Lentivirus-mediated α-melanocyte-stimulating hormone overexpression in the hypothalamus decreases diet induced obesity in mice.

Melanocyte stimulating hormone (MSH) derived from the pro-hormone pro-opiomelanocortin (POMC) has potent effects on metabolism and feeding that lead to reduced body weight in the long-term. To determine the individual roles of POMC derived peptides and their sites of action, we created a method for the delivery of single MSH peptides using lentiviral vectors and studied the long-term anti-obesity effects of hypothalamic α-MSH overexpression in mice. An α-MSH lentivirus (LVi-α-MSH-EGFP) vector carrying the N'-terminal part of POMC and the α-MSH sequence was generated and shown to produce bioactive peptide in an in vitro melanin synthesis assay. Stereotaxis was used to deliver the LVi-α-MSH-EGFP or control LVi-EGFP vector to the arcuate nucleus (ARC) of the hypothalamus of male C57Bl/6N mice fed on a high-fat diet. The effects of 6-week-treatment on body weight, food intake, glucose tolerance and organ weights were determined. Additionally, a 14-day pairfeeding study was conducted to assess whether the weight decreasing effect of the LVi-α-MSH-EGFP treatment is dependent on decreased food intake. The 6-week LVi-α-MSH-EGFP treatment reduced weight gain (8.4 ± 0.4 g versus 12.3 ± 0.6 g; P < 0.05), which was statistically significant starting from 1 week after the injections. The weight of mesenteric fat was decreased and glucose tolerance was improved compared to LVi-EGFP treated mice. Food intake was decreased during the first week in the LVi-α-MSH-EGFP treated mice but subsequently increased to the level of LVi-EGFP treated mice. The LVi-EGFP injected control mice gained more weight even when pairfed to the level of food intake by LVi-α-MSH-EGFP treated mice. We demonstrate that gene transfer of α-MSH, a single peptide product of POMC, into the ARC of the hypothalamus, reduces obesity and improves glucose tolerance, and that factors other than decreased food intake also influence the weight decreasing effects of α-MSH overexpression in the ARC. Furthermore, viral MSH vectors delivered stereotaxically provide a novel tool for further exploration of chronic site-specific effects of POMC peptides.

Randomized pilot study of cabergoline, a dopamine receptor agonist: effects on body weight and glucose tolerance in obese adults.

AIM: Dopaminergic hypofunction and hyperprolactinaemia have been implicated in the pathogenesis of obesity and glucose intolerance. The aim of this pilot study was to determine the efficacy of cabergoline, a dopamine receptor agonist, on body weight and glucose tolerance in obese non-diabetic persons with normal plasma prolactin levels. METHODS: This 16-week double blind, placebo-controlled pilot study randomized non-diabetic obese adults (body mass index 30-42 kg/m(2) ) to placebo or cabergoline (0.25 mg twice weekly for 4 weeks followed by 0.5 mg twice weekly for the next 12 weeks). Of 40 subjects enrolled, 29 completed 16 weeks: 16 randomized to placebo and 13 to cabergoline. All subjects were counselled on a 500 kcal/day calorie deficit diet. A 75-g oral glucose tolerance test was performed at baseline and at 16 weeks. RESULTS: As expected, prolactin levels decreased after cabergoline (p < 0.001). Weight loss was similar after placebo compared with cabergoline treatment: 1.0 vs. 1.2% body weight, respectively. Fasting glucose levels did not differ between groups after treatment, however, 90-min postprandial glucose and insulin decreased in the cabergoline group only (p = 0.029). HOMA-IR (homeostasis model of assessment) increased by 40% after placebo and 1.5% after cabergoline treatment. CONCLUSIONS: This pilot study suggests that cabergoline therapy may improve glucose tolerance independent of weight loss, however, a larger, longer term study of dopamine receptor agonist therapy in obese individuals is warranted to confirm this finding.

Effects of agouti-related protein on metabolism and hypothalamic neuropeptide gene expression.

