Masayuki Ohtaka

New Mitochondrial DNA Homoplasmic Mutations Associated With Japanese Patients With Type 2 Diabetes

Epidemiological studies have shown that patients with type 2 diabetes are more likely to have affected mothers than fathers and that the disease is often transmitted in a mode of maternal inheritance (1,2). Because transmission of mitochondria is exclusively maternal (3), mitochondrial DNA (mtDNA) mutations have been implicated in the maternal inheritance of diabetes (4). However, mtDNA mutation at 3243 has been reported in only 1-1.5% of patients with type 2 diabetes (5,6). Therefore we postulated that other mtDNA mutations may be associated with type 2 diabetes. Recently, using polymerase chain reaction (PCR)-restriction fragment (RF)-single-strand conformation polymorphism (SSCP) analysis (7), we confirmed 56 mtDNA mutations in Japanese subjects (8). In this study, we investigated the prevalence of 5 mtDNA mutations, including 3 mutations that we reported (8) in Japanese subjects. The study was performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects, and the study design was approved by the Ethical Committee of Yamanashi Medical University. Peripheral blood was obtained from 253 unrelated patients (mean age: 55.7 ± 1.8 years) with type 2 diabetes in the order of their visits to outpatient Clinic of Yamanashi Medical University Hospital. We also recruited 345 healthy control subjects (mean age: 43.6 ± 1.6 years) with no family history of diabetes and normal oral glucose tolerance or HbAlc, who visited the Isawa Curehouse or Koseiren Health Center for medical checkups. Genomic DNA was obtained from peripheral leukocytes using a DNAQuick kit (Dainippon Pharmaceutical, Osaka, Japan). mtDNA mutations were detected either by PCR-SSCP or PCR-restriction fragment length polymorphism (RFLP) analyses. Two primer pairs were used: primer pair A, for-

Aldose reductase mRNA expression and its activity are induced by glucose in fetal rat aortic smooth muscle (A10) cells.

The induction of aldose reductase (AR) mRNA expression and its activity by glucose were investigated in fetal rat aortic smooth muscle (A10) cells. The increase in the expression of AR mRNA was observed at 6 h, and reached a maximum (2.3 fold) at 12 h after exposure to 80 mM glucose. On the other hand, increase in AR activity was observed at 12 h and reached a maximum (2 fold) at 48 h after exposure to 80 mM glucose. AR mRNA levels as well as its activity increased almost linearly in a concentration dependent manner up to 80 mM. Although not significant, there was a consistent increase in AR mRNA level and its activity at 23.75 mM glucose. There was a good correlation between AR mRNA level and AR activity. In the presence of 1 microgram/ml actinomycin D, the increase in the expression of AR mRNA level by glucose was almost completely abolished, suggesting its transcriptional regulation by glucose. Other osmolytes were also effective in inducing AR mRNA expression as well as its activity at 80 mOsm/kg. Therefore, the increase in AR mRNA level and its activity by glucose in A10 cells may be a response to the increase in osmolarity.

Glucose modulation of aldose reductase mRNA expression and its activity in cultured calf pulmonary artery endothelial cells

SummaryWe examined the effect of glucose on aldose reductase mRNA expression and its activity in calf pulmonary artery endothelial cells. After the cells were exposed to 18 mmol/l glucose, aldose reductase mRNA expression began to increase at 6 h, reached a maximum (about 2.4-fold increase) at 12 h, and thereafter gradually decreased. Aldose reductase activity was found to strongly correlate with aldose reductase mRNA expression after cells were exposed to 18 mmol/l glucose. In contrast, aldose reductase mRNA expression was significantly decreased following exposure to 55 mmol/l glucose. Aldose reductase activity was also decreased at 24 h after 55 mmol/l glucose. The increase in aldose reductase mRNA level caused by glucose was inhibited by 1 μg/ml of actinomycin D. These phenomena appear to be glucose-specific since neither 3-O-methylglucose nor fructose affected the levels of aldose reductase mRNA. We clearly demonstrate that aldose reductase mRNA level and its activity are modulated by glucose in calf pulmonary artery endothelial cells. Our data suggest that activation of aldose reductase in endothelial cells may contribute to the development of diabetic macroangiopathy.

