Expression of the steroidogenic acute regulatory protein in the corpus luteum of the rabbit: dependence upon the luteotropic hormone, estradiol-17 beta.

The recent characterization of the mitochondrial protein, Steroidogenic Acute Regulatory (StAR) protein, as a rate-limiting protein in steroidogenesis prompted us to investigate whether StAR is expressed in the rabbit corpus luteum and whether the expression of StAR is responsive to estradiol-17 beta, the luteotropic hormone in the rabbit. In rabbits treated continuously with exogenous estradiol through Day 13 of pseudopregnancy (n = 9), immunoblot analysis revealed that luteal expression of StAR was stable, ranging from 8.5 to 9.7 U of corrected integrated optical density. Plasma progesterone concentration (mean +/- SEM) remained elevated in these rabbits (14.3 +/- 2.1 ng/ml). In contrast, expression of StAR decreased in corpora lutea of rabbits deprived of estradiol for the last 48 and 72 h of the experiment (4.9 +/- 2.2 and 0.3 +/- 0.2 U, respectively, n = 3 per group), and was associated with a decline in plasma progesterone (0.8 +/- 0.1 and 0.5 +/- 0.3 ng/ml, respectively). Replacement of estradiol after 48 h of estradiol deprivation (n = 3) stimulated the reappearance of StAR (10.3 +/- 2.6 U) and the restoration of plasma progesterone (10.4 +/- 4.9 ng/ml). [35S]Methionine labeling of proteins in rabbit corpora lutea revealed that several isoforms of StAR protein were specifically synthesized in response to estradiol treatment. Collectively, these observations are consistent with a proposed role for StAR in the mediation of the luteotropic effect of estrogen to promote the synthesis of progesterone in the rabbit.

Estrogen uncouples steroidogenesis from 3',5'-cyclic adenosine monophosphate regulation in the rabbit corpus luteum.

The hypothesis was investigated that estradiol, the luteotrophic hormone in the rabbit, uncouples luteal progesterone production from regulation by LH/cyclic AMP. Progesterone production by corpus luteum (CL) incubated with vehicle, 3-isobutyl-1-methyl-xanthine (IBMX), hCG, or hCG+IBMX was compared in pseudopregnant rabbits treated continuously with estradiol (estradiol-maintained), withdrawn from estradiol for 24-48 h (estradiol-withdrawn), or withdrawn and then replaced with estradiol for 6 or 24 h (estradiol-replaced). Progesterone production in estradiol-maintained rabbits was not altered by hCG and/or IBMX, but was stimulated significantly in estradiol-withdrawn rabbits. This response was reversed (i.e., abolished) in estradiol-replaced (24 h) rabbits. The loss of responsiveness to hCG was not attributable to impaired accumulation of cyclic AMP: basal and hCG-stimulated cyclic AMP concentrations were similar in luteal tissues of estradiol-maintained and estradiol-withdrawn rabbits. The loss of responsiveness to hCG was also not a consequence of maximal progesterone production: CL of estradiol-replaced (6 h) rabbits were also insensitive to hCG, and this occurred before progesterone production attained a maximal rate. We conclude that a striking feature of the luteotrophic action of estrogen is to uncouple the regulation of progesterone production from cyclic AMP.

The autonomy of the rabbit corpus luteum.

