RESUMEN
An increase in circulating immune complexes (AgAb) of medium size has been observed in diabetics with late complications. This increase may be related either to an increased formation or reduced clearance. Alternatively, both mechanisms may be involved. As medium-sized AgAb determined by the C1q solid phase method are mainly removed from circulation by the fixed phagocytes of the reticulo-endothelial system, we investigated the function of these cells using a colloid clearance test in diabetics with various degrees of microangiopathy. Microaggregated iodinated human serum albumin was injected into 30 diabetic volunteers with severe (group 1), moderate (group 2), and absent (group 3) microangiopathy, and into 40 normal volunteers. The colloid clearance was significantly reduced in diabetics with severe microangiopathy in comparison with patients who had no sign of microangiopathy, or with normal subjects. A significant correlation was found between reduced colloid clearance and increased levels of circulating AgAb determined by C1q solid phase method. Results of this study suggest that the increase in circulating AgAb observed in patients with severe microangiopathy may result from an impaired function of mixed phagocytes.
Asunto(s)
Complejo Antígeno-Anticuerpo/análisis , Angiopatías Diabéticas/inmunología , Fagocitos/fisiología , Adulto , Anciano , Enzimas Activadoras de Complemento/análisis , Complemento C1q , Retinopatía Diabética/inmunología , Femenino , Hemoglobina Glucada/análisis , Humanos , Riñón/fisiopatología , Masculino , Persona de Mediana Edad , Albúmina Sérica/análisisRESUMEN
Human sera from 51 recent-onset insulin-dependent (type I) diabetic patients and 47 unrelated control subjects were screened for the possible presence of circulating factors reacting with several anti-pancreatic islet monoclonal antibodies (MoAb.ISL) in solid-phase radioimmunoassay methods (the original goal being the detection of anti-idiotypic islet cell antibodies and/or specific islet cell antigen-bearing immune complexes). MoAbs from the parental myeloma cell line and purified immunoglobulins (Igs) from different animal species were controls. Type I diabetic sera showed significantly increased binding to MoAb.ISL-coated wells compared with normal subjects (P less than .001). However, the same sera also tended to show a higher binding to the control (non-islet-related) MoAb. Sera from type I diabetic patients also reacted with horse, bovine, pig, rabbit, and goat IgG. Displacement of the binding has been obtained by F(ab')2 and/or Fc fragments of IgG. Evidence has been obtained regarding a similar reaction with human IgM. All the sera were negative when tested for rheumatoid factor by nephelometry. The circulating antibodies described have been proven to be different from islet cell autoantibodies. An anti-Ig antibody is thus present in the sera of recent-onset diabetic patients and represents an additional immunological phenomenon with possible physiopathological and clinical significance.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Diabetes Mellitus Tipo 1/inmunología , Inmunoglobulinas/inmunología , Islotes Pancreáticos/inmunología , Adulto , Femenino , Humanos , Inmunidad , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Inmunoglobulinas/metabolismo , Masculino , Factor Reumatoide/análisisRESUMEN
To investigate the role of protein charge in early diabetic proteinuria, the clearance of proteins differing in charge and/or size (anionic and cationic Igs, albumin) was evaluated in 98 insulin-dependent (type I) diabetic patients selected as a representative sample of the 418 patients attending our clinics. Of the patients, 12.9% were microalbuminuric and 4.8% were macroalbuminuric. Anionic and total IgG clearances were significantly increased in 30.6 and 12.2% of patients and were correlated with duration of disease. Anionic IgG4 clearances were increased in patients (9.2%) with normal IgG excretion, suggesting that charge-selectivity impairment is responsible for protein loss. Anionic Ig clearances were also higher in some patients (14.3%) with normal albumin clearance, probably as a result of different glomerular filtration and/or tubular reabsorption. The anionic-cationic IgG clearance ratio tended to increase in parallel with albumin clearance, but once above macroalbuminuric levels, it tended to fall again, indicating the concomitant presence of size-selectivity loss. The anionic IgG clearance and the anionic-cationic IgG ratio, in addition to albumin excretion, may be valuable in assessing early kidney protein charge-selectivity impairment and better characterizing normoalbuminuric patients and those in the preclinical stage of diabetic nephropathy.