The hypothalamic melanocortin system regulates feeding in part through interaction of the appetite stimulating peptide, agouti-related protein (AGRP), and the anorectic peptide, alpha-melanocyte stimulating hormone, a peptide derived from the pro-opiomelanocortin (POMC) polyprotein. Central administration of AGRP induces hyperphagia and increased gain in body weight in rodents, but may also exert metabolic effects even when hyperphagia is prevented. In the present studies, the effects of AGRP on hypothalamic neuropeptide gene expression and metabolism were examined in the rat. Central administration of AGRP for 3- and 7-day periods resulted in hyperphagia, increased body weight and increased plasma leptin and insulin concentrations compared to saline-injected controls. Hypothalamic concentrations of Pomc mRNA were also increased by 27% and 44% (in 3- and 7-day experiments, respectively). The hypothalamic concentration of Agrp mRNA was unchanged after 3 days, but was significantly decreased by 33% after 7 days of AGRP infusion. To determine if these changes were dependent upon AGRP-induced hyperphagia, pair-fed rats with restricted food intake receiving central administration of AGRP were also studied. In the absence of hyperphagia, intracerebralventricular administration of AGRP caused significant increases in plasma leptin and insulin concentrations (two-fold and 1.5-fold, respectively) and fat pad mass. A significant increase in hypothalamic Pomc mRNA concentrations was not detected in pair-fed rats. In contrast, Agrp mRNA concentrations remained suppressed by 45% in the pair-fed group after 7 days of AGRP infusion despite equal body weight compared to saline controls. The ratio of hypothalamic Pomc to Agrp mRNA was elevated two-fold in ad libitum and pair-fed AGRP-injected rats, which is consistent with increased stimulation of central melanocortin signalling pathways. Thus, central administration of AGRP exerts changes in hypothalamic neuropeptide gene expression and metabolic effects that are independent of the effects on food intake and body weight.

Leptin regulation of Agrp and Npy mRNA in the rat hypothalamus.

Agouti-related protein (AGRP) is synthesized in the same neurones in the arcuate nucleus as neuropeptide Y (NPY), another potent orexigenic peptide. AGRP antagonizes the action of alpha-melanocyte stimulating hormone, a derivative of pro-opiomelanocortin (POMC) at the hypothalamic MC4 receptor to increase food intake. Although leptin has been shown to regulate Agrp/Npy and Pomc-expressing neurones, there are differences with respect to Agrp regulation in leptin receptor-deficient mice and rats. Unlike the obese leptin receptor-deficient db/db mouse, which exhibits upregulation of Agrp mRNA expression in the medial basal hypothalamus (MBH) compared to lean controls, the obese leptin receptor-deficient (faf; Koletsky) rat does not exhibit upregulation of Agrp expression. To determine whether this represents a general difference between leptin receptor-deficient mice and rats, neuropeptide gene expression was analysed in the MBH of lean and obese rats segregating for a different leptin receptor mutation, Leprfa (Zucker). Fasting in lean rats (+/fa) for 72 h significantly increased Agrp and Npy mRNA expression, and decreased Pomc mRNA expression as detected by a sensitive solution hybridization/S1 nuclease protection assay. Npy mRNA levels were significantly increased in fed obese fa/fa compared to lean rats, and further increased in the obese animals after fasting. In contrast, Agrp mRNA levels did not differ between fed lean and fed obese rats, and fasting did not significantly change Agrp levels in obese rats. To determine whether the change in Agrp expression that occurs with food deprivation in lean rats could be prevented by leptin replacement, Sprague-Dawley rats were fasted and infused via subcutaneous osmotic micropumps for 48 h with either saline or recombinant mouse leptin. Fasting significantly increased Agrp and Npy, and decreased Pomc mRNA levels. Leptin infusion almost completely reversed these changes such that there was no significant difference between the levels in the fasted rats and those that were fed ad libitum. Thus, in fasted lean rats, Agrp and Npy are upregulated in parallel when leptin levels fall and are downregulated by leptin infusion. By contrast, the absence of a functional leptin receptor results in the upregulation of Npy but not Agrp mRNA.

Method for studying development of colonization and infection of dialysis catheters.

Microbial colonization and infection of dialysis catheters is the major cause of catheter failure. The present study aimed to develop a method to permit the study of microbial biofilm formation and antibiotic prophylaxis and therapy. Standard silicon rubber and silver-impregnated catheters were sectioned into 1 mm slices and placed into 1-cm x 2-cm culture slide chambers. Fresh clinical isolates were obtained from infected patients and suspended in concentrations of 10(3), 10(6), and 10(9) colony forming units (CFU) per milliliter in a variety of liquid suspending culture media, which included serum protein constituents. Antibiotics could be added to the suspending fluid to determine prophylactic activity, or at any time thereafter to determine therapeutic activity. The catheter sections were incubated with the microbial challenge for 6 hours, 12 hours, 24 hours, and 48 hours and then washed in flowing distilled water to remove unattached biofilm. They were then stained with acridine orange, which fluoresces microbial DNA, and were examined by confocal scanning laser microscopy. Biofilm formation, representing colonization and infection, were quantified by comparing the fluorescent pixel analysis of the uninoculated control with the challenged catheter. The method was reproducible and permitted quantitative analysis. Standard silicon rubber catheters demonstrated greater biofilm formation than silver-impregnated catheters. The method examines the factors involved in microbial colonization and infection, and in antibiotic prophylaxis and therapy.

