Neurotensin release from rat hypothalamus in vitro

Neurotensin is a hypothalamic peptide which inhibits secretion of TSH in the rat in vivo. We have demonstrated a calcium-dependent release of neurotensin from incubated rat hypothalamus in response to depolarizing stimuli, as well as a dose-dependent stimulatory effect of tri-iodothyronine (T3) on neurotensin secretion. We suggest that part of the neuroendocrine control of TSH secretion involves the interaction of T3, neurotensin and TSH; the presence of neurotensin in extracts of anterior pituitary gland is further evidence for its hypophysiotrophic role.     

Glucagon induces the hepatic expression of inflammatory markers in vitro and in vivo

Glucagon exerts multiple hepatic actions, including stimulation of glycogenolysis/gluconeogenesis. The liver plays a crucial role in chronic inflammation by synthesizing proinflammatory molecules, which are thought to contribute to insulin resistance and hyperglycaemia. Whether glucagon affects hepatic expression of proinflammatory cytokines and acute-phase reactants is unknown. Herein, we report a positive relationship between fasting glucagon levels and circulating interleukin (IL)-1β (r = 0.252, p = .042), IL-6 (r = 0.230, p = .026), fibrinogen (r = 0.193, p = .031), complement component 3 (r = 0.227, p = .024) and high sensitivity C-reactive protein (r = 0.230, p = .012) in individuals without diabetes. In CD1 mice, 4-week continuous treatment with glucagon induced a significant increase in circulating IL-1β (p = .02), and IL-6 (p = .001), which was countered by the contingent administration of the glucagon receptor antagonist, GRA-II. Consistent with these results, we detected a significant increase in the hepatic activation of inflammatory pathways, such as expression of NLRP3 (p < .02), and the phosphorylation of nuclear factor kappaB (NF-κB; p < .02) and STAT3 (p < .01). In HepG2 cells, we found that glucagon dose-dependently stimulated the expression of IL-1β (p < .002), IL-6 (p < .002), fibrinogen (p < .01), complement component 3 (p < .01) and C-reactive protein (p < .01), stimulated the activation of NLRP3 inflammasome (p < .01) and caspase-1 (p < .05), induced the phosphorylation of TRAF2 (p < .01), NF-κB (p < .01) and STAT3 (p < .01). Preincubating cells with GRA-II inhibited the ability of glucagon to induce an inflammatory response. Using HepaRG cells, we confirmed the dose-dependent ability of glucagon to stimulate the expression of NLRP3, the phosphorylation of NF-κB and STAT3, in the absence of GRA-II. These results suggest that glucagon has proinflammatory effects that may participate in the pathogenesis of hyperglycaemia and unfavourable cardiometabolic risk profile.     

Growth hormone-releasing factor immunoreactivity in the hypothalamus and cortex of the rat: in vivo and in vitro studies

Utilizing a specific RIA for rat (r) GRF, hypothalamus and cerebral cortex from adult rat and long term dissociated fetal rat hypothalamic and cerebral cortical cell cultures were investigated for the presence of rGRF immunoreactivity (IR-GRF). After homogenization in an acidic medium, tissues and cultures were extracted on octadecylsilyl-silica columns, and IR-GRF and somatostatin (IR-SS) were measured by RIA. In extracts from the hypothalamus from the adult rat the content of IR-GRF was 3.02 +/- 0.16 ng ( +/- SE) per hypothalamus. IR-GRF was identical with synthetic rGRF on gel filtration chromatography and by parallel displacement of dilutions of extract in RIA. In extracts from cerebral cortex isolated from the adult rat, no IR-GRF was detected. In extracts from long term dissociated cell cultures from fetal hypothalami, 7.3 +/- 7 pg/10(6) cells of IR-GRF were present and were identical with synthetic rGRF by chromatographic and immunological criteria. In the extracts from cerebral cortical cell cultures cross-reacting material was present which on gel filtration chromatography revealed two peaks of immunoreactivity of higher mol wt than synthetic rGRF. There was nonparallelism on dilution of the extract. The ratio of IR-SS to IR-GRF by weight (IR-SS/IR-GRF) was calculated to compare the relative abundance of IR-GRF in cultured hypothalamic cells. In the hypothalamus isolated from the adult rat the ratio of IR-SS/IR-GRF by weight was 15.8 +/- 1.4 as compared to 48.9 +/- 10.3 in hypothalamic cultures. We conclude that IR-GRF indistinguishable from synthetic rGRF is present in long term dissociated hypothalamic cell cultures, but is relatively less abundant than in the hypothalamus of the adult rat when compared on the basis of IR-SS. No IR-GRF was detectable in cerebral cortex of the adult rat. At least one cross-reacting molecular species is detected in cerebral cortical cultures by the rGRF RIA, but exhibits nonparallelism and has a higher mol wt than synthetic rGRF. The increase of the ratio of IR-SS/IR-GRF in hypothalamic cell cultures in vitro compared to hypothalamus in vivo suggests that the culture conditions change differentially the expression of IR-SS and IR-GRF.     

