Hypox Rats Research Articles

Irreversible perinatal imprinting of adult expression of the principal sex-dependent drug-metabolizing enzyme CYP2C11.

We proposed to determine whether, like other sexual dimorphisms, drug metabolism is permanently imprinted by perinatal hormones, resulting in its irreversible sex-dependent expression. We treated newborn male rats with monosodium glutamate (MSG), a total growth hormone (GH) blocker, and, using cultured hepatocytes, examined expression of adult CYP2C11, the predominant cytochrome-P450 expressed only in males, as well as the signal transduction pathway by which episodic GH solely regulates the isoform's expression. In addition, adolescent hypophysectomized (hypox) male rats served as controls in which GH was eliminated after the critical imprinting period. Whereas renaturalization of the masculine episodic GH profile restored normal male-like levels of CYP2C11, as well as CYP2C12, in hepatocytes from hypox rats, the cells derived from the MSG-treated rats were completely unresponsive. Moreover, GH exposure of hepatocytes from hypox rats resulted in normal induction, activation, nuclear translocation, and binding to the CYP2C11 promoter of the signal transducers mediating GH regulation of CYP2C11 expression, which dramatically contrasted with the complete unresponsiveness of the MSG-derived hepatocytes, also associated with hypermethylation of GH-response elements in the CYP2C11 promoter. Lastly, neonatal MSG treatment had no adverse effect on postnatal and adult testosterone levels. The results demonstrate that the sexually dimorphic expression of CYP2C11 is irreversibly imprinted shortly after birth by a hormone other than the customary testosterone, but likely by GH.

Effects of selective pituitary hormone deficiencies on macrophage phagocytic activity in rat (911.2)

We showed that neurointermediate pituitary lobectomy (NIL) decreases whereas anterior pituitary lobectomy (AL) increases humoral and cell‐mediated immune responses. Studies indicate that PRL and GH are immune stimulatory/modulatory hormones. Macrophages (Mθ) have receptors for AVP, OXT, PRL and GH. In order to obtain a deeper insight into the role of anterior and posterior pituitary hormones on innate immunity, Mθ phagocytic activity was assessed in NIL, AL, HYPOX, sham operated (SHAM) and Control rats. Carbon black (CB) blood clearance, phagocytic index, CB blood half‐life time (T1/2), and histologic changes in liver, spleen and lung were also studied. Results. CB clearance was faster in SHAM > Control > NIL > HYPOX > AL rats. Differences were noted only in AL with slower clearance, suggesting that adenohypophysial but not neurohypophysial hormones are responsible for the faster CB clearance. Phagocytic index was decreased in NIL rats, indicating that AVP and OXT are required to regulate phagocytic activity. Blood CB T1/2 was 10 minutes in Control and SHAM rats, and 20, 17 and 18 minutes in AL, HYPOX and NIL rats. The longer T1/2 suggests that all pituitary hormones participate in CB clearance. Liver, spleen and lung histology showed Mθ markedly loaded with CB. These findings indicate that adenohypophysial and neurohypophysial hormones participate differently in the regulation of Mθ phagocytic activity.Grant Funding Source: CONACYT MEXICO 62317

Role of pituitary in K+ homeostasis: impaired renal responses to altered K+ intake in hypophysectomized rats

