RESUMEN

BACKGROUND: Obesity is a worldwide concern due to its global rapid expansion and remarkable impact on individual's health by predisposing to several other diseases. About twice as many women as men suffer from severe obesity and, in fact, there are stages in a woman's life when weight gain and adiposity can result in greater damage to health. For example, obesity triples the chance of a woman developing gestational diabetes. Many hormones promote the metabolic adaptations of pregnancy, including progesterone, whose role in female obesity is still not well known despite being involved in many physiological and pathological processes. METHODS: Here we investigated whether progesterone treatment at low dose can worsen the glucose metabolism and the morpho functional aspects of adipose tissue and pancreas in obese females. Mice were assigned into four groups: normocaloric diet control (NO-CO), high-fat and -fructose diet control (HFF-CO), normocaloric diet plus progesterone (NO-PG) and high-fat and -fructose diet plus progesterone (HFF-PG) for 10 weeks. Infusion of progesterone (0.25 mg/kg/day) was done by osmotic minipump in the last 21 days of protocol. RESULTS: Animals fed a hypercaloric diet exhibited obesity with increased body weight (p < 0.0001), adipocyte hypertrophy (p < 0.0001), hyperglycemia (p = 0.03), and glucose intolerance (p = 0.001). HFF-CO and HFF-PG groups showed lower adiponectin concentration (p < 0.0001) and glucose-stimulated insulin secretion (p = 0.03), without differences in islet size. Progesterone attenuated glucose intolerance in the HFF-PG group (p = 0.03), however, did not change morphology or endocrine function of adipose tissue and pancreatic islets. CONCLUSIONS: Taken together, our results showed that low dose of progesterone does not worsen the effects of hypercaloric diet in glycemic metabolism, morphology and function of adipose tissue and pancreatic islets in female animals. These results may improve the understanding of the mechanisms underlying the pathogenesis of obesity in women and eventually open new avenues for therapeutic strategies and better comprehension of the interactions between progesterone effects and obesity.

Asunto(s)

Intolerancia a la Glucosa , Islotes Pancreáticos , Humanos , Masculino , Embarazo , Femenino , Ratones , Animales , Progesterona , Intolerancia a la Glucosa/complicaciones , Intolerancia a la Glucosa/patología , Ratones Obesos , Dieta Alta en Grasa/efectos adversos , Obesidad/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Tejido Adiposo/metabolismo , Aumento de Peso , Fructosa , Ratones Endogámicos C57BL , Insulina/metabolismo

RESUMEN

Type 1 diabetes (T1D) is an autoimmune disease that culminates in beta cell destruction in the pancreas and, subsequently, deficiency in insulin production. Cytokines play a crucial role in the development of diabetes, orchestrating the recruitment and action of immune cells, to not only destroy insulin-producing cells but also preserve them. Therefore, the aim of this study was to investigate the effect of orally administered Lactococcus lactis MG1363 FnBPA+ strains carrying plasmids encoding IL-4 and IL-10 in the streptozotocin- (STZ-) induced diabetes model and in nonobese diabetic (NOD) mice. The STZ-induced mice that were treated with combined bacterial strains carrying plasmids encoding IL-4 and IL-10 showed lower incidence of diabetes and more preserved pancreatic islets than the mice that received the individual bacterial strains. Combined administration of L. lactis MG1363 FnBPA+ (pValac::dts::IL-4) and L. lactis MG1363 FnBPA+ (pValac::IL-10) resulted in protection against diabetes in NOD mice. It was shown that the combined treatment with recombinant bacterial by oral route prevented hyperglycemia and reduced the pancreatic islets-destruction in NOD mice. In addition, increased levels of IL-4 and IL-10 in serum and pancreatic tissue revealed a systemic effect of the treatment and also favored an anti-inflammatory microenvironment. Reduced concentrations of IL-12 in pancreas were essential to the regulation of inflammation, resulting in no incidence of diabetes in treated NOD mice. Normal levels of intestinal sIgA after long-term treatment with the L. lactis strains carrying plasmids encoding IL-4 and IL-10 indicate the development of oral tolerance and corroborate the use of this potent tool of mucosal delivery. For the first time, L. lactis MG1363 FnBPA+ strains carrying eukaryotic expression vectors encoding IL-4 and IL-10 are tested in STZ-induced and NOD mouse models. Therefore, our study demonstrates this innovative strategy provides immunomodulatory potential for further investigations in T1D and other autoimmune diseases.

