Endocrinology

Published by Endocrine Society
Online ISSN: 1945-7170
Publications
Plasma concentrations of ACTH (picograms per ml) and cortisol (nanomoles per liter) in six intact llama fetuses and their mothers (white bars) and four denervated llama fetuses and their mothers (black bars). Plasma samples were taken after 45 min of normoxia, after 15 and 45 min of hypoxemia (bar), and after 45 min of recovery. Significant differences (P 0.05) are: a, normoxia vs. hypoxemia/recovery; b, intact vs. denervated; and c, mother vs. fetus.
Cardiac output, adrenal blood flow, adrenal vascular resistance, and ACTH delivery to the adrenals in intact (white bars) and denervated (black bars) llama fetuses during normoxia and after 15 (early) and 45 (late) min of hypoxemia (bars). Values are the mean SEM for each of the experimental periods. *, P 0.05, normoxia vs. hypoxemia.
Maternal blood gases, pH, and hemoglobin in normoxia and hypoxemia in the llama
Fetal blood gases, pH, hemoglobin, blood pressure, and heart rate during the experiment in the llama
Article
This study tested the hypothesis that the fetal llama, a species adapted to the chronic hypoxia of life at high altitude, demonstrates a potent carotid chemoreflex influence on adrenocortical responses during acute hypoxemia. Plasma ACTH and cortisol concentrations, and mesencephalic and adrenal blood flows were measured during a 1-h period of acute hypoxemia in six intact and four carotid sinus-denervated llama fetuses at 0.6-0.7 of gestation. Fetal PaO2 was reduced from approximately 23 to about 14 mm Hg in both intact and carotid-denervated groups during acute hypoxemia. During hypoxemia, fetal plasma ACTH, adrenal blood flow, and, therefore, delivery of ACTH to the adrenals increased to similar extents in both intact and carotid-denervated fetal llamas. Despite this, the increase in plasma cortisol in hypoxemia in intact fetuses was absent in carotid-denervated fetuses. In addition, the increase in delivery of cortisol to the mesencephalon calculated in intact fetuses during hypoxemia did not occur in the carotid-denervated group. These data suggest that the integrity of the carotid chemoreceptors is indispensable to cortisol release during acute hypoxemia in the llama fetus, even at 0.6-0.7 of gestation.
 
Details of animals studied 
Maternal plasma estradiol and progesterone profiles from the serial samples taken during the experimental protocol. Values represent the mean SEM of the hourly plasma samples taken from the pregnant baboons. Group I baboons (A 4 A, n 4) received 10% intralipid iv during Day 1, a low dose of androstenedione iv (0.8 mgkg 1 h 1 ) during Day 2, and a high dose of androstenedione iv (1.6 mgkg 1 h 1 ) during Day 3. Group II baboons (Controls, n 4) received 10% intralipid iv during Days 1, 2, and 3 of the experimental protocol. 
Bars represent the mean SEM of four pregnant baboons treated with androstenedione (Group I, black bars) and four pregnant baboons that were infused with 10% intralipid vehicle (Group II, white bars). Baseline plasma concentrations of progesterone (P 4 , A) and estradiol (E 2 , B) represent the mean of all samples taken before the onset of darkness in the animal's environment during each of the days of the experimental protocol. a P 0.05 represents differences by post hoc analysis, indicating a significant main effect of time; b P 0.05 represents differences by post hoc analysis indicating a significant main effect of treatment (two-way repeated measures ANOVA with Student's-Newman Keuls or Dunnett's tests). 
Hourly change from mean baseline (3 to 0 h) in the estrogen:progesterone ratio (E:P) and in the number of myometrial contractions (mean SEM) during low-and high-dose androstendione treatment (F) or during intralipid infusion (). a P 0.05 represents differences by post hoc analysis indicating a significant main effect of time; b P 0.05 represents differences by post hoc analysis indicating a significant main effect of treatment (two-way repeated measures ANOVA with Student's t test for unpaired data). 
Article
Androstenedione treatment of pregnant monkeys at 0.8 of gestation reproduces endocrine, biophysical, and biochemical changes similar to those measured during spontaneous, term labor in the pregnant monkey. In the pregnant baboon, the spontaneous onset of labor at term has been attributed to a forward shift in the nocturnal estradiol surge relative to that of progesterone in maternal plasma. This study investigated whether androstenedione treatment of the pregnant baboon at 0.7-0.8 of gestation promotes a premature forward shift in the nocturnal surge of maternal plasma estradiol relative to progesterone and whether this shift is associated with premature increases in nocturnal myometrial activity. Eight pregnant baboons were prepared surgically under general anesthesia with vascular catheters and myometrial electromyogram electrodes between 121 and 139 days of gestation (term is ca. 185 days). Catheters were maintained patent by continuous infusion of heparinized saline from the time of surgery until one of two treatments began following at least 9 days of postoperative recovery. In four baboons (Group I), the saline administration was replaced by a continuous infusion of 10% intralipid vehicle during Day 1 of the experimental protocol. During Day 2 and Day 3, the intralipid infusion was switched for a continuous infusion of androstenedione dissolved in intralipid set at a low (0.8 mg x kg(-1) x h(-1)) and at a high (1.6 mg x kg(-1) x h(-1)) dose, each delivered for 24 h. The other four pregnant baboons (Group II) received 10% intralipid vehicle for Days 1, 2, and 3 of the experimental protocol. One baboon from Group I received an additional dose of 0.4 mg x kg(-1) x h(-1) for 24 h before the low and the high dose of androstenedione. In each baboon, during each experimental day, maternal arterial blood samples (1 ml) were taken at 1 h intervals for 12 h, starting 3 h before the onset of darkness in the animal's environment, for measurement of maternal plasma estradiol and progesterone concentrations via RIA. Myometrial contractions were counted during each night-time period of the experimental protocol. All pregnant baboons demonstrated increases in maternal plasma estradiol and progesterone concentrations at night-time. Androstenedione had a dose-dependent effect in elevating day-time maternal plasma estradiol concentrations and in promoting a forward shift in the nocturnal surge of maternal plasma estradiol without affecting the nocturnal progesterone profile in maternal plasma. Maternal treatment with androstenedione also led to an increase in nocturnal myometrial contraction activity. We conclude that androstenedione treatment of the pregnant baboon at 0.7-0.8 of gestation promotes a premature forward shift in the nocturnal estradiol surge relative to that of progesterone in maternal plasma and that this shift is associated with an increase in nocturnal myometrial contraction activity, in a similar way to that measured during spontaneous onset of labor at term in this species.
 
Article
To ascertain if reductions in fetal plasma cortisol cause increases in fetal plasma ACTH, we treated pregnant ewes or their fetuses with aminoglutethimide (10 mg/kg BW) and metyrapone (20 mg/kg BW) and measured the hormonal responses with RIAs. When given to fetuses (n = 9) at 0.90 +/- 0.01 gestation (term-145 days), the steroid synthesis inhibitors reduced fetal plasma cortisol from 35.1 +/- 11.9 to 18.5 +/- 6.2 ng/ml (P less than 0.01) and plasma ACTH increased from 37 +/- 7 to 189 +/- 74 pg/ml (P less than 0.02). Thus, late in gestation cortisol from the fetal adrenal suppresses basal fetal ACTH secretion. Blockade of steroid biosynthesis in pregnant ewes carrying intact fetuses at 0.76 +/- 0.02 gestation (n = 11) or adrenalectomized fetuses at 0.81 +/- 0.01 gestation (n = 6) also reduced cortisol and increased ACTH in fetal plasma. In intact fetuses cortisol declined from 9.4 +/- 2.0 to 3.6 +/- 0.9 ng/ml (P less than 0.05), and ACTH increased from 46 +/- 8 to 183 +/- 67 (P less than 0.01); cortisol declined in adrenalectomized fetuses from 2.1 +/- 0.4 to 1.1 +/- 0.3 ng/ml (P less than 0.01), and ACTH increased from 106 +/- 13 to 400 +/- 104 pg/ml (P less than 0.01). Cortisol infusions into intact and adrenalectomized fetuses prevented both the decline in steroid concentration caused by the biosynthesis inhibitors given to the ewe and the increase in fetal plasma ACTH concentration. These data indicate that reductions in plasma cortisol in adrenalectomized fetuses or intact fetuses at a time in development when the fetal adrenal produces little cortisol cause compensatory increases in fetal plasma ACTH concentration. The simplest explanation for these observations is that from approximately 0.70 gestation, basal fetal ACTH secretion is tonically inhibited by cortisol circulating in fetal plasma. This cortisol can originate from sources other than the fetal adrenal.
 
Article
PTH administration in vivo increases osteoblast number and activity, resulting in increased bone formation, and also increases osteoclast-mediated bone resorption. Studies in vitro, however, have shown that the actions of PTH on osteoblast-like cells are inhibitory and catabolic, as shown by decreases in growth rate and collagen synthesis and increases in collagenase production. The present studies were designed to investigate possible mechanisms for these observations by examining the effects of PTH on the response of osteoblast-like cells to the osteoblast growth factor, epidermal growth factor (EGF). Confluent cultures of UMR 106-01 cells were treated with rat PTH-(1-34) for periods up to 72 h, and EGF receptors were measured with [125I]EGF. PTH, in a dose- and time-dependent manner, increased the number of EGF receptors 2-fold. The half-maximal effect of PTH occurred at a concentration of 1 nM, the same PTH concentration that resulted in half-maximal increases in cAMP generation. The increase in EGF binding was associated with an enhanced biological effect, as shown by augmentation of EGF-stimulated diglyceride production. The effect of PTH could be reproduced by the addition of 8-bromo-cAMP, but not by the phorbol ester phorbol myristate acetate. In the presence of cyclohexamide, the effect of PTH on EGF binding was abolished, suggesting that new protein synthesis was required to increase the number of EGF receptors. Northern blots of total RNA, using a cDNA probe encoding the extracellular domain of the rat EGF receptor, revealed that PTH treatment resulted in a 2- to 3-fold increase in the level of EGF receptor mRNA. These data suggest that the proliferative effects of PTH on the osteoblast may be mediated indirectly by a PTH-induced increase in the number of EGF receptors.
 