Differential effects of estradiol on the adrenocorticotropin responses to interleukin-6 and interleukin-1 in the monkey.

Endotoxin and the inflammatory cytokines interleukin (IL)-1 and IL-6 are potent activators of the hypothalamic-pituitary-adrenal (HPA) axis. Although estradiol (E(2)) has been shown to enhance the HPA response to certain types of stress, previous studies in the rodent have shown that HPA responses to endotoxin and to IL-1 were enhanced by ovariectomy and attenuated by E(2). The mechanisms underlying these observations are unclear, but there is evidence that E(2) may have direct inhibitory effects on IL-6 synthesis and release. Because endotoxin and IL-1 both stimulate IL-6, it is possible that the E(2)-induced suppression of the HPA response to endotoxin and IL-1 results from decreased IL-6 release. We have therefore examined the ACTH response to IL-6 and IL-1beta in six ovariectomized rhesus monkeys with and without 3 weeks of E(2) replacement. In the first study, plasma ACTH levels peaked at 60 min after iv injection of 6 microg recombinant human IL-6. Both the ACTH response, over time, and the area under the ACTH response curve were significantly higher in the E(2)-treated animals (P < 0.05). The peak ACTH level was 66 +/- 16 pg/ml without E(2) vs. 161 +/- 69 pg/ml with E(2). In the second study, iv infusion of recombinant human IL-1beta (400 ng) produced plasma IL-6 levels comparable with those seen after IL-6 injection in the first study. In the IL-1 study, however, there was a significant attenuation of the ACTH response, over time, in the E(2)-treated animals (P < 0.001); the peak ACTH level was 83 +/- 34 pg/ml vs. 13 +/- 4.4 pg/ml after E(2). The IL-6 response was similarly attenuated (P < 0.001); the peak IL-6 level was 614 +/- 168 pg/ml vs. 277 +/- 53 pg/ml after E(2) treatment. Our results demonstrate that physiological levels of E(2) enhance the ACTH response to IL-6 but attenuate the ACTH response to IL-1. The attenuated ACTH response to IL-1 was accompanied by a blunted IL-6 response. Our results suggest that the blunted HPA response to IL-1 can be explained, at least in part, by E(2)-induced alterations in IL-6 release. It remains to be determined whether E(2) affects other inflammatory mediators that also participate in this process.

Estrogen modulates the hypothalamic-pituitary-adrenal and inflammatory cytokine responses to endotoxin in women.

Endotoxin stimulates the release of the inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-alpha, which are potent activators of the hypothalamic-pituitary-adrenal (HPA) axis. Recent studies in the rodent and in the primate have shown that the HPA responses to endotoxin and IL-1 were enhanced by gonadectomy and attenuated by estradiol (E2) replacement. In addition, there is some evidence, in the rodent, that estrogen modulates inflammatory cytokine responses to endotoxin. To determine whether estrogen has similar effects in humans, we studied the cytokine and HPA responses to a low dose of endotoxin (2--3 ng/kg) in six postmenopausal women with and without transdermal E2 (0.1 mg) replacement. Mean E2 levels were 7.3 +/- 0.8 pg/mL in the unreplaced subjects and increased to 102 +/- 13 pg/mL after estrogen replacement. Blood was sampled every 20 min for 1--2 h before, and for 7 h after, iv endotoxin administration. Endotoxin stimulated ACTH, cortisol, and cytokine release in women with and without E2 replacement. E2 significantly attenuated the release of ACTH (P < 0.0001) and of cortisol (P = 0.02). Mean ACTH levels peaked at 190 +/- 91 pg/mL in the E2-replaced group vs. 411 +/- 144 pg/mL in the unreplaced women, whereas the corresponding mean cortisol levels peaked at 27 +/- 2.9 microg/dL with E2 vs. 31 +/- 3.2 microg/dL without E2. Estrogen also attenuated the endotoxin-induced release of IL-6 (P = 0.02), IL-1 receptor antagonist (P = 0.003), and TNF-alpha (P = 0.04). Mean cytokine levels with and without E2 replacement peaked at 341 +/- 94 pg/mL vs. 936 +/- 620 pg/mL for IL-6, 82 +/- 14 ng/mL vs. 133 +/- 24 ng/mL for IL-1 receptor antagonist, and 77 +/- 46 pg/mL vs. 214 +/- 87 pg/mL for TNF-alpha, respectively. We conclude that inflammatory cytokine and HPA responses to a low dose of endotoxin are attenuated in postmenopausal women receiving E2 replacement. These data show, for the first time in the human, that a physiological dose of estrogen can restrain cytokine and neuroendocrine responses to an inflammatory challenge.

