Zinc depletion attenuates growth and decreases circulating IGF-I. To investigate the mechanisms responsible for the IGF-I decline, we determined the effects of dietary zinc (Zn) deficiency on body and organ growth, serum IGF-I, serum GH-binding protein (GHBP), liver GH receptors and liver expression of their corresponding gene. After 1 week of adaptation to a normal zinc diet, a zinc-deficient diet (ZD; Zn, 0 p.p.m.) or a zinc-normal diet (CTR; Zn, 75 p.p.m.) was available ad libitum to 4-week-old Wistar rats for 4 weeks. Pair-fed animals (PF) received the zinc-normal diet in the same absolute amount as that consumed the day before by the ZD group. The food intake of ZD and PF rats was reduced by 32% (P < 0.001) compared with the CTR group. Zinc depletion specifically reduced body weight gain (-22%, P < 0.05), serum IGF-I concentrations (-52%, P < 0.001), hepatic GH receptors (-28%; P < 0.05) and serum GHBP levels (-51%; P < 0.05), compared with the PF group. GH concentrations were reduced in ZD animals compared with CTR rats (P < 0.01). The caloric restriction of PF animals also decreased body weight gain (-50%, P < 0.001), serum IGF-I concentrations (-21%, P < 0.05), liver GH receptors (-38%, P < 0.001) and serum GHBP levels (-38%, P < 0.01), when compared with the CTR group. Both ZD and PF groups had reduced liver IGF-I and GH receptor/GHBP mRNA levels in comparison with the CTR group (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

We have reported that female hypophysectomized (hypox) rats replaced with T4 and cortisone and treated for 7 days with GH injections (4 x 12.5 micrograms/day) had significantly greater growth and increase in serum insulin-like growth factor-I (IGF-I) than did hypox rats continuously infused with GH (50 and 250 micrograms/day), whereas GH binding to liver membranes was increased only by infusion. We now report the effects of hypophysectomy, T4 and cortisone replacement, and the aforementioned continuous vs. intermittent GH treatment on liver IGF-I and GH receptor (GHR)/binding protein (GHBP) gene expression in female rats. Concentrations of IGF-I peptide were measured in acid-extracted sera and liver tissues. Total GH binding to liver membranes was determined in MgCl2-treated homogenates and serum GH binding activity was assessed by gel filtration of serum incubated with 125I-bovine GH. The abundance of messenger RNA (mRNA) transcripts encoding IGF-I, the GHR, and the GHBP was quantified by Northern hybridization analysis of liver poly(A)+ RNA. Hypophysectomy in female animals decreased serum IGF-I and liver IGF-I mRNA concentrations by 95% and 87%, respectively, serum GHBP activity, and total liver GH binding by 50% (P less than 0.001 compared with intact controls), and liver GHR and GHBP mRNA abundance by 30-35% (P less than 0.05 vs. controls). These changes were not reversed by T4 and cortisone treatment. Repeated injections of GH produced a 13-fold increase in liver IGF-I peptide and a 5-fold increase in liver IGF-I mRNA concentrations (vs. saline-treated hypox rats), whereas continuous GH infusions induced only 7-fold and 2-fold increases in IGF-I peptide and mRNA, respectively. Serum GHBP activity was not changed in the GH-injected animals, but rose 2- to 3-fold in the GH-infused rats, an increase similar to that reported for their liver GH binding sites. No major change in liver concentrations of GHR and GHBP mRNAs was seen after repeated GH injections. Differential regulation of the two GHR/GHBP gene products was observed after continuous infusion of GH, with a net 60-70% increase in liver GHBP mRNA abundance contrasting with no apparent change in the GHR mRNA transcript. These results indicate that pulsatile GH administration is more effective than continuous GH infusion in stimulating liver IGF-I gene expression, and this effect is not mediated by an increase in GHR mRNA or protein.(ABSTRACT TRUNCATED AT 400 WORDS)

To further investigate how sex steroids regulate galanin (GAL) in the rat pituitary and hypothalamus, we examined the effects of prepubertal gonadectomy (Gx) and long-term (9 weeks) replacement with estradiol (E2) or testosterone (T) on pituitary and hypothalamic GAL concentrations in Wistar rats (5-6/group). Sham-operated animals served as controls (CTR). Pituitary GAL concentration was markedly higher in random-cycling CTR-females than in CTR-males (1391 +/- 247 vs 39 +/- 5 pg/mg protein, P < 0.01) and decreased after Gx only in females (20 +/- 3 pg/mg protein, P < 0.01). E2 strongly increased pituitary GAL in Gx-females and Gx-males (4470 +/- 365 and 3853 +/- 347 pg/mg protein, P < 0.01), whereas T had no effect. Inversely, hypothalamic GAL was higher in CTR males than in CTR females (5.4 +/- 0.3 vs 4.0 +/- 0.5 ng/mg protein, P < 0.05), and decreased significantly after gonadectomy in males (3.7 +/- 0.2 ng/mg protein, P < 0.01). The only steroid treatment that significantly modified hypothalamic GAL in Gx animals was administration of E2 to females (5.7 +/- 0.4 ng/mg protein, P < 0.01 vs non-treated Gx). We also studied in hypophysectomized (Hx) rats (8/group) the effects of sex steroids on hypothalamic GAL concentration and distribution. The low hypothalamic GAL concentration observed in male and female Hx rats (1.0 +/- 0.1 ng/mg protein) was significantly increased by T in males and in females (respectively, by 40% and by 50%, P < 0.02) and by E2 in males (by 60%, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