Article
The rat osteogenic sarcoma subclone UMR-106-01 is a cell type with osteoblast-like properties. This cell line has been shown to process specific receptors for insulin and insulin-like growth factor I (IGF-I), but not IGF-II. Insulin at physiological concentrations (1-5 ng/ml) in serum-free medium can maintain cell growth, as assessed by protein accumulation, thymidine uptake, and an increase in cell number. IGF-I is less potent than insulin, but, based on relative binding affinities for the insulin receptor, possibly acts via its own receptor. Insulin also enhances PTH-stimulated cAMP accumulation in these cells both by increasing cell number and an effect independent of cell number. Insulin may have a role in bone homeostasis.
 
Article
Insulin modifies the effects of PTH on osteoblast-like cells. However, the basis for this effect is unknown. In bone and kidney cells, the effects of PTH on cellular function are mediated by second messengers generated through both the phospholipase C and adenylate cyclase systems. Therefore, we examined the effects of insulin on PTH second messenger generation in UMR-106-01 rat osteoblastic osteosarcoma cells. PTH produced a rapid, transient increase in intracellular free calcium concentration ([Ca2+]i) which was maximal at 30 sec and was only minimally reduced in the absence of extracellular calcium. Inositol-triphosphate (IP3) production was increased in parallel. PTH stimulation of [Ca2+]i was concentration-dependent from 0.5-1,000 nM, with half-maximal stimulation at approximately 50 nM PTH. A 30-sec exposure to 50 nM PTH produced 32% and 23% increases in IP1 and IP3 production, respectively (both P less than 0.05). Although insulin alone did not significantly alter basal [Ca2+]i, a 1-min exposure to 1-100 nM insulin produced a concentration-dependent suppression of the PTH-stimulated transient increase in [Ca2+]i and IP3 generation. 100 nM insulin decreased 50 nM PTH stimulation of [Ca2+]i and IP3 levels by 84% (P less than 0.02) and 80% (P less than 0.001), respectively. Preexposure to insulin also decreased PTH stimulation of intracellular cAMP levels, but to a lesser degree. A 1-min exposure to 100 nM insulin produced a 32% (P less than 0.01) decrease in PTH-stimulated cAMP generation, but lower insulin concentrations were without significant effects. These results demonstrate that in UMR-106-01 cells, insulin suppresses PTH stimulation of second messengers generated through both the phospholipase C and adenylate cyclase systems, but has a more marked effect on the former.
 
Article
Prostaglandin E2 (PGE2), PTH, and epidermal growth factor (EGF) are potent regulators of osteoblast proliferation. In UMR 106-01 rat osteosarcoma cells with osteoblast-like features, PGE2 and PTH inhibit, while EGF stimulates, mitogenesis. Both PGE2 and PTH increase intracellular cAMP levels, cytosolic calcium, and inositol phosphate turnover. In a variety of cell types, EGF mediates its effects in part via activation of receptor protein-tyrosine kinase and other protein kinases, such as protein kinase-C. The nuclear mechanisms of PGE2, PTH, and EGF regulation of osteoblast proliferation are unknown. Accordingly, we have examined the effects of these agents on mitogenesis, second messenger generation, and primary response genes, which may link second messenger activation to subsequent alterations in gene expression. Northern blot analysis of mRNA from UMR 106-01 cells treated for 3 h with 2 microM PGE2, 10 nM PTH, or 10 ng/ml EGF in the presence of cycloheximide demonstrated that all three agents induced the expression of c-fos and c-jun mRNA. In contrast, only EGF stimulated cellular proliferation and induced Egr-1 mRNA. Also, unlike PGE2 and PTH, EGF did not increase intracellular cAMP levels. c-fos mRNA was induced by treatment with 50 ng/ml tetradecanoyl phorbol acetate or by 40 ng/ml forskolin, while induction of Egr-1 mRNA was stimulated by treatment with tetradecanoyl phorbol acetate, but not forskolin. Thus, EGF signal transduction differs from that of PGE2 and PTH in UMR 106-01 osteoblast-like cells, in that EGF does not stimulate the protein kinase-A second messenger system, but causes activation of Egr-1, a primary response gene that may play a role in the mitogenic effect of EGF.
 
Article
The accumulation of iron or aluminum can cause metabolic bone disease, but the mechanisms by which these agents affect bone metabolism remain uncertain. Since transferrin (Tf) can bind several different metals in plasma, equilibrium radioligand binding studies were performed to identify and characterize the Tf receptor in UMR-106-01 osteoblast-like cells; the role of Tf as a modifier of metal-induced changes in cell proliferation was also examined. Osteoblast-like cells grown in serum-free medium have approximately 40,000 Tf receptors on the cell membrane. Tf receptor expression increases during iron depletion and decreases with iron supplementation; the number of Tf receptors was also inversely related to both cell density and the rate of cell proliferation in vitro. Physiological levels of unsaturated Tf (5 microM) enhanced DNA synthesis in osteoblast-like cells maintained in serum-free medium, as measured by the incorporation of tritiated thymidine into DNA. Although neither 10 microM iron (Fe) nor 10 microM gallium (Ga), a known antiproliferative agent, altered DNA synthesis in UMR-106-01 cells during 48-h incubations in serum-free medium, both agents reduced the rate of DNA synthesis when added to serum-free medium containing 5 microM apo-Tf. Decreases in the incorporation of [3H] thymidine into DNA were also noted in osteoblast-like cells incubated for 48 h with 3 microM partially saturated iron Tf or gallium Tf. The results indicate that osteoblast-like cells have a single class of membrane receptors for Tf and that the regulation of Tf receptor expression in UMR-106-01 cells is similar to that in other cell types. The uptake of iron and gallium via the Tf-receptor complex can affect osteoblast proliferation, and such a mechanism may contribute to the bone cell toxicity of various metals.
 
Article
The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1-34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation.
 
PR mixed agonist activity is promoter dependent. The agonist and antagonist activity of a series of PR ligands was analyzed in PR-containing T47D human breast cancer cells that were transiently transfected with an MMTV-Luciferase reporter plasmid and a CMV-galactosidase expression plasmid for normalization. To assay agonist activity, transfected cells were incubated with (A) either 10 -8 M progesterone or increasing concentrations of the indicated ligands (10 -6 –10 -8 M). Antagonist activity (B) was assessed by incubating cells with either 10 -8 M progesterone alone or together with increasing concentrations of competing ligands as indicated (10 -6 –10 -8 M). Fortyeight hours post transfection, the cells were lysed and assayed for luciferase and -galactosidase activities. The data points are averages of triplicate determinations.  
Receptor binding characteristics
RTI-022 is not a potent inhibitor of dexamethasone induced apoptosis. CEM-C7 cells were seeded at 300,000 cells/ml and grown in the presence of vehicle (EtOH), 1 M dexamethasone, or 1 M dexamethasone (Dex) with increasing concentrations (0.1, 1, or 5 M) of RU486, progesterone (Prog) or RTI-022 (1:10, 1:1 and 5:1) molar ratios compared with dexamethasone (Dex). Following a 72-h incubation, cell viability was measured using trypan blue exclusion. The data are presented as % of viable cells remaining following ligand treatment compared with vehicle alone.
Competitive vs. active inhibition of GR transcriptional activity. Competitive inhibition is a one-step process in which the antagonist competes with the agonist for receptor binding. Competitive inhibitors induce a conformational change in the receptor that is incompatible with DNA binding, preventing the antagonist occupied receptors from competing at the level of DNA binding. The two RTI compounds examined in this study thus function as competitive inhibitors of GR. Active inhibition is a two-step process in which 1) the antagonist competes with the agonist for receptor binding and 2) antagonist occupied receptors compete with agonist occupied receptors for binding to glucocorticoid responsive elements. The ability of PR and GR to recruit the transcriptional corepressors N-CoR and SMRT when occupied by active antagonists is likely to be important also. These proteins are part of a large complex that can de-acetylate histones H3 and H4 resulting in nucleosome condensation and transcriptional silencing.
Article
We have identified two novel compounds (RTI 3021-012 and RTI 3021-022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021-012, and RTI 3021-022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step "active inhibition" model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated by agonists for DNA response elements within target gene promoters. Accordingly, we observed that RU486, RTI 3021-012, and RTI 3021-022, when assayed for PR antagonist activity, accomplished both of these steps. Thus, all three compounds are "active antagonists" of PR function. When assayed on GR, however, RU486 alone functioned as an active antagonist. RTI 3021-012 and RTI 3021-022, on the other hand, functioned solely as "competitive antagonists" since they were capable of high affinity GR binding, but the resulting ligand receptor complex was unable to bind DNA. These results have important pharmaceutical implications supporting the use of mechanism based approaches to identify nuclear receptor modulators. Of equal importance, RTI 3021-012 and RTI 3021-022 are two new antiprogestins that may have clinical utility and are likely to be useful as research reagents with which to separate the effects of antiprogestins and antiglucocorticoids in physiological systems.
 