Pituitary lymphoma presenting as fever of unknown origin.

An 86-yr-old woman presented with fever of unknown origin. When laboratory evaluation revealed partial hypopituitarism, a magnetic resonance imaging scan of the head was performed and revealed a sellar mass consistent with a pituitary adenoma. Only after other possible etiologies for fever were excluded did she undergo transsphenoidal resection of the sellar mass, which proved to be a B-cell lymphoma. Primary central nervous system lymphoma of the pituitary region is a rare cause of a sellar mass, and this is the first reported case of pituitary lymphoma whose presenting manifestation was fever of unknown origin. Several disease processes can manifest themselves as fever and a sellar mass, including lymphomas. In our case, only surgical biopsy could make a diagnosis and distinguish this process from the more common pituitary adenoma.

Testosterone suppresses the response of the hypothalamic-pituitary-adrenal axis to interleukin-6.

OBJECTIVES: Endotoxin and the inflammatory cytokines interleukin (IL)-1 and IL-6 are potent activators of the hypothalamic-pituitary-adrenal (HPA) axis. Previous studies in the rodent and in the primate have shown that the responses of the HPA axis to endotoxin and to IL-1 were enhanced by gonadectomy and attenuated by testosterone or estradiol replacement. The mechanisms underlying these observations are unclear, but there is evidence that gonadal steroids have direct inhibitory effects on IL-6 synthesis and release. Since endotoxin and IL-1 both stimulate IL-6, the question arises as to whether the sex-steroid-induced suppression of the HPA response to endotoxin and IL-1 results solely from decreased IL-6 release, or whether other mediators are involved. METHODS: We have therefore examined the ACTH and corticosterone responses to IL-6 in intact and castrated male rats with and without testosterone replacement. Animals were castrated 2 weeks prior to study; testosterone was replaced by subcutaneous Silastic capsules. Four days prior to study, an indwelling right atrial catheter was implanted. Blood samples for ACTH and corticosterone radioimmunoassays were collected through the catheter 0, 20, 40, 60, 120 and 180 min after intravenous injection of recombinant human IL-6 (500 ng). RESULTS: IL-6 stimulated ACTH and corticosterone release in all groups, with peak stimulation occurring within the first hour. The release of both ACTH and corticosterone was significantly attenuated in the intact (n = 9) and testosterone-replaced (n = 5) animals compared to the castrated animals without replacement (n = 7). Peak ACTH levels were 340 +/- 58 and 133 +/- 41 pg/ml in the intact and testosterone-replaced animals versus 678 +/- 170 pg/ml in the castrated animals (p < 0.02). Peak corticosterone levels were 29 +/- 4.7 and 30 +/- 4.2 microg/dl in the intact and testosterone-replaced animals versus 47 +/- 5.8 microg/dl in the castrated animals (p < 0.05). CONCLUSIONS: We conclude that testosterone attenuates the response of the HPA axis to IL-6 in the rat. This would indicate that other mechanisms, in addition to the inhibition of IL-6 release, are responsible for restraining the HPA response to inflammatory stimuli in the presence of gonadal steroids.

Outcome of radiotherapy for acromegaly using normalization of insulin-like growth factor I to define cure.

