Treatment Of Hypophysectomized Rats Research Articles

Developmental Expression and Endocrine Regulation of CYP1B1 in Rat Testis

Mammalian testis expresses xenobiotic-metabolizing enzymes, including cytochrome P450 1B1 (CYP1B1), which catalyzes the bioactivation of procarcinogens and other chemicals. The factors that control testicular expression of CYP1B1 are largely not known. In the present study, we investigated the influence of age and pituitary, gonadal, and thyroid hormones on CYP1B1 expression in rat testis. Immunoblot analysis showed that testicular CYP1B1 protein was expressed at a level of 5.9+/-2.0 (mean+/-S.E.M.) pmol/mg microsomal protein in prepubertal 22-day-old rats, whereas it was 6.6-fold greater in pubertal rats (34 days old) and 9.6-fold greater in adult rats (84-91 days old). Hypophysectomy decreased testicular CYP1B1 protein levels by 69% in adult rats when compared with intact rats of the same age. Intermittent subcutaneous administration of growth hormone to hypophysectomized adult rats further decreased it by 63%. Luteinizing hormone (LH) and follicle-stimulating hormone increased CYP1B1 expression in hypophysectomized rats, but they did not restore protein levels to those in intact adult male rats. Prolactin treatment alone had no effect; however, it potentiated the increase in CYP1B1 mRNA and protein expression by LH. 3,5,3'-Triiodothyronine, but not thyroxine, resulted in a small increase in testicular CYP1B1 protein levels. Likewise, treatment of hypophysectomized rats with testosterone propionate elicited a small increase in CYP1B1 protein expression. In contrast, treatment of intact adult male rats with 17beta-estradiol benzoate decreased it by 91%. Overall, our findings indicate that rat testicular CYP1B1 protein expression is subject to developmental and endocrine control, with multiple hormones playing a role.

Gonadotropin stimuli increase adrenodoxin immunoreactivity in the mitochondria of rat ovarian cells

Adrenodoxin is a component of the electron transfer system for mitochondrial cytochrome P450 in steroidogenic cells. To elucidate whether the steroidogenic state can modulate the amount and the distribution of adrenodoxin in the mitochondria, we determined immunohistochemically the distribution of adrenodoxin and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in ovaries of rats under hypo- and hyperfunctional states. In the ovaries of control rats, adrenodoxin was distributed in the cristae of round-shaped mitochondria of both interstitial and theca cells. In hypophysectomized rats, no adrenodoxin was found in the mitochondria of atrophied cells. Treatment of hypophysectomized rats with human chorionic gonadotropin (hCG) or Humegon restored immunostaining of adrenodoxin in the interstitial and theca cells, and rendered the vesicular cristae of C- or ring-shaped mitochondria surrounding the lipid droplets adrenodoxin-positive. The distribution of 3betaHSD activity detected by enzyme histochemistry in these ovaries matched well that of adrenodoxin immunostaining. Our results indicate that immunostaining intensity of adrenodoxin in the mitochondria reflects the steroidogenic state in rat ovarian cells.

Overexpression of growth hormone affects alternatively spliced IGF-I mRNA expression in oMt1a-oGH transgenic mice.

Restorative growth hormone (GH) treatment of hypophysectomized rats differentially enhances the transcription of alternative IGF-I mRNA classes in liver. The goal of the present study was to determine the effects of GH overexpression on various classes of hepatic IGF-I mRNA in GH transgenic mice. Unstimulated oMt1a-oGH transgenic mice had low levels of transgene expression, and therefore were used to determine the effects of long-term, slightly elevated GH levels on the abundance on each alternative IGF-I mRNA class. The acute effects of high GH levels on the expression of alternative IGF-I mRNA were studied by gavaging transgenic mice with 25 mM zinc sulfate to activate oMt1a-oGH transgene expression. Long-term, low levels of oGH transgene expression in unstimulated transgenic mice resulted in a 73% down regulation of IGF-I 2Ea mRNA but not 1Ea and 2Eb mRNA. Acute stimulation of transgene expression triggered a rapid, 240% increase in 1Ea mRNA levels within 4 hours of transgene expression while 2Ea mRNA was down regulated to nearly non-detectable levels by 6 hours. IGF-I 2Eb mRNA was not affected by the short-term GH elevation. Our results showed that IGF-I 1Ea and 2Ea mRNA were differentially regulated by chronic low or acute high levels of GH. These results suggest that the regulation of IGF-I 1Ea and 2Ea mRNA transcription involve different postreceptor molecules and/or feedback mechanisms.

