Showing papers in "Endocrinology in 1990"
TL;DR: The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations, and a possible role of GT isoforms in glucose sensing by beta cells is discussed.
Abstract: Two cell lines have been established from insulinomas obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice. These cell lines, designated MIN6 and MIN7, produce insulin and T antigen and have morphological characteristics of pancreatic beta cells. MIN6 cells exhibit glucose-inducible insulin secretion comparable with cultured normal mouse islet cells, whereas MIN7 cells do not. Both cell lines produce liver-type glucose transporter (GT) mRNA at high level. Brain-type GT mRNA is also present at considerable level in MIN7 cells, but is barely detectable in MIN6 cells, suggesting that exclusive expression of the liver-type GT is related to glucose-inducible insulin secretion. MIN6 cells do not express either major histocompatibility (MHC) class I or class II antigens on the cell surface. However, treatment with interferon-gamma induces high levels of MHC class I antigens, and a combination of interferon-gamma and tumor necrosis factor-alpha induces a MHC class II antigen on the cell surface. These results emphasize that the MIN6 cell line retains physiological characteristics of normal beta cells. The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations. We discuss a possible role of GT isoforms in glucose sensing by beta cells.
1,204 citations
TL;DR: The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies.
Abstract: Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the rat and in human prostate. AR was visualized using either of two polyclonal antibodies raised against peptides with sequences derived from rat and human AR. Tissue sections of 6-8 microns, frozen in isopentane and fixed in paraformaldehyde, were stained using immunoglobulin G fractions of immune, preimmune, and peptide-adsorbed immune sera in the avidin-biotin peroxidase procedure. AR was prominent in nuclei of acinar epithelial cells of epididymis, ventral prostate, seminal vesicle, and ductus deferens from the intact rat. Androgen withdrawal, 3 days after castration, resulted in the loss of receptor immunostaining, which was restored within 15 min of androgen administration. Stromal cell staining was absent or weak in the ventral prostate of intact rats, but was more evident in the epididymis. AR was confined to nuclei of cells within and bordering the interstitial compartment of the testis, including Sertoli cells, peritubular myoid cells, and interstitial cells, and was undetectable in germ cells. Submaxillary gland epithelial cells and a population of rat anterior pituitary cells showed strong nuclear staining of AR. In rat brain, AR was present in the medial preoptic, arcurate, and ventromedial nuclei of the hypothalamus, the medial nucleus of the amygdala, the CA-1 hippocampus, and the cortex. AR was prominent in acinar epithelial cells in human benign prostatic hyperplasia and was also present in stroma of fibromuscular benign hyperplasia. Heterogeneous staining was observed in stromal and epithelial cells of prostatic adenocarcinoma. The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies. The presence of AR in tissues correlated with their known androgen responsiveness.
486 citations
TL;DR: The treatment restores the bone marrow cavity virtually absent in the op/op animal and induces the appearance of resorbing osteoclasts and of resident bone marrow macrophages, proving that the deficiency of M-CSF is the cause of the op-op bone disorder and that this cytokine is directly or indirectly necessary for physiological osteoclastogenesis, the resulting bone resorption and for the establishment of bone marrow hemopoiesis.
Abstract: The op/op variant of murine osteopetrosis is a recessive mutation characterized by impaired bone resorption due to lack of osteoclasts. Cultured osteoblasts and fibroblasts from this mutant do not secrete M-CSF activity and resident macrophages are absent in bone marrow. This failure has been related to a mutation within the M-CSF coding region. We report now that the administration of recombinant human M-CSF (rhM-CSF) corrects in vivo the impaired bone resorption in this animal. The treatment restores the bone marrow cavity virtually absent in the op/op animal and induces the appearance of resorbing osteoclasts and of resident bone marrow macrophages. This proves that the deficiency of M-CSF is the cause of the op/op bone disorder and that this cytokine is directly or indirectly necessary for physiological osteoclastogenesis, the resulting bone resorption and for the establishment of bone marrow hemopoiesis.
462 citations
TL;DR: Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography and the distribution of insulin receptor binding was consistent with the distributionof insulin receptor mRNA.
Abstract: Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus.
434 citations
TL;DR: The results suggest that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone resides in its weaker interaction with the androgen receptor, most clearly demonstrable as an increase in the dissociation rate of testosterone from the receptor.
Abstract: Testosterone and dihydrotestosterone are believed to exert their androgenic effects by interacting with a single intracellular receptor protein in androgen target tissues. During fetal life, however, testosterone mediates the virilization of the Wolffian ducts into the epididymis, vas deferens, and seminal vesicles, whereas the urogenital sinus and external genitalia require the in situ conversion of testosterone to dihydrotestosterone to undergo male development. The reason why the signal provided by testosterone needs to be amplified in some androgen target tissues but not in others remains an enigma. To provide insight into the different actions of these androgens we studied their interaction with the human androgen receptor in fibroblasts cultured from the genital skin of a patient with 5α-reductase deficiency. Dihydrotestosterone was formed in negligible amounts in these cells, and in some experiments the residual 5α-reductase activity was further blocked with the 5α-reductase inhibitor finasteride. ...
