17-beta-estradiol treatment maintains differentiative potential of virgin mouse mammary gland in culture and its responsiveness to insulin

Synthesis of the milk proteins, casein and alpha-lactalbumin was not induced in cultured mammary explants from C3H/HeN castrated virgin mice in the presence of lactogenic hormones such as insulin, cortisol and PRL. Replacement therapy with 17-beta-estradiol to the castrated mice completely restored the differentiative potentials of mammary explants, inducing synthesis of the two milk proteins. [3H]thymidine incorporation into DNA synthesized in cultured mammary explants was also decreased by castration to less than 50% (P less than 0.001) of that obtained in intact mice, and 17-beta-estradiol treatment to castrated animals restored DNA synthesis to 90% of the intact level. The addition of insulin to culture medium significantly (P less than 0.001) enhanced [3H]thymidine incorporation into DNA in cultured mammary explants from both intact and 17-beta-estradiol-treated castrated mice but not from castrated animals. Insulin binding sites (1710 +/- 260 sites/cell) to mammary epithelial cells from castrated mice were significantly (P less than 0.05) lower than those from both intact (2870 +/- 300 sites/cell) and 17-beta-estradiol-treated castrated animals (2860 +/- 190 sites/cell). The present findings suggest that 17-beta-estradiol maintains growth and differentiative responses of mammary epithelial cells to insulin, which may be through preserving the number of insulin binding sites in the cells.     

Progesterone action in normal mouse mammary gland

Previously it has been shown that progesterone, as well as estrogen, plays an important role in the growth of the mammary gland. Eighty percent of mammary progesterone receptors (PgR) are estrogen-inducible and are localized in the epithelium; the remaining 20% of PgR are estrogen-independent and appear to be localized in the mammary stroma. The purpose of the present study was to investigate how progestins promote mammary growth in relation to their interactions with epithelial and stromal components of the gland and to assess the role of estrogen in these interactions. Progestins [progesterone, [17 beta-methyl-3H]promogestone (R5020), and medroxy progesterone acetate] alone or in combination with estrogen were combined with Elvax 40P and implanted directly into mammary glands. The effect of hormones on cell proliferation was determined by observing changes in mammary gland morphology and by quantitating DNA synthesis in both epithelial and stromal cells by DNA histoautoradiography. The results demonstrate that in mammary epithelial cells the effects of progestins on mammary gland morphology and DNA synthesis are locally mediated such that proliferative changes in the hormone-implanted glands were greater than in contralateral control glands. Dose-response studies with estrogen and R5020 revealed that the extent of progestin activity was only partially dependent upon the R5020 dose with the major determining factor being the dose of estrogen. Analysis of the effect of estrogen on mammary PgR concentration indicates that the degree and pattern of the morphological response of ductal sidebranching and increases in DNA synthesis are largely due to the increase in estrogen-dependent PgR. The antiprogestin, 11 beta-(4-dimethylamino-phenyl)1-17 beta-hydroxy-17 alpha-(prop-1ynyl)-estra-4,9-diene-3-one (RU486), blocks the proliferation in the epithelium that is mediated through estrogen-dependent PgR. In contrast, in stromal cells progestin activity is not estrogen-dependent, and stimulation of DNA synthesis was not confined to the hormone-implanted glands. Furthermore, RU486 stimulates stromal cell DNA synthesis, and this response is augmented by estrogen. While progestin effects in epithelial cells appear to be mediated by estrogen-dependent PgR, the mechanism operative in stromal cells appears to be different and remains to be elucidated.     

Angiotensin stimulation of bovine adrenocortical cell growth.