Regulation of immunoreactive androgen receptor in the adrenal gland of the adult rat.

The androgen receptor (AR) was measured by an immunoblot assay in adult tissues of both male and female rats. Relatively high levels of AR were detected in tissues of the male urogenital tract and in the adrenal glands and gonads of both sexes. Another group of tissues, including the male levator ani/bulbocavernosus muscles, preputial gland, scrotal skin, and vagina, had low, but detectable, levels of AR. In a third group of tissues, including the uterus, kidney, spleen, liver, gut, heart, lung, pituitary, and hypothalamus, AR was undetectable. In some androgen target tissues, such as the penis, androgens cause an apparent disappearance of AR from the tissue, and in other tissues, such as the ventral prostate, androgen therapy increases the amount of detectable AR. We compared the effect of androgen on AR levels in the adrenal gland and ventral prostate, tissues that differ markedly in their trophic responses to androgen. Castration appeared to have no effect on the amount of detectable AR in the adrenal gland, whereas it caused a profound decrease in AR levels in the ventral prostate. By contrast, 7 days after hypophysectomy, AR levels declined in both the adrenal gland and the ventral prostate. The effects of hypophysectomy plus castration were similar to those of hypophysectomy alone. Administration of ACTH to hypophysectomized rats for 7 days did not reverse the effects of hypophysectomy on adrenal AR, nor did treatment with levothyroxine, dexamethasone, rat GH, or rat PRL. Treatment of hypophysectomized rats with 5alpha-dihydrotestosterone for 7 days caused a dramatic increase in the amount of detectable AR in both the ventral prostate and the adrenal gland, but had a trophic effect only in the ventral prostate. These findings suggest that the amount of immunoreactive AR detected in both the adrenal gland and the ventral prostate is enhanced by androgens: testicular androgens in the case of the ventral prostate and adrenal androgen in the case of the adrenal glands.

Effects of hypophysectomy and thyroxine on the expression of hepatic oestrogen, hydroxysteroid and phenol sulphotransferases

Sulphation in rats, and other mammals, is carried out by a family of sulphotransferase isoenzymes, which can be further subdivided into oestrogen, hydroxysteroid and phenol sulphotransferases. We have examined the effects of hypophysectomy on the activity and expression of representative members of the three major sulphotransferase sub-families in male Wistar rat liver cytosols, and have found that the different sub-families are subject to differential regulation by pituitary hormones. Our data show that in male rat liver hydroxysteroid sulphotransferases activity was increased, oestrogen sulphotransferases activity was not altered and phenol sulphotransferases activity was reduced. Further, we have studied the effect on sulphotransferase expression of administration of thyroxine and dexamethasone to hypophysectomized rats. Treatment of hypophysectomized rats with thyroxine virtually abolished oestrogen sulphotransferase activity in male rat liver but had no effect on hydroxysteroid sulphotransferase or phenol sulphotransferase activity. Treatment of hypophysectomized rats with dexamethasone had no effect on sulphotransferase activities. Quantitative immunoblot analysis of liver cytosols showed that these changes in enzyme activity were related to changes in levels of the respective enzyme proteins.

Rat ovarian insulin-like growth factor-binding protein-6: a hormonally regulated theca-interstitial-selective species with limited antigonadotropic activity.

