Hypophysectomized Female Rats Research Articles

Inherent Sexually Dimorphic Expression of Hepatic CYP2C12 Correlated with Repressed Activation of Growth Hormone-Regulated Signal Transduction in Male Rats

Because of its myriad physiologic functions, it is not surprising that the actions of growth hormone (GH) are mediated by recruiting/activating dozens of signaling molecules involved in numerous transduction pathways. The particular signal transduction pathway activated by the hormone is determined by the affected target cell, the sexually dimorphic secretory GH profile (masculine episodic or feminine continuous) to which the cell is exposed, and the individual's sex. In this regard, expression of female-specific CYP2C12, the most abundant cytochrome P450 in female rat liver, is solely regulated by the feminine GH profile. Sex is a modulating factor in this response in that males are considerably less responsive than females to the CYP2C12-induction effects of continuous GH. Using primary hepatocytes derived from male and female hypophysectomized rats, we have identified several factors in a transduction pathway activated by the feminine GH regime and associated with the induction of hepatic CYP2C12. Elements in the proposed pathway, in their likely order of activation, are the growth hormone receptor, extracellular signal-regulated kinases, the cAMP-response element-binding protein, and hepatocyte nuclear factors 4alpha and 6, which subsequently bind and activate the CYP2C12 promoter. Recruitment and/or activation levels of all of the component factors in the pathway were highly suppressed in male hepatocytes, possibly explaining the dramatically lower induction levels of CYP2C12 in males exposed to the same continuous GH profile as females.

Overexpression of CYP3A aggravates endotoxin‐induced liver injury in hypophysectomized female rats

CYP3A2 is a male-specific isoform of cytochrome P450 enzyme which is expressed abundantly in male rats but not in intact female rats. Having previously reported that hepatic CYP3A2 promotes lipopolysaccharide (LPS)-induced liver injury in male rats, we further examined the impact of CYP3A on LPS-induced liver injury by comparing intact and hypophysectomized female rats. In hypophysectomized female rats, phenobarbital (PB), a cytochrome P450 inducer, markedly increased the hepatic content and activity of CYP3A1/2, but did not do so in intact rats. CYP2B1 increased to similar levels in PB-treated hypophysectomized and intact rats. Rats were administered 10 mg/kg LPS intravenously and some were given PB for three days before LPS injection. Liver injury was analyzed 8 h after LPS injection. PB-LPS increased plasma alanine aminotransferase significantly more in hypophysectomized female rats than in intact female rats. Ketoconazole, a CYP3A inhibitor, inhibited the increase of liver injury. Hepatic 8-hydroxydeoxyguanosine in nuclei and 4-hydroxy-2-nonenal-modified proteins, measured to evaluate oxidative stress by LPS treatment, increased markedly more in PB-treated, hypophysectomized female rats, than in intact female rats. Overexpression of CYP3A aggravated LPS-induced liver injury in rats, apparently through the formation of reactive oxygen species.

Suppression of female-specific murine Cyp2b9 gene expression by growth or glucocorticoid hormones

CYP2B9 is a constitutively and female-specifically expressed P450 isoform in mouse livers. Hypophysectomy-induced CYP2B9 mRNA expression in males to a level similar to that in females, while the operation did not affect females. Twice-daily injection of growth hormone (GH), which mimics the male pattern of GH secretion, significantly repressed hypophysectomy-induced mRNA expression in males. The same treatment completely suppressed expression in intact females. Treatments with synthetic glucocorticoid dexamethasone (DEX) suppressed expression of CYP2B9 mRNA in intact females, but not in GH-treated and un-treated hypophysectomized females. In primary cultured mouse hepatocytes, CYP2B9 mRNA expression was concentration-dependently suppressed by natural glucocorticoids such as hydrocortisone and corticosterone as well as by DEX. Glucocorticoid-mediated suppression was partially inhibited by RU486, a potent antiglucocorticoid. In contrast, RU486 by itself suppressed expression of CYP2B9 mRNA. These observations suggest that the sexually dimorphic expression of CYP2B9 is partly due to suppression by the masculine plasma GH profile and by glucocorticoid hormones.