Asunto(s)
Albuminuria , Diabetes Mellitus Tipo 1/orina , Inmunoglobulina G/orina , Proteinuria , Adulto , Biomarcadores/orina , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/fisiopatología , Nefropatías Diabéticas/diagnóstico , Estudios de Seguimiento , Tasa de Filtración Glomerular , Humanos , Inmunoglobulina G/clasificación , Tasa de Depuración Metabólica , Modelos EstadísticosRESUMEN
The possible influence of diabetes on the higher mnestic and cognitive functions has been investigated. The P300 wave latency, an endogenous electrophysiological event, was explored and compared with the multimodal short-latency evoked potential (EP) recordings (visual [VEP], brainstem auditory [BAEP], and median and tibial nerve somatosensory EPs [mSEP and tSEP, respectively]) and psychometric test measures in 16 insulin-dependent diabetic (IDDM) patients, in 16 age- and (IDDM) sex-matched nondiabetic subjects, and in a large normal reference population. The age of subjects, the duration of IDDM, and the metabolic control of patients were taken into account. P300 values were significantly increased in IDDM versus matched control subjects (P less than 0.001), and 3 patients showed values above the reference value range. Abnormal VEP recordings were present in 1 of 16 patients, BAEP in 3 of 16, mSEP in 7 of 16, and tSEP in 6 of 16. Digit-span backward test results were significantly (P less than 0.02) modified in the diabetic cohort. There was no tendency for anomalies of P300, short-latency EPs, and psychometric test values to be contemporarily present, except in 1 patient. Electrophysiological or psychometric abnormalities were not clearly correlated with the duration of IDDM or the degree of short-term metabolic control. These findings give evidence that 1) higher cognitive functions may be affected in diabetes as documented by P300 analysis and short-term memory tests, 2) endogenous electrophysiological analysis highlights neuropsychological changes not detectable by psychometric tests, 3) an alteration of evoked potentials was present in half of the IDDM subjects studied, and 4) anomalies of the CNS are patchily distributed in diabetes.
Asunto(s)
Tronco Encefálico/fisiopatología , Cognición , Diabetes Mellitus Tipo 1/psicología , Potenciales Evocados Auditivos , Potenciales Evocados Somatosensoriales , Potenciales Evocados Visuales , Nervio Mediano/fisiopatología , Memoria , Nervio Tibial/fisiopatología , Estimulación Acústica , Adulto , Diabetes Mellitus Tipo 1/fisiopatología , Potenciales Evocados , Femenino , Humanos , Masculino , Escalas de WechslerRESUMEN
Recent biochemical studies have shown that the cytoplasmic islet cell-antibody autoantigen has properties of a monosialoganglioside (GM). To characterize islet glycolipids and ascertain whether islets express unique gangliosides, we determined the pattern of ganglioside expression in whole human pancreas and isolated human islets using high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC). The major gangliosides detected in glycolipid extracts of whole human pancreas were GM3, GD3 (disialoganglioside), and in a lesser amount, a GD1a-comigrating ganglioside. In contrast to whole human pancreas, isolated human islets were found to predominantly express GM3, an acidic glycolipid comigrating with GM2, and a ganglioside with mobility between GM2 and GM1 by both HPLC and HPTLC. Quantitation of the major ganglioside UV peaks seen on HPLC gave the following results. In whole pancreas, GM3 represented 66.7% of total gangliosides detected; an asialoglycolipid comigrating with GM2, 2.0%; a ganglioside migrating between GM2 and GM1, 2.6%; GD3, 22.6%; and a GD1a-comigrating ganglioside, 6.1%. In isolated islets, these components were found at the following levels: GM3, 14.9%; GM2-comigrating glycolipid, 74.2%; a ganglioside migrating between GM2 and GM1, 9.8%; GD3, 1.1%; and the GD1a-comigrating ganglioside, not detectable.
Asunto(s)
Gangliósidos/análisis , Islotes Pancreáticos/análisis , Páncreas/análisis , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Gangliósido G(M2)/análisis , Gangliósido G(M3)/análisis , Glucolípidos/análisis , Humanos , Técnicas In VitroRESUMEN
We have studied the functional importance of renal eicosanoids in renal hemodynamics of seven newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients by treatment with two structurally unrelated inhibitors of cyclooxygenase (i.e., piroxicam and sulindac). Glomerular filtration rate (GFR), renal plasma flow (RPF), daily urinary excretion of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha, the stable hydrolysis product of prostacyclin), and thromboxane B2 (TXB2, the stable hydrolysis product of thromboxane A2) were measured before, during, and after piroxicam (all patients) or sulindac (3 patients) treatment. Urinary excretion of 6-keto-PGF1 alpha was significantly increased (P less than .01) in diabetic patients compared with seven healthy subjects, whereas urinary excretion of TXB2 was unchanged. The baseline value of GFR was significantly (P less than .01) higher in diabetic compared with normal volunteers, whereas baseline RPF was comparable in both groups. Piroxicam (20 mg/day) reduced urinary excretion of 6-keto-PGF1 alpha and TXB2 by 65.7 +/- 26 and 64.6 +/- 33%, respectively. These biochemical changes were temporally associated with the approximately 19% decrease in GFR (P less than .01). A week after discontinuation of the drug, GFR and urinary excretion of 6-keto-PGF1 alpha were still significantly (P less than .05) reduced, whereas urinary excretion of TXB2 returned to control values. In contrast, urinary excretion of eicosanoids and renal function were not affected by sulindac (0.4 g/day) treatment. No functional changes were detected in healthy subjects despite a similar suppression of renal cyclooxygenase activity when they were treated with piroxicam.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