Factors controlling proliferation of adrenocortical cells have been studied in monolayer cultures of bovine adrenocortical cells. Angiotensin II stimulated cell proliferation and [3H]thymidine incorporation into DNA with a half-maximal effective concentration of 0.96 +/- 0.27 nM. Similar sensitivity to angiotensin III with reduced sensitivity to angiotensin I and tetradecapeptide renin substrate was observed. Although sensitivity to angiotensin II was equivalent to that for fibroblast growth factor (1.5 nM half-maximal effective concentration), maximal effects of angiotensin were less than for fibroblast growth factor and serum. High concentrations of insulin (1-10 micrometer) also stimulated [3H]thymidine incorporation into DNA and cell proliferation. [Sar1,Ile5,Ile8]Angiotensin II, a competitive antagonist of angiotensin II, blocked angiotensin II stimulation of DNA synthesis but did not affect fibroblast growth factor and insulin stimulation of DNA synthesis. Corticotropin (ACTH) blocked the stimulatory effects of both angiotensin II and fibroblast growth factor. The dose-response curves for angiotensin II stimulation of steroidogenesis were similar to those for stimulation of [3H]thymidine incorporation into DNA. Among the seven cell types examined, only adrenocortical cells responded to angiotension II with stimulation of DNA synthesis.     

Estrogen responsiveness of normal mouse mammary cells in primary cell culture: association of mammary fibroblasts with estrogenic regulation of progesterone receptors

Estrogens enhance proliferation of normal mouse mammary cells in vivo. However, when cultured alone, normal mouse mammary epithelial cells fail to exhibit a proliferative response to estrogen in vitro; the basis for this lack of in vitro responsiveness to estrogen is not known. The purpose of the present study is to determine if cultured normal mouse mammary cells possess estrogen receptors (ER) and/or progesterone receptors (PgR) and if the ER mechanism is functional, as measured by the ability of estrogens to regulate PgR. Recent findings that mammary fibroblasts can influence the behavior of mammary epithelial cells in vitro led us to investigate their effect on epithelial cell responsiveness to estrogen. In these studies, collagenase-dissociated mammary glands of midpregnant BALB/c mice were the source of mixed cultures (containing both epithelial cells and fibroblasts) and epithelial or fibroblast cultures. The purity of epithelial or fibroblast cultures was quantified immunocytochemically using antivimentin antibody as a fibroblast marker. Steroid hormone binding was quantified in intact cultured cells using [3H]R5020 and 17 beta-[3H]estradiol as the ligands. Specific high affinity binding sites for estrogen (Kd = 3.1 +/- 0.8 X 10(-10] and progestins (Kd = 3.3 +/- 1.2 X 10(-9) M) were detected in mixed cultures. To assess the possible role of mammary fibroblasts, we investigated cultures containing only fibroblasts which were derived by differential centrifugation. When 17 beta-estradiol was added to the culture medium, a significant (P less than 0.01) increase in PgR concentration was observed in mixed cultures. While mixed cultures maintain responsiveness to estrogen in vitro, as measured herein, the epithelial cultures, derived by differential centrifugation and Percoll gradient sedimentation, did not. However, estrogenic regulation of PgR appears to be specific to epithelial cells in mixed cultures, since fibroblast cultures neither contained PgR nor displayed estrogen-inducible PgR. The lack of responsiveness of epithelial cultures is not due to a loss or decrease in the ER concentration. Thus, the presence of mammary fibroblasts appears to be associated with epithelial cell responsiveness to estrogen in vitro.     

Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: effect of human placental lactogen, somatomedin-C, multiplication-stimulating activity, and insulin

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.     

Insulin and insulin-like growth factors stimulate deoxyribonucleic acid synthesis in PC12 pheochromocytoma cells

The effects of insulin and insulin-like growth factors (IGFs) on the replication of PC12 pheochromocytoma cells were investigated. Incubation of PC12 cells for 2-3 days in low (0.3%) serum medium decreased [3H]thymidine incorporation into PC12 cell DNA to approximately 30% of that in control (15% serum) medium. Incubation of the cells in low serum medium also slowed the growth of the cultures and increased the percentage of cells in the G0/G1 phase of the cell cycle. Addition of insulin to cells in low serum medium increased [3H]thymidine incorporation into the cells, increased the number of cells in PC12 cultures, and decreased the percentage of cells in the G0/G1 phase of the cell cycle. IGF-I and IGF-II also increased [3H]thymidine incorporation into PC12 cells incubated in low serum medium. IGF-I (EC50, approximately 0.3 nM) was a more potent stimulus of [3H]thymidine incorporation than was insulin (EC50, approximately 3.5 nM). These data suggest that insulin and IGFs are growth factors for PC12 cells, and that the growth-promoting effects of these agents may be mediated by a type I IGF receptor on PC12 cells.     