Radiation therapy (RT) has traditionally been considered a useful additional therapy for patients with acromegaly not achieving biochemical remission after surgery. However, recent evidence has suggested that RT is not curative in most patients with acromegaly when normalization of the serum insulin-like growth factor I (IGF-I) level is used to define remission. Therefore, we evaluated the success of RT based on IGF-I level in the 47 patients who received RT as part of their treatment from the cohort of 161 patients with acromegaly seen by us between 1981 and 1999. Four patients in whom no post-RT IGF-I level was available were excluded from the analysis. Of the remaining 43 patients, 32 patients received external beam RT, 6 received fractionated stereotactic radiosurgery, 4 received gamma-knife RT, and 1 received proton beam RT. The most recent IGF-I levels in these 43 patients, obtained a mean of 5.2 yr post-RT (range, 0.8-13.2 yr), were compared to age-adjusted normal ranges. IGF-I levels were normal in 17 patients (39.5%) without the addition of medical therapy. The percentage of patients with a normal IGF-I level generally increased with time post-RT; 27% of patients less than 6 yr post-RT, but 69.2% of patients 6 yr or more post-RT had normal IGF-I levels. Using the more traditional criterion for cure, a random GH measurement, 74% of patients had a GH level below 5 ng/mL, and 44% had a GH level below 2.5 ng/mL and would have been considered in remission based on these criteria. We conclude that with time RT remains a useful adjunctive treatment for many patients with acromegaly. RT should be considered along with appropriate medical therapy in selected patients who do not achieve normalization of IGF-I level after surgery or for those resistant to medical therapy.

Effects of leptin receptor mutation on Agrp gene expression in fed and fasted lean and obese (LA/N-faf) rats.

Agouti-related protein provides an orexigenic signal, probably through interaction with central melanocortin receptors. Expression of Agrp is markedly increased in the hypothalamus of mice deficient in leptin (Lep(ob)/Lep(ob)) or its receptor (Lepr(db)/Lepr(db)), suggesting that leptin mediates signals suppressing Agouti-related protein production. The regulation of Agrp expression in the rat hypothalamus has not been reported. We, therefore, analyzed the expression of Agrp in the medial basal hypothalamus of lean (+/+, +/fa(f)) and obese leptin receptor-deficient (fa(f)/fa(f)) LA/N rats. Using a sensitive solution hybridization/S1 nuclease protection assay, we found no significant difference in Agrp messenger RNA (mRNA) levels (pg/microg total RNA +/- SEM) in obese rats (n = 5), compared with lean controls (n = 5): 0.46 +/- 0.06 vs. 0.47 +/- 0.06 (P = 0.9). Similarly, no difference in Agrp expression was found using in situ hybridization or semiquantitative RT-PCR. In contrast to Agrp, Pomc mRNA levels were significantly suppressed in the obese, compared with the lean, rats (P = 0.001). Thus, the ratio of Pomc to Agrp mRNA is decreased in the obese rats and may be an important modulator of food intake. To assess the physiological regulation of Agrp in rats, we examined the effect of food deprivation in lean Sprague Dawley (SD) rats. There was a 273% increase in medial basal hypothalamus Agrp mRNA in SD rats fasted for 48 h (n = 8), compared with rats fed ad libitum (n = 8): 0.82 +/- 0.23 vs. 0.30 +/- 0.08 (P = 0.0001). Lean LA/N rats (n = 7) fasted for 48 h also showed a 231% increase in Agrp expression, compared with fed lean controls (n = 8): 0.74 +/- 0.11 vs. 0.32 +/- 0.03 (P = 0.002), whereas Pomc expression was decreased by 32% in fasted animals from the same experiment (0.34 +/- 0.05 vs. 0.50 +/- 0.07; P = 0.03). There were no significant differences in Agrp or Pomc mRNA levels between fasted and fed obese LA/N-fa(f) rats. These results suggest that, in the rat, the Agrp response to fasting may involve leptin-mediated phenomena, but factors in addition to leptin must also be involved in the regulation of Agrp gene expression.

Cyclooxygenase-2 expression is abundant in alveolar type II cells in lung cancer-sensitive mouse strains and in premalignant lesions.