408 citations
TL;DR: Evidence indicates that inhibition depends on fatty acid oxidation, is coupled to decreased glucose oxidation and operates both during normo- and hyperglycemia.
Abstract: Short- and long-term effects of hyperlipidemia with elevated FFA on insulin secretion were investigated Male Sprague-Dawley rats were fed ad libitum and additionally infused with Intralipid 10%, 10 ml/h After 3 h of Intralipid the response to 27 mM glucose in isolated perfused pancreas was enhanced by 86%, P < 002 After 6 h of Intralipid enhancement had subsided After 48 h of Intralipid glucose-induced insulin release was inhibited by 49%, from 1950 ± 177 μU/min after saline to 1003 ± 232 μU/min after Intralipid, P < 002 Inhibition was glucoseselective since responses to other secretagogues (1 mM 3-isobutyl- 1 methylxanthine, 10 mM octanoate, or 5 mM a-ketoisocaproic acid) were unaffected as were pancreatic contents of insulin (2284 ± 111 mU/pancreas after saline, 2566 ± 131 mU/pancreas after Intralipid) In isolated islets from 48 h lipid infused rats production of [14-C]CO2 from D[U-14-C]glucose was decreased (P < 002) in parallel with the insulin response to 27 mM glucose Glucoseinduced secre
396 citations
TL;DR: The pattern of follicular development during artificially lengthened estrous cycles was characterized, with heifers in group 3 having significantly more follicular waves per cycle than groups 1 and 2, and no differences in basal LH concentrations between groups 2 and 3.
Abstract: In cattle the development of ovarian follicles greater than or equal to 5 mm occurs in waves, with either two or three waves per estrous cycle. To increase our understanding of the control of follicular dynamics in cattle, the present study was designed to characterize the pattern of follicular development during artificially lengthened estrous cycles. Cycles were lengthened by intravaginal insertion of Silastic devices containing progesterone [Controlled Internal Drug Release devices (CIDRs)]. Control heifers (group 1) received blank devices, whereas treated heifers received one (group 2) or two CIDRs (group 3) from days 14 to 28 after estrus. In groups 2 and 3, the insertion of CIDRs prevented return to estrus at the normal time and increased cycle length as compared to the control group (30.0 +/- 0.0 and 31.0 +/- 0.3 vs. 21.0 +/- 0.7 days, respectively, P less than 0.05). After natural luteolysis and between days 22 and 28 of cycle, progesterone concentrations were maintained at lower levels in group 2 (range = 0.9-2.1 ng/ml) than in group 3 (range = 3.7-4.9 ng/ml, P less than 0.003). Follicular development and regression were monitored daily by ultrasonography. The number of follicular waves per cycle was identical in groups 1 and 2 (2.7 waves per cycle), despite the significantly longer cycles in group 2. In group 2, the presence of one CIDR altered the normal pattern of follicular development by promoting the prolonged growth of the ovulatory follicle, and associated with it, a complete absence of follicular recruitment. When compared to ovulatory follicles in controls (group 1), ovulatory follicles in group 2 were detected on the ovaries for a longer time (1.8-fold), reached a greater maximal size (1.4-fold), and were dominant for a longer time (3-fold). Heifers in group 3 had significantly more follicular waves per cycle than groups 1 and 2 (3.8 vs. 2.7 waves per cycle, respectively, P less than 0.05), due to the production of additional follicular waves during the lengthened cycle in three of six heifers. The other three heifers in group 3 showed patterns of follicular development similar to those of group 2. All heifers in the control group had normal preovulatory rises in estradiol and LH. During the period of treatment (days 14-28), 17 beta-estradiol concentrations were higher in heifers in group 2 (lower progesterone levels) than in heifers in group 3 (higher progesterone levels; P less than 0.0001). No differences were observed in basal LH concentrations between groups 2 and 3.(ABSTRACT TRUNCATED AT 400 WORDS)
394 citations
TL;DR: Results suggest that 1) a saturable, high affinity binding site for PACAP is present on anterior pituitary membranes; 2) PACAP27 and PACAP38, but not VIP, share this binding site in the anterior pituitsary and possibly the hypothalamus; and 3)PACAP27, PACAP 38, and VIP share a similar or identical binding site on lung membranes and possibly other peripheral tissues.