Asunto(s)

Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/prevención & control , Terapia Genética , Vectores Genéticos , Interleucina-10/genética , Interleucina-4/genética , Lactococcus lactis/genética , Animales , Glucemia/metabolismo , Colon/inmunología , Colon/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Inmunoglobulina A Secretora/metabolismo , Insulina/sangre , Interleucina-10/biosíntesis , Interleucina-10/sangre , Interleucina-4/biosíntesis , Interleucina-4/sangre , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Lactococcus lactis/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD

RESUMEN

Fasting is known to cause physiological changes in the endocrine pancreas, including decreased insulin secretion and increased reactive oxygen species (ROS) production. However, there is no consensus about the long-term effects of intermittent fasting (IF), which can involve up to 24 hours of fasting interspersed with normal feeding days. In the present study, we analyzed the effects of alternate-day IF for 12 weeks in a developing and healthy organism. Female 30-day-old Wistar rats were randomly divided into two groups: control, with free access to standard rodent chow; and IF, subjected to 24-hour fasts intercalated with 24-hours of free access to the same chow. Alternate-day IF decreased weight gain and food intake. Surprisingly, IF also elevated plasma insulin concentrations, both at baseline and after glucose administration collected during oGTT. After 12 weeks of dietary intervention, pancreatic islets displayed increased ROS production and apoptosis. Despite their lower body weight, IF animals had increased fat reserves and decreased muscle mass. Taken together, these findings suggest that alternate-day IF promote ß -cell dysfunction, especially in developing animals. More long-term research is necessary to define the best IF protocol to reduce side effects.

Asunto(s)

Tejido Adiposo/metabolismo , Ingestión de Alimentos , Ayuno/efectos adversos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Pérdida de Peso , Tejido Adiposo/patología , Animales , Apoptosis , Ayuno/fisiología , Femenino , Insulina/sangre , Secreción de Insulina , Músculos/metabolismo , Músculos/patología , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo

RESUMEN

Two siblings from a Mexican family who carried lethal Raine syndrome are presented. A newborn term male (case 1) and his 21 gestational week brother (case 2), with a similar osteosclerotic pattern: generalized osteosclerosis, which is more evident in facial bones and cranial base. Prenatal findings at 21 weeks and histopathological features for case 2 are described. A novel combination of biallelic FAM20C pathogenic variants were detected, a maternal cytosine duplication at position 456 and a paternal deletion of a cytosine in position 474 in exon 1, which change the reading frame with a premature termination at codon 207 and 185 respectively. These changes are in concordance with a negative detection of the protein in liver and kidney as shown in case 2. Necropsy showed absence of pancreatic Langerhans Islets, which are reported here for the first time. Corpus callosum absence is added to the few reported cases of brain defects in Raine syndrome. This report shows two new FAM20C variants not described previously, and negative protein detection in the liver and the kidney. We highlight that lethal Raine syndrome is well defined as early as 21 weeks, including mineralization defects and craniofacial features. Pancreas and brain defects found here in FAM20C deficiency extend the functional spectrum of this protein to previously unknown organs.

Asunto(s)

Anomalías Múltiples/genética , Quinasa de la Caseína I/genética , Fisura del Paladar/genética , Exoftalmia/genética , Proteínas de la Matriz Extracelular/genética , Microcefalia/genética , Osteosclerosis/genética , Anomalías Múltiples/metabolismo , Enfermedades del Desarrollo Óseo , Quinasa de la Caseína I/metabolismo , Fisura del Paladar/metabolismo , Cisteína/genética , Exoftalmia/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Familia , Femenino , Humanos , Recién Nacido , Islotes Pancreáticos/patología , Riñón/patología , Hígado/patología , Masculino , Microcefalia/metabolismo , Mutación , Osteosclerosis/metabolismo , Linaje , Fenotipo , Polimorfismo Genético/genética

RESUMEN

Chronic obesity imposes an organismal state of low-grade inflammation because the physiological resolution of inflammation is progressively repressed giving rise to cellular senescence and its accompanying Senescence-Associated Secretory Phenotype (SASP), which avoids apoptosis but perpetuates the relay of inflammatory signals from adipose tissue toward the rest of the body. Conversely, resolution of inflammation depends on the integrity of heat shock response (HSR) pathway that leads to the expression of cytoprotective and anti-inflammatory protein chaperones of the 70 kDa family (HSP70). However, chronic exposure to the aforementioned injuring factors leads to SASP, which, in turn, suppresses the HSR. A main metabolic tissue severely jeopardized by obesity-related dysfunctions is the endocrine pancreas, particularly ß-cells of the islets of Langerhans. Because exercise is a powerful inducer of HSR and predicted to alleviate negative health outcomes of obesity, we sought whether obesity influence HSP70 expression in pancreatic islets and other metabolic tissues (adipose tissue and skeletal muscle) of adult B6.129SF2/J mice fed on a high-fat diet (HFD) for 13 weeks since the weaning and whether acute exercise as well as moderate-intensity exercise training (8 weeks) could interfere with this scenario. We showed that acute exercise of moderate intensity protects pancreatic islets against cytokine-induced cell death. In addition, acute exercise challenge time-dependently increased islet HSP70 that peaked at 12 h post-exercise in both trained and untrained mice fed on a control diet, suggesting an adequate HSR to exercise training. Unexpectedly, however, neither exercise training nor acute exercise challenges were able to increase islet HSP70 contents in trained mice submitted to HFD, but only in untrained HFD animals. In parallel, HFD disrupted glycemic status which is accompanied by loss of muscular mass resembling sarcopenic obesity that could not be rescued by exercise training. These results suggest that exercise influences HSR in pancreatic islets but obesity undermines islet, muscle and adipose tissue HSR, which is associated with metabolic abnormalities observed in such tissues.