Overexpression of cyclooxygenase-2 (COX-2) is seen in a high percentage of human colon tumors, lung adenocarcinomas and other cancers. Inhibition of this enzyme represses human colon tumorigenesis and decreases lung tumor multiplicity in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-exposed A/J mice. The purpose of this investigation was to characterize the expression of cyclooxygenase-2 (COX-2) during tumor progression in the A/J mouse lung and to compare the results with expression in other cancer-susceptible and several cancer-resistant mouse strains. Analysis of normal A/J mouse lung showed that type II alveolar epithelial cells express high levels of COX-2 protein and mRNA, indicating that COX-2 is present constitutively in this tumor progenitor cell prior to any carcinogen exposure. Examination of lung-cancer-resistant (C3H/HeJ, C57BL/6J, DBA/2J) and other lung-cancer-susceptible (A/WySnJ, SWR/J) strains showed similar levels of COX-2 mRNA expression in the three susceptible strains and lower levels of expression in two of the resistant strains, indicating a possible correlation between COX-2 expression in type II cells and lung cancer susceptibility. COX-2 protein expression was observed in A/J lung tumors at all stages of development. Variation and occasional absence of protein expression were also observed in A/J lung tumors, particularly in adenomas and adenocarcinomas, suggesting that COX-2 is not obligatory for maintenance of the malignant phenotype. In support of this conclusion, treatment of xenografted cell lines derived from malignant murine pulmonary tumors with COX-2 inhibitors produced only a slight repression of growth. However, the frequent expression of COX-2 in early lesions in the A/J mouse lung combined with the known reduction in tumor number in animals treated with COX-2 inhibitors prior to carcinogen exposure indicate that COX-2 could be a promising target for lung cancer chemoprevention. In addition, high levels of COX-2 expression in the normal tumor-progenitor cells of lung-cancer-sensitive mice indicate that COX-2 may play a role in lung cancer susceptibility.

Stimulatory effects of stress on gonadotropin secretion in estrogen-treated women.

Although stress is known to inhibit the hypothalamic-pituitary-gonadal axis, recent studies in the monkey show that, under certain conditions, in the presence of estrogen, stress may actually stimulate LH release. We investigated the effects of a mild inflammatory stress (2.0-3.0 ng/kg endotoxin) on LH release in five postmenopausal women with and without transdermal estradiol (E2, 0.1 mg) replacement. In another five E2-treated women, LH release was studied when the adrenal was stimulated directly by a 3-h ACTH infusion (Cortrosyn, 50 microg/h). Mean E2 levels were less than 12 pg/mL in the unreplaced subjects and were 86 +/- 10 pg/mL and 102 +/- 18 pg/mL in the two groups of E2-replaced subjects. Blood was sampled every 15-20 min for 2 h before and for 7 h after endotoxin or ACTH injection. Mean cortisol and progesterone levels increased in all three groups over time (P < 0.001). In the women without E2 replacement, basal LH was 26.8 +/- 5.3 mIU/mL and did not change significantly, over time, after endotoxin (P = 0.58). In the same women on E2, however, a significant increase in LH occurred after endotoxin (P = 0.02), from a mean hourly baseline of 15.3 +/- 5.4 mIU/mL to a peak of 50.0 +/- 25.2 mIU/mL. During the ACTH infusion, there was a significant stimulation of LH release in the E2-replaced subjects (P < 0.001), from a mean hourly baseline of 13.3 +/- 3.0 mIU/mL to a peak of 44.1 +/- 11.7 mIU/mL. In both groups, this increase occurred 2-4 h after the initial rise in progesterone and persisted to the end. We conclude that, in the presence of sufficient estrogen, activation of the hypothalamic-pituitary-adrenal axis leads to a stimulation of LH release. This is likely related to a rise in adrenal progesterone and its known stimulatory effect on LH release in the presence of E2. These studies provide a potential mechanism in the human by which an acute stress during the follicular phase of the menstrual cycle might lead to a premature LH surge and thereby interfere with follicular maturation and ovulation.

Clinical review 110: Diagnosis and treatment of pituitary tumors.

We are fortunate to have multiple safe and effective therapeutic options available for the treatment of pituitary tumors. These options include medical therapy, transsphenoidal surgery and radiotherapy. The treatment of choice depends on the type of pituitary tumor. The majority, of PRL-secreting tumors can be effectively treated with dopamine agonists. Transsphenoidal surgery is also an effective option for patients who are resistant to or intolerant of these drugs. Transsphenoidal surgery remains the treatment of choice for the majority of patients with GH, ACTH, and TSH-secreting tumors and for large nonsecreting tumors. Medical therapy with somatostatin analogs and/or dopamine agonists should be undertaken in patients with persistent elevations of GH and IGF-I levels; radiotherapy should be considered for patients with significant residual tumor in whom medical therapy is unsuccessful. Radiotherapy is also indicated for ACTH-secreting tumors not cured by surgery; medical therapy with ketoconazole and other adrenal enzyme inhibitors can be used as adjunctive therapy to lower cortisol levels. Postoperative radiotherapy for nonsecreting tumors is also an option if there is considerable residual tumor or evidence of tumor growth on follow-up MRI. Evaluation and treatment of hypopituitarism is an important part of the management of all patients with pituitary tumors. Patients also should be monitored for the development of new deficits, particularly after radiotherapy. The development of new medical therapies, such as GH antagonists, as well as refinements of surgical, radiotherapy, and imaging techniques should continue to improve our management of pituitary tumors.