Lipids and lipid-transporting proteins in Chrysemys picta: role of gonadal steroids and growth hormone in intact and hypophysectomized turtles

In the freshwater turtle, the homeostatic control of plasma lipids and lipid-transporting proteins may be coordinately regulated by ovarian steroids and pituitary hormones such as growth hormone (GH). In order to elucidate the role of these hormones in the regulation of vitellogenesis and ovarian growth, we have investigated lipid metabolic changes in normal male and female turtles, and in hypophysectomized females with and without GH injections, in response to combinations of exogenously administered gonadal steroids (estradiol (E2), progesterone (P), and testosterone (T)). Determinations of total plasma triglycerides, cholesterol, vitellogenin, and apoA-I were performed. We have demonstrated that E2 alone and in combination with P significantly increased plasma apoA-I and triglyceride levels in both intact female and male turtles. Testosterone administered alone to males had no effect on any of the parameters measured. In hypophysectomized females, plasma apoA-I, vitellogenin, and triglyceride levels were all significantly elevated in animals which received GH and E2, compared to controls (sham and hypox) and those which received GH alone.

Hormonal regulation of liver fatty acid-binding protein in vivo and in vitro: effects of growth hormone and insulin.

Liver fatty acid-binding protein (LFABP) is an abundant protein in hepatocytes that binds most of the long chain fatty acids present in the cytosol. It is suggested to be of importance for fatty acid uptake and utilization in the hepatocyte. In the present study, the effects of bovine GH (bGH) and other hormones on the expression of LFABP and its messenger RNA (mRNA) were studied in hypophysectomized rats and in vitro using primary cultures of rat hepatocytes. One injection of bGH increased LFABP mRNA levels about 5-fold after 6 h, but there was no effect of this treatment on LFABP levels. However, 7 days of bGH treatment increased both LFABP mRNA and LFABP protein levels 2- to 5-fold. Female rats had higher levels of LFABP than male rats. Hypophysectomy of female rats, but not that of male rats, decreased LFABP levels markedly. Treatment of hypophysectomized rats with bGH for 7 days as two daily injections or as a continuous infusion increased LFABP levels to a similar degree. This finding indicates that the sex difference in the expression of LFABP is not regulated by the sexually dimorphic secretory pattern of GH. Neither insulin nor insulin-like growth factor I treatment of hypophysectomized rats for 6-7 days had any effect on LFABP mRNA or LFABP levels. In vitro, bGH dose-dependently increased the expression of LFABP mRNA, but only in the presence of insulin. Insulin alone had a marked dose-dependent effect on LFABP mRNA levels and was of importance for maintaining the expression of LFABP mRNA during the culture. Incubation with bGH increased LFABP mRNA levels within 3 h. GH had no effect on LFABP mRNA levels in the presence of actinomycin D, indicating a transcriptional effect of GH. Incubation with glucagon in vitro decreased LFABP mRNA levels markedly, indicating that glucagon, in contrast to GH, has an effect opposite that of insulin on LFABP mRNA expression. It is concluded that GH is an important regulator of LFABP in vivo and in vitro. In contrast to the effect of GH on insulin-like growth factor I mRNA, the presence of insulin was a prerequisite for the effect of GH on LFABP mRNA expression in vitro. The results emphasize the role of GH in the regulation of hepatic fatty acid metabolism.

Role of peripheral-type benzodiazepine receptors in steroidogenesis.

Peripheral-type benzodiazepine (BZ) receptors (PBRs) have been identified in various peripheral tissues as well as in glial cells in in the brain. PBRs are located mainly on the outer mitochondrial membrane and bind with high affinity the BZ Ro 5-4864 (4'-cholorodiazepam) and the non-BZ PK 11195 (an isoquinoline carboxamide derivative), but bind with very low affinity the BZ clonazepam. PBRs have been cloned from various species. PBRs are multimeric receptors composed of the 18-kDa binding site for isoquinolines, the 32-kDa voltage-dependent anion channel, and the 30-kDa adenine nucleotide carrier (which binds BZs). The expression of PBRs is especially high in steroidogenic organs. Steroid administration affects PBR density, whereas depletion of hormones by hypophysectomy in female rats, or castration (surgical or chemical) in male rats, decreases PBR density in endocrine organs, which can be elevated to normal values after administration of the appropriate hormone. PBRs are probably involved in several functions, including cell proliferation, respiration, and steroidogenesis. It has been suggested that PBRs are involved in the translocation of cholesterol from the outer to the inner membrane of the mitochondria and have an effect on the biosynthesis of steroids.