Abstract: A novel bioactive peptide was recently isolated from ovine hypothalamus and was named PACAP (pituitary adenylate cyclase-activating polypeptide). PACAP was present in two bioactive, amidated forms, PACAP27 and PACAP38 (27 and 38 amino acids, respectively), and showed a 68% sequence homology with vasoactive intestinal peptide (VIP) in the Nterminal 28 residues. PACAP38 was at least 1000 times more potent than VIP in stimulating adenylate cyclase in pituitary cells, but both peptides exhibited comparable vasodepressor activity. Thus, we sought to determine whether PACAP acts on specific binding sites in the anterior pituitary or other tissues and whether these binding sites are different from those of VIP. Binding of [125I] PACAP27 to freshly prepared rat anterior pituitary membranes in the presence and absence of 212 nM unlabeled PACAP27 was specific, saturable, and more rapid at 22 C than at 4 C. Scatchard analysis of this binding site using increasing doses of unlabeled PACAP27 revealed a single high aff...
326 citations
TL;DR: It is concluded that, regardless of season, a rise in estradiol to late follicular phase levels initiates a large and abrupt GnRH surge coincident with the onset of the LH surge.
Abstract: Previous studies suggest two roles for estradiol in inducing the LH surge in ewes: a neural action to evoke a sudden release of GnRH and a pituitary action to maximize response to GnRH. We tested two hypotheses: a follicular phase estradiol rise induces a GnRH surge; and the surge-inducing action of estradiol does not vary with season. In the breeding season, ewes in the midluteal phase of the estrous cycle were ovariectomized and treated with implants producing luteal phase levels of estradiol and progesterone, and an apparatus was surgically installed for later sampling of pituitary portal blood. At the normal time of luteolysis (1 week later), progesterone implants were removed, simulating luteal regression. Ewes were divided into two groups: estradiol implants also removed (n = 6) and estradiol implants added 16 h after progesterone removal to produce a rise in estradiol to levels that mimic those that circulate in the late follicular phase (n = 6). In anestrus, the estradiol rise treatment was replicated in ewes (n = 5) after an artificial luteal phase produced by sequential insertion and subsequent removal of progesterone implants. Regardless of season, the LH surge induced by estradiol was invariably accompanied by a massive GnRH surge, ranging from 73- to 394-fold over presurge values. The GnRH and LH surges began together, but the GnRH surge continued well beyond the surge of LH. There was no seasonal difference in time course or amplitude of the GnRH surge. Control ewes not treated with estradiol exhibited regular pulses of LH and GnRH every 1-2 h, but no surge of either hormone. We conclude that, regardless of season, a rise in estradiol to late follicular phase levels initiates a large and abrupt GnRH surge coincident with the onset of the LH surge. The LH surge ends despite continued elevation of GnRH.
324 citations
TL;DR: Data from this study are consistent with the hypothesis that NPY projections within the hypothalamus are involved in regulating feeding behavior and weight gain, and that disturbed regulation of hypothalamic NPY expression may play a role in the etiology of obesity in the genetically obese Zucker rat.
Abstract: Neuropeptide Y (NPY) is a potent orexigenic agent capable of producing hyperphagia and obesity. NPY-containing neurons project from the hypothalmic arcuate nucleus to the paraventricular nucleus, an area known to be sensitive to the orexigenic effects of NPY. In this study we investigated the possibility that preproNPY messenger RNA (mRNA) content may be altered in obese Zucker rats compared to that of their lean littermates. Total RNA was isolated from hypothalamic dissections from male and female, obese and lean Zucker rats. RNA was also isolated from dissections of: olfactory bulb, entorhinal cortex, hippocampus, and striatum of female obese and lean rats. PreproNPY mRNA content was determined by solution hybridization-RNase protection analysis. The results revealed a 2- to 3-fold increase in preproNPY mRNA levels in the hypothalamus of obese animals compared to lean. The increase was observed in both sexes and was specific to the hypothalamus. In situ hybridization localized this increase to the arcuate nucleus. An additional RNase protection study was pursued to investigate the effects of 72 h food deprivation on hypothalamic preproNPY mRNA levels in lean and obese animals. Lean animals displayed an approximate 2-fold increase in preproNPY mRNA content, whereas obese animals showed no significant increase after food deprivation. These data are consistent with the hypothesis that NPY projections within the hypothalamus are involved in regulating feeding behavior and weight gain, and that disturbed regulation of hypothalamic NPY expression may play a role in the etiology of obesity in the genetically obese Zucker rat.
324 citations
TL;DR: It is demonstrated that activin acts as a regulator of spermatogonial proliferation in the male through specific binding of activin A to 2C, but not 4C, germ cells was demonstrated.
Abstract: Activin and inhibin are peptide hormones produced in the gonads which may act as autocrine and/or paracrine regulators of testicular function. Sertoli cells produce inhibin, and it has recently been shown that Leydig cells can produce activin in vitro. To further explore the local actions of activin and inhibin in the testis, Sertoli and germ cells were isolated from immature rats and cocultured in vitro. In these cultures we demonstrate that activin A and activin B, but not inhibin A, stimulated spermatogonial proliferation in vitro. Activin increased [3H]thymidine incorporation 2- to 4-fold in cocultures after 48-72 h of treatment. Using autoradiography, the label was localized in the clusters of spermatogonia adhering to the Sertoli cell monolayer. Additionally, activin stimulated a reaggregation of the cultures into tubule-like structures. Fluorescence-activated cytometry was used to analyze the cell population based on size, DNA content, and lipid content. Sertoli cells were identified using Nile Red...