Asunto(s)

Tejido Adiposo/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Islotes Pancreáticos/metabolismo , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Condicionamiento Físico Animal , Animales , Dieta Alta en Grasa , Respuesta al Choque Térmico , Inflamación/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos

RESUMEN

AIMS: There is a pancreatic islet adaptation in obese subjects, resulting in insulin resistance and diabetes type 2. We studied the effect of intermittent fasting (IntF) on the islet structure of diet-induced obese (DIO) mice. METHODS: Three-month-old male mice fed a control diet (C, 10% Kcal fat) or a high-fat diet (HF, 50% Kcal fat) for two months (n = 20 each group). Then, half of each group did IntF (alternating 24 h fed/24 h fast), continuing in their diets four more weeks: C, C-IntF, HF, HF-IntF. Islets were prepared to microscopy or isolated for molecular analysis. RESULTS: HF group (vs. C group) showed hyperglycemia, hyperinsulinemia, hyperleptinemia, hypoadiponectinemia, glucose intolerance, insulin resistance, and islet hypertrophy with a consequent higher both the alpha-cell and beta-cell masses. In the HF group (vs. C), there was low PDX1 (pancreatic and duodenal homeobox 1), and IntF did not alter PDX1. There was a low p-AKT/AKT ratio (protein kinase B), and IntF enhanced it. Also, tumor suppressor p53 was increased, and IntF decreased it. IL (interleukin) -6 was higher in the HF group (vs. C), and HF-IntF (vs. C-IntF). Any significant change in NFkB was seen among groups. CONCLUSIONS: IntF improves pancreatic islet structure in DIO mice, even with continued HF diet intake, primarily considering on the alpha- and beta-cell masses regulation, then improving insulin signaling and decreasing cell apoptosis. Future research should explore whether the shortening of the IntF extend could maintain the benefits observed in the long term.

Asunto(s)

Ayuno/fisiología , Células Secretoras de Glucagón/patología , Células Secretoras de Insulina/patología , Islotes Pancreáticos/patología , Obesidad/patología , Animales , Recuento de Células , Proliferación Celular , Microambiente Celular/fisiología , Dieta Alta en Grasa , Islotes Pancreáticos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/etiología

RESUMEN

This chapter describes the propagation and characterization of transplantable insulinoma cells as model of insulin-producing pancreatic islet cells in the rat. Here, the cells are propagated by transplantation into rats followed by harvesting after growth for approximately 1 month. The cells are then purified by Percoll density gradient centrifugation and characterized by pulse-chase radiolabelling and immunoprecipitation of the insulin-related peptides. The results show that the transplantable insulinoma cells produce insulin in a manner similar to that found in normal pancreatic islets.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Inmunoprecipitación/métodos , Insulinoma/patología , Neoplasias Pancreáticas/genética , Animales , Proliferación Celular/genética , Humanos , Insulina/genética , Secreción de Insulina/genética , Insulinoma/genética , Islotes Pancreáticos/crecimiento & desarrollo , Islotes Pancreáticos/patología , Neoplasias Pancreáticas/patología , Ratas

RESUMEN

The islets of Langerhans release vital hormones involved in the regulation of blood sugar and other aspects of metabolism. The islets are housed in diffuse clusters of cells within the exocrine pancreas and, therefore, purification of these cells for research or transplant purposes is a difficult undertaking. Here, a detailed protocol is presented for purification of islets from rat pancreas using limited collagenase digestion and step gradient centrifugation techniques. In addition, a method for assessing islet viability is presented using perifusion under both basal and stimulatory glucose conditions, with measurement of the hormone released using an immunoassay for insulin.

