Participation of intestinal hormones in the development of the pancreatic B-cell reactivity of rat fetuses to the action of glucose

The involvement of intestinal hormones in the development of insulin release from rat fetal pancreas was investigated. B-cell responses were determined by changes in the concentration of immunoreactive insulin after glucose addition to the incubation medium. Coincubation of fragments of fetal pancreas and duodenum from adult and newborn rats and from 21.5-day-old fetus has shown that intestinal factors can recover the response of pancreas to glucose in fetuses with experimentally removed hypothalamus and hypophysis. Besides, the intestinal factors in the fetus were found to potentiate the effect of high glucose concentrations on B cells, but had no insulinotropic effect at physiological glucose concentration in the medium. The data obtained suggest that even in the antenatal period the intestinal, along with cephalic factors, can serve as modulators of glucose action on islet B cells.     

EFFECTS OF DEXAMETHASONE ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE AND ADRENALINE IN THE BRAINS AND SUPERIOR CERVICAL GANGLIA OF ADULT AND NEONATAL RATS

Abstract— Phenylethanolamine N -methyltransferase (PNMT) activity assayed by a sensitive radiochemical method was found to be distributed unevenly in the adult rat brain. Highest activities of this enzyme were located in the medulla and the hypothalamus. Small amounts of adrenaline were identified in the hypothalamus using a sensitive enzymatic radiochemical assay procedure, whereas in the medulla and other brain regions the values for adrenaline were at the limits of the sensitivity of the assay for this amine. The daily administration of dexamethasone (1 mg/kg) to adult rats for 13 days significantly increased PNMT activity in medulla and hypothalamus and also increased the adrenaline content of the hypothalamus. Five daily injections of dexamethasone (0·1 mg/kg) to newborn rats did not alter the PNMT activity or the catecholamine content of the brain, but markedly increased the PNMT activity and adrenaline content of superior cervical ganglia. Higher doses of dexamethasone given to newborn rats (6 daily injections of 1 mg/kg) increased PNMT activity both in the medulla and in the hypothalamus.     

The uptake of 3H-serotonin by neural elements of the rat hypothalamus in ontogeny

Development of serotoninergic system of rat hypothalamus has been studied. The level of its maturity was measured by specific absorption of 3H-serotonin by hypothalamus of fetuses (days 16-20 of intra-uterine development), newborn (9 days after birth) and adult rats. Inhibitors of specific serotonin capture by serotoninergic neural elements (cocaine, fluoxetin and cytalopram) were used in control experiments. Specific uptake of 3H-serotonin by hypothalamus was detected on day 16 of intra-uterine development. It increased twice by day 18 and remained unchanged until birth. No statistically significant increase of 3H-serotonin uptake was detected in newborn and adult animals.     

BIOSYNTHESIS OF THYROTROPIN RELEASING FACTOR BY NEWT (TRITURUS VIRIDESCENS) BRAIN IN VITRO. ISOLATION AND CHARACTERIZATION OF THYROTROPIN RELEASING FACTOR

Abstract— The in vitro biosynthesis of thyrotropin releasing factor (TRF) by hypothalamic and forebrain fragments from adult newts ( Triturus viridescens ) was studied by incubating the fragments in the presence of the radioactive amino acid precursors of thyrotropin releasing factor (TRF). The synthesized product was identified by successive chromatography and electrophoresis. A purification scheme was developed which can be applied to the processing of multiple samples labelled with l -[³H]proline and yields a TRF peak which, when derivatized with 1-fluoro-2,4-dinitrobenzene and applied to electrophoresis in acid buffer, yields a single radioactive peak corresponding to the migration of the N^im-Dnp derivative made from standard synthetic [³H]TRF. Both forebrain and hypothalamic fragments of the adult newt are capable of synthesizing a product which migrates with standard TRF after application of this purification scheme. Hypothalamic and forebrain fragments were also assayed for biological activity of TRF and it was determined that one newt hypothalamus contains approximately 200 pg TRF and that an equal amount of TRF is present in a forebrain piece of comparable size. It is concluded that newt TRF is identical to mammalian TRF (pGlu-His-Pro-NH₂) and that the storage and synthesis of this substance is not confined to the hypothalamus in this species.     

Peripolar cell hypertrophy in the renal juxtaglomerular region of newborn sheep

Summary In the renal juxtaglomerular region of newborn sheep, it was found that glomerular peripolar cells and their granules were very much larger than those found in fetal lambs or adult sheep. Similar peripolar cell hypertrophy was triggered in fetal lambs treated in utero with intraperitoneal injections of dexamethasone. Ultrastructurally, granules of peripolar cells from newborn lambs resembled closely the enlarged zymogen granules described in the pancreas of newborn rats. Such peripolar cell hypertrophy may reflect a functional adaptation of the kidney to immediate postnatal life.     

