Association of polymorphism in the NeuroD/BETA2 gene with type 1 diabetes in the Japanese.

NeuroD/BETA2, a transcription factor of the insulin gene, also plays an important role in the development of pancreatic beta-cells. Recently, the NeuroD/BETA2 gene has been mapped to the long arm of human chromosome 2 (2q32) where the IDDM7 gene has previously been mapped, implying its involvement in diabetes. To identify mutations in the NeuroD/BETA2 gene that may predispose patients to develop diabetes, we studied the gene in 50 Japanese subjects with diabetes (4 with type 1 and 46 with type 2) by the polymerase chain reaction (PCR) followed by single-strand conformation polymorphism and sequencing analyses. Further analysis was performed in 392 Japanese subjects (60 with type 1 and 158 with type 2 diabetes and 174 healthy control subjects) by mismatch PCR restriction fragment length polymorphism. We found a DNA polymorphism of the NeuroD/BETA2 gene. A nucleotide G-to-A transition results in the substitution of alanine to threonine at codon 45 (Ala45Thr). The frequencies of heterozygotes for the Ala45Thr variant were 9.8% in the control subjects, 9.5% in the patients with type 2 diabetes, and 25.0% in the patients with type 1 diabetes, a significant difference (P = 0.006). Because the variant of the NeuroD/BETA2 gene (Ala45Thr) is associated with type 1 but not type 2 diabetes, it may be implicated in the loss of pancreatic beta-cells in type 1 diabetes.

Effect of essential hypertension on cardiac autonomic function in type 2 diabetic patients.

OBJECTIVES: The aim of this study was to examine the effects of essential hypertension on cardiac autonomic function in type 2 diabetic patients. BACKGROUND: Hypertension is common in type 2 diabetic patients and is associated with a high mortality. However, the combined effects of type 2 diabetes and essential hypertension on cardiac autonomic function have not been fully elucidated. METHODS: Thirty-three patients with type 2 diabetes were assigned to a hypertensive diabetic group (n = 15; age: 56 +/- 8 years, mean +/- SD) or an age-matched normotensive diabetic group (n = 18, 56 +/- 6 years). Cardiac autonomic function was assessed by baroreflex sensitivity (BRS), heart rate variability (HRV), plasma norepinephrine concentration and cardiac 123I-metaiodobenzylguanidine (MIBG) scintigraphic findings. RESULTS: Baroreflex sensitivity was lower in the hypertensive diabetic group than it was in the normotensive diabetic group (p < 0.05). The early and delayed myocardial uptake of 123I-MIBG was lower (p < 0.01 and p < 0.05, respectively), and the percent washout rate of 123I-MIBG was higher (p < 0.05) in the hypertensive diabetic group. However, the high frequency (HF) power and the ratio of low frequency (LF) power to HF power (LF/HF) of HRV and plasma norepinephrine concentration were not significantly different. The homeostasis model assessment index was higher in the hypertensive diabetic group than it was in the normotensive diabetic group (p < 0.01). CONCLUSIONS: Our results indicate that essential hypertension acts synergistically with type 2 diabetes to depress cardiac reflex vagal and sympathetic function, and the results also suggest that insulin resistance may play a pathogenic role in these processes.

High incidence of rat mammary carcinoma by oral administration of ethyl methanesulphonate.

Ethyl methanesulphonate (EMS), one of the alkylating agents, is known to be a potent mutagen. We tested EMS for its carcinogenicity in female rats by oral administration. EMS was dissolved in drinking tap water at a concentration of 10(-3) M and was given for the first 12 weeks. The subcutaneous tumors were noticed as early as 16 weeks after initiating the experiment. At the 32nd week, all the surviving rats produced tumors; the majority were multiple tumors in the neck, axillar and inguinal areas corresponding to bilateral mammary glands. Histologically, the prevailing feature of the tumors was infiltrating medullary adenocarcinoma consistent with carcinoma of mammary duct origin. Neither regional lymph node involvement nor distant metastases were shown, but intraductal spread of carcinoma was a marked finding during the 32-week period.

Mizoroki-Heck type reaction of organoboron reagents with alkenes and alkynes. A Pd(II)-catalyzed pathway with Cu(OAc)2 as an oxidant.

[reaction: see text]. In contrast to the Pd(0)-catalyzed mechanism by Uemura, Mizoroki-Heck type reaction of boronic acids is found to proceed under a Pd(II)-mediated pathway using a catalytic amount of Pd(OAc)2 in the presence of Cu(OAc)2 as an oxidant. Treatment of a variety of alkenes with boronic acids, boronates, and sodium tetraphenylborate furnishes beta-arylated and alkenylated products in good to excellent yields. The reactions with norbornene, norbornadiene, and diphenylacetylene are also performed to give 1:2 or 2:1 coupling products.