Vitamin A-sensitive tissues in transgenic mice expressing high levels of human cellular retinol-binding protein type I are not altered phenotypically.

The suggested function of cellular retinol-binding protein type I [CRBP(I)] is to carry retinol to esterifying or oxidizing enzymes. The retinyl esters are used in storage or transport, whereas oxidized forms such as all-trans or 9-cis retinoic acid are metabolites used in the mechanism of action of vitamin A. Thus, high expression of human CRBP(I) [hCRBP(I)] in transgenic mice might be expected to increase the production of retinoic acid in tissues, thereby inducing a phenotype resembling vitamin A toxicity. Alternatively, a vitamin A-deficient phenotype could also be envisioned as a result of an increased accumulation of vitamin A in storage cells induced by a high hCRBP(I) level. Signs of vitamin A toxicity or deficiency were therefore examined in tissues from transgenic mice with ectopic expression of hCRBP(I). Testis and intestine, the tissues with the highest expression of the transgene, showed normal gross morphology. Similarly, no abnormalities were observed in other tissues known to be sensitive to vitamin A status such as cornea and retina, and the epithelia in the cervix, trachea and skin. Furthermore, hematologic variables known to be influenced by vitamin A status such as the hemoglobin concentration, hematocrits and the number of red blood cells were within normal ranges in the transgenic mice. In conclusion, these transgenic mice have normal function of vitamin A despite high expression of hCRBP(I) in several tissues.

Intracerebroventricular injection of interleukin-1 stimulates the release of high levels of interleukin-6 and interleukin-1 receptor antagonist into peripheral blood in the primate.

Previous studies in the rodent have shown that the cytokine IL-1 can act within the brain to influence peripheral IL-6 secretion. In order to determine if such an interaction occurs in the primate, we have compared the effects of intracerebroventricular vs. intravenous injection of IL-1beta on the release of IL-6 into the peripheral circulation of the monkey. The effects of i.c.v. IL-1beta on the release of the IL-1 receptor antagonist (IL-1ra) were studied in parallel. For comparison, we have also measured the release of both IL-6 and IL-1ra into lumbar CSF after i.c.v. IL-1beta injection. Ten ovariectomized rhesus monkeys with indwelling lateral ventricular and peripheral venous cannulae were studied. Human rIL-1beta (400 ng) was infused either i.c.v. or i.v. over 30 min and blood samples were collected for IL-6 and IL-1ra measurement by monoclonal human ELISAs. Although both i.c.v. and i.v. IL-1beta stimulated IL-6 and IL-1ra release into peripheral blood, the stimulation was much more profound after i.c.v. injection (p < 0.001). Peak IL-6 levels were 2010 +/- 590 pg/ml after i.c.v. IL-1beta compared to 243 +/- 60 pg/ml after i.v. IL-1beta. Peak IL-1ra levels were 61,310 +/- 16,190 pg/ml after i.c.v. IL-1beta compared to 18,175 +/- 4270 pg/ml after i.v. IL-1beta. There was no significant effect of an i.c.v. saline infusion on peripheral IL-6 or IL-1ra levels. In four animals, lumbar CSF was collected 7 h after i.c.v. IL-1beta injection. The mean concentration of IL-6 in CSF was 103, 570 +/- 13,780 pg/ml after i.c.v. IL-1beta vs. 224 +/- 190 pg/ml after i.c.v. saline injection; IL-1ra was 47,460 +/- 6290 pg/ml vs. 1040 +/- 550 pg/ml. As expected, both i.c.v. and i.v. IL-beta stimulated ACTH and cortisol release; the stimulation was significantly greater after i.c.v. compared to i.v. administration (p < 0.001). Thus, in the monkey, i.c.v. injection of IL-1beta stimulates the release of large amounts of IL-6 and IL-1ra into the CSF and the peripheral circulation. Both IL-6 and IL-1ra were released into the peripheral circulation to a much greater extent after i.c.v. compared to i.v. IL-1beta infusion. These studies provide further support in the primate for a mechanism by which inflammation within the brain could induce a variety of systemic responses.

Endogenous alpha-MSH modulates the hypothalamic-pituitary-adrenal response to the cytokine interleukin-1beta.

