Toshimasa Onaya

Bone mineral density measured by dual energy X-ray absorptiometry in patients with non-insulin-dependent diabetes mellitus

Because of the previous controversial findings in diabetic patients with older methodologies, we assessed bone mineral density (BMD) in 78 patients (38 males and 40 females) with non-insulin-dependent diabetes mellitus using dual energy x-ray absorptiometry (DEXA). BMD was measured in lumbar vertebrae (L2-4). The BMD of each patient was calculated as the percentage of the mean value (%BMD) obtained from a healthy control group matched for sex and age. The %BMD of the patients with diabetes was about 100% for females and 96% for males, as compared with BMD of normal controls. The %BMD of the patients with diabetes was significantly correlated with body mass index and urinary C peptide level, and inversely correlated with age and duration of diabetes within 20 years. No relationships were found between %BMD and serum calcium, phosphorus, or glycosylated hemoglobin A1C levels. These observations suggest that metabolic abnormalities associated with diabetes mellitus alter the BMD, and that such factors as duration of the disease and deficit in insulin secretion are risk factors for decreased BMD.

Increased expression of the sodium/iodide symporter in papillary thyroid carcinomas.

Iodide is concentrated to a much lesser extent by papillary thyroid carcinoma as compared with the normal gland. The Na+/I- symporter (NIS) is primarily responsible for the uptake of iodide into thyroid cells. Our objective was to compare NIS mRNA and protein expression in papillary carcinomas with those in specimens with normal thyroid. Northern blot analysis revealed a 2.8-fold increase in the level of NIS mRNA in specimens with papillary carcinoma versus specimens with normal thyroid. Immunoblot analysis using anti-human NIS antibody that was produced with a glutathione S-transferase fusion protein containing NIS protein (amino acids 466-522) showed the NIS protein at 77 kD. The NIS protein level was elevated in 7 of 17 cases of papillary carcinoma but was not elevated in the normal thyroid. Immunohistochemical staining revealed abundant NIS in 8 of 12 carcinomas, whereas NIS protein was barely detected in specimens with normal thyroid. Although considerable patient-to-patient variation was observed, our results indicate that NIS mRNA is elevated, and its protein tends to be more abundant, in a subset of papillary thyroid carcinomas than in normal thyroid tissue.

The mRNA levels of thyrotropin receptor, thyroglobulin and thyroid peroxidase in neoplastic human thyroid tissues

Expression levels of thyrotropin receptor (TSH-R), thyroglobulin (Tg) and thyroid peroxidase (TPO) mRNA in normal and neoplastic human thyroid tissues (6 adenomas and 7 carcinomas) were investigated by Northern-blot and slot-blot analyses. We found that TSH-R mRNA levels were significantly lower in carcinoma tissues than in normal tissues. The levels of Tg mRNA were also significantly lowered in adenoma and carcinoma tissues as compared to normal tissues. In contrast, no significant difference was observed in the expression levels of TPO mRNA between these tissues. Furthermore, TSH-R mRNA levels were well-correlated with Tg mRNA levels in neoplastic tissues. These results suggest that mRNAs of TSH-R and Tg are expressed in relation to their degree of differentiation.

Anti-platelet action of isoliquiritigenin, an aldose reductase inhibitor in licorice

The mechanism was studied by which isoliquiritigenin, a new aldose reductase inhibitor purified from licorice (Glycyrrhizae radix), inhibits platelet aggregation. This new agent significantly inhibited the phosphorylation of 40,000- and 20,000-dalton proteins, and inhibited the formation of 12 (S)-hydroxy-5,8,10-heptadecatrienoic acid, 12-hydroxyeicosatetraenoic acid and thromboxane B2. The inhibitory effect of isoliquiritigenin on platelet aggregation in vitro was comparable to that of aspirin. Our findings may indicate that isoliquiritigenin elicits an anti-platelet action by inhibiting not only cyclooxygenase but also lipoxygenase or peroxidase activity in platelets. Isoliquiritigenin also showed an anti-platelet action in vivo. Isoliquiritigenin appears to be the only aldose reductase inhibitor with a significant anti-platelet action. Since the hyperaggregability of platelets has been implicated in the pathogenesis of diabetic complications, isoliquiritigenin may offer a unique benefit as an aldose reductase inhibitor.

Immunocytochemical and biochemical localization of parvalbumin in the retina

SummaryThe cellular distribution of parvalbumin-like immunoreactivity (PA-LI) in normal retina of rat, monkey, and human was investigated by immunohistochemical peroxidase antiperoxidase methods, and the levels of PA-LI in normal rat retina and brain were measured by radioimmunoassay. The antibody, raised in rabbits using rat skeletal muscle parvalbumin, did not cross-react with other Ca2+-binding proteins such as calmodulin or S-100 proteins. In rat retina, PA-LI-containing cells are located in the proximal inner nuclear layer and send processes to the external half of the internal plexiform layer, suggesting that they are amacrine cells. In monkey and human retina, PA-LI positive cells exist in the outermost sublayer of inner nuclear layer, and PA-LI-containing fibers that extend horizontally are found in the internal zone of outer plexiform layer. The radioimmunoassay revealed that the rat retina contained 1710±91 ng PA-LI/mg protein, the levels of which were higher than that of brain (881±165 ng PA-LI/mg protein). These results show an additional location for PA-LI outside skeletal muscle and brain, and also provide information on the function of interneurons of retina, which are still poorly understood.

DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells

Aims/hypothesis: The mouse leptin gene, a major hormonal regulator of appetite and fat cell mass, expresses during the differentiation of 3T3-L1 preadipocytes to adipocytes. To determine if DNA methylation is involved in regulating the expression of the leptin gene, we examined the methylation status and methylation-sensitive transcription factors during 3T3-L1 differentiation. Methods: DNase I footprinting, electrophoretic mobility-shift assays, and a Southwestern analysis were carried out using nuclear extracts from preadipocytes and adipocytes. Promoter activity was measured by luciferase assays. The CpG methylation pattern was determined. Results: Transient transfection of reporter constructs with the leptin promoter showed that preadipocytes that do not transcribe the leptin gene show enough transactivation, suggesting the presence of an additional regulatory mechanism. We identified eight CpG sites in the promoter up to nt –161, all of which were highly methylated ( > 92 %) in preadipocytes. Seven of these sites showed a varying degree of demethylation during differentiation, while the site at nt –54 remained methylated. In electrophoretic mobility-shift assays, DNA fragments from nt –115 to nt –70 generated a methylation-sensitive band with nuclear extracts from preadipocytes when the CpG sites were methylated. Southwestern analysis identified a 52 kMr protein that binds strongly to the methylated probes. Promoter activity was reduced by methylation of the CpG sites up to nt –115, but not up to nt –70. Conclusion/interpretation: These results suggest that methylation of specific CpG sites between nt –115 and nt –70 and a methylation-sensitive protein could contribute to leptin gene expression during adipocyte differentiation in 3T3-L1 cells. [Diabetologia (2002) 45: 140–148]

Vitamin D3 stimulates the production of prostacyclin by vascular smooth muscle cells

The effects of vitamin D3 on the production of prostacyclin (PGI2) by cultured rabbit vascular smooth muscle cells (VSMCs) were investigated. PGI2 synthesis by VSMCs was significantly increased in the presence of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 1 alpha hydroxyvitamin D3 (1 alpha(OH)D3) at 48 hours [1,25(OH)2D3 greater than 1 alpha(OH)D3]. Physiological concentration of 1,25(OH)2D3 (10(-10) M) significantly increased the synthesis of PGI2. Further, we observed that treatment with 1,25(OH)2D3 significantly induced the activity of cyclooxygenase without changing the activity of phospholipase A2. These findings suggest that the mechanism of action of 1,25(OH)2D3 on the synthesis of PGI2 is mediated by the cyclooxygenase pathway. It seems possible that vitamin D3 is a vasoactive agent and may play a protective role in the development of atherosclerosis.

The effects of sex hormones on the synthesis of prostacyclin (PGI2) by vascular tissues

The effects of estradiol and testosterone on prostacyclin (PGI2) release (measured as 6-keto-PGF1 alpha) by vascular tissues using rat aortic rings and cultured rabbit aortic smooth muscle cells (SMC) were investigated. Aortic SMC were prepared from either explants of atherosclerotic intima or those of normal media. Aortic rings obtained from male and female rats which had been treated with estradiol resulted in increased PGI2 synthesis. Furthermore, PGI2 synthesis by cultured medial SMC was significantly increased in the presence of estradiol (10(-7), 10(-9) M). An increased tendency in PGI2 synthesis was also observed in intimal SMC. On the other hand, aortic rings obtained from female rats treated with testosterone resulted in a significant decrease in PGI2 synthesis. However, aortic rings from testosterone-treated male rats and cultured medial and intimal SMC treated with testosterone (10(-6), 10(-8) M) for 48 hr did not show any significant changes in PGI2 synthesis. We also found greater PGI2 synthesis by intimal SMC compared with that by medial SMC. These results suggest that estradiol and testosterone may have opposite functions in the development of atherosclerosis, that is, estradiol for anti-atherosclerotic and testosterone for atherogenic, by modulating PGI2 synthesis by vascular tissues.

Parvalbumin in rat cerebrum, cerebellum and retina during postnatal development

Radioimmunoassay and immunohistochemical studies were performed on the occurrence and distribution of parvalbumin-like immunoreactivity (PA-LI) in developing rat cerebrum, cerebellum and retina. No PA-LI was detected in the nervous tissues of the newborn animals. In the cerebrum, the PA-LI appeared in non-pyramidal neurons at the 2nd postnatal week and increased linearly until the 8th week. In the cerebellum, a rapid increase in the PA-LI took place at the 2nd week, with an enrichment of the antigen to Purkinje neurons. In the retina, amacrine cells contained PA-LI, the levels of which increased from the 2nd to 4th week. Regulation of intracellular Ca2+ concentration may be one of the important factors for the maturation of the central nervous system.

The effects of goshajinkigan, a herbal medicine, on subjective symptoms and vibratory threshold in patients with diabetic neuropathy.

Goshajinkigan, a herbal medicine, has long been used in Japan to alleviate the subjective symptoms of diabetic neuropathy; however, its effects have not been confirmed objectively. We evaluated its effects on subjective symptoms and on vibration sensation in patients with diabetic neuropathy. The oral administration of 7.5 g/day of Goshajinkigan for 3 months (treatment period) relieved subjective symptoms of numbness in 9 of 13 patients. When the drug was discontinued for 2 months as a washout period, the subjective symptom worsened in 7 of 13 patients. Chi-square analysis revealed significant effects of Goshajiniagan on subjective symptoms (P < 0.001 for numbness and P < 0.05 for cold sensation). Vibration sensation was evaluated by measuring vibratory threshold using an SMV-5 vibrometer. There were significant changes in vibratory thresholds by paired t-test (P < 0.05) both in the upper and the lower extremities during the treatment and washout periods. Chi-square analysis also revealed a significant effect of Goshajinkigan on vibratory threshold (P < 0.01). There was no significant change in glycosylated hemoglobin as a whole during the study. These observations confirm that Goshajinkigan relieves subjective symptoms and demonstrate that it improves vibration sensation in patients with diabetic neuropathy.