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We have currently studied the changes induced by administration of a fructose-rich diet (FRD) to normal rats in the mass and the endocrine function of abdominal (omental) adipose tissue (AAT). Rats were fed ad libitum a standard commercial chow and tap water, either alone (control diet, CD) or containing fructose (10%, w/vol) (FRD). Three weeks after treatment, circulating metabolic markers and leptin release from adipocytes of AAT were measured. Plasma free fatty acids (FFAs), leptin, adiponectin, and plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in FRD than in CD rats. AAT mass was greater in FRD than in CD rats and their adipocytes were larger, they secreted more leptin and showed impaired insulin sensitivity. While leptin mRNA expression increased in AAT from FRD rats, gene expression of insulin receptor substrate, IRS1 and IRS2 was significantly reduced. Our study demonstrates that administration of a FRD significantly affects insulin sensitivity and several AAT endocrine/metabolic functions. These alterations could be part of a network of interacting abnormalities triggered by FRD-induced oxidative stress at the AAT level. In view of the impaired glucose tolerance observed in FRD rats, these alterations could play a key role in both the development of metabolic syndrome (MS) and β-cell failure. Fil: Alzamendi, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Giovambattista, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Raschia, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Endocrinología Experimental y Aplicada (i); Argentina Fil: Madrid, Viviana Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Endocrinología Experimental y Aplicada (i); Argentina Fil: Gaillard, Rolf C.. University Hospital; Estados Unidos Fil: Rebolledo, Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Endocrinología Experimental y Aplicada (i); Argentina Fil: Gagliardino, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Endocrinología Experimental y Aplicada (i); Argentina Fil: Spinedi, Eduardo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina

After bilateral superior cervical ganglionectomy (SCGx) in adult male rats, norepinephrine content of the pituitary neurointermediate lobe (NIL) decreased at 12-24 h after surgery to attain concentrations 40-60% of controls between 24 and 60 h after surgery. To assess arginine vasopressin (AVP) secretion during this time, plasma and NIL-AVP levels were measured by radioimmunoassay. In sham SCGx controls, plasma AVP increased about 2-fold within 6 h after surgery and decreased thereafter, to attain presurgical values by 60 h after surgery. In SCGx rats, a significant increase in plasma AVP concentration was observed at the 6th h after surgery, as compared to presurgical concentrations, with a decrease to values significantly lower than those of presurgical controls at 16-18 h after SCGx. As compared to sham-operated rats, significantly higher plasma AVP levels 6 h after surgery and significantly lower plasma AVP levels 16-24 h after surgery were found. NIL-AVP concentration in SCGx and sham-operated ras were significantly lower than presurgical levels at 6 h after surgery. SCGx rats had significantly higher amounts of AVP in NIL at 16-24 h after surgery. The changes in plasma and NIL-AVP levels found 6 or 16 h after SCGx or sham SCGx were unaffected by a prior pinealectomy. Two injections of the alpha 1-adrenoceptor blocker prazosin 45 and 90 min before sacrifice, alone or together with the beta-blocker propranolol, prevented the increase in plasma AVP found in SCGx rats 6 h after surgery, and the decrease in plasma AVP and the increase of NIL-AVP found 16 h after SCGx.(ABSTRACT TRUNCATED AT 250 WORDS)