Most but not all of the established insulin-like growth factor-binding proteins (IGFBPs) are expressed in the rat ovary. To continue the process of characterizing these ovarian IGFBPs, a solution hybridization/RNase protection assay was used to study IGFBP-6 gene expression, cellular localization, and hormonal regulation in the immature rat ovary. Total RNA was hybridized with a 458-base long 32P-labeled rat IGFBP-6 cRNA. A single protected fragment (380 bases long) corresponding to IGFBP-6 transcripts was identified in RNA from ovary and lung, but not kidney or liver. The amount of IGFBP-6 transcripts was higher in ovaries from immature rats (25-28 days old) than in ovaries from adult rats and was higher in theca-interstitial than in granulosa cell preparations. Hypophysectomy resulted in a significant (P < 0.05) 2.3 +/- 0.7-fold (mean +/- SD) increase in whole ovarian IGFBP-6 transcripts. This suggests that ovarian IGFBP-6 gene expression is subject to inhibition by one or more pituitary hormones. Therefore, the effect of replacement of FSH, GH, diethylstilbestrol (DES), or combinations thereof was evaluated. Treatment with FSH resulted in a 2.4-fold decrease (P < 0.05) in the abundance of ovarian IGFBP-6 transcripts relative to that in hypophysectomized controls. Provision of DES yielded comparable results. Moreover, the combined provision of FSH and DES resulted in a synergistic decrease (6.0-fold) in ovarian IGFBP-6 transcripts. In contrast, treatment of hypophysectomized rats with GH proved without effect. However, treatment with FSH or DES in addition to GH resulted in a decrease in ovarian IGFBP-6 transcripts (3.9- and 2.7-fold, respectively). To assess the role of ovarian IGFBP-6, its influence on gonadotropin action in primary cultures of rat granulosa cells was also studied. Increasing concentrations (0.01-1 micrograms/ml) of recombinantly expressed human IGFBP-6 did not inhibit the FSH-supported accumulation of either progesterone or estradiol. These findings 1) establish the theca-interstitial compartment of the immature rat ovary as a prominent site of IGFBP-6 gene expression, 2) implicate FSH and DES as inhibitors of IGFBP-6 transcript levels in the whole ovary, 3) and disclose the limited antigonadotropic action of IGBP-6F on the rat granulosa cell.

Characterization of pituitary mediation of stress-induced antinociception in rats

Antinociception, induced by continuous cold-water swims (CCWS) and certain parameters of inescapable foot shock, is reduced in hypophysectomized rats receiving supplements of corticosterone and l-thyroxine. To assess which lobe of the pituitary gland is involved in this effect, the first experiment compared the effects of total hypophysectomy and posterior lobectomy in supplemented rats upon CCWS antinociception on the tail-flick and jump tests and upon continuous inescapable foot shock antinociception on the tail-flick test. Total hypophysectomy, but not posterior lobectomy, significantly reduced CCWS antinociception on both tests in supplemented rats relative to sham surgery. Both total and posterior hypophysectomy either reduced or potentiated foot shock antinociception as functions of shock intensity or duration of exposure in supplemented rats. To assess whether hormonal supplementation is necessary for the observed effects, the second experiment examined CCWS antinociception in sham-operated and hypophysectomized rats that received either no hormonal supplements or corticosterone andor l-thyroxine. These regimens failed to alter CCWS antinociception in sham-operated rats. Treatment of hypophysectomized rats with corticosterone and l-thyroxine either separately or together significantly reduced CCWS antinociception. In contrast, if hypophysectomized rats did not receive supplements, CCWS antinociception was significantly potentiated relative to sham-operated controls. These effects could not be attributed to treatment-induced changes in either body weight or CCWS hypothermia. These data suggest that the anterior lobe of the pituitary gland and adrenal cortex are involved in the mediation and/or maintenance of CCWS antinociception.

Growth hormone promotes early initiation of hepatocyte growth factor gene expression in the liver of hypophysectomized rats after partial hepatectomy.