We and others have previously shown that exogenous alpha-MSH antagonizes the stimulatory effects of the cytokine interleukin (IL)-1 on the hypothalamic-pituitary-adrenal (HPA) axis. It is currently unknown, however, if endogenous alpha-MSH plays a physiological role in regulating the HPA response to IL-1. We have therefore examined the HPA response to IL-1beta in rats pretreated with an affinity purified alpha-MSH antiserum (AS) infused intracerebroventricularly to neutralize endogenous alpha-MSH within the brain. alpha-MSH AS or a similarly purified fraction of normal rabbit serum (NRS) was injected intracerebroventricularly at 16 h and at 1 h prior to the i.c.v. injection of IL-1beta (2 ng or 20 ng) and blood samples were collected through an indwelling atrial catheter. After 2 ng IL-1beta, the adrenocorticotropic hormone (ACTH) response was significantly greater in the alpha-MSH AS treated rats (n = 7) compared to the NRS treated rats (n = 7) (P <0.01); the mean ACTH level rose to a peak of 594+208 pg/ml in the alpha-MSH AS treated rats vs 274+/-122 pg/ml in the NRS treated rats. The area under the ACTH response curve in the alpha-MSH AS treated animals was 181% of that in the NRS treated animals (P<0.05). A significant effect of alpha-MSH AS on the corticosterone response to i.c.v. IL-1beta was also noted during the first 3 h of the study (P<0.05). The mean area under the corticosterone response curve for the first 3 h in the alpha-MSH AS treated animals was 144% of that in the NRS treated animals (P <0.05). After 20 ng IL-1beta, the ACTH response over time was again significantly greater in the alpha-MSH AS treated rats (n=8) compared to the NRS treated rats (n=9) (P<0.02); the mean ACTH level rose to a peak of 673+/-190 pg/ml after alpha-MSH AS vs 490+/-115 pg/ml after NRS. Corticosterone levels rose to a peak of 42+/-3.9 microg/dl in the alpha-MSH AS treated rats vs 37+/-4.6 microg/dl in the NRS treated rats; this difference was not significant. We conclude that the IL-1beta induced stimulation of ACTH is significantly enhanced by antagonizing the activity of alpha-MSH. These results support a physiological role for endogenous alpha-MSH in limiting the HPA response to this inflammatory cytokine.

Regulation of hypothalamic proopiomelanocortin by leptin in lean and obese rats.

The mechanisms by which leptin influences energy homeostasis are not entirely understood. Several observations indicate that proopiomelanocortin (POMC) is involved in the regulation of food intake and may be a mediator of leptin action. To further study this interaction, a sensitive solution hybridization assay was used to compare the levels of POMC mRNA in the medial basal hypothalamus (MBH) of lean (+/+, +/fa(f)) and obese leptin receptor-deficient (fa(f)/fa(f)) rats. POMC peptide products were also measured by RIA in the same animals. Cytoplasmic POMC RNA levels were significantly reduced by 53% in obese rats as compared with lean controls: 0.30 +/- 0.04 vs. 0.64 +/- 0.07 pg/microgram total RNA (p < 0.02). Significant reductions in mean concentrations of hypothalamic POMC-derived peptides from the same dissections were detected in the obese rats vs. lean controls: alpha-MSH 1.77 +/- 0.07 vs. 2.34 +/- 0.10; beta-EP 4.06 +/- 0.24 vs. 5.86 +/- 0.36; gamma(3)-MSH 5.32 +/- 0. 20 vs. 6.52 +/- 0.12 ng/mg protein (p < 0.001). To determine whether leptin stimulates POMC gene transcription, the acute effect of an intracerebroventricular (i.c.v.) injection of leptin (5 microgram) on POMC primary transcript was quantified in the MBH of lean rats after a 16-hour fast. There was a significant 167% increase in mean POMC hnRNA levels 3 h after i.c.v. leptin injection (1.15 +/- 0.22 pg/MBH; p < 0.02), but not after 1 h (0.76 +/- 0.08 pg/MBH), compared to saline controls (0.69 +/- 0.08 pg/MBH). 4 h after the injection of leptin, POMC hnRNA was still increased, but to a lesser extent (140%), as compared with control animals (p = 0.006). These studies demonstrate for the first time in the leptin receptor-deficient rat that there is an associated decrease in POMC gene expression and peptide levels in the MBH. Furthermore, the acute increase in the levels of POMC primary transcript in non-obese rats after a single i.c.v. injection of leptin supports a role for leptin in the regulation of POMC gene transcription. Taken together, these studies provide further evidence that POMC is an important mediator of the effects of leptin on food intake and energy expenditure.