Interleukin-1 stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro.

The number of interstitial macrophages in the testis fluctuates according to age, increasing gradually during prepubertal development to reach 15-20% in the interstitial compartment in the adult rat. These macrophages are in close morphological association with Leydig cells. Macrophage products, interleukin-1 (IL-1) and tumor necrosis factor alpha stimulate and/or inhibit steroid production in cultures of Leydig cells. We have studied the effects of macrophage products on DNA synthesis in rat Leydig cells to investigate a possible paracrine role of testicular macrophage products on the proliferation of Leydig cells. Leydig cells isolated from 10-, 20-, and 70-day-old rats were cultured for 48 h in serum-free medium, washed, and treated with different cytokines for 18 h. The medium was then removed, fresh medium containing 0.5 microCi [3H]thymidine was added, and cells were incubated for 4 h prior to determining the incorporation of [3H]thymidine into DNA. Human recombinant IL-1 beta caused a dose-dependent stimulation in the incorporation of [3H]thymidine into DNA in the Leydig cells from 10- and 20-day-old rats but had no effect on DNA synthesis in interstitial cells from adult rats. Maximum stimulation of DNA synthesis in immature Leydig cells was observed with 1-2 ng/ml IL-1 beta. Autoradiography after incubation with [3H]thymidine showed a dramatic increase in the number of labeled Leydig cells after treatment with IL-1 beta (19.27 +/- 3.77% vs. 1.44 +/- 0.52% in control cultures) indicating that IL-1 beta recruited more cells to enter the cell cycle and initiate DNA synthesis. Human recombinant IL-1 alpha and tumor necrosis factor alpha also caused significant stimulation of DNA synthesis in Leydig cells but these cytokines were much less potent (1-10%) than IL-1 beta. IL-1 beta enhanced the effects of maximally effective concentrations of growth-promoting agents previously known to stimulate DNA synthesis in immature rat Leydig cells, i.e. human CG, steroidogenesis-inducing protein, and transforming growth factor alpha plus insulin. On the basis of these results it is concluded that IL-1 may play an important role in the proliferation of Leydig cells during prepubertal development in immature rats.     

Hormonal control of bone collagen synthesis in vitro. Effects of insulin and glucagon.

The direct effects of porcine insulin and glucagon on bone collagen and non-collagen protein synthesis have been examined in cultures of calvaria obtained from 21-day fetal rats. Bones were incubated for 24 to 96 h and [3H]proline was added for the last 2 h of culture. Incorporation of the label into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Insulin increased the labeling of CDP by 60 to 115% at concentrations of 10(-9) to 10(-6) M. A smaller stimulatory effect was observed on NCP. The effect on CDP appeared after 12 to 24 h of culture, was maintained for 96 h in the continuous presence of the hormone, but was lost within 3 h of removal of insulin from the culture medium. Insulin appeared to have a direct effect on collagen synthesis and not on collagen breakdown. Insulin did not affect the incorporation of [3H]uridine or [3H]thymidine into the RNA and DNA fractions of bone at 24 h. Insulin opposed the inhibitory effects of parathyroid hormone and dibutyryl cyclic-3',5'-adenosine monophosphate and to a lesser extent, the inhibitory effect of isobutylmethylxanthine on the labeling of CDP. Glucagon did not affect the response to insulin and by itself had small and variable inhibitory effects on proline incorporation.     

Magnesium and calcium effects on uptake of hexoses and uridine by chick embryo fibroblasts.