Involvement of growth hormone as a regulating factor in sex differences of mouse hepatic aldehyde oxidase

The participation of circulating growth hormone (GH) as a regulator of sex differences in hepatic aldehyde oxidase (AO) activity in ddy mice was examined. The 2- to 3-fold higher activities in adult male mice compared with adult female mice were decreased to the female levels by neonatal pretreatment with monosodium glutamate (MSG) or monosodium aspartate (MSA), either of which is known to reduce circulating GH levels. A decline of the activities in the MSG-treated male mice was restored nearly to the male control levels by subsequent injections of human GH every 12 hr for 7 days. These changes in AO activities in male mice caused by the excitotoxic amino acids were not observed in females. Hypophysectomy markedly decreased hepatic AO activities in male mice and partially in female mice. The activities in hypophysectomized male mice were restored again to levels similar to the control males by intermittent injections of human GH. Administration of testosterone propionate (TP) significantly increased the activities of hepatic AO in intact female mice, but not in MSA-treated or hypophysectomized females. On the other hand, the AO activities in adult male mice were decreased partially by the administration of estradiol benzoate. These results indicate that the pituitary GH is involved as one of the major regulatory factors of sex differences in the activities of hepatic AO in mice and TP also contributes to maintaining the higher activity in male mice mainly through the hypothalamus-pituitary system.

Sex Differences in Renal Excretion of Drugs.

Sex difference in the elimination of drugs can be due to either a difference in excretion or in metabolism. Sex differences in drug metabolism are well known and occur in both phase I and II metabolism, while the situation with excretion, especially renal excretion, is unclear. Zenarestat, [3-(4-bromo-2-fluorobenzyl)-7-chloro-2, 4-dioxo-1, 2, 3, 4-tetrahydroquinazolin-1-yl] acetic acid is an aldose reductase inhibitor. Rat shows a marked sex difference in the excretion of 14C-zenarestat: only 1 % of the dose was excreted in the urine of males, about 45% of the dose was excreted in the urine of females. 14C in the urine of female rats was almost entirely unchanged drug. Renal clearance experiments, and inhibition of urinary excretion by probenecid, indicated that female rats may possess an active secretory mechanism which is lacking or relatively inactive in male rats. Castration of male rats at 22 days of age abolished th e adult sex difference in urinary excretion of zenarestat, while castration at 5 weeks of age produced urinary excretion of the drug about half that in females. Ovariectomy of females at 22 days or 5 weeks of age had no effect on the urinary excretion of zenarestat. Treatment of male and female gonadectomized rats with testosterone resulted in the urinary excretion of zenarestat characteristic of intact adult male rats. Hypophysectomized male rats and normal female rats has similar urinary excretion of zenarestat which was much greater than that of normal male rats. Treatment of male and female hypophysectomized rats with testosterone resulted in the urinary excretion of zenarestat characteristic of intact adult male rats. Urinary secretion of zenarestat in rats is independent of pituitary hormone and is suppressed only by androgen. Urinary excretion of zenarestat scarcely differed between 3-week-old male and female rats; it decreased in males from 4 weeks of age, but in females urinary excretion of the drug was essentially constant during ageing. Mice show a sex difference in the excretion of zenarestat similar to that seen in rats, but dogs and humans show no significant sex difference. Female rats and mice, and both sexes of dogs and humans, appear to possess an active secretory mechanism in the renal excretion of zenarestat, which is lacking or relatively inactive in male rats and mice. The effect of acute ( ?? 1 week) and chronic ( ?? 3 weeks) streptozocin induced diabetes on the pharmacokinetics of zenarestat was studied in male and female rats. In male acute and chronic diabetic rats, the urinary excretion increased compared with the control after the dose with 14C-zenarestat. It did not change in female acute diabetic rats and decreased in female chronic diabetic rats compared with the control. Renal clearance experiments suggested that the decrease in urinary excretion in female chronic diabetic rats is due to a decrease in active secretion. The increase in the urinary excretion in male rats was probably due to a decrease in testostereone levels in the diabetic state.