GH accelerates hepatic regeneration in the rat. Hepatocyte growth factor (HGF), a potent hepatocyte mitogen in vitro, is considered to be a major regulator of hepatic regeneration. In the present study, the effects of GH and insulin-like growth factor-I (IGF-I) on HGF gene expression in regenerating rat liver was investigated. In hypophysectomized rats treated with GH, hepatic HGF mRNA levels were increased 3 h after partial hepatectomy and reached peak levels after 5 h. In rats with intact pituitaries and in hypophysectomized rats not given GH treatment, HGF mRNA levels in liver were unchanged during the first 5 h following hepatectomy and reached peak levels after 10-18 h. DNA synthesis in the liver of GH-treated rats increased from low levels 10 h after hepatectomy to peak levels after 18 h. In rats without GH treatment the synthesis of DNA was still low 18 h after hepatectomy and was increased after 26 h. Treatment of hypophysectomized rats with IGF-I promoted increases in hepatic HGF mRNA levels and DNA synthesis 3.5 h and 15 h after hepatectomy respectively. HGF mRNA levels were constantly lower after sham-hepatectomy than after partial hepatectomy. In summary, in hypophysectomized rats the responses of hepatic HGF gene expression and DNA synthesis to partial hepatectomy were both accelerated by treatment with GH or IGF-I.

Effects of growth hormone on apolipoprotein-B (apoB) messenger ribonucleic acid editing, and apoB 48 and apoB 100 synthesis and secretion in the rat liver.

Apolipoprotein-B 48 (apoB 48) and apoB 100 expression and the editing of apoB mRNA have previously been shown to be hormonally regulated in rat liver. We have investigated the effects of hypophysectomy and replacement therapy with T4, cortisol (C), and GH in vivo on the proportion of edited apoB mRNA in rat liver and cultured rat hepatocytes as well as the synthesis and secretion of apoB 48 and apoB 100 in cultured rat hepatocytes. Hypophysectomy decreased the proportion of edited apoB mRNA in intact liver from 62% in normal rats to 29% in hypophysectomized rats. Treatment of hypophysectomized rats with T4 and C did not influence the proportion of edited apoB mRNA, whereas treatment with GH, either alone or together with T4 and C, increased the proportion of edited apoB mRNA to the levels observed in normal rats. In cultured hepatocytes isolated from normal rats, the proportion of apoB 48 (percentage of total labeled apoB) was 78% and decreased to 40% in cells isolated from hypophysectomized rats. Treatment of hypophysectomized rats with T4 and C had no effect on the proportion of apoB 48 present in isolated cells, whereas it increased to 60% after treatment with GH together with T4 and C. The proportion of apoB 48 in the medium was affected by hypophysectomy and the various hormonal treatments in a similar way to that observed in the cells. Results from in vivo labeling experiments suggested that GH alone had the capacity to increase the percentage of apoB 48 in hypophysectomized rats. On the contrary, T4 and C was needed, in addition to GH, to increase the proportion of apoB 48 in isolated hepatocytes from hypophysectomized rats. Our results suggest that this discrepancy is due to a difference between the effect of GH alone on apoB mRNA editing in the intact liver and that in isolated hepatocytes. The total secretion of apoB into the cell culture medium was not affected by hypophysectomy and hormonal treatments of the rats. In conclusion, these results indicate that GH is involved in the regulation of editing of apoB mRNA and the proportion of apoB 48 synthesized and secreted in rat liver. Thus, our observations emphasize the importance of GH as a regulator of lipoprotein metabolism.

Rat ovarian insulin-like growth factor binding protein-3: a growth hormone-dependent theca-interstitial cell-derived antigonadotropin.