Asunto(s)

Separación Celular/métodos , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/ultraestructura , Páncreas/metabolismo , Animales , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Páncreas/patología , Ratas

RESUMEN

AIMS: The objective of this study was to assess the mechanisms underlying pancreatic islet adaptation in diabetic mothers and their pups. Additionally, the influence of pancreatic adaptations on maternal reproductive performance was also investigated. MAIN METHODS: Wistar rats were injected with streptozotocin for diabetes induction. At adulthood (3 months), all animals underwent an oral glucose tolerance test (OGTT) for glucose assessment as an inclusion criterion. Following, the animals were mated. At day 18 of pregnancy, the mothers were killed for blood collect ion to determine fasting insulin and glucagon concentrations. The pancreas was removed and processed for the immunohistochemical analysis of insulin, glucagon, somatostatin, Ki-67 and PDX-1, superoxide dismutase 1 (SOD-1), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA). The pregnant uterus was also collected for the evaluation of embryofetal loss. KEY FINDINGS: The diabetic rats showed increased glucose, serum glucagon and insulin concentrations, and embryofetal loss rates. They also showed a reduction in pancreatic islets area and percentage of cells stained for insulin, increased the percentage of non-ß cells (alpha e delta cells) stained for Ki-67, glucagon, and somatostatin. Moreover, the cells stained for somatostatin were spread across the islets and showed stronger staining for MDA and weaker staining for GSH-Px. SIGNIFICANCE: Diabetes leads to adaptive responses from the endocrine pancreas in pregnancy that especially involves non-ß cells, modifying the mantle-core structure. Nonetheless, these adaptations are not enough for glucose homeostasis and affect the maternal environment, which in turn impairs fetal development.

Asunto(s)

Diabetes Mellitus Experimental/fisiopatología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Embarazo en Diabéticas/fisiopatología , Animales , Antioxidantes/metabolismo , Enzimas/metabolismo , Femenino , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Masculino , Estrés Oxidativo , Hormonas Pancreáticas/metabolismo , Embarazo , Ratas Wistar

RESUMEN

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing ß-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse ßTC6 ß-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of ß-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in ßTC6 ß-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human ß-cells, which in turn impacts the sensitivity of ß-cells to cytokine-mediated oxidative stress.

Asunto(s)

Metilación de ADN , Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas de la Membrana/metabolismo , Regiones Promotoras Genéticas , ARN Largo no Codificante/metabolismo , Animales , Proteínas de Unión al Calcio , Línea Celular , Citocinas/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/química , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Diabetes Mellitus Tipo 2/patología , Elementos de Facilitación Genéticos , Epigénesis Genética , Sitios Genéticos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Islotes Pancreáticos/patología , Región de Control de Posición , Proteínas de la Membrana/genética , Ratones , Mutación , Proteínas Nucleares , Estrés Oxidativo/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Bancos de Tejidos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

New compounds with promising antidiabetic activity were synthesized. For the first time, a portion of the glibenclamide molecule was bound to a part of the core structure of thiazolidinedione to evaluate insulin secretagogue activity. Following studies in our laboratory, 4-{2-[2-(3,4-dichlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]ethyl}benzene-1-sulfonamide (DTEBS) was selected to evaluate glycemia using the glucose tolerance test and insulin secretagogue activity by E.L.I.S.A. The mechanism of action of this compound was studied by 45 Ca2+ influx and whole-cell patch-clamp in rat pancreatic isolated islets. Furthermore, AGE formation in vitro was investigated. We herein show that this novel hybrid compound (DTEBS) exhibits an insulinogenic index and a profile of serum insulin secretion able to maintain glucose homeostasis. Its mechanism of action is mediated by ATP-sensitive potassium channels (KATP) and L-type voltage-dependent calcium channels (VDCC) and by activating protein kinase C and A (PKC and PKA). In addition, the stimulatory action of the compound on calcium influx and insulin secretion indicates that the potentiation of voltage-sensitive K+ currents (Kv) is due to the repolarization phase of the action potential after secretagogue excitation-secretion in pancreatic islets. Furthermore, under these experimental conditions, the compound did not induce toxicity and the in vitro late response of the compound to protein glycation reinforces its use to prevent complications of diabetes. DTEBS exerts an insulin secretagogue effect by triggering KATP, VDCC, and Kv ionic currents, possibly via PKC and PKA pathway signal transduction, in beta-cells. Furthermore, DTEBS may hold potential for delaying the late complications of diabetes.

Asunto(s)

Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Compuestos de Sulfonilurea/farmacología , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Ensayo de Inmunoadsorción Enzimática , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Gliburida/química , Gliburida/farmacología , Humanos , Hipoglucemiantes/síntesis química , Insulina/biosíntesis , Secreción de Insulina/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Canales KATP/genética , Técnicas de Placa-Clamp , Proteína Quinasa C/genética , Ratas , Transducción de Señal/efectos de los fármacos , Compuestos de Sulfonilurea/síntesis química , Tiazolidinedionas/síntesis química , Tiazolidinedionas/farmacología