Induction of hepatic estrogen sulfotransferase expression by hypophysectomy in female rats

We have examined the effects of hypophysectomy and treatment with thyroxine (T 4) on enzyme activity and expression (as determined by immunoblot analysis) of members of the three principal sulfotransferase (ST) sub-families (phenol STs, PST; estrogen STs, EST; hydroxysteroid STs, HST) in cytosols prepared from female Wistar rat livers. The results demonstrate that in female rat liver cytosol, EST activity was decreased by treatment with T 4, increased following hypophysectomy and that treatment of hypophysectomized animals with T 4 also greatly reduced EST activity. T 4 had no significant effect on PST or HST activity in normal animals, but it decreased HST activity in hypophysectomized rat liver cytosol. Immunoblot analysis of these cytosols with antibodies recognising HST and PST indicated that where changes in enzyme activity occurred they mirrored changes in enzyme protein expression. In normal adult female rat livers, EST protein is not expressed, and the small residual activity results predominantly from the action of HST. Hypophysectomy induced EST activity and the expression of EST enzyme protein in female rat liver cytosol, and T 4 treatment of hypophysectomized animals reduced the activity to below normal levels without reducing the corresponding enzyme protein levels, indicating that T 4 regulation of EST in females is via a post-translational mechanism.

Induction of proenkephalin in tuberoinfundibular dopaminergic neurons by hyperprolactinemia: the role of sex steroids.

The observation that tuberoinfundibular dopaminergic (TIDA) neurons of pregnant, pseudopregnant, lactating, and aged rats express enkephalins suggested that chronically elevated PRL levels, which are characteristic for these animals, are essential for the induction of proenkephalin gene expression in TIDA neurons. The present studies investigated further the role of PRL in this phenomenon. Elevated PRL levels were achieved either experimentally by implanting anterior pituitaries under the kidney capsule of intact or hypophysectomized female rats or by using lactating rats. For controls, the elevated PRL levels were reduced with bromocryptine, a dopamine receptor agonist. The role of sex steroids in PRL-induced enkephalin gene expression was also studied in cycling, sex hormone-treated, hypophysectomized or ovariectomized rats, pituitary-implanted/sex hormone-treated rats, and ovariectomized mothers. Enkephalin immunoreactivity was detected by immunocytochemistry and enkephalin messenger RNA with in situ hybridization histochemistry using 35S- or digoxigenin-labeled riboprobes. Enkephalin or its messenger RNA was present in TIDA neurons in all experimental animals with elevated PRL levels. Although estradiol had no or only a minor effect on PRL-induced enkephalin gene expression, progesterone supported the effect of PRL. The present observations suggest that the expression of enkephalin in TIDA neurons is PRL dependent and supported by sex steroids, primarily progesterone.

Effects of testosterone, hypophysectomy and growth hormone treatment on clofibrate induction of peroxisomal β-oxidation in female rat liver

Induction of peroxisomal β-oxidation by clofibrate under altered hormonal states was investigated in female rat liver. Treatment of rats with clofibric acid (CPIB) caused a significant increase in hepatic peroxisomal β-oxidation, with female rats being less responsive than males (4.2- vs 12.2-fold increase). However, testosterone treatment following ovariectomy of female rats resulted in an enhanced response to CPIB, giving an induction (11.7-fold) comparable to that seen in male rats. Hypophysectomy of female rats also enhanced the induction (8.2-fold compared with 5.1-fold), suggesting a suppressive effect of a pituitary-dependent factor on CPIB induction of peroxisomal β-oxidation. Continuous infusion of growth hormone to the hypophysectomized female rats suppressed the enhanced induction nearly to the initial level (6.1-fold). The stimulatory effects of testosterone and hypophysectomy on the enzyme induction were additive. These findings suggest the involvement of growth hormone, as well as male sex hormone, in regulating the responsiveness to CPIB induction of peroxisomal β-oxidation in rat liver.

Continuous but not intermittent administration of growth hormone to hypophysectomized rats increases apolipoprotein-E secretion from cultured hepatocytes.

