología , Western Blotting , Colchicina/farmacología , AMP Cíclico , Inmunohistoquímica , Isoproterenol/farmacología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Miografía/métodos , Paclitaxel/farmacología , Ratas , Ratas Endogámicas BB , Receptores Adrenérgicos beta/fisiología , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Poor bone quality, increased fracture risks, and impaired bone healing are orthopedic comorbidities of type 1 diabetes (T1DM). Standard osteogenic growth factor treatments are inadequate in fully rescuing retarded healing of traumatic T1DM long bone injuries where both periosteal and bone marrow niches are disrupted. We test the hypotheses that osteogenesis of bone marrow-derived stromal cells (BMSCs) and periosteum-derived cells (PDCs), two critical skeletal progenitors in long bone healing, are both impaired in T1DM and that they respond differentially to osteogenic bone morphogenetic proteins (BMPs) and/or insulin-like growth factor-1 (IGF-1) rescue. METHODS: BMSCs and PDCs were isolated from Biobreeding Diabetes Prone/Worcester rats acquiring T1DM and normal Wistar rats. Proliferation, osteogenesis, and adipogenesis of the diabetic progenitors were compared with normal controls. Responses of diabetic progenitors to osteogenesis rescue by rhBMP-2/7 heterodimer (45 or 300 ng/ml) and/or rhIGF-1 (15 or 100 ng/ml) in normal and high glucose cultures were examined by alizarin red staining and qPCR. RESULTS: Diabetic BMSCs and PDCs proliferated slower and underwent poorer osteogenesis than nondiabetic controls, and these impairments were exacerbated in high glucose cultures. Osteogenesis of diabetic PDCs was rescued by rhBMP-2/7 or rhBMP-2/7 + rhIGF-1 in both normal and high glucose cultures in a dose-dependent manner. Diabetic BMSCs, however, only responded to 300 ng/nl rhBMP-2/7 with/without 100 ng/ml rhIGF-1 in normal but not high glucose osteogenic culture. IGF-1 alone was insufficient in rescuing the osteogenesis of either diabetic progenitor. Supplementing rhBMP-2/7 in high glucose osteogenic culture significantly enhanced gene expressions of type 1 collagen (Col 1), osteocalcin (OCN), and glucose transporter 1 (GLUT1) while suppressing that of adipogenic marker peroxisome proliferator-activated receptor gamma (PPARγ) in diabetic PDCs. The same treatment in high glucose culture only resulted in a moderate increase in Col 1, but no significant changes in OCN or GLUT1 expressions in diabetic BMSCs. CONCLUSIONS: This study demonstrates more effective osteogenesis rescue of diabetic PDCs than BMSCs by rhBMP-2/7 with/without rhIGF-1 in a hyperglycemia environment, underscoring the necessity to tailor biochemical therapeutics to specific skeletal progenitor niches. Our data also suggest potential benefits of combining growth factor treatment with blood glucose management to optimize orthopedic therapeutic outcomes for T1DM patients.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 7/farmacología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Factor I del Crecimiento Similar a la Insulina/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 1/agonistas , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/genética , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Periostio/efectos de los fármacos , Periostio/metabolismo , Periostio/patología , Cultivo Primario de Células , Ratas , Ratas Endogámicas BB , Ratas Wistar , Proteínas Recombinantes/farmacologíaRESUMEN
Type 1 diabetes is associated with abberations of fat metabolism before and after the clinical onset of disease. It has been hypothesized that the absence of the effect of insulin in the liver contributes to reduced hepatic fat synthesis. We measured hepatic gene expression and serum metabolites before and after the onset of hyperglycemia in a BioBreeding rat model of type 1 diabetes. Functional pathway annotation identified that lipid metabolism was differentially expressed in hyperglycemic rats and that these pathways significantly overlapped with genes regulated by insulin. 17 serum metabolites significantly changed in concentration. All but 2 of the identified metabolites had previously been reported in type 1 diabetes, and carbohydrates were overall the most upregulated class of metabolites. We conclude that lack of insulin in the liver contributes to the changes in fat metabolism observed in type 1 diabetes. Further studies are needed to understand the clinical consequences of a lack of insulin in the liver in patients with type 1 diabetes.
