Metabolic effects of plasmin digests of human growth hormone in the rat and man.

As a first step in our study of structure-function relationships among primate and non-primate growth hormones, human growth hormone (hGH) was subjected to the limited digestive activity of human plasmin. The lyophilized whole digest, containing less than 2% of unchanged hormone, had an average of 2.3 new amino-terminal groups per mole. The digest had the same potency as the native hormone (a) in causing weight gain in hypophysectomized rats; (b) in stimulating somatomedin production in hypophysectomized rats; (c) in stimulating upake of [(3)H]leucine into isolated diaphragm of hypophysectomized rats; (d) in accelerating transport of [(14)C]alpha-aminoisobutyric acid into isolated diaphragm of hypophysectomized rats; (e) in stimulating uptake of [3-0-methyl-(14)C]glucose by isolated adipose tissue of hypophysectomized rats; (f) in accelerating conversion of [(14)C]glucose to (14)CO(2) by isolated epididymal adipose tissue of hypophysectomized rats. The digest also caused glucosuria in partially pancreatectomized rats treated with dexamethasone. These metabolic actions of plasmin-digested hGH in the array of animal tests were confirmed by comparable effects elicited in 11 human subjects (nine pituitary-deficient children and adolescents and two nondeficient adults). A single injection of the plasmin digest caused an increase in plasma free fatty acids and a fall in plasma amino acids. Seven daily injections caused positive balances of nitrogen, phosphorous, sodium, and potassium, gain in body weight, and in two of three subjects impairment of glucose tolerance. The potency of the plasmin digest in producing these metabolic effects in man was comparable to that of native hGH.Thus, 2-3 bonds in the hGH molecule can be cleaved by plasmin without impairing the hormone's growthpromoting, anabolic, diabetogenic, and adipokinetic actions for rat and man.

Anabolic actions of reduced and S-carbamidomethylated human growth hormone and its plasmin digest in man.

Six children aged 12-15 yr, deficient in endogenous growth hormone, were each treated, after a 7-day control period, for 7 days with 0.0168, 0.052, and 0.168 U/kg body wt3/4 human growth (hGH) (doses A, B, and C, respectively) in separate metabolic balance studies. Doses B and C caused a dose-related retention of N, P, K, Na, and Cl in ratios of 1/0.069/4.5/7.5/5.6. These ratios indicate increments in masses of protoplasm/extracellular fluid (ECF)/bone in ratios of 1/2.0/ less than 0.001. Three of the children were also treated with doses A, B, and C of reduced and carbamidomethylated hGH (RCAM-hGH). Doses B and C caused 1.2-2.8 times as much retention of N, P, and K, and 0.3-0.5 times as much retention of Na and Cl, as did the corresponding doses of hGH. The plasmin digest of RCAM-hGH gave results generally similar to RCAM-hGH. For RCAM-hGH and its plasmin digest, N, P, K, Na, and Cl were retained in ratios of about 1/0.14/5.4/2.2/2.1, indicating increments of protoplasm/ECF/bone of about 1/0.8/0.05. These findings indicate that reduction and carbamidomethylation alter the anabolic actions of hGH in man in both quantitative and qualitative manner. RCAM-hGH is more potent in stimulating enlargement of protoplasm and bone, and less potent in stimulating expansion of ECF, than is the native hormone. The profile of anabolic actions of RCAM-hGH in man does not appear to be further altered by digestion with plasmin.

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.

Ability of growth hormone fragments to complete with 125I-iodinated human growth hormone for specific binding to isolated adipocytes of hypophysectomized rats.

Several noncovalent complexes of large fragments of human GH, which are less active than native human GH in stimulating glucose metabolism in adipose tissue of hypophysectomized rats, were tested for their ability to compete with 125I-iodinated human GH for specific binding to isolated adipocytes of hypophysectomized rats. The complexes tested were A (residues 1-134 + residues 141-191; S-carbamidomethylated), B (residues 1-134 + residues 135-191; S-carbamidomethylated) and C (residues 1-134 + residues 135-191; S-carboxymethylated). When compared to native human GH, the complexes were less active in competing with 125I-iodinated human GH for specific binding to adipocytes, and their order of potency in the binding assay (A greater than B greater than C) was similar to that of their respective activities in stimulating glucose metabolism in isolated adipose tissue of hypophysectomized rats.

Trypsin-resistant forms of human growth hormone have diabetogenic and insulin-like activities.

