FAD-linked Glycerophosphate Dehydrogenase Research Articles

Deficient Activity of FAD-Linked Glycerophosphate Dehydrogenase in the Mitochondria of Insulin-Producing Pancreatic Islets B-Cells: A Possible Cause of the Metabolic Syndrome

A deficient activity of mitochondrial FAD-linked glycerophosphate dehydrogenase in insulin-producing pancreatic islet B-cells was recently identified in selected animal models of non-insulin-dependent diabetes mellitus. The incidence of this enzymatic defect remains however to be unambiguously documented in type-2 diabetic patients. The present review mainly aims at speculating that the altered recognition of nutrients, especially D-glucose, by pancreatic B-cells, as resulting from the abovementioned enzymatic perturbation could also eventually lead to the development of the metabolic syndrome (MetS).

P194 “PARAOXONASE (PON1) GLN, ARG 192 POLYMORPHISM” AN INDEPENDENT RISK FACTOR FOR CORONARY ARTERY DISEASE IN NORTH-WEST INDIAN POPULATION

The activities of FAD-linked glycerophosphate dehydrogenase (m-GDH), glutamate dehydrogenase (G1DH), glutamate-pyruvate transaminase (GPT) and glutamate-oxalacetate transaminase (GOT) were measured in purified populations of CD3 + lymphocytes from 55 control subjects, 62 type-2 diabetics and 50 non-diabetic relatives of the latter patients. The activity of m-GDH was measured by both a radioisotopic procedure and colourimetric technique. As judged from these measurements and relative to the paired value for G1DH, the incidence of abnormally low m-GDH activity was significantly higher in type-2 diabetics than in control subjects. Moreover, the paired ratio in reaction velocity between the colourimetric and radioisotopic assay of m-GDH was abnormally high in patients with low m-GDH activity. Low m-GDH activity often coincided with increased GPT activity in plasma or high GPTGOT ratio in lymphocytes. No obvious clustering of these anomalies was found in relatives of diabetic patients. These findings suggest that an inherited or acquired genomic defect of m-GDH in lymphocytes, and possibly in pancreatic B-cells, may participate to the pathogenesis of non-insulin-dependent diabetes mellitus.

Diet and Dysfunctional Metabolism in BB Rats

This review deals with the age- and diet-related metabolic and functional anomalies found in BB rats, currently considered as an animal model of type 1 diabetes mellitus. The metabolism of nutrients, the secretion of insulin, the production of NO, the effects of IL-1β and the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase in pancreatic islets are first considered. The metabolism and mitotic activity in Peyers patch cells and both mesenteric and pancreatic lymph node cells are then evoked. The occurrence of intestinal dysfunction is mentioned, with emphasis on recent findings made in BB rats before weaning. The participation of zonulins and mucins in the perturbation of gut permeability are also discussed. Last, attention is drawn to the perturbation of the anomeric specificity of glucose-induced insulin release, considered as a manifestation of pancreatic islet beta-cell glucotoxicity. Keywords: BioBreeding rats, type 1 diabetes, diet, pancreatic islets, lymph node cells, gut, mucins, anomeric specificity of glucose-induced insulin release

Site-Directed Mutations in the FAD-Binding Domain of Glycerophosphate Dehydrogenase: Catalytic Defects with Preserved Mitochondrial Anchoring of the Enzyme in Transfected COS-7 Cells

Single-strand conformational polymorphism analysis of mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH) gene has revealed mutations in both the calcium- and FAD-binding domains of this enzyme in some diabetic patients. It was now investigated whether site-directed mutations in the FAD-binding domain of the mGDH gene may affect the mitochondrial anchoring and catalytic activity of the enzyme. COS-7 cells were transfected with plasmid cDNA coding for either wild-type or mutated human mGDH (G → A substitutions at positions 352, 355, and 364 and A → C substitution at position 390) fused, when required, at the N-terminus of green fluorescent protein. The activity of mGDH was measured by both radioisotopic (3HOH production from l-[2-3H]glycerol 3-phosphate) and colorimetric (iodoformazan formation) procedures. In cells transfected with the mGDHwt-EGFP or mGDHmut-EGFP constructs, the fused protein was found by confocal microscopy exclusively in the mitochondria, colocalized with a mitochondrial marker. In homogenates of COS-7 cells transfected with mGDHmut, however, the catalytic activity of the enzyme was decreased, this coinciding with low ratios between both the activities measured in the absence/presence of exogenous FAD and the results obtained by the colorimetric/radioisotopic procedure. Thus, although the present site-directed mutations of the mGDH gene failed to impair the mitochondrial anchoring of the enzyme, they led to catalytic defects that were, in some respect, comparable to those previously encountered in the lymphocytes or islets of type 2 diabetic patients.

