Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse.

During early mouse development the homeobox gene Hesx1 is expressed in prospective forebrain tissue, but later becomes restricted to Rathke's pouch, the primordium of the anterior pituitary gland. Mice lacking Hesx1 exhibit variable anterior CNS defects and pituitary dysplasia. Mutants have a reduced prosencephalon, anopthalmia or micropthalmia, defective olfactory development and bifurcations in Rathke's pouch. Neonates exhibit abnormalities in the corpus callosum, the anterior and hippocampal commissures, and the septum pellucidum. A comparable and equally variable phenotype in humans is septo-optic dysplasia (SOD). We have cloned human HESX1 and screened for mutations in affected individuals. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain which destroyed its ability to bind target DNA. These data suggest an important role for Hesx1/HESX1 in forebrain, midline and pituitary development in mouse and human.

Up and down the growth hormone cascade.

In this review we present a personal overview of current research at different levels of the control cascade that regulates growth hormone and insulin-like growth factors, the primary endocrine regulators of post-natal growth and metabolism. Given the broad scope of this topic we have focused on only a few key areas of current interest.

Membrane retrieval in the guinea pig neurohypophysis: biochemical characterization of a retrieval structure.

[3H]Choline and [35S]methionine injected into the guinea pig hypothalamus in vivo were incorporated into the lipids and proteins, respectively, of secretory vesicles transported to the neural lobe. Prolonged in vivo stimulation of hormone secretion by dehydration decreased the [3H]choline content of secretory vesicles, with a concomitant increase in the [3H]choline content of a membrane fraction isolated on sucrose gradients. After stimulation of neural lobes in vitro in the presence of horseradish peroxidase, this extracellular fluid marker was found in the same membrane fraction. SDS electrophoresis of membrane proteins radiolabelled by [35S]methionine in vivo demonstrated that this fraction contained at least one major protein also present in the secretory vesicle membrane. These results suggest that we have isolated a membrane fraction containing the structure(s) involve in membrane retrieval in the neurohypophysis.

Hypothalamic STAT proteins: regulation of somatostatin neurones by growth hormone via STAT5b.

Signal transducers and activators of transcription (STATs) are a family of transcription factors linked to class I cytokine receptors. In the present study, we investigated whether their distribution in the hypothalamus reflects the feedback regulation by growth hormone and what role they might play in the functioning of target neurones. We demonstrate that each of the seven known STATs has a distinct distribution in the hypothalamus. Notably, the STAT5 proteins, that are important in growth hormone (GH) and prolactin signalling in peripheral tissues, were expressed in somatostatin neurones of the periventricular nucleus and dopamine neurones of the arcuate nucleus. Because somatostatin neurones are regulated by feedback from circulating GH, we investigated the importance of STAT5 in these neurones. We demonstrate that STAT5b protein expression, similar to somatostatin mRNA, is sexually dimorphic in the periventricular nucleus of rats and mice. Furthermore, chronic infusion of male dwarf rats with GH increased the expression of STAT5b, while a single injection of GH into similar rats induced the phosphorylation of STAT5 proteins. The cellular abundance of somatostatin mRNA in STAT5b-deficient mice was significantly reduced in the periventricular nucleus, effectively reducing the sexually dimorphic expression. These results are consistent with the hypothesis that STAT5 proteins are involved in the feedback regulation of somatostatin neurones by GH, and that these neurones may respond to patterned GH secretion to reinforce sexual dimorphism in the GH axis.

Single cell enzyme activity and proliferation in the growth plate: effects of growth hormone.