TL;DR: The identification of IL- 1 receptors in brain with characteristics similar to IL-1 receptors in immune and neuroendocrine tissues provides further support for a physiological role for IL-2 to regulate central nervous system activity.
Abstract: The cytokine interleukin-1 (IL-1) has a variety of effects in brain, including induction of fever, alteration of slow wave sleep, and alteration of neuroendocrine activity. To examine the potential sites of action of IL-1 in brain, we used iodine-125-labeled recombinant human interleukin-1 [( 125I]IL-1) to identify and characterize IL-1 receptors in crude membrane preparations of mouse (C57BL/6) hippocampus and to study the distribution of IL-1-binding sites in brain using autoradiography. In preliminary homogenate binding and autoradiographic studies, [125I]IL-1 alpha showed significantly higher specific binding than [125I]IL-1 beta. Thus, [125I]IL-1 alpha was used in all subsequent assays. The binding of [125I]IL-1 alpha was linear over a broad range of membrane protein concentrations, saturable, reversible, and of high affinity, with an equilibrium dissociation constant value of 114 +/- 35 pM and a maximum number of binding sites of 2.5 +/- 0.4 fmol/mg protein. In competition studies, recombinant human IL-1 alpha, recombinant human IL-1 beta, and a weak IL-1 beta analog. IL-1 beta +, inhibited [125I]IL-1 alpha binding to mouse hippocampus in parallel with their relative bioactivities in the T-cell comitogenesis assay, with inhibitory binding affinity constants of 55 +/- 18, 76 +/- 20, and 2940 +/- 742 pM, respectively; rat/human CRF and human tumor necrosis factor showed no effect on [125I]IL-1 alpha binding. Autoradiographic localization studies revealed very low densities of [125I]IL-1 alpha-binding sites throughout the brain, with highest densities present in the molecular and granular layers of the dentate gyrus of the hippocampus and in the choroid plexus. Quinolinic acid lesion studies demonstrated that the [125I]IL-1 alpha-binding sites in the hippocampus were localized to intrinsic neurons. After hypophysectomy, homogenate binding and autoradiographic studies showed that there was no apparent change in the relative density of IL-1 receptors in the hippocampus. The identification of IL-1 receptors in brain with characteristics similar to IL-1 receptors in immune and neuroendocrine tissues provides further support for a physiological role for IL-1 to regulate central nervous system activity.
TL;DR: The results demonstrate the expression of VEGF in the CL but not in mural granulosa cells, suggesting a temporal relation between V EGF expression and growth of capillary vessels, and suggest that VegF is involved in the process of CL angiogenesis.
Abstract: In the course of the development of the ovarian follicle and differentiation of granulosa cells into corpus luteum (CL), extensive changes in the microvasculature of these structures take place. This suggests the local release of angiogenic factors. In the present work we examined whether a newly described secreted vascular endothelial growth factor (VEGF) is expressed in normal rat ovary by in situ hybridization. Our results demonstrate the expression of VEGF in the CL but not in mural granulosa cells, suggesting a temporal relation between VEGF expression and growth of capillary vessels. The hybridization pattern in the CL was consistent with localization of VEGF message to luteal cells. Expression of VEGF was detected also in cumulus oophorus cells. These findings suggest that VEGF is involved in the process of CL angiogenesis.
TL;DR: Evidence suggests that inhibin and activin act in a paracrine manner to regulate follicular development, inhibin as a follicular growth signal and Activin as an atretagenic signal.
Abstract: The role of inhibin and activin in the initiation of follicular development, growth, and atresia was examined. Human recombinant inhibin (1 microgram) was unilaterally injected into the ovarian intrabursal space of 25-day-old rats. The contralateral ovary served as a control. Recruited growing follicles (350-500 microns) were observed 24 h after injection. The accumulation of follicles was greater in the inhibin-treated ovaries than in contralateral control ovaries. Moreover, the size distribution of the follicles was similar to the distribution of follicles recruited by systemic exogenous PMSG treatment. The effect of inhibin plus PMSG on follicular development was not different from that of PMSG treatment alone. Injection of human recombinant activin (1 microgram) into the ovarian bursa caused follicular atresia. Activin therapy blocked the follicular development caused by PMSG treatment. The effect of inhibin and activin on follicular development was further characterized by measuring the incorporation of [3H]thymidine into dividing cells. Inhibin enhanced follicular thymidine incorporation, while activin decreased granulosa cell proliferation. Furthermore, receptors for inhibin-A (6.4 x 10(3) receptors/cell) and activin-A (2.3 X 10(4) receptors/cell) were identified on granulosa cells. The evidence suggests that inhibin and activin act in a paracrine manner to regulate follicular development, inhibin as a follicular growth signal and activin as an atretagenic signal.
