Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture.

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positive influence of tetracycline on human fetal kidney in serum-free organ culture

Summary Human fetal kidney explants can be maintained during 5 days in Leibovitz’s L15, a basic serum-free medium. Because culture conditions are minimal for growth and differentiation, DNA synthesis drastically decreases during the first 48 h, but stabilizes thereafter. The addition of insulin plus transferrin significantly restores this important cellular function in kidneys of fetuses younger than 16 wk. However, renal explants from older fetuses are more difficult to culture: they respond less to growth factors and are more prone to necrosis. The objective of this study was to verify the influence of tetracycline, an antibiotic with anti-collagenase potential, on cultured kidney explants aged 17 to 20 wk. The addition of 20μg/ml tetracycline did not influence DNA synthesis nor the effectiveness of insulin plus transferrin on cell proliferation. Nor did it change the activities of alkaline phosphatase and γ-glutamyltransferase, two enzymic markers of brush border differentiation. After 5 days in L15 alone, explants often showed necrosis and an important reduction in both weight and volume. Insulin plus transferrin significantly restored these parameters to control values observed at Day 0, but evidence of necrosis was still present. Tetracycline alone markedly reduced explant necrosis resulting in a significant increase in weight and volume. The effectiveness of insulin plus transferrin on explant morphometry was not improved when tetracycline was added as third factor. These results indicate that insulin plus transferrin restores explant mass through cell proliferation, whereas tetracycline does so possibly through a reduction in extracellular matrix degradation. The two effects are not additive in cultured mid-term fetal kidneys.     

Synergistic influence of epidermal growth factor, insulin and transferrin on human fetal kidney in culture

In human fetal kidneys (15-21 weeks of gestation) maintained in serum-free organ culture, protein synthesis remained relatively constant, but DNA synthesis decreased dramatically after 2 days. The addition of transferrin alone had no influence, but insulin and epidermal growth factor (EGF) both significantly stimulated DNA and protein synthesis. When supplemented in combination, transferrin strongly potentiated the insulin effect and after 5 days of culture DNA synthesis was practically restored to values observed in control uncultured renal explants (day 0). When EGF, a potent mitogen, was added as a third factor, the stimulating effectiveness of the (insulin plus transferrin) combination was significantly reduced. However, EGF had no such inhibiting influence on protein synthesis. Differentiation of brush border membranes, as evaluated by hydrolase activities, was not importantly induced nor retarded by any of the three factors supplemented either alone or in combination. The present results indicate that the individual effects of the three factors are not additive, but suggest that they rather act synergistically through a complex mechanism of receptor cross-talk. In our laboratory, there is convincing indication that the response of fetal organs varies according to age, proliferative state of tissues as well as stage of differentiation.     

Effects on induction of tyrosine aminotransferase in fetal mouse liver in vitro of prednisolone,insulin and thyroxine

The hormonal requirements for formation of tyrosine aminotransferase (EC 2.6.1.5) in fetal mouse liver were investigated in organ culture using chemically defined medium. The hormones tested were insulin, thyroxine and prednisolone. Prednisolone alone resulted in a two-fold increase in tyrosine amino-transferase activity in explanted liver in hormone-free medium on day 6, and its effect was dose dependent, but neither insulin nor thyroxine alone induced the enzyme. Addition of prednisolone plus thyroxine and prednisolone plus insulin increased the enzyme activity 1.4- and 1.3-fold, respectively, over that of explants with prednisolone alone. These three hormones together had the greatest effect, causing induction of 1.5-fold more activity than that with prednisolone plus insulin or plus thyroxine. The three hormones were not all needed continuously during the culture period: prednisolone and insulin were required during the early part of cultivation and thyroxine during the later part. The effects of these hormones were blocked by actinomycin D or puromycin, suggesting that these hormones increase de novo synthesis of tyrosine aminotransferase. Phase-contrast microscopy showed that prednisolone stimulated liver epithelial cell outgrowth, probably acting with insulin.     

Hormonal control of casein synthesis in mammary explants from pregnant goats

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.     