CRF activity in fetal rat hypothalamus, in late pregnancy.

The corticosterone content of the adrenal glands was determined in 21-day-old rat fetuses: (1) before and after encephalectomy or decapitation (hypophysectomy); (2) after ACTH treatment of the encephalectomized or decapitated (hypophysectomized) fetuses; and (3) after administration of crude extracts (0.1 N HCl) of hypothalamic or cortical tissue from 20-day-old rat fetuses. A peak in the corticosterone content of the adrenals was observed 10 min after ACTH injection to enceaphlectomized or decapitated fetuses. A rise in corticosterone concentration was noted 5 and 10 min after the encephalectomized fetuses were given an injection of hypothalamic extract. This extract was devoid of appreciable ACTH activity when tested in decapitated fetuses. Cortical extract was inactive in encephalectomized or decapitated fetuses. These data suggest that fetal hypothalamic extract contains some CRF activity and that the fetal pituitary gland is responsive to the CRF.     

Glucagon secretion in rats with non-insulin-dependent diabetes: an in vivo and in vitro study

Non-insulin-dependent diabetes ( NIDD ) was obtained in adult rats following a neonatal streptozotocin injection. Rats with NIDD exhibited a chronic low-insulin response to glucose in vivo, slightly elevated basal plasma glucose values (less than 2 g/l) and low pancreatic insulin stores (50% of the controls). Glucagon secretion was studied in this model, in vivo and in vitro using the isolated perfused pancreas technique. Normal basal plasma glucagon levels were observed in the fed state and were in accordance with normal basal glucagon release in vitro. The pancreatic glucagon stores were normal in the diabetics. In experiments with the perfused pancreas, the increased glucose concentration suppressed glucagon release as readily in the diabetics as in the controls. Moreover 5.5 mM glucose suppressed glucagon release stimulated by 19 mM arginine to the same extent in both groups. These data indicate that the suppression of A cell function by glucose is normal in rats with NIDD . Theophylline and isoproterenol also produced normal glucagon release in diabetics. By contrast, the glucagon secretion in response to arginine was lower in the diabetics. This was observed either in vivo (arginine infusion) or in vitro in the presence or the absence of glucose in the perfusate. But in the presence of theophylline the response to arginine was normalized in the diabetics. Impairment of A cell function of the diabetics is not limited to recognition of amino-acids, since acetylcholine evoked a lower glucagon response in the diabetics than in the controls. These defects are different from those described in their B cells.     

Control of proglucagon-derived peptide synthesis and secretion in fetal rat hypothalamus.

The hypothalamus has the highest concentration of proglucagon-derived peptides (Pgdp's) in the brain, however, the control of the synthesis and secretion of these peptides is not understood. The goal of our studies was to examine in detail the regulation of synthesis and secretion of Pgdp's in the hypothalamus. Hypothalamic cultures were prepared from fetal rats on day 19-21 of gestation and Pgdp's in media and cells were determined by radioimmunoassay after treatment with test agents. Dibutyryl cyclic AMP or forskolin, activators of protein kinase A, markedly stimulated both Pgdp synthesis (by 5-fold) and secretion (by 10-fold) after 24 h of treatment (p < 0.05). The effects of protein kinase A stimulation on Pgdp's in the hypothalamus were greater than seen in our previous studies with the Pgdp-producing pancreatic A and intestinal L cells. Therefore there are tissue-specific differences with regard to the magnitude of the response of Pgdp's to protein kinase A stimulation. Consistent with an involvement of protein kinase A in hypothalamic Pgdp synthesis and secretion, somatostatin-14, an inhibitor of protein kinase A, was found to inhibit Pgdp synthesis and secretion in a dose-dependent fashion (p < 0.05). Phorbol myristate acetate (PMA), a stimulator of protein kinase C, did not significantly affect the synthesis or secretion of Pgdp's at 6 h, but significantly stimulated Pgdp secretion after 24 h (p < 0.05). The inactive phorbol ester, phorbol triacetate was without effect on Pgdp synthesis or secretion after 24 h of incubation (p > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠胚胎垂体生长激素细胞的体外诱导研究