Longitudinal growth is a result of proliferation and differentiation of chondrocytes in the growth plate. Growth hormone (GH) stimulates longitudinal growth, and GH receptors have been shown on growth plate chondrocytes, but the effects of GH on chondrocytes of different cell layers are not clear. To study the effect of GH on chondrocyte activity, in situ biochemical techniques were used to measure enzyme activities, which are associated with cell differentiation (alkaline phosphatase [ALP]) and osteoclast activity (tartrate-resistant acid phosphatase [TRAP]), within single cells of the growth plate. Uptake of bromodeoxyuridine (BrdU) was used as a parameter for proliferative activity. In addition, glucose-6-phosphate dehydrogenase (G6PD) was measured since increased proliferation has been associated with increased G6PD activity. The role of GH was studied in a model of isolated GH deficiency (dwarf rat) and complete pituitary deficiency (hypophysectomized rat). Groups of GH-deficient dwarf rats were infused with recombinant human GH in either a continuous or a pulsatile manner, since the pattern of GH secretion is an important regulator of growth in the rat. After 7 days, G6PD activity in proliferative chondrocytes and TRAP activity in osteoclasts was increased, while ALP activity in hypertrophic chondrocytes was decreased. GH not only increased the number of chondrocytes that incorporated BrdU but also the total number of chondrocytes in the proliferative zone; therefore, its ratio, the labeling index (an indicator of proliferative rate), was not increased. The widths of the proliferative and hypertrophic zones were increased by both patterns of GH administration. The width of the resting zone was unaffected by continuous GH but decreased by pulsatile GH. ALP and TRAP activities were, respectively, higher and lower in hypophysectomized rats compared with the GH-deficient animals. Hypophysectomized rats had smaller growth plates than dwarf rats with a disproportionally wide resting zone, which, like BrdU uptake, was not affected by GH. GH treatment resulted in increased TRAP and decreased ALP activity. These results indicate that GH stimulates the commitment of chondrocytes within the resting/germinal layer to a proliferative phenotype (as opposed to stimulating the rate of chondrocyte proliferation) but only in the presence of other pituitary hormones. Furthermore, this study shows that enzyme activities within single chondrocytes and osteoclasts are GH-sensitive. The extent to which these effects are direct or mediated by systemic or local growth factors remains to be clarified.

Paradoxical growth-promoting effects induced by patterned infusions of somatostatin in female rats.

In previous studies with iv infusions of GH or its releasing factor (GRF), we showed that a pulsatile pattern of GH was more effective than continuous GH exposure in stimulating growth in the rat. Since GH release is profoundly affected by its inhibitory factor, somatostatin (SS), we were interested to know whether the effects of SS on GH secretion and growth were also dependent on its pattern of administration. SS infusions were given iv to conscious chronically cannulated female rats through programmable multichannel infusion pumps. Multiple blood samples were obtained with the use of an automated system of pumps, solenoid fluid valves, and a fraction collector, all controlled by a microcomputer. SS infusions (5, 25, or 50 micrograms/h) suppressed GH secretion and elicited a rapid, short-lived rebound release of GH after stopping the infusion. Sinusoidal SS infusions in female rats produced cyclic episodes of GH secretion, but a male type of regular 3-hourly secretory pulses of GH was best achieved by prolonged infusions in which the delivery of SS was interrupted for a short period every 3 h. This intermittent SS infusion pattern elicited a repetitive series of rebound bursts of GH secretion, which increased body weight gain and pituitary GH content. In contrast, continuous infusions of equivalent amounts of SS had no effect on body weight gain and reduced bone growth significantly. Thus the effects of SS on growth do depend on the pattern in which it is administered, and this peptide, which itself powerfully inhibits GH secretion, paradoxically stimulates weight gain in a normal animal when given in a manner that promotes a more pulsatile GH secretory pattern.

Activation of the hypothalamo-pituitary-adrenal axis by the growth hormone (GH) secretagogue, GH-releasing peptide-6, in rats.

