Yoshitaka Nagasawa

Streptozocin- and Alloxan-Induced H2O2 Generation and DNA Fragmentation in Pancreatic Islets: H2O2 as Mediator for DNA Fragmentation

Streptozocin (STZ) and alloxan (ALX) exhibit the most potent diabetogenicity and are used for induction of experimental diabetes mellitus. An understanding of the mechanisms of action of the typical diabetogenic agents is important for elucidating the causes of diabetes. Okamoto proposed a model in which DNA fragmentation plays an important role in the development of diabetes. DNA fragmentation supposedly results from the accumulation of superoxide or hydroxyl radicals. However, direct evidence for this accumulation is lacking. With isolated rat pancreatic islets in vitro, we demonstrated that STZ and ALX stimulated H2O2 generation and caused DNA fragmentation. Addition of STZ or ALX resulted in an increase in H2O2 generation. On DNA analysis, when incubated without STZ or ALX, DNA sedimented as a single peak; when incubated with STZ or ALX, DNA sedimented slower as a broad peak and was fragmented. Graded doses of STZ and ALX stimulated H2O2 generation and induced DNA fragmentation; their effects on H2O2 generation and DNA fragmentation were evident at a concentration of 0.1 mM and were maximal at 1 mM. Administration of STX or ALX to rats in vivo stimulated H2O2 generation and caused DNA fragmentation in pancreatic islets. H2O2 itself also induced DNA fragmentation. These findings may support Okamotos proposal that STZ and ALX induce diabetes through the following biochemical events: STZ and ALX→H2O2 generation→DNA fragmentation→β-cell destruction. This study may constitute the first demonstration of STZ- and ALX-stimulated H2O2 generation, which probably acts as a mediator of STZ- and ALX-induced DNA fragmentation.

Defective Fatty Acid-mediated -Cell Compensation in Zucker Diabetic Fatty Rats PATHOGENIC IMPLICATIONS FOR OBESITY-DEPENDENT DIABETES

Although obesity is associated with insulin resistance, most obese humans and rodents remain normoglycemic because of compensatory hyperinsulinemia. This has been attributed to β-cell hyperplasia and increased low K glucose metabolism of islets. Since free fatty acids (FFA) can induce these same β-cell changes in normal islets of Wistar rats and since plasma FFA are increased in obesity, FFA could be the signal from adipocytes that elicits β-cell compensation sufficient to prevent diabetes. To determine if FFA-induced compensation is impaired in islets of rats with a diabetogenic mutation, the Zucker diabetic fatty (ZDF) rat, we cultured islets from 6-week-old obese (fa/fa) rats that had compensated for obesity and apparently normal islets from lean ZDF rats (fa/+) in 0, 1, or 2 mM FFA. Low K glucose usage rose 2.5-fold in FFA-cultured control islets from age-matched Wistar rats, but failed to rise in either the precompensated islets of ZDF rats or in islets of lean ZDF rats. Bromodeoxyuridine incorporation increased 3.2-fold in Wistar islets but not in islets from obese or lean ZDF rats. Insulin secretion doubled in normal islets cultured in 2 mM FFA (p < 0.01) but increased only slightly in islets from lean ZDF rats (not significant) and declined in islets from obese ZDF rats (p < 0.05). We conclude that, unlike the islets of age-matched Wistar rats, islets of 6-week-old heterozygous and homozygous ZDF rats lack the capacity for FFA-induced enhancement of β-cell function.