The kidneys maintain extracellular K⁺ homeostasis by altering K⁺ excretion to match K⁺ intake. Because this can occur without changes in plasma K⁺ concentrations ([K⁺]), how the kidneys sense K⁺ intake is unclear. We tested the hypothesis that the pituitary plays a critical role in signaling K⁺ intake to the kidneys. If this hypothesis is true, hypophysectomy would impair kidney responses to altered K⁺ intake. Hypophysectomized (Hypox) and sham-operated control rats (n = 8 each) were compared for their abilities to adjust K⁺ excretion during a transition from normal to reduced (to one-third of normal) K⁺ intake, followed by a reversal to normal K⁺ intake. Food was provided only at night, and renal K⁺ excretion was determined both for absorptive (night or feeding) and postabsorptive (day or nonfeeding) periods. In normal rats, both absorptive and postabsorptive renal K⁺ excretion were changed in parallel to the changes in K⁺ intake, indicating a rapid adaptation of normal kidneys to altered K⁺ intake. In Hypox rats, whereas absorptive renal K⁺ excretion was changed in response to changes in K⁺ intake, postabsorptive K⁺ excretion was not responsive (P < 0.001), indicating impaired renal responses to altered K⁺ intake. In addition, Hypox rats, compared with control rats, showed K⁺ intolerance (increases in plasma [K⁺]) upon feeding (i.e., K⁺ intake) at night or following an intravenous K⁺ infusion (P < 0.01), indicating an impairment of acute renal responses to K⁺ intake. These data support that the pituitary plays a key role in the signaling of K⁺ intake to the kidneys (and kidney responses to altered K⁺ intake).

Bone and hypopituitarism: not only a mass issue

Bone and hypopituitarism: not only a mass issue

Growth hormone regulates the balance between bone formation and bone marrow adiposity

Cancellous bone decreases and bone marrow fat content increases with age. Osteoblasts and adipocytes are derived from a common precursor, and growth hormone (GH), a key hormone in integration of energy metabolism, regulates the differentiation and function of both cell lineages. Since an age-related decline in GH is associated with bone loss, we investigated the relationship between GH and bone marrow adiposity in hypophysectomized (HYPOX) rats and in mice with defects in GH signaling. HYPOX dramatically reduced body weight gain, bone growth and mineralizing perimeter, serum insulin-like growth factor 1 (IGF-1) levels, and mRNA levels for IGF-1 in liver and bone. Despite reduced body mass and adipocyte precursor pool size, HYPOX resulted in a dramatic increase in bone lipid levels, as reflected by increased bone marrow adiposity and bone triglyceride and cholesterol content. GH replacement normalized bone marrow adiposity and precursor pool size, as well as mineralizing perimeter in HYPOX rats. In contrast, 17β -estradiol, IGF-1, thyroxine, and cortisone were ineffective. Parathyroid hormone (PTH) reversed the inhibitory effects of HYPOX on mineralizing perimeter but had no effect on adiposity. Finally, bone marrow adiposity was increased in mice deficient in GH and IGF-1 but not in mice deficient in serum IGF-1. Taken together, our findings indicate that the reciprocal changes in bone and fat mass in GH signaling-deficient rodents are not directly coupled with one another. Rather, GH enhances adipocyte as well as osteoblast precursor pool size. However, GH increases osteoblast differentiation while suppressing bone marrow lipid accumulation. © 2010 American Society for Bone and Mineral Research

Effects of alcohol on skeletal response to growth hormone in hypophysectomized rats

Chronic alcohol abuse is an established risk factor for osteoporosis. However, the precise mechanisms for the bone loss are largely unknown. Alcohol decreases skeletal expression of insulin-like growth factor-I (IGF-I), an important growth hormone (GH)-regulated skeletal growth factor. Therefore, we investigated the effects of alcohol on the skeletal response to GH in male Sprague–Dawley rats made GH-deficient by hypophysectomy (HYPOX). Four groups of sexually mature (3-month-old) rats were studied: pituitary-intact (control), HYPOX, HYPOX + GH, and HYPOX + alcohol + GH. All animals were transferred to a liquid diet 6 days following surgery. The alcohol-fed group was adapted to a graded increase in alcohol beginning 11 days following surgery. GH or vehicle was administered during the final 8 days of study and all animals were sacrificed 25 days following surgery. HYPOX resulted in cessation of body weight gain and tibial growth. Compared to controls, longitudinal bone growth and cancellous bone formation were lower following HYPOX. The latter was associated with lower mineralizing perimeter/bone perimeter. Bone marrow adiposity was higher following HYPOX. Compared to HYPOX, GH treatment increased body weight gain and bone formation rate, and decreased bone marrow adiposity. In contrast to the effects of GH treatment without alcohol, bone marrow adiposity did not differ between HYPOX and alcohol-fed GH-treated HYPOX rats. Alcohol did not alter GH-induced weight gain or increases in serum IGF-I levels, but significantly impaired the effects of GH on tibial growth and cancellous bone formation. We conclude that the detrimental skeletal effects of alcohol abuse observed in this experiment are mediated, at least in part, by skeletal resistance to GH.