Article
Two receptors [estrogen receptor (ER)alpha and ERbeta] mediate the manifold effects of estrogens throughout the body. Although a clear role has been established for ERalpha in the classical effects of estrogen activity, the physiological role of ERbeta is less well understood. A small-molecule ERbeta selective agonist, ERB-041, has potent antiinflammatory activity in the Lewis rat model of adjuvant-induced arthritis. To characterize the response of target organs and pathways responsible for this antiinflammatory effect, mRNA expression profiling of the spleen, lymph node, and liver was performed, in conjunction with a global analysis of the plasma proteome. We find that the expression of a large number of genes and proteins are altered in the disease model and the majority of these are partially or fully reversed by ERB-041 treatment. Regulated pathways include the acute-phase response, eicosanoid synthesis, fatty acid metabolism, and iron metabolism. In addition, many of the regulated genes and proteins are known to be dysregulated in human rheumatoid arthritis, providing further evidence that the manifestations of the Lewis rat adjuvant-induced arthritis model bear similarity to the human disease.
 
Article
Thiazolidinedione analogs are new antidiabetic agents that attenuate peripheral insulin resistance in noninsulin-dependent diabetic patients; however, the effects of these agents on insulin secretion are not known. We determined the short-term and long-term effects of troglitazone (CS-045) on insulin secretion in a Syrian hamster clonal beta-cell line, HIT-T 15 cells. The direct effect of troglitazone (CS-045: 10(-6)-10(-4) M) on insulin secretion was examined in F-12 K incubation medium containing 7 mM glucose. CS-045 significantly stimulated insulin secretion within 10 min at the concentration of 10(-4) M and dose dependently stimulated insulin secretion within 60 min at the concentration of 10(-6)-10(-4) M. The addition of 10(-5) M CS-045 showed an immediate increase of cytoplasmic free Ca2+ concentrations ([Ca2+]i). Removal of extracellular Ca2+ by the addition of 1.5 mM EGTA completely abolished the 10(-4) M CS-045-induced insulin secretion for 10-min. Long-term incubation (24 h) with 10(-4) M CS-045 significantly decreased beta-cell insulin content and inhibited insulin secretion. During a 5-day incubation, CS-045 showed a dose-dependent reduction of insulin secretion measured during the final 24 h. Long-term incubation with CS-045 over 3 days inhibited the beta-cell proliferation rate, assessed with [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. CS-045 dose dependently increased the amount of DNA fragmentation measured by ELISA. The addition of nifedipine failed to attenuate the reduction of beta-cell proliferation rate and insulin secretion by CS-045, nifedipine antagonized an increase in the amount of DNA fragmentation caused by 10(-4) M CS-045. The present studies provide evidence that CS-045 inhibits beta-cell function following an acute stimulation of insulin secretion in HIT-T 15 cells. The immediate stimulation of insulin secretion by CS-045 may be mediated by an increase in Ca2+ influx from extracellular space. The induction of apoptosis may partially involves the reduction of beta-cell number by CS-045.
 
Article
Recent evidence suggests that IL-1beta-mediated glucotoxicity plays a critical role in type 2 diabetes mellitus. Although previous work has shown that inhibiting IL-1beta can lead to improvements in glucose control and beta-cell function, we hypothesized that more efficient targeting of IL-1beta with a novel monoclonal antibody, XOMA 052, would reveal an effect on additional parameters affecting metabolic disease. In the diet-induced obesity model, XOMA 052 was administered to mice fed either normal or high-fat diet (HFD) for up to 19 wk. XOMA 052 was administered as a prophylactic treatment or as a therapy. Mice were analyzed for glucose tolerance, insulin tolerance, insulin secretion, and lipid profile. In addition, the pancreata were analyzed for beta-cell apoptosis, proliferation, and beta-cell mass. Mice on HFD exhibited elevated glucose and glycated hemoglobin levels, impaired glucose tolerance and insulin secretion, and elevated lipid profile, which were prevented by XOMA 052. XOMA 052 also reduced beta-cell apoptosis and increased beta-cell proliferation. XOMA 052 maintained the HFD-induced compensatory increase in beta-cell mass, while also preventing the loss in beta-cell mass seen with extended HFD feeding. Analysis of fasting insulin and glucose levels suggests that XOMA 052 prevented HFD-induced insulin resistance. These studies provide new evidence that targeting IL-1beta in vivo could improve insulin sensitivity and lead to beta-cell sparing. This is in addition to previously reported benefits on glycemic control. Taken together, the data presented suggest that XOMA 052 could be effective for treating many aspects of type 2 diabetes mellitus.
 
Article
The UMR 106-06 rat osteosarcoma osteoblast-like cell line possesses calcitonin (CT) receptors in addition to expressing PTH receptors and a highly osteoblast-like phenotype, and may represent an intermediate developmental stage between early osteoblast precursors and mature osteoblasts. Therefore, we examined the effects of CT and PTH on second messenger generation and osteoblastic function in these cells. In UMR-106-06 cells, 10-1000 nM CT produced a dose-dependent stimulation of intracellular free calcium concentration ([Ca2+]i), which reached a plateau between 2-3 min. This stimulatory effect was abolished in the absence of extracellular Ca2+ ([Ca2+]o) and was mimicked by forskolin and (Bu)2cAMP. One hundred nanomolar CT also produced a slight but significant increase in inositol triphosphate production (13%, P less than 0.05) but did not produce a rapid, transient increase in [Ca2+]i. In contrast, PTH produced a rapid, transient increase in [Ca2+]i, which reached a maximum within 30 sec. This stimulatory effect of PTH on [Ca2+]i signal was dose-dependent and accompanied by a parallel stimulation of inositol triphosphate production. PTH, forskolin, and (Bu)2cAMP all produced a marked dose-related suppression of both DNA and collagen synthesis, which paralleled their stimulatory effects on intracellular cAMP levels. In marked contrast, CT only minimally reduced DNA and collagen synthesis despite producing comparable increases in intracellular cAMP. One hundred nanomolar CT also stimulated alkaline phosphatase specific activity by 33% (P less than 0.05). Thus, CT stimulates cAMP, [Ca2+]i, and inositol phosphate second messengers in UMR 106-06 cells. However, in contrast to other agents which elevate intracellular cAMP levels, CT does not suppress DNA synthesis. These results suggest that the linkage of CT receptor second messengers to effects on cell function differ from those of PTH and/or that CT may produce additional second messenger(s) which antagonize the antiproliferative effect of increased cAMP levels in UMR-106-06 cells.
 
Article
MK-0677, a spiroindoline sulfonamide, is a novel, orally active GH secretagogue. The effects of MK-0677 on serum GH and other hormones after oral and iv single dose administrations in beagles were evaluated. After oral administration in a balanced eight-dog crossover study, treatment with MK-0677 significantly increased peak GH concentrations, with a 5.3-fold increase (mean +/- SEM, 10.5 +/- 1.9 ng/ml) at the 0.25 mg/kg dose, a 9.0-fold increase (18.0 +/- 3.3 ng/ml) at the 0.50 mg/kg dose, and a 15.8-fold increase (31.6 +/- 5.8 ng/ml) at the 1.0 mg/kg dose. Total GH release, expressed as the area under the curve, showed similar significant increases over the effect of the water placebo. A single oral 1 mg/kg dose in three dogs induced a mean GH peak of 27.6 +/- 1.5 ng/ml at 120 min, and GH levels remained elevated up to 360 min after treatment. Insulin-like growth factor I (IGF-I) levels were significantly increased by 30% at 480 min after treatment. Cortisol levels were increased 2.4-fold over pretreatment levels. After i.v. administration, compared to the saline control group which had a mean (+/- SEM) serum GH peak of 3.8 +/- 0.7 ng/ml, MK-0677 at 0.25 mg/kg significantly increased (P < 0.05) peak GH concentrations 20.4-fold (77.4 +/- 13.7 ng/ml). Total GH release, expressed as the area under the curve, showed a similar increase. The mean peak GH level was recorded 10 min after treatment, with GH levels elevated up to 180 min after treatment. IGF-I levels were significantly elevated by 25% 360 min after the administration of MK-0677. Cortisol levels were increased 2.3-fold over pretreatment levels. Insulin and glucose levels were higher, LH and PRL levels were unaltered, and T4 levels were marginally lower; the levels of each of these hormones remained within the normal ranges for dogs throughout the experiment. In summary, MK-0677 is a potent GH secretagogue that induces an immediate, large, long lasting increase in GH levels when administered orally or i.v. In contrast to GH-releasing peptide-6 and benzolactam secretagogues, GH levels were elevated up to 360 min after treatment, and this was associated with a significant increase in IGF-I levels. Cortisol levels were increased; however, the increases were modest compared to those in GH.
 
Article
It has been well established that the spiroindoline sulfonamide MK-0677 stimulates GH secretion from the pituitary both in vitro and in vivo. MK-0677 has also been shown to increase serum insulin-like growth factor I (IGF-I) and cortisol levels in vivo; these increases are assumed to be driven by the increased serum GH and ACTH levels, respectively. However, such increases could also be due to a direct stimulatory action of MK-0677 at the level of the liver and adrenal cortex. To address this possibility, we investigated whether MK-0677 increased IGF-I and cortisol levels in hypophysectomized dogs. Baseline GH, IGF-I, and cortisol responses to MK-0677 (1 mg/kg, orally) were initially determined. Hypophysectomy (hypox; n = 7) or sham surgery (sham; n = 5) was then carried out. Six days postsurgery, the GH and cortisol responses to MK-0677 were reevaluated in each dog. In addition, each dog was treated with porcine GH (PST; 0.1 IU/kg, s.c.) to confirm the responsiveness of the GH-IGF-I axis. The mean peak GH increases in response to MK-0677 in the presham dogs (83.7 +/- 19.2 ng/ml), post-sham dogs (108 +/- 26.2 ng/ml), and pre-hypox dogs (121.2 +/- 13.6 ng/ml) were not significantly different. Mean peak GH levels were unchanged after MK-0677 administration in the hypox dogs (2.3 +/- 0.7 ng/ml). Before surgery, serum IGF-I levels increased to 243 +/- 27 and 224 +/- 47 ng/ml in the sham and hypox groups, respectively, after MK-0677 administration. Surgery was associated with a marked (> or =50%) decrease in serum IGF-I levels. MK-0677 administration increased IGF-I levels in the sham dogs from 78 +/- 14 to 187 +/- 31 ng/ml, whereas IGF-I levels remained unchanged (17.7 +/- 2.4 ng/ml) in the-hypox dogs. PST treatment increased IGF-I levels in the sham dogs from 162 +/- 30 to 325 +/- 32 ng/ml. In the hypox dogs PST treatment restored IGF-I to physiological levels (from 17.7 +/- 2.4 to 199 +/- 41 ng/ml). Cortisol was increased after MK-0677 administration 3.7-fold in the pre-sham, 3.6-fold in the post-sham, and 3.6-fold in the pre-hypox dogs, but no increase was seen in the post-hypox dogs. ACTH GEL administration (2.2 U/kg, i.m.) to hypox dogs returned cortisol to normal physiological levels, demonstrating the functional integrity of the adrenal cortex. This study demonstrates that the GH secretagogue MK-0677 does not directly stimulate an increase in serum IGF-I or cortisol levels, but depends upon the presence of an intact pituitary.
 
