Yasunori Iwashima

Increased frequency of apolipoprotein ε2 allele in non‐insulin dependent diabetic (NIDDM) patients with nephropathy

The genetic polymorphism of apolipoprotein E (ε2, ε3 and ε4) is associated with lipid abnormalities. It has been suggested that lipid abnormalities may contribute to the development and progression of kidney diseases, including diabetic nephropathy. Thus, in this study we compared the apo E allele frequencies among 146 non‐insulin‐dependent diabetic (NIDDM) patients with nephropathy, 135 NIDDM patients without nephropathy and 576 of the general Japanese population. The ε2 allele frequency was significantly higher in diabetic patients with nephropathy (7.2%) and with renal failure (9.7%) than in diabetic patients without nephropathy (2.6%) and in the general Japanese population (3.7%). It is concluded that there is a possibility that the ε2 allele is associated with nephropathy in NIDDM.

Detection of the association between a deletion polymorphism in the gene encoding angiotensin I-converting enzyme and advanced diabetic retinopathy

We investigated the relationship between advanced diabetic retinopathy (ADR) and an angiotensin-converting enzyme (ACE) gene polymorphism in subjects with type 2 diabetes and ADR, pre-proliferative (PrePDR) or proliferative diabetic retinopathy (PDR) without overt nephropathy. Polymerase chain reactions were used to detect insertion/deletion (I/D) polymorphisms of the ACE gene. There was no difference in the frequency of II, ID, or DD genotypes, or of I and D alleles among subjects with type 2 diabetes without diabetic retinopathy (NDR) or with simple diabetic retinopathy (SDR) and non-diabetic controls. There was also no difference in the frequency of ACE genotypes among subjects with type 2 diabetes with NDR, or SDR and ADR. However, the frequency of the ACE DD genotype in ADR was significantly higher than that in controls (chi(2)=6.64, P=0.036). On the other hand, the frequency of the D allele in ADR was significantly higher than that in controls (chi(2)=6.33, P=0.012), NDR (chi(2)=4.18, P=0.041) and SDR (chi(2)=4. 89, P=0.027), respectively. These results indicate a significant relationship between the presence of the D allele polymorphism in the ACE gene and ADR in Japanese subjects with type 2 diabetes and no overt nephropathy.

Effects of probucol on plasma lipids and lipoproteins in familial hypercholesterolemic patients with and without apolipoprotein E4

It has been reported that total cholesterol (Chol) response to probucol is greater in familial hypercholesterolemic (FH) patients with apo E4 than in those without apo E4. We further examined the effect of probucol on plasma triglyceride (TG) and lipoprotein-Chol levels as well as total Chol levels in heterozygous FH patients with apo E4 (n = 14 for apo E4/3, n = 1 for apo E4/4) and without apo E4 (n = 31 for apo E3/3). Probucol was administered in a dosage of 500 mg twice daily for 3 months. The reduction in total Chol levels was significantly greater in FH patients with apo E4 (-90 mg/dl, -27.5%) than in those without apo E4 (-41 mg/dl, -13.7%). The reduction in low density lipoprotein (LDL)-Chol levels was also significantly greater in FH patients with apo E4 (-73 mg/dl vs. -34 mg/dl). There was a significant difference in the change in TG and very low density lipoprotein (VLDL)-Chol levels with treatment between the FH patients with apo E4 (-37 and -8 mg/dl, respectively) and without apo E4 (+8 and +2 mg/dl, respectively). However, there was no significant difference in the reduction in HDL-Chol levels between the 2 groups (-9 mg/dl vs. -9 mg/dl). It is concluded that FH patients with apo E4 showed the greater reduction in plasma TG levels as well as total Chol levels with probucol treatment than those without apo E4, and that the greater reduction in total Chol levels in them, as reported previously, was mainly due to the greater reduction in LDL-Chol levels and slightly due to that in VLDL-Chol levels.

Effects of the prostanoids on the proliferation or hypertrophy of cultured murine aortic smooth muscle cells

