RESUMEN
AIMS/HYPOTHESIS: Cyclin-dependent kinase 4 (Cdk4) is crucial for beta cell development. A mutation in the gene encoding for Cdk4, Cdk4R24C, causes this kinase to be insensitive to INK4 cell cycle inhibitors and induces beta cell hyperplasia in Cdk4R24C knockin mice. We aimed to determine whether this Cdk4R24C mutation also affects proper islet function, and whether it promotes proliferation in human islets lentivirally transduced with Cdk4R24C cDNA. METHODS: Our study was conducted on wild-type and Cdk4R24C knockin mice. Pancreases were morphometrically analysed. Intraperitoneal glucose tolerance tests and intravenous insulin tolerance tests were performed on wild-type and Cdk4R24C mice. We also did in vitro islet perifusion studies and islet metabolic labelling analysis. Human islets were transduced with Cdk4R24C cDNA. RESULTS: Pancreatic islets from Cdk4R24C knockin mice exhibit a larger insulin-producing beta cell area and a higher insulin content than islets from wild-type littermates. Insulin secretion in response to glucose is faster and reaches a higher peak in Cdk4R24C mice without leading to hypoglycaemia. Conversion of pro-insulin into insulin and its intermediates is similar in Cdk4R24C and wild-type mice. Glucose utilisation and oxidation measured per islet were similar in both experimental groups. Insulin secretion was faster and enhanced in Cdk4R24C islets perifused with 16.7 mmol/l glucose, with slower decay kinetics when glucose returned to 2.8 mmol/l. Moreover, human islets expressing Cdk4R24C cDNA exhibited higher beta cell proliferation. CONCLUSIONS/INTERPRETATION: Despite their hyperplastic growth, Cdk4R24C insulin-producing islet cells behave like differentiated beta cells with regard to insulin production, insulin secretion in response to glucose, and islet glucose metabolism. Therefore Cdk4 could possibly be used to engineer a source of beta cell mass for islet transplantation.
Asunto(s)
Quinasas Ciclina-Dependientes/genética , Diabetes Mellitus Tipo 1/terapia , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Proteínas Proto-Oncogénicas/genética , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Quinasa 4 Dependiente de la Ciclina , Activación Enzimática/fisiología , Terapia Genética , Vectores Genéticos , Glucosa/metabolismo , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/genética , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Lentivirus/genética , Ratones , Ratones Transgénicos , Tamaño de los Órganos , Oxidación-Reducción , Proinsulina/biosíntesis , Proinsulina/genética , Regeneración/genéticaRESUMEN
In a new experimental type 2 diabetic syndrome, a 40% reduction of pancreatic beta cells was observed by morphometric analysis. In diabetic islets, as compared to control islets, insulin release was decreased in response to high glucose but not to other stimuli, and total glucose oxidation and utilization were unchanged or slightly reduced. The extent of metabolic and functional impairment appeared proportional to the beta-cell loss. However, a substantial decrease was found in protein level and activity (by 77 and 60%, respectively, versus controls) of mitochondrial FAD-glycerophosphate dehydrogenase (mGDH), the key enzyme of the glycerophosphate shuttle. Interestingly, in diabetic islets, as recently reported for mGDH-deficient transgenic mice, definite functional alterations (mainly in response to D-glyceraldehyde) were only obtained upon pharmacological blockade of the second shuttle (i.e. malate-aspartate) responsible for mitochondrial transfer of reducing equivalents. In conclusion, in this diabetes model with reduction of beta-cell mass, the islets, despite decreased mGDH amount and activity, appear metabolically and functionally active in vitro, likely through the intervention of adaptive mechanisms, yet prone to failure in challenging situations.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Islotes Pancreáticos/patología , Ácido Aminooxiacético/farmacología , Animales , Diabetes Mellitus Tipo 2/inducido químicamente , Diabetes Mellitus Tipo 2/etiología , Glucosa/metabolismo , Glucosa/farmacología , Glicerolfosfato Deshidrogenasa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Masculino , Mitocondrias/enzimología , Niacinamida , Ratas , Ratas Wistar , EstreptozocinaRESUMEN
Ca2+-responsive mitochondrial FAD-linked glycerophosphate dehydrogenase (mGPDH) is a key component of the pancreatic beta-cell glucose-sensing device. The purpose of this study was to examine the association of mutations in the cDNA coding for the FAD-binding domain of mGPDH and to explore the functional consequences of these mutations in vitro. To investigate this association in type 2 diabetes mellitus, we studied a cohort of 168 patients with type 2 diabetes and 179 glucose-tolerant control subjects of Spanish Caucasian origin by single-stranded conformational polymorphism analysis. In vitro site-directed mutagenesis was performed in the mGPDH cDNA sequence to reproduce those mutations that produce amino acid changes in a patient with type 2 diabetes. We detected mutations in the mGPDH FAD-binding domain in a single patient, resulting in a Gly to Arg amino acid change at positions 77, 78, and 81 and a Thr to Pro at position 90. In vitro expression of the mutated constructs in Xenopus oocytes resulted in a significantly lower enzymatic activity than in cells expressing the wild-type form of the enzyme. Our results indicate that although mutations in the mGPDH gene do not appear to have a major role in type 2 diabetes mellitus, the reduction in mGPDH enzymatic activity associated with the newly described mGPDH mutations suggests that they may contribute to the disease in some patients.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Flavina-Adenina Dinucleótido/metabolismo , Glicerolfosfato Deshidrogenasa/genética , Glicerolfosfato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Mutación , Anciano , Animales , Células Cultivadas , Estudios de Cohortes , ADN Complementario/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oocitos , Estructura Terciaria de Proteína/genética , XenopusRESUMEN
