A test for hormonal responsiveness in a mammary epithelial cell line,NMuMg

Summary The NMuMG cell line derived from normal mouse mammary epithelial cells was tested for responsiveness to hormones. The hormones studied included insulin, glucocorticoids (cortisol and dexamethasone), and prolactin. In addition to membrane bound insulin receptors and prolactin receptors, the cells had 2 × 10⁴ cytoplasmic glucocorticoid receptors per cell. Morphological changes were observed in response to hormones. Clusters of cells appeared with greatly increased diameter, and the number of cells per plate was reduced. The rate of DNA synthesis, corrected by cell number, indicates that cell division, and hence cell turnover, was increased by the combination of all three hormones. Insulin greatly enhanced protein synthesis, but glucocorticoid and prolactin did not further increase the rate. The combination of the three hormones produced a change in the synthesis of histones, consistent with the increase in cell turnover. There were substantial responses of enzyme activities to hormonal treatment of the cells. Insulin by itself induced a doubling of the activity of glyceraldehyde phosphate dehydrogenase and perhaps a modest increase in NADH-cytochromec reductase. Lactose synthetase activity showed a three- to fourfold induction of both A and B subunits of the enzyme when the cells were treated with insulin, glucocorticoid, and prolactin, and the effect of the latter two hormones was shown to be additional to that of insulin. This work was supported by Contract N01-CB-43866 from the National Cancer Institute, by Grants GB-38658 from the National Science Foundation and GMS-20338 from the National Institutes of Health, and by the Agricultural Experimental Station at the University of California.     

Role of insulin receptors in the changing metabolism of adipose tissue during pregnancy and lactation in the rat.

Changes in the volume, the rates of fatty acid synthesis and synthesis of the glycerol moiety of acylglycerols, the activity of lipoprotein lipase, and the number and affinity of insulin receptors of adipocytes, and concentrations of serum insulin, prolactin and progesterone were determined in virgin rats and in rats at various stages of pregnancy and lactation. Changes in the metabolic activities of adipose tissue appeared to be synchronized and primarily comprised a marked decrease in anabolic activity around parturition. In contrast, the number of insulin receptors (Kd 1.5 nM) per adipocyte doubled during pregnancy before returning to normal values around parturition. It is postulated that the increase in the number of insulin receptors is an adaptation to counteract the effects of insulin-antagonistic hormones during pregnancy and that the decrease in the number of receptors is primarily responsible for the loss of anabolic activity around parturition.     

Cortisol 21-mesylate exerts glucocorticoid action on the induction of milk protein synthesis in cultured mammary gland

Cortisol 21-mesylate, an alkylating derivatives of cortisol, was previously shown to exert an anti-glucocorticoid action in rat hepatoma cell culture (Simons, Thompson and Johnson 1980). In this study the effect of cortisol 21-mesylate on milk protein synthesis induced in cultured mouse mammary gland by glucocorticoid, insulin, and prolactin was investigated. Addition of cortisol 21-mesylate at concentrations ranging from 10(-8) M to 10(-6) M produced no inhibition of casein synthesis that was induced by glucocorticoid, insulin and prolactin in mammary explants from midpregnant mice. On the other hand, cortisol 21-mesylate in combination with insulin and prolactin stimulated casein synthesis in cultured tissue. The potency of cortisol mesylate was about 1/10 to 1/30th of that of cortisol. Cortisol 21-mesylate, like cortisol, also augmented the accumulation of alpha-lactalbumin in midpregnant rat mammary tissue cultured in the presence of insulin and prolactin. A cell-free competition study of glucocorticoid receptors using cytoplasmic extracts from mouse mammary tissue showed that cortisol 21-mesylate competitively inhibited the binding of dexamethasone on glucocorticoid receptors. The apparent affinity of cortisol 21-mesylate for glucocorticoid receptors is about 1/10th of that of cortisol. These results indicate that cortisol 21-mesylate acts as a glucocorticoid but not as an antiglucocorticoid in the mammary gland.     

