flavin-adenine-dinucleotide and Diabetes-Mellitus--Type-2

The mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (m-GDH) is thought to play a key role in the glucose-sensing mechanism of the insulin-producing B-cell. It catalyses a rate-limiting step of the glycerol phosphate shuttle in pancreatic islets. Its activation by Ca2+ accounts for the preferential stimulation of oxidative glycolysis and, hence, pyruvate oxidation in glucose-stimulated islets. Reduced activity of m-GDH was recently observed in islet, but not liver, homogenates from rats injected with streptozotocin during the neonatal period and in two models of inherited diabetes, i.e. GK rats and db/db mice. In the streptozotocin-injected and GK rats the m-GDH islet defect coincided, in intact islets, with an abnormally low ratio between oxidative and total glycolysis. Decreased activity of m-GDH in T-lymphocytes was also observed in 12 of 32 type 2 (non-insulin-dependent) diabetic patients, but only once among 26 other subjects including 11 healthy volunteers, 9 non-diabetics and 6 patients with either type 1 (insulin-dependent) or symptomatic diabetes. In the T-lymphocytes of type 2 diabetics the m-GDH deficiency occasionally coincided with an abnormally high ratio between glutamate-pyruvate and glutamate-oxaloacetate transaminase activities, as also observed in islets from streptozotocin-injected or GK rats. It is speculated that an islet m-GDH defect could represent a far from uncommon factor contributing to the pathogenesis of type 2 diabetes mellitus.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Flavin-Adenine Dinucleotide; Glycerolphosphate Dehydrogenase; Humans; Islets of Langerhans; Mitochondria; Models, Biological

Topics: Alloxan; Animals; Antigens, Surface; Cell Separation; Cells, Cultured; Cyclic AMP; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Flavin-Adenine Dinucleotide; Flow Cytometry; Glucagon; Glucose; Insulin; Insulin Secretion; Islets of Langerhans; Islets of Langerhans Transplantation; NADP; Somatostatin; Streptozocin

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.

Topics: Aged; Animals; Cells, Cultured; Cohort Studies; Diabetes Mellitus, Type 2; DNA, Complementary; Female; Flavin-Adenine Dinucleotide; Glycerolphosphate Dehydrogenase; Humans; Male; Middle Aged; Mitochondria; Mutation; Oocytes; Protein Structure, Tertiary; Xenopus

To evaluate if potential defects in the FAD-binding domain of the mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) gene could contribute to susceptibility to type 2 diabetes mellitus, we have screened 151 type 2 DM patients for mutations using PCR single-strand conformational polymorphism. Both a single substitution (T to A) at position 18 and a 6-base-pair deletion (TTTTAA) at position 26 of intron 3 have been detected in five type 2 DM patients and in one control subject. The evolution time of diabetes was longer in patients with these mutations than in patients without (24.2 +/- 11.1 vs 12.6 +/- 8.7 years, p < 0.02). These mutations generate a cryptic site that may have functional significance in the correct mechanism of the FAD-binding domain. In the process of PCR amplification of the mGPDH gene we also unexpectedly amplified the mGPDH retropseudogene. Subsequently, we decided to further characterize and completely sequence 2213 bp of this mGPDH retropseudogene. Our results suggest that two previously reported mGPDH pseudogene partial sequences may be identical copies of the mGPDH gene inserted in two different genomic locations and provide information about the alternative 5'- and 3'-untranslated regions. The data obtained are also important in order to avoid artifactual amplification of the mGPDH pseudogene in the process of screening for mGPDH mutations in diabetic patients.

Topics: Base Sequence; Binding Sites; Diabetes Mellitus, Type 2; Exons; Female; Flavin-Adenine Dinucleotide; Flavoproteins; Genetic Testing; Genetic Variation; Glycerolphosphate Dehydrogenase; Humans; Introns; Male; Meiosis; Mitochondria; Molecular Sequence Data; Pedigree; Polymorphism, Single-Stranded Conformational; Pseudogenes; Reverse Transcriptase Polymerase Chain Reaction; Spain; Untranslated Regions; White People