The rabbit corpus luteum possesses LH receptors that are coupled to adenylyl cyclase, but paradoxically it does not require LH as a luteotrophic factor for the maintenance of progesterone secretion. This suggests that rabbit luteal cells may not respond physiologically to LH. Therefore, the present study was undertaken to investigate the responsiveness of the rabbit corpus luteum of pseudopregnancy to human chorionic gonadotrophin (hCG) which acts on the same receptor as LH. Pseudopregnancy was induced by injection of 40 IU pregnant mare serum gonadotrophin followed 50 h later by an injection of 40 IU hCG (day 0). On days 7 and 11 of pseudopregnancy, corpora lutea were obtained and incubated for 2 or 5 h in the presence of either 0.1 or 1 microgram/ml hCG or 1 mM monobutyryl cyclic AMP (bcAMP). Neither hCG nor bcAMP stimulated progesterone production by the isolated corpus luteum, despite a sustained high rate of progesterone production by the tissue throughout the incubation period. By contrast, Graafian follicles removed from the same ovaries and incubated under the same conditions responded both to hCG and bcAMP with large increases in progesterone production. To determine whether the cyclic AMP content of the corpus luteum was altered by in vitro exposure to hCG, day 7 and day 11 corpora lutea were incubated for 5 or 15 min with various concentrations of hCG, and cyclic AMP in the tissue was then measured. Even at the highest concentration of hCG tested (10 micrograms/ml), the cyclic AMP content of the corpus luteum was unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Pioglitazone inhibits the diabetogenic action of growth hormone, but not its ability to promote growth.

Analogs of thiazolidinedione improve the responsiveness of insulin-resistant animals to insulin. One such analog, pioglitazone (5-(4-[2-(5-ethyl-2-pyridinyl)ethoxy]benzyl)thiazolidine-2,4-dione hydrochloride), when fed to insulin-resistant animals such as the obese (ob/ob) mouse, reduces blood glucose and lipids and also lowers the plasma insulin level. Because GH can produce insulin resistance in humans and animals such as the ob/ob mouse, the present study was conducted to determine whether feeding pioglitazone can 1) inhibit the ability of GH to induce enhanced insulin resistance in obese mice, 2) ameliorate or reverse GH-induced insulin resistance once it has been induced in ob/ob mice, and 3) alter the ability of GH to promote growth in hypophysectomized rats. Female ob/ob mice were fed a control diet or a diet containing pioglitazone (20 mg/kg animal.day) for 4 days. During the last 3 days of the feeding period, the mice also received a daily sc injection of either saline or 200 micrograms S-carboxymethylated human GH (RCM-hGH), which is a GH derivative having mainly diabetogenic activity. In control-fed mice, RCM-hGH increased blood glucose and plasma insulin levels, which is an expected response to GH-induced insulin resistance. By contrast, the ability of RCM-hGH to increase blood glucose and plasma insulin levels was totally blocked in pioglitazone-fed mice. To determine whether pioglitazone can ameliorate GH-induced insulin resistance once it has been established, ob/ob mice were treated sc with either saline or 200 micrograms RCM-hGH for 3 days. Half of the saline-treated and half of the hormone-treated mice were then fed pioglitazone, whereas the remaining animals were continued on the control diet. After 48 h on the diets, the blood glucose and plasma insulin levels of the RCM-hGH treated mice fed the control diet remained elevated with respect to those in the saline-treated controls. On the other hand, the blood glucose and plasma insulin levels of the RCM-hGH treated mice fed pioglitazone were markedly reduced compared to those of the RCM-hGH-treated control-fed animals. Thus, these results suggest that pioglitazone can ameliorate GH-induced insulin resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

The stimulatory effect of insulin on diacylglycerol generation in adipocyte membranes from ob/ob mice is impaired by growth hormone.

Physiologically, the action of insulin on carbohydrate and lipid metabolism is opposed by several hormones, including glucocorticoids, glucagon, catecholamines, and pituitary GH. Perhaps least is known about the mechanism(s) involved in the antiinsulin action of GH. Since the generation of diacylglycerol (DAG) appears to be an early event in the insulin-signaling cascade, it was of interest to determine whether GH would interfere with this effect of insulin. Experiments were conducted to determine whether insulin would stimulate the generation of DAG in adipocytes of the obese (ob/ob) mouse, and whether this response could be blocked by the diabetogenic GH derivative S-carboxymethylated human GH (RCM-hGH). Isolated adipocytes of the ob/ob mouse were used for these studies, because unlike normal rodents, the ob/ob mouse responds predictably to the antiinsulin action of GH. Insulin produced a rapid biphasic increase in the amount of DAG in a crude membrane fraction of the adipocytes. The first peak in DAG mass occurred within 5 min of exposure of the cells to insulin, and the second peak occurred after 30 min. The first peak in DAG mass did not occur in adipocytes that had been incubated with pertussis toxin before exposure to insulin. Also, adipocytes isolated from ob/ob mice that had been treated with RCM-hGH failed to respond to insulin with an increase in DAG mass. RCM-hGH blocked both the first and second insulin-induced peaks in DAG mass within 6 h of its administration. This is the time at which ob/ob mouse adipocytes exhibit increased insulin resistance in response to RCM-hGH. Neither exposure to insulin nor treatment with RCM-hGH had any appreciable effect on the fatty acid composition of the DAG present in the adipocyte membranes. These findings are compatible with the idea that GH produces some defect in the insulin-signaling cascade that is proximal to the events that result in the generation of DAG in the adipocyte.