To further the identification and characterization of insulin-like growth factor binding proteins at the level of the immature rat ovary, we have set out to study the ovarian expression, cellular localization, and hormonal regulation of the insulin-like growth factor binding protein (IGFBP)-3. To this end, use was made of a solution hybridization/RNAse protection assay wherein ovarian total RNA from immature (21-23 days old) female rats was hybridized with a 343 bases-long [32P]-labeled rat IGFBP-3 riboprobe. As in liver, a single protected fragment (315 bases-long) corresponding to IGFBP-3 transcripts was identified in whole ovarian material. Cellular localization studies revealed the IGFBP-3 gene to be exclusively expressed in the theca-interstitial rather than the granulosa cell compartment. To confirm presence and cellular distribution of the IGFBP-3 protein, media conditioned by cultured granulosa cells, theca-interstitial cells, and whole ovarian dispersates were subjected to Western Ligand Blotting. Importantly, media conditioned by cultured theca-interstitial (but not granulosa) cells displayed an IGFBP the size of rat IGFBP-3 (46kDa) as determined by comigration with a rat serum standard. A similarly-sized band was apparent in media conditioned by cultured whole ovarian dispersates reflecting in all likelihood the contribution of the theca-interstitial cell component. Significantly, deglycosylation of media conditioned by cultured theca-interstitial cells revealed the glycosylated nature of the 46kDa IGFBP species as judged by the apparent reduction in its molecular size to 35kDa. Similar alterations were noted in corresponding rat serum samples. Hypophysectomy of immature rats resulted in a modest but statistically insignificant decrease in the relative (densitometrically-quantified) abundance of ovarian IGFBP-3 transcripts, an effect further augmented by the systemic provision of either FSH or diethylstilbestrol (DES). In contrast, systemic treatment of hypophysectomized rats with GH produced a marked (3.2-fold) increase (P less than 0.05) in the steady state levels of ovarian (as well as hepatic) IGFBP-3 transcripts. However, the concurrent provision of either FSH or DES resulted in substantial (P less than 0.05) attenuation (78 and 57% inhibition, respectively) of the upregulatory GH effect. These findings document the highly compartmentalized expression of the IGFBP-3 gene at the level of the immature rat ovary, implicate the theca-interstitial cell as the sole source of its generation, reveal its pituitary dependence, and disclose its diametrically-opposed (indeed antagonistic) regulation by FSH (or estrogens) and GH.(ABSTRACT TRUNCATED AT 400 WORDS)

Pituitary regulation of the expression of the farnesyl pyrophosphate synthetase gene in the testes of the sexually maturing rat.

While investigating the coordinate regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and farnesyl pyrophosphate synthetase, the authors observed that rat testes contained high levels of a farnesyl pyrophosphate synthetase mRNA that was larger than that found in most other tissues. This mRNA contains upstream AUG codons that may alter its rate of translation. The developmental and hormonal regulation of this testicular mRNA were investigated. Testicular levels of farnesyl pyrophosphate synthetase mRNA increased in rats between 30 and 40 days of age and remained elevated. Significant increases in serum testosterone concentrations and secondary sexual organ weights first occurred at 50 days of age. Hypophysectomy resulted in nearly undetectable levels of testicular farnesyl pyrophosphate synthetase mRNA. Treatment of hypophysectomized rats with gonadotropins increased the levels of this mRNA toward normal. These data indicate that an increase in farnesyl pyrophosphate synthetase mRNA takes place in testes just before the onset of puberty. This may be induced by the peripubertal rise in follicle-stimulating hormone.

Modulation of Hepatic Level of Microsomal Testosterone 7α-Hydroxylase, P-450a (P450IIA), by Thyroid Hormone and Growth Hormone in Rat Liver1

The effects of thyroid hormone and growth hormone on microsomal testosterone 7 alpha-hydroxylase, P-450a, were studied to understand the interaction of these hormone-mediated regulations in rats. In Western blots using anti-P-450a IgG, 1.7-fold higher content of P-450a was observed in livers of female than male adult rats, while no appreciable sex-related difference was detected in prepubertal rats and rats of 24 months of age. Treatment with n-propyl-2-thiouracil or thyroidectomy of male rats increased by 2-fold the hepatic content of P-450a, but neither regimen had a significant effect on the content in female rats. Levels of P-450a in both sexes of thyroidectomized rats were decreased by the supplementation of triiodothyronine (T3, 50 micrograms per kg, i.p. for 7 days) to levels similar to that observed in normal male rats. Hypophysectomy also caused an increase in microsomal P-450a content in male rats. Continuous infusion of human growth hormone, which mimicked the female secretion, further significantly increased the content in hypophysectomized rats to a level similar to that observed in normal female rats. In contrast, hepatic level of P-450a in hypophysectomized male and female rats was reduced by intermittent injection, which mimicked the male secretion. Clear suppression on the level of hepatic P-450a was also observed by the treatment of hypophysectomized rats with 5 or 50 micrograms/kg of T3 and of hGH-infused hypophysectomized rat with 50 micrograms/kg of T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone and thyroxine affect lipoprotein metabolism in hypothyroid and hypophysectomized rats

