Mototaka Yoshinari

Association between a polymorphism in the angiotensin-converting enzyme gene and microvascular complications in Japanese patients with NIDDM

SummaryThe relationship between diabetic nephropathy and an insertion (I)/deletion (D) polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene is still under debate. The association of ACE gene polymorphism with nephropathy and retinopathy was therefore examined in 362 Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM) and 105 healthy control subjects. Distribution of the ACE genotype did not differ between healthy control subjects and diabetic patients without complications. However, the frequency of the D allele was significantly higher in the diabetic subjects with nephropathy than in those without (0.32 in normoalbuminuric patients vs 0.44 in albuminuria patients with albuminuria) (χ2=7.7; p=0.006). There was no significant association between ACE genotype and retinopathy. These observations thus demonstrate a significant association of the ACE gene polymorphism with nephropathy, but not with retinopathy, in Japanese patients with NIDDM.

Peroxisome proliferator-activated receptor γ1 (PPARγ1) expresses in rat mesangial cells and PPARγ agonists modulate its differentiation

Thiazolidinediones, synthetic ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), are reported to have direct beneficial effects on diabetic nephropathy without lowering blood glucose levels in human and rat. We hypothesized these effects of thiazolidinediones might be derived from PPARgamma activation of kidney cells, and we examined the expression of PPARgamma and the effect of PPARgamma agonists, troglitazone and 15-deoxy-delta-prostaglandin J2 (15d-PGJ2), on the proliferation and differentiation in rat mesangial cells. A single band of mRNA of PPARgamma with a predicted size was detected in reverse transcription-polymerase chain reaction products (RT-PCR) using established PCR probes of PPARgamma. PPARgamma protein in rat mesangial cells was identified as PPARgamma1 by a Western blot. In a gel mobility shift assay to determine a binding activity of PPARgamma, the nuclear protein from rat mesangial cells bound to a (32)P-labeled oligonucleotide probe, including PPAR response elements. A synthetic and a natural ligand of PPARgamma, troglitazone and 15d-PGJ2, decreased thymidine incorporation in a dose dependent manner. After 7 days incubation with troglitazone and 15d-PGJ2, alpha-smooth muscle actin expression, a marker of mesangial cell de-differentiation, was decreased significantly compared to that of control. These results indicate that PPARgamma1 is expressing in rat mesangial cells, and PPARgamma1 activation with its agonists modulates the proliferation and differentiation of cultured rat mesangial cells.

A Sensitive Thyroid Stimulating Hormone Assay for Screening of Thyroid Functional Disorder in Elderly Japanese

The use of a screening test for thyroid functional disorder by sensitive thyroid stimulating hormone assay in the elderly was investigated. The basal thyroid stimulating hormone levels predicted the response of thyroid stimulating hormone to thyrotropin releasing hormone; it was suppressed in 99 (99.0%) of 100 hyperthyroid patients. Therefore, not only primary hypothyroidism but also hyperthyroidism can be excluded when the serum thyroid stimulating hormone levels are normal.

Polymorphism of the angiotensin-converting enzyme (ACE) gene in patients with thrombotic brain infarction.

The relationship between cerebrovascular disease and an insertion/deletion (I/D) polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene is still being debated. We examined its role as a risk factor in patients with thrombotic brain infarction. The association between ACE polymorphism and ischemic stroke was examined in 181 patients with thrombotic brain infarction and 271 controls without strokes. The I/D polymorphism was examined using the polymerase chain reaction. Distributions of the ACE genotypes and alleles did not differ between the infarcted patients and the controls. Both distributions in patients with onset at age 60 years or younger were significantly higher than those in younger controls (genotype: chi 2 = 7.6, P = 0.02; allele: chi 2 = 5.6, P = 0.02). There were no significant differences in the distributions of ACE genotypes and alleles between the patients with lacunar infarcts and with cortical infarcts in all ages. There were also significant differences in the distribution of ACE genotypes and alleles between the younger and the elderly subgroup of patients with brain infarction (genotype: chi 2 = 12.9, P = 0.002; allele: chi 2 = 11.1, P = 0.0009). Furthermore, there was a significant decline in the frequency of the ACE D allele with increasing age in all patients with thrombotic brain infarction. These observations demonstrated a significant association between the ACE gene polymorphism and thrombotic brain infarction in patients age 60 years or younger in a Japanese population. Furthermore, there may be an association between the ACE D allele and mortality after cerebral infarction.