Evidence for secretion of 7B2 by A- and B-cells of hamster pancreatic islets.

7B2 is a neuroendocrine protein, and in the pancreatic islets the presence of 7B2 in A- and B-cells was immunohistochemically demonstrated. In order to examine 7B2 secretion by A- and B-cells of pancreatic islets, we prepared isolated hamster pancreatic islet cells as well as an A-cell-rich culture, and studied 7B2 secretion under certain stimulations. 7B2 was secreted by isolated hamster pancreatic islet cells. This secretion was stimulated by theophylline and arginine, but glucose had a weak effect on the 7B2 secretion. Such a response of 7B2 to the stimulations was different from that of insulin or glucagon. 7B2 secretion was also noted in the A-cell-rich culture. These results suggest that 7B2 is secreted by both A- and B-cells of the hamster pancreatic islets and its secretion is regulated under certain conditions.

Transplantation of normal islets into the portal vein of Otsuka Long Evans Tokushima Fatty rats prevents diabetic progression.

To investigate the long-term effects of normal pancreatic islet transplantation on progression of obese type 2 diabetes mellitus (DM), 1500 normal islets (per rat) from Wistar King A rats at 8 weeks of age were transplanted into the liver through the portal vein of Otsuka Long Evans Tokushima Fatty (OLETF) rats, an animal model of obese type 2 DM, at 12 weeks of age. Body weight in the transplanted OLETF (IT) rats 8 and 28 weeks after islet transplantation did not differ from that in the corresponding sham-operated (SO) rats, but was greater than that in lean littermates (LETO rats; P < 0.05 for each group). In the early phase, 8 weeks after transplantation, rats in both IT and SO groups were normoglycemic, but hyperinsulinemic (P < 0.05 for each compared with LETO rats), probably resulting from increased body weight. In the late phase, 28 weeks after transplantation, hyperglycemia in the IT group was greatly attenuated compared with the SO group (P < 0.05), but hyperinsulinemia remained in both the IT and the SO groups compared with that in the LETO group (P < 0.05 for each). Immunohistochemical studies demonstrated that hypertrophic and fibrotic changes in pancreatic islets, together with mesangial proliferation of the glomerular matrix, an indicator for diabetic nephropathy, were attenuated predominantly in the IT group at the late phase after transplantation compared with those in the corresponding phase of the SO group. Islet transplantation into the liver of OLETF rats thus prevented further progression of obese type 2 DM. A possible mechanism is that islet transplantation may prevent development of hyperglycemia by improving abnormal hepatic glucose metabolism and consequently insulin resistance, which may lead to blockade of a vicious cycle between advancing damage to pancreatic islet cells and increased demand for insulin secretion, thus sparing original pancreatic cells from exhaustion induced by increased demand for insulin secretion.

Effects of glucose and insulin on cultured human microvascular endothelial cells.

The prolonged effects of glucose and insulin on cultured human microvascular endothelial cells from omental tissue (HOMEC) were observed to identify the contribution of sustained hyperglycemia and/or hyperinsulinemia to the pathogenesis of diabetic microangiopathy. When the cells were cultured for 10 days in Medium 199 with 100 or 500 mg/dl glucose, the number of cells was reduced to 78% in the culture of 500 mg/dl glucose as opposed to that of 100 mg/dl glucose. The difference in the number of cells between these two groups became obvious between the 5th and the 7th culture days. The replacement of D-glucose with L-glucose did not show any reduction in the number of cells, indicating the impertinence of high osmolarity, induced by high glucose (305 mOsm/kg) to the number of cells. This reduction resulted from the cellular damage during the culture period rather than the retardation of growth, according to the experiments of [3H]thymidine uptake and the 51Cr release assay. Since the uptake of glucose, measured as the uptake of 3-O-methyl-alpha-D-glucose, was higher and the Na+/K+ pump activity decreased in high glucose condition, it is suggested that the excessive intracellular accumulation of glucose caused the damage of cells through the disturbance of ion exchange. Insulin augmented the reduction in the number of cells induced by high glucose when supplemented together for 10 days at concentrations of 10(-6)-10(-12)M. The uptake of glucose increased further to 154% by the addition of insulin to high glucose as compared to that of high glucose alone, however, the decreased Na+/K+ pump activity by high glucose was restored to the control level by insulin. The aggravating effect of insulin to the cellular damage induced by high glucose seems to be mediation via the mechanism other than the decreased Na+/K+ pump activity. In conclusion, HOMEC were gradually damaged by high glucose and by insulin, and hyperglycemia and hyperinsulinemia would be of pathogenetic importance for diabetic microangiopathy.