目的探讨地塞米松和生长激素释放激素(GHRH)对大鼠胚胎垂体生长激素(GH)细胞分化的作用,为将来选择性细胞移植治疗生长激素缺乏症(GHD)奠定基础。方法利用免疫组织化学方法和放射免疫分析方法,在大鼠胚胎垂体组织的原代培养中,对糖皮质激素诱导GH细胞分化的最佳浓度、诱导GH细胞发生分化所需要的最短时间以及GHRH在大鼠胚胎垂体细胞分化中的作用进行实验研究。结果地塞米松诱导GH细胞分化的最佳浓度为50nmol/L(P<0.01)。体外培养的胚胎垂体细胞经地塞米松诱导16h,GH细胞开始分化,诱导48h,分化更明显,上清液中GH水平也明显增加(P<0.01);当GHRH的浓度达到10-7mol/L时,可以增强地塞米松对GH细胞的诱导分化作用,明显提高GH细胞的百分比和GH的分泌量(P<0.01)。结论地塞米松可以诱导生长激素前体细胞最终分化为具有功能活性的GH细胞,GHRH与地塞米松发挥协同作用,调节GH细胞的分化与GH分泌。     

Puberty-related increase in episodic LHRH release from rat hypothalamus in vitro

The retrochiasmatic hypothalamus (RCH) was removed from brains of male rats between 12 and 50 days of age, and immediately studied in vitro. The release of LHRH from the RCH was evaluated by periodic (7.5-min) collections of culture medium and subsequent RIA. With synthetic LHRH in the experimental system, the mean (+/- 1 SD) recovery was 94 +/- 7% with a variation coefficient of 14 +/- 3%. An increase in LHRH release was considered to be significant when it exceeded 6 pg/7.5 min. Biological viability of RCH in vitro was assessed by an increased release of LHRH in response to the depolarizing effect of veratridine. As age increased, from 12 to 50 days, the hypothalamic LHRH content steadily increased. However, a significant increase in veratridine - induced release of LHRH occurred only at 23 days and thereafter. At various ages, single hypothalami were studied during a mean 112-min period to evaluate the spontaneous release of LHRH. In all age groups, the in vitro LHRH release occurred in pulses. However, mean pulse frequency increased significantly with age: in 12- and 17-day-old rats, 0.3 pulse/112 min was observed; at 23, 25 and 27 days, this frequency varied between 1.8 and 3.0 pulses/112 min. At 50 days of age, the observed frequency was within the same range. We conclude that the RCH obtained from rats of various ages may retain in vitro its capacity to release LHRH episodically and that the frequency of these episodic pulses markedly increases with age to the time of the onset of puberty in male rats.     

Comparative study in vivo and in vitro of the differentiation of immunoreactive corticotropic cells in fetal rat anterior pituitary

In order to study the mechanisms of the differentiation of adenohypophysial corticotropic cells, an immuno-cytological study was performed in fetal rat anterior pituitary in vivo and in vitro with antisera against beta-(1-24) and alpha-(17-39) ACTH and beta-LPH, alpha- and beta-endorphins. In vivo, these cells appeared at 16 days of gestation without any difference in the timing of appearance of the two immunoreactivities. The same immunoreactivity was also detected in adenohypophysial primordia explanted from 12 to 15 days of gestation and maintained in organ culture until 21 days by using either medium containing fetal calf serum or medium containing insulin and transferrin instead of fetal calf serum. These immunoreactive cells were first detected in the different experimental primordia after a minimal period of culture, corresponding to a final equivalent of 16 days as in vivo. However, the mean cytoplasmic area of immunoreactive cells increased in relation to the day of explantation whatever the duration of culture. These data suggest: (1) the nature of culture medium used in this study has no influence on the differentiation of the corticotropic cells; (2) this cell type seems to be committed precociously (before day 12) by one or several substances of unknown origin; (3) the normal development seems to require the presence of factors (before day 14) whose nature and origin remain to be elucidated.     