GH-releasing hexapeptide (GHRP-6) is a synthetic secretagogue that stimulates the release of GH by acting at both hypothalamic and pituitary sites. GHRPs also consistently elicit small, but significant, increases in plasma concentrations of ACTH and adrenal steroids. As these secretagogues do not release ACTH directly, they probably interact with the hypothalamic peptidergic systems controlling ACTH release, such as CRH and arginine vasopressin (AVP). We have now examined the activation of the hypothalamo-pituitary-adrenal axis by GHRP-6 in conscious rats. In a series of experiments, rats were injected i.v. with 10 microg GHRP-6, 2 microg CRH, 0.5 microg AVP, or saline, alone or in combination, and serial plasma samples withdrawn and assayed for ACTH, corticosterone, and GH. CRH and AVP increased plasma ACTH levels in all rats, whereas ACTH and corticosterone responses to GHRP-6 were variable and were dependent on the prevailing activity of the hypothalamo-pituitary-adrenal axis. GHRP-6 stimulated the largest ACTH responses in rats that had the lowest basal plasma ACTH and corticosterone levels before GHRP-6 administration. GHRP-6 given in combination with CRH did not increase ACTH levels beyond the response to CRH alone (change in ACTH, 1570 +/- 207 vs. 1714 +/- 245 pg/ml), whereas the combination of GHRP-6 and AVP markedly increased ACTH levels compared with the effects of AVP alone (change in ACTH, 5587 +/- 669 vs. 2338 +/- 451 pg/ml; P < 0.05). The GH responses to GHRP-6 were significantly greater in rats with low basal plasma ACTH and corticosterone levels than in rats with elevated ACTH and corticosterone levels (change in GH response, 119 +/- 27 vs. 29 +/- 7 ng/ml; P < 0.01). CRH alone significantly inhibited GH release (pre- vs. 40 min post-CRH, 11.9 +/- 3.8 vs. 1.7 +/- 0.4 ng/ml; P < 0.05), whereas AVP alone had no effect on GH levels. Neither CRH nor AVP had any effect on the GH response to GHRP-6. We suggest that GHRP-6 acts via the hypothalamus to mediate the release of ACTH, and that these effects are probably mediated at least in part via the release of endogenous CRH and are subject to regulation by circulating glucocorticoids.

Effect of food withdrawal and insulin on growth hormone secretion in the guinea pig.

The guinea pig is unusual in that its postnatal growth appears to be independent of GH even though its pituitary gland produces a GH molecule. The effects of fasting on the GH secretory pattern and the GH responses to insulin, GH-releasing factor (GRF), and somatostatin (SS) during fasting have now been studied by automatic microsampling of blood in chronically cannulated normal guinea pigs. Withdrawal of food in both male and female guinea pigs changed the GH secretory pattern dramatically. The normal episodic GH secretory pattern [large GH peaks occurring at 3.6 +/- 0.4-h intervals over a low (approximately 0.5-1.5 ng/ml) baseline secretion] was altered to a pattern of more continuous GH output, characterized by a 10-fold elevated baseline secretion (5-15 ng/ml) with no large secretory episodes or troughs. Glucose injections (three injections of 600 mg, iv, at hourly intervals) in fasted guinea pigs lowered their elevated blood GH levels significantly (from 9.1 +/- 1.1 to 6.5 +/- 0.9 ng/ml). Insulin injections (1, 2, or 6 U, iv) inhibited spontaneous GH pulses in normally fed animals, but had little effect on the high continuous GH tone during fasting. The elevated GH secretion in fasted animals could be inhibited by continuous infusion of SS or a single iv injection of a long-acting SS analog. The secretion of GH during fasting could be further increased, either by injections of GRF (two injections of 2 micrograms, iv, 90 min apart), producing peak levels of 102 +/- 16 and 68 +/- 21 ng/ml (above a baseline output of 8.8 +/- 2.2 ng/ml), or by a continuous iv infusion of GRF (12 micrograms/h). Because the GH secretory pattern in the guinea pig is so sensitive to nutrition and insulin, this species may provide an interesting model in which to study selectively the metabolic, as opposed to growth-promoting, actions and regulation of GH.

Growth hormone binding protein in the rat: effects of gonadal steroids.

In normal rats, females have higher circulating GH-binding protein (GHBP) levels than males, whereas in the GH-deficient dwarf (Dw) rat, there is no sexual dimorphism in plasma GHBP, suggesting that GH secretion may be involved in this difference. In order to study the relationship between gonadal steroids and GH on GHBP and GH receptor regulation, the levels of plasma GHBP, hepatic bovine GH, and human GH (hGH) binding as well as GHBP and GH receptor messenger RNA (mRNA) have now been studied in normal, Dw, hypophysectomized (Hx), or ovariectomized (Ovx) rats, subjected to different GH and gonadal steroid exposure. In normal male rats, estradiol (E2, 12.5-25 micrograms/day for 1 or 2 weeks) markedly increased plasma GHBP and hepatic hGH, and bGH binding. These effects of E2 were diminished in Dw rats, absent in Hx rats, but restored in Hx rats given exogenous hGH. Plasma GHBP rose in female rats given E2, and fell in females given the anti-estrogen tamoxifen. Ovx animals had lower plasma GHBP and hepatic GH binding which was reversed by E2, but not testosterone treatment. Continuous hGH infusions in Ovx rats restored hepatic GH binding, and increased plasma GHBP. In Dw males, hGH increased plasma GHBP and hepatic GH binding, whereas testosterone had no effect on GHBP or GH receptors and did not affect their up-regulation by hGH. Hepatic levels of GHBP-, and GH receptor mRNA transcripts showed the same trends in response to steroid or GH treatment, but the differences were rarely significant, except in Ovx animals which had higher GHBP mRNA transcripts after GH or E2 treatment. Thus E2 and GH increase both plasma GHBP and hepatic GH receptor binding. GH up-regulates GHBP in the absence of E2, whereas E2 treatment does not raise GHBP in the absence of GH. Whereas some of the effects of estrogen could be mediated via alterations in GH secretion, estrogen may also directly influence GHBP production at the liver, but only in the presence of GH.