Evidence that the N-terminus of human growth hormone is involved in expression of its growth promoting, diabetogenic, and insulin-like activities.

Recent work with various point and deletion mutants of human GH (hGH) has suggested that the proximal N-terminal end of the hormone molecule is important for its growth promoting action. This study was conducted to examine the growth promoting, diabetogenic, and insulin-like activities of two N-terminal mutants of hGH, the deletion mutant Des-7 hGH (met8, ala11), and a chimeric mutant of bovine GH (bGH) and hGH containing the N-terminal 13 amino acids of bGH (met, ala 1-13/14-191, asp11). The CD spectra of these mutants are similar to that of wild-type hGH and they retain lactogenic activity on Nb2 lymphoma cells, whereas their ability to bind to somatogenic receptors on IM-9 lymphocytes and bovine liver membranes is markedly reduced. In this study, growth promoting activity of the mutants was assessed using the 9-day weight gain test in hypophysectomized rats. Des-7 hGH had a potency of 0.03 IU/mg protein in this assay, whereas the potency of the bGH/hGH chimera was 0.71 IU/mg. Diabetogenic activity was tested in the ob/ob mouse, using the elevation of fasting blood glucose and the worsening of glucose tolerance after a 3-day course of treatment as end-points. Both Des-7 hGH and the bGH/hGH chimera had reduced diabetogenic activity compared to that of biosynthetic wild-type hGH, consistent with their reduced growth activity. Insulin-like activity was assessed by testing the in vitro ability of the mutants to stimulate [14C] glucose oxidation by epididymal adipose tissue of hypophysectomized rats. Des-7 hGH had about 1% the activity of wild-type hGH, whereas the chimera was about 20% as active. When Des-7 hGH was added to the incubation medium along with wild-type hGH in ratios of 5, 12.5, or 25:1 (Des-7 hGH:hGH), the insulin-like action of hGH was significantly inhibited, indicating that the mutant is a modest antagonist of the insulin-like action of hGH. When the ability of Des-7 hGH to compete with [125I] hGH for binding to isolated rat adipocytes was tested, the mutant was about 10% as effective as wild-type hGH. Thus, Des-7 hGH appears to be more effective in binding to adipocyte GH receptors than in triggering an insulin-like response, perhaps accounting for its modest antagonistic activity. The results of this study suggest that the proximal N-terminal end of the hGH molecule is involved in the expression of the growth promoting, diabetogenic and insulin-like activities of GH.

Metabolic interfaces between growth and reproduction. IV. Chronic pulsatile administration of growth hormone and the timing of puberty in the female sheep.