6-Cetoprostaglandina F1 alfa/orina , Diabetes Mellitus Tipo 1/orina , Tromboxano A2/orina , Adolescente , Adulto , Creatinina/orina , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/fisiopatología , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Humanos , Riñón/fisiopatología , Masculino , Piroxicam/uso terapéutico , Sulindac/uso terapéuticoRESUMEN
Nonenzymatic glycation has been implicated in the pathogenesis of the dysregulated tissue remodeling that characterizes diabetic glomerulopathy, via the formation of advanced glycation end products (AGEs) and their binding to cell surface receptors. Several AGE-binding proteins have been identified so far, including p60, p90, and the adhesive and growth-regulating lectin galectin-3 (Gal-3), the components of the so-called AGE-receptor complex. This study aimed to evaluate the mesangial expression of the AGE-receptor complex and its modulation by the diabetic milieu, both in vivo, in non-diabetic versus streptozotocin-induced diabetic rats, and in vitro, in mesangial cells exposed to either normal glucose (NG) levels (5.5 mmol/l), as compared with high glucose (HG) levels (30 mmol/l) and iso-osmolar mannitol (M), or to native bovine serum albumin (BSA), as compared with glycated BSA with AGE formation (BSA-AGE) and glycated BSA in which AGE formation was prevented by aminoguanidine (BSA-AM). In vivo, Gal-3 protein and mRNA were not detectable in glomeruli from nondiabetic rats until 12 months after initiating the study. On the contrary, in diabetic rats, Gal-3 expression was observed at 2 months of disease duration, and it increased thereafter. Both p60 and p90 immunoreactivities were observed at the glomerular level with slightly increased expression of p90, but not p60, in diabetic versus nondiabetic animals. In vitro, Gal-3 was not detectable in mesangial cells cultured in NG (although it became evident after a certain number of passages in culture), whereas Gal-3 was detectable in cells grown on BSA. Prolonged exposure (2-4 weeks) of mesangial cells to HG but not to M, as well as growing cells on BSA-AGE and, to a lesser extent, BSA-AM, induced or significantly increased the expression of Gal-3, both protein (up to 2.65-fold) and mRNA (up to 3.10-fold) and its secretion in the medium (by approximately 50%). Both p60 and p90 were demonstrated in mesangial cells under NG conditions, and the expression of p90, but not p60, was upregulated by approximately 20% by HG or BSA-AGE. These results indicate that 1) under basal conditions, Gal-3, unlike p90 and p60, is not detectable in the mesangium but becomes expressed with aging and 2) the diabetic milieu induces or upregulates Gal-3 production, whereas it increases only slightly the expression of p90, but not p60. Gal-3 expression or overexpression may modulate the AGE-receptor-mediated events by modifying the function of the AGE-receptor complex. Additionally, it may exert direct effects on tissue remodeling by virtue of its adhesive and growth-regulating properties.
Asunto(s)
Antígenos de Diferenciación/genética , Diabetes Mellitus Experimental/fisiopatología , Regulación de la Expresión Génica/fisiología , Mesangio Glomerular/metabolismo , Glucosa/farmacología , Productos Finales de Glicación Avanzada/farmacología , Albúmina Sérica Bovina/farmacología , Envejecimiento/fisiología , Animales , Antígenos de Diferenciación/biosíntesis , Bovinos , Células Cultivadas , Galectina 3 , Regulación de la Expresión Génica/efectos de los fármacos , Mesangio Glomerular/citología , Mesangio Glomerular/patología , Humanos , Masculino , Manitol/farmacología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaRESUMEN
Enhanced advanced glycosylation end product (AGE) formation has been shown to participate in the pathogenesis of diabetes-induced glomerular injury by mediating the increased extracellular matrix (ECM) deposition and altered cell growth and turnover leading to mesangial expansion. These effects could be exerted via an AGE-receptor-mediated upregulation of growth factors, such as the IGFs and transforming growth factor-beta (TGF-beta). We tested this hypothesis in human and rat mesangial cells grown on nonglycated or native bovine serum albumin (BSA), glycated BSA with AGE formation (BSA-AGE), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM), in the presence or absence of an antibody, alpha-p60, directed against the p60/OST protein named AGE-receptor 1 (AGE-R1), or normal control (pre-immune) serum. The mRNA and/or protein levels of IGF-I, IGF-II, IGF receptors, IGF binding proteins (IGFBPs), TGF-beta1 and the ECM components fibronectin, laminin, and collagen IV were measured, together with cell proliferation. Both human and rat mesangial cells grown on BSA-AGE showed increased IGF-I and total and bioactive TGF-beta medium levels and enhanced IGF-I, IGF-II, and TGF-beta1 gene expression, compared with cells grown on BSA, whereas total IGFBP and IGFBP-3 medium content, IGF receptor density and affinity, and IGF-I receptor transcripts were unchanged. Moreover, cells grown on BSA-AGE showed increased ECM protein and mRNA levels versus cells cultured on BSA, whereas cell proliferation was unchanged in human mesangial cells and slightly reduced in rat mesangial cells. Growing cells on BSA-AM did not affect any of the measured parameters. Co-incubation of BSA-AGE with anti-AGE-R1, but not with pre-immune serum, prevented AGE-induced increases in IGF-I, TGF-beta1, and ECM production or gene expression; anti-AGE-R1 also reduced growth factor and matrix synthesis in cells grown on BSA. These results demonstrate that mesangial IGF and TGF-beta1 synthesis is upregulated by AGE-modified proteins through an AGE-receptor-mediated mechanism. The parallelism with increased ECM production raises the speculation that the enhanced synthesis of these growth factors resulting from advanced nonenzymatic glycation participates in the pathogenesis of hyperglycemia-induced mesangial expansion.