In rats, neonatal treatment with monosodium L-glutamate (MSG) induces several metabolic and neuroendocrine abnormalities, which result in hyperadiposity. No data exist, however, regarding neuroendocrine, immune and metabolic responses to acute endotoxemia in the MSG-damaged rat. We studied the consequences of MSG treatment during the acute phase response of inflammatory stress. Neonatal male rats were treated with MSG or vehicle (controls, CTR) and studied at age 90 days. Pituitary, adrenal, adipo-insular axis, immune, metabolic and gonadal functions were explored before and up to 5 h after single sub-lethal i.p. injection of bacterial lipopolysaccharide (LPS; 150 μg/kg). Our results showed that, during the acute phase response of inflammatory stress in MSG rats: (1) the corticotrope-adrenal, leptin, insulin and triglyceride responses were higher than in CTR rats, (2) pro-inflammatory (TNFα) cytokine response was impaired and anti-inflammatory (IL-10) cytokine response was normal, and (3) changes in peripheral estradiol and testosterone levels after LPS varied as in CTR rats. These data indicate that metabolic and neroendocrine-immune functions are altered in MSG-damaged rats. Our study also suggests that the enhanced corticotrope-corticoadrenal activity in MSG animals could be responsible, at least in part, for the immune and metabolic derangements characterizing hypothalamic obesity. Fil: Castrogiovanni, Daniel Cayetano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Gaillard, Rolf C.. Centre Hospitalier Universitaire Vaudois; Suiza Fil: Giovambattista, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Spinedi, Eduardo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina

Background/Aim: We have reported that neonatal treatment with monosodium L-glutamate (MSG), which causes damage to the arcuate nucleus, leads to severe hyperleptinemia and reduced adrenal leptin receptor (ob-Rb) expression in adulthood. As a result, rats given MSG neonatally display corticoadrenal leptin-resistance, a defect that is overridden by normalization of corticoadrenal hyperfunction. The aim of the present study was to determine whether negative energy conditions could correct corticoadrenal cell dysfunction in rats given MSG neonatally. Methods: Normal (CTR) and MSG-treated female rats were subjected to food removal for 1-5 days, or prolonged (24-61 days) food restriction (FR). Plasma levels of several biomarkers and in vitro corticoadrenal function were evaluated following starvation or FR. Results: Fasting for 1-5 days reduced plasma leptin levels in CTR and MSG rats, compared to levels in the respective groups fed ad libitum(p < 0.05), but adrenal leptin-resistance was unchanged. With prolonged FR, isolated adrenal cells from MSG rats became sensitive to leptin, which lowered ACTH-induced glucocorticoid release. This restoration of leptin response was associated with normalization of adrenal ob-Rb gene expression. Conclusion: Dietary restriction in some leptin-resistant obese phenotypes may normalize adrenocortical function. Fil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Castrogiovanni, Daniel Cayetano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Giovambattista, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Gaillard, Rolf C.. Université de Lausanne; Suiza Fil: Spinedi, Eduardo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina

This work analyzes the 24-hour changes of hypothalamic–pituitary–adrenal (HPA) axis activity and leptin release in aged rats. Three- and 22-month-old male Wistar rats were killed at 6 time intervals during a 24- hour cycle (n= 8–10 rats/group). Aging augmented plasma ACTH while it decreased plasma and adrenal gland corticosterone levels. Plasma and adrenal corticosterone levels attained high levels during all the scotophase, concomitantly with the maxima in ACTH levels, whereas in aged rats only a brief plasma corticosterone peak at the early scotophase and no time of day variations of adrenal corticosterone were observed. Aging augmented circulating leptin, with a significant interaction “age × time” in the factorial ANOVA, i.e. only in young rats time of day changes were significant, with the lowest values of leptin at the middle of the light period and higher values at night. When plasma leptin was expressed on body weight basis, the age-related differences became not significant but the daily pattern of plasma leptin found in young rats persisted. Plasma and adrenal corticosterone levels correlated significantly with plasma ACTH only in young rats. Likewise, plasma leptin correlated with plasma corticosterone only in young rats. These changes can be attributed to a disrupting effect of aging on the homeostatic mechanisms modulating HPA activity and leptin release. Fil: Cano, Pilar. Universidad Complutense de Madrid; España Fil: Cardinali, Daniel Pedro. Universidad Complutense de Madrid. Facultad de Medicina. Departamento de Fisiología; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Spinedi, Eduardo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Esquifino, Ana I.. Universidad Complutense de Madrid; España