Effects of growth hormone secretagogues on prolactin release in anesthetized dwarf (dw/dw) rats.

In addition to stimulating GH release, GH secretagogues such as GH-releasing peptide-6 (GHRP-6) stimulate small amounts of ACTH and PRL release. Although the effects on ACTH have recently been studied, there is little information about the effects of GHRP-6 on PRL. We have now studied GHRP-6-induced GH and PRL release and their regulation by estrogen (E2) in anesthetized male and female rats and in GH-deficient dwarf (dw/dw) rats that maintain high pituitary PRL stores and show elevated hypothalamic GH secretagogue receptor expression. Whereas GHRP-6 (0.1-2.5 microg, i.v.) did not induce PRL release in normal male or female rats, significant PRL responses were observed in dw/dw females. These responses were abolished by ovariectomy and could be strongly induced in male dw/dw rats by E2 treatment. These effects could be dissociated from GHRP-6-induced GH release in the same animals, but not from PRL release induced by TRH, which was also abolished by ovariectomy and induced in males by E2 treatment. However, the effects of GHRP-6 on PRL were unlikely to be mediated by TRH because in the same animals, TSH levels were unaffected by GHRP-6 whereas they were increased by TRH. The increased PRL response could reflect an increase in GH secretagogue receptor expression that was observed in the arcuate and ventromedial nuclei of E2-treated rats. Our results suggest that the minimal PRL-releasing activity of GHRP-6 in normal rats becomes prominent in GH-deficient female dw/dw rats and is probably exerted directly at the pituitary; these GHRP-6 actions may be modulated by E2 at both hypothalamic and pituitary sites.

Growth hormone (GH) binding protein and GH interactions in vivo in the guinea pig.

GH binding proteins (GHBPs) are present in the blood of several species and by complexing with circulating GH may alter its clearance and distribution. The actions of human GHBP (hGHBP) have hitherto been studied indirectly via their effects on the clearance of hGH. In the present experiments, recombinant preparations of hGHBP and a shorter variant lacking exon 3 (delta 3hGHBP) were tested in vivo, either alone or after preincubation with recombinant hGH, recombinant 20K methionyl-hGH, or rat GH. Multiple serial blood sampling was performed in conscious chronically cannulated guinea pigs and both GHBP and GH variants monitored in the same samples by specific RIAs, unaffected by endogenous activities in this species. hGHBP, delta 3hGHBP, hGH, and 20K met-hGH (10-40 micrograms) were all cleared rapidly when injected alone (t1/2 = 11-20 min). However, when the same amounts of hGHBP and hGH were incubated together for 60 min and then injected, the clearance of both proteins was greatly prolonged (t1/2 hGHBP = 75-94 min, hGH = 80-137 min). Similar results were obtained for hGH complexed with delta 3hGHBP, and over a range of GH:GHBP ratios from 0.3:1 to 4:1. The effect was specific for 22K hGH; neither rat GH nor 20K met-hGH altered hGHBP clearance, nor were their clearances slowed by preincubation with hGHBP. Rapid complex formation also could be demonstrated in vivo. Injections of hGH 30 min after delta 3hGHBP, or vice versa, altered the established plasma disappearance curve of hGHBP, and hGH clearance was slowed in parallel. During a continuous infusion of hGH to steady state, injections of delta 3hGHBP increased plateau hGH concentrations by forming a delta 3hGHBP/hGH complex in the circulation. These experiments show that the conscious chronically cannulated guinea pig provides a useful model in which the in vivo interaction between hGHBP and hGH can be studied dynamically. The results imply that the location, extent, and rate of complex formation between GHBP and GH may be an important determinant of the passage of GH between the intra- and extravascular compartments, which could affect the pattern of tissue exposure to GH.