Effects of the prostanoids on the growth of cultured aortic vascular smooth muscle cells (VSMCs) were examined using mice lacking prostanoid receptors. Proliferation of VSMCs was assessed by measuring [3H]‐thymidine incorporation and the cell number, and their hypertrophy by [14C]‐leucine incorporation and protein content. In VSMCs from wild‐type mice, expressions of mRNAs for the EP4 and TP were most abundant, followed by those for the IP, EP3 and FP, when examined by competitive reverse transcriptase‐PCR. Those for the EP1, EP2 and DP, however, could not be detected. AE1‐329, an EP4 agonist, and cicaprost, an IP agonist, inhibited platelet derived growth factor (PDGF)‐induced proliferation of VSMCs from wild‐type mice; these inhibitory effects disappeared completely in VSMCs from EP4−/− and IP−/− mice, respectively. In accordance with these effects, AE1‐329 and cicaprost stimulated cAMP production in VSMCs from wild‐type mice, which were absent in VSMCs from EP4−/− and IP−/− mice, respectively. Effects of PGE2 on cell proliferation and adenylate cyclase were almost similar with those of AE1‐329 in VSMCs from wild‐type mice, which disappeared in VSMCs from EP4−/− mice. PGD2 inhibited PDGF‐induced proliferation of VSMCs from both wild‐type and DP−/− mice to a similar extent. This action of PGD2 was also observed in VSMCs from EP4−/− and IP−/− mice. In VSMCs from wild‐type mice, I‐BOP, a TP agonist, showed potentiation of PDGF‐induced hypertrophy. I‐BOP failed to show this action in VSMCs from TP−/− mice. The specific agonists for the EP1, EP2 or EP3, and PGF2α showed little effect on the growth of VSMCs. These results show that PGE2, PGI2 and TXA2 modulate PDGF‐induced proliferation or hypertrophy of VSMCs via the EP4, IP and TP, respectively, and that the inhibitory effect of PGD2 on PDGF‐induced proliferation is not mediated by the DP, EP4 or IP.

Metabolic and morphological changes of the heart in Chinese hamsters (CHAD strain) with spontaneous long-term diabetes

We studied the effect of spontaneous long-term (9-10 months) diabetes on the heart of Chinese hamsters (CHAD strain) to elucidate the relationship between diabetes mellitus and cardiomyopathy. The diabetic hamsters, aged approximately 11 months, showed body weight loss, hyperglycemia (mean fasting plasma glucose 402 mg/dl), hypoinsulinemia, hyperlipidemia and ketonemia. The diabetic hamsters showed reduced activities of cytoplasmic glycolytic key enzymes; hexokinase, pyruvate kinase and phosphofructokinase, increases in cardiac glycogen and glucose-6-phosphate contents and a 40% decrease in cardiac ATP content, indicating decreased energy production. An accumulation of myocardial triglyceride and cholesterol was found in the diabetic hamsters. In addition, the cardiac norepinephrine content was increased in the diabetic hamsters, suggesting the presence of autonomic nervous disorder. Increased heart weight and thickening of the septum and both ventricular walls were found in the diabetic hamsters. Light-microscopic analysis revealed that 42.9% of the diabetic hamsters had myocardial degeneration without any vascular lesion of extramural large and intramural small vessels, whereas the non-diabetic controls had no myocardial or vascular lesions. These data suggest that the diabetic Chinese hamsters had cardiomyopathy, which is possibly caused by extravascular factors such as metabolic or autonomic nervous disorder although conclusive evidence is lacking.

Type III hyperlipoproteinema with apolipoprotein E2/2 genotype in Japan.

Limited information is available concerning type III hyperlipoproteinemia (HLP) in the Asian population. Therefore, clinical and biochemical characteristics of type III HLP were examined in 16 Japanese patients. Mean plasma triglyceride (TG) and total cholesterol (chol) levels were 381 mg/dl and 253 mg/dl, respectively, and the mean very low density lipoprotein (VLDL)‐chol/plasma TG ratio was 0.27, which were lower than those reported in Western countries. Eighty percent of the patients had high plasma remnant‐like particles (RLP)‐chol levels above 50 mg/dl and a high RLP‐chol/plasma TG ratio above 0.1. Twelve patients (75.0%) were obese. Seven patients (43.8%) had type 2 diabetes mellitus and four patients (25.0%) had impaired glucose tolerance. Six patients (37.5%) had coronary heart disease (CHD), but none had peripheral vascular disease or xanthomas. TG‐rich lipoproteins from type III HLP patients with diabetes mellitus stimulated cholesteryl ester synthesis by human macrophages significantly (p < 0.001) more than those from type III HLP patients without diabetes mellitus. In conclusion, the Japanese type III HLP patients had lower plasma TG and total chol levels and a lower VLDL‐chol/plasma TG ratio, but CHD was more common. The patients were characterized by a high frequency of obesity and/or glucose intolerance. The TG‐rich lipoproteins from type III HLP patients with diabetes mellitus were more atherogenic.