Asunto(s)
Proteínas de la Matriz Extracelular/biosíntesis , Matriz Extracelular/fisiología , Regulación de la Expresión Génica , Mesangio Glomerular/metabolismo , Productos Finales de Glicación Avanzada/farmacología , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Receptores Inmunológicos/fisiología , Albúmina Sérica Bovina/farmacología , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Colágeno/biosíntesis , Matriz Extracelular/efectos de los fármacos , Fibronectinas/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Guanidinas/farmacología , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Cinética , Laminina/biosíntesis , ARN Mensajero/biosíntesis , Ratas , Receptor para Productos Finales de Glicación Avanzada , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 2/biosíntesis , Receptores Inmunológicos/efectos de los fármacos , Albúmina Sérica/farmacología , Transcripción Genética/efectos de los fármacos , Regulación hacia ArribaRESUMEN
In this study, we investigated 1) whether long-term restoration of euglycemia by means of pancreatic islet transplants is capable of preventing and/or reversing renal functional and structural alterations in an experimental model of insulin-deficient diabetes, and 2) whether changes in extracellular matrix (ECM) and cell turnover at the glomerular level and biochemical abnormalities associated with hyperglycemia correlate with the renal outcome after transplantation. Male Lewis rats, rendered diabetic by intravenous injection of streptozotocin, underwent homologous islet transplantation via the portal vein at 2 weeks (study A), at 4 months (study B), and at 8 months (study C) after the induction of diabetes and killed 12 months after transplantation in study A and 4 months after transplantation in studies B and C. Age-matched nondiabetic and untreated diabetic rats were used as control animals and were studied at 4, 8, and 12 months. In the untreated diabetic animals, metabolic derangement was associated with increased erythrocyte polyol and fructose levels, tail-tendon content of advanced glycation end products (AGEs), total proteinuria, albuminuria, kidney weight, and mean glomerular volume as well as with marked glomerular and extraglomerular lesions. Glomerular gene expression for the ECM components fibronectin and collagen IV and for TGF-beta was also increased, whereas glomerular cell proliferation was unaffected by diabetes. In study A, changes in renal function and structure observed in diabetic rats at 12 months were completely prevented by successful islet transplants. In study B, all functional and structural abnormalities detected in diabetic rats at 4 months of disease duration were virtually reversed by 4 months of euglycemia in transplanted animals, whereas they progressed further in untreated diabetic rats. In study C, the course of functional and structural changes observed in untreated diabetic rats was not reversed by islet transplantation. Likewise, tissue AGE accumulation and particularly upregulation of glomerular ECM and transforming growth factor (TGF)-beta gene expression, which are believed to play a role in the pathogenesis of altered renal function and structure in diabetes, were normalized in transplanted rats from study A and study B, but not in those from study C. These experiments show that restoration of euglycemia by islet transplants is capable of preventing experimental diabetic glomerulopathy and reversing early changes in renal function and structure induced by diabetes. In a later phase of the disease, when glomerular matrix gene expression becomes independent of hyperglycemia, possibly because of the persistent increase in tissue AGE accumulation, metabolic control is not capable of reversing renal abnormalities.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/cirugía , Proteínas de la Matriz Extracelular/genética , Expresión Génica/genética , Trasplante de Islotes Pancreáticos , Glomérulos Renales/fisiopatología , Animales , Estudios de Cohortes , Colágeno/análisis , Colágeno/genética , Colágeno/metabolismo , Creatinina/sangre , Creatinina/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Experimental/prevención & control , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/análisis , Fibronectinas/genética , Fibronectinas/metabolismo , Glomérulos Renales/metabolismo , Glomérulos Renales/ultraestructura , Masculino , Estudios Prospectivos , Proteinuria/metabolismo , Proteinuria/orina , ARN Mensajero/análisis , ARN Mensajero/genética , Distribución Aleatoria , Ratas , Ratas Endogámicas Lew , Factores de Tiempo , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Recently, the GM2-1 pancreatic islet ganglioside, proposed as a potential autoantigen in type I diabetes autoimmunity, has been biochemically characterized and found to be a novel ganglioside structure. In the present study, we aimed to determine whether an autoimmune response toward this novel islet molecule is 1) present in type I diabetes and is specifically directed against this molecule and not to gangliosides in general and 2) predictive