Hypothalamic receptors of sex hormones in the mechanism of the sexual differentiation of the brain in rats

Studies have been made on the content of receptors of estradiol (E2) and testosterone (T) in cytoplasmic and nuclear fractions of the hypothalamus of male and female rats during neonatal development, as well as in adult females after androgenization in neonatal period and adult males castrated within 3 days of postnatal life. It was shown that both E2 and T are present in the blood serum of male and female newborn rats. In female hypothalamus, only E2 receptors were found, whereas in males both types of receptors were revealed, their content being higher than in females. In adult animals subjected to changes in the level of sex hormones in the blood during early neonatal period, changes in concentration of the receptors in the hypothalamic centres of regulation of tonic and cyclic secretion of gonadotropins were found. The data obtained presumably reveal the role of receptors of sex hormones in sex differentiation of the brain.     

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.     

Serotonin contents in the hypothalamus of adult males, females and males castrated during the neonatal period

The aim of the present work was to study the character of the change in serotonin level in the anterior and medial basal hypothalamus of adult rats after the effect of testicular hormones had been switched off on the first day of postnatal life. It was shown in our work that in males serotonin level was significantly lower than that in females by 67 and 46% in the anterior and medial basal hypothalamus, respectively. Castration of newborn males resulted in a significant increase in serotonin level in both anterior and medial basal hypothalamus-up to the level observed in females. It is supposed that the male sex hormones affect differentiation of serotoninergic system of the brain.     

Starvation increases gastrointestinal somatostatin in normal and obese Zucker rats: a possible regulatory mechanism

Normal, male Sprague-Dawley (S-D) rats and female, lean and obese Zucker rats were studied in the fed state and after 48 hours of food deprivation. Somatostatin-like immunoreactivity (SLI) was measured from acetic acid extracts of oesophagus-cardia, stomach, small and large intestine, pancreas, hypothalamus, pituitary and cerebellum. Within the CNS, the highest levels of SLI were found in the hypothalamus, while in the gut, these levels were highest in the stomach and pancreas. All Zucker rats displayed higher hypothalamic levels of SLI than did S-D rats. Obese Zucker rats in the fed state differed from their lean littermates in that SLI levels were lower in oesophagus-cardia, stomach and hypothalamus, while being higher in pancreas and pituitary. The response to starvation in both obese and lean Zucker rats was qualitatively similar, and included significant increases in stomach and oesophagus-cardia SLI, but with a significant fall hypothalamic SLI. We have concluded that the increase in gastrointestinal SLI with starvation in Zucker as well as in S-D rats may represent a significant regulatory mechanism in nutrient homeostasis. We postulate that gastric SLI may decrease the availability of intestinal insulin secretagogues in the fasting state. This adaptive mechanism appears to be intact in the obese Zucker rat.     

Anogenital distance at birth as a predictor of volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus and pituitary responsiveness in castrated adult rats.

Variation in anogenital distance (AGD) in female newborn rats depends upon androgenization secondary to transplacental/transmembraneous testosterone from adjacent intrauterine male siblings. Since the size of the sexually dimorphic nucleus of the preoptic area of the hypothalamus (SDN-POA) and the degree of pituitary sensitivity to GnRH are neuroendocrine markers of neonatal androgenization, we compared these to AGD in castrated adult male and female rats. Compared to 1-day-old female rats with short AGD (less than or equal to 1.4 mm), 1-day-old female rats with long AGD (greater than 1.4 mm) had significantly larger SDN-POA volumes as adults. In contrast, LH secretion following GnRH injection did not differ in the two subgroups. Our results emphasize that some endpoints of central nervous system sexual differentiation in the adult rat are predicted by the appearance of a masculinized genital tract at birth. It follows that the complete evaluation of potential androgenizing agents will require systematic assessment of multiple morphologic and functional endpoints.     

Rhythmic variations in acid phosphatase activity in the liver of newborn rats

The rhythm of acid phosphatase activity in liver homogenates of newborn rats (aged about 14 days) was compared with a similar rhythm in adult rats (aged 4.5 months). Serial chromatographic investigations demonstrating isoenzyme patterns demonstrated age-related changes of this rhythm connected with the synthesis of the enzyme in newborn rats. The averaged activity of the enzyme in the liver homogenates of newborn rats was about 4 times lower than in adult rats. The maximal values of total enzyme activity of both isoenzymes after chromatographic separation in newborn rats were shifted by about 7 hours in relation to adult animals. Similar changes were observed in the case of the greatest maximal values of the activity ratios--subunit: both isoenzymes, and isoenzyme II: isoenzyme I. In adult rats these maximal values appeared during the night hours and in newborn rats during the day.     