Previous reports concerning the regulation of glucokinase expression in beta cells have been done using cell models from rodent origin. Evidence is lacking so far to implicate the same regulatory mechanisms in human cells. In this study, we investigate the effects of glucose on the expression of glucokinase using isolated human pancreatic islets. High glucose (16.7 mM), in a time-dependent manner, increases the amount of immunoreactive glucokinase (+150% after 7 days culture, P < 0.01) without apparent changes in glucokinase gene expression, suggesting that glucose exerts its effect at a posttranscriptional level. Mannose, but not the nonmetabolized hexoses, 3-O-methylglucose or 2-deoxyglucose, increases glucokinase protein content. Even though these findings are compatible with an involvement of signals derived from glucose metabolism, additional data argue against this hypothesis: (i) a glucokinase inhibitor (mannoheptulose) does not block glucose-induced increase in glucokinase content and (ii) other metabolic fuels (amino acids) are ineffective. We suggest that the glucose molecule, by mechanisms yet to be defined, but probably not involving its metabolism, regulates human glucokinase expression.
Asunto(s)
Glucoquinasa/genética , Glucosa/fisiología , Islotes Pancreáticos/enzimología , Regulación Enzimológica de la Expresión Génica , Glucoquinasa/biosíntesis , Glucosa/metabolismo , Humanos , Técnicas In Vitro , Islotes Pancreáticos/metabolismoRESUMEN
Human pancreatic islets were cultured for 63 hr at 2.8 or 16.7 mM D-glucose in the absence or presence of dexamethasone. In the 1.0 to 10 microM range, dexamethasone caused a concentration-related decrease in the FAD (flavin adenine dinucleotide)-linked mitochondrial glycerophosphate dehydrogenase (mGDH) mRNA content of the islets, and decreased both the mGDH content of the islets and the catalytic activity of the enzyme in islet homogenates, these effects being often more marked in islets cultured at 16.7 mM, rather than 2.8 mM, D-glucose. Even after culture in the presence of no more than 10 nM dexamethasone, namely under conditions in which the mGDH mRNA content and activity were both virtually unaffected, the corticosteroid restored the capacity of the beta-cells to display an increase in insulin output in response to a rise in D-glucose concentration in islets first cultured at 2.8 mM D-glucose but suppressed the insulinotropic action of the hexose in islets first cultured at 16.7 mM D-glucose. Whilst revealing an untoward effect of high concentrations of dexamethasone upon mGDH mRNA, content and activity in human islets, these findings also document a dual effect of a low concentration of the corticosteroid (10 nM) upon the secretory responsiveness of human islets to D-glucose, independently of any significant change in mGDH gene expression. It is proposed that such a dual action may account, in part at least, for both the well known increase in insulin output found in hypercorticism and the more recently discovered unfavourable direct effect of corticosteroid hormones on the secretory activity of islet beta-cells.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Glicerolfosfato Deshidrogenasa/genética , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , ARN Mensajero/metabolismo , Adulto , Anciano , Northern Blotting , Western Blotting , Femenino , Flavina-Adenina Dinucleótido/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/ultraestructura , Persona de Mediana Edad , Mitocondrias/enzimologíaRESUMEN
COS-7 cells were transfected with the green fluorescent protein (GFP) of Aequorea victoria, human mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH), a mGDHwt-EGFP construct, or two mutant mGDH-proteins fused with EGFP. The site of mutation was selected to affect cationic amino acids in the peptide signal sequence currently believed to play a key role in the subcellular distribution of mitochondrial proteins. All proteins were suitably expressed in the COS-7 cells. However, an increase in mGDH enzymatic activity above the control value in non-transfected COS-7 cell homogenates was only observed in cells transfected with mGDH, indicating that the catalytic activity of mGDH was masked in fused proteins. Confocal microscopy documented that, in the cells transfected with the mGDHwt-EGFP construct, the fusion protein was located exclusively in mitochondria, this contrasting with the nuclear labelling of cells expressing the green fluorescent protein alone. The mitochondrial anchoring of the mutated mGDH fused protein was altered, this alteration being most obvious in the mGDH313233-EGFP mutant. These findings raise the idea that a conformation change of the mGDH protein, as resulting from either an inherited or acquired alteration of its amino acid sequence, may affect its subcellular distribution and, hence, modify its immunogenic potential.
