Tatsuo Tamagawa

Insulin release independent of a rise in cytosolic free Ca2+ by forskolin and phorbol ester

The role of cytosolic free Ca2+ in insulin release was evaluated using isolated rat pancreatic islets permeabilized with digitonin and incubated in Ca‐EGTA buffers to fix free Ca2+ concentration at arbitrary levels. Ca2+ induced insulin release in a concentration‐dependent manner with the threshold being between 0.1 and 1 μM. The hormone release was increased by forskolin and 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), a potent activator of adenylate cyclase and that of protein kinase C, respectively. The findings suggest that activation of both protein kinase A and protein kinase C modulate insulin release without a concomitant increase in cytosolic free Ca2+.

Neostigmine-induced hyperglycemia is mediated by central muscarinic receptor in fed rats

We previously reported that neostigmine injected into the third cerebral ventricle stimulated adrenal secretion of epinephrine, secretion of glucagon from the pancreas, and direct neural innervation of the liver, resulting in hepatic venous plasma hyperglycemia in anesthetized fed rats. However, receptor type of these 3 mechanisms is not known. Therefore, we examined the effects of intraventricularly injected cholinergic or adrenergic antagonists on neostigmine-induced catecholamines in intact rats, glucagon secretion which is mediated by direct neural innervation of pancreas in bilateral adrenalectomized (ADX) rats, and hepatic venous hyperglycemia which is mediated by direct neural innervation of liver in ADX rats receiving constant infusion of somatostatin from femoral vein. Atropine injected into the third cerebral ventricle suppressed epinephrine secretion and dose-dependently inhibited hepatic venous hyperglycemia induced by neostigmine in intact rats. The neostigmine-induced glucagon secretion which occurs in ADX rats was suppressed by atropine. Atropine also prevented the neostigmine-induced hyperglycemia in ADX rats receiving constant somatostatin infusion through femoral vein (ADX-Somato rats). On the other hand, phentolamine, propranolol and hexamethonium showed no significant inhibitory effect on neostigmine-induced hyperglycemia, epinephrine and glucagon secretion in intact rats, glucagon secretion in ADX rats, or hyperglycemia in ADX-Somato rats. These results suggest that neostigmine-induced epinephrine and glucagon secretion and increased hepatic glucose output stimulated by direct neural innervation to liver is mediated by central muscarinic receptor in fed rats.

Mechanism of intrahippocampal neostigmine-induced hyperglycemia in fed rats

We previously reported that the injection of neostigmine, an acetylcholine esterase inhibitor, into the dorsal hippocampus produced hepatic venous plasma hyperglycemia associated with an increase of epinephrine and glucagon in anesthetized fed rats. To evaluate the relative contribution of these glucoregulatory hormones and the nervous system to the net hyperglycemic response, we unilaterally injected neostigmine (5 x 10(-8) mol) into the dorsal hippocampus in the following groups of rats: intact rats with bilateral adrenalectomy to eliminate the action of epinephrine, and rats receiving a constant infusion of somatostatin and insulin to prevent the glucagon response and to maintain the basal insulin level. Hepatic venous plasma levels of glucose, immunoreactive glucagon, immunoreactive insulin, epinephrine, and norepinephrine were determined. The area under the glucose curve during the 120-min period following the injection of neostigmine was compared between groups. The areas under the glucose curve for rats receiving somatostatin and insulin, adrenalectomy rats, and adrenalectomy rats receiving somatostatin and insulin were, respectively, 82, 31, and 61% of that for intact rats. The fashion of hippocampal stimulated hyperglycemia with neostigmine was similar to that after injection of neostigmine into the third cerebral ventricle. Therefore, we investigated hyperglycemia in rats with lesions of ventromedial hypothalamus and found that the response to hippocampal neostigmine was significantly inhibited by the hypothalamic lesion. These findings suggest that the glucoregulatory hippocampal activity evoked by neostigmine may be transmitted to peripheral organs via the ventromedial hypothalamus.

Central nervous system control of glycogenolysis and gluconeogenesis in fed and fasted rat liver.

