Takayuki Asahina

Abnormal glutathione metabolism and increased cytotoxicity caused by H2O2 in human umbilical vein endothelial cells cultured in high glucose medium

SummaryTo determine whether increased oxidative stress in diabetes mellitus is due to an impaired freeradical scavenger function in endothelial cells, GSH-dependent H2O2 degradation in human umbilical vein endothelial cells was studied. The GSH-dependent, NaN3-uninhibitable H2O2-degradation in endothelial cells was reduced by 48% (p <0.001) when the cells were exposed to 33 mmol/l d-glucose vs 5.5 mmol/l d-glucose. This impairment was dependent not only on the d-glucose concentration in the medium but also on d-glucose specific metabolism, since neither 27.5 mmol/l l-glucose nor 27.5 mmol/l d-raffinose had any effect on the peroxide degradation activity. Activation of the glutathione redox cycle by H2O2 in cells exposed to high glucose concentrations was attenuated as compared with 5.5 mmol/l d-glucose because of: 1) a 42% decrease (p <0.001) in intracellular NADPH content, and 2) a 34% reduction (p <0.01) in glutathione release into the media. This results in an accumulation of GSSG in the cells following exposure to H2O2. Both H2O2-evoked 51Cr-release and H2O2-induced endothelial cell damage were significantly (p <0.01) greater in the 33 mmol/l d-glucose group than in the 5.5 mmol/l d-glucose group. These results indicate that the abnormal glutathione redox cycle observed in endothelial cells is induced by high glucose concentrations in the medium, resulting in an impairment of reduced GSH-dependent H2O2-degradation. These abnormalities may associate with the increased cellular damage following an exogenous exposure to H2O2.

Significance of fructose-induced protein oxidation and formation of advanced glycation end product

To investigate the significance of fructose-induced protein modification, we examined both fructose- and glucose-induced protein oxidation and the formation of advanced glycation end products (AGE) in vitro. Albumin incubated in the presence of 100 mM fructose at 37 degrees C for 1 week showed 5.1-fold and 3.1-fold increases in the content of carbonyl, which is a marker for oxidized protein, when compared with either control incubated without sugar or with 100 mM glucose. Similarly, the same incubation with fructose increased the fluorescence intensity over 100-fold and 15-fold formation compared with that of no sugar and glucose controls, respectively. Both fructose-induced fluorescence and protein oxidation were almost completely suppressed in the presence of the iron chelator; deferoxamine (100 microM), the hydroxyl radical scavenger; MK-447 (1 mM), or aminoguanidine (200 mM), which is an inhibitor of AGE formation. In contrast, the fructose-induced formation of fluorescent albumin was potentiated in the presence of 100 microM FeCl2. This was completely inhibited in the presence of 60 microM or more deferoxamine. These results suggest that fructose promotes both AGE formation and protein oxidation possibly through the formation of hydroxyl radicals.

Glycation, oxidative stress, and scavenger activity: glucose metabolism and radical scavenger dysfunction in endothelial cells.

It has been reported that oxidative stress is increased in vivo in the diabetic state. Increased oxidative stress is caused not only by accelerated production of oxygen-free radicals but also by decreased scavenging of those molecules. Endothelial cells are extremely sensitive to oxidative stress, resulting in impairments of various endothelial cell function. In this report, we studied the association of intracellular glucose metabolism and oxygen radical scavenging function via the glutathione redox (GR) cycle in cells exposed to high-glucose conditions using cultured human umbilical vein endothelial cells. Glutathione-dependent H2O2 degradation in cells exposed to 33 mmol/1 glucose (HG) for 5–7 days was reduced by 48% vs. 5.5 mmol/1 glucose (NG). This impairment under the oxidative stress was D-glucose-specific and concentration-dependent and was also associated with a 42% decrease in intracellular NADPH content. Exposure of cells to 200 μmol/1 H2O2 stimulated the GR cycle and the pentose phosphate pathway (PPP) at the same time. In the HG condition, activation of PPP was reduced by 50%, which was consistent with a decrease in NADPH content. Inhibition of glycolysis by H2O2 was less marked in HG cells versus NG cells. Activation of polyol pathway in HG cells is not responsible for the decrease in intracellular NADPH content. These results indicate that activation of the PPP and NADPH supply to the GR cycle is impaired in HG cells exposed to H2O2, which may result in increased oxidative stress to endothelial cells.