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Diabetes Mellitus Tipo 1/genética , Hiperglucemia/genética , Metabolismo de los Lípidos/genética , Metaboloma , Transcriptoma/genética , Animales , Glucemia/metabolismo , Cruzamiento , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Cromatografía de Gases y Espectrometría de Masas , Perfilación de la Expresión Génica/métodos , Hiperglucemia/sangre , Hiperglucemia/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Masculino , Metabolómica/métodos , Ratas Endogámicas BB , Ratas Endogámicas F344 , Transducción de Señal/genética , Factores de TiempoRESUMEN
BACKGROUND: Ghrelin is a peptide hormone with pleiotropic effects. It stimulates cell proliferation and inhibits apoptosis-mediated cell death. It prevents diabetes mellitus in several models of chemical, surgical and biological toxic insults to pancreas in both in vivo and in vitro models and promotes glucose-stimulated insulin secretion under cytotoxic conditions. It has not yet been tested in vivo in an autoimmune model of diabetes with a persistent insult to the ß-cell. Given the immunomodulating effects of ghrelin and its trophic effects on ß-cells, we hypothesized that ghrelin treatment during the early stages of insulitis would delay diabetes onset. METHODS: BioBreeding/Worcester male rats received ghrelin (10 ng/kg/day) before insulitis development. Glucose metabolism was characterized by glucose and insulin tolerance tests. ß-cell mass, islet area, islet number, ß-cell clusters, proliferation and apoptosis and degree of insulitis were analysed by histomorphometry. A Kaplan-Meier survival curve was plotted and analysed applying the log-rank (Mantel-Cox) test. RESULTS: Ghrelin treatment significantly reduced the probability of developing diabetes in our model (p < 0.0001). It decreased islet infiltration and partially prevented ß-cell mass loss, enabling the maintenance of ß-cell neogenesis and proliferation rates. Furthermore, ghrelin treatment did not induce any metabolic perturbations. CONCLUSIONS: These findings support the hypothesis that ghrelin delays the development of autoimmune diabetes by attenuating insulitis and supporting ß-cell mass. GENERAL SIGNIFICANCE: Ghrelin promotes ß-cell viability and function through diverse mechanisms that may have significant implications for diabetes prevention, therapy and also transplant success of both islets and complete pancreas. Copyright © 2016 John Wiley & Sons, Ltd.
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Diabetes Mellitus Tipo 1/prevención & control , Modelos Animales de Enfermedad , Ghrelina/farmacología , Resistencia a la Insulina , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Hipoglucemiantes/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratas , Ratas Endogámicas BBRESUMEN
BACKGROUND & AIMS: Patients with diabetes have defects in the vagal afferent pathway that result in abnormal gastrointestinal function. We investigated whether selective increased activation of the 2-pore domain potassium channel TRESK (2-pore-domain weak inward-rectifying potassium channel-related spinal cord potassium channel) contributes to nodose ganglia (NG) malfunction, disrupting gastrointestinal function in diabetic rats. METHODS: We conducted whole-cell current-clamp and single-unit recordings in NG neurons from diabetes-prone BioBreeding/Worcester rats and streptozotocin-induced diabetic (STZ-D) rats and compared them with control rats. NG neurons in rats or cultured NG neurons were exposed to pharmacologic antagonists and/or transfected with short hairpin or small interfering RNAs that reduced expression of TRESK. We then made electrophysiologic recordings and studied gastrointestinal functions. RESULTS: We observed reduced input resistance, hyperpolarized membrane potential, and increased current threshold to elicit action potentiation in NG neurons of STZ-D rats compared with controls. NG neuron excitability was similarly altered in diabetes-prone rats. In vivo single-unit NG neuronal discharges in response to 30 and 60 pmol cholecystokinin octapeptide were significantly lower in STZ-D rats compared with controls. Reducing expression of the TRESK K+ channel restored NG excitability in vitro and in vivo, as well as cholecystokinin 8-stimulated secretion of pancreatic enzymes and secretin-induced gastrointestinal motility, which are mediated by vago-vagal reflexes. These abnormalities resulted from increased intracellular Ca2+ in the NG, activating calcineurin, which, in turn, bound to an nuclear factor of activated T cell-like docking site on the TRESK protein, resulting in neuronal membrane hyperpolarization. CONCLUSIONS: In 2 rate models of diabetes, we found that activation of the TRESK K+ channel reduced NG excitability and disrupted gastrointestinal functions.