The influence of brain cholinergic activation on hepatic glycogenolysis and gluconeogenesis was studied in fed and 48-hour fasted rats. Neostigmine was injected into the third cerebral ventricle and hepatic venous plasma glucose, glucagon, insulin, and epinephrine were measured. The activity of hepatic phosphorylase-a and phosphoenolpyruvate-carboxykinase (PEP-CK) was also measured. Experimental groups: 1, intact rats; 2, rats infused with somatostatin through the femoral vein; 3, bilateral adrenodemedullated (ADMX) rats; 4, somatostatin infused ADMX rats; 5, 5-methoxyindole-2-carboxylic acid (MICA) was injected intraperitoneally 30 minutes before injection of neostigmine into the third cerebral ventricle of intact rats. MICA treatment completely suppressed the increase in hepatic glucose in fasted rats, but had no effect in fed rats. Phosphorylase-a activity was not changed in fasted rats, but increased in fed rats, intact rats, somatostatin-infused rats, somatostatin-infused ADMX rats, and ADMX rats in that order. PEP-CK was not changed in fed rats, but increased at 60 and 120 minutes after neostigmine injection into the third cerebral ventricle in fasted rats. We conclude that, in fed states, brain cholinergic activation causes glycogenolysis by epinephrine, glucagon, and direct neural innervation. In fasted states, on the other hand, gluconeogenesis is dependent on epinephrine alone to increase hepatic glucose output.

Dissociation of hyperthermic and hyperglycemic effects of central prostaglandin F2α

We previously reported that intraventricular prostaglandins (PGs) produced hyperthermia and hyperglycemia in anesthetized rats. However, the relationship of them is little known. We examined the relationship between hyperthermia and hyperglycemia induced by intraventricular PGF2 alpha using curarized and adrenal demedullated rats. Iv curare completely prevented the PGF2 alpha-induced hyperthermia, but enhanced the hyperglycemic effect of PGF2 alpha. Adrenal demedullation completely prevented the hyperglycemia, but did not affect the hyperthermic effect of PGF2 alpha. To further assess the site of action concerned with PGF2 alpha-induced thermoregulation and glucoregulation in the central nervous system (CNS), we injected saline or PGF2 alpha into the preoptic area of the anterior hypothalamus (POA) in intact rats. After microinjection of PGF2 alpha into the POA, the rectal temperature rose, but the plasma glucose level did not increase significantly, as compared with saline-treated control rats. These results suggest that PGF2 alpha causes the central nervous system to produce hyperthermia via shivering, stimulated the somatic motor system, and to produce hyperglycemia by stimulating central sympathetic outflow to the adrenal medulla, but these operate independently under different neural regulation, and these sensitive sites are organically dissociated in the CNS.

Neither adrenergic nor cholinergic antagonists in the central nervous system affect 2-deoxy-D-glucose(2-DG)-induced hyperglycemia.

To investigate whether the brain adrenergic and cholinergic neurotransmitter systems are involved in the regulation of 2-deoxy-D-glucose (2-DG)-induced hyperglycemia, we studied the effects of adrenergic and cholinergic antagonists on 2-DG-induced secretion of epinephrine and glucagon, and hyperglycemia, in anesthetized fed rats. When 2-DG (10 mg/10 microliters) was injected into the third cerebral ventricle, hepatic venous plasma glucose, glucagon, and epinephrine concentrations were significantly increased. Co-administration of phentolamine, propranolol, atropine and hexamethonium (1 X 10(-7) mol) with 2-DG did not modify the hyperglycemia and hormonal responses normally observed after the administration of 2-DG alone. From this evidence we concluded that neither brain adrenoceptive nor cholinoceptive neurons are involved in the regulation of 2-DG-induced hyperglycemia.

Effects of imidazoline antagonists of α2-adrenoceptors on endogenous adrenaline-induced inhibition of insulin release