Growth responses in a mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor I.

A new mutant GH-deficient dwarf rat has been used to study the effects of iv infusions of human GH (hGH) and recombinant human insulin-like growth factor I (hIGF-I). This animal has only about 5% of normal pituitary GH content, low circulating GH levels, and no regular GH surges. The defect seems to be specific for GH. Infusions of hIGF-I at 180 micrograms/day for 9 days elevated serum IGF-I concentrations significantly over those in the saline-infused controls (713 +/- 20 ng/ml vs. 395 +/- 31 ng/ml); hGH infusions did not raise IGF-I levels significantly (435 +/- 20 ng/ml). Gel filtration of serum samples showed that the high-dose hIGF-I infusions increased free IGF concentrations, without apparently altering the pattern of IGF-I binding whereas hGH infusions increased the amount of high mol wt IGF-I binding protein. Neither IGF-I nor hGH infusions affected the small amounts of rat GH present in the dwarf rat pituitary glands. Continuous iv infusions of hGH (200 mU/day for 9 days) stimulated body wt gain (2.1 +/- 0.2 g/day) and bone growth (96 +/- 9 microns/day) significantly compared to saline-infused dwarf rats (1.2 +/- 0.3 g/day and 43 +/- 3 microns/day). Infusions of hIGF-I at 180 micrograms/day produced a body wt gain (2.1 +/- 0.5 g/day) similar to that seen in the hGH-infused group but a significantly smaller stimulation of bone growth (63 +/- 3 microns/day). Infusion of a 5-fold lower dose of hIGF-I (36 micrograms/day for 9 days) had no effect on body wt or bone growth. Food intake was unaffected by either hGH or hIGF-I infusions. The pattern of tissue growth was affected differentially by hGH and IGF-I infusions that produced the same overall body wt gain. hGH induced a relatively proportional growth in most of the organs studied, whereas hIGF-I infusion at 180 micrograms/day stimulated a disproportionately greater growth of the kidney, adrenals, and spleen. In some of the animals, tissues were extracted for RIA of IGF-I; the amounts of IGF-I in the liver were similar in control, hGH, or IGF-I-infused animals, whereas kidney and adrenals from IGF-I infused animals contained larger amounts of immunoreactive IGF-I than did those tissues from hGH-treated rats. Thus, both hGH and hIGF-I can promote growth in the mutant dwarf rat, but they differ both quantitatively and qualitatively in their pattern of actions.

The sensitivity of hepatic CYP2C gene expression to baseline growth hormone (GH) bioactivity in dwarf rats: effects of GH-binding protein in vivo.

Hepatic mRNA transcripts for the steroid-metabolizing enzymes cytochrome P4502C11 (male specific) and P4502C12 (female specific) differ in abundance by 10- to 20-fold in male and female rats and are regulated by their different patterns of GH secretion. This sex difference is also found in dwarf rats with low GH secretion, implying that these transcripts may be very sensitive to low level GH exposure. This has now been characterized in normal and dwarf rats. Continuous i.v. infusion of recombinant human (h) GH (0, 3, 12, and 48 micrograms/day) in both dwarf and normal male rats caused a dose-dependent decrease in P4502C11 and an increase in P4502C12, so that the 2C11/2C12 ratio fell from 17.9 +/- 1.3 to 1.5 +/- 1.0 in normal males and from 6.5 +/- 0.9 to 0.4 +/- 0.3 in dwarf males (0 vs. 48 micrograms hGH/day); over this dose range of hGH, body weight gain, total hepatic insulin-like growth factor-I mRNA levels, and plasma GHBP levels were largely unaffected. These effects of hGH were pattern dependent. The 2C11/2C12 ratio in dwarf males was feminized (from 11.9 +/- 1.3 to 0.08 +/- 0.03) by continuous infusion of hGH (36 micrograms/day), whereas a pulsatile infusion (3-min pulses every 3 h) of the same daily hGH dose was much less effective. Neither continuous nor pulsatile hGH affected P4502C11 or P4502C12 transcripts in dwarf females, although pulsatile hGH infusion caused a significant weight gain. To test whether baseline GH levels could be modified by circulating GH-binding protein (GHBP), hGH infusions were given with and without recombinant hGHBP in different patterns. Pulsatile infusions of recombinant hGHBP (42 micrograms/day, i.v.) did not prevent the feminizing effect of continuously infused hGH (36 micrograms/day, sc) in dwarf males (2C11/2C12 ratios were 0.08 +/- 0.01 and 0.09 +/- 0.01 for hGH vs. hGH plus hGHBP, respectively). This suggested that intermittent complex formation with GHBP did not prevent continuous access of hGH to the hepatic GH receptors. Furthermore, pulses of hGH complexed with GHBP significantly reduced the 2C11/2C12 ratio in dwarf males (from 21.5 +/- 3.9 with pulsatile hGH alone to 9.2 +/- 2.5 with pulses of hGH plus hGHBP), indicating that GHBP prolongs the exposure to hGH. Thus, 2C11/2C12 expression is very sensitive to basal GH levels in dwarf rats, and GHBP can alter hepatic gene expression by modifying the pattern of GH exposure.