Exacerbation of Autoimmune Thyroid Dysfunction after Unilateral Adrenalectomy in Patients with Cushing's Syndrome Due to an Adrenocortical Adenoma

Little is known about the factors that cause exacerbations of autoimmune thyroid dysfunction. One possibility is an alteration in adrenocortical function, since glucocorticoids are known to alter both pituitary-thyroid and immunologic function. We encountered three patients in whom overt autoimmune thyroid disease developed after unilateral adrenalectomy for Cushings syndrome due to an adrenocortical adenoma. We compared the postoperative changes in thyroid function in these patients with those in 21 other patients with Cushings syndrome who underwent the same treatment. After unilateral adrenalectomy, one of the three patients had transient hyperthyroidism and a low thyroid uptake of 131I, indicative of silent thyroiditis. After the same surgical procedure, the second patient had hypothyroidism, where-as the third patient had transient hyperthyroidism at first, and hypothyroidism then gradually developed. All three patients had serum antithyroid antibodies, the titers of which increased after surgery. In the remaining 21 patients (only 2 of whom had antithyroid antibodies initially), the serum concentrations of thyroxine, triiodothyronine, and thyroxine-binding globulin and the secretion of thyroid-stimulating hormone increased after surgery from values that were low or near the lower limit of normal to values still well within the normal range. None of these patients had clinically evident thyroid disease or increased antithyroid-antibody titers. We conclude that reductions in the secretion of glucocorticoid may exacerbate subclinical autoimmune thyroid disease. Patients with Cushings syndrome due to adrenocortical adenoma who have thyroid antibodies should be followed closely after treatment, because thyroid dysfunction may develop.

An abnormal sodium metabolism in Japanese patients with essential hypertension, judged by serum sodium distribution, renal function and the renin-aldosterone system

Objective The role of the renin–aldosterone system and the ability of renal sodium reabsorption to facilitate pressure natriuresis were analyzed by using a sufficient number of Japanese patients with essential hypertension. Methods We studied 3222 normal Japanese subjects (610 in Kashiwa City Hospital and 2612 in Shinshu University Hospital), 741 Japanese patients with essential hypertension (256 in Kashiwa City Hospital and 485 in Shinshu University Hospital), 20 patients with aldosterone-producing adenomas and 11 patients with idiopathic hyperaldosteronism to determine the possible roles of sodium, renal function, and plasma aldosterone concentration (PAC) on blood pressure elevation. Inappropriate elevation of aldosterone levels [elevation of the aldosterone: plasma renin activity (PRA) ratio] was used to assess aldosterone action. Results The peak of the serum sodium distribution curve was approximately 2 mmol/l higher in the patients with essential hypertension than it was in controls. The prevalence of higher serum sodium concentrations (≥ 147 mmol/l) also was increased significantly hypertensive patients. Age-related deterioration of renal function did not explain the hypertension and abnormal sodium metabolism in the hypertensive patients. In stepwise regression analysis, the serum sodium concentration was related inversely to the PRA and positively to the PAC:PRA ratio. Although there was an inverse relationship between urinary sodium excretion (representing sodium intake) and the PRA, urinary sodium excretion proved not to be significant as a source of variation in the PAC or in the PAC:PRA ratio in the hypertensive patients. Although the PAC was within the normal range in patients with serum sodium concentrations of 147 mmol/l or more and an elevated PAC:PRA ratio, it was inappropriately high for the stimulus applied, as indicated by the PRA; this is similar to the situation with aldosterone-producing adenomas or idiopathic hyperaldosteronism. Conclusion Serum sodium distribution patterns differed between normal subjects and patients with essential hypertension in this Japanese population. The deterioration of renal function and increased sodium intake did not explain this abnormal sodium metabolism. A higher serum sodium concentration is related to an elevated blood pressure, and, in some patients, an inappropriate elevation of plasma aldosterone levels. Of the Japanese hypertensive patients, 10–14% exhibited serum sodium concentrations of 147 mmol/l or more and inappropriate elevations of aldosterone level (suppressed PRA and normal aldosterone level). The defect in these patients presumably lies in the inappropriately high secretion of aldosterone.

Test for recovery from hypothyroidism during thyroxine therapy in Hashimoto's thyroiditis.