Article
Rats (200-275 g) were rendered pseudopregnant on the morning of estrus (day 1) by cervical stimulation. On day 5 of pseudopregnancy (PS), the rats (8-12/group) were subjected to either hysterectomy (HX) or unilateral or bilateral decidual cell reactions (uni- or bi-DCR). Control pseudopregnant (C-PS) rats were sham operated on day 5. On days 8 and 12, the rats were killed by decapitation. Blood was collected, and serum was saved for RIA of estradiol (E2), progesterone (P), androgen (A), and gonadotropins. Corpoa lutea (CL) and the nonluteal ovarian compartment (NLO) were incubated separately in vitro for 2 h in Krebs-Ringer bicarbonate buffer, and steroid concentrations were determined in Cl and NLS (before and after incubation) and in incubation media. On day 8 of PS, P and A levels in the serum in bi-DCR rats were higher than those in C-PS but not in uni-DCR and HX-PSP rats; however, on day 12, serum P and A levels in bi-DCR rats were higher than the levels of other PS groups, with the exception of C-PS rats whose serum A levels were the same as those in bi-DCR rats. No differences were observed in serum E2 levels throughout the experimental period. On days 8 and 12 the luteal P concentration of bi-DCR rats was higher than that of any other PS group, i.e. uni-DCR, HX-PS, and C-PS rats. The higher concentrations of luteal P in bi-DCR rats were associated with neither concomitant increases of luteal A and E2 nor higher serum levels of FSH, LH, and PRL, and they are therefore attributed to the large amount of decidual tissue (DT) in bi-DCR rats. In bi-DCR uni-DCR, and HX-PS rats, no differences were observed in the luteal concentration of A on day 8 or 12; however, E2 levels in CL of all PS groups were higher on day 12 than on day 8. The reasons for that difference is unclear, but it may be related to the onset of LH dependency of these Cl and, therefore, causally related to luteal maintenance. Collectively, these results indicate the possibility that DT selectively stimulates luteal P production in vivo without altering luteal A and E2, and that this effect of DT may be quantitatively related to the amount of DT. In vitro, CL of bi-DCR rats produced P, A, and E2 in quantities similar to other PS groups, with the exception of C-PS rats on day 12 whose CL were regressed and synthesized less P and more E2 than bi-DCR rats; therefore, a DCR effect on steroidogenesis in vitro was not observed. On day 12 of PS, a significant increase in vitro A and E2 production by NLO was observed, indicating that antral follicles may become highly competent to produce A and E2 during the latter portion of PS.
 
Article
The mechanism(s) of action of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on rat (r) GH release was studied in primary rat pituitary cell cultures. TPA stimulated rGH release (3.2- to 4.1-fold above control value) and rTSH and rLH release (1.4- and 1.7-fold above control values, respectively), but not rPRL release. The ED50 of TPA on rGH secretion was 1.3 X 10(-9) M compared to 4.5 X 10(-11) M for human pancreatic GH-releasing factor [hpGRF-(1-44)]. If maximally effective doses of TPA or hpGRF-(1-44) were added to the cells, the magnitudes of the increase in rGH release were quite similar for both agents when the incubation period was less than 12 h. When (Bu)2cAMP was added simultaneously with various doses of TPA, (Bu)2cAMP increased rGH release beyond the maximal effect of TPA. There was an additive effect when hpGRF-(1-44) and TPA were used to stimulate rGH release. These results indicate that TPA enhances rGH release through a different pathway than hpGRF-(1-44). TPA failed to increase the formation of intra- and extracellular cAMP, whereas hpGRF-(1-44) increased both, suggesting that TPA stimulates rGH release through an cAMP-independent pathway(s). Protein kinase C has been postulated to be a receptor for TPA in human platelets. When phospholipase C, which activates protein kinase C via the formation of diacylglycerol, was added to the cells, rGH release was stimulated in a dose-dependent manner. This effect was not blocked by indomethacin. These results may suggest that activation of protein kinase C leads to rGH release. The observations are consistent with the hypothesis that TPA activates protein kinase C and causes the release of rGH in normal pituitary cells in culture. These findings indicate that the mechanism(s) of action of TPA on rGH release is different from that of hpGRF-(1-44).
 
Article
The present studies were designed to determine the effects of ethane 1,1 diphosphoric acid (EHDP(TM)), an analog of pyrophosphate, on the hypocalcemic and hypophosphatemic actions of salmon calcitonin (SCT). Rats were pretreated for 3 or 4 days with daily subcutaneous (sc) injections of EHDP (40 mg/kg body wt). 45Ca was administered (intraperitoneally) 10 days prior to EHDP treatment and 32P (intravenously) the day following the last injection of the drug. SCT (0.4 MRC mU/g body wt) was injected sc 1 hr after 32P administration. Preliminary tests with this drug had confirmed that the dosage used at least partially inhibited the 45Ca removal from bone stimulated by low calcium diet or parathyroid hormone administration, and at least partially reduced the uptake of both 45Ca and 32P by bone, during the first 5 hr after intravenous administration of isotopes. Following SCT injection plasma calcium concentrations fell only 5% in EHDP treated rats compared to 25% in rats not treated with EHDP. Plasma phosphate concentrations and 32P plasma values, however, fell markedly and similarly in rats given SCT regardless of whether or not they had been pretreated with EHDP. These studies demonstrate that the hypophosphatemic effect of SCT was produced independently of the hypocalcemic action of the hormone and the hypophosphatemia did not result solely from a reduction of phosphate release from bone but rather was due to an increased exit of phosphate out of the plasma.
 
Effects of various concentrations of HPTE on an E 2 doseresponse curve with ER. A, Experiments were performed as described in Fig. 1 with 10 11-10 5 M E 2 , either alone (f) or in combination with 10 7 (‚), 10 6 (), and 10 5 M HPTE () in HepG2 cells transfected with expression plasmids for human ER, C3-Luc, and-galactosidase. Values represent the mean SE normalized luciferase activity from three separate assays. B, Schild regression analysis of the data shown in A. The dose ratio (dr) is [A] [A], where [A] and [A] refer to equiactive concentrations of E 2 in the presence and absence of HPTE, respectively. 
Activities of E 2 and HPTE with estrogen-responsive MMTV and vt-ERE promoters. Experiments were performed as described in Fig. 1. HepG2 cells (A-D), and HeLa cells (E and F) were transfected with either human ER or ER plus either ERE-MMTV-luciferase (EREMMTV-Luc) or vtERE-luciferase (ERE-Luc) reporter plasmids plus-galactosidase expression plasmid. Values represent the mean of three or four separate experiments and are presented as the percent response, with 100% activity defined as the activity achieved with the plateau E 2 concentration. 
Activities of E 2 and HPTE with coexpression of ER and ER. Experiments were performed as described in Fig. 1. HepG2 cells were transfected with 0-400 ng/well human ER plasmid alone (A) or in combination with 40 ng/well ER plasmid plus C3-Luc and-galactosidase expression plasmids (B). C, Cells were cotransfected with 80 ng/well each of ER and ER. Values represent the mean SE normalized luciferase activity from three separate experiments. 
Article
Concern that some chemicals in our environment may affect human health by disrupting normal endocrine function has prompted research on interactions of environmental contaminants with steroid hormone receptors. We compared the activity of 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), an estrogenic metabolite of the organochlorine pesticide methoxychlor, at estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). Human hepatoma cells (HepG2) were transiently transfected with either human or rat ERalpha or ERbeta plus an estrogen-responsive, complement 3-luciferase construct containing a complement 3 gene promoter sequence linked to a luciferase reporter gene. After transfection, cells were treated with various concentrations of HPTE in the presence (for detecting antagonism) or absence (for detecting agonism) of 17beta-estradiol. HPTE was a potent ERalpha agonist in HepG2 cells, with EC50 values of approximately 5 x 10(-8) and 10(-8) M for human and rat ERalpha, respectively. In contrast, HPTE had minimal agonist activity with either human or rat ERbeta and almost completely abolished 17beta-estradiol-induced ERbeta-mediated activity. Moreover, HPTE behaved as an ERalpha agonist and an ERbeta antagonist with other estrogen-responsive promoters (ERE-MMTV and vtERE) in HepG2 and HeLa cells. This study demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that may act as agonists or antagonists through one or more hormone receptors.
 