Puberty in the female lamb is accompanied by an increased frequency of LH pulses, and during normal development this is preceded by a decline in GH. Conversely, in the growth-retarded lamb, when LH levels are depressed by low nutrition, GH secretion is elevated. Based upon this inverse relationship, we tested the hypothesis that GH may act as a metabolic signal from the brain to inhibit the secretion of LH, and that the decline in GH times puberty. Our approach was to extend high circulating GH levels far beyond the early postnatal period, in a physiological pattern and level, in an attempt to block the pubertal LH rise. To evaluate the pattern of LH as a continuous variable under conditions of constant estradiol negative feedback, the gonadotropin was measured in blood samples collected by jugular venipuncture twice weekly; the lambs were ovariectomized and treated chronically with estradiol (Silastic capsule) beginning at 3 weeks of age. Nine lambs served as untreated controls, and 7 were infused iv with pituitary-derived bovine GH (bGH) between 5 and 28 weeks of age. A programmable backpack infusion pump delivered bGH as hourly pulses, with a total dose of 18 micrograms/kg.24 h, to maintain a physiological pattern and level of GH. At various ages, blood samples were collected at 12-min intervals for 6 h to monitor patterns and levels of peripheral LH and GH. Circulating GH in untreated and treated lambs averaged 7.7 +/- 1.5 ng/ml over a 6-h period at 4 weeks of age and declined to 1.1 +/- 0.2 ng/ml by 19 weeks in the untreated lambs; in contrast, bGH-infused lambs averaged 10.4 +/- 0.9 ng/ml at 19 weeks. Although body weights did not differ, back fat depth and quantity of perirenal fat were reduced in bGH-treated females compared to that in controls. Moreover, insulin-like growth factor-I levels were higher in bGH-treated compared with control lambs, and the bGH-treated lambs exhibited glucose intolerance, thus confirming that infused bGH was biologically active. Neuroendocrine sexual maturity, however, was not different in bGH-treated and control lambs, and it occurred at 21-22 weeks of age. The results do not support our hypothesis that decreasing GH secretion is a requirement for puberty in the sheep. Moreover, unlike in children with delayed puberty, exogenous bGH did not advance normal puberty in the lamb.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factor-I stimulates steroidogenesis in rabbit luteal cells.

A potential role of insulin-like growth factor I (IGF-I) in the regulation of steroidogenesis in the rabbit corpus luteum was investigated using a primary culture of luteal cells obtained 3 days after ovulation. Dissociated cells were cultured for 1 day in the presence of medium 199 and 10% fetal bovine serum; thereafter, the cells were cultured in medium 199 containing 0.1% BSA, gentamicin (50 micrograms/ml), and hormones or growth factors, and without serum. IGF-I (human recombinant, 100 ng/ml) was as effective as LH (ovine, 10 ng/ml) in maintaining progesterone accumulation through 4 days of culture. Estradiol (10(-8) M), either alone or in combination with LH or IGF-I failed to stimulate progesterone accumulation, which was not surprising since these cells did not possess estrogen receptors. The stimulation of progesterone by IGF-I was not detectable until 24-36 h after introduction of the growth factor to the cultures, whereas stimulation by LH was observed within 2 h. The steroidogenic effect of IGF-I was not attributable to increased cell number, as DNA values or [3H]thymidine incorporation were unchanged by IGF-I. IGF-I increased functional enzymatic activity, observed as increased progesterone accumulation in the presence of 25-hydroxycholesterol used as exogenous substrate. These data indicate that luteal cells have the capacity to respond to IGF-I, raising the possibility that IGF-I has a role in the regulation of steroidogenesis in the rabbit corpus luteum.

Growth hormone receptors and action in BC3H-1 myocytes.

The muscles of the body are generally regarded as major targets for the actions of growth hormone (GH), but these tissues are difficult to utilize for in vitro studies of rapid cellular events. Therefore, the present study was conducted to determine whether the BC3H-1 cultured myocyte cell line possesses GH receptors and whether it exhibits metabolic responsiveness to GH. It was found that the myocytes possess receptors, which appear to be specific for GH and GH derivatives but which do not bind prolactin. The degree of GH binding observed was small, suggesting that receptors for GH are present in rather low abundance on these cells. To determine whether BC3H-1 myocytes are metabolically responsive to GH, the insulin-like activity of GH (i.e. the ability of GH to stimulate glucose metabolism) was examined, since this effect of GH is readily demonstrable when isolated rat muscle is incubated directly with the hormone. GH was found to stimulate glucose oxidation by the myocytes indicating that the receptors for GH on these cells are functional.

