Keiko Uchimura

Changes in Superoxide Dismutase Activities and Concentrations and Myeloperoxidase Activities in Leukocytes from Patients with Diabetes Mellitus

To investigate whether the two free-radical scavengers, Cu, Zn- and Mn-superoxide dismutase (SOD), are changed in leukocytes of diabetic patients, and the alteration of these enzymes correlates with the diabetic state, we measured the activity and concentration of these enzymes in leukocytes from diabetic patients. Both Cu, Zn-SOD and Mn-SOD activities in neutrophils and lymphocytes were significantly lower in patients with non-insulin-dependent diabetes mellitus than in healthy controls. The concentrations of these enzymes in leukocytes from diabetic patients, however, did not differ from those in controls. Cu, Zn-SOD and Mn-SOD activities in neutrophils inversely correlated with HbA(1c) concentrations. Myeloperoxidase activity in leukocytes was significantly reduced in NIDDM patients. These findings suggest that changes in these enzymes may affect the susceptibility to infection and immunocompetence of patients with diabetes.

Scavenging effect of nicorandil on free radicals and lipid peroxide in streptozotocin-induced diabetic rats.

Free radicals and lipid peroxide (LPO), easily formed in the diabetic state, play an important role in the development of diabetic complications. Potentially, nicorandil may reduce the production of free radicals and LPO in various organs. In fact, increased LPO levels in the serum, kidney, and cardiac muscle of diabetic (DM) rats were reduced by nicorandil treatment (N treatment). Xanthine oxidase (XOD), which produces free radicals, was decreased in the liver and increased in the kidney of DM rats compared with control rats, and these changes were prevented by N treatment. The concentration of Cu, Zn-superoxide dismutase (SOD) decreased in the cardiac muscle and increased in the kidney of DM rats, and these changes returned to normal after N treatment. The decreased concentration of Mn-SOD in the liver, kidney, and cardiac muscle from DM rats was also reversed by N treatment. The changes in catalase and glutathione peroxidase (GSH-PX) activities in DM rats were not improved effectively by N treatment. Another K-adenosine triphosphate (K-ATP) channel opener, tilisolol hydrochloride, had an effect similar to that of nicorandil. The effects of nicorandil and tilisolol were studied only in DM rats. These data imply that N treatment, as an antioxidative therapy, may be beneficial in preventing diabetic complications due to lipoperoxidation and free radicals in DM rats.

Reduction of activity, but no decrease in concentration, of erythrocyte Cu, Zn-superoxide dismutase by hyperglycaemia in diabetic patients

Cu,Zn‐superoxide dismutase (SOD) activity in erythrocytes is affected by various diseases, including diabetes mellitus (DM). We investigated changes in the Cu,Zn‐SOD activity compared to changes in the Cu,Zn‐SOD concentration in erythrocytes obtained from patients with Type 2 (non‐insulin‐dependent) diabetes mellitus. Cu,Zn‐SOD activity in erythrocytes was significantly lower in Type 2 DM patients than in healthy non‐diabetic subjects. The activity correlated negatively with HbA1c, but not with other indicators of metabolic control, such as fasting blood glucose or plasma cholesterol or triglyceride. However, there was no statistically significant difference in erythrocyte concentration of Cu,Zn‐SOD between diabetic and control samples. Concentration did not correlate with enzymatic activity or HbA1c. These findings indicate that the inactivation of Cu,Zn‐SOD activity in erythrocytes of Type 2 DM patients by hyperglycaemia may be slow, because there is a negative correlation between the enzyme activities and HbA1c levels, but not fasting blood glucose levels. This is consistent with glycosylation of the active site of Cu,Zn‐SOD, without any effect of hyperglycaemia on the concentration of Cu,Zn‐SOD.

Effects of thyroid hormone on the sorbitol pathway in streptozotocin-induced diabetic rats

