Human growth hormone deletion mutant (hGH44-191) binds with high affinity to lactogenic receptors but not to somatogenic receptors.

The carboxyl-terminal hGH fragment, hGH44-191, displays diabetogenic activity. To understand whether this biological activity is mediated through somatogenic or lactogenic receptors, we investigated the ability of hGH44-191 to bind both receptor classes. We found that hGH44-191 could not compete with [125I]hGH or [125I]bGH for bovine liver somatogenic binding sites. Additionally, hGH44-191 could not displace [125I]hGH from the somatogenic receptor sites when human liver microsomes were used as the receptor source. In contrast, hGH44-191 effectively competed with [125I]hGH for lactogenic receptor sites of lactating mammary gland microsomes and of bovine liver microsomes. In summary, hGH44-191 does not bind to somatogenic receptors but does not bind to lactogenic receptors. These data suggest that the biological actions of hGH44-191 could be mediated through lactogenic receptors.

Luteotropic actions of placental lactogens at midpregnancy in the mouse.

In this study the luteotropic activity of mouse placental lactogen I (mPL-I) at midpregnancy was assessed using in vivo and in vitro methodologies. Ovaries from 10-day pregnant mice were enzymatically dispersed and plated on fibronectin-coated wells in serum-free medium. The percentage of ovarian cells that stained for the presence of 3 beta-hydroxysteroid dehydrogenase activity was 24.4 +/- 2.7% at the time of plating and remained constant (26.1 +/- 5.0%) after a 20-h attachment period. Two types of 3 beta-hydroxysteroid dehydrogenase-staining cells, with distinct differences in size and morphology, were present in the culture. Large luteal cells (26-45 microns) were characterized by a small round nucleus and spherical shape with abundant cytoplasm. In contrast, small luteal cells (< 20 microns) were stellate, with little cytoplasm and a large oval nucleus. Basal progesterone secretion was maintained without a change in cellular DNA content and cell number for 168 h of culture. Treatment of ovarian cells with mPL-I (0.05-10 micrograms/ml) caused a dose-dependent increase in the progesterone concentration in the medium. The magnitude and time course of mPL-I-stimulated progesterone accumulation in culture were dependent on the time after plating that mPL-I treatment was initiated. The effects of mPL-II and mouse PRL (mPRL) on progesterone production were similar to those of mPL-I. The ability of sera from 10-, 14-, and 17-day pregnant mice to maintain progesterone production in bromocryptine-treated hysterectomized mice was also examined. Mice were hysterectomized on day 9 of pregnancy, and serum progesterone, mPL-I, mPL-II, and mPRL concentrations were measured 72 h later. Twice daily injections of 0.5 ml day 10 pregnancy serum maintained the circulating progesterone concentration at values not different from those present at the time of hysterectomy. In contrast, serum progesterone concentrations were not maintained in mice treated with serum of 14- or 17-day pregnant mice or with saline. Depletion of mPL-I from day 10 pregnancy serum by affinity chromatography on an anti-mPL-I column removed all luteotropic activity, as determined by the inability of this modified serum to maintain the serum progesterone concentration in bromocryptine-treated hysterectomized mice. A similar pool of day 10 pregnancy serum chromatographed on a nonspecific IgG control column did maintain progesterone production, but at somewhat lower concentrations than those present at the time of surgery. These studies offer direct evidence that mPL-I and mPL-II are luteotropic and support progesterone production at midpregnancy in the mouse.

Stimulation of body weight gain of the mature female rat by bovine GH and bovine placental lactogen.