Growth hormone inhibits activation of phosphatidylinositol phospholipase C in adipose plasma membranes: evidence for a growth hormone-induced change in G protein function.

Pituitary growth hormone (GH) functions physiologically to oppose the actions of insulin on carbohydrate and lipid metabolism by interfering with metabolic events that occur after insulin binds to its receptor. Which postreceptor effects are involved is presently unknown. Recently, we found that insulin rapidly stimulates a phosphatidylinositol phospholipase C (PI-PLC) in adipose tissue of obese (ob/ob) mice and that this effect of insulin is blocked by treatment of the animals with S-carboxymethylated human GH (RCM-hGH), a derivative having mainly anti-insulin activity. The activation of this PI-PLC by insulin is also inhibited by pertussis toxin. Thus, this study was performed to examine whether the inhibitory effect of GH on the activation of this PI-PLC is exerted at the level of signal transmission by guanine nucleotide binding proteins (G proteins). We found that the nonhydrolyzable GTP analogue, guanosine 5'-[gamma-thio]triphosphate, stimulated basal PI-PLC activity in plasma membranes of adipose tissue of saline-treated ob/ob mice, but it did not stimulate the enzyme in adipose membranes from RCM-hGH-treated mice. Also, RCM-hGH treatment markedly inhibited pertussis toxin-catalyzed ADP ribosylation of G protein alpha subunits in the membranes, suggesting some modification of the G proteins by GH. Immunoblot analysis of adipose membranes from saline- and RCM-hGH-treated mice using antiserum AS/7 (anti-Gi1 alpha and anti-Gi2 alpha) or antiserum EC/2 (anti-Gi3 alpha) showed no difference in the amount of Gi alpha-like protein between the groups. These findings suggest that GH interferes with the ability of a putative Gi-like protein to mediate the activation of PI-PLC in adipose membranes without altering the expression of the G protein.

The biosynthesis of cholesterol side-chain cleavage cytochrome P-450 in the rabbit corpus luteum depends upon estrogen.

To gain a better understanding of the luteotropic action of estrogen, we have investigated the effect of estrogen on the synthesis of the enzyme, cholesterol side-chain cleavage cytochrome P-450 (P-450scc) in the rabbit corpus luteum. Using an established protocol, rabbits were treated with estradiol, and the estradiol was then withdrawn on day 9 of pseudopregnancy, which caused an 88% fall in serum progesterone within 48 h. In other rabbits, estradiol was replaced at 48 h which stimulated a 6.6-fold increase in serum progesterone concentration within the next 24 h. Luteal tissues were incubated with [35S]methionine and homogenized, and a mitochondrial fraction lysate was obtained. Equal trichloroacetic acid-precipitable radioactivity was taken for immunoprecipitation using a well-characterized polyclonal antiserum against bovine adrenal P-450scc. The immunoisolated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radioactivity was visualized by autofluorography. The results indicate that the rate of synthesis of P-450scc in 48 h-estradiol withdrawn animals was markedly reduced, and by 72 h of withdrawal was barely detectable. When estradiol was reintroduced, the synthesis of P-450scc was increased. Despite the prominent changes in P-450scc synthesis, immunoblotting revealed only a minimal (approximately 30%) decrease in relative P-450scc content by 72 h after estradiol withdrawal. Analyses of DNA and protein contents of luteal tissues revealed an increase in DNA per mg luteal tissue, a decline in total tissue protein/DNA ratio, but no change in mitochondrial fraction protein/DNA ratio after estrogen withdrawal. The results indicate that de novo synthesis of P-450scc in the corpus luteum is sensitive to estrogen; however, the estrogen-sensitive rate-limiting step(s) for steroidogenesis are at other sites in the steroid biosynthetic pathway.

Isolated adipocytes from growth hormone-treated obese (ob/ob) mice exhibit insulin resistance.

