Takaya Nakane

Antioxidants in the serum of children with insulin-dependent diabetes mellitus

To determine whether alteration in serum antioxidant status is related to the increased oxidative stress as a cause of diabetic angiopathy, we measured both the antioxidant activity (AOA) and total peroxyl radical-trapping antioxidant parameter (TRAP), and their component individual antioxidants in serum of children with insulin-dependent diabetes mellitus (IDDM). The AOA was measured as the ability to inhibit lipid autoxidation in brain homogenates. TRAP was assayed as the ability to delay lipid peroxidation induced by an azo initiator. Antioxidants measured were ceruloplasmin, transferrin, and albumin as components of AOA; and ascorbic acid, uric acid, protein sulfhydryl, and alpha-tocopherol as components of TRAP. Serum AOA appeared to be decreased in the diabetics in relation to poor glycemic control, corresponding to the decrease in transferrin and albumin. Serum haptoglobin level was also decreased in the diabetics. Similarly, the directly measured TRAP value was decreased in the diabetic serum mainly due to the decreased contribution of unidentified chain-breaking antioxidants, despite the increase in ascorbic acid and alpha-tocopherol. The decrease in both types of antioxidant activity in the diabetic serum, as new findings, suggests that a defective serum antioxidant status contributes to the increased oxidative stress in IDDM.

Threshold values of visceral fat measures and their anthropometric alternatives for metabolic derangement in Japanese obese boys.

OBJECTIVE: To determine whether the direct measure of visceral adipose tissue (VAT) by computed tomography (CT) is a superior diagnostic criterion to the anthropometric surrogates and more classical criteria of obesity.DESIGN: Cross-sectional, clinical study. Obese boys were classified according to the occurrence of abnormal values in either serum triglyceride, alanine aminotransferase or insulin level. A threshold value of each criterion for such metabolic derangement was calculated, using the analysis of receiver operating characteristic (ROC) curve.SUBJECTS: Seventy-five consecutive outpatient Japanese obese boys, ranging in age from 6 to 14 y, were studied.MEASUREMENTS: Anthropometric indices measured were height, body weight, waist girth, hip girth, triceps and subscapular skinfold thicknesses. Classical criteria for obesity used were percentage overweight (POW), body mass index (BMI) and percentage body fat. Waist girth, sagittal diameter by CT and waist–hip ratio (WHR) were evaluated as anthropometric surrogates for VAT. The areas of total abdominal fat (TAF), VAT and subcutaneous adipose tissue (SAT) were measured by CT at the level of the umbilicus. Clinical blood biochemistry was analyzed in fasting blood samples of obese boys.RESULTS: Thirty-three boys were classified into a no-complication group, and 42 into a complication group. TAF, VAT and SAT areas were closely associated with age, body size and degree of overweight and adiposity, while VAT/SAT was not. VAT area, sagittal diameter, TAF area and waist girth were closely correlated with alanine aminotransferase, insulin, TG and HDL-C. VAT/SAT, BMI, SAT area, WHR, percentage body fat and POW were less closely associated with these biochemical indices. The descending order of the values of area under the curve for the ROC curves were as follows: VAT>sagittal diameter>TAF>VAT/SAT>waist girth>BMI>WHR>percentage body fat>POW. Both VAT area and VAT/SAT gave >80% of sensitivity and specificity. Among the anthropometric indices studied, the sagittal diameter was the best surrogate of visceral fat measure. The sensitivity and specificity for the rest of the anthropometric indices were in an unsatisfactory range. The threshold values for VAT area, VAT/SAT and sagittal diameter were 58.0 cm2, 0.276 and 19.2 cm, respectively.CONCLUSION: The threshold values for VAT area, VAT/SAT and sagittal diameter for detecting biochemical complication in Japanese obese boys were lower than the respective values reported in adults. These values can be used for classifying the obese boys into two types: those with medical problem and those without.

