Seiichi Tagami

Weakened cellular scavenging activity against oxidative stress in diabetes mellitus: regulation of glutathione synthesis and efflux

SummaryGlutathione functions to scavenge oxidants or xenobiotics by covalently binding them and transporting the resulting metabolites through an adenosine 5′-triphosphate-dependent transport system. It has been reported that the intracellular concentration of glutathione decreases in diabetes mellitus. In order to elucidate the physiological significance and the regulation of anti-oxidants in diabetic patients, changes in the activity of the glutathione-synthesizing enzyme, γ-glutamylcysteine synthetase, and transport of thiol [S-(2,4-dinitrophenyl)glutathione] were studied in erythrocytes from patients with non-insulin-dependent diabetes and K562 cells cultured with 27 mmol/l glucose for 7 days. The activity of γ-glutamylcysteine synthetase, the concentration of glutathione, and the thiol transport were 77%, 77% and 69%, respectively in erythrocytes from diabetic patients compared to normal control subjects. Treatment of patients with an antidiabetic agent for 6 months resulted in the restoration of γ-glutamylcysteine synthetase activity, the concentration of glutathione, and the thiol transport. A similar impairment of glutathione metabolism was observed in K562 cells with high glucose levels. The cytotoxicity by a xenobiotic (1-chloro-2,4-dinitrobenzene) was higher in K562 cells with high glucose than in control subjects (50% of inhibitory concentration. 300±24 Μmol/l vs 840±29 Μmol/l, p<0.01). Expression of γ-glutamylcysteine synthetase protein was augmented in K562 cells with high glucose, while enzymatic activity and expression of mRNA were lower than those in the control subjects. These results suggest that inactivation of glutathione synthesis and thiol transport in diabetic patients increases the sensitivity of the cells to oxidative stresses, and these changes may lead to the development of some complications in diabetes mellitus.

Effect of insulin on impaired antioxidant activities in aortic endothelial cells from diabetic rabbits

The defense system of aortic endothelial cells against oxidative stress was studied in alloxan-induced diabetic rabbits, and the effect of insulin on the antioxidant activities was estimated. Endothelial cells were prepared from 10 diabetic rabbits, 18 diabetic rabbits treated with insulin, and 10 age-matched controls after 17 days of diabetes. These cells were used for the estimation of glutathione (GSH) levels and its related enzyme activities. The antioxidant activities in these endothelial cells from diabetic rabbits were compared with those from control subjects. The concentration of GSH decreased in diabetic rabbits (1.6 +/- 0.2 nmol/mg protein [mean +/- SD] v 3.7 +/- 0.6 nmol/mg protein). Decreases in the activities of Cu, Zn-superoxide dismutase (Cu,Zn-SOD) (62.7 +/- 11.0 U/mg protein v 172.9 +/- 20.2 U/mg protein), catalase (7.6 +/- 2.1 U/mg protein v 12.3 +/- 3.2 U/mg protein), and GSH peroxidase (134.0 +/- 27.0 mU/mg protein v 179.1 +/- 26.2 mU/mg protein) were observed. The activities of other GSH-related enzymes such as GSH S-transferase or GSH reductase did not change in endothelial cells from diabetic rabbits. Most of these antioxidant activities were prevented when diabetic rabbits were treated with insulin (1 to 2 U/kg/d). These antioxidant activities were also determined in the diabetic liver and kidney. Similar decreases in the cellular defense activities and prevention of the decrease in activities by insulin were observed in the diabetic liver, while these antioxidant enzyme activities in the kidney were resistant to diabetic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating levels of ganglioside GM3 in metabolic syndrome: A pilot study