RESUMEN

Low levels of estrogens are associated with obesity-related comorbidities. Mice with lower levels of estrogens are thereby more sensitive to the effects of a high-fat-diet (HFD) for the development of glucose intolerance and insulin resistance. Studies in vivo have demonstrated that taurine (TAU) supplementation prevents glucose and insulin resistance. Thus, we aimed to investigate the potential beneficial effects of TAU supplementation on glucose homeostasis of mice with low levels of estrogens fed with a HFD. 3-month-old female C57BL/6J mice underwent bilateral ovariectomy (OVX). After 1 week of recovery, mice were divided into 4 groups and either received: a standard chow diet (OVXC), chow diet plus drinking water enriched with 3% of TAU (OVXCT), HFD (OVXH), and HFD plus supplementation of TAU (OVXHT) for 14 weeks. Exposure to the HFD increased adiposity and plasma levels of glucose and insulin. Contrary to our prediction, the addition of TAU enhanced the deleterious effects of the HFD. Glucose and insulin tolerance tests (ipGTT and ipITT) indicated that mice maintained on the HFD + TAU had worse glucose intolerance and insulin resistance that was linked to lower insulin signaling in skeletal muscle and liver. Insulin secretion of isolated pancreatic islets of OVXH mice was higher than OVXC, and the addition of TAU associated with a HFD did not modulate insulin secretion, suggesting a failure of pancreatic ß cells of OVXHT mice. These results suggest that despite the beneficial reports of TAU, it should be used cautiously in situations where the levels of estrogens are low.

Asunto(s)

Suplementos Dietéticos , Glucosa/metabolismo , Obesidad/tratamiento farmacológico , Taurina/administración & dosificación , Animales , Glucemia/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Estrógenos/metabolismo , Homeostasis , Humanos , Insulina/metabolismo , Resistencia a la Insulina/genética , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Ovariectomía

RESUMEN

SUMMARY: Hyperglycaemia is one of the main causes for the endothelial cell (EC) damage in diabetic patients. Even though circulating endothelial progenitor cells (CEPC) could be used as a prognosis for microvascular complications, there is very little information on the islet microvasculature. We analysed by immunohistochemistry and by flow cytometric immunophenotyping, the expression of CD34 on EC and the expressions of CD31, CD34, CD45 and CD133 on CEPC in Streptozotocin (STZ)-induced diabetic rats. Peripheral blood and tissue specimens were obtained from rats of different treatment regimens: STZ treatment, control saline (NS) and sodium citrate (CB) treatments. Blood cells were exposed to flow cytometric immunophenotyping for CD133, CD31, CD34, CD45 and CD133. While tissues from the pancreas, liver and kidney were routinely processed and stained immunohistochemically for CD34. There was a tendency of an increased in CD45-/CD133+/CD31+/CD34+ cells (0.04 ± 0.11 %) in diabetic rats compared to the controls (CB: 0.03 ± 0.04 %; Saline: 0.01 ± 0.03 %). But there was no significant statistical difference between them. The expression pattern of CD34 on the EC in the organs' vascular beds including arterioles, venules, capillaries and sinusoids was extremely heterogeneous across and within treatment regimens. The ECs in the sinusoids of the liver presented similar CD34 expression patterns across different treatment regimens, while the expression of CD34 on the ECs of sinusoidal capillaries in the pancreas vary with the treatment regimen. We conclude that the degree of endothelial cell damage is not uniform across organs' vascular beds in the rat, contrary to mice and humans. Furthermore, the sinusoids in the pancreas and the kidney may have the same degree of endothelial damage when exposed to the same deleterious causes.

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RESUMEN: La hiperglucemia es una de las principales causas del daño de las células endoteliales (EC) en pacientes diabéticos. A pesar de que las células progenitoras endoteliales circulantes (CEPC) podrían utilizarse como pronóstico de las complicaciones microvasculares, hay muy poca información sobre la microvasculatura de los islotes. Se analizaron por inmunohistoquímica y por inmunofenotipificación citométrica de flujo, la expresión de CD34 en EC y las expresiones de CD31, CD34, CD45 y CD133 en CEPC en ratas diabéticas inducidas por estreptozotocina (STZ). Se obtuvieron muestras de sangre y tejidos periféricos a partir de ratas de diferentes regímenes de tratamiento: tratamiento con STZ, solución salina control (NS) y citrato de sodio (CB). Las células sanguíneas fueron expuestas a inmunofenotipado por citometría de flujo para CD133, CD31, CD34, CD45 y CD133. Mientras que los tejidos del páncreas, el hígado y el riñón fueron rutinariamente procesados y teñidos inmunohistoquímicamente para CD34. Se observó una tendencia a un aumento en las células CD45- / CD133 + / CD31 + / CD34 + (0,04 ± 0,11 %) en ratas diabéticas en comparación con los controles (CB: 0,03 ± 0,04 %; Salino: 0,01 ± 0,03 %). Pero no hubo diferencias estadísticamente significativas entre ellos. El patrón de expresión de CD34 en la EC en los lechos vasculares de los órganos incluyendo arteriolas, vénulas, capilares y sinusoides fue extremadamente heterogéneo a través de y dentro de los regímenes de tratamiento. Las EC en los sinusoides del hígado presentaron patrones de expresión de CD34 similares a través de diferentes regímenes de tratamiento, mientras que la expresión de CD34 en las CE de capilares sinusoidales en el páncreas varía con el régimen de tratamiento. Concluimos que el grado de daño de las células endoteliales no es uniforme en los lechos vasculares de los órganos en la rata, en comparación de los ratones y los seres humanos. Además, los sinusoides en el páncreas y el riñón pueden tener el mismo grado de daño endotelial cuando se exponen a las mismas causas deletéreas.