Article
1,10-Phenanthroline, a metal ion chelator, inhibits the binding of previously activated (25 C for 30 min) rat hepatic [3H]triamcinolone acetonide (3H-labeled 9-fluoro-11 beta, 21-dihydroxy-16 alpha, 17-1-[1-metylethylidenebis(oxy)]pregna-1,4-diene-3,20-dione ([3H]TA)-receptor complexes to DNA-cellulose. The observed inhibition increases as the temperature of the preincubation with chelator is increased from 0 to 25 C. Fifty percent of the maximal inhibition (greater than 90%) detected at 25 C is achieved with 1 mM 1,10-phenanthroline. The observed inhibition is not the consequence of DNA degradation by 1,10-phenanthroline-Cu2+ complexes, since preincubation of activated cytosol with neocuproine (2,9-dimethyl-1,10-phenanthroline), a potent Cu2+ chelator, fails to block the subsequent inhibition of DNA-cellulose binding by 1,10-phenanthroline. The failure of other chelators which complex siilar metal ions (alpha, alpha'-dipyridyl,8-hydroxyquinoline, 2,2',2"-tripyridine, EDTA, EGTA, and Na azide) to inhibit DNA-cellulose binding suggests that the effectiveness of 1,10-phenanthroline does not result from removal of a required free metal ion(s) but, rather, from a specific interaction with a metal ion(s) which may be located within the activated receptor protein. The observed inhibition is dependent on the metal chelating properties of 1,10-phenanthroline, since preincubation with several divalent metal cations (Zn2+, Co2+, and Ni2+) which are known to be chelated by this compound block its subsequent inhibitory effect. Ferroin (1,10-phenanthroline-ferrous sulfate complex) and 1,7-phenanthroline (nonchelating isomer) also fail to inhibit DNA-cellulose binding. The inhibition mediated by 1,10-phenanthroline persists after gel filtration, suggesting that 1,10-phenanthroline associated with a macromolecule is the effective form of the inhibitor, rather than free 1,10-phenanthroline. Finally, 1,10-phenanthroline appears to interact directly with activated [3H]TA-receptor complexes, since it alters their net charge and results in their elution from DEAE-cellulose at a salt concentration characteristic of unactivated complexes. Collectively, the data suggest that the activated [3H]TA-receptor complex is a metalloprotein and that the metal ion(s) may be associated directly with the DNA-binding site or may regulate this site indirectly through an allostreic mechanism.
 
Article
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was examined for a possible stimulative effect on osteoblastic MC3T3-E1 cells. During the early period of culture, 1,25-(OH)2D3 had a stimulative effect. During the growth phase, however, the steroid had little effect on either the protein or DNA content of the cultures. 1,25-(OH)2D3 increased bone-liver-kidney-type alkaline phosphatase activity in a dose-related manner up to a concentration of 5 pg/ml; the increase was 2.2-fold over the control value. Studies on the effect of actinomycin D or cycloheximide treatment indicated that the vitamin may enhance de novo synthesis of ALP. The steroid also stimulated type I collagen production dose dependently via an increase in collagen synthesis rather than by inhibition of collagen degradation. MC3T3-E1 cells have a specific receptor for 1,25-(OH)2D3 which has a dissociation constant of 4.17 X 10(-11) M and a sedimentation coefficient of 3.67S. The receptor concentration varied with the period of culture, being higher during the growth phase and lower at confluence, but its affinity did not change. The results indicate that 1,25-(OH)2D3 has a direct specific anabolic effect on osteoblastic cells in vitro during the growth phase and that this effect is related to receptor concentration.
 
Article
The transmembrane signaling events of GH were investigated in the liver, a major target organ of GH action. Recombinant human GH when added to freshly isolated rat hepatocytes rapidly stimulated the production of sn-1,2-diacylglycerol (DAG). The generation of DAG was biphasic with the first transient peak observed at 2 min and the second peak at 15 min (1.2-fold and 1.4-fold over control, respectively). Levels of DAG continued to be elevated above those in control cells at 30 min. The response was dose-dependent with an EC50 of 0.15 nM. Both bovine GH and rat GH, which bind to the rat GH receptor but not to the PRL receptor, also stimulated DAG production. Similarly, human PRL, which binds to the PRL but not GH receptor, stimulated DAG formation to a comparable extent. These results suggest that production of DAG may be an early signaling event mediated by hormone stimulation of both the GH and PRL receptors.
 
Article
Based on in vitro studies, an insulin-mediated increase in muscle 1,2-diacylglycerol (DAG) content has been proposed as a signal for the insulin induced stimulation of glucose transport. A recent study [Turinsky, J., Bayly, B.P. and O'Sullivan, D.M. (1990) J. Biol. Chem. 265, 7933-7938] challenged this hypothesis because no increase in muscle 1,2-diacylglycerol was observed after in vivo infusions of insulin at doses which markedly stimulated muscle glucose transport. We observed a 30-45% increase in DAG in rat gastrocnemius and diaphragm muscles, 5-15 min after intramuscular or intravenous injections of 1-3 U of insulin per rat, doses which would be expected to activate insulin receptors more fully. The effects on DAG were similar whether or not hypoglycemia was prevented by co-injection of glucose.
 
Article
In bovine adrenal glomerulosa cells, angiotensin-II (AII) induced a biphasic increase in 1,2-sn-diacylglycerol (DAG), with an initial peak at 10 sec followed by a transient decrease at 30 sec. The second increase was much higher in magnitude than the first peak and reached its maximum after 1 h of stimulation. Such kinetics of DAG formation resemble those with which AII stimulates the formation of inositol-1,4,5-trisphosphate. The protein synthesis inhibitor cycloheximide, which prevents hormone-induced de novo phospholipid synthesis in adrenal fasciculata cells, had no effect on the DAG response to Aii. The first phase of signal generation of both inositol-1,4,5-trisphosphate and DAG was not affected by incubation in calcium-deficient extracellular medium. However, the second phase of the inositol phosphate response was almost completely inhibited in low calcium medium, while the DAG response was reduced by only one third. Pertussis toxin (150 ng/ml) and the voltage-sensitive calcium channel inhibitors, nifedipine (1 microM) and Ni2+ (100 microM), had no effect on the DAG response to AII. The retention of a substantial DAG response to AII in low calcium medium, with concomitant diminution of the inositol phosphate response, indicates that a major part of the DAG formed during the sustained phase of hormonal stimulation is derived from sources other than phosphoinositides. The DAGs produced from different phospholipids could have distinctive fatty acid compositions and membrane localizations, which, in turn, could result in the differential activation of protein kinase-C. In this way, the increased complexity of the hormonally induced signalling pathway could allow for a greater diversity of responses in hormone-stimulated target cells.
 
Article
The localization of radioactivity in the brain of immature intact and mature castrated Sprague—Dawley rats was investigated by drymount autoradiography at 1 hr after the injection of l,2–³H-testosterone. Radioactivity is found to be selectively concentrated and retained in specific neurons in the nucleus (n.) arcuatus and the n. ventromedialis hypothalami, the n. preopticus medialis, the n. interstitialis striae terminalis, the n. septi lateralis, the hippocampus and the amygdala. The topographic distribution of androgenconcentrating neurons agrees well with areas in the brain that have been associated with the regulation of gonadotropin secretion and male sex behavior. The autoradiographic data are consistent with the hypothesis that androgen feedback occurs at hypothalamic and extrahypothalamic sites in the central nervous system. (Endocrinology 92: 251, 1973)
 
Article
Experiments were designed to evaluate the role of activators of protein kinase C, such as 1,2-diacylglycerol and phorbol esters, on the release of all the anterior pituitary (AP) hormones in vitro. Dispersed rat AP cells were incubated in the presence of 1,2-didecanoylglycerol (DiC10), a synthetic diacylglycerol, or phorbol 12,13-dibutyrate (PDBu), a tumor-promoting phorbol ester, at different concentrations and for varying periods of time. ACTH and beta-endorphin (beta-End) secretion were enhanced by DiC10 in a concentration-dependent manner, with a minimal effective concentration of 5 microM. PDBu at 5 nM produced a significant release of both ACTH and beta-End. The effect of DiC10 and PDBu was time dependent, with maximal responses occurring at 15-30 min for DiC10 and 30-60 min for PDBu. Release of GH was also enhanced significantly by DiC10 and PDBu, with minimal effective concentrations of 1 microM and 1 nM, respectively. Maximal release of GH was already attained within 15 min with DiC10 or 60 min with PDBu. In additional experiments, the effects of DiC10 and PDBu on secretion of LH, FSH, PRL, and TSH were evaluated. The results indicate that 5-25 microM DiC10 produced a concentration-dependent release of each of those hormones, and that 5 microM was the minimal effective concentration in every case. Nearly maximal stimulation was achieved within 15 min for each hormone. PDBu (50 nM) significantly enhanced LH, FSH, PRL, and TSH release within 30 min. Although qualitatively all hormones were similarly stimulated, both with respect to time and concentration, some quantitative differences were observed. ACTH and beta-End release were enhanced 100% by DiC10 and 300% by PDBu, whereas the increase in other hormones was of a lesser magnitude. The present study indicates that two specific stimulators of protein kinase C, diacylglycerol and phorbol ester, can enhance secretion of all AP hormones in a concentration- and time-dependent manner. This suggests that formation of endogenous 1,2-diacylglycerol may represent a physiological intracellular messenger in the events leading to AP peptide hormone release.
 
Article
The effects of adult-onset hypothyroidism on the metabolic compartmentation of the cerebral tricarboxylic acid cycle and the gamma-aminobutyric acid (GABA) shunt have been investigated by 13C nuclear magnetic resonance spectroscopy. Rats thyroidectomized as adults and age-matched controls were infused in the right jugular vein with unlabeled or (1,2-13C2) acetate solutions for 60 min. At the end of the infusion, the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by 13C nuclear magnetic resonance and reverse-phase HPLC. Thyroidectomized animals showed a decrease in the incorporation of 13C from (1,2-13C2) acetate in cerebral metabolites and an increase in the concentrations of unlabeled glutamate and GABA. Computer-assisted interpretation of the 13C multiplets observed for the carbons of glutamate, glutamine, and GABA indicated that adult-onset hypothyroidism produced 1) a decrease in the contribution of infused (1,2-13C2) acetate to the glial tricarboxylic acid cycle; 2) an increase in the contribution of unlabeled acetyl-CoA to the neuronal tricarboxylic acid cycle; and 3) impairments in the exchange of glutamate, glutamine, and GABA between the neuronal and glial compartments. Despite the fact that the adult brain has often been considered metabolically unresponsive to thyroid hormone status, present results show metabolic alterations in the neuronal and glial compartments that are reversible with substitution therapy.
 