Growth, growth hormone (GH)-binding protein, and GH receptors are differentially regulated by peak and trough components of the GH secretory pattern in the rat.

Body growth, GH secretory pattern, hepatic GH receptor (GHR), and plasma GH-binding protein (GHBP) levels are all sexually dimorphic in the rat. Male rats grow faster than females, and in GH-deficient animals, GH therapy is more effective when given in a pulsatile pattern rather than a continuous infusion. This contrasts with GHBP and hepatic GHR levels, which are lower in males than in females and raised by continuous but not pulsatile GH therapy. One possible explanation is that growth is primarily regulated by GH pulses, whereas GHR and GHBP are regulated mostly by the trough levels (which are lower in males than in females). To test this hypothesis directly, GH-deficient dwarf rats were given patterned iv infusions of hGH in which the relative contributions of the peak and trough components of the GH pattern were systematically varied, independently of dose, and their effects on weight and length gain, plasma GHBP, and hepatic GHR binding were measured. We found that the dose-response curves for GH given by pulsatile vs. continuous infusion were significantly nonparallel, and that growth was primarily stimulated by the pulsatile component of a mixed GH infusion pattern; doubling the GH dose by adding a continuous (c) infusion to a series of pulses (p) neither enhanced nor inhibited weight gain (36 micrograms hGH/day pulses (36p) vs. 36 micrograms hGH/day pulses + 36 micrograms hGH/day continuously (36p + 36c):0.9 +/- 0.2 g/day vs. 1.1 +/- 0.2 g/day), whereas doubling the GH dose by adding a pulsatile component significantly enhanced growth (72p:2.1 +/- 0.2 g/day, P < 0.01). Conversely, hepatic GHR and plasma GHBP levels were highly sensitive to the continuous element of the mixed infusion pattern and were totally unaffected by varying the pulsatile component over a wide range of doses. These results strongly suggest that growth and hepatic GHR/plasma GHBP respond differentially to the peak and trough components of the GH secretion pattern in the rat.

Differential regulation of the growth hormone receptor gene: effects of dexamethasone and estradiol.

GH receptor (GHR) expression differs during development between central and peripheral tissues. Peripheral GHR expression is known to be sensitive to gonadal and adrenal steroids, but little is known about their effects on GHR in the central nervous system. We have now studied the effects of estradiol (E2) or dexamethasone on GHR expression in rat arcuate nucleus (ARC) and hippocampus, using quantitative in situ hybridization. Dexamethasone, which strongly down-regulates hepatic GHR expression, had no effect on central GHR transcript abundance, whereas E2 treatment, which stimulates hepatic GHR expression, significantly reduced ARC GHR messenger RNA (mRNA) levels. E2 also increased somatostatin (SS) expression significantly in both ARC and periventricular nuclei but did not reduce ARC GH-releasing hormone (GHRH) mRNA levels. Ovariectomy stimulated GHR and GHRH mRNA levels in the ARC, whereas it lowered ARC SS expression. E2 replacement in ovariectomized animals restored GHRH and SS mRNA levels to control values. Hippocampal GHR mRNA transcripts showed the same response to these endocrine manipulations as seen in the ARC. The induction of hepatic GHR expression by E2 is known to involve the transcription of an alternate 5' untranslated first exon, GHR1. This was readily detectable in the liver using a specific GHR1 probe but could not be detected in any CNS area. Our results show that GHR expression in the CNS is sensitive to regulation by peripheral steroids but that CNS and hepatic expression of GHR is differentially regulated by the same treatments.