Although diabetogenic and insulin-like activities are intrinsic properties of the growth hormone (GH) molecule, it has been frequently suggested that the hormone must be proteolytically processed for these activities to be expressed. If this is correct, then derivatives of GH having resistance to appropriate proteolytic attack might not have diabetogenic and/or insulin-like activity. The purpose of the present study was to prepare derivatives of human GH that are resistant to digestion by trypsin and to determine whether they possess diabetogenic or insulin-like activity. Three derivatives were prepared from purified native human GH in which lysine residues were modified with methyl acetimidate, citraconic anhydride or S-ethyl-thioltrifluoroacetate, and one in which arginine residues were modified with camphorquinone-10-sulfonic acid. Comparisons of peptide maps of tryptic digests of these derivatives with that of unmodified human GH indicated that all four were resistant to proteolysis by trypsin. All of these trypsin-resistant forms of human GH were found to possess significant growth-promoting, diabetogenic and insulin-like activities, although all activities were attenuated to some extent in each derivative. The relative potencies of the human GH derivatives in a radioimmunoassay for human GH were somewhat similar to their order of potency in the growth-promoting and diabetogenic assays. These results suggest that if proteolytic processing of the GH molecule is involved in the expression of one or more of its biological activities, such processing probably does not involve a trypsin-like proteinase.

Possible evidence for the existence of a growth hormone fragment in porcine pituitary.

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126-180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of 125I-labelled fragment B by procine growth hormone required a 10(3) M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of 125I-labelled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possible representing a growth hormone fragment.

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.

Influence of age on responsiveness to diabetogenic action of growth hormone.

Responsiveness to the growth-promoting action of growth hormone (GH) develops gradually during post-natal life, and as the organism ages, sensitivity to the hormone declines. Our study was undertaken to determine whether there is a similar age-related pattern of sensitivity to the diabetogenic action of purified native human GH (hGH) in the obese (ob/ob) mouse. The ob/ob mouse was used because adults of this strain respond to chronic GH treatment with increases in fasting plasma insulin and blood glucose concentrations and with glucose intolerance. When hGH was given subcutaneously to adult (4-mo-old) female mice at doses of 5, 10, or 25 micrograms/day for 3 days, a significant increase in fasting blood glucose concentration and an impairment in glucose tolerance were produced by the 10-micrograms/day dose. Larger effects were obtained with the 25-micrograms/day dose. A threefold increase in fasting plasma insulin concentration was also produced with this dose of the hormone. By contrast, 25 micrograms/day of hGH had no effect on fasting plasma insulin, blood glucose, or glucose tolerance in 1-mo-old ob/ob mice. When a dose of 100 micrograms/day of hGH was given to 1-mo-old animals, a significant increase in fasting plasma insulin concentration occurred, but again there were no effects on fasting blood glucose or glucose tolerance. Mice 1.5 mo old showed marginal changes in fasting blood glucose and glucose tolerance when given hGH at doses of 25 or 100 micrograms/day. Mice 9 or 12 mo old exhibited responsiveness similar to that of 4-mo-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered profiles of biological activity of growth hormone fragments on adipocyte metabolism.

A highly purified preparation of human GH (hGH) and two large fragment complexes (Da1 and Dc2) isolated from plasmin digests of reduced and S-carbamidomethylated hGH were tested for their ability to produce five different biological responses in segments of epididymal fat obtained from hypophysectomized rats. Judging by the minimal concentration of hormone needed to produce a statistically significant response, hGH was 3-10 times as potent as Da1 and Dc2 in increasing the oxidation of [U-14C]glucose to 14CO2, hGH and Da1 were equipotent in stimulating the oxidation of L-[1-14C]leucine to 14CO2, antagonizing the lipolytic effect of epinephrine, and inducing refractoriness to the insulin-like action of hGH. At least 3 times as much Dc2 was required to produce significant responses. Da1 was 10 times more active than hGH in producing delayed lipolysis in the presence of theophylline and 30 times as effective as Dc2. These findings suggest that the various responses to GH that can be measured in adipose tissue may result from more than one kind of interaction between GH and adipocytes. (Endocrinology 108: 553, 1981)

Nonmammalian growth hormones have diabetogenic and insulin-like activities.