Differences in spleen mRNA ILs expression in anterior, posterior and total hypophysectomized rats with experimental autoimmune encephalomyelitis (EAE) or adjuvant arthritis (AA); the vasopressin role

Neurointermediate pituitary lobectomy (NIL) in Lewis rats causes mild signs of EAE, while hypophysectomy (HYPOX) and anterior pituitary lobectomy (AL) worse the disease, and desmopressin (vasopressin analog) aggravates the EAE in NIL and HYPOX rats. Besides, the AIA severity is low in NIL and no signs of AIA occur in HYPOX animals, while AL induces severe arthritis. Here we compare the splenic expression (RT‐PCR) of mRNA of ILs 1, 2, 6, 10, 12 and INF‐¦Ã between sham operated (SHAM), AL, NIL, and HYPOX groups immunized for EAE (females) or AIA (males). Intact control groups were included for basal IL levels. Rats were operated 3 weeks before immunizations and sacrificed at day 15. Results showed over expression of IL‐2 in AL‐EAE group. Similar augments of IL‐1 and 2 in SHAM, NIL and HYPOX‐EAE groups, and low expression in ILs 6, 10 and INF‐¦Ã in NIL, AL and HYPOX groups. IL‐12 was higher in HYPOX‐EAE rats with no changes in the remaining groups. In AL‐AIA group, significant increases in ILs 1, 2, 6 and INF‐¦Ã occurred, while in NIL‐AIA group ILs 2, 12 and INF‐¦Ã were low respect the remaining groups. HYPOX group did not developed clinical signs of AIA and ILs 2, 6, 10, 12 and INF‐¦Ã levels were similar to the SHAM group. We conclude that neurohypophysial hormones are important in the control of autoimmune diseases and that the ILs involved in the immune responses are different for EAE and AIA.CONACYT. M̈¦xico

Two Novel Bovine Somatotropin Species Generated from a Common Dehydroalanine Intermediate

Under stressed conditions such as prolonged exposure to high pH, the C-terminal disulfide bridge in bovine somatotropin (bST) is susceptible to a base catalyzed beta-elimination reaction. This reaction converts the disulfide bond to a dehydroalanine residue with loss of a sulphur atom. Two altered species were isolated in pure form and determined to be generated from this dehydroalanine intermediate. One is a monomeric lanthionyl bST (L-bST) with a thioether linkage, and the other is an inter-molecular disulfide linked dimer containing a lysinoalanine. These two novel structures were unambiguously determined using various techniques including enzymatic digestion, amino acid sequencing and analysis, and mass spectrometry. The monomeric L-bST was demonstrated to be equipotent to normal bST in a hypox rat assay, thus showing that formation of lanthionine in place of this disulfide bond does not affect it bioactivity.

Comparative Study of the Effects of Different Growth Hormone Doses on Growth and Spatial Performance of Hypophysectomized Rats