Effect of selenium deficiency on cellular and extracellular glutathione peroxidases: immunochemical detection and mRNA analysis in rat kidney and serum

To determine the effect of selenium (Se) deficiency on expression of glutathione peroxidase (GSH-Px) 1 and 2, we measured GSH-Px activity in rat serum, liver and kidneys, serum immunoreactive GSH-Px 2, and the mRNAs of kidney GSH-Px 1 and 2. We purified rat GSH-Px 2 and raised polyclonal antibodies. Immunoreactive GSH-Px 2 was measured by rocket immunoelectrophoresis. GSH-Px 2 was purified 1470-fold with a specific activity of 250 units/mg. Immunoblotting detected only GSH-Px 2 in rat serum, and much less GSH-Px 2 than GSH-Px 1 in kidney. Immunoblot signal of kidney GSH-Px 1 and 2 decreased progressively in Se deficient rats. Serum GSH-Px activity in Se deficient rats at 1, 2, 3, and 4 weeks declined to 33, 20, 10, and 9% of the control, while the serum level of immunoreactive GSH-Px 2 was 58, 24, 15, and 10% of the control, suggesting the presence of an inactive protein at week 1. GSH-Px activity declined to 4 and 11% of the control in the liver and kidney at 4 weeks. The mRNAs of kidney GSH-Px 1 and 2 showed similar decreases, and were 24 and 23% of the control at 4 weeks. GSH-Px mRNA levels were better preserved than GSH-Px activity, suggesting that GSH-Px expression was regulated at both pre-translational and translational levels.

Increased plasma cholesteryl ester transfer activity in obese children

To determine whether enhanced activity of cholesteryl ester transfer protein (CETP) contributes to the development of atherogenic lipoprotein profiles in obese children, plasma CETP activity was assayed according to a micro-method, by co-incubating lipoprotein-deficient samples with exogenous donor and acceptor lipoproteins. The study subjects were 31 obese children (14 males and 17 females). Serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), TC:high-density lipoprotein (HDL)-C, LDL-C:HDL-C, apolipoprotein (apo) B, and apo B:apo Al were increased in obese children. Thus they appeared to exhibit an atherogenic lipoprotein profile, with a relative decrease in cholesterol carried by HDL compared with the cholesterol in the other lipoprotein fractions. The mean fasting plasma insulin level was also increased. CETP activity was significantly higher in the obese children than in nonobese control children, and was correlated with LDL-C, TC:HDL-C, LDL-C:HDL-C, and apo B:apo Al. These results suggest that an increase in plasma CETP activity results in atherogenic change in lipoprotein metabolism in obese children. The increase in CETP may be due to the adiposity or insulin resistance. Alternatively, dyslipidemia per se, physical inactivity or excessive fat intake, that are commonly found in obese children, may contribute to the increase in CETP activity.

Increased peroxisomal fatty acid beta-oxidation and enhanced expression of peroxisome proliferator-activated receptor-alpha in diabetic rat liver.

To determine whether the increased fatty acid β-oxidation in the peroxisomes of diabetic rat liver is mediated by a common peroxisome proliferation mechanism, we measured the activation of long-chain (LC) and very long chain (VLC) fatty acids catalyzed by palmitoyl CoA ligase (PAL) and lignoceryl CoA ligase and oxidation of LC (palmitic acid) and VLC (lignoceric acid) fatty acids by isotopic methods. Immunoblot analysis of acyl-CoA oxidase (ACO), and Northern blot analysis of peroxisome proliferator-activated receptor (PPAR-α), ACO, and PAL were also performed. The PAL activity increased in peroxisomes and mitochondria from the liver of diabetic rats by 2.6-fold and 2.1-fold, respectively. The lignoceroyl-CoA ligase activity increased by 2.6-fold in diabetic peroxisomes. Palmitic acid oxidation increased in the diabetic peroxisomes and mitochondria by 2.5-fold and 2.7-fold, respectively, while lignoceric acid oxidation increased by 2.0-fold in the peroxisomes. Immunoreactive ACO protein increased by 2-fold in the diabetic group. The mRNA levels for PPAR-α, ACO and PAL increased 2.9-, 2.8- and 1.6-fold, respectively, in the diabetic group. These results suggest that the increased supply of fatty acids to liver in diabetic state stimulates the expression of PPAR-α and its target genes responsible for the metabolism of fatty acids.

Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.

Effect of obesity and troglitazone on expression of two glutathione peroxidases: cellular and extracellular types in serum, kidney and adipose tissue.