The effect of ethanol, alone and in combination with the glucocorticoids and insulin, on glucose-6-phosphate dehydrogenase synthesis and mRNA in primary cultures of hepatocytes.

The hormonal regulation of the relative rate of synthesis and mRNA of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of adult-rat liver parenchymal cells maintained in a chemically defined medium. Maintenance of hepatocytes from starved animals in a culture medium devoid of any hormones resulted in a 4-fold increase in the relative rate of G6PDH synthesis in 48 h. Parallel cultures treated with glucocorticoids alone exhibited a rate of G6PDH synthesis comparable with that in the control cultures, whereas insulin alone caused a 6.5-fold increase in the rate of synthesis in 48 h. However, if the cultures were treated with glucocorticoids and insulin simultaneously, a 13-fold increase in the rate of synthesis was observed. The effect of ethanol, alone and in combination with the hormones, on the relative rate of G6PDH synthesis was studied also. Ethanol alone caused an 8-fold increase in the rate of synthesis in 48 h, whereas the combination of ethanol, glucocorticoid and insulin caused a 25-fold increase. The amount of functional mRNA encoding G6PDH, as measured in a cell-free translation system, was compared with enzyme activity and relative rate of enzyme synthesis. The increases in G6PDH activity and relative rate of synthesis in primary cultures of hepatocytes treated with ethanol, alone and in combination with the glucocorticoids and insulin, were paralleled by comparable increases in G6PDH mRNA. The results of this study show that the glucocorticoids acted in a permissive manner to amplify the insulin stimulation of G6PDH synthesis and that insulin, glucocorticoids and ethanol interact to stimulate synthesis of G6PDH primarily by increasing the concentration of functional G6PDH mRNA.     

Hormonal regulation of the synthesis of casein and alpha-lactalbumin in a primary mammary cell culture system

The addition of 5 micrograms/ml of both insulin and prolactin, 3 microM cortisol and 5% fetal bovine serum stimulated casein synthesis during a 5 day culture of mammary epithelium from lactating mice using a floating collagen gel as a culture substratum. Omission of any of the three hormones or serum decreased casein synthesis substantially. The use of 10% serum or the attached gel culture system also decreased casein synthesis. Cells cultured with the combination of the three hormones and 5% serum contained a low level of casein mRNA on day 2, but it increased to much higher levels on day 4 and 5, amounting to over 30% of total mRNA on day 5. In contrast to casein synthesis, the maximal increase in alpha-lactalbumin synthesis required the presence of 0.03 microM cortisol. The combination of insulin, prolactin and 3 microM cortisol or insulin and prolactin elicited smaller increases. The translatable mRNA for alpha-lactalbumin in cells cultured with insulin, cortisol and prolactin for 5 days was detected, but not in cells with insulin and cortisol. Both a high and low concentration of cortisol in combination with insulin increased prolactin binding capacity of cultured cells to the same extent, whereas cells cultured with insulin alone contained much lower levels of prolactin binding. The difference in the capacity of prolactin binding between cells cultured with insulin alone and those cultured with insulin and cortisol correlated well with their ability to synthesize casein in response to prolactin.     

Glucocorticoids increase insulin binding and the amount of insulin-receptor mRNA in human cultured lymphocytes.

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.     

Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland

Milk production during lactation is a consequence of the suckling stimulus and the presence of glucocorticoids, prolactin, and insulin. After weaning the glucocorticoid hormone level drops, secretory mammary epithelial cells die by programmed cell death and the gland is prepared for a new pregnancy. We studied the role of steroid hormones and prolactin on the mammary gland structure, milk protein synthesis, and on programmed cell death. Slow-release plastic pellets containing individual hormones were implanted into a single mammary gland at lactation. At the same time the pups were removed and the consequences of the release of hormones were investigated histologically and biochemically. We found a local inhibition of involution in the vicinity of deoxycorticosterone- and progesterone-release pellets while prolactin-release pellets were ineffective. Dexamethasone, a very stable and potent glucocorticoid hormone analogue, inhibited involution and programmed cell death in all the mammary glands. It led to an accumulation of milk in the glands and was accompanied by an induction of protein kinase A, AP-1 DNA binding activity and elevated c-fos, junB, and junD mRNA levels. Several potential target genes of AP-1 such as stromelysin-1, c-jun, and SGP-2 that are induced during normal involution were strongly inhibited in dexamethasone-treated animals. Our results suggest that the cross-talk between steroid hormone receptors and AP-1 previously described in cells in culture leads to an impairment of AP-1 activity and to an inhibition of involution in the mammary gland implying that programmed cell death in the postlactational mammary gland depends on functional AP-1.     

Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland.

Mouse mammary gland contains choline kinase activity that can be stimulated by polyamines. Developmental studies show that the activity of choline kinase in mammary gland is low in both virgin and nonpregnant primiparous animals but increases severalfold during pregnancy and reaches a maximal level during the lactation period. Similar increases in enzyme activity are observed by cultivation of tissue explants in the presence of insulin, cortisol, and prolactin, a combination of hormones which induces the ultrastructural and biochemical changes associated with the development of mammary gland during pregnancy and lactation. The increase in enzyme activity in cultured explants is dependent only on the actions of both insulin and cortisol and parallels the formation of rough endoplasmic reticulum, which is effected by the same combination of hormones. The hormonal stimulation of choline kinase activity appears to involve the action of spermidine, a polyamine which accumulates in the cells under the influence of cortisol and mimicks the effect of cortisol on milk-protein synthesis in cultured explants.     

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.     

Growth hormone and insulin binding to isolated hepatocytes in the genetically dwarf mouse

The interaction of growth hormone with its specific receptors in dwarf mice was investigated. (1) The interaction of 125I-labeled human growth hormone with isolated mouse liver cells is a specific, time-dependent and saturable process. Hepataocytes of male and female dw/dw mice bound only 10-20% as much growth hormone per unit of cell surface area as those of their litter mates. Scatchard analysis suggested that this decrease in binding was due to a decreased number of receptor sites in th liver cell of the dwarf mouse. (2) In contrast to the marked decrease in growth hormone receptors, the binding of insulin is higher in dwarf mice than in litter mates, at low hormone concentration. (3) Competition and stoichiometric studies indicate that growth hormone and prolactin bind to the same type of binding site in female and male mouse hepatocytes. These results indicate that dwarfism in this animal was associated with a loss in the number of growth hormone binding sites. The decrease in growth hormone receptors and the increase in insulin receptors correlate well with the respective biological activity of these two hormones.     

Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal alpha-glucosidase

The levels of functional mRNA encoding glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) were examined in hepatocytes from fasted and fasted/carbohydrate-refed rats and in hepatocytes inoculated into primary culture. Functional G6PDH mRNA was assessed in a cell-free protein synthesis system in vitro. We observed that hepatocytes from fasted/carbohydrate-refed rats had a 12-fold higher level of mRNA than did hepatocytes from fasted rats. The possibility that the adrenal glucocorticoids and insulin were responsible for the increase in G6PDH mRNA in refed rats was examined by studying the effect of insulin and the synthetic glucocorticoid, dexamethasone, on the level of functional G6PDH mRNA in primary cultures of rat hepatocytes maintained in a chemically defined medium. Hepatocytes from fasted rats were inoculated into primary culture and maintained for 48 h either in the absence of hormones or in the presence of insulin alone, dexamethasone alone or both hormones together. We observed that dexamethasone alone caused a fourfold increase in G6PDH mRNA while insulin caused about a twofold increase. Both hormones together elicited an increase that was additive. A comparison of functional G6PDH mRNA levels with the effect of the hormones on G6PDH activity and relative rate of enzyme synthesis suggests that the glucocorticoid elevates the level of G6PDH mRNA within the cell without causing a concommitant increase in the rate of synthesis of the enzyme or the level of G6PDH activity. The results obtained with the primary cultures of hepatocytes indicate that insulin and the glucocorticoids are probably involved with the regulation of hepatic G6PDH mRNA. However, involvement of other hormones, such as thyroid hormone, seems likely since the induced levels of G6PDH mRNA in hepatocytes in culture was one-third of that observed in refed rats.     

