Masato Tawata

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.

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.

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]

Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene

The insulin-receptor genes from a 16-year-old girl with type A insulin resistance, who presented with fasting hyperinsulinaemia, acanthosis nigricans, and reduced insulin binding, and from her family were examined. One allele of her insulin-receptor gene inherited from her mother contained a 1.2 kb deletion arising from a recombination between two Alu elements. The deletion removed the 14th exon in the beta subunit and altered the reading frame, to produce a stop codon after aminoacid 867. Pedigree analysis indicated that this mutation alone will not cause diabetes, and the proband is possibly a compound heterozygote. 4 other members of her family were heterozygous for the same mutation; all 4 had a decrease in insulin binding and slight impairment of glucose tolerance. Perhaps the same mutation is an underlying feature of some cases of non-insulin-dependent diabetes mellitus.

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 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.

Z-4 allele upstream of the aldose reductase gene is associated with proliferative retinopathy in Japanese patients with NIDDM, and elevated luciferase gene transcription in vitro.

We determined by PCR the number of (A-C)n repeats in the 2. 1 kb upstream of the aldose reductase (AR2) gene in healthy control subjects and in patients with NIDDM in Japanese. Sixty-one patients were recruited based on the severity of retinopathy and subdivided into two groups with proliferative retinopathy and without retinopathy. Japanese exhibited 10 different alleles in this region. The most prevalent allele was designated Z ((A-C)24 repeats) allele. The Z-4 allele was significantly associated with patients with proliferative retinopathy, whereas the Z+2 allele was significantly associated with patients without retinopathy. Erythrocyte AR2 protein levels were significantly elevated in patients exhibiting the Z-4 allele compared to those exhibiting other alleles. When Z-4 allele was ligated in transfection experiments to luciferase vector containing the promoter region of the AR2 gene, the construct showed significantly higher transcription of the reporter gene compared to constructs without (A-C) repeat or with Z-2, Z or Z+2 alleles. Our results suggest that the Z-4 allele in the 2. 1 kb upstream of the AR2 gene may enhance gene transcription and may be a genetic risk factor, which determines the predisposition to retinopathy in Japanese patients with NIDDM.

Bone mineral density in patients with hyperthyroidism measured by dual energy X-ray absorptiometry

OBJECTIVE We assessed the changes of bone mass in patients with hyperthyroidism by measuring bone mineral density using a new method, dual energy X‐ray absorptiometry.

Phosphorylation of Myosin Light Chain in Resting Platelets From NIDDM Patients Is Enhanced: Correlation With Spontaneous Aggregation

Platelet function in patients with NIDDM is enhanced. We have found that spontaneous aggregation (i.e., the formation of small-sized aggregates in the absence of agonist stimulation) occurs at a high rate in platelets from NIDDM patients. We then investigated basal myosin light chain 20 (MLC) phosphorylation, which plays a key role in platelet shape change and aggregation, using a monoclonal antibody against a phosphorylation site (serine 19 residue) in the MLC molecule in platelets from these patients. Standard calibration curves obtained from purified MLC or the phosphorylated form of myosin light chain 20 (MLC-P) were linear within the range of 0–150 ng for MLC and 0–3 ng for MLC-P. The amount of MLC or MLC-P in platelets was estimated, and basal MLC phosphorylation was calculated. Platelets were obtained from 9 young healthy control subjects, 13 age- and sex-matched nondiabetic control subjects, and 13 patients with NIDDM. The basal MLC phosphorylation in platelets was significantly higher in the NIDDM patients than in the control subjects, irrespective of age. These findings suggest that platelets from NIDDM patients are activated in vivo. Platelets obtained from NIDDM patients generated spontaneous aggregation, the degree of which was significantly higher than that in control subjects. Platelet spontaneous aggregation correlated well with basal MLC phosphorylation. These findings suggest that increases in basal MLC in platelets may be one factor leading to hyperaggregability of platelets in these patients.

Clinical efficacy of a stable prostacyclin analog, iloprost, in diabetic neuropathy

Iloprost, a stable prostacyclin analog, was evaluated clinically for its ability to ameliorate the symptoms of peripheral neuropathy associated with diabetes. In an open, nonrandomized trial, 13 diabetic patients with neuropathy but without proliferative retinopathy received an intravenous infusion of Iloprost at a dose of 10 micrograms, at a rate of 0.1 micrograms/kg/h, twice daily for two weeks. The administration of Iloprost relieved the majority of such subjective symptoms as pain, numbness or sensation of cold and to a lesser extent, such autonomic symptoms as dizziness. In contrast, there was little evidence of objective improvement, e.g., in motor nerve conduction velocity. Iloprost treatment significantly inhibited the platelet aggregation rate stimulated by collagen in vitro. In the one patient tested, thermography revealed an increase in skin temperature by more than 2 degrees C. Side effects associated with Iloprost included headache (3 patients) or aggravation of pain in the extremities (2 patients) and could be ameliorated by slowing the infusion rate or by discontinuing the drug (one patient). Iloprost appears to be safe and effective for relieving the symptoms of diabetic neuropathy. Our results provide the rationale for a double-blind, clinical trial in larger populations of diabetics with peripheral neuropathy.