Fatty acid synthesis in the fetal lung: relationship to surfactant lipids

The aims of this study were to investigate the control of fatty acid synthesis and its relationship to surfactant production in the fetal lung during alteration of hormonal and substrate conditions. Lung explants from 18 day fetuses (term = 22 days) which were cultured 2 days in the presence of 10 mM lactate showed parallel acceleration of de novo fatty acid synthesis (3H2O incorporation) and [14C]choline incorporation into disaturated phosphatidylcholine (DSPC) compared to culture of explants in glucose. Both the cultured and fresh explants were resistant to the classical short term (4 h) cAMP inhibition of fatty acid synthesis with 3 mM dibutyryl cAMP or 0.5 mM aminophylline. In the cultured explants short term cAMP elevation increased DSPC production, and long term (2 day) cAMP elevation caused a further increase in DSPC synthesis and also stimulated fatty acid synthesis. In cultured explants from 17 day fetuses, dexamethasone (0.1 microM) caused a synergistic increase with aminophylline in both fatty acid synthesis and DSPC production whereas, in explants from 18 day fetuses, dexamethasone inhibited both processes and reduced the level of stimulation of DSPC and fatty acid synthesis seen with aminophylline alone. Dexamethasone also reduced the stimulation of both DSPC and fatty acid synthesis produced in the culture of 18 day explants with bacitracin (0.5 mg/ml), whereas the combination of bacitracin and aminophylline resulted in a synergistic increase in DSPC production. Culture with glucagon (0.1 microM) also stimulated DSPC synthesis but at physiological levels insulin had no effect on either DSPC or fatty acid synthesis. These data show that lung fatty acid synthesis exhibits unique features of fatty acid synthesis regulation compared to other lipogenic tissues and also suggest a link between de novo fatty acid synthesis and surfactant production during the critical period of accelerated lung maturation.     

Induction of lipid synthesis in mammary organ cultures from mature virgin and pregnant goats.

The hormones needed to induce lipogenesis in mammary organ cultures from mature virgin and pregnant goats were studied. In tissues from both mature virgin goats and goats at week 10 of pregnancy, cultivated in Waymouth medium without hormones, the rate of the incorporation of (1-(14C))-acetate into the lipids was low and decreased throughout culture. In the presence of insulin, the rate of acetate incorporation was maintained at a higher level. Cortisol acted synergistically with insulin, to produce a rate of lipid synthesis higher than that using insulin alone. The further addition of prolactin had little effect on the incorporation of acetate into the lipids of mammary explants from mature virgin goats, but markedly stimulated it in tissue from animals at weeks 9--10 of pregnancy. The maximum increase in the rate of lipid synthesis was achieved in the presence of 0.5 microgram prolactin/ml, whereas with growth hormone 50 microgram/ml was needed for the maximum effect. The initial rate of acetate incorporation into mammary explants from goats at weeks 13 and 18 of pregnancy was high. It was not stimulated by the hormones during culture, however, and decreased more rapidly in the absence of hormones than in their presence. The rate of acetate incorporation into the lipids was in agreement with the histological evaluation of the secretory response of the mammary explants after cultivation. The secretory response to prolactin and the rate of the incorporation of acetate into the lipids were highest in goats at weeks 9--10 of pregnancy while in tissues from goats at weeks 13 and 18 were not stimulated and decreased during culture.     

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.     

Serum-free organ culture of suckling rat jejunum: Effect of regulatory hormones

Summary To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when cultured under hyperbaric conditions for 24 h in serum-free Dulbecco’s modified Eagle’s medium/F12 medium than in RPMI 1640 plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium. Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture. However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after 48 h culture. Dexamethasone increased specific activities of alkaline phosphatase (30%,P<0.001) and lactase (15%,P<0.001), and reduced shedding of alkaline phosphatase into the medium (P<0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the rate of protein or DNA synthesis but did increase villus height (P=0.04) and crypt depth (P=0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II, des-(1–3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity of explants after 24 or 48 h culture. In conclusion, histology, enzyme activity, protein, and DNA synthesis of suckling rat jejunal explants were equivalent or better in serum-free than in serum-containing organ culture systems. Furthermore, biological responsiveness was demonstrated by dexamethasone and insulin altering the explants morphologically or biochemically. None of the IGFs or GH had any biological effects, raising doubts about their direct biological action on the developing intestinal epithelium.     

