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Search for other papers by C. Y. Lee in
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ABSTRACT
Untreated serum exhibited two forms of insulin-like growth factor-I (IGF-I)-binding protein complexes during gel chromatography: one of M r 150 000 and the other of M r 40 000–45 000. The majority of the immunoreactive IGF-I was associated with the M r 150 000 complex. Following acid-ethanol extraction of serum, the binding activity at M r 150 000 disappeared and a reduced binding activity appeared in the albumin size range. Acid incubation of serum was slightly less effective than acid-ethanol extraction in reducing the binding activity. Acid-ethanol-extracted or acid-incubated serum were parallel to IGF-I standard in the dose–response displacement of iodinated IGF-I. Gel filtration of serum with 1 mol acetic acid/l almost completely separated IGF-I and the binding proteins. Binding-protein fractions from gel filtration interfered with the immunoreactivity of IGF-I with its antibodies, causing a non-parallel displacement curve in the radioimmunoassay (RIA). Serum IGF-I could be isolated as a single peak by high performance C18 reverse-phase liquid chromatography (HPLC). The concentrations of IGF-I measured in bovine sera by RIA were similar between acid gel filtration and HPLC; the concentrations by acid-ethanol extraction and acid incubation being about 30% smaller than those measured with former methods. The lower concentration of IGF-I measured in bovine serum with acid-ethanol extraction or acid incubation appears to be due to interference of IGF-binding proteins not removed by either treatment.
Journal of Endocrinology (1990) 127, 139–148
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Search for other papers by S H Shin in
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Abstract
The effects of somatostatin (SRIF) on prolactin (PRL) synthesis and release were examined in primary cultured pituitary cells derived from normal and estradiol (E2)-primed male rat pituitaries. The cells were continuously incubated in a pulse medium containing [3H]leucine with or without 10−6 mol/l SRIF for a period of 15, 30, 60, 180 or 360 min. Following incubation, the medium was recovered and the cells were fractionated into cytosolic and granular fractions. PRL was isolated by SDS-PAGE and newly synthesized PRL ([3H]PRL) was identified by coincident peaks of tritium activities and PRL contents. The specific activity (SA, c.p.m./ng), a ratio of [3H]PRL to total PRL, was determined for the granular, cytosolic and medium fractions.
In control and SRIF-treated groups of non-primed pituitary cells, SAs of all three fractions significantly increased during the 6-h incubation. Cytosolic and granular SAs showed similar profiles of increasing rate in comparison to control. Medium SAs showed a significantly higher value in the SRIF-treated group than in the control group only at 180 min. These observations indicate that, in the non-primed condition, PRL synthesis is not inhibited by SRIF. Medium SAs in the E2-primed group were significantly higher than SAs in the non-primed control cells during the initial 3 h of incubation, and cytosolic and granular SAs were significantly higher than those of the non-primed control during the 3- to 6-h incubation period. These observations demonstrate that E2 enhances PRL synthesis and secretion of newly synthesized PRL. SRIF treatment of E2-primed lactotrophs resulted in a significant decrease in SAs of all three fractions as compared with those of the E2-primed control. Our results indicate that in normal male rat pituitary cells SRIF does not inhibit PRL synthesis but effectively inhibits PRL synthesis in E-primed lactotrophs. This suggests that the inhibitory action of SRIF on PRL synthesis is estrogen dependent.
Journal of Endocrinology (1996) 148, 69–76
Search for other papers by AUDREY E. LEE in
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Search for other papers by P. C. WILLIAMS in
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SUMMARY
Spayed mice drinking dilute aqueous solutions of oestrone (40 μg./1.) maintain a state of vaginal cornification for several weeks.
A single injection of progesterone inhibits the vaginal response and the degree of inhibition can be measured. The ED 50 of progesterone by this method is about 75 μg.
A satisfactory 3 + 3-point assay of megestrol acetate, using progesterone as a standard, is reported.
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Search for other papers by C Lee in
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Keratinocyte growth factor (KGF/FGF-7) is a stromally derived factor which exerts proliferative and differentiating effects on a variety of epithelial cells. Results of recent studies utilizing in vitro methods such as tissue culture and organ culture have suggested that KGF may act as a paracrine mediator of androgen-induced growth and development of the prostate and seminal vesicle. We undertook the present study to determine the distribution of KGF in relation to the functional regions of the rat prostatic ductal system, and whether KGF expression is influenced by androgen in vivo. Immunohistochemical staining revealed KGF to be present in the stroma throughout the prostate, regardless of the functional region, and staining for KGF remained high through 21 days post-castration. Message for KGF could also be detected by reverse transcriptase-PCR analysis of prostate stromal cells isolated from 4- and 21-day castrated animals, and no gross change in message level was observed following castration. Furthermore, no significant change in either stromal staining or message for KGF was observed in newborn rat prostates 10 days after castration, suggesting a similar regulatory mechanism for KGF in the adult and immature prostate. Epithelial staining for KGF decreased following castration, and greatly increased upon androgen replacement, possibly indicating a change in KGF internalization. These observations suggest that the presence of KGF protein is not related to functional differences in the prostate epithelium, and that expression of KGF in vivo is not greatly influenced by androgen.