Insulin and glucagon response of the diabetic Chinese hamster in the Asahikawa colony

The relationship between pancreatic hormone content and pattern of hormone release has not been completely elucidated because of heterogeneity in diabetes. Accordingly, this study was performed to establish the relationship, using spontaneously diabetic Chinese hamsters in the Asahikawa colony, a newly discovered experimental model resembling insulin-deficient diabetes in humans. As a result of investigations of insulin and glucagon responses to glucose or arginine in vivo and in vitro using isolated islets obtained by the collagenase procedure, a decreased insulin response and paradoxical glucagon response to glucose, and an excessive glucagon response to arginine were found in the diabetic animals. While the yield of isolated islets tended to decrease, a decreased pancreatic insulin content and increased pancreatic glucagon content were found as the diabetic state advanced. It may be suggested, therefore, that the relationship between pancreatic hormone content and pattern of hormone release in diabetic animals in the Asahikawa colony is based on the disruption of islets, disruption or dysfunction of B-cells and hyperplasia or hypertrophy of A-cells by some cause genetically determined.

Changes in the pancreatic A-, B- and D-cell populations during development of diabetes in spontaneously diabetic Chinese hamsters of the Asahikawa colony (CHAD).

We investigated the pathological changes in pancreatic islets during the development of diabetes in spontaneously diabetic Chinese hamsters of the Asahikawa colony (CHAD), using morphometric analysis and specific immunocytochemical methods. We also investigated the relationships between changes in islet cell composition and the hormonal changes in the plasma and pancreas. Plasma and pancreatic insulin levels were significantly lower in diabetic hamsters than in pre-diabetic hamsters. However, plasma insulin levels in the pre-diabetic hamsters were significantly higher than those in the hamsters from the non-diabetic control strain, although the pancreatic insulin content in the pre-diabetics was significantly lower than that in the non-diabetics. Since even a severely diabetic CHAD is alive for many months after the onset of the disease without injections of insulin, its clinical course seems to be close to that of type 2 human diabetes. In contrast, plasma and pancreatic glucagon levels were significantly higher in diabetic hamsters than in non-diabetics and pre-diabetics. There were significantly positive correlations between plasma and pancreatic insulin, and plasma and pancreatic glucagon levels in CHAD (P less than 0.01). On the other hand, no significant differences in the pancreatic somatostatin content were found among the non-diabetics, pre-diabetics, and severe diabetics. Significant correlations were found between plasma and pancreatic hormone levels (except for somatostatin) and the advance of diabetes in CHAD (P less than 0.01). Morphometric analysis by planimeter revealed that islets in the severe diabetics were 25% smaller than in the pre-diabetics. Significantly less B-cell area within the diabetic islets was found when compared with the non-diabetic and pre-diabetic islets. Significantly larger A- and D-cell areas within the diabetic islets were found compared with the non-diabetic and pre-diabetic islets. There was a significant correlation between the areas of the three types of cell within the islets and the severity of diabetes (P less than 0.01). It is suggested, therefore, that the pancreatic islet function in CHAD is closely associated with the morphologic changes in islet endocrine cells. The elevation of plasma and pancreatic glucagon levels and the marked increase of the A-cell area within the islets from severely diabetic CHAD may reveal an absolute increase of A-cell numbers.

Polyol pathway in tissues of spontaneously diabetic Chinese hamsters (Cricetulus griseus) and the effect of an aldose reductase inhibitor, ONO-2235

1. Sorbitol and fructose levels were significantly elevated in the lens, the sciatic nerve, the retina and the kidney of diabetic Chinese hamsters and inositol level was significantly decreased in the lens and sciatic nerve of diabetics. 2. The activity of an aldose reductase in the kidney was not different between normal and diabetic Chinese hamsters. 3. An aldose reductase inhibitor (ONO-2235) had no effect in sorbitol, fructose and inositol contents of all these tissues from diabetic Chinese hamsters. 4. These results suggest that diabetic Chinese hamsters produce polyol accumulation in tissues but that there is a clear species-specific difference to inhibition of aldose reductase.

Paradoxical glucagon response in perifused islets of the diabetic Chinese hamster

Dynamic insulin and glucagon response to glucose was examined in the perifusion system to investigate the relationship between pancreatic hormone content and the pattern of hormone secretion in diabetic Chinese hamsters of the Asahikawa colony (CHA). Isolated islets of normals and diabetics from the CHA were perifused. When the medium was changed to high glucose (500 mg/dl), a low insulin response and paradoxical glucagon response were seen in diabetics compared with normals. Positive correlations were found between pancreatic insulin and the amount of perifusate insulin, and glucagon content and glucagon release, respectively. It is suggested, accordingly, that pancreatic hormone content is related to the amount of hormone release in CHA. A negative correlation between the amount of perifusate insulin and glucagon release was found. It is suggested, therefore, that an impaired suppression of glucagon release in the diabetic CHA animals could be attributed at least to insulin deficiency. These findings agree with the histological discovery of decreased B-cells and increased A-cells in the diabetic islets. Both decreased B-cells and islet numbers could be the cause of the low insulin response to glucose. Increased numbers of A-cells with hyperfunction resulting from local insulin deficiency could be the cause of the paradoxical glucagon response.