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Insulin-induced hypoglycemia stimulates both adrenaline and noradrenaline release from adrenal medulla in 21-day-old rats.

It has been well-established that insulin-induced hypoglycemia evokes preferential adrenaline release from the adrenal medulla in fasted adult rats. The present study examined the responsiveness to hypoglycemia in fasted 21-day-old and 8-week-old rats. The recovery of adrenaline in the chromaffin granule fraction prepared from the 8-week-old rat adrenal homogenate decreased 30 min after subcutaneous injection of 3 U/kg insulin, whereas the recovery of both adrenaline and noradrenaline was diminished in 21-day-old rats. In electron microscopy, omega-shaped profiles, indicative of exocytosis, were frequently observed in adrenaline- and noradrenaline-storing cells of 21-day-old rats. These results indicate that the responsiveness of the noradrenaline-storing cells to hypoglycemia in 21-day-old rats is different from that in young adult rats.

The importance of the sympathoadrenal system of fetuses and neonates has been emphasized for survival against various stresses such as hypoxia, asphyxia and hypoglycemia (1)(2)(3). It has been well-established that splanchnic nerves innervating the adrenal medulla are mainly responsible for the catecholamine release from adrenal chromaffin cells. In rats, the adrenal chromaffin cells are not yet innervated by splanchnic nerves at birth (4,5). In 4-day-old rats, innervation of cholinergic nerves occurs (6). The number of innervations increases threefold during the first postnatal week (7). It has been reported that the function of the cholinergic nerves in the catecholamine biosynthesis and release in the chromaffin cells becomes fully mature in 10-day-old rats (4,5). In the previous study, we demonstrated that nicotine hardly elicits catecholamine release from the adrenal medulla of 21-day-old rats (8). The responsiveness of the adrenal chromaffin cells of infant rats to various stimulations may be different from that in adult rats.
Insulin-induced hypoglycemia causes the preferential release of adrenaline from the adrenal medulla in various animals including rats (9)(10)(11)(12)(13)(14). This release is blocked by transection of the splanchnic nerves (15). In the present study, the response of the adrenal chromaffin cells to hypoglycemia in fasted 21-day-old and 8-week-old rats was examined. Catecholamine release from the adrenal chromaffin cells was detected by biochemical methods and observation of ,2-shaped profiles, indicative of exocytosis, using the electron microscope. Swelling of rough endoplasmic reticulum (rER) that indicates enhanced synthesis of secretory proteins, probably resulting in chromaffin granule regeneration, was also observed carefully. Insulin (3 U/kg) was injected subcutaneously. This is a dose that produces severe hypoglycemia in fasted adult rats 3 hr after the injection (14). As a result, hypoglycemia induced both adrenaline and noradrenaline release from the adrenal chromaffin cells of 21-day-old rats 30 min after the injection, whereas adrenaline release solely occurred in 8-week-old rats.

Isolation of the adrenal chromaffin granule fraction
Male Wistar-Imamichi 21-day-old and 8-week-old rats (The Institute for Animal Reproduction, Ibaraki) were used after 1-week housing in our animal room for adaptation. The animals were kept under lighting conditions of 12L -12D, at 24.h2 C , and they had free access to food and water. They were fasted from 16:00 pm to the next morning at 10:00 am; Then, 3 U/kg insulin, dissolved in saline, was injected subcutaneously. The animals were killed by decapitation 30 min or 3 hr after the injection. Adrenals were dissected immediately from the animals and homogenized with a Teflon pestle homogenizer in a medium composed of 0.3 M sucrose, 20 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer (pH 5.9), 2 mM EGTA and 1 mM phenylmethylsulfonyl fluoride (PMSF). The chromaffin granule fraction was prepared from a mixture of equal volumes of the homogenates obtained from 6 rats (10 mg protein in total) by linear sucrose gradient centrifugation, as reported previously (16). All procedures described above were carried out at 41C.
Electron microscopy Adrenal glands were excised after decapitation as soon as possible. Segments of the adrenal medulla were processed for electron microscopic study as previously reported (8).