Effects of growth hormone secretagogues in the transgenic growth-retarded (Tgr) rat.

Exogenous GH inhibits endogenous GH release by hypothalamic feedback. We have recently exploited this to generate transgenic growth-retarded (Tgr) rats, in which human GH is expressed in the hypothalamus, under the control of the rat GRF gene promoter. These rats show reduced pituitary size, GH deficiency, and dominant dwarfism, but are large enough for serial blood sampling studies to examine their spontaneous GH secretion and responses to GRF, somatostatin, and GH-releasing peptide-6 (GHRP-6). Like their normal wild-type littermates, Tgr rats show a sexually dimorphic pattern of GH secretion; males secrete GH in 3-h episodes, whereas females exhibit a more continuous irregular output, with higher baseline GH levels. In anesthetized male Tgr rats, the GH responses to GRF or GHRP-6 were markedly reduced compared with those of their nontransgenic littermates, but the differences were smaller in females. Despite the reduction in pituitary GH, peak plasma GH responses to serial GRF injections in conscious Tgr males or intermittent somatostatin infusions in conscious Tgr females were indistinguishable from the responses in their wild-type littermates. Furthermore, 7-day iv infusions of GRF (12.5-100 micrograms/day), given either continuously or as a pulsatile infusion stimulated growth in Tgr rats, as did pulsatile infusions of GHRP-6. Thus, despite their pituitary GH deficiency and dwarfism, Tgr rats maintain a sexually dimorphic pattern of GH release and can produce large GH secretory responses to exogenous secretagogues. They represent the first genetic model of GH deficiency in the rat in which dwarfism can be corrected by treatment with exogenous GH secretagogues.

Neurohypophyseal and fluid homeostasis in transgenic rats expressing a tagged rat vasopressin prepropeptide in hypothalamic neurons.

We have developed a transgenic system that, for the first time, facilitates monitoring of the regulatory dynamics of a central peptidergic system from transcription of a neuropeptide gene to the storage and release of the mature secretory product. A rat vasopressin (VP) transgene (5-VCAT-3), the expression of which is restricted to hypothalamic vasopressinergic magnocellular neurons in rats, contains a sequence that, if translated, would place a unique hexadecapeptide (DRSAGYYGLFKDRKEK, abbreviated to DR-12-EK) at the C-terminus of the VP precursor. We have raised an antibody against this "tag" and, using immunohistochemistry, electron microscopy, RIA, and HPLC, have shown for the first time that a VP transgene RNA is translated into a protein product found, in a processed form, in secretory granules in the posterior pituitaries of transgenic rats. Disruption of the C-terminus of the VP precursor by the peptide tag is well tolerated and does not disrupt VP production or disturb salt and water balance. An osmotic stimulus increased hypothalamic DR-12-EK levels, but changes in posterior pituitary DR-12-EK levels were more complex. After 5 days of salt-loading, DR-12-EK levels fell, as would be expected if its release was coordinate with that of VP. However, after 10 days of salt-loading, posterior pituitary DR-12-EK levels increased, despite the lower level of VP. This probably reflects the greater response of the transgene to osmotic challenge at the RNA level, increasing the proportion of DR-12-EK-containing translation products transported to the posterior pituitary relative to those derived from the endogenous gene. The exaggerated response of the tagged transgene to osmotic challenge at both RNA and protein levels affords a new opportunity to study the regulatory dynamics of the VP system at the molecular level, but within the physiologically advantageous context of the intact animal.

Steroid regulation of growth hormone (GH) receptor and GH-binding protein messenger ribonucleic acids in the rat.