Characterization of secretory responses of a glucagon-producing In-R1-G9 cell line.

The In-R1-G9 cell line is one of the clones derived from the In-111-R1 hamster insulinoma cell line and produces glucagon. The secretory responses of In-R1-G9 cells were further examined to characterize the nature of the cells. Vincristine had no effect on glucagon secretion and colchicine enhanced glucagon secretion slightly after a short incubation. Two calmodulin inhibitors, trifluoperazine and chlorpromazine, did not affect glucagon secretion. Monensin at 10(-8) M suppressed glucagon secretion by 50%. Secretion of glucagon was calcium-dependent. The addition of A23187 to the incubation medium resulted in a 180% increase over control for 1 h and calcium deprivation from the medium suppressed glucagon secretion markedly. Theophylline, a phosphodiesterase inhibitor, caused a 230% increase in glucagon secretion. An experiment using cycloheximide suggested that newly synthesized glucagon appears in the medium at 30 min. This cell line should be useful for various experiments in many fields of research.

Microencapsulation of pancreatic islets. A technique and its application to culture and transplantation.

Hamster pancreatic islets were encapsulated by a biocompatible membrane composed of the molecular sequence of alginate-polylysine-alginate. The encapsulated islets released insulin into the culture medium in response to secretagogues in short-term incubation. In long-term culture, the encapsulated islets maintained their insulin-releasing capacity for 28 days at a level similar to that of the unencapsulated islets. No overgrowth of fibroblastic cells was observed inside the capsule even after 70 days of culture. Further, the encapsulated hamster islets were xenotransplanted to streptozotocin-induced diabetic rats intraperitoneally. Some of the encapsulated islets, which were recovered from a recipient 27 days after transplantation, were found to be viable, although prolonged normalization of fasting plasma glucose levels of the recipients could not been achieved. On the contrary, the unencapsulated islets were replaced by massive connective tissue elements and insulin-positive B cells were hardly detected within the grafts 22 days after transplantation. The results of this study seem to confirm the potential of the application of the encapsulating technique to primary culture of parenchymal cells and to transplantation of pancreatic islets.

Vascular cell adhesion molecule-1 expression in the renal interstitium of diabetic KKAy mice.

To investigate the mechanism of interstitial inflammation in diabetic nephropathy, we used spontaneously diabetic KKAy mice. Twelve KKAy mice were divided into two groups; six mice were fed standard mouse chow ad libitum and six mice were placed on a diet (i.e. they received the same amount of chow as six control C57BL mice). Diabetic KKAy mice developed hypercholesterolemia and albuminuria. Animals were killed at 16 weeks of age and renal tissues were immunostained for vascular cell adhesion molecule-1 (VCAM-1). In diabetic KKAy mice, the renal interstitium was infiltrated by monocytes, lymphocytes, plasma cells, and other cells. The walls of venules near the infiltrating cells were more intensely stained for VCAM-1 when compared with other sites. In contrast, the VCAM-1 staining of arterioles and peritubular capillaries was not significantly increased. There was weak VCAM-1 staining of the infiltrating cells, including lymphocytes, monocytes, and other cells. Electron microscopy demonstrated immunolabeling for VCAM-1 on the cell surface and in the cytoplasm of both infiltrating cells and vascular endothelial cells. In KKAy mice placed on a diet, there was less staining for VCAM-1 and cellular infiltration was also decreased. Thus, increased expression of VCAM-1 by the endothelial cells of venules and VCAM-1 expression by infiltrating cells were demonstrated in the interstitium of kidneys from diabetic mice. These results suggest that increased expression of VCAM-1 by endothelial cells and infiltrating cells contributes to interstitial inflammation in diabetic nephropathy.

[Diabetes and atherosclerosis].

Recently, the number of diabetic patients in Japan has increased and reached 6 millions, and it was estimated that 1.5 million diabetic patients were suffering from diabetic complications of microangiopathy (neuropathy, retinopathy and nephropathy) and macroangiopathy. According to the study for the causes of death among Japanese diabetic patients during 10 years from 1981 to 1990, mean longevity of diabetic patients was shorter of 9.4 years in men and 13.5 years in women than those of non-diabetics. Forty percent of diabetic patients died from the vascular diseases (ischemic heart disease 14.6%, cerebrovascular disease 13.5% and renal disease 11.2%). The frequency of death due to ischemic heart disease was almost double in diabetic patients in comparison to non-diabetics in Japan. From the data obtained from the study of Japanese-American, more than 50% of them showed abnormal glucose tolerance and the frequency of ischemic heart disease was higher twice than that of Japanese. Diabetes has been recognized as one of the important risk factors for atherosclerosis, and so many factors, such as hyperglycemia, glycation, dyslipidemia, hyperinsulinemia, insulin resistance, hypertension and obesity in diabetes, are related to atherosclerosis. The relation of these factors will be introduced. Clinically, it is very important to make a check list of these factors and make an effort to diminish them for prevention of atherosclerosis of diabetic patients.