We studied the effects of adrenoceptor antagonists and imidazoline derivatives on endogenous adrenaline-induced inhibition of insulin release in anesthetized rats. The intracerebroventricular injection of neostigmine increased plasma levels of catecholamines and glucose but not insulin. Pretreatment with an i.p. injection with phentolamine caused a dose-dependent increase in insulin secretion. When atropine was coadministered with phentolamine, the phentolamine-induced increase in insulin secretion was inhibited. Neither phentolamine nor atropine affected plasma levels of catecholamine. Yohimbine and idazoxan, which are alpha 2-adrenoceptor antagonists, and tolazoline, a non-selective alpha-adrenoceptor antagonist, also reversed adrenaline-induced inhibition of insulin secretion. Phenoxybenzamine, prazosin, propranolol, and antazoline, an imidazoline without alpha 2-adrenoceptor activity, did not affect insulin levels. When agents were preinjected i.p. in rats that were given saline into the third cerebral ventricle, phentolamine and antazoline, but not yohimbine and idazoxan, increased plasma levels of insulin. The results suggest that the inhibition of insulin release induced by adrenaline was reversed by antagonism of alpha 2-adrenoceptors. Phentolamine and antazoline, both of which are imidazoline derivatives, induced insulin secretion independently of the adrenoceptors only under the resting conditions.

Intrahypothalamic, but not hippocampal, administration of muscimol suppresses hyperglycemia induced by hippocampal neostigmine in anesthetized rats

We investigated the effects of intrahypothalamic or hippocampal injection of GABA receptor agonists on hyperglycemia induced by hippocampal neostigmine. Prior to the injection of neostigmine (50 nmol) into the hippocampus (HPC), muscimol (0.01-1 nmol) or baclofen (1 nmol) was injected into the bilateral ventromedial hypothalamus (VMH). Muscimol suppressed the hyperglycemia in a dose-dependent manner, but baclofen affected it only minimally. In contrast, neither hippocampal muscimol (1 or 2.5 nmol) nor baclofen (1 nmol) suppressed the hippocampal neostigmine-dependent hyperglycemia. Intrahypothalamic muscimol (1 nmol) also decreased the changes in hepatic venous plasma glucagon and epinephrine significantly. These results indicate that intrahypothalamic muscimol suppresses hyperglycemia caused by cholinergic neurons originating from the HPC, indicating existence of the location specificity.

Role of brain histamine H1- and H2-receptors in neostigmine-induced hyperglycemia in rats

We previously reported that when neostigmine, an inhibitor of acetylcholine esterase, was injected into the third cerebral ventricle, the concentration of hepatic venous plasma glucose was increased via central muscarinic receptors in anesthetized rats. To determine whether brain histamine receptors are involved in cholinergic system transmission with regard to central nervous system (CNS)-mediated glucoregulation, we examined the effects of the H1 receptor antagonist pyrilamine and the H2 receptor antagonist ranitidine on neostigmine-induced hyperglycemia in anesthetized rats. The injection of pyrilamine (5 x 10(-9)-5 x 10(-7) mol) into the third cerebral ventricle suppressed hyperglycemia induced by intraventricular injection of neostigmine (1 x 10(-9) mol) in a dose-dependent manner. Injection of ranitidine (5 x 10(-9)-5 x 10(-7) mol) into the third cerebral ventricle did not suppress the hyperglycemia induced by neostigmine, but enhanced it in a dose-dependent manner. These findings suggest that neostigmine-induced CNS-mediated hyperglycemia is transmitted by not only brain cholinergic muscarinic receptors but also in part by histamine H1 receptors.

Activation of GABAA receptors in hypothalamus modulates PGF2α- or PGE2-induced catecholamine secretion in rats

We previously reported that injection of PGF2 alpha into the third cerebral ventricle produces hyperglycemia and hyperthermia associated with catecholamine secretion in anesthetized rats. We have also studied the potency of catecholamine secretion induced by injecting PGE2 or PGF2 alpha into the third cerebral ventricle and the effect of the GABA-selective agonist, muscimol, on the catecholamine secretion induced by PGE2 or PGF2 alpha. Administration of 50 micrograms of PGE2 into the third cerebral ventricle increased norepinephrine secretion to a greater extent than the same dose of PGF2 alpha, whereas the latter increased epinephrine secretion to a greater degree. These effects paralleled the potencies of the hyperglycemic and hyperthermic effects of PGF2 alpha and PGE2, respectively. Simultaneous injection of 2.5 nmol of muscimol into the third cerebral ventricle with 50 micrograms of PGF2 alpha or PGE2 completely suppressed epinephrine and norepinephrine secretion induced by PGF2 alpha or PGE2. These findings suggest that central PGF2 alpha and PGE2 stimulate epinephrine and norepinephrine secretion with different potencies, and that brain GABAA receptors suppress catecholamine secretion induced by PGF2 alpha or PGE2.