It is known that ghrelin and des-N-octanoyl (desacyl) ghrelin modulate food intake and adipogenesis in vivo. However, desacyl ghrelin represents the majority of ghrelin forms found in the circulation. The present study explored whether ghrelin gene-derived compounds could modulate, in vitro, adipocyte endocrine function and preadipocyte differentiation. Retroperitoneal (RP) adipocytes were cultured in the absence or presence of either ghrelin or desacyl ghrelin and in combination with either inhibitors of protein synthesis, insulin, dexamethasone (DXM), or GHSR1a antagonist. The results indicate that both ghrelin forms possess a direct leptin-releasing activity (LRA) on RP adipocytes and significantly enhanced adipocyte ob mRNA expression. These activities were related and unrelated to the activation of GHSR1a after coincubation with ghrelin and desacyl ghrelin, respectively. Moreover, desacyl ghrelin facilitated RP preadipocyte differentiation. Desacyl ghrelin enhanced cell lipid content, and PPARgamma2, and LPL mRNAs expression. The LRAs developed by different substances tested followed a rank order: ghrelindesacyl ghrelin = insulinor = DXM. Additionally, desacyl ghrelin was able to enhance medium glucose consumption by mature adipocytes in culture. These data strongly support that adipogenesis and adipocyte function are processes directly and positively modulated by ghrelin gene-derived peptides, thus further indicating that, besides their effects on food intake, ghrelin gene-derived peptides could play an important role on adiposity for maintaining homeostasis.

A link is known to exist between hyperandrogenicity and insulin resistance in mammals. We explored whether androgenization, early in reproductive life, in the female rat has any impact on later peripheral insulin sensitivity and parametrial (PM) fat function. Female, 60 day-old, rats were injected (i.m.) with 100 mul of sterile corn oil either alone (CT) or containing 2 mg of testosterone propionate (TP); rats were then used for experimentation at age 120 days. Daily food intake and body weight were recorded. Different groups of CT and TP rats were subjected to a high glucose load test or 24 h fasting for evaluation of changes in circulating levels of several metabolites and body composition. In vitro experiments were run to study the impact of androgenization on isolated PM adipocyte response to insulin. Finally, the direct effect of testosterone on insulin-induced leptin secretion by normal PM adipocytes was also evaluated. Androgenization induced a significant increase in daily food intake and body weight for the first 20 days after treatment. In vivo experiments indicate that TP rats released more (P0.05) insulin than CT animals after high glucose load in order to maintain similar circulating glucose levels, a characteristic accompanied by decreased (P0.05) overall corticoadrenal response in TP rats. Several metabolic responses to fasting were similar in both groups, although impaired adrenal response and changes in body composition were observed only in TP rats. Interestingly, cultured PM adipocytes from TP rats were less (P0.05) sensitive than CT cells to insulin-induced leptin secretion. Also, we found that 48 h exposure of normal PM adipocytes to high testosterone concentration also impaired adipocyte endocrine function. Our study strongly supports that development of insulin resistance, in the female gender, can be established after an early, even transient, hyperandrogenemia.

There are few data for hormonal levels and testis structure and function during postnatal development in rats neonatally treated with monosodium L-glutamate (MSG). In our study, newborn male pups were ip injected with MSG (4 mg/g body weight) every 2 d up to 10 d of age and investigated at prepubertal and adult ages. Plasma levels of leptin, LH, FSH, prolactin, testosterone (T), corticosterone, and free T4 (FT4) were measured. MSG rats displayed elevated circulating levels of corticosterone and hyperadiposity/ hyperleptinemia, regardless of the age examined; conversely, circulating prolactin levels were not affected. Moreover, prepubertal MSG rats revealed a significant (P < 0.05) reduction in testis weight and the number of Sertoli (SC) and Leydig cells per testis. Leptin plasma levels were severalfold higher (2.41 vs. 8.07; P < 0.05) in prepubertal MSG rats, and these animals displayed plasma LH, FSH, T, and FT4 levels significantly decreased (P < 0.05). Taken together, these data indicate that testis development, as well as SC and Leydig cell proliferation, were disturbed in prepubertal MSG rats. Adult MSG rats also displayed significantly higher leptin plasma levels (7.26 vs. 27.04; P < 0.05) and lower (P < 0.05) LH and FSH plasma levels. However, T and FT4 plasma levels were normal, and no apparent alterations were observed in testis structure of MSG rats. Only the number of SCs per testis was significantly (P < 0.05) reduced in the adult MSG rats. In conclusion, although early installed hyperadipose/hyperleptinemia phenotype was probably responsible for the reproductive axis damages in MSG animals, it remains to be investigated whether this condition is the main factor for hypothalamus-pituitary-gonadal axis dysfunction in MSG rats., Instituto Multidisciplinario de Biología Celular