Ontogeny of ovine fetal liver and kidney plasma membrane insulin receptors and fetal growth

This investigation was performed to define certain characteristics of insulin-receptor interaction during the last 2 months of gestation in fetal sheep liver and kidney. Twenty-one sheep carrying a total of 46 fetuses were sacrificed at various gestational ages from 94 days to term; fetal and maternal livers and kidneys were analyzed by a radioreceptor assay for insulin binding characteristics. Specific binding of insulin to partially purified ovine fetal liver and kidney plasma membranes increased as gestation approached term, at which time specific binding was two- to fourfold greater to fetal than to maternal tissues. Associated with increased specific binding were late gestational increases in affinity of insulin for receptors in both fetal liver and kidney and an earlier increase in insulin receptor concentration in fetal kidney. These observations in fetal sheep liver and kidney are similar to reported observations in other species. However, the increase in specific binding of insulin to male fetal liver membranes was exponential; in contrast, there was no apparent increase in specific binding to female fetal liver membranes during the gestational interval surveyed. Both the weights and the vertebral column lengths of these fetuses were shown by multivariate analysis to be significantly affected by the interaction between specific binding of insulin and fetal sex. However, in 30 additional sheep fetuses we observed no difference between male and female fetuses in the increase with time in liver glycogen content. The lack of sex difference in this postreceptor event is consonant with the demonstrated dissociation between liver insulin receptors and glycogen synthesis in the late fetal rat. Our observations suggest that late gestational differences between male and female sheep fetuses in insulin specific binding to liver and, possibly, to other tissues such as cartilage, muscle, and/or fat, that are coupled to postreceptor events may account for differences in fetal growth between the sexes.     

Casein turnover in rabbit mammary explants in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.     

Effect of somatotropin on adipose tissue net glucose-stimulated lipogenesis in young goats.

Net glucose-stimulated lipogenesis (NGSL: the rate of lipogenesis in the presence of glucose minus the rate of lipogenesis in the absence of glucose) in omental adipose tissue explants from young castrated male goats was evaluated in control animals (n = 3; placebo-treated) and in animals treated with the sustained release of recombinant bovine somatotropin (n = 4; bST; 100 mg at 7-day intervals in a 147 days lasting experiment). The rate of fatty acid synthesis was determined in acute incubations in both freshly prepared and chronically cultured explants. Adipose explants remained metabolically active and retained their ability to respond to hormones when maintained in a tissue culture medium. NGSL in explants cultured for 24 h in the presence of insulin alone or bST alone, was non-significantly increased (more in the controls) and decreased (more in bST-treated animals), respectively. However, cortisol alone decreased (P<0.05) NGSL in explants from both control and bST-treated animals. In tissues from bST-treated animals, cortisol acted synergistically with insulin to produce a higher rate of NGSL than that observed in cultures with insulin alone. bST inhibited insulin plus cortisol-stimulated lipogenesis significantly (P<0.05) in explants from bST-treated animals but non-significantly in control animals. The rates of NGSL were decreased (P<0.05) by catecholamines in explants from both control and bST-treated animals. Norepinephrine (NE) and isoprenaline (ISO) were equally effective in the controls, whereas isoprenaline was more effective than norepinephrine in bST-treated animals.     

Epidermal growth factor (EGF) influences DNA synthesis in human fetal kidneys maturing in serum-free organ culture

Human fetal kidney explants (13-17 weeks of gestation) were maintained in serum-free organ culture. The influence of epidermal growth factor (EGF) was determined after 2 and 5 days by evaluating DNA and protein synthesis as well as the activities of five brush border hydrolases. During the studied period the overall morphology was preserved and the analysed parameters remained constant. Only DNA synthesis decreased after 2 days. The addition of EGF to the medium did not change any of the cell activities, except DNA synthesis. In fact, the incorporation of [3H]thymidine was significantly stimulated by 105% in 5-day explants cultured in the presence of the growth factor. These results indicate that EGF directly influences proliferation but not maturation of brush border enzymes in fetal human kidneys in culture.     