In vivo studies of somatostatin-14 and somatostatin-28 biosynthesis in rat hypothalamus

The biosynthesis of somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28) was studied in rat hypothalamus after injection of 35S-labeled cysteine into the third ventricle. Cysteine specific activity was quantitated, and found to decline rapidly after injection of the labeled amino acid: less than 0.1% of the injected label remained as free cysteine in the hypothalamus 30 min post injection. Incorporation of label into SRIF-14 and SRIF-28 reached maximum values 8 h post injection, compared with a labeling maximum at 1-2 h for acid-precipitable protein. Within 2 h after [35S]cysteine injection, nearly half of the total labeled hypothalamic SRIF appeared in the medial basal hypothalamus; 24-h post injection this percentage reached approximately 75%. Colchicine administration dramatically reduced the appearance of labeled SRIF in medial basal hypothalamus, but had no apparent effect on the total incorporation of [35S]cysteine into SRIF-14, SRIF-28, or acid-precipitable protein, or on radioimmunoassayable SRIF levels. These results suggest that 1) the technique employed for administering [35S] cysteine delivers the label as a pulse; 2) the timing of the appearance of labeled amino acid in SRIF-14, SRIF-28, and acid-precipitable protein is consistent with initial synthesis of a larger prohormone, followed by conversion to peptide products; and 3) the newly synthesized peptides are rapidly transported to the medial basal hypothalamus by a colchicine sensitive mechanism.     

Immunoreactive prolactin in the rat hypothalamus: in vitro release and subcellular localization

Immunocytochemical studies have identified immunoreactive prolactin (IR-PRL) in the hypothalamus and other areas of the rat brain. However, neither the release of IR-PRL from the hypothalamus nor its subcellular localization have been demonstrated. In this study, the release of IR-PRL from hypothalami obtained from female rats was examined using hypothalamic units incubated in vitro in Krebs-Ringer bicarbonate-glucose buffer. Hypothalamic tissue spontaneously released IR-PRL, and this release was increased by depolarizing concentrations of potassium by a calcium-dependent mechanism. Hypothalamic IR-PRL was also released from hypothalamic tissue obtained from hypophysectomized rats (14 days). The subcellular localization of IR-PRL was investigated using equilibrium-density centrifugation. Tissue homogenates from intact or hypophysectomized rats were centrifuged at 150 g at 4 degrees C for 10 min, and the supernatants were layered onto continuous sucrose gradients (1.00-1.27 g/ml) and centrifuged at 100,000 g (max.) for 16 h. IR-PRL in pituitary supernatants showed a high equilibrium-density peak with a modal density of 1.23 g/ml. Fractionation of the supernatant from ventral or dorsal hypothalamic tissue resulted in two high-equilibrium density peaks, a primary peak with a modal density of 1.23 g/ml and a smaller peak with a modal density of 1.10 g/ml. Both high-density peaks were maintained in tissue obtained from hypophysectomized rats and were disrupted by homogenization in hypo-osmotic medium. Together, these data suggest that hypothalamic IR-PRL is stored in membrane-bound particles which have densities similar to those of secretory granules and is released by a calcium-dependent mechanism when the tissue is depolarized.     

Concomitant changes in the in vitro and in vivo release of opioid peptides and luteinizing hormone-releasing hormone from the hypothalamus following blockade of receptors for corticotropin-releasing factor

In vitro and in vivo perfusion techniques were used to examine the changes in the release of beta-endorphin, methionine-enkephalin (met-enkephalin), dynorphin and luteinizing hormone releasing hormone (LHRH) in response to the corticotropin releasing factor (CRF) receptor antagonist, alpha-helical CRF9-41. All four peptides were measured in the same sample collected at each time interval by specific radioimmunoassay methods. In vitro release experiments were conducted using slices of hypothalami obtained from male rats whereas the in vivo release of these peptides was assessed in push-pull perfusates of the arcuate-median eminence (ARC-ME) region of the medial basal hypothalamus of chloral hydrate-anaesthetized male rats. Treatment of rat hypothalamic slices in vitro with alpha-helical CRF9-41 (10(-6) M) resulted in a significant suppression of the release of beta-endorphin and met-enkephalin within 10 min of application of the antagonist and a coincident significant increase in the release of LHRH. The levels of dynorphin were reduced but these changes were not significant. Within 10 min of withdrawal of the receptor antagonist and perfusion with normal (antagonist-free) medium the levels of these peptides returned to pretreatment values, i.e. the levels of beta-endorphin, met-enkephalin and dynorphin rose while those of LHRH fell. Comparable results were obtained in vivo during push-pull perfusion of the ARC-ME region with alpha-helical CRF9-41.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attempts to demonstrate peptide localization and secretion in primary cell cultures of fetal rat hypothalamus

Primary cultures of dispersed hypothalamic cells were prepared from 18- to 19-day-old rat fetuses. Morphological studies revealed two types of cells having typical glial and neuronal appearances. Immunostaining of cells in culture was positive for neurophysin. The incubation medium contained radioimmunoassayable luteinizing hormone-releasing hormone, vasopressin, beta-endorphin and adrenocorticotropin hormone. The present data suggest that hypothalamic cells in primary culture secrete immunoreactive hormones or peptides; however, they do not seem to store significant amounts of these peptides.     

