Masaaki Osegawa

Effect of Hypophysectomy and Growth Hormone Administration on Somatostatin Content in the Rat Hypothalamus

The effect of hypophysectomy and bovine GH administration on somatostatin (SRIF) content in the rat hypothalamus was investigated. SRIF content was determined by a specific radioimmunoassay method described elsewhere. The total SRIF content of the rat hypothalamus as well as its content per milligram wet weight had decreased 4 weeks after hypophysectomy but was restored significantly in those rats which were subjected to bovine GH administration for 7 days 3 weeks after hypophysectomy. Furthermore, in nonoperated rats, increase of hypothalamic SRIF content was observed after 7 days GH administration. These results indicate that growth hormone may influence the SRIF content of hypothalamus and it seems likely that a feedback mechanism between pituitary GH and hypothalamic SRIF exists.

Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells

The effects of sulfonylureas and a biguanide on membrane-bound low Km cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 microM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 microM). Tolbutamide (0.5-10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. Km, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 microM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.

Augmented gastrin responses in diabetic patients with vagal neuropathy

SummaryWe evaluated serum gastrin responses to a test meal in normal subjects and diabetic patients with or without vagal neuropathy. Vagal neuropathy was defined as a heart rate variation during deep breathing of < 9 beats/min. Forty-three percent (54 out of 124) of the diabetic patients had abnormal heart rate variation, compared with 5% (3 out of 53) of the normal subjects. Serum gastrin responses to a test meal were examined in 17 normal subjects, 20 out of 70 diabetic patients without vagal neuropathy and 17 out of 54 diabetic patients with vagal neuropathy. Meal-stimulated gastrin levels were significantly higher in the diabetic patients with vagal neuropathy than in the normal subjects, while the findings in the diabetic patients without vagal neuropathy were similar to those in normal subjects. These data suggest that augmented gastrin responses are due to vagal denervation induced by autonomic neuropathy.

Activation of insulin-sensitive phosphodiesterase by lectins and insulin-dextran complex in rat fat cells☆

Membrane-bound low-Km cAMP phosphodiesterase was activated by concanavalin A, wheat germ agglutinin, and insulin-dextran complex under conditions of incubation with intact rat fat cells. Concanavalin A rapidly stimulated the enzyme activities and maximum was reached at 10 to 15 minutes. As little as 10 micrograms/mL concanavalin A activated the enzyme and a maximal response was obtained at 100 to 300 micrograms/mL, but concanavalin A and wheat germ agglutinin were less potent than insulin. Specific saccharide inhibitors completely abolished activation of the enzyme by lectins, but had no effect on the activation of insulin. Digestion of fat cells with 1 mg/mL trypsin for 15 minutes completely inhibited activation of the enzyme by insulin. However, concanavalin A was less sensitive to trypsinization. The insulin-dextran complex, which did not penetrate the plasma membrane, activated the enzyme and was one tenth as effective as the native insulin. These results suggest that the insulin-like actions of these lectins are provoked through coupling with the carbohydrate moiety on the cell membrane close to insulin receptors.

Effects of dithiothreitol on insulin-sensitive phosphodiesterase in rat fat cells.

Dithiothreitol activates the low-Km membrane-bound cyclic AMP phosphodiesterase when incubated with the enzyme in a cell-free system. To investigate the mechanism of its activation, we studied the effect of protease inhibitors. Isolated fat cells obtained from Sprague-Dawley rats were incubated in Krebs-Henseleit Hepes buffer, pH 7.4, at 37 degrees C with and without insulin (2 nM, 10 min). A crude microsomal fraction prepared by differential centrifugation was suspended in 0.25 M sucrose containing 10 mM Tes buffer, pH 7.5, with and without 2 mM dithiothreitol and protease inhibitors at 4 degrees C for 48 h. Dithiothreitol stimulated the phosphodiesterase, in a time-dependent manner. As little as 0.02 mM dithiothreitol activated the enzyme, and the maximally effective dose was 2-10 mM. Among the various protease inhibitors tested, antipain, leupeptin, chymostatin and E-64 were the most effective in preventing activation of the enzyme by dithiothreitol. Antipain also inhibited release of the enzyme from the bound fraction. These results suggest that activation of the low-Km phosphodiesterase by dithiothreitol may be provoked by stimulation of an endogenous thiol protease.

Effects of changes in serum insulin in response to dexamethasone and adrenalectomy on insulin-sensitive phosphodiesterase in rat fat cells

The effects of dexamethasone administration and adrenalectomy on insulin-sensitive phosphodiesterase were studied in rat fat cells. Isolated fat cells were incubated at 37 degrees C for ten minutes or without insulin. A crude microsomal fraction prepared by differential centrifugation was used for the determination of phosphodiesterase level. With dexamethasone treatment (400 micrograms/kg/day) for seven days, specific activity of the enzyme and its sensitivity (ED50) to insulin were decreased, as was the maximal responsiveness to insulin. Under conditions of adrenalectomy, the specific activity and the sensitivity (ED50) were increased while the maximal responsiveness to insulin was decreased. Following dexamethasone treatment specific insulin binding was decreased, and after adrenalectomy it increased. These findings were attributed to changes in the number of insulin receptors per cell rather than to changes in affinity. Alterations in insulin sensitivity (ED50) of the enzyme seemed to be due to alterations in insulin binding to the receptor. The reduction in maximal insulin responsiveness suggested postreceptor defects in both experimental groups. The mechanism related to alterations in the specific activity was not thoroughly clarified; however, serum insulin levels may specifically affect the enzyme activity.

Somatostatin and insulin secretion from pancreatic islets: Studies on the effect of high K+, 9-aminoacridine and valinomycin

We attempted to determine whether a decrease in the potassium permeability of the D cell membrane plays a role in the stimulus-secretion coupling, as it does in the pancreatic B cell. Elevation in the extracellular potassium concentration from 5.5 to 16.5 mM, or 0.2 mM 9-aminoacridine, which decreases potassium permeability in plasma membrane, stimulated the release of somatostatin as well as insulin from the isolated rat pancreatic islets. Valinomycin (1 microM), a potassium ionophore inhibited the secretion in response to high glucose, high extracellular potassium or 9-aminoacridine. These findings indicate that a reduction in potassium permeability in the D cell membrane, as induced by glucose or other stimulants, may be a major step in secretion of somatostatin.

Hypotonic activation of insulin-sensitive phosphodiesterase in rat fat cells

The effect of hypotonic treatment on the low Km membrane-bound cyclic AMP phosphodiesterase was investigated. Isolated fat cells obtained from Sprague-Dawley rats were incubated at 37 degrees C with an without 2 nM insulin. A crude microsomal fraction prepared by differential centrifugation was suspended in a hypotonic buffer at 4 degrees C, with and without protease inhibitors. Following solubilization from the particulate fraction, hypotonic treatment stimulated the phosphodiesterase in a time-dependent manner. Among the protease inhibitors, E-64, leupeptin and antipain were effective in preventing hypotonic activation of the enzyme. The release of the enzyme from the particulate fraction was partially inhibited by antipain. Kinetic analysis of the enzyme from hypotonic activation was much the same as that of the enzyme from the isotonic buffer. These results suggest that hypotonic activation of the phosphodiesterase may be the result of stimulation of an endogenous thiol protease of lysosomal origin.