Cultures of chick embryo fibroblasts were incubated for varying periods in media containing different concentrations of Ca2+ and Mg2+-Mg2+ deprivation produced a gradual decrease in the Vmax of the glucose transport system for the D-glucose analogues 3-O-[3H]methyl-D-glucose and 2-deoxy-D-[3H]glucose and a parallel decrease in the rate of production of lactate from glucose in the medium. It greatly reduced the rates of [3H]uridine uptake and incorporation by decreasing the Vmax of the uridine transport system. Addition of Mg2+ to Mg2+-deprived cultures rapidly increased the rate of [3H]uridine uptake without requiring protein synthesis and increased the rate of 2-deoxy-D-[3H]glucose uptake without requiring RNA synthesis. These effects of changes in Mg2+ concentration qualitatively reproduce the effects of such variables as cell density and serum and insulin concentrations. Ca2+ deprivation resulted in similar, though much smaller, changes in the activities of the two transport systems, but also greatly increased the "leakiness" of the cells to the nontransported hexose L-[3H]glucose.     

Steroidogenesis-inducing protein promotes deoxyribonucleic acid synthesis in Leydig cells from immature rats.

A protein [steroidogenesis-inducing protein (SIP)] has been isolated from human ovarian follicular fluid and shown previously to stimulate steroidogenesis in Leydig cells, adrenal cells, and early luteal cells. Since proteins and peptides known to regulate steroidogenesis, such as gonadotropins and growth factors, also influence the growth of gonadal cells, the present study was designed to assess the effects of SIP on the synthesis of DNA by Leydig cells in vitro. Leydig cells were isolated from 10- and 20-day-old rats and cultured in serum-free medium for 48 h. The cells were then treated with the test materials for 18 h. Incorporation of [3H]thymidine into DNA was measured during the final 4 h of the culture. SIP significantly stimulated DNA synthesis in Leydig cells in a dose- and time-dependent manner, and the response to SIP was higher than that obtained with maximal concentrations of LH/hCG. The stimulatory effects of SIP were significantly enhanced when the cell cultures were preincubated in the presence of low levels of ovine LH (2 ng/ml). Cultures treated with SIP, followed by incubation with [3H]thymidine, contained 22 times as many labeled cells as control cultures, as assessed by autoradiography. The cells that were labeled were identified morphologically as Leydig cells. Insulin/insulin-like growth factor-I and/or transforming growth factor-alpha alone stimulated DNA synthesis and enhanced the effects of SIP on DNA synthesis. Dramatic changes in the morphology of cultured Leydig cells treated with SIP were observed; cells became flattened and developed extended projections which connected adjacent cells. LH/hCG, insulin, and transforming growth factor-alpha did not induce effects comparable to those of SIP on the morphology of Leydig cells. The effects of SIP on the synthesis of DNA and the morphology of Leydig cells were blocked in the presence of cycloheximide. It is concluded that SIP not only stimulates steroid production in Leydig cells, as shown previously, but also stimulates DNA synthesis and induces morphological changes in these cells. The latter properties of SIP combined with the magnitude of the responses elicited identify SIP as a unique gonadal protein.     

Mammary growth and function and pituitary prolactin secretion in female nude mice

Mammary structural growth in the wholemount preparation, content and synthesis of mammary DNA and RNA estimated by the incorporation of [3H]thymidine and [14C]uridine, pituitary and plasma levels of prolactin and weights and histological structures of some organs of female nude mice (nu/nu) were compared to those of the control (nu/+) with the same genetical background (BALB/c). Both at 3 months of age and on day 1 of lactation, the weights per 100 g body weight of adrenals, spleen and liver of nu/nu mice were significantly higher than those of nu/+ mice. Mammary growth stimulation by pituitary graft was much more marked in nu/nu mice than in nu/+ mice. Slight differences between groups were found in the pituitary and plasma levels of prolactin, in the histological structures of ovaries as well as of the adrenals and thyroids and in the pattern of oestrous cycles. On the other hand, the content and synthesis of mammary DNA at 3 months of age and content and synthesis of both DNA and RNA and RNA/DNA ratio on day 1 of lactation were significantly higher in nu/+mice than in nu/nu mice. All findings suggest that thymus deficient immunosuppression has deleterious effects on mammary growth and function without its alteration in the secretion of prolactin and oestrogen and probably through its decrease in mammary responsiveness to mammotrophins.     

Glucocorticoid-induced formation of tight junctions in mouse mammary epithelial cells in vitro.