In the rat, the GH receptor (GHR) and the GH-binding protein (GHBP), which arise from alternative splicing of the same gene, show a sexually dimorphic and GH-dependent expression pattern. Multiple alternative 5'-untranslated regions (UTRs) are present in GHR and GHBP transcripts in the rat, one of which, GHR1, has recently been shown to be liver specific and found at higher levels in females. We have measured the hepatic GHR1, GHR, and GHBP transcript levels, by RNase protection and solution hybridization assay, in animals with differing hormonal status, in which hepatic GHR binding and plasma GHBP have been previously assayed. Estradiol (E2) induced GHR1 in males, whereas ovariectomy or the antiestrogen tamoxifen reduced GHR1 expression in females. The induction of GHR1 by E2 was GH dependent, being lower in GH-deficient dwarf rats and absent in hypophysectomized rats, paralleling previous measurements of plasma GHBP and hepatic GHR binding in these animals. Significant changes in GHR1 could explain the trends seen in the same extracts when coding region probes were used. Short-term adrenalectomy had no effect on GHR and GHBP expression, but dexamethasone markedly reduced both protein and messenger RNA (mRNA) levels. Corticosterone treatment had no effect alone but reduced the E2-induced increase in GHR1 levels, whereas methylprednisolone administered orally reduced hepatic GH binding, plasma GHBP, and GHR1 mRNA levels. Thus, 5'-UTRs, encoded by different first exons, are involved in the regulation of hepatic GHR and GHBP expression and need to be considered when comparing effects of hormonal manipulation on the mRNA transcripts and protein products of the GHR gene. Previous studies have found discrepancies between levels of protein expression and mRNA transcripts measured only with coding region probes. Our results suggest that posttranscriptional differences related to 5'-UTR heterogeneity in the GHR gene explain some of these discrepancies.

The obese growth hormone (GH)-deficient dwarf rat: body fat responses to patterned delivery of GH and insulin-like growth factor-I.

We describe a new animal model of obesity and GH deficiency and report the effects on body fat of administering (GH) and insulin-like growth factor (IGF-I) in the model. Female GH-deficient dwarf rats fed a high-fat diet became obese and insulin-resistant compared with chow-fed controls. They were treated with recombinant human GH (rhGH 100-500 micrograms/day, s.c. for 14 days) by daily injection or minipump infusion with or without rhIGF-I (200 micrograms/day, sc infusion). Injections of rhGH increased body weight; infusions of rhGH caused weight loss. RhIGF-I by itself, or rhIGF-I plus GH injections had little effect, whereas rhGH infusions plus rhIGF-I caused a weight loss equivalent to the weight gained during the high-fat feeding and a decrease in fat pad weight. For some responses (serum IGF-1 and GHBP), the obese rats were GH resistant. Fat was lost from the internal fat pads when obese rats were returned to a chow diet, and injections of rhGH surprisingly attenuated this loss of fat. In obese dwarf rats, the lipolytic effects of rhGH are dose-regime dependent. By itself IGF-I is not insulin-like, but in the presence of GH it has antiinsulin actions causing a powerful net lipolysis. If GH plus IGF-I have similar effects in humans they may be useful for reducing body fat.

Enhancement of growth hormone bioactivity by zinc in the eluted stain assay system.

The effects of ionic zinc (Zn2+) on human (h) GH bioactivity have been examined using a lactogenic bioassay. The potencies of pituitary-derived hGH (IRP 80/505), recombinant 22K hGH (IRP 88/624), pituitary-derived human PRL (IRP 84/500), and a recombinant methionyl 20-kilodalton variant of hGH in the presence of selected concentrations of ZnCl2 were investigated with an eluted stain assay that uses Nb2 rat lymphoma cells. This precise colorimetric bioassay is based upon the reduction of a yellow tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-di-phenyl-tetrazolium bromide, to its purple formazan by lactogen-activated Nb2 cells. Zinc (6-100 microM) enhanced the bioactivity of low doses (< 0.045 nM) of both pituitary-derived and recombinant 22K hGH, although the magnitude of enhancement was considerably less than might have been anticipated from previous binding studies (13). Higher concentrations of pituitary-derived hGH (> 0.045 nM) were inhibited by Zn2+. The bioactivity of recombinant methionyl 20K hGH was greatly enhanced by zinc (3-100 microM). In contrast to hGH, the bioactivity of hPRL was not potentiated by Zn2+. These discriminatory effects of Zn2+ when stimulating via the lactogenic receptor are in concordance with the results of previous radioligand binding studies (13). The striking enhancement of 20K hGH lactogenic bioactivity was observed at Zn2+ concentrations within the physiological range for normal human serum (5-20 microM).