The effect of prostaglandin E1·αCD on vibratory threshold determined with the SMV-5 vibrometer in patients with diabetic neuropathy

We studied the effect of prostaglandin E1.alpha CD (PGE1) on diabetic peripheral neuropathy by evaluating subjective symptoms and vibration sensation using a new vibrometer (SMV-5). Patients with diabetic neuropathy (n = 38) were divided into three groups; group A received no drugs (control), group B was treated with 1500 micrograms/day of oral methyl vitamin B12 (VB12) for four weeks, and group C received 1.2 micrograms/kg/day PGE1 intravenously for four weeks. There was a close relationship between symptom scores and vibratory threshold (VT). The effect of PGE1 on subjective symptoms and VT were compared with those in groups A and B. Patients who received PGE1 showed a significant improvement rate in pain and hypesthesia compared to patients in groups A and B, and in numbness compared to group A. During the study period, there was no significant change in VT in groups A and B, whereas VT was significantly improved at styloid process (P < 0.05) and at medial malleolus (P < 0.001) in group C. Our results confirmed that PGE1 significantly improved both subjective symptoms and VT, indicating that PGE1 therapy may be useful in diabetic neuropathy.

Iloprost decreases urinary albumin excretion rate in patients with diabetic nephropathy

We conducted an open clinical trial to determine whether administration of iloprost, a stable prostacyclin analog, has any effect on urinary albumin excretion and other parameters associated with non-insulin-dependent diabetes mellitus (NIDDM) patients. Twenty-three NIDDM patients with nephropathy were divided into groups A and B which were matched in terms of sex, age, duration of diabetes and blood glucose control. After 2 weeks of observation, 11 patients in group A received an intravenous infusion of iloprost (10 micrograms at a rate of 0.075 microgram/kg per h) once daily for 2 weeks, while 12 untreated diabetic patients in group B served as controls. In group A, iloprost significantly reduced the urinary albumin excretion rate, the urinary albumin-creatinine ratio and N-acetyl-beta-D-glucosaminidase without decreasing creatinine clearance during the treatment period (P < 0.05, respectively). However, none of these parameters changed significantly in group B. Urinary beta 2-microglobulin, blood pressure, heart rate, serum electrolytes, BUN and serum creatinine were not significantly altered by iloprost during the treatment period. Side effects associated with iloprost were mild and could be ameliorated by slowing the infusion rate. We conclude that iloprost appears to be safe and has an apparent effect on the urinary albumin excretion rate and N-acetyl-beta-D-glucosaminidase.

Variant forms of glucokinase gene in Japanese patients with late-onset type 2 diabetes

We have applied the technique of single-strand conformation polymorphism analysis to detect mutations of the glucokinase gene in 50 Japanese patients with lateonset type 2 diabetes and in 50 normal Japanese subjects. Out of the 50 patients with late-onset type 2 diabetes, we observed three kinds of variant patterns: one in exon 1b, one in exon 4, and one in exon 5. The incidence of these patterns was one in exon 1b, two in exon 4 and one in exon 5. Direct sequencing of exon 1b and exon 5 revealed mutations in intron areas at the 12th nucleotide downstream from the 5′ splice points in two cases. Direct sequencing of exon 4 revealed a heterozygous silent mutation, CCP[Pro]→CCG[Pro] at codon 145. In contrast, 50 normal Japanese subjects showed no variant patterns in any exons. Our results showed that although 8% (4 out of 50) of Japanese patients with late-onset type 2 diabetes have variant forms of the glucokinase gene, none is expected to cause apparent qualitative changes in glucokinase. We think that the frequency of mutations of the glucokinase gene which could cause qualitative change is very low in Japanese patients with late-onset type 2 diabetes.