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Search for other papers by CH Lee in
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Search for other papers by C Chinpaisal in
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Search for other papers by LN Wei in
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The orphan nuclear receptor TR2 and its truncated isoform deleted in the ligand binding domain (LBD) were localized exclusively in the nuclei as revealed by two methods of detection. An anti-hemagglutinin (HA) antibody detected specific nuclear localization of HA-tagged receptors and the green fluorescent protein (GFP)-tagged receptors were found to be distributed in the nuclei of living cells. By deletion analyses, the sequence responsible for targeting this receptor into the nucleus was defined. A stretch of 20 amino acid residues (KDCVINKHHRNRCQYCRLQR) within the second zinc-finger of this receptor is required for its nuclear localization and this signal is constitutively active. No nuclear localization signal was found in the N-terminus or the LBD. The GFP-tagged receptor remained biologically active, as evidenced by its repressive activity on the reporter that carried a binding site for this receptor, a direct repeat-5 (DR5). An electrophoretic mobility shift assay was performed to characterize the binding property of TR2 and its truncated isoform. TR2 bound to the DR5 as dimers whereas its truncated isoform bound as monomers.
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Search for other papers by C. C. LEE in
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Search for other papers by S. S. YOU in
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Search for other papers by C. Y. Lee in
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Search for other papers by F. W. Bazer in
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Search for other papers by F. A. Simmen in
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ABSTRACT
To gain insight into the involvement and interactions of the insulin-like growth factors (IGFs) and oestrogen in mammary growth and differentiation, the temporal expression of mammary mRNAs encoding components of the IGF system in pregnant and pseudopregnant pigs was examined. Pseudopregnant pigs received 5 mg oestradiol valerate or vehicle daily from day 45 after oestrus and underwent mammary biopsy on days 60, 90 or 112. In mammary tissue of pregnant pigs, steady-state levels of the mRNAs encoding IGF-I, IGF-II and type-I IGF receptor as well as the levels of the membrane-associated type-II IGF receptor were higher during the early phase of mammogenesis (≤day 45) than during the subsequent stages of mammary development. Mammary IGF-I, IGF-II and type-I receptor mRNAs were expressed at their lowest levels around day 90 of pregnancy (20–40% of those for day 30 of pregnancy) coincident with the onset of β-casein mRNA accumulation. Mammary IGF-binding protein-2 (IGFBP-2) mRNA levels increased twofold during the latter half of pregnancy, whereas the amount of IGFBP-3 mRNA declined after day 30 to undetectable levels by midpregnancy. Pseudopregnant pigs had reduced levels of these mRNAs (except for IGF-II) relative to their pregnant counterparts and this was associated with premature differentiation of mammary tissue as reflected by an earlier onset of β-casein mRNA accumulation in the former. The administration of oestradiol valerate decreased the levels of IGF-I and type-I IGF receptor mRNAs by day 60 of pseudopregnancy, but the reverse was evident by day 112. Oestradiol administration increased β-casein mRNA levels in pseudopregnant pigs, but had no effect on mammary IGFBP-2 and IGFBP-3 mRNA levels. Mammary IGF content was greater in late pregnancy (≥day 90) and pseudopregnancy than at early pregnancy. Serum IGF-I and IGF-II levels declined steadily during pregnancy and this was similar to, but not correlated with, mammary IGF mRNA levels, whereas in pseudopregnant pigs, serum IGF concentrations did not change temporally or in response to oestradiol. Serum IGFBP-2 levels were unaltered during pregnancy or pseudopregnancy, but serum IGFBP-3 levels declined after day 60 of pregnancy. In pseudopregnant pigs, serum IGFBP-3 levels did not change temporally, but declined after oestradiol treatment. Results indicate that mammary IGF-I and type-I IGF receptor systems are down-regulated during pregnancy-associated differentiation of this tissue and in response to oestrogen. Locally produced (autocrine and paracrine) IGFs are likely to mediate mammogenesis, whereas oestrogen stimulates mammary differentiation and lactogenesis in the pig. However, the high mammary IGF content and the reciprocal expression of mammary IGFBP-2 and IGFBP-3 mRNAs during late pregnancy suggests the involvement of IGFs in lactogenesis as well.