Asunto(s)

Animales , Masculino , Ratas , Diabetes Mellitus Experimental/patología , Células Progenitoras Endoteliales/patología , Islotes Pancreáticos/patología , Glucemia , Peso Corporal , Diabetes Mellitus Experimental/inmunología , Inmunofenotipificación , Islotes Pancreáticos/irrigación sanguínea , Riñón/patología , Hígado/patología , Ratas Wistar

RESUMEN

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in the pathogenesis of type 2 diabetes mellitus (T2DM). Although the effect of high glucose on liver function has been described, the role of MIF in hepatic mitochondrial function during T2DM has not been studied. OBJECTIVE: We examine the influence of MIF to hepatic mitochondrial function in T2DM mouse model. METHODS: WT and Mif-/- BALB/c mice were treated with a single dose of streptozotocin (STZ). After an 8-week follow-up, serum glucose, proinflammatory cytokines, C-reactive protein (CRP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme quantification, and liver histological analyses were performed. Liver mitochondria were extracted, and mitochondrial function was evaluated by oximetry, swelling and peroxide production. RESULTS: Following treatment with STZ, WT mice (WT/STZ) developed significant hyperglycemia and high serum levels of MIF, tumor necrosis factor (TNF)-α, interleukin-ß (IL-ß), and CRP. Liver damage enzymes ALT and AST were found at high levels. In contrast, Mif-/-STZ lacked serum MIF levels and showed smaller increases in blood glucose, less TNF-α, IL-1ß, CPR, ALT and AST, and failure to develop clinical signs of disease compared to the WT/STZ group. Mitochondria extracted from the Mif-/-STZ liver showed similar respiratory control (RC) to WT/STZ or healthy mice with glutamate/malate or succinate as substrates. The four respiratory chain complexes also had comparable activities. WT/STZ-isolated mitochondria showed low swelling with calcium compared to mitochondria from Mif-/-STZ or healthy mice. Peroxide production was comparable in all groups. CONCLUSION: These results show although high systemic levels of MIF contribute to the development of T2DM pathology, the liver mitochondria remain unaltered. Importantly, the absence of MIF reduced the pathology of T2DM, also without altering liver mitochondrial function. These support MIF as a therapeutic target for the treatment of this disease in humans.

Asunto(s)

Diabetes Mellitus Tipo 2/metabolismo , Mediadores de Inflamación/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Hígado/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Mitocondrias/metabolismo , Animales , Proteína C-Reactiva/metabolismo , Respiración de la Célula , Citocromos/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/patología , Electrodos , Hiperglucemia/complicaciones , Hiperglucemia/patología , Interleucina-1beta/sangre , Oxidorreductasas Intramoleculares/deficiencia , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Hígado/patología , Factores Inhibidores de la Migración de Macrófagos/deficiencia , Masculino , Ratones Endogámicos BALB C , Dilatación Mitocondrial , Oxígeno/metabolismo , Peróxidos/metabolismo , Estreptozocina , Transaminasas/metabolismo , Factor de Necrosis Tumoral alfa/sangre

RESUMEN

Several studies indicated that pancreatic ß-cell death occurs in both type 1 and type 2 diabetes. This experimental study was designed to determine the effect of gestational diabetes on the ß-cells in 16-week-old rat offspring. By this aim, adult Wistar rats aged 10-12 weeks were randomly allocated in control and diabetic groups. The diabetic group received 40 mg/kg/body weight of streptozotocin (STZ) on day zero of gestation. After delivery, diabetic offspring of GDM mothers and controls at the age of 16 weeks were sacrificed and pancreases harvested and fixed. The number of ß-cells and were counted by Gomori's method staining. Also, apoptosis in pancreas tissue of diabetic and control offspring was detected by TUNEL assay. Results showed a significant reduction in ß-cell number in offspring of GDM (p<0.05). TUNEL assay showed that the number of apoptotic cells increased in GDM compared to controls (P<0.05). This study revealed that gestational diabetes induces pancreatic beta-cells apoptosis in 16-week-old rat offspring.