Hypophysectomy of female rats has been shown to decrease the serum levels of apolipoprotein E (apoE). Continuous but not intermittent administration of GH to hypophysectomized (HX) rats increases these levels to those of normal rats, indicating that the sexually dimorphic secretion of GH is important in the regulation of apoE metabolism. In this study, these effects of GH were further investigated by studying the biosynthesis and secretion of apoE from isolated hepatocytes. Hepatocytes were isolated from HX rats as well as from HX rats that had received hormonal treatment with T4 and cortisol (C) or T4 and C together with GH given either as two daily sc injections (GH x 2) or as a continuous infusion (GHc). Hypophysectomy decreased by 47% the amount of apoE present in the culture medium after a 4-h incubation. Treatment of HX rats with T4 and C alone or in combination with GH x 2 did not influence the amount apoE present in the medium, whereas treatment with T4, C, and GHc increased the amount of apoE to that of normal controls. The different levels of apoE in the medium was not due to differences in the disappearance of apoE, indicating that it was caused by changes in the rate of apoE secretion. Consistent with this, hypophysectomy decreased the rate of intracellular accumulation of apoE measured by incubation of the cells with [35S]methionine for 0, 8, and 20 min. Treatment with T4, C, and GHc increased the rate of accumulation, but T4, C, and GH x 2 had no effect. The differences in the initial rate of intracellular accumulation of apoE were not due to variations in apoE messenger RNA pools or to differences in the degradation of apoE at a step early in the secretory pathway. These results indicate that the differences in the initial rate of accumulation of apoE results from differences in the translational rate. The major amount of apoE that was secreted to the medium appeared in the high-density lipoprotein fraction, whereas small amounts were present in the very-low-density lipoprotein fraction (VLDL). Hypophysectomy decreased the amount of newly secreted apoE in the VLDL fraction. Only therapy with T4, C, and GHc could restore the normal distribution of apoE in the VLDL fraction. In conclusion, the results indicate that the secretory pattern of GH is involved in the regulation of the apoE secretion by influencing the rate of translation.

Down-regulation of multiple cytochrome p450 gene products by inflammatory mediators in vivo: Independence from the hypothalamo-pituitary axis

Inflammatory stimuli suppress constitutive hepatic expression of the CYP2C11 and CYP2C12 genes in male and female rat livers, respectively. Because growth hormone (GH) is an important regulator of P450 gene expression in the rat, the effects of bacterial endotoxin injection in hypophysectomized rats were compared to those in normal animals. In intact females, 0.2 mg kg endotoxin suppressed total P450 and hepatic expression of CYP2C6, CYP2C7, CYP2C12, and CYP2E1 mRNAs, as well as CYP2C12 and CYP2E1 proteins, measured 16 hr later. CYP2C7 and CYP2E1 mRNAs were most affected (17 and 13% of untreated levels, respectively). Endotoxin treatment also induced the mRNA for the hepatic acute phase protein, haptoglobin, to 260% of control female levels. In hypophysectomized females supplemented with GH infusion, endotoxin caused the same or greater effects on expression of the P450 and haptoglobin gene products than were observed in the intact animals. It is concluded that the P450 suppression observed after endotoxin administration can occur independently of an effect on pituitary hormone secretion.

Effects of hypophysectomy and testosterone treatment on the urinary excretion of zenarestat in rats.

1. The effects of hypophysectomy and androgen treatment on the urinary excretion of zenarestat were investigated in Sprague-Dawley rats. 2. Hypophysectomized male rats and normal female rats has similar urinary excretion of zenarestat which was much greater than that of normal male rats. 3. Treatment of male and female hypophysectomized rats with testosterone resulted in the urinary excretion of zenarestat characteristic of intact adult male rats. 4. Urinary secretion of zenarestat in rats is independent of pituitary hormone and is suppressed only by androgen.

Comparativein Vitroandin VivoStudies on the Biological Characteristics of Recombinant Human Follicle-Stimulating Hormone