To understand better the relationship between hypothalamic galaninergic neurons and the pituitary gland, we studied the effects of hypophysectomy on hypothalamic galanin (GAL) content and distribution by radioimmunoassay and immunohistochemistry, and on GAL mRNA by Northern blot analysis. Three weeks after hypophysectomy, performed at 5 or 8 weeks of age, the hypothalamic concentrations of GAL and GAL mRNA were reduced by 30-50% in both male and female rats, compared to age- and sex-matched controls. Similar reverse-phase HPLC retention times of hypothalamic GAL were observed in intact and hypophysectomized rats. The reduction of hypothalamic GAL concentration following hypophysectomy was time-dependent, as peptide levels were unaffected one week after surgery. Immunohistochemistry showed regional differences in the effect of hypophysectomy on galaninergic neurons. In the hypophysiotropic hypothalamus, the scarce GAL immunoreactivity normally observed in the arcuate nuclei was no longer detectable in hypophysectomized rats, and the intense GAL immunoreactivity of the external zone of the median eminence progressively decreased and completely disappeared 3 and 6 weeks after hypophysectomy. In contrast, in the neurohypophyseal system, there was an increase of GAL labelling of the perikarya and emerging axons in the supraoptic and lateral-paraventricular nuclei, 1 and 3 weeks after hypophysectomy, that disappeared 6 weeks after hypophysectomy. An increase of GAL immunoreactivity was also observed in the internal zone of the median eminence 1 week but not 3 weeks after hypophysectomy. We conclude that hypophysectomy reduces the content of GAL and GAL mRNA in the rat hypothalamus. These changes are time-dependent and clearly detected after 3 weeks. The neurohypophyseal and hypophysiotropic galaninergic systems respond differently to hypophysectomy. In the neurohypophyseal system, the transient increase of GAL immunoreactivity could be related to axonal injury, diabetes insipidus or decreased neurosecretion. In contrast, the marked decrease of the GAL immunoreactivity found in the hypophysiotropic system, mainly in the external zone of the median eminence, accounts for the overall changes in hypothalamic peptide content and likely results from the loss of feedback up-regulation by pituitary or pituitary-dependent factors.

Administration of GH complexed with monoclonal antibodies (MABs) potentiates the in vivo actions of the hormone. In particular, growth and serum IGF-I concentrations of GH-treated hypophysectomized rats are increased by concomitant injection of anti-GH MABs. Among 37 anti-bovine GH (bGH) MABs, we selected one MAB with the most potentiating effects to investigate the mechanisms responsible for this phenomenon. Hypophysectomized rats were killed 18 h after a single s.c. injection of bGH (100 micrograms/rat), alone or complexed with increasing doses of MAB (4, 40, 400 micrograms/rat; MAB:bGH molar ratio: 0.005, 0.05, 0.5). IGF-I was measured by radioimmunoassay in acid-extracted sera and livers, whereas liver IGF-I mRNA was quantified by Northern blot hybridization. The in vivo occupancy of liver somatogenic (GH) receptors was derived from the determinations of total and free 125I-labelled bGH binding to liver homogenates treated with 4 mol MgCl2/l or water. Injection of MAB-bGH complexes enhanced body weight gain and raised serum IGF-I, liver IGF-I and liver IGF-I mRNA more than bGH alone (1.6-, 6-, 10- and 7-fold increases at the highest dose of MAB, compared with bGH alone; P < 0.001). These potentiating effects of the MAB were dose-dependent and significant potentiation of the growth response was already observed with the lowest dose of MAB. In vivo occupancy of liver GH receptors was markedly higher 18 h after injection of MAB-bGH complexes than after bGH alone, and this effect was also dose-dependent (receptor occupancy of 28%, 37% and 83% after 4, 40 and 400 micrograms of MAB respectively compared with 6% after bGH alone; P < 0.05, 0.05 and 0.001 respectively). In contrast, the in vitro binding of 125I-labelled bGH to liver homogenates was decreased in the presence of high doses of MAB. We conclude that low amounts of MABs complexed with bGH potentiate the stimulation by the hormone of liver IGF-I synthesis and secretion in a dose-dependent manner. These effects are mediated, at least in part, through changes in hormone-receptor interaction in vivo, leading to enhanced and/or prolonged binding of bGH to its somatogenic receptors.