Somatostatin release from rat hypothalamus in vitro: effects of melatonin and serotonin

The release of immunoreactive somatostatin (SRIF) from explants of rat medial basal hypothalamus, which were maintained in culture for 24 h, was quantitated by a sensitive RIA. Validity of the specific SRIF assay has been previously established by chromatographic criteria using gel and high pressure systems and by the demonstration of immunological parallelism. After 24 h of culture in medium containing heat-inactivated fetal calf serum, hypothalamic fragments were incubated in serum-free medium, and the release of immunoreactive SRIF was quantitated. Melatonin at concentrations of 10(--8) and 10(--7) M stimulated SRIF release, and no significant increases were observed at concentrations of 10(--9) M or less or at concentrations of 10(--6) M or greater. Serotonin oxalate at concentrations of 10(--8)--10(--5) M significantly inhibited SRIF release. The serotonin antagonist cyproheptadine at a concentration of 10(--5)M had no effect on basal SRIF release but abolished the inhibitory effect of 10(--7) M serotonin. Finally, when hypothalami were incubated with melatonin and serotonin, each at 10(--7) M, SRIF release was unchanged compared to control values. The results suggest that the brain indoleamines, melatonin and serotonin, may modulate GH secretion by effects on SRIF release at a hypothalamic level.     

Glucose affects in vitro maturation of fetal rat islets

Fetal pancreatic islets (21.5 days old) were cultured in RPMI 1640 containing either 2.8 or 11.1 mM glucose for 7 days. After the 7-day culture period, islets cultured in 2.8 mM glucose demonstrated a minimal first phase of insulin secretion in response to acute glucose stimulation, whereas islets cultured in 11.1 mM glucose demonstrated a biphasic insulin secretory pattern. Islets cultured in 11.1 mM glucose initiated insulin secretion at 4.4 +/- 0.1 mM glucose and plateaued at 11.6 mM glucose when exposed to a linear gradient. In addition, culture in 11.1 mM glucose increased DNA content (P less than 0.01) and [3H]thymidine incorporation (P less than 0.05) in fetal islets. However, ultrastructural morphometric analysis indicated that the actual number of beta-cells within islets cultured in either 2.8 or 11.1 mM glucose did not increase. The insulin contents of islets cultured in 2.8 and 11.1 mM glucose were 0.46 +/- 0.06 and 1.14 +/- 0.10 mU/islet, respectively. During subsequent glucose stimulation, islets cultured in 2.8 and 11.1 mM glucose released 3% and 5.6% of their total insulin content, respectively. Ultrastructural morphometric analysis indicated that 11.1 mM glucose stimulated an increase in the volume of individual beta-cells, i.e. hypertrophy. The hypertrophy of beta-cells within islets cultured in 11.1 mM glucose resulted in a concomitant increase in islet volume. Finally, the hypertrophy of beta-cells within islets cultured in 11.1 mM glucose was a result of increased volumes of mitochondria, secretory granules, and, to the greatest extent, endoplasmic reticulum. These findings indicate that glucose is a potent factor in the maturation of cultured fetal rat islets.     

Luteinizing hormone-releasing hormone (LH-RH) neurons in cultures of fetal rat hypothalamus.

Hypothalamic fragments from 21-day-old fetal rats were cultured in Maximow double-coverslip assemblies for 1 to 2 months. Neurons containing LH-RH were demonstrated immunohistochemically using an antiserum to LH-RH (Dermody; 1:500--1:4,000). LH-RH was demonstrable only in neuronal perikarya (8--13 micrometer) and in small (less than 1 micrometer) round structures nearby, primarily in explants of the median eminence-arcuate nucleus region. Reactive neurons were not found in explants of the preoptic area and could not be demonstrated in fetal hypothalami at the time of explantation. The presence of mature-looking LH-RH containing neurons in these cultures suggests that this tissue culture system can be used for the study of hypothalamic development.