The in vitro and in vivo activities of recombinant human FSH (recFSH) produced by a Chinese hamster ovary cell line were studied and compared with those of natural FSH preparations. The specific FSH activities of recFSH established by immunoassay and in vivo bioassay were greater than 10,000 IU/mg protein and considerably higher than the activities of tested urinary FSH references, while the in vivo bio/immuno ratios of these preparations were not significantly different. Compared to a highly purified pituitary standard (IS 83/575), recFSH had a comparable high specific in vivo bioactivity, but the specific immunoreactivity of IS 83/575 was about 2 times lower. In receptor displacement and in vitro bioassay studies recFSH provided dose-response curves parallel to those of pituitary and urinary FSH references. When equal amounts of immunoreactivity FSH were tested, recFSH and urinary and pituitary FSH displayed comparable activities in both assays. The in vitro bioactivity of recFSH could be neutralized effectively by each of three monoclonal antibodies raised against recFSH (alpha-specific), urinary FSH (beta-specific), and pituitary FSH (alpha beta-specific), respectively. Moreover, 50% inhibition of comparable responses induced by recFSH, urinary "pure" FSH, or pituitary FSH was established by the same amount of monoclonal antibody. These results support the structural and functional similarity of recFSH and natural FSH. To test whether recFSH is capable of inducing LH-specific biological responses, the in vitro induction of testosterone production in mouse Leydig cells was assessed. At least 16 IU recFSH/ml incubate were needed to increase testosterone production, indicating that the intrinsic LH bioactivity of recFSH is negligible (less than 0.025 mIU LH/IU FSH). The in vivo efficacy of recFSH was examined by treating immature female hypophysectomized rats during 4 days with recFSH only or with recFSH supplemented with hCG. RecFSH only treatment increased ovarian weight and aromatase activity in a dose-dependent manner. When recFSH dosages providing submaximal responses were supplemented with 1 IU hCG, both ovarian weight and aromatase activity were largely augmented. Neither recFSH nor urinary pure FSH, administered in a high dose was able to increase plasma estradiol levels, while ovarian weight and aromatase activity were increased to the same extent. However, when recFSH was supplemented with only 0.1 IU hCG, a 3-fold increase in median plasma estradiol levels was obtained. These findings support the two-cell two-gonadotropin theory, holding that both FSH and LH are required for estrogen biosynthesis, but also reveal that only very small amounts of LH activity are sufficient to increase estrogen secretion up to measurable plasma levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of Expression of Male-Specific Rat Liver Microsomal 3β-Hydroxysteroid Dehydrogenase

In the steroidogenic pathways present in the gonads and adrenal cortex, 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta HSD) is a key enzyme which controls the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. Herein, we used an antibody against human placental 3 beta HSD and a rat testicular 3 beta HSD cDNA probe to study the expression of rat liver 3 beta HSD mRNA and protein. Rat liver microsomal 3 beta HSD activity has been previously reported to exhibit a significant sex difference, with much higher activity in the male. We have shown an age-dependent increase in levels of immunoreactive 3 beta HSD through the time of maturation of the male rat. The immunoreactive protein, of similar molecular size to the human placental and rat testicular 3 beta HSD, was localized to the microsomal fraction of liver and was concentrated in pericentral locations. Immunoreactive protein was not detected in liver of immature (before 25 days of age) rats of either sex or in adult female liver. Northern blot analysis of liver and testicular RNA with a rat testicular 3 beta HSD cDNA probe revealed the presence of a 1.6-kilobase mRNA species in addition to the major 2.1-kilobase mRNA species in adult male liver, neither of which was detected in immature or adult female liver RNA. Hypophysectomy of female rats or treatment with testosterone implants caused induction of liver 3 beta HSD protein, while continuous infusion of GH to male rats decreased the level of 3 beta HSD protein. Similarly, the levels of the mRNA species were decreased after GH treatment. Using [3 alpha-3H]dehydroepiandrosterone as substrate for 3 beta HSD activity, we determined the apparent Km for liver microsomal NAD(+)-dependent 3 beta HSD activity to be 20 microM in both adult male and female liver and was much greater than the Km of rat Leydig tumor 3 beta HSD activity (0.2 microM). Liver 3 beta HSD activity was inhibited by trilostane, a proven inhibitor of gonadal and adrenal 3 beta HSD activity. A rat liver 3 beta HSD cDNA was isolated from a male liver cDNA library that was closely related to the type II 3 beta HSD form of rat ovary but different from type III liver 3 beta HSD. The enzyme obtained upon expression of this cDNA had properties characteristic of male-specific NAD(+)-dependent liver microsomal 3 beta HSD (i.e. high apparent Km for dehydroepiandrosterone) and distinct from those of the high affinity gonadal type I 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of growth hormone on rat hepatic cytochrome P-450 f mRNA: a new mode of regulation