TL;DR: The view that PACAP may play a multifunctional role, including that of a hypophysiotropic hormone, neurotransmitter, neuromodulator, and vasoregulator, is supported.
Abstract: We recently reported isolation, characterization and synthesis of a novel ovine hypothalamic peptide with 38 residues which stimulates accumulation of cAMP in rat anterior pituitary cell cultures. The peptide was named PACAP38 (pituitary adenylate cyclase-activating polypeptide with 38 residues). The presence of another peptide corresponding to the N-terminal 1-27 residues (PACAP27) was also demonstrated. Both PACAP38 and PACAP27 have an amidated C-terminus. Antisera against synthetic PACAP27 were generated in rabbits. These antisera were tested for titer and specificity in enzyme-linked immunosorbent assay. One of the antisera (no. 88121-3) exhibited a high titer of antibody, which was specific to PACAP27 and PACAP38 with exception of slight cross-reactivity with ovine CRF (oCRF). Therefore, the antibodies against oCRF were removed from the antiserum using a solid phase method. Removal of oCRF antibodies was confirmed by enzyme-linked immunosorbent assay. A dense immunoreactive fiber network was found in both external and internal zones of the median eminence and pituitary stalk. The fibers were demonstrated to be in close contact with the hypophysial portal capillaries. The preabsorption of antiserum with vasoactive intestinal polypeptide or with the mixture containing TRH, LHRH, oCRF, ovine GH-releasing factor, somatostatin, and bovine thyroglobulin did not affect the immunostaining. On the other hand, the preabsorption of antiserum with an excess of PACAP27 or PACAP38 abolished the immunostaining. Therefore, the staining is considered specific for PACAP27 and PACAP38. Stained fibers were also present in the posterior pituitary. A dense fiber network was observed and the lateral hypothalamus the fibers appeared to cling to unstained neuronal cell bodies and their dendrites. In the lateral septum the fibers surrounded some blood vessels. Immunolabeled cell bodies were found in the paraventricular and supraoptic nuclei. These findings support the view that PACAP may play a multifunctional role, including that of a hypophysiotropic hormone, neurotransmitter, neuromodulator, and vasoregulator.
TL;DR: There is localized 11 beta-OHSD mRNA expression and enzyme bioactivity in rat brain, which corresponds to areas of reduced glucocorticoid or mineraloc Corticosterone receptor affinity for corticosterone, and may regulate the access of cortic testosterone to cerebral mineralocORTicoid and/or glucOCorticoids receptors and thus modulate corticosteroid effects on brain function.
Abstract: In peripheral aldosterone target sites (e.g., kidney), 11β-hydroxysteroid dehydrogenase (11β-OHSD) metabolizes corticosterone to inactive 11-dehydrocorticosterone and thus protects mineralocorticoid receptors from exposure to corticosterone in vivo. We have investigated whether 11β-OHSD could account for the site-specific differences in corticosteroid receptor sensitivity to corticosterone in rat brain. Enzyme activity, estimated as the percentage conversion of [3H]corticosterone to [3H] 11-dehydrocorticosterone in the presence of NADP+(200 μM), was: hippocampus, 55.8 ± 2.7%; cortex, 52 ± 3.1%; pituitary; 40 ± 2%, hypothalamus, 26.1 ± 1.2%; brain stem, 21.4 ± 1.7%; and spinal cord, 12.3 ± 1.8%. Northern blots, using [32P]dCTPlabeled probes from an llβ-OHSD cDNA clone derived from rat liver, showed expression of a single mRNA species in all brain areas, of identical size to 11β-OHSD mRNA in liver and kidney. Highest expression was found in hippocampus and cortex. In situ hybridization, using [35S]UTP-label...
TL;DR: In this paper, the authors used an autoradiographic method based on the incorporation of [3H] proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21-day-old fetal rat calvariae.
Abstract: Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGFβ) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H] proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21- day-old fetal rat calvariae. IGF I, PDGF, and TGFβ increased bone matrix apposition in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGFβ was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest whe...
TL;DR: The results suggest that TNF alpha represents one of the immune response mediators that directly or via stimulation of other cytokines act as activators of the HPA axis during immune/inflammatory reactions.
