Thyroxine (T4)
The plasma level of thyroxine is very low two weeks after hypophysectomy [145] due to the lost action of thyroid-stimulating hormone. This was substituted for by giving the hypophysectomised rats a daily subcutaneous injection of 10 µg L-thyroxine/kg/day (Nycomed, Oslo, Norway) diluted in saline. This dose results in somewhat higher thyroxine levels than in normal rats [139, 146], but has been shown to be within the physiological range as measured by the effect of different thyroxine doses on longitudinal bone growth [147]. However, the level of the active form of thyroxine, i.e. T3, has not been measured after this substitution.
Glucocorticoids
To substitute for the lack of adrenocorticotropic hormone, all Hx rats were given a daily subcutaneous injection of 400 µg cortisol phosphate/kg/day (Solo-Cortef,
Upjohn, Puurs, Belgium) diluted in saline. A replacement dose of 500 µg cortisone/kg/day has been shown to be within the physiological range with respect to body growth and longitudinal bone growth [148] as well as GH binding to adipocytes [149]. The dose of cortisol was adjusted to 400 µg/kg/day due to the higher potency of cortisol than cortisone.
In the in vitro studies, 1 nM dexamethasone was added to the medium since glucocorticoids exert a permissive action on some GH effects [150, 151]. The dose of 1 nM dexamethasone, or 0.39 ng/ml, corresponds to 32.5 ng/ml corticosterone after correction for the different potencies of these glucocorticoids. This dose of dexamethasone is somewhat below the physiological range of corticosterone concentration in serum (50-150 ng/ml) [152].
Growth hormone
Recombinant bovine GH was given in a dose of 0.5-1.5 mg/kg/day diluted in 0.05 M phosphate buffer (pH 8.6) containing 1.6% glycerol and 0.02% sodium azide. Bovine GH was chosen rather than human GH, since bovine GH only binds to the GH receptor in contrast to human GH that also binds the prolactin receptor. Compared to rat GH, bovine GH is more stable and also much more available on the market.
GH were administered to the hypophysectomised rats either as a continuous infusion via an osmotic mini-pump (Alza Corp., Palo Alto, CA, USA) implanted subcutaneously between the scapulae, or as two daily subcutaneous injections at 12-h intervals (0800 and 2000 h). These modes of GH administration have been shown to experimentally imitate the female and the male GH secretory pattern, respectively, with respect to feminisation and masculinisation of P450 enzyme levels in rat liver [101, 153]. GH injections, however, will result in fluctuations of GH plasma levels during the day with the possible outcome that GH effects on mRNA species with high turnover are not detected. The hepatic expression was therefore analysed both at 2 and 6 hours after the last GH injection.
The normal secretory rate of GH in 50-60 days old female rats is 1.3 mg/kg/day as calculated from the GH clearance rate (1.19 ml/min) [154] and the normal mean plasma level of GH (135 ng/ml) [155]. Thus, both the in vitro dose of GH (100 ng/ml, Paper II) and the continuous infusion of 1.5 mg GH/kg/day to Hx rats (Paper I) are within the physiological range. When the hypophysectomy model was used to investigate the regulatory role of the feminine and masculine GH secretory pattern, a lower dose of GH (0.7 mg/kg/day) was administered (Paper II and III). This dose was chosen to assure very low or undetectable GH levels between the GH injections analogous to the GH secretory pattern of the male. Although lower than the normal
GH secretion rate, this GH restitution is sufficient as indicated by the increased final body weight and body weight gain in hypophysectomised rats ([156] and Paper II).
To assess the role of the different GH secretory patterns without giving GH injections that result in slow diurnal variations in GH concentrations, a low dose of GH (0.5 mg/kg/day) was administered as a continuous infusion to intact rats. This mode of GH administration will feminise male rats with respect to the GH secretory pattern without any major changes in the total mean plasma level of GH [157, 158]. The final body weight, body weight gain and IGF-I mRNA expression did not change by this GH treatment in Paper II and III, indicating the total GH exposure was not particularly affected in our studies.
Insulin
A slow-release form of insulin (Insulatard, 100 IU/ml, Novo Nordisk A/S, Denmark) diluted in saline was given as a daily subcutaneous injection at 1600 h to the Hx animals in Paper I. To avoid insulin-induced fatal hypoglycemia, the dose of insulin was gradually increased from 1 IU to 2 IU/day. This insulin treatment has been shown to result in serum insulin levels similar to those in sham-operated animals [159].
In the in vitro studies, the cells were plated in a medium containing 16 nM insulin and then cultured in the presence of 3 nM insulin. The insulin concentration in the portal blood of fasting rats is 0.34 nM [160] and is several-fold higher in fed rats. The in vitro doses of insulin are therefore near or above the physiological range.
Sex steroids
Testosterone was diluted in propylene glycol and given at a dose of 0.5 mg/kg/day as a daily subcutaneous injection to Gx male rats (Paper III). A similar dose of testosterone has been shown to increase the level of plasma testosterone in Gx rats to that of intact male rats [161], and also to masculinise the secretory pattern of GH [155]. 17β-estradiol was diluted in propylene glycol and administered at a dose of 0.1 mg/kg/day as a daily subcutaneous injection to female Gx rats (Paper III). The serum level of estradiol in intact female rats varies between 2 and 50 pg/ml throughout the cycle [162]. As treatment of intact rats with 0.01 mg estradiol/kg/day increases the plasma estradiol level to approximately 51 pg/ml [163], our higher dose of estradiol (0.1 mg/kg/day) would therefore probably result in supraphysiological serum levels of estradiol in Gx rats.