This study was designed to examine the effects of recombinant human growth hormone replacement on somatic growth and cognitive function in hypophysectomized (HYPOX) female Sprague-Dawley rats. Rats (5 per group) were randomized by weight to 3 experimental groups: group 1, administered 200 µg/kg of GH once daily for 9 days; group 2, administered 200 µg/kg of GH twice daily; and group 3, administered saline daily. Somatic growth was evaluated by measurement of body weight daily and of the width of the proximal tibial growth plate of the HYPOX rats. Cognitive function was evaluated using the Morris water maze (MWM) test. The results indicated that GH replacement therapy in HYPOX rats promoted an increase in the body weight and the width of the tibial growth plate in a dose-dependent manner. On the third day of the MWM test, the escape latency in the GH-treated groups 1 and 2 was significantly shorter than that in the control rats (P<0.001 and P=0.032, respectively), suggesting that rhGH improved spatial memory acquisition in the MWM test. Therefore it is concluded that rhGH replacement therapy in HYPOX rats stimulates an increase in somatic growth in a dose-dependent manner and also has beneficial effects on cognitive functions.

Role of Pituitary Hormones on 17α-Ethinylestradiol-Induced Cholestasis in Rat

Estrogens cause intrahepatic cholestasis in susceptible women during pregnancy, after administration of oral contraceptives, or during postmenopausal hormone replacement therapy. 17alpha-Ethinylestradiol (EE) is a synthetic estrogen widely used to cause experimental cholestasis in rodents with the aim of examining molecular mechanisms involved in this disease. EE actions on the liver are thought to be mediated by estrogen receptor alpha (ERalpha) and pituitary hormones. We tested this hypothesis by analyzing metabolic changes induced by EE in livers from hypophysectomized (HYPOX) and hypothyroid rats. Microarray studies revealed that the number of genes regulated by EE was increased almost 4-fold in HYPOX rat livers compared with intact males. Little overlap was apparent between the effects of EE in intact and HYPOX rats, demonstrating that pituitary hormones play a critical role in the hepatic effects of EE. Consistently, hypophysectomy protects the liver against induction by EE of serum bilirubin and alkaline phosphatase, two markers of cholestasis and hepatotoxicity and modulates the effects of EE on several genes involved in bile acid homeostasis (e.g., FXR, SHP, BSEP, and Cyp8b1). Finally, we demonstrate a novel mechanism of action of EE through binding and negative regulation of glucocorticoid receptor-mediated transcription. In summary, pituitary- and ERalpha-independent mechanisms contribute to development of EE-induced changes in liver transcriptome. Such mechanisms may be relevant when this model of EE-induced cholestasis is evaluated. The observation that the pharmacological effects of estrogen in liver differ in the absence or presence of the pituitary could be clinically relevant, because different drugs that block actions of pituitary hormones are now available.

Hypophysectomy and Neurointermediate Pituitary Lobectomy Reduce Serum Immunoglobulin M (IgM) and IgG and Intestinal IgA Responses toSalmonella entericaSerovar Typhimurium Infection in Rats

The influence of anterior pituitary hormones on the gastrointestinal tract of humans and animals has been reported. Hypophysectomy (HYPOX) in the rat causes atrophy of the intestinal mucosa, reduction of gastric secretion and intestinal absorption, and increased susceptibility to infections. To our knowledge, there are no studies on the humoral immune response of the gut-associated lymphoid tissue after HYPOX. We have reported that decreased secretion of vasopressin and oxytocin due to neurointermediate pituitary lobectomy (NIL) diminishes humoral and cell-mediated immune responses. However, no data have been published on whether NIL can affect intestinal immune responses. We analyzed the effects of HYPOX and NIL on bacterial colonization of the intestinal lumen, Peyer's patches, and spleen as well as the serum immunoglobulin G (IgG) and IgM and specific intestinal IgA levels in response to Salmonella enterica serovar Typhimurium oral infection. Results showed the following: (i) Salmonella serovar Typhimurium was eliminated from the intestinal lumen at the same rate in rats that underwent a sham operation, HYPOX, and NIL; (ii) Salmonella serovar Typhimurium colonization of Peyer's patches and spleen was significantly higher in both HYPOX and NIL rats than in sham-operated rats; (iii) serum IgG and IgM and intestinal IgA against surface proteins of Salmonella serovar Typhimurium were significantly lower in HYPOX and NIL rats than in sham-operated rats; and (iv) compared to NIL rats, higher Peyer's patch and spleen bacterial colonization and decreased IgG, IgM, and IgA production were observed in HYPOX rats. We conclude that hormones from each pituitary lobe affect the systemic and gastrointestinal humoral immune responses through different mechanisms.