Protein anabolic effects of insulin and IGF-I in the ovine fetus

We determined the effect of insulin and/or recombinant human (rh)IGF-I infusion on ovine fetal phenylalanine kinetics, protein synthesis, and phenylalanine accretion. The chronically catheterized fetal lamb model was used at 130 days gestation. All studies were performed while fetal glucose and amino acid concentrations were held constant. Experimental infusates were 1). saline, 2). rhIGF-I plus a replacement dose of insulin (40 nmol), 3). insulin (890 mIU/h), and 4). IGF-I plus insulin (40 nmol IGF-I/h and 890 mIU insulin/h). Both hormones increased glucose and amino acid utilization, with insulin having a greater effect. The major effect on phenylalanine kinetics was a pronounced fall in phenylalanine hydroxylation, again with insulin having the greatest effect. Whole body protein breakdown was not significantly altered by either hormone; whole body protein synthesis was significantly increased during the combined infusion. Protein accretion was increased by both hormones, with the greatest increase during combined infusion. The fractional synthetic rate (FSR) of circulating albumin was increased by IGF-I but not by insulin. Both hormones significantly increased skeletal muscle FSR without a synergistic effect. The anabolic effects of insulin and IGF-I were more pronounced in these studies than in previous studies where amino acid concentrations were not maintained. The present data also suggest that insulin and IGF-I promote fetal growth through distinct, organ-specific mechanisms.     

Maintenance of glucokinase activity in primary hepatocyte cultures

When primary cultures of hepatocytes are maintained for 2 weeks from the time of perfusion, the activity of the enzyme glucokinase decreases rapidly, so that the activity can no longer be detected after the fourth day in culture. Concomitantly, there occurs an increase in the activity of hexokinases, the low-K_M isozymes, which predominate in fetal liver. We have made several modifications of the culture medium in an attempt to prevent the decrease in glucokinase activity. When the medium was supplemented with a mixture of insulin, thyroxine, glucagon, dexamethasone, testosterone, and estradiol, the activity of the enzyme in the hepatocytes was present at approximately 15% of in vivo levels after 2 weeks in culture. When this hormone mixture was present during the first 4 hrs of culture and when the hepatocytes were allowed to attach to the collagen support and were maintained thereafter in medium supplemented with fetal bovine serum, insulin, and dexamethasone, the activity of glucokinase increased after an initial decrease for 3 days and was maintained thereafter at levels comparable to those observed in vivo. This effect of the hormone mixture was found to be the result of the presence of glucagon in the mixture, since the presence of glucagon with no other hormones added, except insulin, during the attachment period produced the same pattern of increased glucokinase activity. Immunoprecipitation of glucokinase from the hepatocytes, using monospecific antibody, indicated that the increase in enzyme activity was the result of increased glucokinase enzyme protein and not an increased synthesis of the other hexokinase isozymes. These studies demonstrate the specific hormonal requirements for the maintenance of glucokinase levels in primary hepatocyte culture at those seen in vivo and lends support to the hypothesis that fetal gene expression in primary hepatocyte cultures is selectively regulated rather than being a general effect with a common regulatory mechanism.     

Muscle and cartilage differentiation in axolotl limb regeneration blastema cultures

A tissue culture system is described for explants of mesenchyme from Ambystoma mexicanum limb regeneration blastemas. Explants were cultured on collagen substrate for 3 weeks in minimal essential medium supplemented with the hormones insulin, thyroxine, somatotropin, and hydrocortisone, plus beef embryo extract (EE), 2%. This medium supported extensive cell migration onto the substrate followed by cell proliferation and differentiation of both cartilage matrix and myotubes. Cultures on plastic substrate, rather than on collagen, displayed similar cell outgrowth and cartilage formation, but relatively little myotube formation. Differentiation in EE-supplemented medium was compared with that in two defined media: Explants in medium containing only the hormones showed little outgrowth or cartilage development and never formed myotubes; medium containing the hormones plus fibroblast growth factor, 50 ng/ml, supported an intermediate degree of outgrowth and cartilage development and occasional myotube formation. Explant size was also a factor: Smaller explants survived and formed myotubes less frequently, even when on collagen in EE-supplemented medium.     

Chronic effects of somatotropin treatment in vivo and in vitro on lipogenic activity of goat adipose tissue in a glucose-free buffer during acute incubation.