Article
The precise roles of the calcium and lipid pathways in TRH-stimulated PRL secretion from rat pituitary (GH3) cells are controversial. In particular, it is debated whether elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i) is sufficient to cause burst secretion (0-2 min) or whether an increase in 1,2-diacylglycerol must accompany the Ca2+ elevation. In this study, the effects of TRH, which elevates 1,2-diacylglycerol, on [Ca2+]i and stimulation of burst secretion were compared with those of depolarization by high extracellular K+, which does not increase 1,2-diacylglycerol. A maximal concentration of TRH (1 microM) and depolarization by 17.5 mM K+ caused elevation of [Ca2+]i from the resting level of 140 +/- 20 nM to 470 +/- 70 nM and 514 +/- 60 nM, respectively, and stimulated burst secretion from 0.6 +/- 0.2 ng/10(6) cells/min to 3.3 +/- 0.8 and 3.1 +/- 0.4 ng/10(6) cells/min, respectively, when a small component of TRH-stimulated secretion that is independent of elevation of [Ca2+]i was subtracted. A detailed comparison of multiple levels to which [Ca2+]i was elevated (up to 600 nM) and the degree of stimulation of burst phase secretion demonstrated the same positive linear correlation (correlation coefficient = 0.96) for TRH and K+ depolarization. Hence, elevation of [Ca2+]i is sufficient to cause burst secretion irrespective of elevation of 1,2-diacylglycerol. Optimal stimulation by TRH of sustained secretion of PRL did not depend on elevation of [Ca2+]i; sustained PRL secretion stimulated by 10 nM TRH was 2.6 +/- 0.4 and 2.7 +/- 0.2 ng/10(6) cells/min in control cells and arachidonic acid-pretreated cells in which [Ca2+]i was not elevated, respectively. The data from this and previous studies demonstrate that elevation of [Ca2+]i and 1,2-diacylglycerol may act coordinately, but not synergistically, to mediate TRH stimulation of PRL secretion from GH3 cells.
 
Article
In the present study the involvement of protein kinase-C (PKC) in the regulation of the vitamin D receptor (VDR) and interaction of PKC with cAMP-induced up-regulation of VDR in osteoblast-like cells were examined. Activation of PKC by incubation for 4 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a comparable dose-dependent decrease in 1,25-dihydroxyvitamin D3 binding in the osteoblast-like cell lines UMR 106 and ROS 17/2.8, with a maximum inhibition at 100 nM and an IC50 at 5 nM PMA. Time-course studies revealed that in both UMR 106 and ROS 17/2.8 cells, 24-h incubation with PMA caused an increase in 1,25-dihydroxyvitamin D3 binding. This can be related to down-regulation of PKC. Scatchard analysis demonstrated that activation of PKC resulted not in a change in receptor affinity, but, rather, in an increase in VDR number. This is supported by Northern blot analysis, which shows at 2 h a decrease and at 24 h an increase in VDR mRNA. At 4 h, when activation of the cAMP pathway results in an increase in VDR, activation of PKC results in a decrease in VDR. Coincubation for 4 h with PMA caused a decrease in PTH- and forskolin-induced up-regulation of VDR. This inhibition is not due to a reduction in cAMP production, as PTH-stimulated cAMP production was potentiated by PMA. The effect of activation of PKC on VDR is not a general effect, as PMA does not affect basal ornithine decarboxylase activity and potentiates PTH-induced ornithine decarboxylase activity. The present study demonstrates that PKC is involved in the regulation of VDR in UMR 106 and ROS 17/2.8 and that PKC interacts with cAMP in the regulation of VDR. The current data point to a negative controlling role for PKC in the regulation of VDR. Moreover, two different cAMP-regulated actions in UMR 106 cells (VDR up-regulation and ornithine decarboxylase activity) are differently modulated by PKC. Although the precise mechanism by which PKC represses and stimulates gene expression is not yet clear, this study demonstrates the important regulatory role for PKC in two osteoblast-like sarcoma cell lines.
 
Article
We reported that TSH, which stimulates cAMP accumulation and proliferation of FRTL-5 thyroid cells, chronically increases the 1,2-diacylglycerol (1,2-DG) content of FRTL-5 cells. Because activation of inositol lipid hydrolysis by a phospholipase-C enzyme would generate 1,2-DG, we compared the effects of TSH on inositol lipid metabolism to TSH-induced increases in 1,2-DG content and stimulation of cAMP accumulation and cell growth. Acute stimulation of inositol lipid hydrolysis did not occur with doses of 1000 microU/ml or lower, but did occur with TSH doses of 3000 microU/ml and higher, with rates between 1-4%/h. More importantly, in cells chronically exposed to TSH, the rate of inositol lipid hydrolysis was increased only at TSH doses of 10,000 microU/ml or greater, and the maximum rate was 4-5%/h. When cells were growth arrested by TSH deprivation, there was no change in the content of inositol phosphates or polyphosphoinositides. In contrast to the high doses of TSH required to stimulate inositol lipid hydrolysis, TSH-induced elevation of 1,2-DG content and stimulation of cAMP accumulation and growth occurred at physiological TSH concentrations, with minimal effective doses in the range of 1-10 microU TSH/ml, and half-maximally effective doses between 50-200 microU TSH/ml. These data suggest that inositol lipid hydrolysis does not mediate the proliferative response to TSH in FRTL-5 cells and is not the mechanism by which increases in 1,2-DG content occur at physiological TSH concentrations.
 
Article
A study was made of the influence of certain hormones and synthetic hormonal compounds on the uptake of radioactivity by rat seminal vesicles and prostate gland following the administration of testosterone-l,2-3H in vivo. Androstenedione, 17β-methyltestosterone, 17βmethyl-β-nortestosterone (SK & F 7690), progesterone and corticosterone reduced the uptake of radioactivity. 17β-Estradiol and diethylstilbestrol did not reduce the uptake; indeed, in the lateral prostate an increased accumulation of radioactivity was observed. One hr after administration of testosterone-3H, unchanged testosterone represented 3.0–17.1& of the total radioactivity in the accessory sex organs. 5a- Androstan-17β-ol-3-one was the main metabolite, constituting about 70% of the total radioactivity. (Endocrinology 85: 683, 1969)
 
Article
Activation of calcium-stimulated phospholipid-dependent protein kinase C (PKC) by diacylglycerols and phorbol esters has been shown to mediate the release of secretory proteins in several systems. To determine whether PKC activation is involved in regulation of the release of human placental lactogen (hPL) from the placenta, we examined the effects of various acylglycerols and phorbol esters on the release of hPL from cultured human trophoblast cells. Sn-1,2-dioctanoylglycerol (diC8) and phorbol-12-myristate-13-acetate (PMA), both of which stimulate placental protein kinase C activity, caused dose-dependent increases in hPL release over a 0.5-h period. The maximal amounts of hPL released in response to diC8 (300 microM) and PMA (10(-8) M) were 200-300% and 150-225% greater, respectively, than that released in response to diluent alone. Acylglycerols and phorbol esters, which are less potent stimulators of PKC activity in other systems, stimulated hPL release to a lesser extent than either diC8 or PMA. PKC-inactive acylglycerols and phorbol esters were without effect. After 0.5 h of exposure, diC8 (300 microM)- and PMA (10(-8) M)-exposed cells synthesized 257.5% and 250.3% more hPL than control cells. Cycloheximide at a dose (50 micrograms/ml) that inhibited the synthesis of trichloroacetic acid-precipitable [35S]methionyl placental proteins by more than 80% completely blocked the stimulatory effects of diC8 and PMA on hPL synthesis and release. Although diC8 and PMA stimulated the synthesis and release of hPL, these compounds had no effect on the release of hCG and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. The demonstration that acylglycerols and phorbol esters stimulate the synthesis and release of hPL strongly implicates protein kinase C activation in the mechanisms of hPL synthesis and release.
 
Article
Side-chain modified vitamin D analogs including 20-Epi-22-oxa-24a,26a,27a-trihomo-1alpha,2 5-dihydroxyvitamin D3 (KH1060), and 1,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (MC903) were originally designed to aid in the treatment of hyperproliferative disorders including psoriasis and cancer. Here we demonstrate that these analogs, as well as the 6-cis-locked conformer, 1alpha,25-dihydroxy-lumisterol3 (JN) prime NB4 cells for monocytic differentiation. Previously, the action of MC903 and KH1060 was presumed to be mediated by the nuclear vitamin D receptor (VDRnuc). Differentiation in response to all analogs was shown to be inhibited by 1beta,25-dihydroxyvitamin D3 (HL), the antagonist to the nongenomic activities of 1,25D3. These data suggest that although MC903 and KH1060 may bind the VDRnuc, that the differentiative activities of these agents requires nongenomic signaling pathways. Here we show that 1alpha,25(OH)2-d5-previtamin D3 (HF), JN, KH1060, and MC903 induce expression of PKC alpha and PKC delta and translocation of both isoforms to the particulate fraction, and PKC alpha to the nuclear fraction. The full differentiation response with combinations of analogs and TPA was inhibited 50% by the membrane permeable Ca2+ chelator, 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) or calpain inhibitor I. These data demonstrate that intracellular free calcium and the calcium-dependent protease, calpain play critical roles in monocytic differentiation. Intracellular calcium appears to be most critical in the 1,25D3-priming stage of differentiation, while calpain is essential in the TPA maturation response.
 