of disease in high-risk subjects. To this end, the following patients have been studied: 1) 24 newly diagnosed type I diabetic subjects, 20 islet cell autoantibody (ICA)-negative first-degree relatives of type I diabetic subjects, and 25 age-matched normal control individuals; and 2) 31 prospectively evaluated ICA+ first-degree relatives of type I diabetic subjects who were followed for up to 10 years, during which 14 of them developed type I diabetes. A direct assay for autoantibodies to GM2-1 and to other pancreatic gangliosides (GM3, GD3, GD1a) was developed using an indirect immunoperoxidase technique performed directly on thin layer chromatography plates. Anti-GM2-1 autoantibodies (all belonging to the IgG class) were expressed in a high percentage of newly diagnosed type I diabetic subjects (71%), while no significant difference was found in the expression of antibodies directed against other pancreatic gangliosides (GM3, GD3, GD1a) among the different groups studied. Anti-GM2-1 autoantibodies were also present in ICA+ relatives (64%) (P < 0.001 vs. control subjects and ICA-relatives): in this group, life table analysis of progression to diabetes showed that anti-GM2-1 autoantibodies were significantly (P < 0.001) associated with disease, occurring in all relatives developing type I diabetes within 5 years and thus identifying a cohort of ICA+ subjects with markedly increased diabetes risk.
Asunto(s)
Autoanticuerpos/sangre , Autoantígenos/inmunología , Autoinmunidad , Diabetes Mellitus Tipo 1/inmunología , Gangliósido G(M2)/inmunología , Islotes Pancreáticos/inmunología , Estado Prediabético/inmunología , Autoantígenos/aislamiento & purificación , Diabetes Mellitus Tipo 1/genética , Familia , Gangliósido G(M2)/aislamiento & purificación , Gangliósidos/inmunología , Humanos , Islotes Pancreáticos/química , Valores de ReferenciaRESUMEN
The elucidation of mechanisms regulating the regeneration and survival of pancreatic beta cells has fundamental implications in the cell therapy of type 1 diabetes. The present study had the following three aims: 1. to investigate whether pancreatic ductal epithelial cells can be induced to differentiate into insulin-producing cells by exposing them to hepatocyte growth factor (HGF); 2. to characterize some of the molecular events leading to their differentiation toward a beta-cell-like phenotype; 3. to evaluate the susceptibility of newly differentiated insulin-secreting cells to cytokine-induced apoptosis, a mechanism of beta-cell destruction occurring in type 1 diabetes. We demonstrated that HGF-treated rat pancreatic ductal cell line (ARIP) cells acquired the capability to transcribe the insulin gene and translate its counterpart protein. HGF-treated cells also exhibited a glucose-dependent capability to secrete insulin into the cultured medium. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent transcription of neurogenin-3 and Neuro-D in response to HGF. Finally, we determined the susceptibility to proinflammatory cytokine (PTh1)-induced apoptosis by incubating HGF-treated and untreated ARIP cells with a cocktail of interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Such treatment induced apoptotic death, as determined by the TUNEL technique, in about 40% of HGF-treated, insulin-secreting ARIP cells, while untreated ARIP cells were resistant to PTh1-induced apoptosis. In conclusion, we showed that HGF promotes the differentiation of ARIP cells into pancreatic beta-cell-like cells, and that the differentiation toward an insulin-secreting phenotype is associated with the appearance of susceptibility to cytokine-induced apoptosis.
Asunto(s)
Apoptosis/fisiología , Citocinas/farmacología , Células Epiteliales/efectos de los fármacos , Factor de Crecimiento de Hepatocito/farmacología , Insulina/metabolismo , Conductos Pancreáticos/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular/fisiología , Células Cultivadas , Citocinas/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Glucosa/metabolismo , Humanos , Insulina/genética , Secreción de Insulina , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Ratas , Receptor Notch3 , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Notch , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We used random peptide libraries displayed on phage to search for ligands to insulin dependent diabetes mellitus-related antibodies and were able to identify several candidate disease-related peptides. One of them, clone 92, showed a significant difference in the frequency of reactivity with the sera of patients and normal controls. Human immunoglobulins immunopurified on phage 92 specifically stained the islets on human pancreatic sections. When injected into rabbits, the selected peptide elicited antibodies that also stained human and rat pancreatic sections, with a pattern similar to that observed with immunoglobulins purified from the sera of patients. No reactivity was observed in other tissues. Our results indicate that the peptide identified in this work mimics a novel, diabetes-related self-antigen.