Enzymic activities in two populations of purified rat islet β-cells

In terms of glucose sensing by pancreatic islet beta-cells, emphasis is currently placed on both the role of glucokinase, with negligible activity of low-Km hexokinase(s), and the prevalence of the oxidative over non-oxidative modality of glycolysis, a situation tentatively attributed, in part at least, to a low activity of lactate dehydrogenase. Conflicting information is available, however, on the activity of both low-Km hexokinase(s) and lactate dehydrogenase in purified beta-cell homogenates. This issue was reinvestigated, therefore, in two populations of purified rat islet beta-cells selected on the basis of their low (betaL) or high (betaH) content in reduced pyridine nucleotides. The size and protein content of betaH cells represented about twice that of betaL cells. Such was also the case for low-Km hexokinase(s), lactate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, glutamate dehydrogenase and glutamate-alanine and glutamate-aspartate transaminases. Whether in betaH or betaL cells, the activity of low-Km hexokinase(s) was at least as high as or higher than that of glucokinase. In both betaH and betaL, the activity of lactate dehydrogenase exceeded that required to catalyze the full reduction of glucose-derived pyruvate to L-lactate, as estimated from the rate of D-glucose phosphorylation under physiological conditions. These findings thus argue against a low expression of either low-Km hexokinase(s) or lactate dehydrogenase as major determinants of the glucose-sensing device in beta-cells.

Distinct effect of diazoxide on insulin secretion stimulated by protein kinase A and protein kinase C in rat pancreatic islets

Protein kinase activation is known to stimulate glucose-induced insulin secretion in the presence of diazoxide. Diazoxide opens the ATP-sensitive K + channel and inhibits FAD-linked glycerophosphate dehydrogenase activity in a concentration-dependent manner. In the present study, we examined the effect of lower (100 μM) and higher (250 μM) concentrations of diazoxide on insulin release by protein kinase A (PKA) and protein kinase C (PKC) activation. Forced depolarization by a high potassium concentration, augmented the intracellular Ca 2+ concentration ([Ca 2+] i ) similarly in the presence of both concentrations of diazoxide. Under this condition, 250 μM diazoxide inhibited insulin release enhanced by PKA activation but not that by PKC. Under a basal concentration of [Ca 2+] i , PKC activation elicited glucose-induced insulin secretion at 100 and 250 μM diazoxide, while PKA activation did so only at 100 μM. These augmentations were completely inhibited by mannoheptulose, a glucokinase inhibitor. Glyceraldehyde, in place of glucose, enhanced insulin secretion by PKC activation under both concentrations of diazoxide. On the other hand, it did not affect PKA-stimulated insulin release under either conditions, but in the case of 100 μM, glucose augmented the insulin secretion in the presence of glyceraldehyde and db-cAMP concentration-dependently. These data suggest that insulin release stimulated by PKA and PKC activation under diazoxide is dependent on glucose metabolism, and that a signal derived from proximal steps in glycolysis may be necessary for the secretion by PKA activation.

Identification and functional analysis of mutations in FAD-binding domain of mitochondrial glycerophosphate dehydrogenase in caucasian patients with type 2 diabetes mellitus.

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.

Effects of high extracellular K + concentrations, diazoxide and/or Ca 2+ deprivation upon d-glucose metabolism in pancreatic islets

A rise in d-glucose concentration may augment insulin release independently of changes in K + conductance or Ca 2+ influx in pancreatic islet cells, the insulinotropic action of the hexose remaining dependent on an increased generation of high-energy phosphates. In the present study, therefore, it was investigated to which extent the procedures currently used to assess the modalities of the secretory response to d-glucose independent of its effect on ATP-sensitive K + channels and Ca 2+ inflow may themselves affect the catabolism of the hexose in isolated rat pancreatic islets. A rise in the extracellular K + concentration from 5 to 30 or 60 mM failed to significantly affect the metabolism of d-glucose. At 90 mM K +, however, the maximal velocity of the glycolytic flux was decreased and the apparent K m for d-glucose lowered, without an obvious alteration of the preferential stimulation of oxidative mitochondrial events in response to a rise in d-glucose concentration. Such a preferential stimulation was abolished, however, either by diazoxide at a low, but not high, K + concentration or by Ca 2+ deprivation, in the absence or presence of diazoxide, at a high K + concentration. It is speculated that these metabolic changes may be attributable, in part at least, to an altered activity of key cytosolic (e.g. pyruvate kinase) and mitochondrial (e.g. FAD-linked glycerophosphate dehydrogenase) enzymes.

Site-Directed Mutations of the FAD-Linked Glycerophosphate Dehydrogenase Gene Impairs the Mitochondrial Anchoring of the Enzyme in Transfected COS-7 Cells

COS-7 cells were transfected with the green fluorescent protein (GFP) ofAequorea 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.