Young castrated male goats (n = 8) were used to investigate the effect of long-term treatment with recombinant methionyl bovine somatotropin in a sustained release vehicle (bST; 100 mg at seven-day intervals in a 147-day experiment) and chronic culture (24 h) of omental adipose tissue in the presence of various hormones on lipogenic responses to catecholamines during acute incubation (2 h) in a sodium acetate supplemented glucose-free buffer. The rate of fatty acid synthesis in freshly-prepared adipose explants was low and did not differ from those cultured in the absence of hormones for 24 h. Hormonal combination of insulin (17 nmol.l(-1)) plus cortisol (138 nmol.l(-1)) or insulin plus recombinant enterokinase linker bST (4.5 nmol.l(-1) increased lipogenesis (P<0.05). Further addition of bST or cortisol decreased lipogenesis significantly (P<0.05) in the controls but not significantly in bST-treated animals. Cultured explants from either control or bST-treated animals showed significant inhibition of lipogenesis by both norepinephrine (10 micromol.l(-1)) and isoprenaline (10 micromol.l(-1)). BST treatment in vivo did not increase the responsiveness of cultured explants to norepinephrine in vitro, however, the responsiveness to isoprenaline(inhibition of lipogenesis) was greater in bST-treated animals than in the controls.     

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.     

Induction of surfactant protein in fetal lung. Effects of cAMP and dexamethasone on SAP-35 RNA and synthesis

Synthesis of surfactant-associated glycoprotein of Mr = 30,000-35,000 (SAP-35) was induced in explant culture of human fetal lung obtained from 8 to 24 weeks of gestation. SAP-35 synthesis and content increased markedly during 1-5 days in organ culture in association with the morphologic maturation of Type II epithelial cells and the appearance of lamellar bodies. [35S] Methionine labeling of the explants and subsequent immunoprecipitation of SAP-35 demonstrated distinct high-mannose precursors and sialylated SAP-35 forms as early in culture as SAP-35 synthesis was detectable. The increase in SAP-35 synthesis was associated with increased SAP-35 RNA of 2.1 kilobases as assessed by hybridization assay with [32P]cDNA specific for human SAP-35. Specific SAP-35 RNA increased during organ culture and both SAP-35 content and SAP-35 RNA increased in the absence of exogenous hormones in 2% carbon-stripped fetal calf serum. SAP-35 content and synthesis was stimulated by 8-Br-cAMP. Addition of 100 microM 8-Br-cAMP, enhanced both the concentration of SAP-35 protein and the SAP-35 RNA as assessed by hybridization assay. In contrast, treatment of the explants with dexamethasone was associated with decreased SAP-35 protein synthesis, SAP-35 content, and decreased SAP-35 RNA levels compared to untreated explants. Inhibition by dexamethasone occurred at all gestational ages tested, was dose-dependent, and detectable within 24-48 h during organ culture. Dexamethasone significantly inhibited both basal and cAMP-induced SAP-35 synthesis. Induction of pulmonary surfactant protein (SAP-35) synthesis during organ culture of human fetal lung was associated with increased SAP-35 RNA. SAP-35 synthesis and SAP-35 RNA were inhibited by dexamethasone and enhanced cAMP.     

Hormonal control of net glucose-stimulated lipogenesis during transition from brown to white adipose tissue in the goat

Perinatal (1-2 days of age) and one-month-old (24-32 days of age) male goats were used to investigate the effect of age and long-term culture (24 h) of perirenal and omental adipose explants in the presence of insulin, cortisol and bovine somatotropin (alone or in different combinations) on net glucose-stimulated lipogenesis (NGSL, i.e. the rate of lipogenesis in the presence of glucose minus the rate of lipogenesis in the absence of glucose) in the absence and in the presence of catecholamines in acute incubations (2 h). Mean values of NGSL in both freshly prepared and cultured explants were consistently lower in perinatal than in one-month-old goats. Cortisol alone decreased and combinations of insulin plus cortisol increased NGSL in perirenal explants of one-month-old animals. When perirenal explants from these one-month-old goats were cultured in the presence of insulin plus cortisol plus bovine somatotropin, the rates of lipogenesis were lower than those in cultures with insulin plus cortisol. No such effects of these hormones were noted in omental explants of both perinatal and one-month-old animals. In freshly prepared perirenal and omental explants, the rates of NGSL were inhibited by isoprenaline in tissues of both groups of animals and by noradrenaline in omental tissues of animals of the older group only. The mean values of NGSL in cultured explants of perinatal animals were not affected by noradrenaline. Isoprenaline inhibited NGSL in omental but not in perirenal tissue. In older animals the rates of NGSL were decreased by both noradrenaline and isoprenaline in perirenal and omental adipose tissues. Isoprenaline was more effective than noradrenaline in perirenal adipose tissue.