Advanced glycosylation end products induced tissue factor expression in human monocyte-like U937 cells and increased tissue factor expression in monocytes from diabetic patients

Tissue factor (TF) plays a central role in the initial activation of the extrinsic coagulation pathway and is thought to be involved in the development of atherosclerosis and thrombosis. The effect of advanced glycosylation end products (AGEs) on TF expression and its mechanism were assessed by flow cytometric analysis. Human macrophage-like U937 cells, which were shown to contain mRNA encoding the receptors of advanced glycosylation end products (RAGE), expressed TF in a dose-dependent manner on incubation with AGE-albumin. AGE-albumin-induced TF expression was completely inhibited by the anti-oxidant agents, catalase and probucol. TF expression in peripheral monocytes from normal volunteers was also increased by AGE-albumin. Finally, TF expression in monocytes from individuals with diabetes mellitus, in whom the concentration of circulating AGEs is reported to be increased, was higher than that in monocytes from normal controls. These results suggest that AGE-induced TF expression in macrophages/monocytes is mediated by oxidant stress. AGEs may promote thrombosis and the development of atherosclerosis by inducing TF expression in monocytes in patients with diabetes mellitus.

A new diabetes model induced by neonatal alloxan treatment in rats

Rats treated with streptozotocin (STZ) during the neonatal period have been used as a model of non-insulin-dependent diabetes mellitus. The present study was designed to produce another diabetes model by substituting alloxan for STZ. Male Sprague-Dawley rats of 2, 4 or 6 days of age were injected intraperitoneally with 200 mg/kg of alloxan monohydrate after 16 h fast. Control rats received vehicle alone at 6 days of age. Non-fasting plasma glucose levels in alloxan-treated rats significantly increased after 8 weeks as compared with control, as the age of alloxan treatment advanced (6.6 +/- 0.2 (S.E.M.) mM in control, 8.3 +/- 0.3 mM in 2 days, P < 0.05, 9.8 +/- 0.9 mM in 4 days, P < 0.05, 17.1 +/- 3.5 mM in 6 days, P < 0.05). For the long-term observation, alloxan-treated rats were divided into mild and severe diabetes groups. Hyperglycemia persisted in both groups until 52 weeks (6.5 +/- 0.1 mM in control, 10.3 +/- 0.7 mM in mild diabetes group, 25.3 +/- 3.6 mM in severe group), but significant albuminuria developed only in severe diabetes group. The diabetogenicity of alloxan rapidly increased during the neonatal period, and the neonatal alloxan diabetes model may be useful for studying chronic diabetic complications.

Stroke topography in diabetic and nondiabetic patients by magnetic resonance imaging

Stroke is more frequent in diabetic than in nondiabetic patients. We studied lesion topography in 61 stroke patients with diabetes mellitus and 77 without diabetes using magnetic resonance imaging. Location, number and size of infarcts were investigated. Prevalence of hypertension did not differ between diabetic and nondiabetic patients. In elderly subgroups (> or = 65 years), infratentorial infarcts > or = 5 mm in diameter were more frequent in diabetic than in nondiabetic patients (32 vs. 12%, P < 0.05), while supratentorial infarcts were not different (56 vs. 68%, ns). Asymptomatic hemorrhage combined with silent infarction was significantly less frequent in diabetic than in nondiabetic patients (5 vs. 22%, P < 0.05). In nondiabetic patients with significant stenosis in the carotid system, supratentorial infarction measuring > or = 15 mm was more frequent than in those without stenotic lesions, but the number of infarcts per patient irrespective of size was smaller when stenosis was present. On the other hand, the number of infarcts in diabetic patients was similar between those with and without carotid system stenosis. The present study demonstrated that infratentorial infarcts were more common in elderly diabetic patients, suggesting the vulnerability of vertebrobasilar circulation in diabetes.