Determination
of catecholamines, serum glucose and protein concentration Catecholamines were analyzed by HPLC as reported previously (16). Serum glucose concentration was determined by the enzymatical method using a commercially available kit (F-kit glucose; Boehringer Mannheim, Germany). Protein concentration was measured by the method of Bradford (17) with bovine r-globulin as the standard.
Chemicals EGTA, MES and PMSF were purchased from Sigma Chem., Co., St. Louis, MO, USA. The dye reagent for protein determination and r-globulin were from Bio-Rad, Richmond, CA, USA. Pig insulin was the product of Yamanouchi Pharmaceutical Co., Tokyo. Glutaraldehyde, paraformaldehyde and OSO4 were from TAAB Laboratories Equipment, Ltd., Berks, England. Other chemicals were all analytical or electron microscopic grade.

Statistical analyses
Data are expressed as means ±S.E. To define statistically significant differences in the catecholamine concentration between the control and insulin-treated rats, the data were analyzed by Student's t-test.

Effect of hypoglycemia on adrenal chromaffin cells in 8-
week-old rats Although the behavioral activity of fasted 8-week-old rats seemed to become somewhat low 30 min after subcutaneous injection of 3 U/kg insulin, convulsion was not evoked. The animals were killed by cervical dislocation 30 min after the injection. As shown in Table 1, serum glucose concentration was decreased significantly by insulin treatment. Liver weight also decreased markedly.
The adrenaline and noradrenaline concentration of the adrenal homogenate tended to decrease, but this was not statistically significant As indicated in Fig. 1 (a and b), the adrenaline-and noradrenaline-storing chromaffin granule fractions were obtained by linear sucrose gradient centrifugation in fraction numbers 2 to 6. The recovery of adrenaline from 10 mg protein of the adrenal homogenate in the chromaffin granule fraction was decreased in insulin-treated animals (Fig la) The recovery of noradrenaline was scarcely changed by the treatment Three hours after insulin injection, severe convulsion was evoked in almost all rats. In these animals, preferential decrease of the recovery of adrenaline in the chromaffin granule fraction occurred (Fig 3 a and b). In electron microscopy, the number of chromaffin granules was decreased markedly, and a remarkable number of vacuoles were observed in the adrenaline-storing chromaffin cells, but there were hardly any morphological changes in the noradrenaline-storing cells (data not shown). In the cell membrane of the synapse buttons of cholinergic nerve fibers, innervated to the noradrenaline-storing cells of insulin-treated animals, Q-shaped profiles formed by acetylcholine release were not present at 30 min and 3 hr after the injection (data not shown).

Effect of hypoglycemia on adrenal chromaffin cells in 21-day-old rats
The behavioral activity of insulin-treated 21-day-old rats was almost the same as that in the control animals 30 min after the injection. As indicated in Table 2, the serum glucose concentration of 21-day-old fasted rats was significantly reduced 30 min after the insulin injection. In these animals, liver weight also decreased markedly. Both the adrenaline and noradrenaline concentration in the adrenal homogenate was decreased significantly by insulin treatment (P<0.005 and P<0.05 vs control, respectively). As indicated in Fig. 4 (a and b), the adrenalineand noradrenaline-storing chromaffin granule fractions were obtained by sucrose gradient centrifugation in fraction numbers 3 to 7. The adrenaline and noradrenaline contents in the chromaffin granule fraction were also depleted markedly by the injection.
As shown in Fig. 5, adrenaline-and noradrenalinestoring chromaffin cells in 21-day-old control rats are morphologically very similar to those in the 8-week-old control animals (Fig. 2a). As indicated with a large arrowhead in the figure, an Q-shaped profile is found, although this is a rare case. In this photograph, a synapse button of a cholinergic nerve innervated to the noradrenaline-storing cell is present (n). Several very small acetylcholine-storing secretory granules, that are less electron dense than the adrenaline-storing chromaffin granules, exist in the synapse button (small arrowheads). An Q-shaped profile formed by acetylcholine release is observed in the cell membrane of the synapse button facing the synaptic cleft (arrow). Figure 6 shows an electron micrograph of adrenalinestoring and noradrenaline-storing cells of an insulintreated animal. In both cells, Q-shaped profiles are frequently observed (arrowheads).
Sometimes large Qshaped profiles are present (arrow), probably produced by the fusion of several chromaffin granules that had occurred just before exocytosis.
As indicated in Fig. 7, very large size granules were sometimes observed in the noradrenaline-storing cells of 21-day-old insulin-treated rats (arrowheads). In these granules, an electron-dense core was present. Swelling of the rER was also found in these cells (arrows).