Article
In young rats, PTH markedly stimulates the renal conversion of 25-hydroxyvitamin D3 (25OHD3) to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active form of vitamin D3. With increasing age, serum 1,25-(OH)2D3 decreases while serum PTH increases. Therefore, the effect of PTH on the renal metabolism of 25OHD3 to 24,25-(OH)2D3 or 1,25-(OH)2D3 was compared in young and adult rats. Rats were housed in the dark and fed a low Ca, vitamin D-deficient diet for 4-6 weeks, and thyroparathyroidectomy was performed. Renal 25OHD3 metabolism was measured in vitro by incubating renal cortical slices with tritiated 25OHD3 and quantifying tritiated metabolites by high pressure liquid chromatography. When young (2 months old) thyroparathyroidectomized (TPTX) rats were repleted with PTH by ip injection, 1,25-(OH)2D3 production increased 61%, and 24,25-(OH)2D3 production decreased to 40%. When adult (13 months old) TPTX rats were repleted with PTH, there was no increase in 1,25-(OH)2D3, but 24,25-(OH)2D3 production decreased to 43%. When PTH was added in vitro by incubating renal slices from young TPTX rats for 4 h, 1,25-(OH)2D3 production increased 68%, and 24,25-(OH)2D3 production decreased to 71%. In slices from adult rats, 24,25-(OH)2D3 production was decreased significantly to 71%, and 1,25-(OH)2D3 production was unaffected by PTH. The PTH-stimulated increase in the cAMP content of renal slices from adult rats was 75% that of slices from young rats. These studies demonstrate that PTH modulates renal 24,25-(OH)2D3 production in the adult. However, PTH does not modulate renal 1,25-(OH)2D3 production in the adult under the same conditions that produce a PTH effect in the young animal.
 
Article
The 1,25-dihydroxyvitamin D3 (vitamin D) receptor (VDR) is a key trans-activating protein that mediates calcium regulation as well as cellular proliferation and differentiation. Phosphorylation of the VDR contributes significantly to its functional activity, but the specific mechanisms that mediate this regulation are not well understood. Phosphorylation may influence DNA binding, ligand binding, and protein-protein interactions, including heterodimerization and/or transactivation functions. We used a protein kinase C inhibitor, staurosporine (ST), and an inhibitor of serine-threonine phosphatases, okadaic acid (OA), to elucidate the contribution of VDR phosphorylation to vitamin D-mediated transcription of the osteocalcin (OC) gene. Vitamin D-induced transcription was assayed in transfected ROS 17/2.8 osteosarcoma cells using chloraminphenicol acetyltransferase constructs containing the vitamin D-responsive element (VDRE) at its native locus in the rat OC promoter as well as fused to a heterologous promoter. Both ST and OA inhibit VDRE-mediated and vitamin D-dependent enhancement of OC gene transcription as well as OC biosynthesis, as assessed by RIAs. Results from gel mobility shift and Western blot analyses using nuclear proteins from ROS 17/2.8 cells show that binding of the VDR-retinoid-X receptor heterodimer complex to the OC VDRE is not inhibited in the presence of ST. In contrast, OA does inhibit the formation of complexes interacting with both the OC and osteopontin VDREs; immunoprecipitation studies using 32P-labeled ROS 17/2.8 cells reveal that OA treatment result in ligand-independent hyperphosphorylation of the VDR. Our results suggest that two distinct phosphorylation events modulate rat VDR function. One event is related to transactivation, and the other is also critical to the VDRE-binding activity of VDR-retinoid X receptor-DNA complexes with consequential effects on transactivation.
 
Article
Calcium and vitamin D metabolism were studied in streptozotocin-treated rats up to 10 days after the induction of diabetes. Proteinuria, hypercalciuria, and hyperphosphaturia appeared as early as 3 days after diabetes induction and were reversed by insulin. The serum proteins and fasting calcium concentrations were decreased in untreated diabetic rats. The concentration of serum vitamin D binding protein (DBP) was higher in male than in female control rats (mean +/- SD; 555 +/- 73 vs. 348 +/- 28 mg/liter, P less than 0.001). When sequentially measured in male untreated diabetic rats, DBP concentration steadily decreased. Compared with control values, DBP was reduced 19%, 28%, and 32% on days 3, 6, and 10, respectively, after induction of diabetes in male rats. In female animals, DBP was reduced 22% on day 10 of diabetes. DBP concentration was corrected by insulin treatment of diabetic rats and remained normal in streptozotocin-treated animals that did not develop diabetes. The serum concentration of 25-hydroxyvitamin D3 was similar in both sexes and was not affected by diabetes. Like DBP, the concentration of total 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was higher in male than in female control rats (120 +/- 24 vs. 96 +/- 17 ng/liter, P less than 0.001), but 10 days after induction of diabetes this concentration decreased by 37% and 29% in male and female rats, respectively. The free 1,25-(OH)2D3 concentration, estimated from the molar 1,25-(OH)2D3/DBP ratio, was similar in both sexes and was not decreased by diabetes. We conclude that experimental diabetes in the rat induces a decrease in DBP concentration and a concomitant decrease in total but not in free 1,25-(OH)2D3 concentrations. This may indicate that diabetes decreases circulating 1,25-(OH)2D3 concentrations through alterations in DBP levels.
 
Article
Receptors for 1,25 dihydroxyvitamin D3 [1,25-(OH)2D3] have been reported in breast tissue; however, the presence of multiple binding sites and limited availability of human tumor tissue have precluded complete biochemical characterization of the receptor in breast cancer. In the present study, binding proteins for 1,25-(OH)2D3 in breast tumor tissue were analyzed using a rat model of breast cancer. Breast tumors were induced in adult female rats with the carcinogen nitrosomethylurea. Such tumors previously have been shown to possess high levels of estrogen receptors and are estrogen dependent. Binding proteins for 1,25-(OH)2D3 in 0.3 M KCl extracts of tumor tissue were analyzed on sucrose density gradients. Two binding proteins were detected: one sedimenting at 5-6 S representing binding of 1,25-(OH)2D3 to the 25 hydroxyvitamin D3 (25OHD3) binding protein and a second moiety sedimenting, like the rat intestinal receptor, at 3.3 S. Binding of the dihydroxy metabolite to the faster sedimenting protein could be eliminated by inclusion of radioinert 25OHD3 in the incubation medium. Receptor content of rat breast tumor was investigated using an hydroxylapatite assay by incubating tumor extracts with a saturating concentration of 1,25-(OH)2-[3H]D3, plus unlabeled 25OHD3 to eliminate binding of the hormone to the 5-6 S species. Scatchard analysis of 1,25-(OH)2D3 binding to the tumor extracts yielded an apparent dissociation constant (Kd) of 0.33 nM. In summary, breast tumors induced in rats by nitrosomethylurea were shown to contain high affinity 1,25-(OH)2D3 receptors with properties very similar to those reported for the receptor in other mammalian target organs. The presence of receptors for 1,25-(OH)2D3 in these rat breast tumors implies that the tissue is potentially responsive to the hormone.
 
Article
We have previously shown that protein S, a vitamin K-dependent protein, is a bone matrix component synthesized and secreted by osteoblasts. Because protein S is a cofactor of protein C in inhibiting factor Va and VIIIa, we have looked for the presence of the proteins related to the anticoagulant protein C system in human MG 63 osteosarcoma cells and in human adult osteoblast-like cells. Using immunoblotting, we have shown that protein C, factor V, and C4b binding protein are not secreted by these cells. We have shown by enzyme-linked immunoassay, immunocytochemistry, and immunoprecipitation of labeled proteins that thrombomodulin, a transmembrane glycoprotein involved with thrombin in the activation of protein C, is present at the cell surface of osteoblasts. Moreover, using a protein C activation system where thrombin and protein C are added to the cells, we have shown that protein C could be activated at the osteoblast cell surface. This activation of exogenous protein C, reflecting the activity of thrombomodulin, as well as the expression of the thrombomodulin antigen, is regulated by some bone resorption-enhancing factors. 1,25-dihydroxyvitamin D3 and retinoic acid increase thrombomodulin expression and activity in a dose-dependent manner whereas tumor necrosis factor alpha and interleukin 1 decrease these parameters. Because thrombomodulin is known to inhibit single-chain urokinase-type plasminogen activator, a molecule present in the osteoblast microenvironment, these findings suggest that thrombomodulin could play a role in the regulation of bone resorption by modulating the plasmin system.
 
Article
The findings of specific binding of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in normal rat pituitary tissue and selective effects of 1,25-(OH)2D3 on gene expression in clonal pituitary tumour cells have suggested that vitamin D may regulate pituitary function. Therefore, the in vitro effect of 1,25-(OH)2D3 on normal pituitary cells was investigated. Primary anterior pituitary cell cultures prepared from female rats were maintained in experimental medium +/- 10(-8) M 1,25-(OH)2D3 for up to 24 h and then incubated with fresh experimental medium containing TRH (10(-10)-10(-8) M) or vehicle for 1 h. Pretreatment with 1,25-(OH)2D3 for 24 h led to increased TSH release at all TRH concentrations tested (P less than 0.0001), a decrease in the half-maximal stimulatory dose of TRH for TSH release from 2 X 10(-9) M to 0.4 X 10(-9) M, a 22% increase in maximal TSH release (P less than 0.01), and an 81% increase in TSH release at 10(-9) M TRH (P less than 0.001). 1 X 10(-9) M 1,25-(OH)2D3 increased TRH (10(-9) M)-induced TSH release by 20% (P less than 0.05) but 10(-7) M and 10(-6) M 25-hydroxyvitamin D3 (25-OH D3) had no effect. The effect of 1,25-(OH)2D3 on TRH (10(-9) M)-induced TSH release was evident within 8 h and was maximal by 16 h. There was no effect on basal TSH release, TSH accumulation in the medium in the preceding 24 h nor on cell-associated TSH. 1,25-(OH)2D3 pretreatment had no effect on TRH-induced PRL secretion, PRL accumulation in the medium nor on cell-associated PRL. We have shown that 1,25-(OH)2D3 acts selectively on the thyrotroph to enhance in vitro responsiveness to physiologically relevant concentrations of TRH. These findings are consistent with the reported autoradiographic localization of [3H]-1,25-(OH)2D3 in the thyrotroph and support a permissive or regulatory role of vitamin D in the normal pituitary gland.
 