The aim of this study was to evaluate the effect of thyroxine (T4) and GH replacement therapy on serum lipoproteins in rats with primary and secondary hypothyroidism and to see whether recovery of GH activity was associated with normalization of serum lipoproteins. In both the primary and secondary hypoproteins. In both the primary and secondary hypothyroid rats, total cholesterol (TC) was higher than in normal controls, due to an increase in low-density lipoprotein cholesterol (LDL-c) and to a lesser extent also in high-density lipoprotein cholesterol (HDL-c). Treatment of hypophysectomized rats with GH induced a decrease in the concentration of TC, LDL-c and HDL-c, while liver lipase activity (LLA) increased. The effect of GH on HDL-c was correlated with the increase in LLA. T4 replacement therapy of hypophysectomized rats normalized LDL-c, but HDL-c and LLA were unaffected. During primary hypothyroidism, T4 treatment induced GH activity, increased LLA and reduced the HDL-c concentration. GH treatment of primary hypothyroid rats had a similar influence on the lipid levels and LLA as in hypophysectomized animals, although the lowering of HDL-c was less prominent. These results demonstrate that GH determines serum lipoproteins during both primary and secondary hypothyroidism.

Different Tissue Distribution and Hormonal Regulation of Messenger RNAs Encoding Rat Insulin-Like Growth Factor-Binding Proteins-1 and -2

The insulin-like growth factor-binding proteins IGFBP-1 and IGFBP-2 are low mol wt IGFBPs that are similar in structure. They are not glycosylated and have a homologous amino acid sequence, including the number and position of 18 cysteine residues and a carboxyl-terminal Arg-Gly-Asp sequence that can be recognized by cell adhesion receptors. The present study demonstrates that expression of mRNAs encoding the two BPs differs in some fetal rat tissues and in the livers of adult rats after hypophysectomy, fasting, or streptozotocin-induced diabetes. As determined by Northern blot hybridization using cDNA probes for rat IGFBP-2 or human IGFBP-1, both mRNAs are expressed at high levels in liver of 21-day gestation and 1-day-old rats and at lower levels in 21- and 65-day-old rat liver. Levels of both mRNAs are higher in liver than in other fetal rat tissues. The relative abundance of the two mRNAs in most fetal tissues is similar to that in liver, except that kidney and brain have 8-fold and more than 25-fold higher relative levels of IGFBP-2 mRNA, respectively. IGFBP-2 mRNA is about 10- to 20-fold increased after hypophysectomy or fasting, whereas IGFBP-1 mRNA is relatively unchanged. IGFBP-2 mRNA levels are decreased completely by refeeding fasted rats for 3 days, but only partially decreased by treatment of hypophysectomized rats with GH, cortisone acetate, T4, and testosterone for 4 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanisms of ACTH-induced inhibition of aldosterone biosynthesis in the rat adrenal cortex

In rats, chronic treatment with high doses of ACTH (10-40 micrograms/100 g body weight per day) leads to a marked reduction in aldosterone synthesis by adrenal capsules. The possibility that this inhibition is secondary to a decrease in plasma potassium levels or in renin angiotensin system (RAS) activity has been explored. The effects of chronic ACTH treatment were compared in (I) animals in which the endogenous RAS activity was stimulated by restricting dietary sodium intake, (II) animals in which plasma angiotensin II was increased by infusion from implanted osmotic minipumps and (III) animals which received supplementary potassium and in which hypokalaemia was prevented. In all cases, rates of aldosterone biosynthesis in vitro by adrenal capsules were decreased in ACTH-treated animals to an extent similar to those in untreated controls. In addition, ACTH treatment of hypophysectomized rats resulted in a similar inhibition of aldosterone biosynthesis to that found in sham-operated controls. It may be concluded that the ACTH-induced reduction of aldosterone biosynthesis is independent of the secretion of other pituitary hormones, and cannot be simply ascribed to either a reduction in RAS activity or in plasma potassium levels. The results are consistent with the view that the effects of chronic ACTH treatment are mediated by a direct action on the zona glomerulosa cell, which leads to its transformation into a zona fasciculata-like form.