Asunto(s)
Autoantígenos/análisis , Diabetes Mellitus Tipo 1/inmunología , Epítopos/análisis , Islotes Pancreáticos/inmunología , Estado Prediabético/inmunología , Animales , Especificidad de Anticuerpos , Autoanticuerpos , Diabetes Mellitus Tipo 1/genética , Epítopos/genética , Femenino , Humanos , Sueros Inmunes , Biblioteca de Péptidos , Péptidos/análisis , Péptidos/genética , Péptidos/inmunología , Conejos , RatasRESUMEN
Several molecules were shown to bind advanced glycation end products (AGEs) in vitro, but it is not known whether they all serve as AGE receptors and which functional role they play in vivo. We investigated the role of galectin-3, a multifunctional lectin with (anti)adhesive and growth-regulating properties, as an AGE receptor and its contribution to the development of diabetic glomerular disease, using a knockout mouse model. Galectin-3 knockout mice obtained by gene ablation and the corresponding wild-type mice were rendered diabetic with streptozotocin and killed 4 months later, together with age-matched nondiabetic controls. Despite a comparable degree of metabolic derangement, galectin-3-deficient mice developed accelerated glomerulopathy vs. the wild-type animals, as evidenced by the more pronounced increase in proteinuria, extracellular matrix gene expression, and mesangial expansion. This was associated with a more marked renal/glomerular AGE accumulation, indicating it was attributable to the lack of galectin-3 AGE receptor function. The galectin-3-deficient genotype was associated with reduced expression of receptors implicated in AGE removal (macrophage scavenger receptor A and AGE-R1) and increased expression of those mediating cell activation (RAGE and AGE-R2). These results show that the galectin-3-regulated AGE receptor pathway is operating in vivo and protects toward AGE-induced tissue injury in contrast to that through RAGE.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Nefropatías Diabéticas/etiología , Receptores Inmunológicos/metabolismo , Animales , Antígenos de Diferenciación/genética , Glucemia/metabolismo , Peso Corporal , Colágeno Tipo IV/genética , Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/fisiopatología , Fibronectinas/genética , Galectina 3 , Expresión Génica , Genotipo , Hemoglobina Glucada/metabolismo , Riñón/metabolismo , Riñón/patología , Riñón/fisiopatología , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/sangre , Receptores Inmunológicos/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1RESUMEN
OBJECTIVE: Adrenomedullin (AM) is a potent hypotensive peptide which may be implicated in the insulin regulatory system. Acute hyperinsulinemia exerts no influence on plasma AM in normal subjects while no data on obese subjects has been reported. PURPOSE: The aim of the study was to investigate the effect of acute hyperinsulinemia on the plasma AM concentration in patients with uncomplicated obesity. RESEARCH METHODS: We measured the plasma AM levels in 23 obese subjects (BMI 41.9 +/- 9.8 kg/m2), 21 females and 2 males (mean age 31 +/- 7.2 years), before and during a euglycemic hyperinsulinemic clamp. The control group consisted of 43 healthy subjects (HS) (22 males and 21 females; mean age 38 +/- 12 years; BMI 23.3 +/- 3.2 kg/m2). RESULTS: Baseline plasma AM was found to be higher in obese subjects (20.4 +/- 8.4 pg/ml) than in normal subjects (11.3 +/- 0.8 pg/ml) (p < 0.001). A significant increase in the plasma AM levels was observed in obese subjects during acute hyperinsulinemia (from 20.4 +/- 8.4 pg/ml at 0 min to 26 +/- 8.9 pg/ml at 120 min, p < 0.02). Plasma AM concentrations were significantly correlated with insulin levels at 30 min (r = 0.44; p = 0.04) and 120 min (r = 0.40, p = 0.05) during the clamp. DISCUSSION: In conclusion, acute hyperinsulinemia induced a significant increase in the plasma levels of AM in uncomplicated obese subjects. Hyperinsulinemia may, at least in part, regulate levels of AM in obesity, explaining the high levels of the peptide in these subjects.