Mature female rats (200 g) were treated for 10 days with either recombinant bovine GH (bGH) or recombinant bovine placental lactogen (bPL) to compare the somatogenic responses elicited by these hormones. The treatments were administered by daily s.c. injection at four dose levels (0.19, 0.56, 1.67 and 5.0 mg/day). Both bGH and bPL stimulated significant increases in weight gain, but the slopes of the dose-response curves were different (P less than 0.05). Bovine PL was more potent than bGH (P less than 0.01) at the lowest dose, although there were no differences between treatment groups at the three higher doses. Feed consumption was stimulated more by bPL than bGH at all doses (P less than 0.001). The concentration of insulin-like growth factor-I (IGF-I) in blood plasma was increased by bGH in a dose-responsive manner and was higher than control at doses of 1.67 and 5 mg/day (P less than 0.05). Low doses of bPL stimulated increases in IGF-I similar to those with bGH. At the highest dose of bPL, however, there was no concomitant increase in plasma IGF-I. Nevertheless, the growth rate of the animals in this group matched that of the group given the highest dose of bGH. Receptor binding studies indicated that bPL bound to both GH and prolactin receptors. This is consistent with the growth data which suggests that bPL stimulated weight gain through a somatogenic mechanism as well as by another route, possibly mediated by lactogenic receptors.

Distinct placental lactogen and prolactin (lactogen) receptors in bovine endometrium.

Specific binding sites for bovine placental lactogen (bPL) and the lactogenic hormone, prolactin, have been detected in endometrial membranes isolated from uteri of mid-pregnant heifers. The specific binding of human growth hormone (hGH) (used to monitor the presence of lactogenic binding sites) and of bPL was increased approximately 4-fold following treatment of the membranes with 4 M MgCl2. Binding was found to be ligand specific, membrane protein concentration-, time- and temperature-dependent and reversible. Scatchard analysis of bPL and hGH competition binding data revealed curvilinear plots with dissociation constants for the high affinity sites of 4.1 x 10(-11) M and 6.4 x 10(-11) M, respectively. The maximum capacity of binding of bPL at the high affinity site was 21 fmol/mg). membrane protein while approximately twice the level of binding was measured for hGH (39 fmol/mg). Both hGH and bGH, but not ovine prolactin, competed with [125I]bPL for binding. The concentrations of hGH and bGH needed to effectively compete were however 100-fold higher than those required for unlabeled bPL. No specific binding of radiolabeled bGH was detected in endometrial tissue suggesting the absence of bGH receptors. Preferential competition of [125I]hGH binding was observed by prolactin and bPL. From these data it may be inferred that hGH binding is indicative of the presence of both lactogenic (prolactin) and bPL binding sites in endometrial tissue. The presence of distinct bPL receptors in the endometrium from mid-pregnant cows suggests a possible role for bPL in the maintenance of pregnancy.

A recombinant-DNA-derived modification of human growth hormone (hGH44-191) with enhanced diabetogenic activity.

A modified human growth hormone (hGH) that lacks the first 43 residues of the intact hormone was prepared by recombinant-DNA technology. For preparative purposes an additional alanine was made the amino terminal residue. Sequence analysis and tryptic peptide mapping combined with amino acid analyses confirmed the structure of the polypeptide. Less than 2% N-terminal methionine was detected. The hGH44-191 was estimated to be at least 10 times more active than hGH in producing glucose intolerance in obese yellow mice (Avy/A) and was equipotent to hGH in increasing serum free fatty acids in fasted, hypophysectomized rats. The peptide did not promote growth in hypophysectomized rats nor did it exhibit early (1h) insulin-like activity in fasted, hypophysectomized rats, as indicated by its failure to lower blood glucose and fatty acids. The modified hGH was inactive in the Nb-2 cell assay but was about one-third as active as hGH in stimulating the pigeon crop sac. In radioimmunoassays using 125I-labeled hGH and polyclonal antibodies to intact hGH, cross-reactivity of hGH44-191 was less than 1%. We conclude that removal of the amino terminal portion of hGH enhances its diabetogenic properties, and that this activity does not depend upon the ability to promote growth. Furthermore, the insulin-like activity can be separated from its diabetogenic action by deletion of the first 43 amino terminal residues. This is the first report of a modified hGH that has anti-insulin effects greater than hGH itself.