Asunto(s)
Glicerolfosfato Deshidrogenasa/química , Glicerolfosfato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Glicerolfosfato Deshidrogenasa/biosíntesis , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/biosíntesis , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NAD/metabolismo , Oligodesoxirribonucleótidos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Escifozoos , TransfecciónRESUMEN
The mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (mGDH) plays a key role in the recognition of glucose as a stimulus for insulin release from the pancreatic islet B-cell. In the present study, an ELISA procedure was used for the measurement of mGDH antibodies in both insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetic patients. Positive readings, exceeding the upper limit of the normal range, were recorded in 7 out of 12 IDDM patients, as distinct (P < 0.01) from 2 out of 12 nondiabetic subjects of comparable age. The study conducted in 41 NIDDM patients and 15 control subjects of similar age indicated that the incidence of mGDH-positive cases was not significantly different in the diabetic (4/41) and control (1/15) groups, the measurement of optical density in the positive cases barely exceeding the upper limit of the normal range. These findings indicate that the mitochondrial enzyme mGDH often acts as an antigenic determinant in IDDM, but not in NIDDM, patients.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/inmunología , Glicerolfosfato Deshidrogenasa/inmunología , Mitocondrias/enzimología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Autoantígenos/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana EdadRESUMEN
The mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (mGDH) plays a key role in the recognition of D-glucose as a stimulus for insulin release from the pancreatic islet B-cell. This study reveals that autoantibodies against this enzyme are not uncommonly found in patients with insulin-dependent diabetes mellitus (IDDM) examined at the onset of the disease. Antibodies reacting with a recombinant mGDH fragment product were observed in the serum of four out of 15 type-1 diabetics, but in none of 15 control subjects. The serum of patients positive for the recombinant mGDH fragment also recognized native mGDH in a rat testis extract, provided that the enzymatic protein was first exposed to an anti-mGDH rabbit serum. Antibodies against mGDH were also found in four out 12 patients with autoimmune thyroiditis. These findings reveal that a mitochondrial enzyme, that represents an essential component of the islet B-cell glucose-sensing device, may act as an antigenic determinant in patients with IDDM or other autoimmune diseases.
Asunto(s)
Autoanticuerpos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Glicerolfosfato Deshidrogenasa/inmunología , Adolescente , Adulto , Anciano , Animales , Diabetes Mellitus Tipo 1/enzimología , Femenino , Glutatión Transferasa/inmunología , Humanos , Sueros Inmunes/inmunología , Immunoblotting , Masculino , Persona de Mediana Edad , Mitocondrias/enzimología , Conejos/inmunología , Ratas , Proteínas Recombinantes/inmunología , Testículo/enzimología , Testículo/inmunología , Tiroiditis Autoinmune/enzimología , Tiroiditis Autoinmune/inmunologíaRESUMEN
The mitochondrial enzyme glycerophosphate dehydrogenase (mGDH) plays an essential role in the B-cell glucose-sensing device and its activity in islet homogenates is impaired in several animal models of type 2 diabetes. We have now developed a polyclonal antibody, raised against a recombinant mGDH fragment product, that could be used for the immunodetection of mGDH. Total RNA was isolated from rat pancreatic islets and used in the synthesis of cDNA. Specific primers were designed that corresponded to the FAD binding domain of mGDH. The PCR product was purified and cloned into an appropriate expression vector used for transformation of Escherichia coli cells. The fusion protein was extracted from the transformed cells, further purified, and used for immunization of rabbits. The antibody recognized a single band of 72 kDa in rat islets and testis. The recombinant mGDH product was also recognized as a single band with the expected 65-kDa reference. An ELISA procedure was designed for detection of antibodies against the recombinant mGDH fragment product. The availability of the mGDH antibody opens the way to a number of further applications such as immunocytochemis- try and mGDH quantification in biological material.
Asunto(s)
Anticuerpos/inmunología , Glicerolfosfato Deshidrogenasa/metabolismo , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes/inmunología , Animales , Diabetes Mellitus Tipo 1/metabolismo , Glicerolfosfato Deshidrogenasa/química , Humanos , Reacción en Cadena de la Polimerasa , RatasRESUMEN
Western blotting of pancreatic islet extracts from either hereditarily diabetic Goto-Kakizaki rats (GK rats) or animals injected with streptozotocin during the neonatal period (STZ rats) demonstrated a pronounced decrease of immunoreactive mitochondrial glycerophosphate dehydrogenase (m-GDH), when compared to results obtained in islets from control rats. By contrast, the islet glucokinase protein content was either unaffected (GK rats) or much less severely decreased than that of m-GDH (STZ rats). These findings indicate that the impaired activity of m-GDH previously documented in islet homogenates from diabetic rats coincides with a decreased content of this enzyme in the endocrine pancreas.