Usefulness of Revised Fasting Plasma Glucose Criterion and Characteristics of the Insulin Response to an Oral Glucose Load in Newly Diagnosed Japanese Diabetic Subjects

OBJECTIVE To examine the usefulness of the revised criterion for fasting plasma glucose (FPG) in the diagnosis of diabetes recommended by the American Diabetic Association (ADA) (126 mg/dl, 7 mmol/1), and to characterize insulin response during the 75-g oral glucose tolerance test (OGTT) in newly diagnosed Japanese diabetic subjects. RESEARCH DESIGN AND METHODS A series of 2,121 Japanese subjects underwent a 75-g OGTT (0–3 h) and were divided into three groups (normal glucose tolerance [NGT], impaired glucose tolerance [IGT], and diabetes mellitus [DM]) according to the current World Health Organization criteria. After the cutoff values of FPG that distinguish NGT and IGT from diabetes were analyzed, the usefulness of the ADA criterion for FPG was examined by comparing diagnostic parameters (sensitivity, specificity, and accuracy) with those for the cutoff value of 140 mg/dl. To assess insulin response, both the insulinogenic index (Islx), a marker of early secretion, and the area under the insulin response curve (AUCIns), a marker of total secretion, were compared between the DM, NGT, and 1GT groups. RESULTS First, the FPG cutoff value distinguishing NGT from diabetes was 109 mg/dl. An FPG of 126 mg/dl showed a higher sensitivity (0.52 vs. 0.31), the same specificity (1.00), and a higher accuracy (0.82 vs. 0.74) than an FPG of 140 mg/dl, and it had a higher specificity (1.00 vs. 0.86) with a slightly lower accuracy (0.82 vs. 0.85) than an FPG of 109 mg/dl. Second, the FPG cutoff value differentiating IGT from diabetes was 113 mg/dl. An FPG of 126 mg/dl showed a higher sensitivity (0.52 vs. 0.31) and accuracy (0.80 vs. 0.74) and a similar specificity (0.97 vs. 1.00) compared with an FPG of 140 mg/dl, and it had a higher specificity (0.97 vs. 0.82) with the same accuracy (0.80) as an FPG of 113 mg/dl. Third, the DM group showed the lowest Islx among the three groups at all FPG values. The AUCIns in the DM group increased along with FPG, reached the maximum level at an FPG of 110 mg/dl, and declined thereafter. AUCIns was higher in the DM group than in the NGT group at FPG values ≥100 mg/dl. CONCLUSIONS The revised ADA criterion for FPG of 126 mg/dl may improve diagnostic sensitivity without loss of specificity in Japanese diabetic subjects when compared with an FPG criterion of 140 mg/dl. Although early insulin secretion was impaired, total insulin secretion did not seem to be reduced in newly diagnosed Japanese diabetic subjects.

Diabetes Associated With a Novel 3264 Mitochondrial tRNALeu(UUR) mutation

OBJECTIVE To present a novel mitochondrial DNA mutation in a diabetic family RESEARCH DESIGN AND METHODS The proband was a 64-year-old man. In the family, diabetes was maternally inherited. He had diabetes, cerebellar ataxia, cervical lipoma, hearing loss, olfactory dysfunction, ophthalmoplegia, and facial nerve bilateral palsy. On examination, early insulin secretion was blunted, and the M value on glucose clamp test was low. In muscle, ragged red fibers were not found. T-to-C mutation at position 3264 was detected in the proband (0.5% mutant DNAs in leukocyte and 30% in muscle), but was not detected in 201 normal individuals. RESULTS Heteroplasmy of mutation, maternal inheritance of diabetes, and symptoms related to mitochondrial dysfunction suggest the pathogenecity of this 3264 mutation. As for diabetes etiology, both impaired insulin secretion and decreased insulin sensitivity seem to be important. In phenotypic characteristics, the combination of cerebellar ataxia and lipoma is a symptom sometimes found in myoclonic epilepsy and ragged red fibers (MERRFs). Ophthamoplegia is a symptom of chronic progressive external ophthalmoplegia (CPEO). These suggest that our proband had phenotypic overlap with MERRF and CPEO. Conversely, facial nerve bilateral palsy is a rare finding. The pictures that focused on his cranial nerves were thus unique, suggesting the heterogeneity of mitochondrial DNA (mtDNA)-related diabetes. CONCLUSIONS A novel 3264 mitochondrial DNA mutation in diabetes gives new insight to the etiology of mitochondrial diabetes. Its pathogenecity supports the belief that the tRNA(Leu)(UUR) gene is an etiological hot spot of mitochondrial diseases.