Asunto(s)
Hiperinsulinismo/sangre , Obesidad/sangre , Péptidos/sangre , Enfermedad Aguda , Adrenomedulina , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Concentración OsmolarRESUMEN
Maturity-onset diabetes of the young (MODY), an autosomal dominant, early-onset form of type-2 diabetes, is caused by mutations in five different genes all leading to defect(s) in the pancreatic beta cell. However, some patients with this form of diabetes do not bear a mutation in any of the known (MODY1-MODY5) loci, a notion prompting the search for new MODY genes. Clinical and genetic data point toward a defect in beta cell function in the majority of patients with MODY, and partners of the glucose-sensing device are reasonable functional candidates. The high-capacity glucose transporter GLUT2 has the ideal kinetic features for performing this task. However, complete GLUT2 deficiency in humans leads to hepato-renal glycogenosis (Fanconi-Bickel syndrome), and heterozygous GLUT2 mutations apparently behave in a recessive manner. Furthermore, in the human beta cell GLUT1 mRNA is predominant when compared to GLUT2 and glucose influx appears to be largely mediated by this low-Km transporter. Thus, we looked for the presence of sequence variants by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) within the GLUT1 gene in 90 Italian pedigrees negative at the search for mutations in glucokinase (MODY2) and hepatocyte nuclear factor-1alpha (MODY3), the two genes responsible for about 60% of MODY cases in Italian children. We found three already described silent mutations and a new single base deletion in position -173 of the 5' regulatory region. The -173de1A variant, which was detected in the heterozygous or homozygous state in 30.8% of MODY patients examined and is located in a Nuclear Factor Y binding sequence, is not associated with hyperglycemia in affected relatives of MODY probands. In conclusion, it appears from these results that the glucose transporter gene GLUT1 is unlikely to play a major role in the etiology of MODY diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Proteínas de Transporte de Monosacáridos/genética , Polimorfismo Conformacional Retorcido-Simple , Adolescente , Secuencia de Bases , Factor de Unión a CCAAT/metabolismo , Niño , Preescolar , Secuencia de Consenso/genética , Exones/genética , Transportador de Glucosa de Tipo 1 , Humanos , Italia , Mutación/genética , Reacción en Cadena de la Polimerasa , Elementos de Respuesta/genética , Factores de Transcripción/metabolismoRESUMEN
Type 1 diabetes is caused by the immune-mediated destruction of islet insulin-secreting beta-cells. This chronic destructive process is associated with both cellular and humoral immune changes in the peripheral blood that can be detected months or even years before the onset of clinical diabetes. Throughout this prediabetic period, metabolic changes, including altered glucose tolerance and reduced insulin secretion, deteriorate at variable rates and eventually result in clinical diabetes. A fraction of individuals with humoral immunological changes have clinical diabetes that initially is not insulin-requiring. The onset of diabetes in these patients is usually in adult life, and because their diabetes is at least initially not insulin-requiring, they appear clinically to be affected by type 2 diabetes. Such patients probably have the same disease process as patients with type 1 diabetes in that they have similar HLA genetic susceptibility as well as autoantibodies to islet antigens, low insulin secretion, and a higher rate of progression to insulin dependency. These patients are defined as being affected by an autoimmune type of diabetes not requiring insulin at diagnosis, which is also named latent autoimmune diabetes of the adult (LADA). Special attention should be paid to diagnose such patients because therapy may influence the speed of progression toward insulin dependency, and in this respect, efforts should be made to protect residual C-peptide secretion. LADA can serve as a model for designing new strategies for prevention of type 1 diabetes but also as a target group for prevention in its own right.
Asunto(s)
Diabetes Mellitus Tipo 1/clasificación , Diabetes Mellitus Tipo 1/fisiopatología , Islotes Pancreáticos/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Diabetes Mellitus Tipo 1/terapia , Humanos , Insulina/uso terapéutico , Islotes Pancreáticos/patologíaRESUMEN
Biologic and immunogenic activities of semisynthetic human monocomponent insulins were examined in insulin-dependent diabetic patients (group 1). Patients treated with porcine monocomponent (group 2) and conventional (group 3) insulins were studied for control purposes. The patients were examined before the beginning of insulin treatment and for a 6-mo follow-up period. The data collected during the study show that insulin antibody levels were significantly lower in group 1 than in groups 2 and 3. Furthermore, the prevalence of immune complexes assays with the C1q solid phase technique failed to reveal any differences between the three groups. When the conglutinin binding test was used, the prevalence of immune complexes showed a slight but not significant reduction in group 1 and a significant increase in group 3. The metabolic control was similar in the three groups during follow-up and the insulin requirement was lower, but not significantly, in group 1 than in groups 2 and 3. These data suggest that with human monocomponent insulins equivalent glycemic control may be achieved at similar doses than those required with porcine monocomponent insulins. Furthermore, human insulin is the least immunogenic of the present available insulins.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Insulina/normas , Adulto , Animales , Complejo Antígeno-Anticuerpo/análisis , Unión Competitiva , Enzimas Activadoras de Complemento/metabolismo , Complemento C1q , Diabetes Mellitus Tipo 1/inmunología , Femenino , Estudios de Seguimiento , Hemoglobina Glucada/análisis , Humanos , Insulina/inmunología , Insulina/metabolismo , Masculino , Porcinos , Factores de TiempoRESUMEN
Clarification of the extent and mechanisms of damage to the central nervous system in diabetes is a frontier of current neurological research. Our aim was to obtain ample electrophysiological documentation of possible neurological abnormalities in both insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetic patients with a short duration of disease and without overt complications, taking into account metabolic control. Group 1 comprised 11 IDDM patients, and group 2 included 14 NIDDM patients treated with diet alone; the duration of disease was less than 4 yr, and no concomitant clinical complications were present. Age- and sex-matched normal subjects formed groups 3 and 4. Pattern visual evoked potentials (VEP), brain stem auditory evoked potentials (BAEP), and somatosensory evoked potentials (SEP; after the stimulation of both median and tibial nerves) were recorded in all subjects, and metabolic control was evaluated in terms of glycemia and glycosylated hemoglobin. In group 1, significant abnormalities were found in the latency values of VEP, median SEP, and tibial SEP compared with control subjects. Similar latency abnormalities were shown in group 2 for VEP, median SEP, and tibial SEP values and for wave I latency of BAEP. Glycosylated hemoglobin values were correlated with BAEP and SEP abnormalities in many patients in both groups. Furthermore, in group 2, glycemic values correlated with SEP abnormalities. We therefore conclude that neurophysiological abnormalities are present at different levels in IDDM and NIDDM patients only a few years after clinical diagnosis and before the appearance of overt complications, and these abnormalities seem to be correlated with metabolic control status.