Involvement of Thyroxine in Ovarian Toxicity of Di-(2-ethylhexyl) Phthalate

Forced ovulation induced by the administration of exogenous gonadotropin is a useful marker for studying the ovarian toxicity of chemicals in experimental animals. We examined the toxicity of di-(2-ethylhexyl) phthalate (DEHP) in the ovaries of immature F344 female rats. Superovulation was induced by injections of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) in rats dosed with 125, 250, 500, 1,000 or 2,000 mg/kg body weight of DEHP for 4 consecutive days. The number of ova shed during superovulation significantly decreased in rats treated with DEHP at 500 mg/kg as compared with control, but no changes were observed in the number of ova in groups given other doses of DEHP. In control rats treated with olive oil, hypophysectomy reduced significantly the number of ovulated ova. When 2,000 mg DEHP was given to hypophysectomized (hypox) rats, the number of ova in the hypox group was significantly smaller than that in the intact group administered with the same doses of DEHP. In contrast, the numbers of ova of the intact and hypox groups did not significantly differ in rats given 500 mg DEHP. The levels of circulating thyroxine (T4) were significantly decreased by 2,000 mg DEHP in intact rats, and a tendency for T4 to decrease in T4 was also observed in hypox rats given 2,000 mg DEHP. These results suggest that daily administration of 500 mg DEHP suppressed superovulation in immature F344 rats by disrupting the hypothalamic-pituitary-ovarian axis in a manner similar to that of hypophysectomy. Decreased circulating T4 levels seemed to negate this disruption as observed in recovered superovulation after treatment with 2,000 mg DEHP.

The effect of hypophysectomy on pancreatic islet hormone and insulin-like growth factor I content and mRNA expression in rat

The growth arrest after hypophysectomy in rats is mainly due to growth hormone (GH) deficiency because replacement of GH or insulin-like growth factor (IGF) I, the mediator of GH action, leads to resumption of growth despite the lack of other pituitary hormones. Hypophysectomized (hypox) rats have, therefore, often been used to study metabolic consequences of GH deficiency and its effects on tissues concerned with growth. The present study was undertaken to assess the effects of hypophysectomy on the serum and pancreatic levels of the three major islet hormones insulin, glucagon, and somatostatin, as well as on IGF-I. Immunohistochemistry (IHC), in situ hybridization (ISH), radioimmunoassays (RIA), and Northern blot analysis were used to localize and quantify the hormones in the pancreas at the peptide and mRNA levels. IHC showed slightly decreased insulin levels in the beta cells of hypox compared with normal, age-matched rats whereas glucagon in alpha cells and somatostatin in delta cells showed increase. IGF-I, which localized to alpha cells, showed decrease. ISH detected a slightly higher expression of insulin mRNA and markedly stronger signals for glucagon and somatostatin mRNA in the islets of hypox rats. Serum glucose concentrations did not differ between the two groups although serum insulin and C-peptide were lower and serum glucagon was higher in the hypox animals. These changes were accompanied by a more than tenfold drop in serum IGF-I. The pancreatic insulin content per gram of tissue was not significantly different in hypox and normal rats. Pancreatic glucagon and somatostatin per gram of tissue were higher in the hypox animals. The pancreatic IGF-I content of hypox rats was significantly reduced. Northern blot analysis gave a 2.6-, 4.5-, and 2.2-fold increase in pancreatic insulin, glucagon, and somatostatin mRNA levels, respectively, in hypox rats, and a 2.3-fold decrease in IGF-I mRNA levels. Our results show that the fall of serum IGF-I after hypophysectomy is accompanied by a decrease in pancreatic IGF-I peptide and mRNA but by partly discordant changes in the serum concentrations of insulin and glucagon and the islet peptide and/or mRNA content of the three major islet hormones. It appears that GH deficiency resulting in a "low IGF-I state" affects translational efficiency of these hormones as well as their secretory responses. The maintenance of normoglycemia in the presence of reduced insulin and elevated glucagon serum levels, both of which would be expected to raise blood glucose, may result mainly from the enhanced insulin sensitivity, possibly due to GH deficiency and the subsequent decrease in IGF-I production.

