A simple dot blot assay adaptable for field use in the diagnosis of onchocerciasis: preparation of an adult worm antigen fraction which enhances sensitivity and specificity.

The lack of a convenient diagnostic method for onchocerciasis has motivated attempts to develop better detection techniques. Preliminary results indicate that a dot blot assay of the total immunoglobulin G (IgG) response to an easily available and simply prepared Tris buffer soluble fraction (TSF) of whole female worms may be applicable in the field as a first screening method. The specificity of the assay is improved by using a partially purified fraction of TSF of which the dominant component is a 23 kDa protein antigen, PakF. The IgG response to PakF was 100% sensitive and at least as specific as the IgG4 response to TSF. Sera from 189 individuals including onchocerciasis patients and apparently uninfected control subjects from an endemic area and from an urban area in Ghana were screened. The specificity of the assay with urban and endemic control sera was 93% and 68% respectively. The dot blot assay has advantages over the existing skin snip and enzyme-linked immunosorbent assay techniques in terms of simplicity, cost, consumption of antigen, risk of spreading other infections, and patient comfort.

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.

Effects of exogenous growth hormone on lipid metabolism in the isolated epididymal fat pad of the growth hormone-deficient little mouse.

The effects of two preparations of highly purified human GH (hGH) on lipid metabolism were studied in the GH-deficient little mouse (50-60 days old). Marked decreases in incorporation of [14C]glucose into fatty acid and in the activity of acetyl-CoA carboxylase in the epididymal fat pads were observed after i.p. injection of hGH at a dose of 1.0 microgram/g body weight or after continuous infusion of hGH by osmotic minipump. The rate of glucose incorporation into fatty acid decreased from 107.0 +/- 27.6 (S.E.M.) to 38.1 +/- 19.6 mumol/g tissue per h after a single injection of hGH and from 174.1 +/- 28.5 to 56.3 +/- 20.3 mumol/g tissue per h after continuous infusion of hGH for 2 days. Activity of the lipogenic enzyme acetyl-CoA carboxylase was also reduced by more than 50% in the epididymal fat pad from hGH-treated mice in comparison with the corresponding control animals. Incubation of isolated fat pads with hGH (0.1 microgram/ml) revealed similar inhibitory effects of the hormone on fatty acid synthesis and acetyl-CoA carboxylase activity. No lipolytic effect of hGH was found as determined by the rate of glycerol release from epididymal fat pads of little mice following hormone treatment in vivo or in vitro. The results lend strong support to the conclusion that GH inhibits lipogenesis but has no effect on lipolysis in adipose tissues, and indicate that the physiological role of GH in lipid metabolism is concerned mainly with the regulation of anabolic rather than catabolic processes.

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.

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.

Metabolic basis for the diabetogenic action of growth hormone in the obese (ob/ob) mouse.

The ob/ob mouse responds predictably to chronic treatment with large doses of pituitary GH with marked hyperglycemia and decreased glucose tolerance. The purpose of the present study was to characterize the metabolic alterations produced by GH that lead to this diabetogenic response in the ob/ob mouse in order to determine whether this animal might serve as a useful model for the study of the cellular mechanisms involved in the diabetogenic action of GH. Female ob/ob mice were treated sc for 3 days with either saline or 200 micrograms/day S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative lacking significant growth-promoting or insulin-like activities. Six hours before the start of the experiment, the animals were given a sc injection of 2 micrograms dexamethasone and deprived of food. RCM-hGH treatment produced marked increases in fasting blood glucose and plasma insulin concentrations, but had no effect on plasma glucagon or serum insulin-like growth factor I levels. It had no effect on liver glycogen level or in vitro hepatic glucose production in the absence or presence of pyruvate and lactate added to the incubation medium. By contrast, the in vitro stimulatory effects of insulin on [14C] glucose oxidation by isolated soleus muscle or segments of parametrial fat were greatly attenuated by RCM-hGH treatment, without changes in rates of basal glucose oxidation. This change in peripheral tissue responsiveness to insulin does not appear to involve glucose transport, since the in vitro stimulation by insulin of 3-O-[14C]methylglucose transport into isolated diaphragm muscle was not altered by RCM-hGH treatment. Moreover, the RCM-hGH-induced reduction in adipose tissue responsiveness to insulin does not appear to be mediated by a reduction in insulin binding, since [125I]iodoinsulin binding to adipocytes isolated from RCM-hGH-treated mice was similar to that to cells from saline-treated animals. Interestingly, the reduction in responsiveness to insulin seen with segments of adipose tissue from RCM-hGH-treated animals was not found with isolated adipocytes prepared from such tissue by collagenase digestion. These results suggest that the hyperglycemia and glucose intolerance produced in ob/ob mice by chronic GH treatment result primarily from increased peripheral tissue insulin resistance. Therefore, the ob/ob mouse provides a useful model to elucidate the cellular mechanism(s) of this aspect of the diabetogenic action of GH.

Effects of growth hormone on lipogenesis and glucose oxidation in genetically GH-deficient mice.

Late inhibitory effects of growth hormone were observed when the hormone (0.1-1.0 microgram/ml) was incubated with adipose tissue from GH-deficient and normal mice. Early insulin-like effects of GH occurred only when glucose was used as precursor. Adult GH-deficient mice have a much higher basal acetyl-CoA carboxylase activity than normal mice. Administration of GH caused a marked reduction of enzyme activity in both groups.