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Varios estudios indican que la muerte de las células ß del páncreas se produce tanto en la diabetes Tipo 1 como en la Tipo 2. Este estudio experimental fue diseñado para determinar el efecto de la diabetes gestacional en las células ß del páncreas en crías de ratas de 16 semanas. Para ello, ratas Wistar adultas de entre 10-12 semanas fueron asignadas al azar en dos grupos: control y diabetes. El grupo diabetes recibió 40 mg / kg / peso corporal de estreptozotocina (STZ) en el día cero de la gestación. Después del parto, a las 16 semanas, las crías de las madres diabéticas y controles de madres con diabetes gestacional (MDG), fueron sacrificadas para la extracción del páncreas, el cual posteriormente fue fijado. Se contó el número de células ß del páncreas mediante tinción con el método de Gomori. Además, se detectó apoptosis en el tejido del páncreas de la descendencia diabética y el grupo control mediante un ensayo TUNEL. Los resultados mostraron una reducción significativa en el número de células b en la descendencia de MDG (p <0,05). El ensayo TUNEL mostró que el número de células apoptóticas aumentó en MDG en comparación con los controles (P <0,05). Este estudio reveló que la diabetes gestacional induce apoptosis de células ß en el páncreas de crías de ratas de 16 semanas.

Asunto(s)

Animales , Ratas , Apoptosis , Diabetes Gestacional/patología , Islotes Pancreáticos/patología , Glucemia/análisis , Etiquetado Corte-Fin in Situ , Páncreas/patología , Ratas Wistar

RESUMEN

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing ß-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B-chain amino acids 9-23 (B:9-23), which are known to be a critical self-antigen-driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment.

Asunto(s)

Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Insulina/inmunología , Islotes Pancreáticos/inmunología , Fragmentos de Péptidos/inmunología , Proinsulina/inmunología , Precursores de Proteínas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Adolescente , Péptido C/inmunología , Niño , Femenino , Antígenos HLA-DQ/inmunología , Antígenos HLA-DR/inmunología , Humanos , Células Secretoras de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/patología , Receptores de Antígenos de Linfocitos T/genética , Adulto Joven

RESUMEN

Macrophage migration inhibitory factor (Mif) is highly expressed in type 1 diabetes mellitus (T1DM). However, there is limited information about how Mif influences the activation of macrophages (Mφ) and dendritic cells (DC) in T1DM. To address this issue, we induced T1DM by administering multiple low doses of streptozotocin (STZ) to Mif-/- or wild-type (Wt) BALB/c mice. We found that Mif-/- mice treated with STZ (Mif-/-STZ) developed lower levels of hyperglycemia, inflammatory cytokines, and specific pancreatic islet antigen- (PIAg-) IgG and displayed reduced cellular infiltration into the pancreatic islets compared to Wt mice treated with STZ (WtSTZ). Moreover, Mφ and DC from Mif-/-STZ displayed lower expression of MHC-II, costimulatory molecules CD80, CD86, and CD40, Toll-like receptor- (TLR-) 2, and TLR-4 than WtSTZ. These changes were associated with a reduced capacity of Mφ and DC from Mif-/-STZ to induce proliferation in ovalbumin-specific T cells. All the deficiencies observed in Mif-/-STZ were recovered by exogenous administration of recombinant Mif. These findings suggest that Mif plays a role in the molecular mechanisms of Mφ and DC activation and drives T cell responses involved in the pathology of T1DM. Therefore, Mif is a potential therapeutic target to reduce the pathology of T1DM.

Asunto(s)

Células Dendríticas/inmunología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/inmunología , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Macrófagos/inmunología , Células TH1/inmunología , Animales , Autoanticuerpos/inmunología , Antígeno B7-1/inmunología , Antígeno B7-2/inmunología , Glucemia/metabolismo , Antígenos CD40/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Inmunoglobulina G , Interferón gamma/inmunología , Oxidorreductasas Intramoleculares/inmunología , Islotes Pancreáticos/patología , Factores Inhibidores de la Migración de Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Índice de Severidad de la Enfermedad , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

Neuromedin B (NB) and gastrin-releasing peptide (GRP) are bombesin-like peptides, found in the gastrointestinal tube and pancreas, among other tissues. Consistent data proposed that GRP stimulates insulin secretion, acting directly in pancreatic cells or in the release of gastrointestinal hormones that are incretins. However, the role of NB remains unclear. We examined the glucose homeostasis in mice with deletion of NB receptor (NBR-KO). Female NBR-KO exhibited similar fasting basal glucose with lower insulinemia (48.4%) and lower homeostasis model assessment of insulin resistance index (50.5%) than wild type (WT). Additionally, they were more tolerant to oral glucose, demonstrated by a decrease in the area under the glucose curve (18%). In addition, 15 min after an oral glucose load, female and male NBR-KO showed lower insulin serum levels (45.6 and 26.8%, respectively) than WT, even though blood glucose rose to similar levels in both groups. Single injection of NB, one hour before the oral glucose administration, tended to induce higher serum insulin in WT (28.9%, p=0.3), however the same did not occur in NBR-KO. They showed no changes in fasting insulin content in pancreatic islets by immunohistochemistry, however, the fasting serum levels of glucagon-like peptide, a potent incretin, exhibited a strong trend to reduction (40%, p=0.07). Collectively, mice with deletion of NB receptor have lower insulinemia, especially in response to oral glucose, and females also exhibited a better glucose tolerance, suggesting the involvement of NB and its receptor in regulation of insulin secretion induced by incretins, and also, in insulin sensitivity.