Abstract: We studied the effects of tumor necrosis factor-alpha (TNF alpha), a macrophage-derived pleiotropic cytokine produced during the inflammatory/immune response, on the function of the hypothalamic-pituitary-adrenal (HPA) axis of the rat. Intravenous injections of TNF alpha stimulated plasma ACTH and corticosterone secretion in a dose-dependent fashion. This effect was inhibited by a rat CRH antiserum that was administered to the rats 1 h before the TNF alpha injections. This suggested that CRH is a major mediator of the HPA axis response to TNF alpha. We subsequently evaluated the ability of TNF alpha to influence CRH and ACTH secretion in vitro by explanted rat hypothalami in organ culture and by dispersed rat anterior pituicytes in primary culture respectively. Hypothalami were incubated for 40 min with graded concentrations of TNF alpha (10 pM to 1 microM). This cytokine stimulated CRH secretion in a dose-dependent fashion, with an EC50 of 6.7 x 10 pM (P less than 0.05). Preincubation of hypothalamic explants with dexamethasone, indomethacin (1 microM), eicosatetraynoic acid (10 microM), or nordihydroguaiaretic acid (30 microM) resulted in inhibition of TNF alpha-stimulated CRH secretion (P less than 0.05). Interestingly, 4-h incubation with TNF alpha had no effect on ACTH secretion from rat anterior pituicytes at a concentration of 10 nM. Higher concentrations of TNF alpha (100 nM and 1 microM), however, elicited a dose-dependent increase in the ACTH concentration in the medium. Our results suggest that TNF alpha represents one of the immune response mediators that directly or via stimulation of other cytokines act as activators of the HPA axis during immune/inflammatory reactions. This effect appears to be glucocorticoid suppressible and eicosanoid mediated. The primary site of action of TNF alpha appears to by the hypothalamic CRH-secreting neuron. Some pituitary and adrenal effects of TNF alpha, however, cannot be excluded.
TL;DR: The results suggest that committed mononuclear osteoclast precursors are derived from CFU-GM, the committed granulocyte-macrophage progenitor, have a distinct polygonal morphology and cross-react with monoclonal antibodies that recognize mature osteoclasts and are capable of forming multinucleated cells which fulfill the functional criteria for osteoclast.
Abstract: Nonadherent marrow mononuclear cells enriched for hematopoietic progenitor cells were cultured in semisolid medium with recombinant human granulocyte-macrophage colony-stimulating factor for 9 days to form colony forming unitgranulocyte macrophage (CFU-GM) colonies. 1,25-Dihydroxyvitamin D was then gently layered over the cultures. After 2 weeks, approximately 30% of the colonies that formed were composed of cells with a unique polygonal morphology. One hundred percent of the polygonal cells in these colonies crossreacted with the monoclonal antibody 23c6, which preferentially recognizes osteoclasts. Homogenous populations of these polygonal cells formed multinucleated cells (MNC) in suspension culture, 100% of which cross-reacted with the 23c6 monoclonal antibody, and greater than 90% of the MNC contracted in response to calcitonin. Approximately 20% of these MNC formed resorption lacunae on calcified matrices. These results suggest that 1) early osteoclast precursors are derived from CFUGM, the committe...
TL;DR: It is demonstrated that blockade of NMDA receptors can prevent the development of enhanced LH secretion in female rats undergoing sexual maturation, and this results support the view that activation ofNMDA receptors significantly contributes to the physiological initiation of the pubertal process.
Abstract: The physiological role of N-methyl-D-aspartate (NMDA) receptors in controlling LH secretion and the initiation of puberty was investigated using two specific antagonists, MK-801 and DL-2-amino-5-phosphono valeric acid (AP-5). Single daily sc injections of MK-801 (0.1-0.2 mg/kg BW), a noncompetitive NMDA receptor antagonist, given to prepubertal rats significantly delayed but did not prevent the timing of puberty, as determined by the age at vaginal opening and first ovulation. Infusion of MK-801 (5 μg/h) via osmotic minipumps for 4 days inhibited the postovarieclomy rise of LH secretion in prepubertal rats. Both MK-801 (0.2 mg/kg BW, sc) and AP-5 (4 × 30 mg, iv), a competitive NMDA receptor antagonist, blocked the estradiol-induced LH surge in prepubertal ovariectomized rats. These results demonstrate that blockade of NMDA receptors can prevent the development of enhanced LH secretion in female rats undergoing sexual maturation. Moreover, they support the view that activation of NMDA receptors significant...
TL;DR: Comparing the effects of recombinant native human IGF-I on primary cultures of osteoblasts in the presence and absence of GH shows that the accumulation of BP-3 correlates with enhanced hIGF-I activity on osteoblastic cells, suggesting that BPs may act locally to augment the effects in bone.
Abstract: Insulin-like growth factors (IGFs) are bound in the circulation to specific binding proteins (BP)- The predominant BP is a GH-dependent glycosylated protein of 42-49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BP-3), whereas nonglycosylated GH-independent IGFBPs of 32 kDa and less are minor constituents. Primary cultures of rat osteoblastic cells constitutively produce IGFBP species of 32 kDa, while GH induces the accumulation of BP-3. To examine whether BP-3 could regulate the biological activity of IGF-I on osteoblasts, we compared the effects of recombinant native human IGF-I (hIGF-I) on primary cultures of osteoblasts in the presence and absence of GH. hIGF-I stimulated cell replication and c*i(I) collagen gene expression in a dose-dependent manner, and these effects were potentiated by the presence of GH, which increased the accumulation of BP-3. To further examine this correlation, we compared the effects of two IGF-I peptides on the osteoblastic cell line PyMS, which constitut...