Effect of prolactin on maintenance of prelactating human mammary gland in vitro

Summary Human mammary tissue from a female at the end of the second trimester of pregnancy was studied in organ culture in a chemically defined medium. Sampling was carried out at 1, 2 and 3 weeks. Without hormones, there was nearly total lobuloalveolar degeneration inall specimens at all times. Addition of insulin, hydrocortisone and ovine prolactin, in combination at a concentration of 5 μg per ml each, did not affect the extent of degeneration. Raising the concentration of prolactin to 50 μg per ml resulted in greatly improved lobulo-alveolar maintenance inall specimens and continued epithelial cell DNA synthesis for up to 3 weeks in vitro. This work was supported by grant no. CA11536 from the National Cancer Institute.     

Uptake and receptor binding of dexamethasone in cultured 7800 C1 hepatoma cells in relation to regulation of cell growth and peroxisomal beta-oxidation

1. Uptake and binding of dexamethasone to glucocorticoid receptor has been studied in Morris hepatoma 7800 C1 cells in relation to its effect on cell growth and peroxisomal beta-oxidation. 2. Intact cells showed saturable, specific dexamethasone binding of limited capacity and Scatchard analysis revealed one single class of binding sites with equilibrium dissociation constant (Kd) of 0.24 nM similar to other glucocorticoid receptors. However, the binding capacity of 24 fmol/mg cell protein is less than 5% of previously reported values. 3. Uptake of [3H]dexamethasone by intact cells was temperature dependent giving a linear Arrhenius plot with a calculated energy of activation of 58.5 kJ mol-1 x degree-1. 4. Cytosol fractions had specific binding proteins for glucocorticoid hormones with sedimentation coefficient of ca 7S. No specific binding sites for [3H]dexamethasone was demonstrated in purified membrane fractions. 5. Dexamethasone and the synthetic fatty acid analogue tetradecylthio acetic acid (TTA) both inhibited the growth of the 7800 C1 cells and induced the peroxisomal acyl-CoA oxidase activity. A combination of the two compounds gave additive effects. Both these effects of dexamethasone and TTA were counteracted by insulin. 6. We conclude that dexamethasone induces growth inhibition and enzyme induction by binding to functional intracellular glucocorticoid receptors. The action of dexamethasone is consistent with a dissolution in the membrane from where it diffuses passively into the cell and binds to specific receptors in an energy dependent step. 6. The synergistic action of dexamethasone and TTA and the counteraction exerted by insulin are not due to changes in the dexamethasone receptor affinity or binding capacity.     

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.     

Cortisol and prolactin modulation of caudal neurosecretory system activity in the euryhaline flounder Platichthys flesus

Previous studies have shown roles for cortisol and prolactin in osmoregulatory adaptation to seawater and freshwater, respectively, in euryhaline fish. This study of the European flounder investigated the potential for these hormones to modulate activity of the caudal neurosecretory system (CNSS), which is thought to be involved in physiological adaptation to changing external salinity. Superfusion of isolated CNSS with either cortisol or prolactin (10 microM; 15 min) led to changes in firing activity in neuroendocrine Dahlgren cells, recorded extracellularly. Cortisol evoked a modest increase in overall firing activity, with the response delayed by 4 h after treatment. The response to prolactin was short latency, continued to build up over the subsequent 4-h wash period, and comprised increased firing activity together with recruitment of previously silent Dahlgren cells. Immunoreactivity for glucocorticoid and prolactin receptors was localised to Dahlgren cells. The CNSS expression level for glucocorticoid-2 receptor mRNA, measured by Q-PCR, was significantly lower in fish fully acclimated to freshwater, compared to seawater. No differences were seen between these two states for prolactin receptor mRNA expression. These results provide evidence for a modulatory action of both hormones on the neurosecretory function of the CNSS.     