Enzyme-Linked Immunosorbent Assay of Autoantibodies against Mitochondrial Glycerophosphate Dehydrogenase in Insulin-Dependent and Non-Insulin-Dependent Diabetic Subjects

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.

Autoantibodies against mitochondrial glycerophosphate dehydrogenase in patients with IDDM

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.

Effects of Dehydroepiandrosterone in Rats Injected with Streptozotocin during the Neonatal Period

Control rats and diabetic animals injected with streptozotocin during the neonatal period were either maintained on a standard diet or given access to food supplemented with dehydroepiandrosterone (DHEA, 0.2%) for 11 days before sacrifice. In both control and diabetic rats, DHEA feeding augmented the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase and cytosolic NADP-linked malate dehydrogenase in liver, but not so in either the parotid gland or pancreatic islets. DHEA lowered, in both control and diabetic rats, the ratio betweend-glucose oxidation and utilization and the rate of insulin release in pancreatic islets exposed to a high concentration ofd-glucose, as well as the insulin concentration and insulin/glucose ratio in plasma. These findings support the view that, in diabetes, DHEA, by increasing sensitivity to insulin, may allow islet B-cells to avoid the otherwise unfavorable consequences of chronic hyperactivity.

Detection of variants in the mitochondrial glycerophosphate dehydrogenase gene in Japanese NIDDM patients

Mitochondrial FAD-linked glycerophosphate dehydrogenase (mGPDH) is thought to be an important factor for glucose sensing in pancreatic beta cells. To evaluate the significance of the mGPDH gene in the development of non-insulin-dependent diabetes mellitus (NIDDM), we set up primers and conditions for polymerase chain reaction (PCR) amplification of the coding exons and flanking regions. Screening of 100 Japanese NIDDM patients for mutations using the PCR-single strand conformation polymorphism (SSCP) method revealed four variants (ACA:Thr243-ACG:Thr243, CAT:His264-CGT:Arg264, GCA:Ala305-GCC:Ala305, GCA:Ala 306-TCA:Ser306). The His264-Arg264 variant was found in 36 patients, while the other variants were found in only one patient each. Neither the genotypic (chi 2 = 3.15, p = 0.21) nor the allelic (chi 2 = 2.27, p = 0.13) frequency of the His264-Arg264 mutation differed between 253 Japanese NIDDM patients and 157 non-diabetic subjects. In addition, in NIDDM patients, neither the treatment modality nor body mass index differed between those with and without this mutation. These results suggest that inherited defects at this locus do not make a major contribution to genetic susceptibility to NIDDM in the Japanese population.

Mutation in the Calcium-Binding Domain of the Mitochondrial Glycerophosphate Dehydrogenase Gene in a Family of Diabetic Subjects

The Ca2+-sensitive and mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (m-GDH) represents an essential component of the pancreatic B-cell glucose-sensing device. This report deals with the first identified case of mutation in the calcium-binding domain of the m-GDH gene in a patient with type-2 diabetes and his glucose-intolerant half sister. Single strand conformation polymorphism analysis indeed revealed an abnormal mobility of the32P-labelled polymerase chain reaction product in these two subjects. The corresponding base pair mutations and amino acid changes were documented. In the diabetic proband, the relative extent of the Ca2+-induced activation of m-GDH in CD3+T-lymphocytes was lower than in his brother with a normal m-GDH gene sequence.

Low mitochondrial glycerophosphate dehydrogenase activity in lymphocytes of women with gestational diabetes.

An abnormally low activity of mitochondrial FAD-linked glycerophosphate dehydrogenase (m-GDH), relative to the paired measurement of glutamate dehydrogenase, was found in CD3+ lymphocytes from 4 out of 14 mothers with gestational diabetes mellitus, but in none of 36 control mothers. The low m-GDH activity coincided with an abnormally high incidence of familial history for non-insulin-dependent diabetes. These findings are compatible with the view that an inherited or acquired defect of m-GDH may participate to the pathogenesis of beta-cell dysfunction in a subgroup of patients with gestational diabetes.

Nutrient Metabolism in Pancreatic Islets from Protein Malnourished Rats

The metabolism ofD-[5-3H]glucose,D-[3,4-14C]glu- cose, [2-3H]glycerol,L-[U-14C]glutamine,L-[1-14C]leucine, andL-[U-14C]leucine was investigated in pancreatic islets isolated from either control rats or animals fed a low-protein isocaloric diet, containing 8% instead of 20% protein, during both fetal and postnatal life. In the latter animals, decreases in body weight, plasma insulin concentration, and insulinogenic index were associated with two major anomalies of islet nutrient metabolism. First, an imbalance between oxidative and anaerobic glycolysis was found in the islets of rats fed the low-protein isocaloric diet. It coincided with a decreased circulation in the glycerol phosphate shuttle, as judged by the generation of3HOH from [2-3H]glycerol, and was probably attributable to the deficiency of mitochondrial FAD-linked glycerophosphate dehydrogenase previously documented in islet homogenates of the rats fed low protein. Second, the transamination ofL-leucine to 2-ketoisocaproate was decreased in the low-protein-fed rats, while the oxidative decarboxylation of the 2-keto acid and the further catabolism of isovaleryl CoA occurred at normal rates when expressed relative to the initial transamination rate. These metabolic anomalies may account, in part at least, for the impairment of insulin release in protein malnutrition.