TL;DR: TGF beta 1 may be a physiological intermediate in the programmed cell death of rat prostatic glandular cells activated after androgen ablation, as demonstrated by results in androgen-maintained ventral prostate organ cultures in vitro.
Abstract: The ability of transforming growth factor B1 (TGFβ1) to inhibit proliferation and activate death of rat ventral prostatic glandular cells was tested both in vivo and in vitro. In vivo administration of 50 ng TGFβ1/day directly to the regressed ventral prostate of previously castrated male rats had no effect on the proliferative regrowth of the prostatic glandular cells induced by exogeneous androgen replacement. In addition, androgen- stimulated ventral prostatic cell proliferation in vitro in organ culture was not affected by exposure to 0.1–20 ng/ml TGFβ1. In contrast in vivo administration of 50 ng TGFβ1/day directly to the ventral prostate of intact noncastrated male rats resulted in the death of about 25% of the prostatic glandular cells within 7 days of treatment. Such TGFjSi treatment did not lower serum testosterone, nor did it affect the size or DNA content of the seminal vesicles, demonstrating the local nature of the response. Likewise, in androgen-maintained ventral prostate organ cultures in ...
TL;DR: TNF alpha inhibits the secretion of pituitary hormones and particularly suppresses the response of the corticotroph cells, and this inhibitory effect may contribute to the increased mortality observed in cases of severe septic shock with high circulating TNF alpha levels.
Abstract: Tumor necrosis factor α (TNFα), a monokine produced by activated macrophages and monocytes, may be an essential mediator of the pathogenesis and of the hormonal response to endotoxic shock. It has been suggested that an elevated level of TNFα is a marker for morbidity and mortality during septic shock, and that treatment with antibodies against TNFα decreases mortality. Because monokines have been shown to interact at the hypothalamic-pituitary level, we have studied the effect of TNFα on basal and stimulated hormone release from normal rat anterior pituitary cells. After 3 days of incubation, primary cultures of rat anterior pituitary cells were stimulated with either 0.5 ng/ml CRF, 3 ng/ml AVP, 10 ng/ml angiotensin II (All), 10-6 M TRF, 10-8 M LHRH, or 10-8 M GHRH, alone or in the presence of 20 or 50 ng/ml human or murine recombinant TNFα. The culture media were analyzed for ACTH, GH, LH, and PRL content. Each experiment was performed in triplicate and was repeated 3 to 8 times. Timecourse experiments ...
TL;DR: The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase is reported, showing that the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.
Abstract: The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3β- hydroxysteroid dehydrogenase/δ5→4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the Nterminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3β-hydroxysteroid dehydrogenase/ Aδ5→4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17α-hydroxypregnenolone to 17α-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandro3- terone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5α-androstan- 3β,17β-diol to 5α-dihydrotestosterone and, upon addition of NADH, reduced 5α-dihydrotestosterone to 5α-androstan- 3β,17β-diol. Thus, the ...
TL;DR: In this article, the effect of androgens on the regulation of lipolysis in male rats was studied, and it was shown that androgens enhance the lipolytic capacity of these cells by increasing the apparent number of beta-adrenoceptors (T only) and the activity of adenylate cyclase.
Abstract: Adipose precursor cells from male rats were exposed in primary culture to testosterone (T) or dihydrotestosterone (DHT), and their effects on the regulation of lipolysis were studied. T, but not DHT, stimulated catecholamine-induced lipolysis in a dose-dependent manner, including physiological concentrations. The effect was equally pronounced with isoproterenol (a pure beta-adrenergic agonist) and norepinephrine (a mixed alpha 2- and beta-adrenergic agonist). The higher lipolytic capacity of catecholamines on T-treated cells was paralleled by a similar increase in the number of beta-adrenoceptors in the cells, without a change in the receptor affinity, suggesting that T induced new synthesis or externalization of beta-adrenoceptors. Both T and DHT stimulated forskolin-induced lipolysis, suggesting an androgen effect at the level of the catalytic subunit of adenylate cyclase. The pertussis toxin-stimulated lipolysis was not influenced by the presence of androgens in the culture medium, and no effect was seen on the antilipolytic effect of insulin. These effects did not disappear in the presence of an aromatase inhibitor, suggesting that the T effects were not mediated by conversion to estrogens. These cells showed specific saturable binding for androgens, with a Kd in the range of androgen concentrations shown to be active. In conclusion, androgens enhance the lipolytic capacity of these cells by increasing the apparent number of beta-adrenoceptors (T only) and the activity of adenylate cyclase (both T and DHT). These changes are not mediated by conversion to estrogens. These effects probably occur via binding to specific androgen receptors.
TL;DR: High levels of cortisol decrease skeletal IGF-I synthesis by reducing IGF- I transcript levels, and this effect probably contributes to the inhibitory influence of cortisol on bone formation.