Enhancement of α-lactalbumin-like activity in mammary explants from pregnant rats in the absence of exogenous prolactin

Mammary explants from pregnant rats showed a progressive increase in α-lactalbumin activity during culture with insulin, hydrocortisone and prolactin. Unexpectedly, culture with only insulin and hydrocortisone produced a similar rate of increase of α-lactalbumin-like activity, but this increase commenced about 24 hr later. The delay suggests that the enhanced activity effected by insulin and hydrocortisone is not a reflection of carry-over of endogenous mammotrophic hormones. Insulin plus hydrocortisone did not stimulate casein or fatty acid synthesis by pregnancy tissue, and did not enhance α-lactalbumin-like activity in virgin rat mammary explants. Enhancement of this activity by insulin plus hydrocortisone in pregnant tissue was constant over a wide range of glucocorticoid concentrations, but was inhibited by progesterone. Available evidence indicates that the active factor in extracts from insulin-hydrocortisone-explants is a heat-stable protein which is either α-lactalbumin itself, or another molecule with similar specifier properties.     

The influence of somatotropin, prolactin and insulin on granulosa cells from bovine atretic follicles

An influence of somatotropin, prolactin and insulin on destructive processes in bovine granulosa cells from small antral follicles following atresia in vivo was studied in vitro. As compared to control, the addition of the studied hormones to serum-free suspension system was shown to result in increase in number of cells without signs of chromosome degeneration after 24 and 48 hrs of incubation. The revealed inhibitory action of somatotropin, prolactin and insulin on chromatin degeneration in granulosa cells was not due to the hormonal influence on proliferative activity of the cells. The stimulatory action of insulin on the viability and estrogen-secretory activity of granulosa cells cultured for 1 day was also found. At the same time, somatotropin and prolactin did not affect the estradiol and progesterone production by the cells. The data obtained suggest that the inhibitory action of somatotropin and prolactin on destructive processes in cultured granulosa cells is not related to the hormonal regulation of the steroidogenic activity of the cells, whereas the similar action of insulin may be partially due to its stimulatory influence on the estradiol secretion.     

The study of differentiative potential of the lactating mouse mammary gland in organ culture

Summary The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and α-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. The results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.     

Super-active forms of placental lactogen and prolactin

Both placental lactogen and prolactin can be converted into super-active forms. These super-active hormones, in combination with insulin and hydrocortisone, stimulate accumulation of α-lactalbumin and increase RNA synthesis in explants from mouse mammary glands to an extent greater than the maximal level obtained with the native hormones. Also, they are able to stimulate RNA synthesis by suspensions of mammary epithelial cells which have lost the ability to respond to native lactogen and prolactin.     

Precocious induction of arginase in primary cultures of fetal rat hepatocytes

Summary Fetal rat hepatocytes were isolated and cultured in primary culture to investigate activity changes of arginase under defined conditions. In hormone-free medium, cultured cells maintained the enzyme activity at levels equal to that of freshly isolated cells for at least 4 d. Arginase activity could be induced by dexamethasone in hepatocytes isolated from 16.5-d-old fetuses although cells were competent to respond to glucagon only at the stage of 18.5 d. The combination of the two hormones induced greater levels of arginase activity than the individual compounds. These findings indicate that glucocorticoid and glucagon receptors appear early and sequentially before birth and reveal that cultured fetal hepatocytes provide a suitable system for the investigation of the role of hormones in the initiation of enzyme synthesis. This work was supported by the Institut National Scientifique et de la Recherche Médicale through Grant 85.80.117.