The genetically obese (ob/ob) mouse is a useful model for the study of the diabetogenic action of growth hormone (GH), because treatment of these animals with GH results in decreased responsiveness of their adipose tissue to insulin in vitro. Studies of the mechanisms involved in GH-induced insulin resistance using isolated adipocytes of ob/ob mice have not been possible, however, because of their extreme fragility and the lack of an adequate system for the maintenance of these cells. This study describes a new method for the isolation of ob/ob mouse adipocytes. The isolated cells are stable, viable and metabolically responsive to insulin. In addition, these adipocytes have been maintained in primary culture, in serum-free medium, for up to 3 days. During culture, the cells exhibit large increases in 125I-hGH binding (10-20-fold) and porcine 125I-insulin binding (5-10-fold). The induction of insulin resistance by GH has also been demonstrated in these freshly isolated ob/ob mouse adipocytes. The studies to date indicate that the ob/ob mouse adipocyte system should provide a useful model for detailed studies of the cellular and molecular mechanisms of GH induced insulin resistance.

Lipolytic and antilipolytic effects of human growth hormone, its 20-kilodalton variant, a reduced and carboxymethylated derivative, and human placental lactogen on chicken adipose tissue in vitro.

The lipolytic and antilipolytic effects of human growth hormone (22K-hGH), its 20-kilodalton variant (20K-hGH), a reduced and S-carboxymethylated derivative (RCM-hGH), and human placental lactogen were examined using chicken adipose tissue explants in vitro. Lipolysis, as determined by glycerol release, was stimulated by 22K-hGH (biosynthetic and pituitary derived), 20K-hGH (pituitary derived), and RCM-hGH (modified biosynthetic). These growth hormone preparations also exhibited similar antilipolytic activity (i.e., transient inhibition of glucagon-induced lipolysis). However, unlike human growth hormone, human placental lactogen neither stimulated lipolysis nor inhibited glucagon-stimulated lipolysis. Some augmentation of glucagon-stimulated lipolysis was observed in the presence of human placental lactogen. These results indicate that the disulfide bridges (Cys53----Cys165; Cys182----Cys189) and amino acid residues 32-46 of hGH are not required for lipolytic or antilipolytic activities of human growth hormone on chicken adipose tissue.

Growth hormone inhibits activation of phosphatidylinositol phospholipase C by insulin in ob/ob mouse adipose tissue.

The cellular mechanism(s) by which GH produces insulin resistance in peripheral tissues is poorly understood. Recent evidence suggests that insulin exerts certain of its intracellular actions by rapidly activating phosphatidylinositol-specific phospholipase C(s) (PI-PLC) in the plasma membranes of target cells. Therefore, the present study was conducted to determine whether insulin can activate PI-PLC in adipose tissue of the genetically obese (ob/ob) mouse, an animal that responds markedly to GH with enhanced peripheral insulin resistance. Also, experiments were performed to determine whether the activation of PI-PLC by insulin could be blocked by S-carboxymethylated human GH (RCM-hGH), a GH derivative possessing mainly diabetogenic activity. Isolated adipose segments were incubated for various periods with insulin (10 mU/ml), homogenized and centrifuged to obtain a 150,000 x g pellet, and the latter was assayed for the ability to produce [3H]inositol phosphate from phosphatidyl[3H]inositol. PI-PLC activity was significantly stimulated 5 min after exposure of the segments to insulin. By 10 min, the insulin effect was no longer apparent, and after 30 min, insulin reduced the activity of the enzyme. One hour after exposure to insulin, PI-PLC activity returned to the control level. When adipose segments of RCM-hGH-treated mice (200 micrograms/day for 3 days sc) were incubated for 5 min with insulin, the ability of insulin to activate PI-PLC was abolished. However, RCM-hGH did not alter basal PI-PLC, indicating that its action involves the mechanism by which the enzyme is activated by insulin. Also, studies utilizing acute RCM-hGH treatment showed that its inhibitory effect on insulin activation of PI-PLC occurs within the same time frame as the onset of enhanced insulin resistance in the adipose tissue. Thus, the ability of GH to inhibit the activation of PI-PLC by insulin in adipocytes may account, at least in part, for its ability to induce insulin resistance in these cells.