Asunto(s)

Eliminación de Gen , Glucosa/administración & dosificación , Glucosa/farmacología , Insulina/metabolismo , Receptores de Bombesina/metabolismo , Administración Oral , Animales , Ayuno , Femenino , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Prueba de Tolerancia a la Glucosa , Homeostasis/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroquinina B/administración & dosificación , Neuroquinina B/análogos & derivados , Neuroquinina B/farmacología , Receptores de Bombesina/deficiencia

RESUMEN

Pancreatic beta cell (ß) dysfunction is an outcome of malnutrition. We assessed the role of the amplifying pathway (AMP PATH) in ß cells in malnourished obese mice. C57Bl-6 mice were fed a control (C) or a low-protein diet (R). The groups were then fed a high-fat diet (CH and RH). AMP PATH contribution to insulin secretion was assessed upon incubating islets with diazoxide and KCl. CH and RH displayed increased glucose intolerance, insulin resistance and glucose-stimulated insulin secretion. Only RH showed a higher contribution of the AMP PATH. The mitochondrial membrane potential of RH was decreased, and ATP flux was unaltered. In RH islets, glutamate dehydrogenase (GDH) protein content and activity increased, and the AMP PATH contribution was reestablished when GDH was blunted. Thus, protein malnutrition induces mitochondrial dysfunction in ß cells, leading to an increased contribution of the AMP PATH to insulin secretion through the enhancement of GDH content and activity.

Asunto(s)

Envejecimiento/patología , Insulina/metabolismo , Desnutrición Proteico-Calórica/metabolismo , Animales , Intolerancia a la Glucosa/complicaciones , Intolerancia a la Glucosa/metabolismo , Intolerancia a la Glucosa/patología , Glutamato Deshidrogenasa/metabolismo , Resistencia a la Insulina , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones Endogámicos C57BL , Ratones Obesos , Mitocondrias/metabolismo , Desnutrición Proteico-Calórica/complicaciones , Desnutrición Proteico-Calórica/patología

RESUMEN

Pyranocoumarins are compounds with an important pharmacological profile, such as anti-inflammatory, antioxidant, cytotoxic, antiviral, antibacterial, and hypoglycemic effects. These molecules have a widespread presence as secondary metabolites in medicinal plants used to treat Diabetes Mellitus (DM). The aim of this work was to evaluate antidiabetic activity in Streptozotocin (STZ)-induced diabetic rats and the antioxidant effects of 3',4'-Di-O-acetyl-cis-khellactone (DOAcK), as well as its toxic potential. We obtained DOAcK with an enantiomeric excess of 70% by chemical synthesis. Our results showed that this compound exerts an important antidiabetic effect: blood glucose decreased in groups treated with DOAcK by 60.9% at dose of 15mg/kg (p<0.05) compared with the diabetic control group, and demonstrated a statistically significant increase in weight gain (45.7±9.7 in the group treated with DOAcK vs. -23.0±33.1 in the group with diabetes). In a biochemical profile, DOAcK did not modify lipid metabolism and did not cause damage at the renal level. DOAcK administration increased the activities of Catalase (CAT), Glutathione Peroxidase (GPx), and Super Oxide Dismutase (SOD) to levels near those of the healthy group. Histopathological analysis exhibited morphology similar to that of the healthy group and the group treated with DOAcK. DOAcK is not mutagenic by Ames test for Salmonella typhimurium strains TA98, TA100, or TA102, and is not genotoxic by Micronucleus assay; median lethal dose (LD50) >2000mg/kg and, at this dose, no signs of toxicity or death were reported after 14days of observation. These results indicate that DOAcK can improve glucose metabolism, which may be due to the increased antioxidant activity of CAT, GPx and SOD. In addition, DOAcK is not toxic in the studies tested.

Asunto(s)

Antioxidantes/química , Cumarinas/química , Hipoglucemiantes/química , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Glucemia/análisis , Peso Corporal/efectos de los fármacos , Catalasa/metabolismo , Cumarinas/farmacología , Cumarinas/uso terapéutico , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Hígado/efectos de los fármacos , Hígado/patología , Pruebas de Mutagenicidad , Estrés Oxidativo/efectos de los fármacos , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Estreptozocina/toxicidad , Superóxido Dismutasa/metabolismo