Purified GHs isolated from ostrich, sea turtle, snapping turtle, bullfrog, Tilapia, and sturgeon were tested for in vivo diabetogenic activity in the hereditarily obese ob/ob mouse and for in vitro insulin-like activity in isolated adipose tissue from hypophysectomized rats. GHs from all species exhibited significant diabetogenic activity, causing fasting hyperglycemia and decreased glucose tolerance when administered at doses of 100 micrograms/day (ostrich, bullfrog, and sturgeon) or 200 micrograms/day (sea turtle, snapping turtle, and Tilapia) for 3 days. Similar responses were obtained when purified human GH was administered at a dose of 10 micrograms/day for 3 days. GHs from most species also exhibited significant insulin-like activity, stimulating increased [14 C]glucose oxidation to 14CO2 by isolated adipose tissue from hypophysectomized rats when employed at concentrations of 50 nM (bullfrog), 250 nM (sturgeon), 500 nM (ostrich), or 2500 nM (sea turtle and Tilapia). Purified human GH gave similar responses at concentrations of 2.5-5 nM in this assay. These results support the hypothesis that diabetogenic and insulin-like activities are intrinsic properties of GH and provide strong evidence that the structural determinants for diabetogenic and insulin-like activities arose early in the evolution of the GH molecule.

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)

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.

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.

Correlation between the biological and immunological activities of growth hormone circulating in rats bearing MtTW15 tumors.

Growth hormone-like activity was demonstrated in diluted plasma of rats bearing the GH-secreting tumor, MtTW15, using an in vitro bioassay. The bioassay used depends upon the ability of GH in vitro to stimulate the uptake of 3-0-methyl glucose (3-OMG) into the isolated diaphragm of the hypophysectomized rat. It was found that the effect of the diluted plasma on this system was qualitatively like that of rat pituitary GH. Theophylline, a drug which blocks the action of GH on 3-OMG uptake by the diaphragm but has no effect on the actions of insulin or somatomedin on this process, completely abolished the stimulatory action of the plasma on the transport of the sugar. Furthermore, antibodies against rat pituitary GH (ArGH) completely abolished the biological activity of the diluted plasma. When plasma was diluted to give specific concentrations of rat GH (rGH), as determined by radioimmunoassay (RIA), the plasma produced responses in the 3-OMG assay equivalent to those obtained with similar concentrations of pituitary rGH. A good correlation was also observed between the concentration of rGH in the plasma of tumor-bearing rats measured by RIA and by an in vivo bioassay in which the effects were determined of injections of diluted plasma into hypophysectomized rats on the subsequent incorporation of [3H]thymidine into costal cartilage. This in vivo biological activity was abolished by incubation of the plasma with ArGH prior to its injection into the test animals. Thus, these studies indicate that in rats bearing the MtTW15 tumor there is a good correlation between the biological and immunological activities of the rGH circulating the bloodstream.

A hybrid noncovalent complex of fragments of porcine and human growth hormones with diabetogenic activity.

Although porcine GH (pGH) and human GH (hGH) are structurally related, their structures differ to the extent that, unlike hGH, pGH is not active in primates and does not exhibit lactogenic activity. Therefore, it was of interest to determine whether hybrid noncovalent complexes could be formed by complementation of large fragments of pGH with fragments of hGH and to study the immunological and biological properties of such hybrids. For this purpose, pGH was digested with bovine thrombin conjugated to Sepharose. After reduction and S-carbamidomethylation of the digested hormone, two large fragments consisting of residues 1-133 (p1-133T) and 134-191 (p134-191T) were isolated by gel filtration. The human GH fragments used in this work were S-carbamidomethylated peptides 1-134 (h1-134T) and 135-191 (h135-191T) isolated from thrombin-digested hGH. Noncovalent complementation of the peptide fragments was attempted by dissolving equimolar amounts of the materials in 0.5% (wt/vol) ammonium bicarbonate solution containing 6 M guanidine-HCl and then gradually removing the guanidine-HCl by dialysis. Using this method, a hybrid noncovalent complex of peptides p1-133T and h135-191T was produced in 50% yield by weight of starting material. Attempts to produce the reciprocal complex between h1-134T and p134-191T, as well as attempts to recombine the contiguous pGH fragments, p1-133T, and p134-191T, were unsuccessful. The hybrid complex (p1-133T + h135-191T) had 40% the cross-reactivity of native pGH in a rat GH RIA, but it was inert in an hGH RIA. It had very low growth-promoting activity in the 9-day weight gain test in hypophysectomized rats and no detectable in vitro ability to stimulate [14C]phenylalanine incorporation into the protein of the diaphragm of the hypophysectomized rat. When tested for insulin-like activity (stimulation of glucose oxidation in vitro in adipose tissue of hypophysectomized rats), the hybrid complex had less than 1% the activity of native pGH. In contrast, the hybrid complex had approximately 10-20% the diabetogenic activity of native pGH in the ob/ob mouse. The fact that the hybrid complex has substantial diabetogenic activity, although lacking significant anabolic or insulin-like activities, suggests that the structural requirements differ for the expression of these various activities of GH.

Complementation of human growth hormone (GH) peptide 1-134 with C-terminal fragments of human GH produced by digestion with bromelain.