Different growth hormone-receptor interactions mediate insulin-like and lipolytic responses of rat adipose tissue.

The GH receptor in adipocytes is a glycoprotein that has a half-life of less than 1 h. After 2 h of treatment with the alkaloid swainsonine, which interferes with carbohydrate processing, virtually all of the GH receptors on the surface of adipocytes are replaced with receptors whose carbohydrate side-chains are incomplete. We examined the effects of swainsonine on the responsiveness of adipose tissue to GH to determine whether these receptors, which bind GH normally, retain biological competence. In the concentration range of 100-300 ng/ml human (h) GH rapidly evokes insulin-like responses in adipose tissue or adipocytes that have been deprived of GH for at least 3 h. hGH, at concentrations ranging from 1-10 ng/ml, also increases lipolysis after a delay of at least 2 h. Pretreatment with 50 micrograms/ml swainsonine failed to influence insulin-like responsiveness to hGH, as judged by increased glucose oxidation, but nearly completely abolished the lipolytic response. Pretreatment with swainsonine, however, did not reduce lipolysis in response to isoproterenol, suggesting that signal transmission rather than the lipolytic apparatus per se had been affected. To determine whether the same receptors mediate lipolytic and insulin-like responses, the binding properties of hGH were compared to those of Da1, a chemically modified form of hGH, whose insulin-like potency is reduced relative to its lipolytic potency. Da1 and hGH were equipotent in promoting lipolysis and had an ED50 of about 3 ng/ml, but hGH was at least 6 times as potent as Da1 in promoting glucose oxidation (ED50 of 65 vs. 400 ng/ml). Scatchard plots of both Da1 and hGH binding data were linear, consistent with a single class of binding sites whose affinity for hGH was about 3.5 times higher for hGH than Da1. hGH and Da1 both produced half-maximal stimulation of glucose oxidation when about 90% of the GH receptors were occupied. In contrast, half-maximal lipolysis was produced by Da1 when 8% of GH receptors were occupied, but 21% occupancy was required for a similar effect of hGH. If a subclass of GH receptors mediates lipolysis, it is likely to comprise 10% or less of the total receptor population.

Acute effects of S-carboxymethylated human growth hormone on insulin resistance in the obese (ob/ob) mouse.

Chronic treatment of ob/ob mice with growth hormone (GH) increases plasma insulin and blood glucose concentrations, and enhances insulin resistance in peripheral tissues. The purpose of the present study was: (1) to determine the length of time required for the development of increased circulating insulin concentration and adipose tissue insulin resistance in response to GH in the ob/ob mouse, (2) to examine the relationship between the rise in insulin concentration and the development of insulin resistance, and (3) to test whether the hormone derivative could enhance insulin resistance in isolated adipose tissue when added in vitro. Female ob/ob mice were injected intraperitoneally (ip) with either saline or 200 micrograms of S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative, which lacks significant insulinlike or growth-promoting activities. A threefold increase in plasma insulin concentration was observed three and six hours after RCM-hGH injection, but increased hyperglycemia was evident only after six hours. The in vitro stimulatory effect of insulin on [14C]glucose oxidation by parametrial adipose tissue was unchanged at three hours but suppressed six hours following administration of RCM-hGH. When the plasma insulin level of ob/ob mice was increased threefold by the administration of neutral protamine Hagedorn (NPH) insulin, the in vitro stimulatory effect of insulin on [14C]glucose oxidation by adipose tissue isolated from these animals was not altered, suggesting that insulin-induced receptor or postreceptor changes do not account for the increased insulin resistance produced by RCM-hGH.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of cycloheximide on acute diabetogenic effects of S-carboxymethylated human growth hormone in obese (ob/ob) mice.