SUMMARY BACKGROUND Insulin resistance is a characteristic feature of metabolic syndrome. Ganglioside GM3 [α-Neu5Ac-(2-3)-β-Gal-(1-4)-β-Glc-(1-1)-ceramide] may impair insulin sensitivity in adipose tissue. We investigated the relationship between serum GM3 levels and adiposity indices, as well as between serum GM3 levels and metabolic risk variables. METHODS Study 1: we assessed serum GM3 levels in normal subjects and in patients with hyperglycemia and/or hyperlipidemia (HL). Study 2: we investigated the relationship between serum GM3 levels and metabolic risk variables in patients with type 2 diabetes. RESULTS Study 1: serum GM3 levels were higher in hyperglycemic patients (1.4-fold), hyperlipidemic patients (1.4-fold) and hyperglycemic patients with hyperlipidemia (1.6-fold), than in normal subjects. Study 2: serum GM3 levels were significantly increased in type 2 diabetic patients with severe obesity (visceral fat area (VFA) >200 cm(2), BMI > 30). The GM3 level was positively correlated with LDL-c (0.403, p = 0.012) in type 2 diabetes mellitus, but not affected by blood pressure. In addition, the high levels of small dense LDL (>10 mg/dL) were associated with the elevation of GM3. CONCLUSIONS Serum GM3 levels was affected by glucose and lipid metabolism abnormalities and by visceral obesity. GM3 may be a useful marker for severity of metabolic syndrome.

Effects of cold stress on glutathione and related enzymes in rat erythrocytes

Effects of acute and chronic cold stress on glutathione and related enzymes in rat erythrocytes were investigated. Blood from both cold-acclimated (CA) and cold-adapted (CG) rats had significantly lower concentrations of glutathione than blood from control animals. Superoxide dismutase activity was increased significantly in CA rats and tended to rise in CG rats. Activity of glutathione peroxidase in erythrocytes was inconsistent in that it tended to increase in CA rats but decreased significantly in CG rats. The results may imply that CG rats suffered deleterious effects of hydrogen peroxide. On the other hand, there were marked decreases in glutathione peroxidase and glutathione reductase activities in acutely cold-exposed rats in conjunction with unchanged levels of glutathione. In all treatments the state of riboflavin metabolism was estimated to be adequate, since no increases were observed in the erythrocyte glutathione reductase activity coefficient.

GM3 synthase gene is a novel biomarker for histological classification and drug sensitivity against epidermal growth factor receptor tyrosine kinase inhibitors in non‐small cell lung cancer

Expression of gangliosides and alterations in their composition have been observed during cell proliferation and differentiation and in certain cell cycle phases, brain development and cancer malignancy. To investigate the characteristics of GM3 synthase, SAT‐I mRNA and ganglioside GM3 expression levels in lung cancer, we examined the expression levels of SAT‐I mRNA as well as GM3 in 40 tumor tissues surgically removed from non‐small cell lung cancer patients. Adenocarcinoma tissues expressed SAT‐I mRNA levels that were significantly higher than those of squamous and other carcinomas (P < 0.0001). Moreover, the SAT‐I mRNA levels were high in the bronchioalveolar carcinoma subtype and low in the solid and mucin subtypes of adenocarcinomas (P = 0.049, 0.049 and 0.013, respectively). To clarify the relationship between SAT‐I mRNA and epidermal growth factor receptor (EGFR)‐tyrosine kinase (TK) inhibitor sensitivity, we carried out drug sensitivity tests for the EGFR‐TK inhibitors gefitinib and AG1478 using eight adenocarcinoma cell lines expressing no EGFR mutations. The IC50 values for gefitinib and AG1478 decreased dramatically with increasing SAT‐I mRNA levels (R2 = 0.81 and 0.59, respectively), representing a wide range of drug sensitivities among adenocarcinoma cell lines. To explore a possible mechanism of how GM3 could enhance the sensitivity to EGFR‐TK inhibitors, the SAT‐I gene was introduced stably into a GM3‐negative clone of murine 3LL lung cancer cells to produce GM3‐reconstituted clones. We found an increase in EGFR protein levels and gefitinib sensitivity in GM3‐reconstituted cells, suggesting the involvement of GM3 in the turnover of EGFR protein. Therefore, it is highly expected that, by measuring the expression levels of SAT‐I mRNA in lung biopsy samples from non‐small cell lung cancer patients, enhanced pathological identification and individualized chemotherapeutic strategies can be established for the appropriate use of EGFR‐TK inhibitors. (Cancer Sci 2007; 98: 1625–1632)