The digestion of human GH (hGH) with the proteolytic enzyme, bromelain, results in a major product consisting of a mixture of three large fragments, i.e. residues 1-135 + 143-191, 1-135 + 145-191, and 1-135 + 146-191. In the case of each fragment, the N-terminal peptide is joined to the C-terminal fragment by the disulfide bridge between residues 56 and 165. A C-terminal fragment mixture consisting of peptides 143-191, 145-191, and 146-191 was isolated from this major digestion product after reduction and S-carbamidomethylation of its disulfide bonds. In the present study, the noncovalent complementation of the peptides in this mixture with S-carbamidomethylated peptide 1-134 derived from thrombin-digested hGH was investigated. Noncovalent complementation of these peptides was accomplished by dissolving equimolar amounts of the materials in 0.5% ammonium bicarbonate-6 M guanidine-HCl and dialyzing the mixture slowly to remove the guanidine-HCl. The recombinant mixture was recovered in 26% yield by gel filtration of the peptide mixture and was found to contain three noncovalent recombinant species, i.e. peptides 1-134 + 143-191, 1-134 + 145-191, and 1-134 + 146-191. Thus it would appear that residues 135-145 are not required to obtain noncovalent complementation between the N- and C-terminal regions of the hGH molecule. In an RIA for hGH the recombinant mixture was found to possess approximately 40% the cross-reactivity of the native hormone. In contrast, it had only about 10% the activity of native hGH in the weight gain test in hypophysectomized rats, in stimulating phenylalanine incorporation into the protein of the isolated hypophysectomized rat diaphragm, and in stimulating glucose oxidation by isolated adipose tissue of hypophysectomized rats. The limited biological activity of the recombinant mixture is of interest, since the major bromelain digestion product from which the C-terminal peptides were derived consists of a mixture of rather similar molecules (i.e. peptides 1-135 + 143-191, 1-135 + 145-191, 1-135 + 146-191; and with intact disulfide bridges), which exhibits substantial growth-promoting and insulin-like activities.

Relationship between the biological and immunological activities of growth hormone circulating in normal rats.

The ability of GH to induce refractoriness to its own insulin-like effect in adipose tissue is highly specific for GH. Moreover, refractoriness to the insulin-like action of GH can be induced with low concentrations of GH in the range of 10 ng/ml. In the present study, the ability of plasma from normal rats to induce refractoriness to the effect of GH on the production of 14CO2 from [14C]glucose in epididymal fat pads from hypophysectomized rats was examined as a measure of a GH-specific biological effect of the plasma. Plasma levels of GH were measured by RIA. Without exception, preincubation of fat pads with plasma having immunoreactive rat GH (rGH) levels over 50 ng/ml induced refractoriness to GH, whereas plasma having immunoreactive rGH levels below 15 ng/ml did not exert this effect. In fat pads preincubated with plasma having immunoreactive rGH levels between 10-50 ng/ml, the effect of a second exposure to GH varied in a dose-dependent manner. Also, plasma pools that were diluted with buffer or plasma from hypophysectomized rats were unable to induce refractoriness when the calculated or measured levels of immunoreactive rGH were below 15 ng/ml. The results suggest that in terms of the ability of GH to induce refractoriness to the insulin-like effect of the hormone, a very good correlation exists between the biological and immunological activities of GH in plasma from normal undisturbed rats.

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.

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.

Isolation and characterization of fragments of reduced and S-carbamidomethylated human growth hormone produced by plasmin digestion. I. Chemistry.

Reduced and S-carbamidomethylated human GH (RCAM-hGH) was digested with human plasmin, yielding a mixture of products. These were partially separated by chromatography on DEAE-cellulose, yielding three major fractions: Da and Db, which were equipotent with native hGH in the weight gain test; and Dc, which was about half as active as hGH. Each of these was further purified by gel filtration, yielding a number of subfractions which were characterized as follows: Da1 is a very stable noncovalent complex of residues 1--134 and 141--191 of RCAM-hGH; Da2 represents residues 20--41; Db1 is very similar to Da1, but appears to have lost one or more amide groups; Db3 represents residues 95--134; Dc2 is a heterogenous fraction containing a further deamidated version of Da1 and Db1 plus a similar complex of residues 42--134 and 141--191, apparently with some carboxyterminal heterogeneity; Dc3, like Db3, represents residues 95--134. The biological activities of these fragments are discussed in the accompanying paper. Earlier work has shown that native hGH, upon digestion with plasmin, is cleaved primarily at residues 134 and 140. It is shown here that when RCAM-hGH is digested with plasmin, in about 87% of the molecules at least one cleavage takes place in addition to those at residues 134 and 140.