Journal of Endocrinology (1993) 137, 473–483
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Search for other papers by R L Heisler in
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Search for other papers by C S-S Lee in
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Abstract
The neurohypophysial hormones, oxytocin and vasopressin, are present as non-covalently bound complexes with their designated neurophysin in the secretory granules of the posterior pituitary. The neurophysins are generally considered to be biologically inert carrier proteins for oxytocin and vasopressin. We have examined the actions of bovine neurophysin-I (bNP-I), bovine neurophysin-II (bNP-II), rat neurophysin (rat NP) and oxytocin on prolactin release using primary cultured rat pituitary cells. A dynamic perifusion system was chosen to test their stimulatory actions. The rat NP and bNP-II stimulated prolactin release. It is a new observation that rat NP and bNP-II stimulate prolactin release from primary cultured rat pituitary cells. The maximum sensitivities, the lowest concentration which stimulate prolactin release, of rat NP, bNP-II, bNP-I and oxytocin in primary cultured cells were 1 nmol/l, 1 nmol/l, 1000 nmol/l and 1 nmol/l respectively. The maximum sensitivities of rat NP and bNP-II were within the physiologically relevant concentrations.
Journal of Endocrinology (1995) 144, 225–231
Search for other papers by T. C. LEE in
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Search for other papers by B. van der WAL in
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Search for other papers by D. de WIED in
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SUMMARY
Studies of the rate of aldosterone production in vitro of adrenals of rats hypophysectomized before dietary sodium restriction showed that hypophysectomy not only prevented the increases in aldosterone production observed in intact, Na-deprived rats, but also depressed the level of aldosterone production to below that of intact rats maintained on a normal diet. Rats hypophysectomized for a similar period of time but maintained on the normal diet showed a similar decrease.
Experiments on adeno- and neuro-hypophysectomized rats indicated that the pituitary factor required for the normal mineralocorticoid response to dietary sodium restriction resides in the anterior pituitary.
Treatment of hypophysectomized rats during dietary sodium restriction with doses of a long-acting corticotrophin (ACTH) prevented adrenal atrophy and maintained a normal glucocorticoid response to intravenous injections of ACTH, but failed to increase aldosterone production rates in vitro to levels above that of intact rats on a normal diet; it also failed to restore the enhanced adrenocortical sensitivity to the stimulating effect of aldosterone production of intravenously injected ACTH which is characteristic of acutely hypophysectomized, Na-deficient rats. Treatment with anterior pituitary powder (8–12 mg./day) for similar periods, however, restored the aldosterone production of adrenals in vitro of hypophysectomized, Na-deprived rats to levels nearly indistinguishable from those of acutely hypophysectomized, Na-deprived controls. The same doses of anterior pituitary powder were shown not to have any demonstrable effect on the aldosterone production of adrenals in vitro of intact rats on a normal diet.
These results are interpreted as indicating the existence of a pituitary factor other than ACTH which stimulates aldosterone secretion. This factor does not appear to act directly on the adrenal cortex or to stimulate the secretion of specific glomerulotropic substances, but probably exerts its effect by maintaining the normal functional capacity of some as yet undefined tissues which secrete glomerulotropic substances in response to dietary sodium restriction.
Search for other papers by Simon C Lee in
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Search for other papers by Christine A Robson-Doucette in
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Currently, the physiological function of uncoupling protein-2 (UCP2) in pancreatic islets and its role in the development of diabetes is a matter of great debate. To further investigate the impact of UCP2 on diabetes development, we used streptozotocin (STZ) to experimentally generate diabetes in both wild-type (WT) and UCP2-knockout (UCP2KO) mice. While multiple low-dose STZ injections led to hyperglycemia development over a 14-day period in both WT and UCP2KO mice, we found the development of hyperglycemia to be significantly less severe in the UCP2KO mice. Measurement of insulin and glucagon secretion (in vitro), as well as their plasma concentrations (in vivo), indicated that UCP2-deficiency showed enhanced insulin secretion but impaired α-cell function. Glucagon secretion was attenuated, despite reduced insulin secretion after exposure to STZ, which together contributed to less severe hyperglycemia development in UCP2KO mice. Further experimentation revealed that UCP2-deficient α- and β-cells had chronically higher cellular reactive oxygen species (ROS) levels than the WT prior to STZ application, which correlated with increased basal β- and α-cell mass. Overall, we suggest that increased chronic ROS signaling as a result of UCP2-deficiency contributes to enhanced β-cell function and impairment of α-cell function, leading to an attenuation of STZ-induced hyperglycemia development.