Rat ovarian insulin-like growth factor I (IGF-I) gene expression is granulosa cell-selective: 5'-untranslated mRNA variant representation and hormonal regulation.

We, and others, have recently reported that the ovary is a site of insulin-like growth factor (IGF)I gene expression. It was the objective of the present studies to assess the relative ovarian abundance of IGF-I transcripts with alternative 5'-untranslated (UT) regions, their cellular localization, and hormonal regulation. To this end, a solution hybridization/RNase protection assay was employed wherein total rat ovarian RNA was hybridized with a 404-base 32P-labelled rat IGF-I riboprobe corresponding to the Class A 5'UT variant. As in liver, three protected bands [322 (Class A), 297 (Class B), and 242 (Class C) bases long] were noted, in keeping with established alternative 5' UT transcripts. The ovarian (as the hepatic) Class C variant proved the most abundant. The ovarian Class B variant was barely detectable. Cellular localization studies revealed these ovarian IGF-I transcripts to be primarily, if not exclusively, of granulosa but not theca-interstitial cell origin. Treatment of immature (21-23 days old) hypophysectomized rats with a diethylstilbestrol (DES)-containing subcutaneous silastic implant for a total of 5 days resulted in a 2-fold increase in the (densitometrically quantified) abundance of ovarian IGF-I transcripts, a diametrically-opposed effect (2.6-fold decrease) being noted at the level of the liver. Whereas treatment of hypophysectomized rats with oGH by itself (150 micrograms, qd, sc x5 days) resulted in a 5-fold increase in hepatic IGF-I gene expression, a limited, albeit distinct inhibitory effect was observed on the steady-state levels of ovarian IGF-I mRNA. In contrast, combined treatment with oGH and DES yielded a 3-fold increase in the abundance of ovarian IGF-I transcripts, there being no net alteration in hepatic IGF-I gene expression. Taken together, these findings reveal ovarian expression of the 3 known 5'-UT IGF-I mRNA variants, document the granulosa cell as the main somatic ovarian cell of IGF-I mRNA generation, and indicate that hepatic and ovarian IGF-I gene expression are differentially regulated in diametrically opposed directions.

Cytochrome P450 in livers of diabetic rats: Regulation by growth hormone and insulin

The effects of pituitary and pancreatic hormones on the change in hepatic cytochrome P450s were studied in alloxan- or streptozotocin-induced male rats. In two major sex-specific forms, P450-male and P450 6β-1, the former was decreased in chronic (5 week) diabetes to only less than one-third of controls and the latter was also reduced in early (1 week) diabetes. In contrast, a main phenobarbital-inducible form, P450b, was enhanced 25- to 30-fold in these diabetic rats. 3-Methylcholanthrene-inducible P448H was also elevated 3-fold in alloxan-induced diabetes. These changes in hepatic contents of P450-male, P450- 6β-1, and P450b, which are under the regulation of pituitary growth hormone, associated well with the reported results of time-dependent changes in growth hormone levels in diabetes ( G. S. Tannenbaum (1981) Endocrinology 108,76–82) , suggesting that the change in growth hormone level is a factor responsible for alterations in hepatic cytochrome P450s. Normalizing effects of insulin on these forms were also studied. Treatment of diabetic rats with insulin reversed the decreased amounts of both P450-male protein and mRNA. Insulin also normalized hepatic contents of P450b, P450 6β-1, and P448H. However, the treatment of hypophysectomized rats with insulin had no effect, and treatment of diabetic rats with growth hormone or a suppressing agent of somatostatin, cysteamine, showed trivial effects on P450-male and P450b. These results suggest that insulin does not act directly as a substitute of growth hormone, but exerts its effect indirectly through the normalization of a growth hormone-mediated process(es) in diabetic rats.