Growth hormone (GH) is known to be involved in the control of rat hepatic drug and steroid metabolism through its action on cytochrome P-450 s. To examine the role of GH in the regulation of cytochrome P-450 f ( P-450 f), a full-length cDNA clone corresponding to P-450 f was isolated and the 3'-non-coding region was utilized for Northern and slot-blot analyses. P-450f mRNA levels were low in neonates, increased after age 4 weeks in male and female rats, and were approximately 3 times higher in the liver of adult female rats than male rats. Hypophysectomy caused a significant decrease in P-450 f mRNA levels in male and female rats. Intermittent injection with human growth hormone (hGH) to mimic the male secretory pattern of GH caused a 9-fold increase in P-450f mRNA in hypophysectomized male rats to levels near male control levels, whereas continuous administration of hGH to mimic the female secretory pattern caused a greater increase in P-450f mRNA levels in male and female hypophysectomized rats (25-fold and 9-fold respectively) to levels nearer female control levels. The responses of the other GH-stimulated P-450 s, P-450- male and P-450- female, to the different modes of hGH treatment were different from that of P-450 f. Since sex hormones are known to affect the regulation of other P-450 s, the effect of sex hormones on P-450f mRNA was studied. Ovariectomy caused a 2.4-fold reduction in P-450f mRNA which was partially reversed by estradiol treatment. These results demonstrate a role for GH and sex steroids in the regulation of P-450f mRNA. The mode of regulation of P-450f mRNA by GH was distinct from other known P-450 s.

Structural and Regulatory Analysis of the Male-Specific Rat Liver Cytochrome P-450 g: Repression by Continuous Growth Hormone Administration.

Complementary DNA clones encoding the male-specific rat liver cytochrome P-450 g have been isolated by cross-hybridization with sequences from the female-specific rat liver cytochrome P-450 15 beta. Tissue distribution analysis indicates the liver as the organ with major expression of this cytochrome P-450 gene. Minimal P-450 g expression was also detected in prostate, kidney, heart, and brain. A developmental analysis reveals liver expression in the 8-week-old male and to a lesser extent in the 4-week-old male, but no detectable expression is seen in females of these ages or in 1- and 2-week-old rats from both sexes. Hypophysectomy of female rats dramatically increases hepatic expression of P-450 g, whereas continuous GH administration represses hepatic expression in male or female hypophysectomized rats. In similarity to P-450 15 beta and P-450 16 alpha, therefore, the cytochrome P-450 g gene in liver is GH regulated.

Recombinant DNA-derived human insulin-like growth factor II (hIGF-II) stimulates growth in hypophysectomized rats.

Sustained subcutaneous administration of recombinant DNA-derived insulin-like growth factor II to immature female hypophysectomized rats stimulated significant increases in body weight gain, tibial epiphyseal cartilage width, femur hydroxyproline concentrations and a significant decrease in serum urea nitrogen concentrations. Recombinant DNA-derived human growth hormone (Humatrope), administered in the same manner produced similar biological effects. The data support the contention that hIGF-II has anabolic effects when administered to hypophysectomized rats and may be a locally acting mediator of pituitary hormone actions.

The lack of a growth-promoting effect of orally administered bovine somatotropin in the rat body-weight-gain bioassay

Bovine somatotropin (bSt) was given either orally or subcutaneously to groups of female hypophysectomized rats daily for 9 days. Ten rats per dose group were given oral dosages of 0 (buffered-water vehicle control), 40, 400, 2000, and 4000 μg of bSt per day. Similar groups of ten rats each received subcutaneous doses of 0 (buffered-water vehicle control), 15, 30, and 60 μg of bSt per day. Rats were weighed daily to observe their body-weight gain, which is a measure of the biological activity of bSt in the hypophysectomized rat. At study termination, serum of treated rats was monitored for the presence of bSt and antibody to bSt. Bovine somatotropin was detected in the serum of the subcutaneously treated rats, but not in those rats treated orally. Of 18 rats treated subcutaneously with bSt, 14 developed antibodies to bSt, whereas of 38 rats treated orally with bSt, 11 developed antibodies. Subcutaneously treated rats grew in a dose-related manner as expected in this assay. Orally administered bSt failed to elicit a growth response at any dose in this sensitive bioassay system. The data suggest that neither bSt nor growth-promoting fragments of bSt are absorbed after oral administration of doses up to 40,000 μg/kg/day in the hypophysectomized rat.