Abstract: Supraphysiological levels of cortisol inhibit bone cell replication and matrix synthesis, but its mechanism of action is unknown and could be secondary to an inhibition of local growth factor synthesis. These inhibitory effects of cortisol are the converse of the observed anabolic influences of the endogenously produced insulin-like growth factor-I (IGF-I); therefore, cortisol was examined for its effect on the production of IGF-I in osteoblast (Ob)- and fibroblast/preosteoblast-enriched cell cultures prepared from fetal rat parietal bone. Synthesis of IGF-I was monitored by Northern blot analysis to determine steady state IGF-I mRNA levels and by an IGF-I-specific RIA to quantitate polypeptide levels in acidified and fractionated culture medium. Cortisol at 100 nM decreased IGF-I transcript levels by 60% or more in Ob cultures within 6 h of treatment, and the concentration of immunoreactive IGF-I by 50% after 24 h; these effects were observed in the absence of a change in cellular DNA content. In Ob cultures, PTH at 10 nM increased IGF-I transcripts at 6 h and polypeptide levels at 24 h by 2.5- and 4.1-fold, respectively, and cortisol opposed this effect. The inhibitory effect of cortisol was not specific for the Ob cell population, since at 100 nM it also decreased IGF-I transcript and immunoreactive IGF-I levels in fibroblast/preosteoblast cultures and opposed the stimulation of IGF-I synthesis after treatment with 100 ng/ml GH. In conclusion, high levels of cortisol decrease skeletal IGF-I synthesis by reducing IGF-I transcript levels, and this effect probably contributes to the inhibitory influence of cortisol on bone formation.
TL;DR: It is demonstrated that gonadal steroids, administered in a paradigm that predictably produces timed stimulation of LH release, induce c-fos in LHRH neurons and provides a potentially useful and powerful tool for studying L HRH activation at the cellular level.
Abstract: Immature female rats received implants containing 17β-estradiol on postnatal day 28 at 0900 h, followed 24 h later by either blank capsules or progesterone. Between 1500– 1600 h on the day of progesterone (or blank capsule) implantation, these rats, a group of unoperated or sham controls, and a group of estrogen-progesterone-treated immature male rats were killed and perfused, and their brains processed for immunocytochemistry of c-fos antigen and LHRH. LHRH neurons consistently expressed c-fos after estrogen-progesterone treatment in females but not males; in only one of four females examined was c-fos induced after estrogen treatment. No fos was associated with LHRH neurons in the control groups. The LHRH neurons that expressed c-fos were located in the preoptic area and anterior hypothalamus; more rostral LHRH cells did not appear stimulated. These data demonstrate that gonadal steroids, administered in a paradigm that predictably produces timed stimulation of LH release, induce c-fos in LHRH neurons. ...
TL;DR: The possible involvement of opiate-dependent pathways in mediating the inhibitory action of Il-1 beta on reproductive processes was tested by implanting naloxone pellets 16-24 h before lymphokine treatment, and it was observed that the icv injection of endotoxin, also interfered with ovulation.
Abstract: Alterations of immune activity are often accompanied by reproductive disorders. Because interleukins mediate the host's response to immune activation, we first examined the effect of the central injection of several lymphokines on LH secretion by gonadectomized rats. We then studied the ability of the most potent lymphokine in this system, interleukin-1β (11-1β), to interfere with the proestrous LH surge and ovulation in the intact female rat as well as the dependence of this effect on the activation of opiate receptors. Finally, we investigated the possibility that increased brain levels of Us, as induced by the central administration of a bacterial endotoxin, might also alter the normal ovulatory process. After intracerebroventricular (icv) injection, 11-1β, 11-6, and tumor necrosis factor all lowered plasma LH levels in castrated rats. On a molar basis, 11-1β was the most potent inhibitor of LH secretion. In gonadectomized animals, 2.5 and 10 ng 11-1β administered icv significantly (P ≤ 0.01) decreased...
TL;DR: Analysis of differences in organ growth among these mice suggests that GH and IGF-I also have growth promoting actions that are independent of one another; GH appears to be necessary for the attainment of normal liver size, while IGF-i can stimulate brain growth.
Abstract: A line of transgenic mice expressing insulin-like growth factor-I (IGF-I) under the control of the mouse metallothionien- 1 promoter was crossed to a line of dwarf transgenic mice lacking GH expressing cells that were genetically ablated by diphtheria toxin expression. Mice generated from this cross that carry both transgenes express IGF-I in the absence of GH. These mice grew larger than their GH-deficient transgenic littermates and exhibited weight and linear growth indistinguishable from that of their nontransgenic siblings. These results confirm the suspected role of IGF-I in mediating GH’s stimulation of somatic growth, including that of long bones, and illustrates the essential role of GH and IGF-I in the modulation of postnatal growth. Analysis of differences in organ growth among these mice, however, suggests that GH and IGF-I also have growth promoting actions that are independent of one another; GH appears to be necessary for the attainment of normal liver size, while IGF-I can stimulate brain g...