Phenotypically stable cultures of untransformed mouse mammary epithelial cells (denoted 31EG4) were established and utilized to investigate the lactogenic hormone (glucocorticoids, insulin, and prolactin) regulation of tight junction formation. When 31EG4 cells were grown on permeable supports for 4 days in medium containing the synthetic glucocorticoid dexamethasone and insulin, confluent cell monolayers obtained a transepithelial electrical resistance (TER) of 1000-3000 omega.cm2. In contrast, over the same time period, confluent monolayers treated with insulin or insulin and prolactin maintained a low TER (35-150 omega.cm2). Consistent with the formation of tight junctions, apical to basolateral paracellular permeability was decreased from 12% to 1% for [14C]mannitol and 3.3% to 0.3% for [3H]inulin when cells were cultured in dexamethasone. This effect of dexamethasone on TER required extracellular calcium, de novo protein synthesis, dose-dependently correlated with glucocorticoid receptor occupancy, and was not due to an increase in cell density. As shown by direct and indirect immunofluorescence microscopy, dexamethasone treatment did not modulate the production or location of filamentous actin, the tight junction protein ZO-1, or the cell adhesion protein E-cadherin. Our results suggest that glucocorticoids play a fundamental role in the function and maintenance of cell-cell contact in the mammary epithelia by inducing the formation of tight junctions.     

Neoplastic transformation of rat mammary cells exposed to 7,12-dimethylbenz[alpha]anthracene or N-nitrosomethylurea in cell culture.

Primary cultures of mammary cells from virgin Lewis rats were seeded at 5 X 10(5) cells per cm2 in medium 199 supplemented with 10% fetal calf serum, insulin (5 microgram/ml), prolactin (5 microgram/ml), estradiol (5 ng/ml), progesterone (0.5 microgram/ml), and hydrocortisone (0.5 microgram/ml). On the second or third day of culture, cells were exposed to either 7,12-dimethylbenz[alpha]anthracene (0.1 microgram/ml for 24 hr) or N-nitrosomethylurea (80 microgram/ml for 2 hr). The cells were later assayed for transformation by transplanting 10(6) or 10(5) cells into gland-free mammary fat pads of 3-week-old female hosts. Untreated cells produced only normal mammary outgrowths when transplanted. Cells treated with 7,12-dimethylbenz[alpha]anthracene or N-nitrosomethylurea produced abnormal outgrowths in 11% of the transplants. These abnormal outgrowths ranged from rapidly growing adenocarcinoma to alveolar and ductal hyperplastic lesions. The results indicate that rat mammary epithelial cells can be transformed by exposure to chemical carcinogens in culture and thus represent a potential in vitro model for epithelial cell transformation.     

Regulation of Y-organ ecdysteroidogenesis by molt-inhibiting hormone in crabs: involvement of cyclic AMP-mediated protein synthesis

The crustacean neuropeptide, molt-inhibiting hormone (MIH), directly inhibits Y-organ ecdysteroidogenesis, an effect mediated by cyclic AMP (cAMP) and antagonized by calcium-calmodulin. We investigated regulation of Y-organ protein. RNA, and DNA syntheses by MIH, cAMP, and calcium in relation to steroidogenesis in vitro. Ecdysteroid production and [3H]leucine incorporation into protein were inhibited 50-60 and 80-90%, respectively, by MIH activity in eyestalk extracts (4 eyestalk equivalents), 10(-6) M forskolin, or a combination of 10(-2) M dibutyryl cAMP and 10(-4) M 3-isobutyl-1-methylxanthine (dbcAMP-IBMX). Calcium ionophore A23187 (10(-4) M) stimulated ecdysteroidogenesis two-fold, did not affect the relatively high basal (control) rate of protein synthesis, and reduced the inhibitory effects of forskolin on steroidogenesis and protein synthesis. Incorporation of [3H]uridine into RNA was unaffected by MIH, forskolin, or A23187 but was reduced 50% by dbcAMP-IBMX. Basal rates of [3H]thymidine incorporation into DNA were low and were not affected by treatments. The effects of MIH were specific; extracts of brain or muscle did not alter Y-organ steroidogenesis or protein synthesis, while muscle extract increased precursor incorporation into RNA. Eyestalk extract did not affect [3H]leucine incorporation into protein of brain, muscle, or gill. Cycloheximide (5 micrograms/ml) depressed protein synthesis 90% and steroidogenesis 60%, enhanced the inhibition induced by MIH, and blocked the stimulation of steroidogenesis induced by A23187; effects on basal steroidogenesis were evident after 1 hr. Actinomycin D (1 microgram/ml) depressed RNA synthesis 86% but did not alter basal, MIH-inhibited, or A23187-stimulated ecdysteroidogenesis during incubations. These results indicate that MIH suppresses Y-organ steroidogenesis in part by inhibiting protein synthesis at the translational level; the effect is mediated by cAMP. The stimulation of steroidogenesis by calcium, mediated by lowering cAMP, also appears to depend in part upon protein synthesis.     