Renal complications in patients with diabetes mellitus associated with an A to G mutation of mitochondrial DNA at the 3243 position of leucine tRNA

The substitution of guanine for adenine at position 3243 of the leucine tRNA gene of mitochondrial DNA was originally described in association with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). Diabetes mellitus associated with the mutation (mitochondrial diabetes) is a different phenotype from MELAS. We identified 11 patients with the mutation among 385 Japanese diabetic patients: two had MELAS and nine had mitochondrial diabetes. We present data on a male patient with mitochondrial diabetes who developed the nephrotic syndrome at the age of 23. Light microscopy revealed mesangial expansion, PAS-positive deposits and segmental sclerosis in the glomeruli. Scattered mesangial electron-dense deposits and thickening of the basement membrane were found on electron microscopy, suggesting that diabetic glomerulosclerosis accompanied by focal glomerulosclerosis (FGS). Mitochondrial diabetes may pre-dispose patients to renal complications, including forms of glomerulonephritis, such as FGS.

Effect of glycated collagen on proliferation of human smooth muscle cells in vitro

SummaryWhile non-enzymatic glycation of long-lived tissue proteins such as collagen has been implicated in chronic complications of diabetes mellitus, its role in the aetiology of diabetic macroangiopathy has not been elucidated. To test the hypothesis that glycation of collagen abolishes the inhibitory effect of native collagen on the proliferation of human smooth muscle cells, we obtained smooth muscle cells from human gastric arteries and cultured them on dishes coated with glycated or non-glycated collagen. The proliferation of human smooth muscle cells in the presence of 10 % fetal calf serum or platelet derived growth factor-BB (10 ng/ml) was inhibited by type 1 collagen coated on the dishes. Glycation of collagen with glucose 6-phosphate for 7 days abolished the growth-inhibitory effect of native collagen. Succinylation of collagen, which like glycation blocked the lysyl residues in collagen, also abolished the growth-inhibitory effect. Adhesion of human smooth muscle cells to collagen-coated dishes was not affected by glycation of collagen. Addition of glycated albumin to the medium did not affect the growth of human smooth muscle cells on plastic dishes. The inhibition of human smooth muscle cell proliferation by collagen was not reversed by the glycation of collagen in the presence of aminoguanidine. Results suggest that early glycation abolishes the inhibitory effect of collagen on human smooth muscle cell proliferation and may thus participate in the progression of macro-angiopathy in diabetes.

Dilazep and fenofibric acid inhibit MCP-1 mRNA expression in glycoxidized LDL-stimulated human endothelial cells

We previously reported that glycoxidized low-density lipoprotein (glycoxidized LDL) enhanced monocyte chemoattractant protein-1 (MCP-1) mRNA expression through activation of nuclear factor-kappaB (NF-kappaB). Here we investigated the effects of dilazep, an anti-platelet agent, and fenofibric acid, an active metabolite of fenofibrate, on glycoxidized low-density lipoprotein-(LDL)-enhanced MCP-1 mRNA expression. Both 10 microg/ml dilazep and 100 microM fenofibric acid abrogated MCP-1 mRNA expression. ZM241385, an A2a adenosine receptor antagonist, partially inhibited the suppressive effect of dilazep. NF-kappaB activity was also suppressed by 1 microg/ml dilazep and 10 microM fenofibric acid. The antioxidative activity of these drugs on glycation to native LDL or oxidation to glycated LDL was measured using lipid peroxidation and lyso-phosphatidylcholine contents in LDL. Dilazep but not fenofibric acid exhibited antioxidative activity. Although the mechanisms of anti-atherogenic effects of the two drugs on glycoxidized LDL are different, both dilazep and fenofibric acid could potentially prevent atherosclerosis in diabetes mellitus.