Neurointermediate pituitary lobectomy decreases the incidence and severity of experimental autoimmune encephalomyelitis in Lewis rats

Acute experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system, mediated by T lymphocytes. Immunization of Lewis rats with myelin antigens suspended in complete Freund's adjuvant induces EAE. In a previous study on rats we have found that neurointermediate pituitary lobectomy (NIL) decreased both the humoral and cell-mediated immune responses. Here we investigated the effect of NIL on the incidence and severity of EAE and on the function of the hypothalamic-pituitary-adrenal axis in Lewis rats. NIL, hypophysectomized (Hypox) and sham-operated (Sham) rats were immunized s.c. with guinea-pig brain extract suspended in complete Freund's adjuvant. Untreated rats were used as controls. Water intake, body weight gain, clinical and histopathologic incidence and severity of EAE were evaluated in the operated groups. On killing, plasma adrenocorticotropin and corticosterone levels were measured and adrenals, thymuses and spleens were weighed. Histopathologic lesions were counted in the brain and spinal cord. Water intake and body weight gain were significantly decreased in Sham and Hypox animals with EAE whereas higher intakes persisted in the NIL group. Plasma levels of adrenocorticotropin were within the normal range whereas corticosterone levels increased in Sham and occasionally in NIL animals. Thymus weights were decreased in NIL and Hypox groups. The clinical and histopathologic incidence and severity of EAE were significantly decreased in NIL animals as compared with Sham and Hypox rats. We concluded that NIL affects the cell-mediated immune response and plays a role in the development and progression of EAE in the Lewis rat.

Influence of muscle strength and total work on exercise-induced plasma growth hormone isoforms in women

The purpose of this investigation was to determine the influence of physical strength and the ability to do more total work on human growth hormone (GH) variants to a heavy resistance exercise protocol in untrained women. From a distribution of 100 healthy, untrained women, the strongest 10 women (S) and the weakest 10 women (W) were compared for GH responses pre- and post an acute heavy resistance exercise test (AHRET, 6 sets of 10 RM squats, 2 minutes rest between sets). Blood samples were obtained pre-exercise and immediately post-exercise and subsequently analysed in total as well as fractionated by Sephacryl S-100R column chromatography into three molecular weight size classes: fraction A: > 60 kD, fraction B: 30-60 kD, fraction C: < 30 kD. For each total sample as well as each fraction, immunoreactive GH was measured via the Nichols IRMA, while bioactive GH was measured via the hypox rat tibial line bioassay and Diagnostic Systems Laboratory's immunofunctional GH ELISA. No exercise-induced changes or differences between groups were observed in the tibial line bioassay. However, the S group displayed a significantly higher pre-exercise resting value in the total fraction than the W group. Conversely, the W group exhibited a significantly higher pre-exercise value in the smaller molecular weight fraction C. With regards to the immunofunctional and immunoreactive assays, the total fraction, fraction A, and fraction B demonstrated significant (P < or = 0.05) exercise-induced increases in both the S and W group despite no group differences. For the Nichols and immunofunctional assays significant exercise-induced changes were observed in the smaller molecular weight C fraction in the W group but not the S group. However, the S group displayed a significantly higher pre-exercise value in fraction C relative to the W group. These data demonstrate for the first time that differences exist in the GH molecular weight variants between strong and weak untrained women, with the lower molecular weight variants seemingly less responsive to greater amounts of exercise in stronger women, thus suggesting differential regulation of GH molecular weight variants during resistance exercise due to pre-existing physical parameters.