Epidermal growth factor and insulin-induced deoxyribonucleic acid synthesis in primary rat hepatocytes is phosphatidylinositol 3-kinase dependent and dissociated from protooncogene induction

The mitogenic response to insulin and epidermal growth factor (EGF) was studied in subconfluent and confluent cultures of primary rat hepatocytes. In subconfluent cultures, wortmannin, LY294002, and rapamycin reversed insulin- and EGF-induced [3H]thymidine incorporation into DNA. The mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) inhibitor PD98059 was without significant effect on either insulin- or EGF-induced [3H]thymidine incorporation. Insulin treatment did not alter levels of messenger RNAs (mRNAs) for c-fos, c-jun, and c-myc. EGF induced an increase in c-myc, but not c-fos or c-jun, mRNA levels in subconfluent hepatocyte cultures. This increase in c-myc mRNA was abolished by PD98059. In confluent cells that could not be induced to synthesize DNA, EGF treatment also promoted an increase in c-myc mRNA to levels seen in subconfluent cultures. This increase was also abrogated by PD98059. These data indicate that in primary rat hepatocyte cultures, 1) the phosphoinositol 3-kinase pathway, perhaps through p70s6k activation, regulates DNA synthesis in response to insulin and EGF; 2) the MAPKpathway is not involved in insulin- and EGF-induced DNA synthesis; and 3) p44/42 MAPKs are involved the induction of c-myc mRNA levels, although this induction is not required for DNA synthesis. These studies define two distinct signal transduction pathways that independently mediate growth-related responses in a physiologically relevant, normal cell system.     

Temporal sequence of prolactin actions on phospholipid biosynthesis in mouse mammary gland explants

Studies were carried out to determine the effect of PRL on the metabolic fate of 32PO4 in cultured mammary gland explants derived from 12 to 14-day pregnant mice. Explants were initially cultured for 24-36 h with 10(-7) M cortisol and 1 microgram/ml insulin. PRL (1 microgram/ml) was then added to certain of the cultures and incubation continued for 2-24 h. Tissues were pulse labeled with 5 microCi/ml 32PO4 during the final 2 h of culture. Tissues were fractionated by the method of Bligh-Dyer. Radioactivity was determined in the organic fraction (containing phospholipids), the aqueous fraction, and an insoluble fraction containing macromolecules. In all these fractions, PRL effected a greater than 2-fold increase in radioactivity content; the onset of the PRL responses was 8-12 h after PRL exposure and PRL effected responses at concentrations of 2.5 ng/ml and above. The enhanced rate of 32P incorporation in the macromolecular fraction was found to occur in both the RNA and phosphoproteins in that fraction. As determined by TLC, PRL was also found to increase 32P incorporation into all phospholipid fractions. This was confirmed by observing that [3H]inositol and [3H]choline incorporation into their respective phospholipids was also increased by PRL; the time sequence of response was similar to that when 32PO4 incorporation was determined. The magnitude of the PRL stimulation of 32PO4 incorporation, however, was about 2-fold higher than either the [3H]inositol or [3H]choline incorporation. The magnified response when 32PO4 was employed may reflect a PRL effect on phosphate uptake into the mammary cells; this is supported by the fact that the radioactive content of the aqueous fraction was significantly elevated in the Bligh-Dyer extract. The effect of PRL on phospholipid synthesis probably reflects the initiation of the packaging process involved in the assimilation of milk products.     