Usefulness of stable HbA1c for supportive marker to diagnose diabetes mellitus in Japanese subjects

To evaluate the adequacy and usefulness of the stable glycated hemoglobin (HbA(1c)) value of 6.5% suggested by the Japan Diabetic Society in 1999 for supportive diagnostic marker of diabetes, we assessed the sensitivity and specificity of an HbA(1c) value of 6.5% in patients who were newly diagnosed by the 75 g oral glucose tolerance test (75g-OGTT). A total of 866 Japanese subjects underwent the 75g-OGTT and HbA(1c) measurement (normal range: 4.3-5.8%). They were divided into three groups [normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and diabetes mellitus (DM)], using the WHO criteria, since no subject with impaired fasting glycemia (IFG) was observed. The cut-off value of HbA(1c) separating DM from NGT or DM from IGT on cumulative distribution curve analysis was 5.9% (sensitivity 0.76 and specificity 0.86) and 5.9% (sensitivity 0.76 and specificity 0.77), respectively. The sensitivity of an HbA(1c) of 6.5% for separation of DM from NGT or IGT by the same analysis was 0.49 and 0.49, respectively. Similarly, the specificity for separation of DM from NGT or IGT was 0.98 and 0.98, respectively. These results mean that 49% of diabetic subjects show an HbA(1c)> or =6.5%, and 51% have an HbA(1c) less than 6.5%, while only 2% of NGT and IGT subjects have an HbA(1c)> or =6.5%, and 98% have a value less than 6.5%. Therefore, the sensitivity of an HbA(1c) value of 6.5% in separating DM from NGT or IGT is low, and thus 6.5% is too high value to use when screening for diabetes. However, the specificity is very high, so an HbA(1c) of 6.5% is a useful supportive marker to diagnose diabetes.

Increase in cardiac muscle fructose content in streptozotocin-induced diabetic rats.

To evaluate the activation of the sorbitol pathway in cardiac muscle in diabetic rats, we measured sorbitol, fructose, and myo-inositol content in cardiac tissue obtained from control and streptozotocin-diabetic rats, with or without an 8-week insulin treatment, using gas chromatography-mass spectrometry (GC-MS). Cardiac fructose and sorbitol content in 10-week diabetic rats increased by 60-fold and 3.9-fold of those of control rats, respectively (P less than .001). In contrast, cardiac myo-inositol content in 10-week diabetic rats decreased to 56% (P less than .025) of the control value. The abnormalities in cardiac fructose, sorbitol, and myo-inositol content were completely normalized by the 8-week insulin treatment, which was initiated 2 weeks after the induction of diabetes. There was no difference in cardiac aldose reductase activity between control and diabetic rats. However, cardiac sorbitol dehydrogenase activity in diabetic rats was 151% (P less than .005) higher than that of control rats, although hepatic sorbitol dehydrogenase activity was not different between the two groups. These results indicate that the sorbitol pathway is significantly activated in cardiac tissue obtained from streptozotocin-induced diabetic rats, which results in the marked cardiac accumulation of fructose.