Effect of growth hormone on high plasma levels of glucagon-like peptide-1 (GLP-1) in hypophysectomized rats.

Fasting plasma GLP-1 levels were significantly higher in hypophysectomized (hypox) rats (n = 6) than in intact (normal) rats (n = 7) (54.3 +/- 5.2 vs. 33.3 +/- 2.4 pmol/L, p < 0.001). To examine the influence of pituitary hormones on plasma GLP-1 levels, concentrations of plasma glucose, insulin and GLP-1 after an oral glucose load to hypox rats that were given either rat growth hormone (rGH) (n = 7), cortisol and thyroxine (n = 7) or no substitution (n = 6) were compared with those of normal rats (n = 7). Plasma glucose levels in the fasting state and after the glucose ingestion were significantly lower in hypox rats, but the hormonal replacements to hypox rats increased their total glucose levels to those of normal rats, although the increasing patterns were different from those in normal rats. Insulin levels both in the fasting state and after the glucose ingestion were significantly decreased in hypox rats and the fasting and total GLP-1 levels were significantly increased in those rats. rGH substitution significantly increased the total insulin levels in hypox rats and decreased the fasting and total GLP-1 levels closely to levels in normal rats, while substitution with cortisol and thyroxine failed to introduce such a significant effect. These results suggested that secretion of GLP-1 might be influenced by the function of GH.

Vaginocervical Stimulation Releases Oxytocin within the Spinal Cord in Rats

Vaginocervical stimulation (VS) significantly elevated the concentration of oxytocin (OT) in spinal cord superfusates of 8 intact urethane-anesthetized rats measured 10–15 min after VS (median [interquartile range]: 1.7 [1.00–3.37] pg/ml) compared to that measured 10–15 min before VS (1.1 [1.01–1.40] pg/ml). When VS was administered once (n = 8), it produced a 55% increase over baseline values; when administered a second time 45 min later (n = 6), it produced only a 22% increase over pre-VS values. The effects of estrogen on the VS-induced release of OT were then investigated using ovariectomized rats that were treated either with estradiol benzoate (EB; 10 µg/100 g bw) (n = 6) or with an oil vehicle (n = 6) subcutaneously for 3 days. The EB treatment significantly elevated the basal levels of OT released into spinal cord superfusates above vehicle control levels. Within 5–10 min after the onset of VS, OT concentrations in the superfusates were significantly higher in EB-treated than in vehicle-treated rats. The vehicle-treated rats did not show a significant elevation in OT concentration following VS. To rule out the possibility that the posterior pituitary gland was the source of this OT, the effect of hypophysectomy (HYPOX) was assessed on the VS-induced release of OT into spinal cord superfusates and plasma. The concentration of OT in spinal cord superfusates of both the HYPOX (n = 5) and intact rats (n = 6) increased significantly from 5.8 [4.4–6.5] pg/ml pre-VS to 7.9 [6.7–10.3] pg/ml immediately after VS, and from 4.4 [3.8–5] pg/ml pre-VS to 5.1 [4.6–5.7] pg/ml immediately after VS, respectively. There was no significant difference in baseline levels of OT in cerebrospinal fluid between the two groups. By contrast, plasma OT levels, while significantly elevated in response to VS from 3.42 [2.9–5.34] pg/ml baseline to 7.25 [5.33–15.77] pg/ml in the intact group, failed to respond significantly to VS in the HYPOX group (n = 5). The present findings provide evidence of a direct estrogen-dependent release of OT within the spinal cord in response to VS, presumably via descending oxytocinergic neurons.

Feeding-entrained circadian rhythms in hypophysectomized rats with suprachiasmatic nucleus lesions.