Glucose-stimulated insulin secretion is impaired in GK (Goto-Kakizaki) rats, perhaps because of abnormalities in glucose metabolism in pancreatic islet beta cells. The glycerol phosphate shuttle plays a major role in glucose metabolism by reoxidizing cytosolic NADH generated by glycolysis. In the pancreatic islets of GK rats, the activity of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate shuttle, is decreased and this abnormality may be responsible, at least in part, for impaired glucose-stimulated insulin secretion. To investigate this possibility, we overexpressed mGPDH in islets isolated from GK rats via recombinant adenovirus-mediated gene transduction, and examined glucose-stimulated insulin secretion. In islets isolated from diabetic GK rats at 8 to 10 weeks of age, glucose-stimulated insulin secretion was severely impaired, and mGPDH activity was decreased to 79 % of that in non-diabetic Wistar rats. When mGPDH was overexpressed in islets from GK rats, enzyme activity and protein content increased 2- and 6-fold, respectively. Basal (3 mmol/l glucose) and glucose-stimulated (20 mmol/l) insulin secretion from the Adex1CAlacZ-infected GK rat islets were, respectively, 4.4 +/- 0.7 and 8.1 +/- 0.7 ng. x islet(-1) x 30 min(-1), and those from mGPDH-overexpressed GK rat islets 4.7 +/- 0.3 and 9.1 +/- 0.8 ng x islet(-1) x 30 min(-1), in contrast to those from the AdexlCAlacZ-infected non-diabetic Wistar rat islets (4.7 +/- 1.6 and 47.6 +/- 11.9 ng x islet(-1) x 30 min(-1)). Thus, glucose-stimulated insulin secretion is severely impaired in GK rats even in the stage when mGPDH activity is modestly decreased, and at this stage, overexpression of mGPDH cannot restore glucose-stimulated insulin secretion. We conclude that decreased mGPDH activity in GK rat islets is not the defect primarily responsible for impaired glucose-stimulated insulin secretion.

Topics: Adenoviridae; Animals; Blotting, Western; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Disease Models, Animal; Female; Flavin-Adenine Dinucleotide; Gene Expression; Gene Expression Regulation, Enzymologic; Glucose; Glycerolphosphate Dehydrogenase; Insulin; Insulin Secretion; Islets of Langerhans; Lac Operon; Male; Mitochondria; Rats; Rats, Inbred Strains; Rats, Wistar; Recombinant Fusion Proteins; Transcriptional Activation; Transfection

In 12 out of 32 non-insulin-dependent diabetic subjects, the activity of FAD-linked glycerophosphate dehydrogenase in T lymphocyte homogenates was abnormally low when measured by both a colorimetric and radioisotopic procedure. A comparable situation characterized by a deficient activity of FAD-linked glycerophosphate dehydrogenase in both the colorimetric and radioisotopic assays was only observed once among 26 other subjects including 11 healthy volunteers, 9 non-diabetic patients, 5 type-1 (insulin-dependent) diabetics, and 1 pancreatectomized diabetic. By analogy, it is speculated that an impaired activity of FAD-linked glycerophosphate dehydrogenase in the insulin-producing pancreatic B-cell could represent a far-from-uncommon contributive factor in the pathogenesis of non-insulin-dependent diabetes mellitus.

Topics: Adolescent; Adult; Aged; Colorimetry; Coloring Agents; Diabetes Mellitus, Type 2; Female; Flavin-Adenine Dinucleotide; Glycerolphosphate Dehydrogenase; Glycerophosphates; Humans; Male; Middle Aged; Monocytes; T-Lymphocytes; Tetrazolium Salts

The activity of FAD-linked glycerophosphate dehydrogenase (m-GDH), as well as that of glutamate dehydrogenase and both glutamate-oxalacetate and glutamate-pyruvate transaminases, were measured in islet, liver, and splenocyte homogenates from 6- to 7-week-old female nonobese diabetic mice (NOD) and age- and sex-matched control mice. Despite incipient insulitis and euglycemia, the NOD mice displayed both high islet insulin content and elevated insulinemia. The activity of m-GDH, expressed relative to protein content, was not decreased in islets of NOD mice, despite the fact that such a specific activity is lower in splenic lymphocytes than islet cells. In liver homogenates, the activity of m-GDH was even higher in NOD than control mice. It is proposed, therefore, that in this model of insulin-dependent diabetes no primary decrease in islet m-GDH activity occurs, at variance with the situation recently documented in several animal models of non-insulin-dependent diabetes.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Diabetes Mellitus, Type 2; Female; Flavin-Adenine Dinucleotide; Glycerolphosphate Dehydrogenase; Immunoassay; Insulin; Islets of Langerhans; Liver; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Spleen