Regulation, perturbation, and correction of metabolic events in pancreatic islets.

The physiological regulation of nutrient catabolism in islet cells, its perturbation in non-insulin-dependent diabetes mellitus, and the tools available to compensate for such a perturbation are reviewed. In terms of physiology, emphasis is placed on the relevance of glucokinase to hexose-induced insulin release, protein-to-protein interaction and enzyme-to-enzyme channelling, and the preferential stimulation of mitochondrial oxidative events in glucose-stimulated B-cells. In terms of pathology, attention is drawn to the deficiency of FAD-linked mitochondrial glycerophosphate dehydrogenase. Last, as far as therapeutic aspects are concerned, the potential usefulness of hypoglycemic sulfonylureas and meglitinide analogs, adenosine analogs, non-glucidic nutrients, and GLP-1 is underlined.

FAD-glycerophosphate dehydrogenase activity in lymphocytes of type-2 diabetic patients and their relatives

The activities of FAD-linked glycerophosphate dehydrogenase (m-GDH), glutamate dehydrogenase (G1DH), glutamate-pyruvate transaminase (GPT) and glutamate-oxalacetate transaminase (GOT) were measured in purified populations of CD3 + lymphocytes from 55 control subjects, 62 type-2 diabetics and 50 non-diabetic relatives of the latter patients. The activity of m-GDH was measured by both a radioisotopic procedure and colourimetric technique. As judged from these measurements and relative to the paired value for G1DH, the incidence of abnormally low m-GDH activity was significantly higher in type-2 diabetics than in control subjects. Moreover, the paired ratio in reaction velocity between the colourimetric and radioisotopic assay of m-GDH was abnormally high in patients with low m-GDH activity. Low m-GDH activity often coincided with increased GPT activity in plasma or high GPT GOT ratio in lymphocytes. No obvious clustering of these anomalies was found in relatives of diabetic patients. These findings suggest that an inherited or acquired genomic defect of m-GDH in lymphocytes, and possibly in pancreatic B-cells, may participate to the pathogenesis of non-insulin-dependent diabetes mellitus.

Relevance of Lactate Dehydrogenase Activity to the Control of Oxidative Glycolysis in Pancreatic Islet B-Cells

The activities of hexokinase isoenzymes, lactate dehydrogenase, cytosolic NAD-linked glycerophosphate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, and glutamate dehydrogenase were measured in homogenates of rat purified pancreatic B and non-B islet cells. In B cell homogenates, the maximal activity of hexokinase and glucokinase was one to two orders of magnitude lower than that of lactate dehydrogenase. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase was also much lower than that of the cytosolic NAD-linked glycerophosphate dehydrogenase. A comparable hierarchy in the activity of these enzymes was observed in non-B islet cells. These findings reinforce the view that the preferential stimulation of oxidative glycolysis observed in insulin-producing cells, when exposed to high concentrations ofD-glucose, is attributable to a Ca2+-induced activation of the mitochondrial FAD-linked glycerophosphate dehydrogenase, rather than to saturation of the catalytic activity of lactate dehydrogenase.

Beta-cell stimulus - secretion coupling defects in rodent models of obesity.

Hyperinsulinemia accompanies obesity in human patients and experimental rodent models and exacerbates insulin resistance, but the causes of increased insulin secretion remain obscure. This review examines progress in defining biochemical and molecular beta-cell defects that have elucidated in the past 5 years. Some defects, such as decreased glucose transport, decreased mitochondrial FAD-linked glycerophosphate dehydrogenase activity, and altered anomeric specificity for glucose, become evident only after onset of non-insulin-dependent diabetes mellitus. Thus, these defects are unlikely to play a role in the pathogenesis of hyperinsulinemia in obesity. Other biochemical changes, including increased glucokinase and (or) hexokinase function, increased glucose cycling, and altered regulation of intracellular Ca2+ are present in obese nondiabetic animals and may therefore contribute to development of hyperinsulinemia. Few developmental studies have been performed to correlate onset of defects with environmentally and genetically mediated control mechanisms of beta-cell function. However, the availability of new molecular biology techniques should facilitate identification of factors causing hyperinsulinemia in obesity.