Caerulein-induced desensitization of enzyme secretion fails in neonatal rat pancreas

Pancreatic segments of 1-, 3-, 5-, 10-day-old and adult female OFA (Sprague-Dowley strain) rats were superfused with graded concentrations of caerulein (10(-12)-10(-7) M) to establish concentration-response relation of amylase release. Furthermore, pancreatic segments of 3-, 5-, 10-day-old and adult rats were superfused with 10(-10) or 10(-8) M caerulein and then superfusion was repeated with 10(-10) M concentration of caerulein to show whether the phenomenon of desensitization of amylase release can be induced in the postnatal period. The 1-day-old pancreas was found practically insensitive to caerulein. The 3- and 5-day-old gland was by one order of magnitude less sensitive (EDmax = 10(-8) M) than the adult pancreas (EDmax = 10(-9) M). Repeated superfusion of the 3- and 5-day-old pancreas with 10(-10) M caerulein after the first 10(-8) M caerulein superfusion failed to cause desensitization, while the same (10(-10) M) repeated superfusion of the 10-day-old adult pancreatic segments after the first 10(-8) M caerulein superfusion evoked desensitization of enzyme release. The authors suggest that the failure of desensitization of enzyme secretion for caerulein may be due to the maturation process of newborn rat pancreatic acinar cells at receptorial and postreceptorial level.     

Effect of electrostimulation of the hypothalamus on protein biosynthesis in organs of adult and aged rats

The effect of hypothalamus electrical stimulation on total protein biosynthesis was studied in skeletal muscle, heart, liver, adrenal cortex and thyroid gland of adult rats. In adult animals hypothalamus stimulation provokes a pronounced increase in 3H-leucine incorporation into total protein of all tissues, as well as into liver chromatin proteins. No significant changes were observed in protein biosynthesis when hypothalamus of old rats was stimulated. This can serve as evidence of age-related decrease in the ability of the hypothalamus to stimulate protein synthesis in peripheral tissues.     

Metabolism of thyrotropin releasing factor in two clonal cell lines of nervous system origin

Immunoreactive thyrotropin releasing factor (TRF) was detected in homogenates of two clonal cell lines, BN1010-1 and BN1010-3, derived from a rat central nervous system tumor. TRF was present in logarithmically-growing cells; daily medium changes with slightly acid culture medium (pH 6.8) greatly increased the TRF content of these cells. In contrast, TRF could not be detected in stationary phase cells. TRF peptidases were <1% as active in homogenates of BN1010 cells as those in homogenates of guinea pig brain or hypothalamus. It is expected that these cells will provide an excellent model system for the study of various aspects of TRF metabolism.     

Histogenesis of the endocrine pancreas in newborn rats after destruction by streptozotocin. An immunocytochemical study

Endocrine pancreatic tissue in newborn rats was studied 1 to 17 days after the destruction of B cells by an injection of streptozotocin. Regeneration of insulin cells was observed four days after streptozotocin injection, which was followed by recovery from the diabetic state and an increased pancreatic insulin content. Regeneration was characterised by new islets budding from small ducts. The pancreas of newborn rats, like the embryonic pancreas, thus retains a capacity to form endocrine tissue, although some degree of reduplication of preexisting B cells may also be involved in the process. Newborn rats injected with streptozotocin constitute an interesting model for the study of factors which may act on the regenerative potential of pancreatic endocrine tissue in the diabetic state.     

Ontogeny of secretory function and cholecystokinin binding capacity in immature rat pancreas

Isolated acini were prepared from the pancreas of immature rats (age less than 1 hr. - 48 hrs) in order to study the development of the secretory process. The ultrastructural integrity of the acinar cells was maintained after digestion and stimulation with secretagogues. Acini prepared from rats aged 24 - 48 hours responded to both CCK-8 and carbachol with significant increases in amylase release. Although typical biphasic dose response curves were obtained, the curves were shifted to the right by 1 - 2 log units, compared to the responses of adult acini. At ages younger than 24 hours, acini were insensitive to secretagogues but were sensitive to the calcium ionophore A23187. CCK receptors were virtually absent from membranes prepared from newborn pancreases, but binding of CCK, although small, was measurable at 12 hours and slowly increased up to 48 hours. A greater amount of binding was seen at 72 hours, which appeared constant up to 14 days. At 21 days, adult levels of binding were found. These results confirm previous studies that the rat pancreas is insensitive to secretagogues in the first 24 hours of life. After age 24 hours the secretory process is intact but less sensitive to secretory agents than the more mature pancreas. In the case of CCK, this may be due to lesser numbers of CCK receptors and/or affinity of CCK for its receptor.     