Pyruvate Improves Deleterious Effects of High Glucose on Activation of Pentose Phosphate Pathway and Glutathione Redox Cycle in Endothelial Cells

In our previous study (Diabetes 44:520–526, 1995), endothelial cells cultured in high glucose condition showed impairment of an oxidant-induced activation of the pentose phosphate pathway (PPP) and a reduced supply of NADPH to the glutathione redox cycle. To gain insight into the mechanisms of this impairment, the protective effect of pyruvate was studied in human umbilical vein endothelial cells cultured in either 5.5 mmol/l glucose (normal glucose [NG] condition) or 33 mmol/l glucose (high glucose [HG] condition). Through pretreatment of cells with 0.2 mmol/l pyruvate for 5–7 days in the HG condition, glucose oxidation through the PPP and total cellular NADPH content in the presence of 0.2 mmol/l H2O2 were increased by 54 (P < 0.05) and 34%, respectively, and glutathione-dependent degradation of H2O2 in HG cells was enhanced by 41% (P < 0.01), when compared with those cells to which pyruvate was not added. The addition of pyruvate significantly reduced the fructose 1,6-bisphosphate (FDP) content and free cytoplasmic NADH/NAD ratio, estimated by increased pyruvate/lactate ratio in NG and HG cells exposed to H2O2. Furthermore, the addition of pyruvate also showed a 46% reduction (P < 0.01) of endothelial cell damage induced by H2O2 in HG cells. These results indicate that abnormalities in PPP activation and glutathione redox cycle activity induced by H2O2 in HG cells are compensated, and that the accentuated reductive stress is improved by an addition of pyruvate. These pyruvate effects are associated with protection against an oxidant-induced endothelial cell injury in the high glucose condition.

Increase in [3H]PN 200-110 binding to cardiac muscle membrane in streptozocin-induced diabetic rats.

Voltage-sensitive Ca2+ channels in cardiac left ventricular muscle membranes isolated from nondiabetic control and diabetic rats were measured with [3H]PN 200-110, a dihydropyridine derivative, as a ligand. The binding site (Bmax) of [3H]PN 200-110 in cardiac membranes isolated from streptozocin-induced diabetic (STZ-D) rats (128 +/- 10 fmol/mg protein) significantly (P less than 0.01) increased by 64% compared with that of control rats (78 +/- 4 fmol/mg protein) 10 wk after STZ administration without a significant change in Kd. However, the significant increase in Bmax of [3H]PN 200-110 binding in diabetic rats depended on the duration of diabetes such that the increase was not found until 6 wk after STZ injection. An 8-wk intensive insulin treatment, which was initiated 2 wk after STZ injection, normalized the increase in [3H]PN 200-110 binding in STZ-D rats to control levels (85 +/- 4 fmol/mg protein). Furthermore, [3H]PN 200-110 binding to control cardiac membranes was dose-dependently inhibited in the presence of verapamil, a phenylalkylamine Ca2+ antagonist, but that was not the case in cardiac membranes isolated from STZ-D rats. These results indicate that voltage-sensitive Ca2+ channels in cardiac muscle isolated from STZ-D rats are quantitatively and qualitatively altered, because the course of diabetes and the increase in the channels can be prevented by treatment with insulin.

Insulin edema in diabetes mellitus associated with the 3243 mitochondrial tRNALeU(UUR) mutation; Case reports

We encountered a patient with diabetes mellitus due to the 3243 mitochondrial tRNA mutation(DM-Mt3243), who developed insulin edema and hepatic dysfunction after starting insulin. Such a rare phenomenon was unlikely to be a fortuitous coincidence in mitochondrial diabetes, as none in 197 non-mutant NIDDM patients had same episode. Moreover, similar leg edema was noticed in another DM-Mt3243 patient, and other two DM-Mt3243 patients had leg edema which responded to coenzyme Q10. These observations suggest further a role of mitochondrial function on leg edema. The mechanism of his insulin edema may involve vasomotor changes induced by the rapidly glycemic control, because our case of insulin edema had a prominent increase of strong succinate dehydrogenase reactive vessels. Alternatively, myocardial dysfunction might have produced leg edema and hepatic dysfunction, because he had subclinical myocardial dysfunction, judged by imaging with beta-methyl-p-(123I)-iodophenyl-pentadecanoic acid. The third explanation is that a rapid improvement of glycemic control might have induced hepatic reoxygenation and the production of reactive oxygen species in the liver that contributed to cell damage. Thus, although we cannot draw definite conclusion, our experiences here suggest that mitochondrial dysfunction is important in the etiology of insulin edema.