Simple method for the collection of pancreatic islets by the use of Ficoll-Conray gradient.

Simple method for the collection of large numbers of viable pancreatic islets from normal rats is described. This method involves collagenase digestion of the pancreas, followed by the use of Ficoll-Conray discontinuous gradient for the separation of isolated islets from unwanted acinal debris. Ficoll-Conray A solution was prepared by mixing undialyzed 12.5% Ficoll and 33.4% Conray in the ratio 2 : 1, to make a specific gravity of 1.095 and osmolarity of 396 mOsm/l. The solution B, C, and D, with specific gravities 1.084, 1.072, and 1.048, respectively were obtained by diluting A solution with distilled water. Using Ficoll-Conray gradient, about 200 viable islets which maintained excellently their morphology and function, were collected consistently from a young adult rat pancreas.

Electrocardiographic changes related to hypersecretion of catecholamine in a patient with fulminant hepatitis.

A 48 year-old-man, with fulminant hepatitis complicated with myocarditis was treated. Despite intensive care, he died of fulminant hepatitis associated with hepatitis B virus infection. Electrocardiography (ECG) showed myocardial infarction-like changes when he went into a deep coma. Microscopically, scattered foci of myocardial cell damage and cell death associated with clusters of inflammatory cells were present in the heart at autopsy. However, there were no findings related to myocardial infarction and staining for hepatitis B surface antigen and core antigen were nil. The concentration of plasma catecholamine was elevated concomitantly with high level of ECG changes. We consider that abnormal ECGs may reflect a hypersecretion of catecholamine and suggest that our patient had a catecholamine cardiopathy.

Cryopreservation of pancreatic islet cells.

Our study describes a useful procedure for cryopreservation of pancreatic islet cells. The pancreatic islets of adult hamsters were collected by collagenase digestion succeeded by gradient centrifugation and were dispersed by EDTA-Dispase treatment. The dispersed cells were suspended in medium consisting of 90% Dulbecco's modified Eagle's medium and 10% fetal bovine serum, supplemented with 10% dimethyl sulfoxide or glycerol. One milliliter each of the cell suspensions containing 10(6) cells was distributed into 2 ml polypropylene tubes, processed for freezing under six cooling conditions, and stored in liquid nitrogen (-196 degrees C). Of the various conditions tested, the cells suspended in dimethyl sulfoxide-containing medium and cooled in a program freezer at a rate of 0.5 degrees C/min down to -40 degrees C, succeeded by a rate of 5 degrees C/min down to -70 degrees C, resulted in the highest recovery rate of cells, 61.2% +/- 3.1%. This rate was comparable to those of several tissue culture cell lines frozen under similar conditions. Recovered cells preserved their morphologic characteristics under light and phase-contrast microscopy and formed cell sheets in culture. Response of insulin secretion to 3 mg/ml glucose appeared 6 hours after thawing, and the response to both 3 mg/ml glucose and 10 mmol/L theophylline was recovery to the same level as nonfrozen islet cells after 12 hours. The applicability of cryopreserved cells for the detection of islet cell surface antibody was demonstrated by the indirect method of immunofluorescence using islet cell surface antibody-positive human sera.

Adult human pancreatic endocrine cells in culture. Insulin and glucagon release and influence of cold storage.

Adult human pancreatic tissue was minced and digested with collagenase. Resulting cell clusters, including both exocrine and endocrine components, were suspended in the medium and inoculated into 35 mm Petri dishes. Viable cell clusters became spherical 4 or 5 hr after inoculation and attached to the bottom of the dish within 24 hr. They spread out to form a pavementlike epithelial cell sheet by the seventh day of culture. Insulin and glucagon release were determined on the culture day 3. Insulin release stimulated by 500 mg/dl glucose, 10 mM theophylline, and 200 micrograms/ml tolbutamide was increased 1.5- to 3.7-fold in comparison with that stimulated by 100 mg/dl glucose. A 20 mM arginine concentration did not affect insulin release. Glucagon release, stimulated by 10 mM theophylline and 20 mM arginine, showed 1.7- to 2.7-fold increase and was suppressed significantly by 500 mg/dl glucose. Tolbutamide did not affect glucagon release significantly. Ultrastructural studies demonstrated that islet cells were preserved excellently. Chopped pancreatic tissue could be stored at 4 degrees C for at least 12 hr, with retention of normal following subsequent in vitro culture of islet cells.