Sorbitol accumulation plays an important role in diabetic complications involving the kidney, nerves, retina, lens and cardiac muscle. To investigate the influence of thyroid hormone on the sorbitol pathway, we studied the effects of thyroid hormone on polyol metabolism in normal and diabetic rats. Rats were divided into three groups: controls, streptozotocin (STZ)-induced diabetic euthyroid rats (DM) and STZ-induced diabetic hyperthyroid (thyroxine-injected) rats (DM+HT). The sorbitol (Sor) concentrations in the kidney, liver and sciatic nerve (2.53+/-0.74, 0.97+/-0.16 and 24.0+/-5.1 nmol/mg protein, respectively) of the DM rats were significantly higher than those (1.48+/-0.31, 0.58+/-0.13 and 3. 1+/-0.6 nmol/mg protein) of the control rats. The Sor concentrations in the kidney and sciatic nerve of the DM+HT rats (1.26+/-0.29 and 9. 40+/-1.2 nmol/mg protein) were significantly lower than those in the DM rats. These values were reduced in the liver, unchanged in the kidney, and increased in the sciatic nerve from the hyperthyroid rats without diabetes. Thyroid hormone reduced the aldose reductase (AR) activities in the kidney, liver and sciatic nerve of the DM rats, and similarly reduced AR in the kidney and liver, but not in the sciatic nerve, of the non-diabetic rats. The sorbitol dehydrogenase (SDH) activities were decreased by thyroid hormone in the kidney and liver but not the sciatic nerve of DM rats. In the non-diabetic rats, this enzyme activity was decreased in liver, but not in kidney or sciatic nerve. A positive correlation between the Sor concentration and AR activity was observed in the kidney and liver but not in the sciatic nerve from control, DM and DM+HT rats. A negative correlation was observed between the Sor concentration and SDH activities in the same organs. These data suggest that thyroid hormone affects the sorbitol pathway, but the detailed mechanism whereby this hormone reduces the sorbitol content (especially in diabetic rats) remains to be clarified.

The effects of CD40‐ and interleukin (IL‐4)‐activated CD23+ cells on the production of IL‐10 by mononuclear cells in Graves’ disease: the role of CD8+ cells

The possible roles of CD8+ cells in the abnormal T cell‐dependent B‐cell activation in Graves’ disease were investigated by analysing lymphocyte subsets in peripheral blood mononuclear cells (PBMC) and their production of soluble factors and cytokines such as IL‐10 in patients with Graves’ disease, Hashimoto’s thyroiditis and normal controls. The PBMC were separated into CD8+ and CD8‐depleted cells by magnetic separation columns, and cultured for 7 days with or without anti‐CD40 monoclonal antibodies and IL‐4. The culture supernatant was assayed for sCD23 and IL‐10 using EIA, and the remaining cells were analysed by flow cytometry. Stimulation with anti‐CD40 antibody together with IL‐4 increased sCD23 levels and the number of CD23+ cells. The latter was further augmented by depletion of CD8+ cells. This combination of B cell stimulants increased production of IL‐10 by PBMC from patients with Graves’ disease. The CD40‐ and IL‐4‐activated production of IL‐10 was decreased by CD8+ cell depletion. In contrast, constitutive production of IL‐10 was increased after CD8+ cell depletion in a group of patients with low basal secretion levels (<35 ng/ml). It was, however, decreased in a group with higher basal production levels, but such a relationship was not found in the normal control group. Thus, T cell‐dependent B‐cell activation via a CD40 pathway activates CD23+ cells, leading to over‐production of IL‐10 and a shift of the Th1/Th2 balance to Th2 dominance, while CD8+ cells may suppress this activation to counteract the Th2 deviation in Graves’ disease.

Surface expression and release of soluble forms of CD8 and CD23 in CD40- and IL-4-activated mononuclear cells from patients with Graves' disease (GD).

We investigated the effect of T cell‐dependent B cell activation on the surface expression and release of the soluble forms of CD8 and CD23 by peripheral blood mononuclear cells (PBMC) obtained from patients with GD, versus patients with Hashimotos thyroiditis, and normal controls. Incubating the PBMC with anti‐CD40 MoAbs and IL‐4 increased the soluble CD23 levels in cells from all three groups. An increase in the number of CD23+ cells was observed in the PBMC from the patients with GD, but not in PBMC from Hashimotos thyroiditis or controls. Less soluble CD8 was released from anti‐CD40 antibody and IL‐4‐stimulated PBMC obtained from patients with GD relative to those from the controls. In addition, the number of CD8+ cells was significantly reduced in stimulated PBMC from the GD patients relative to those from controls. Incubation of PBMC with anti‐CD40 antibody plus IL‐4 did not affect the proportions of CD4+, CD20+, Fas+CD4+, and Fas+CD8+ cells. The addition of T3 to cultured PBMC from controls did not reproduce the changes in CD23+ and CD8+ cells noted in the samples from GD patients. Thus, T cell‐dependent B cell activation, mediated by a CD40 pathway, may reduce the number of CD8+ cells, causing exacerbation of GD.

Changes in Erythrocyte Sorbitol Concentrations Measured Using an Improved Assay System in Patients With Diabetic Complications and Treated With Aldose Reductase Inhibitor

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