Effects of growth hormone on cytochrome P-450 j

Hypophysectomy of male and female rats resulted in a 5 and 10-fold increase respectively, in cytochrome P-450j as determined by immunoblotting. Treatment of hypophysectomized rats with growth hormone resulted in a decrease in P-450j. Microsomal N-nitrosodimethylamine demethylase activity was increased approximately 23 and 75 per cent in male and female rats respectively, over the activity in microsomes of intact animals. These studies suggest that hypophysectomy results in an increase in cytochrome P-450j and that growth hormone acts as a repressive factor for constitutive P-450j.

Neural and humoral factors separately regulate neuropeptide Y, enkephalin, and chromogranin A and B mRNA levels in rat adrenal medulla.

The influence of neurogenic versus humoral factors on mRNA levels of several secretory proteins of rat adrenal medulla was studied in vivo. Increased splanchnic nerve activity was generated (reflexly) with insulin treatment. Twenty-four hours after insulin injection, levels of mRNAs encoding neuropeptides (enkephalin and neuropeptide Y) were increased 6.5-fold, whereas those of mRNAs for the major secretory proteins (chromogranins A and B) were unchanged. Bilateral transection of the splanchnic nerves completely prevented this increase. Hypophysectomy decreased levels of chromogranin A mRNA to 32% of control, suggesting a dependence on hormones of the pituitary-adrenal axis. Treatment of hypophysectomized rats with dexamethasone restored chromogranin A mRNA to basal levels. Chromogranin B mRNA levels were not changed by either insulin treatment or hypophysectomy. These results demonstrate (i) that different classes of secretory proteins present in chromaffin granules are regulated by different mechanisms, (ii) that this regulation occurs at a pretranslational site, and (iii) that the relative concentration of secretory constituents of chromaffin granules may vary. The significance of an altered composition of secretory-granule constituents, which may be important in hypotension or stress, is discussed.

Effects of thyrotropin on the vascular conductance of the thyroid gland.

It is well established that TSH from the anterior pituitary is the principal stimulatory agent in the physiological regulation of the thyroid gland. Chronic elevations of plasma TSH induce hyperplasia and hypertrophy of thyroid follicular cells and enlargement of blood capillaries. At low plasma TSH levels the thyroid gland atrophies. We have examined the vascular conductance (C = blood flow/mean arterial pressure) of the thyroid gland and several other tissues over a wide range of endogenous plasma TSH concentrations and after treatment with bovine TSH (bTSH) in rats. Tissue blood flows were determined using 15 +/- 5-microns diameter 141Ce-labeled microspheres in a modification of the reference sample microsphere technique. The microspheres were injected directly into the left cardiac ventricle via a 23-gauge needle passed through the chest wall, while the reference blood sample was collected and systemic arterial blood pressure was monitored through femoral arterial catheters. After the animals were killed, tissues were cleaned and weighed, and the tissue radioactivity was determined. Blood samples for determination of plasma hormone levels were obtained from the jugular vein before the injection of microspheres. In the first series of experiments, the vascular C per mass of thyroid gland was significantly decreased 4 and 8 days after hypophysectomy. Treatment of hypophysectomized rats with bTSH (185 mU/100 g.day as a continuous iv infusion for 2 or 6 days) restored thyroid vascular C per mass of tissue to control levels. In the second series of experiments, we manipulated circulating plasma TSH levels in intact rats by 6 days of treatment with propylthiouracil (2.0 mg/day, ip), thyroid hormones (1.5 micrograms T4, 0.4 micrograms T3 or 3.0 micrograms T4, plus 0.8 micrograms T3/100 g.day, sc by continuous infusion), TRH (240 micrograms/day, iv, by continuous infusion), bTSH (800 mU/day, iv, by continuous infusion), or combinations of these treatments. The vascular C per mass of thyroid gland was significantly decreased at very low chronic plasma TSH levels and increased at very high chronic plasma TSH levels. Thyroid vascular C per mass was unchanged, however, over a broad intermediate range of plasma TSH concentrations encompassing normal values, despite alterations in the size and function of the thyroid gland. At these intermediate levels of TSH stimulation, the thyroid gland may respond by adding or subtracting functional units without changing the blood flow per unit. The amount of blood flow per functional unit may be altered only at very high or very low levels of TSH stimulation.