Effects of insulin-like growth factors on chick embryo hepatocytes

Biological effects of insulin-like growth factors (IGF) I and II on primary cultures of chick embryo liver cells have been investigated and compared 1) with the biological effect of insulin and 2) with competitive binding of the three hormones to their respective binding sites. IGF I and II stimulate the incorporation of D[U-14C]-glucose into liver cell glycogen in a time- and dose-dependent manner, but with a 5-10-fold lower potency than insulin. Both IGFs also lead to enhanced incorporation of 5-[3H]uridine and L[U-14C]valine into trichloroacetic acid (TCA) insoluble material and to activation of ornithine decarboxylase activity. Their potency in stimulating RNA synthesis and ornithine decarboxylase activity is comparable to that of insulin. Protein synthesis is maximally stimulated at 3 nM by all three hormones. In the competitive binding studies, IGF I and II are 10-fold less potent than insulin in competing for [125I]insulin binding, but 100-fold more potent than insulin in competing for [125I]IGF I or II binding. These studies show that IGF I and II stimulate the same metabolic indices as insulin in the chick embryo liver. By comparing these biological effects with competitive binding data it appears that IGFs act on glucose metabolism in the chick embryo liver via the insulin receptor, whereas stimulation of growth indices by IGFs and insulin appears to be mediated by their own specific receptors.     

Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes.

Adult rat hepatocytes have been previously isolated and maintained in monolayer culture, but attempts to stimulate DNA synthesis have been unsuccessful. Hormonal conditions are now described which induce DNA synthesis in cultured hepatocytes from partially hepatectomized rats. DNA synthesis was determined autoradiographically by the incorporation of [3H]thymidine into nuclei of morphologically distinct hepatocytes. Insulin (4-4000 nM) or epidermal growth factor (10 ng/ml) alone caused significant increases in the labeling index. The two hormones together acted synergistically to produce labeling indices of 35-50% on the third day of culture, compared with 2-7% in control cultures. The addition of glucagon (400 nM) further increased the labeling indes. Dexamethasone (80 ng/ml) inhibited DNA synthesis but, under certain conditions, enhanced cell attachment. Growth hormone and triiodothyronine had no significant effect on DNA synthesis. The mixture of epidermal growth factor, insulin, and glucagon also stimulated incorporation of [3H]thymidine into phenol-extracted DNA. Although DNA synthesis was stimulated, cell division occurred infrequently. These data suggest a prominent role for epidermal growth factor in promoting hepatic DNA synthesis by acting in concert with insulin and glucagon.     

The effects of actinomycin D, puromycin, cycloheximide and hydroxyurea on 3',5,3-triiodo-L-thyronine stimulated 2-deoxy-D-glucose uptake in chick embryo heart cells in vitro

The uptake of 2-deoxy-D-[1-3H]glucose ([1-3H]DOG) by cultured chick embryo heart cells was stimulated by L-triiodothyronine (T3). This stimulation began with the exposure to T3, rose linearly for about 8 h and reached a plateau around 24h. Actinomycin D, puromycin and cyclohexmide had no effect on the stimulated sugar uptake during the first 6h. These agents prevented the further increase in the rate of [1-3H]DOG uptake in T3-treated cultures that occurred between 6 and 24h. In the presence of hydroxyurea the uptake of [1-3H]DOG in both control and T3-treated cells was unchanged. These results suggest that some of the effects of T3 may not require new synthesis of either RNA, proteins or DNA.     

Cyclic AMP: a mitogenic signal for Swiss 3T3 cells.

Addition of cholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal growth factor, and fibroblast-derived growth factor to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose--response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15--500 microM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5--100 microM), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase which are potent inhibitors of cyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ml) and insulin at 10 micrograms/ml (4- to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 microM (35-fold increase in cAMP level), DNA synthesis began after a lag of 16 hr. These results indicate that cAMP acts as a mitogenic signal for Swiss 3T3 cells and differ from the widely held view that cyclic AMP inhibits the proliferation of fibroblast cells.