Several pituitary hormones are important in the regulation of metabolism, and their release appears to be controlled by a circadian clock. Consequently, they may be involved in feeding-entrained circadian rhythms. Hypophysectomized (Hypox) and sham-operated male Sprague-Dawley rats had access to food for 4 h each day. Food-anticipatory activity (FAA) and core body temperature (T(b)) were monitored. Both groups entrained to the daily meal with an increase in activity in the 4 h preceding meal access and quickly reentrained after an 8-h phase advance of food access. FAA was not disrupted in either group after suprachiasmatic lesions were added. Core T(b) increased in the sham-operated subjects before mealtime, but Hypox rats failed to show this effect. Rather, T(b) declined during anticipation and throughout the food access period. Respiratory quotient (RQ), an indirect measure of metabolic rate, was measured for 24 h in some subjects. Sham-operated rats showed a dramatic downturn in RQ 1 h before mealtime, whereas Hypox rats showed a steadily decreasing RQ throughout the day. The results show that the pituitary hormones are not necessary for FAA and that in Hypox rats the anticipatory rise in T(b) and changes in RQ become dissociated from anticipatory behavior, indicating that these functions are separate outputs of the food-entrainable circadian oscillator.

Growth hormone suppression of insulin-like growth factor binding protein-1 promoter activity

Hepatic transcription of insulin-like growth factor binding protein-1 (IGFBP-1) is rapidly downregulated by growth hormone (GH) which is also known to induce expression of c-fos and c-jun. Co-expression of c-fos or c-jun in rat hepatocytes, individually or together, suppresses IGFBP-1 promoter activity by ≈60%. When hepatic nuclear extracts from sham-operated, hypophysectomized (hypox) and GH-treated hypox rats were analyzed by DNase-1 footprinting, differences in the protection pattern were identified in three regions of the IGFBP-1 promoter. F1 corresponding to −660 to −640 bp showed acute changes in response to GH administration. In additional regions, F2 and F3, representing −758 to −748 bp and −477 to −447 bp, respectively, differences were apparent between nuclear extracts from the hypox and sham-operated rats. When F1 and F2 were removed by deletion of the region from −824 to−557 bp, the GH response was lost but suppression by co-expression of c-fos and c-jun was preserved. A putative AP-1 binding site was present in the F3 footprint region, however removal of F3 did not affect the GH responsiveness. These data indicate that several distinct sequences, other than the putative AP-1 site are involved in mediating the GH effects on IGFBP-1 transcription.

Augmentation of hormonal activities with antibodies from cattle immunized with a combination of synthetic and recombinant growth Hormone Peptide

Antibodies generated against a synthetic growth hormone (GH) peptide in a number of animal species were shown to enhance the efficacy of GH. However, the ability to produce the effective antibodies diminished over time and repeated boosters failed to overcome the hurdle. Therefore, this study was designed to address the issue on the failed antibody responses by employing different GH peptide antigen preparations in cattle. Holstein steers were repeatedly immunized with a synthetic peptide corresponding to an amino acid sequence 54‐95 of porcine GH (pGH). The peptide was conjugated to ovalbumin (OVA) as a carrier. Animals initially responded to the antigen well and elicited antibodies specific to the peptide. However, the 4th challenge with the same OVA‐peptide antigen rendered animals unresponsive, resulting in a decline in antibody production. This unresponsiveness was overcome by switching the antigen at the 5th immunization from OVA‐peptide to a recombinant peptide preparation which was composed of maltose binding protein (MBP) as a carrier. Antibodies generated in cattle after the 5th immunization recognized not only the pGH(54‐95) peptide, but also bovine GH (bGH) and pGH. These antibodies were not immunoreactive with an unrelated control peptide. Hypophysectomized (hypox) rats were used for functional analysis and bGH was active in promoting the growth of these GH‐deficient rats. The growth‐promoting effect of bGH was significantly enhanced by mixing it with bovine anti‐peptide antibodies prior to administration. Therefore, the present findings suggest that peptide 54‐95 induces cattle to elicit antibodies capable of not only recognizing bGH but also augmenting the somatogenic effectiveness of bGH in hypox rats.