Asunto(s)
Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Neuropatías Diabéticas/diagnóstico , Potenciales Evocados Auditivos , Potenciales Evocados Somatosensoriales , Potenciales Evocados Visuales , Adulto , Glucemia/análisis , Tronco Encefálico/fisiopatología , Neuropatías Diabéticas/fisiopatología , Neuropatías Diabéticas/prevención & control , Femenino , Hemoglobina Glucada/análisis , Humanos , Masculino , Persona de Mediana Edad , Valores de ReferenciaRESUMEN
Recent observations have shown that the presumed target antigen of cytoplasmic islet cell antibodies (ICA) has properties of a monosialo-ganglioside migrating between GM2 and GM1 standards (GM2-1) and that ICA binding is higher in nonobese diabetic (NOD) than in C57BL/10SnJ mouse pancreatic frozen sections. This study aimed to characterize the ganglioside expression in NOD mouse islets in comparison with the control C57BL/10SnJ strain, taking into account possible sex differences, variations with age, and changes after autoimmune beta-cell destruction. Thus, acidic glycolipid composition was analyzed 1) in isolated islets from 11-week-old female and male NOD mice and age-matched female and male C57BL/10SnJ mice, and 2) in whole pancreas of both NOD and control mouse strains at different ages (4, 8, and 18 weeks) and of female NOD mice before and after diabetes onset. The acidic glycolipid GM2-1 is expressed in isolated female NOD islets, male NOD islets, and C57BL/10SnJ mouse islets, but quantitative analysis showed an increased amount of GM2-1 in NOD vs. C57BL/10 islets. GM3 is a ganglioside fraction expressed in female and male NOD mice and not in the C57BL/10 strain, whereas GD3 characterizes the C57BL/10 strain islets. GM2-1 is the sole ganglioside fraction in the whole pancreas to clearly decrease with age in the NOD mouse, and diabetes onset in this strain is associated with a significant decrease in the expression of this component as well as of GM3, whereas other pancreatic ganglioside (GD3, GD1a, and GT1b) levels did not significantly decrease; no age-related ganglioside change was observed in the C57BL/10SnJ mouse. Interestingly, the observed increased ICA binding in NOD islets is paralleled by the increased expression of GM2-1 islet ganglioside, and beta-cell destruction in NOD mice is associated with a significant decrease in the amount of this ganglioside in the pancreas.
Asunto(s)
Autoinmunidad , Diabetes Mellitus Tipo 1/metabolismo , Gangliósidos/análisis , Islotes Pancreáticos/química , Ratones Endogámicos NOD/metabolismo , Factores de Edad , Animales , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Femenino , Islotes Pancreáticos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Páncreas/química , Factores SexualesRESUMEN
Murine monoclonal antibodies (MAbs) HISL-5, -9, and -14, generated after immunization of mice with human pancreatic islet cell preparations, recognize a differentiation antigen expressed by the pancreatic islet cells. These MAbs react strongly with all endocrine cell subtypes of human pancreatic islets, but minimally if at all with the exocrine acinar cells, vascular cells, and stromal connective tissue cells of the pancreas. The antigen is located on the cell surface (plasma membranes), as indicated by immunofluorescence staining of viable cell preparations. Besides the pancreatic islets, HISL-5, -9, and -14 antigenic determinants are also expressed by thyroid follicular cells, parathyroid chief cells, and anterior pituitary cells, other commonly involved targets in organ-specific autoimmune disorders. Preliminary biochemical findings indicated that the MAb-defined epitope(s) is trypsin sensitive and resistant to periodate oxidation and exposure to chloroform-methanol. Further biochemical studies, including single step MAb immunoaffinity chromatographic purification, indicate that the antigen recognized by the MAbs HISL-5, -9, and -14 is a 100 K glycoprotein.