Acute treatment of ob/ob mice with S-carboxymethylated hGH (RCM-hGH), a diabetogenic derivative of GH which lacks significant insulin-like and growth-promoting activities, results in an increase in fasting plasma insulin and blood glucose levels and enhanced peripheral tissue insulin resistance. Plasma insulin level increases within 3 h after RCM-hGH is administered, whereas increased blood glucose concentration and enhanced peripheral tissue insulin resistance became evident 6 h after the hormone derivative is given. The lag period seen in the manifestation of these diabetogenic effects of RCM-hGH is consistent with the time required for gene expression. Therefore, the present study was undertaken to determine whether the above acute responses to the diabetogenic action of RCM-hGH would be expressed in ob/ob mice in which protein synthesis was blocked with cycloheximide. Female ob/ob mice were given either saline or cycloheximide (0.1 mg/g BW) ip and 1 h later were fasted and treated with either saline or 200 micrograms RCM-hGH ip. The mice were given a second injection of cycloheximide during the middle of the hormone treatment period to insure that protein synthesis remained blocked for the entire 6 h. In the animals not receiving cycloheximide, fasting plasma insulin level and blood glucose concentration were markedly elevated 6 h after the injection of RCM-hGH. Also, the GH derivative attenuated the ability of insulin added in vitro to stimulate glucose oxidation by adipose tissue segments isolated from the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological characterization of charge isomers of human growth hormone.

Since deamidation of the human GH molecule may alter the manner and extent to which the hormone is cleaved by proteases, and since it has been repeatedly suggested that proteolytic processing is required for the expression of certain of the activities of GH, the present study was conducted to determine whether the biological activity profiles of more acidic forms of human GH are altered. Three charge isomers, GH-b, GH-c and GH-d, representing primarily deamidated forms, were isolated from a native human GH preparation (Crescormon) in amounts adequate for characterization of their biological activities. All three were essentially equipotent in a radioimmunoassay for human GH. When assessed for growth-promoting activity in the hypophysectomized rat, the isomers were again equipotent with each other and with the GH preparation from which they were derived. The charge isomers also had significant in vitro insulin-like activity on isolated rat adipose tissue and diabetogenic activity in the ob/ob mouse. Thus, the biological activity profiles of these charge isomers of human GH do not differ greatly from one another.

The acute effects of growth hormone on amino acid transport and protein synthesis are due to its insulin-like action.

GH has acute stimulatory effects on amino acid transport and protein synthesis in a variety of tissues, but it has not been established whether these effects are expressions of the growth-promoting property of GH or of its separate insulin-like action. The 20,000-dalton structural variant of human GH (20K hGH) has been shown to have a high ratio of growth-promoting to insulin-like activity compared to native hGH (22K hGH), suggesting that it could be used as a tool to address the above question. Therefore, experiments were conducted to compare the relative abilities of native 22K hGH and 20K hGH, when added in vitro, to stimulate amino acid transport and protein synthesis in the isolated diaphragm of the female hypophysectomized rat. Paired intact hemidiaphragms were preincubated for 1 h in the absence or presence of various concentrations of 22K or 20K hGH. Then, 3-O-[14C]methylglucose was added to the medium to measure sugar transport as a test of insulin-like activity, and either alpha-[3H]aminoisobutyric acid acid or [3H] phenylalanine was also added to measure amino acid transport or protein synthesis, respectively, during a final hour of incubation. When the responses to the various concentrations of 22K and 20K were compared, 20K hGH was only about 20% as effective as 22K in stimulating 3-O-methylglucose transport, reflecting its markedly attenuated insulin-like activity on the diaphragm. Similarly, 20K hGH was only 20% as effective as 22K hGH in stimulating alpha-aminoisobutyric acid transport and phenylalanine incorporation into protein in the same muscles. Therefore, these findings support the idea that the rapid stimulatory effects of GH on amino acid transport and protein synthesis are expressions of the insulin-like action of GH and are not components of the response of target cells to its growth-promoting action.

Biological characterization of purified native 20-kDa human growth hormone.

Because of the propensity of the 20-kDa variant of human growth hormone (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa GH was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.