Effect of streptozotocin administration on somatostatin content of pancreas and hypothalamus in rats.

A radioimmunoassay (RIA) method for somatostatin (SRIF) utilizing rabbit antiserum against synthetic SRIF coupled with human serum alpha-globulin is described. Synthetic N alpha-tyrosylated SRIF was labelled with 125I using the lactoperoxidase method and purified on a Sephadex G-10 column. This assay system was highly specific for SRIF and did not cross-react with hypothalamic trophic hormones, pituitary trophic hormones or gastrointestinal hormones. The effect of streptozotocin induced diabetes on the SRIF content was examined in the pancreas, the pancreatic islets, as well as the hypothalamus of rats. SRIF content in both the pancreas and islets of the diabetic rats was shown by RIA to have significantly increased. However, content in the hypothalamus of the diabetic rats did not differ from that of the control. The physiological and pathophysiological significance of the SRIF changes remains to determined.     

Effects of ammonia,chloroquine, and monensin on the vacuolar apparatus of an absorptive epithelium

Summary The distribution of two major immunoreactive forms of somatostatin, somatostatin-14 and somatostatin-34, within the brain, pancreas and intestine of adult lampreys, Petromyzon marinus, was identified using antisera raised against these peptides. Immunostaining of the brain is similar in juveniles and upstream migrants, and somatostatin-14 is the major somatostatin form demonstrated. A few somatostatin-34-containing cells are localized within the olfactory bulbs, thalamus and hypothalamus, but cells immunoreactive to anti-somatostatin-34 in the hypothalamus and thalamus do not co-localize somatostatin-14. Immunostaining of pinealocytes within the pineal pellucida with anti-somatostatin-14 may infer a novel function for this structure. Somatostatin-14 and somatostatin-34 are co-localized within D-cells of the cranial pancreas and caudal pancreas of juveniles and upstream migrants. Numerous somatostatin-34-immunoreactive cells are distributed within the epithelial mucosa of the anterior intestine but not all of these cells cross-react with anti-somatostatin-14. It appears that somatostatin-34 is the major somatostatin in the pancreo-gastrointestinal system of adult lampreys.     

Age-related changes in the sympathetic innervation of the pancreas

Using immunohistochemical methods, the morphological features of the sympathetic nerve structures in the pancreas of newborn, pubescent, and aging rats have been studied. The neural composition of intramural ganglia has been described. The intramural ganglia were shown to include chromaffin cells. In many ganglia of the pancreas, two types of pericellular nerve apparatuses have been detected simultaneously: tyrosine hydroxylase-containing catecholaminergic synaptic terminals and PGP 9.5-immunopositive cholinergic synapses. It was established that the density of catecholaminergic structures in the pancreas of rats decreases with age.     

Ontogeny of the pig hypothalamic neurosecretory system with particular reference to the distribution of neurophysin

The immunoperoxidase cytochemical reaction was applied to the localization of neurophysin-containing elements in the fetal and adult pig hypothalamus. In the 60 day fetal pig, cells of the supraoptic nucleus (SON) were the only structues in the hypothalamus in which neurophysin was detected. However, by 87 days the cell bodies in both the SON and paraventricular nucleus (PVN) contained neurophysin-like material. The distribution of immunoreactive material in the 111 day fetal animal was similar to that found in the adult pig. In transverse section of the mature pig the SON exists in two discrete components; an antero-lateral group of cells connected by scattered cells to a smaller postero-medial group. Anteriorly, the PVN appears as a line of cells bordering the third ventricle but as we proceed posteriorly the dorsal aspect expands laterally to give a wedge-shaped group of cells. In mid-sagittal sections, the cells of the PVN are distributed over a wide area of the anterior hypothalamus in a triangular profile. The borders between the SON and PVN became more difficult to define in medial sections than in lateral sections. Continuous gradient polyacrylamide gel electrophoresis was carried out on the neural lobe extracts from fetal, newborn and adult pigs. Proteins with an electrophoretic mobility similar to that of porcine neurophysins-I, -II and -III were present in the newborn and 98 day fetal pig. It is concluded that material immunoreactive with anti-neurophysin serum is present in the hypothalamus of the 60 day fetal pig. Furthermore, at late fetal development and during the postnatal period it is tentatively suggested that the neurophysin present in the pituitaries of these animals is chemically identical with that of adult neurophysin.