DISCUSSION
In the present study, Sl-shaped profiles were found in the cell membrane of synapse buttons of cholinergic nerve terminals innervating the adrenal medulla in 21-day-old rats. This indicates that acetylcholine release from the nerve terminals occurs to some extent in 21-day-old rats. The cholinergic nerves are at least functionally active, although the degree of maturity is not clear from the present data.
In the present study, serum glucose levels of 21-day-old and 8-week-old rats were remarkably decreased 30 min after insulin injection. No convulsions were evoked by hypoglycemia in these animals. It has been reported that excitatory amino acids such as aspartate and glutamate are increased in synaptic clefts of the central nervous system by hypoglycemia, resulting from a decrease in cellular uptake due to energy depletion, and that these amino acids are neurotoxic (18). These increased excitatory amino acids in synaptic clefts of the central nervous system probably result in severe convulsion and catecholamine output from the adrenal medulla. The decrease of the recovery of catecholamines in the chromaffin granule fraction observed 30 min after insulin treatment in 21day-old and 8-week-old rats is probably not induced by the effect of aspartate and glutamate in the central nervous system, because no convulsions were evoked by hypoglycemia in these animals.
In the present study, not only adrenaline but also noradrenaline was released from the adrenal medulla by insulin treatment in 21-day-old rats, whereas preferential adrenaline release occurred in 8-week-old rats. The present results indicate that the responsiveness of the noradrenaline-storing cells of infant rats against hypoglycemia is somewhat different from that in young adult rats. It is well-known that the serum glucose level is elevated more effectively by adrenaline than noradrenaline and that noradrenaline is a more potent vasopressor when compared to adrenaline. The merit of the noradrenaline release in the protection against hypoglycemia for the survival of infant rats is unknown at present.
In the previous study, the catecholamine concentration of the adrenals in 8-week-old rats was not significantly changed by 5 mg/kg nicotine stimulation, whereas the contents in the chromaffin granule fraction decreased markedly (8). The data of the previous report suggest that the chromaffin granules recovered in the chromaffin granule fraction are mature ones and that these are mainly linked to catecholamine secretion. The recovery of adrenaline in this fraction from the homogenate of adrenal medulla in control animals estimated in the previous study was about 66% (8). The other portion of the granules is probably not involved in catecholamine release. Therefore, the magnitude of the decrease in this fraction is more apparent than that in the homogenate. In the present study, insulin did not significantly reduce the adrenaline concentration of the adrenals in 8-week-old rats, while the recovery in the chromaffin granule fraction was remarkably decreased by the administration. This is probably because insulin-treatment only induces exocytosis in the mature chromaffin granules. In the present study, large granules were occasionally found in noradrenaline-storing cells of insulin-treated 21day-old animals. These granules had an electron-dense core. The core has been observed in noradrenaline-storing chromaffin granules, which were pre-fixed by glutaraldehyde and post-fixed by OS04 and then stained with uranyl acetate and lead citrate (19). Therefore, the granules seen in the present study were probably not vacuoles but chromaffin granules. Fusion of several chromaffin granules at high Ca 21 concentration (about 1 1tM) has been reported in in vitro studies (20,21). These large chromaffin granules, observed in the present study, may be produced by a high cytosolic Ca2+ concentration, resulting from Ca2+ influx caused by acetylcholine release from cholinergic nerve terminals innervating the noradrenaline-storing cells. In these cells, swelling of the rER was simultaneously found. This indicates that synthesis of secretory proteins such as chromogranin A is enhanced by insulintreatment, probably resulting in chromafl'in granule regeneration. Fig. 7. Electron micrograph of a noradrenaline-storing cell in a 21-day-old insulin-treated rat. Animals were killed 30 min after insulin injection. NA: noradrenaline-storing cell, E: endothelial cell, p: perivascular space. Note that very large granules that have an electron-dense core are present (arrowheads) and that swelling of the rER is observed (arrows). The bar indicates 1 pm.