To determine the effect of obesity on expression of cellular- (C-) and extracellular (EC-) glutathione peroxidase (GPX) in serum, kidney and adipose tissue, we measured GPX in serum, kidneys and adipose tissue of the obese Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat and its lean counterpart (LETO). We also investigated the effect of troglitazone. Five each of OLETF and LETO rats were fed diet with or without 0.2% troglitazone for 10 days. Final body weight, kidney weight, blood glucose and serum tumor necrosis factor-α (TNF-α) level were higher in OLETF rats than in LETO rats. Serum and kidney GPX activities were higher, but adipose tissue GPX activity was lower, in OLETF rats than in LETO rats. Troglitazone treatment decreased adipose tissue GPX activity and abolished overproduction of TNF-α in OLETF rats. Immunoblot analysis, for the first time, revealed that both obesity and troglitazone suppressed the protein signals for C-GPX and EC-GPX in adipose tissue. Serum protein carbonyl groups were increased in OLETF rats and troglitazone completely blocked this increase. Increased serum GPX activity in obese rat was due to the increased secretion of EC-GPX from the kidney. Troglitazone protected against the enhanced oxidative stress induced by obesity independently of the serum GPX concentration.

Troglitazone inhibits the expression of inducible nitric oxide synthase in adipocytes in vitro and in vivo: Study in 3T3-L1 cells and Otsuka Long-Evans Tokushima Fatty rats

The aim of this study was to determine the mechanism of troglitazone action on nitric oxide (NO) production via inducible NO synthase (iNOS) in adipocytes in vitro and in vivo. The treatment of 3T3-L1 adipocytes with the combination of lipopolysaccharide (LPS), tumor necrosis factor-alpha and interferon-gamma synergistically induced de novo iNOS expression leading to enhanced NO production. The NO production was inhibited by co-treatment with aminoguanidine or N-nitro-L-arginine methylester hydrochloride. Troglitazone inhibited the NO production in a dose dependent manner by the suppression of iNOS expression. In the 24 week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, the mean weight and the blood glucose were 21% and 30%, respectively, higher than in their lean counterparts. The serum nitrite concentration was increased after injection of LPS (4 mg/kg, i.p.), more markedly in OLETF rats than in the lean rats. The epididymal fats from LPS-injected groups, but not the ones from the non-injected groups, expressed mRNA and protein of iNOS. Troglitazone pre-treatment blocked the LPS-induced expression of iNOS in adipose tissue and the increase in serum nitrite concentration. These results suggest that troglitazone inhibits the cytokine-induced NO production in adipocytes by blocking iNOS expression both in vitro and in vivo.

Relationships between an index of body fat distribution (based on waist and hip circumferences) and stature, and biochemical complications in obese children.

OBJECTIVE: To determine the clinical utility of a new age-adjusted measure of body fat distribution (based on waist and hip circumferences) and stature, in relation to biochemical complications in obese children.DESIGN: Cross-sectional, clinical study. The formula to calculate the common standard deviation score (SDS) of waist-to-hip ratio/height (WHR/Ht) was obtained from the data of control children. The relationship between WHR/Ht SDS, as the age-adjusted measure, in obese children and their clinical laboratory data was evaluated.SUBJECTS: Outpatient obese Japanese children (102 boys and 75 girls) and control children (508 boys and 549 girls), ranging in age from 6–15 y.MEASUREMENTS: Height, body weight, waist girth, hip girth, triceps and subscapular skinfold thicknesses, as anthropometric measures. Percent overweight, percent body fat, waist girth, WHR and WHR/Ht SDS as criteria for obesity. Clinical laboratory analyses for fasting blood samples of obese children.RESULTS: The WHR/Ht SDS closely correlated with age in obese children, thus reflecting the progress of abdominal obesity during growth. The obese boys were more hyperlipidaemic than the girls were, although the percent overweight was similar in both genders. The percent overweight, percent body fat, waist girth and WHR/Ht SDS all correlated well with triglyceride (TG), alanine aminotransferase (ALT) and insulin in boys, whereas only waist girth and WHR/Ht SDS showed a close correlation with TG and insulin in girls. The obese subjects were subdivided according to the number of abnormal values observed in TG, ALT and insulin. For obese boys, all five indices of obesity were higher in the groups with complications than in the group without. In the girls, only the WHR/Ht SDS constantly differed between subgroups. WHR/Ht SDS most obviously distinguished the groups with complications from the other group with a wide margin of difference (2-fold in boys and >2 -fold in girls) in the mean values.CONCLUSION: The WHR/Ht SDS can serve as an index predicting the occurrence of biochemical complications in obese children ranging from the age of 6–15 y.

Induction of glutathione peroxidase in response to inactivation by nitric oxide.

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 μg/ml) and tumor necrosis factor-α (TNF-α, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-α exerted a transient effect on the GPX activity. The treatment with SNAP (200 μM) or sodium nitroprusside (200 μM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-α, and aminoguanidine blocked the effect. In cells metabolically labeled with 75Se, LPS + TNF-α accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.