Hypothyroid patients with Hashimotos thyroiditis usually receive lifelong thyroxine therapy. Some are known to recover thyroid function, but identification of these patients during continued thyroxine therapy has been impossible. 92 patients with hypothyroidism after Hashimotos thyroiditis and 70 normal controls were studied. All controls but not patient before thyroxine was started had a normal thyroid response to thyroid stimulating hormone (TSH), circulating concentrations of which were increased by administration of 500 micrograms thyrotropin releasing hormone (TRH). During treatment with thyroxine, 22 patients recovered thyroid responsiveness to TSH, and when treatment was stopped these patients have remained euthyroid for 1-8 years, whereas all 70 who did not recover thyroid TSH responsiveness became hypothyroid within 3 months. Over 20% of patients with hypothyroidism after Hashimotos thyroiditis may recover satisfactory thyroid function, and can be identified during thyroxine treatment by their thyroid response to TSH in a TRH test.

Interrelationship between insulin-like growth factor I-induced activation of the Na+H+-antiporter and intracellular Ca2+-mobilization in thyroid cells

Insulin-like growth factor I (IGF-I) increased cytoplamic pH (pHi) and cytoplasmic Ca2+ [( Ca2+]i) in cultured porcine thyroid cells. Inhibition of the Na+/H(+)-antiporter by dimethylamiloride or a reduction of external Na(+)-concentrations attenuates the increases in pHi and [Ca2+]i. The [Ca2+]i response to IGF-I is a pHi-dependent process. IGF-I activates Na+/H(+)-antiporter and alkalinizes thyroid cells. The resulting increase in pHi facilitates the [Ca2+]i response by adjusting the pHi closer to the pHi-optimum of the intracellular Ca(2+)-mobilizing system. One of the biological functions of IGF-I-induced activation of the Na+/H(+)-antiporter is to shift the pHi to an optimal value for the [Ca2+]i response.

Hypouricemia in NIDDM Patients

I n the epidemiological survey conducted by Herman et al. (1), serum uric acid levels were significantly lower in non-insulin-dependent diabetic (NIDDM) patients than in normal subjects. Gotfredsen et al. (2) also reported a decrease in serum uric acid levels in insulin-dependent diabetic patients, and increases in urinary uric acid excretion and uric acid clearance (Curate). In this study, we investigated the serum and urinary uric acid levels in NIDDM patients and discuss the mechanism of hypouricemia in diabetic patients. Two hundred and four nonnephrotic NIDDM patients (95 men, 109 women) and 44 normal controls (20 men, 24 women) were studied. All participants were admitted to the hospital between January 1984 and September 1987. Diagnosis of diabetes was based on the 1982 Japan Diabetes Society criteria (3). Patients with >25 mg/dl of proteinuria in morning urine samples, measured with a Combistix (Ames-Sankyo, Tokyo), were excluded from the study. Patients whose insulin dosage was >0.6 U/kg of body weight and who were receiving diuretics, antigout or antituberculous agents, antibiotics, aspirin, or anti-inflammatory agents were also excluded. Forty-nine of the diabetic patients were treated with insulin with the mean dosage consisting of 22.7 U/day. Forty-five patients received oral hypoglycemics, and the remainder were treated by diet therapy. Ageand body-weight-matched subjects with no major diseases were used as control subjects. Medical problems were either thyroid adenoma or simple goiter with normal thyroid function. Diabetic patients maintained their prescribed diets containing 25-30 kcal/kg of ideal body weight with 17% protein, 23% fat, and 60% carbohydrate. Control subjects maintained a normal hospital diet containing 2200 kcal/day with the same composition of protein, fat, and carbohydrate as diabetic patients. Blood and urine samples were collected at 0800 after at least 10 h of fasting. A 2-h postprandial plasma glucose level was measured at 1030. In 100 of the diabetic patients (49 men, 51 women) and all the controls, urine samples were collected for 3 days after the blood sampling day. From the 3-day measurements, the average data per 24 h were used for analysis. Serum and urinary uric acid levels were measured by a colorimetric procedure using an enzymatic (uricase) method, and plasma and urinary glucose levels were measured by the glucose oxidase method. Other serum chemicals were analyzed by an autoanalyzer (SMAC, Technicon Japan, Tokyo). Table 1 shows 13 clinical parameters in NIDDM patients and controls. Diabetic patients had a signifi-