Article
Physiological studies of the Atlantic cod, Gadus morhua, have suggested a role for the vitamin D3 system in this marine teleost similar to that in other vertebrates. Accordingly, the present study was carried out to assess the plasma concentrations of vitamin D3, 25-hydroxyvitamin D3 (25OHD3), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in this fish. Additionally, the presence of binding proteins in plasma and target-specific tissue receptors for these vitamin D3 metabolites was studied in organs normally associated with calcium regulation. Plasma levels of 25-OHD3 (undetectable to 148 pg/ml; n = 5) were comparatively low (20-30 ng/ml), whereas the levels of vitamin D3 (approximately 30 ng/ml) and 1,25-(OH)2D3 (approximately 50 pg/ml) were comparable to levels reported in higher vertebrates. Cod plasma contained a protein that bound both 25OHD3 and 1,25-(OH)2D3. This plasma binding protein revealed low affinity for 25OHD3, did not bind G-actin, and had a sedimentation coefficient of 3.4S. High affinity 1,25-(OH)2D3 receptors [Kd, 1.02 +/- 0.36 (n = 6), 1.02 +/- 0.3 (n = 5), and 0.95 +/- 0.51 (n = 5) nM; mean +/- SEM] were found in high salt cytosols from intestine, liver, and gills, respectively, and had sedimentation coefficients (3.6-3.8S in 0.3 M KCl sucrose gradients) similar to those in higher vertebrates. No specific 1,25-(OH)2D3 binding was found in kidney, ultimobranchial glands, corpuscles of Stannius, or bone. The finding of significant quantities of 1,25-(OH)2D3 in the plasma, the presence of plasma binding proteins that bind this seco-steroid, and the localization of specific high affinity receptors for this metabolite in calcium regulatory tissues in teleosts are all consistent with a physiological role for the vitamin D3 system in the calcium regulation of the cod.
 
Article
Although many patients with humoral hypercalcemia of malignancy exhibit reduced serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels, N-terminal fragments of recently identified PTH-related protein as well as PTH itself elevate serum 1,25-(OH)2D concentrations. In the present study, the effect of tumor extracts from human tumor-implanted hypercalcemic nude rat models with high and low serum 1,25-(OH)2D on renal 1,25-(OH)2D3 production was examined using rat kidney cells in culture. Whereas tumors from rats with high serum 1,25-(OH)2D levels (OCC rats) contained only a single peak of cAMP production-stimulating activity (CPSA) in osteogenic sarcoma cells on reverse phase HPLC, tumor extracts from rats with low serum 1,25-(OH)2D levels (UCC rats) contained at least two peaks of CPSA. The main peak (peak A) was estimated to be approximately 17K by gel permeation chromatography, which was the same as the molecular size of the hitherto identified PTH-related protein, and a minor peak of CPSA (peak B) was estimated to be about 25K. When peak A or crude extracts of OCC tumors as well as human PTH-(1-34) were added to primary cultures of rat kidney cells, the production of 1,25-(OH)2D3 was significantly stimulated. In contrast, although peak B or crude UCC tumor extracts had no effect on 1,25-(OH)2D3 production in themselves, when they were added together with peak A or human PTH-(1-34) the stimulation of 1,25-(OH)2D3 production was almost completely inhibited. Both peak A and peak B enhanced cAMP production in cultured kidney cells, and the cAMP production by peak A was not affected by peak B. These results are consistent with the possibility that elaboration of an additional factor from tumor cells may be the mechanism by which serum 1,25-(OH)2D levels are suppressed in patients with humoral hypercalcemia of malignancy. The nature as well as the mechanism of action of this factor remain to be elucidated.
 
Article
1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is known to modulate the development of bone and other mesenchymal cell types. Since osteoblasts and adipocytes are thought to arise in bone marrow from a common progenitor, this work examined the effects of 1,25-(OH)2D3 on adipocyte development, and in particular on the expression of lipoprotein lipase (LPL), which is an early marker for the differentiated adipocyte. 3T3-L1 preadipocytes were cultured in the presence of 1,25-(OH)2D3 (10(-9) to 10(-7) M) for up to 7 days. LPL activity was measured in the medium and cell extracts, and LPL messenger RNA levels were measured by Northern blotting. When compared to control cells, 10(-7) M 1,25-(OH)2D3 increased medium LPL activity by 2- to 3-fold and cellular LPL by 1.5-fold. Significant increases in medium and cellular LPL were observed at 10(-9) M and were maximal at 10(-7) M. Along with the increase in LPL activity, there was an increase in LPL messenger RNA by 2-fold at 5 days, and by 5-fold at 7 days. In addition to an increase in LPL, 1,25-(OH)2D3 increased expression of aP2, an adipocyte-specific marker associated with differentiation. After the addition of 1,25-(OH)2D3, there was a decrease in 3T3-L1 cell number, which is consistent with differentiation, and a decrease in vitamin D receptors. Finally, these cells developed a different morphology. 1,25-(OH)2D3-treated cells assumed a rounded appearance, although without detachment from the dish and without the degree of lipid accumulation usually associated with the addition of insulin, isbutylmethylxanthine, and dexamethasone. It is concluded that 1,25-(OH)2D3 induced LPL expression in 3T3-L1 cells through an induction of differentiation-dependent mechanism(s). These findings suggest an important role for 1,25-(OH)2D3 in normal adipocyte differentiation.
 
Article
Skeletal unloading results in osteopenia. To examine the involvement of vitamin D in this process, the rear limbs of growing rats were unloaded, and alterations in bone calcium and bone histology were related to changes in serum calcium (Ca), inorganic phosphorus, 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D [24,25-(OH)2D], and 1,25-dihydroxyvitamin D [1,25-(OH)2D]. Acute skeletal unloading induced a transitory inhibition of Ca accumulation in unloaded bones. This was accompanied by a transitory rise in serum Ca, a 21% decrease in longitudinal bone growth (P less than 0.01), a 32% decrease in bone surface lined with osteoblasts (P less than 0.05), no change in bone surface lined with osteoclasts, and a decrease in circulating 1,25-(OH)2D from 130 +/- 10 to 53 +/- 11 pg/ml. No significant changes in the serum concentrations of inorganic phosphorus, 25-hydroxyvitamin D, or 24,25-(OH)2D were observed. After 2 weeks of unloading, bone Ca stabilized at approximately 70% of control values, and serum Ca and 1,25-(OH)2D returned to control values. Maintenance of a constant serum 1,25-(OH)2D concentration by chronic infusion of 1,25-(OH)2D (Alza osmotic minipump) throughout the study period did not prevent the bone changes induced by acute unloading. These results suggest that acute skeletal unloading in the growing rat produces a transitory inhibition of bone formation, which, in turn, produces a transitory hypercalcemia, leading to a temporary decrease in serum 1,25-(OH)2D. No evidence could be found for a direct involvement of 1,25-(OH)2D in the bone changes induced by skeletal unloading.
 
Article
Previous studies demonstrated that 1,25-dihydroxyvitamin D [1,25-(OH)2D] treatment in vivo stimulates [125I]calmodulin (CaM) binding to several proteins (detected by [125I]CaM gel overlay) in cytosol preparations from rat kidney. This study establishes the sizes of the principal stimulated forms and physiological aspects of their stimulation by the hormone. Densitometric analysis of the 1,25-(OH)2D-stimulated [125I]CaM binding activities demonstrated induction of two major bands, M(r) = 110 +/- 2.4 and 94 +/- 1.2 K. This analysis also revealed induction of a previously existing band at 150 +/- 2.7 K and induction of a 74 +/- 1.1 K band. 1,25-(OH)2D-induction of the [125I]CaM binding activities (CaMBP-Ds) was observed in both vitamin D-deficient and normal vitamin D-sufficient rats. The [125I]CaM binding activities were abolished by incubation with 1000-fold excess CaM, but not calbindin-D28, troponin C, parvalbumin, or alpha-lactalbumin. 1,25-(OH)2D induction of the [125I]CaM binding activities exhibited a graded dose response at 5-100 ng/day, and 5-7 days treatment was required for strong induction. The [125I]CaM binding activities in the kidney exhibited differential subcellular distributions: 150 K CaMBPs were present in crude preparations of nuclei, microsomes, and mitochondria; a 110 K CaMBP was present in the microsomal preparation; and the 94 and 74 K CaMBPs were restricted to the cytosol. 1,25-(OH)2D treatment resulted in the induction of the microsomal 110 K CaMBP and possibly the nuclear (but not in mitochondrial or microsomal) 150 K CaMBPs. In conclusion, there are at least four 1,25-(OH)2D-induced [125I]CaM binding activities in the rat kidney, with some variations in subcellular distribution. Moreover, their pattern of induction suggests that 1,25-(OH)2D regulation of the [125I]CaM binding activities is not a part of the immediate 1,25-(OH)2D signal transduction pathway, but rather may result from altered genomic activity after hormone treatment.
 
Top-cited authors
Jan-Ake Gustafsson
  • Nova Scotia Museum
Kaj Grandien
Bo Carlsson
  • Stockholm University
Allan E Herbison
  • University of Otago
Manuel Tena-Sempere
  • University of Cordoba (Spain)