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Diabetes Mellitus Experimental/fisiopatología , Motilidad Gastrointestinal/fisiología , Ganglio Nudoso/fisiopatología , Canales de Potasio/metabolismo , Animales , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/metabolismo , Masculino , Potenciales de la Membrana , Técnicas de Placa-Clamp , Ratas , Ratas Endogámicas BB , ReflejoRESUMEN
OBJECTIVES: The aging kidney exhibits a progressive decline in renal function with characteristic histopathologic changes and is a risk factor for renal transplant. However, the degree to which the kidney exhibits this decline depends on several factors that vary from one individual to the next. Optical coherence tomography is an evolving noninvasive imaging technology that has recently been used to evaluate acute tubular necrosis of living-human donor kidneys before their transplant. With the increasing use of kidneys from older individuals, it is important to determine whether optical coherence tomography also can distinguish the histopathology associated with aging. MATERIALS AND METHODS: In this investigation, we used Munich-Wistar rats to evaluate the ability of optical coherence tomography to detect histopathologic changes associated with aging. Optical coherence tomography observations were correlated with renal function and conventional light microscopic evaluation of these same kidneys. RESULTS: With the onset of severe proteinuria at 10 to 12 months of age, optical coherence tomography revealed tubular necrosis/atrophy, interstitial fibrosis, tubular dilation, and glomerulosclerosis. With a further deterioration in kidney function at 16 to 18 months of age (as indicated by rising creatinine levels), optical coherence tomography revealed more extensive interstitial fibrosis and tubular atrophy, increased tubular dilation with cyst formation and more sclerotic glomeruli. CONCLUSIONS: The foregoing observations suggest that optical coherence tomography can be used to detect the histopathology of progressive nephropathy associated with aging.
Asunto(s)
Envejecimiento , Riñón/diagnóstico por imagen , Tomografía de Coherencia Óptica , Animales , Masculino , Ratas , Ratas Endogámicas BBRESUMEN
Type 1 Diabetes (T1D) is an autoimmune disease characterized by the pancreatic infiltration of immune cells resulting in T cell-mediated destruction of the insulin-producing beta cells. The successes of the Non-Obese Diabetic (NOD) mouse model have come in multiple forms including identifying key genetic and environmental risk factors e.g. Idd loci and effects of microorganisms including the gut microbiota, respectively, and how they may contribute to disease susceptibility and pathogenesis. Furthermore, the NOD model also provides insights into the roles of the innate immune cells as well as the B cells in contributing to the T cell-mediated disease. Unlike many autoimmune disease models, the NOD mouse develops spontaneous disease and has many similarities to human T1D. Through exploiting these similarities many targets have been identified for immune-intervention strategies. Although many of these immunotherapies did not have a significant impact on human T1D, they have been shown to be effective in the NOD mouse in early stage disease, which is not equivalent to trials in newly-diagnosed patients with diabetes. However, the continued development of humanized NOD mice would enable further clinical developments, bringing T1D research to a new translational level. Therefore, it is the aim of this review to discuss the importance of the NOD model in identifying the roles of the innate immune system and the interaction with the gut microbiota in modifying diabetes susceptibility. In addition, the role of the B cells will also be discussed with new insights gained through B cell depletion experiments and the impact on translational developments. Finally, this review will also discuss the future of the NOD mouse and the development of humanized NOD mice, providing novel insights into human T1D.