Objective: To explore the effects of transient correction of enhanced corticoadrenal activity in monosodium L-glutamate (MSG)-damaged female rats on peripheral insulin sensitivity and in vitro retroperitoneal (RP) adipocyte function. Designs: A dose of 4 mg/g body weight (BW) of MSG or vehicle (CTR) was i.p. injected, once every 2 days, between days 2 and 10 of age, in female rats. Intact and 21 day-operated (sham or adrenal enucleation (AE)) rats from both (CTR and MSG) groups were used for experimentation on day 120 of age. Circulating levels of several hormones, in basal and after i.v. high-glucose load conditions, and RP adiposity morphology and function were then evaluated. Results: MSG rats developed increased adrenocortical function, hyperadiposity, hyperleptinemia, hyperinsulinemia and decreased peripheral insulin sensitivity. These characteristics were fully reversed after transient correction of corticoadrenal hyperactivity induced by AE. In addition, in vitro experimentation with isolated RP adipocytes indicated that cells from intact MSG animals displayed decreased sensitivity to insulin and dexamethasone stimulation of leptin secretion. Interestingly, adipocyte dysfunction in MSG rats was fully abrogated after AE-induced transient correction of insulinemia, leptinemia and adrenocortical activity. Importantly, the reversion of these metabolic abnormalities, induced by AE for 21 days, in MSG animals did occur, despite no significant changes in BW values. Conclusion: Our results support that the changes in adipocyte characteristics and peripheral insulin resistance, developed in this pseudo-obese female rat model, are mainly due to increased glucocorticoid production. Importantly, appropriate correction of the enhanced adrenocortical activity fully reversed these abnormal functions., Instituto Multidisciplinario de Biología Celular, Facultad de Ciencias Médicas, Facultad de Ciencias Exactas

This work analyzes the effect of social isolation of growing male rats on 24-h changes of plasma prolactin, growth hormone, ACTH and leptin, and on plasma and adrenal corticosterone concentrations. At 35 days of life, rats were either individually caged or kept in groups (6-8 animals per cage) under a 12:12 h light/dark schedule (lights on at 08:00 h). A significant arrest of body weight gain regardless of unchanged daily food intake was found in isolated rats after 2 weeks of isolation. On the 4th week, rats were killed at 6 time intervals during a 24-h cycle, beginning at 09:00 h. In isolated rats the 24-h pattern of all parameters tested became distorted, as assessed by Cosinor analysis. When analyzed as a main factor in a factorial analysis of variance, isolation decreased plasma prolactin and growth hormone, increased plasma leptin and corticosterone while decreased adrenal corticosterone. Plasma corticosterone levels correlated significantly with plasma ACTH and with adrenal corticosterone levels in group-caged rats only. These changes can be attributed to an effect of mild stress on the endogenous clock that modulates the circadian hormone release. Fil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Chacon, Fernando. Universidad Complutense de Madrid; España Fil: Cardinali, Daniel Pedro. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Cátedra de Fisiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Esquifino, Ana I.. Universidad Complutense de Madrid; España Fil: Spinedi, Eduardo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina

Neonatal administration of monosodium glutamate (MSG) to rats produces severe lesions in certain hypothalamic nuclei, with repercussions in different neuroendocrine axes, and serves as a model for their study. In addition, adipose tissue, as a target organ, is known to be directly related to several neurondocrine axes. We used 21-day-old female Sprague-Dawley rats that had received a neonatal treatment with MSG (4 mg/g body weight, i.p., from day 2 up to day 10 of age) in addition to control rats (injected with 10% NaCl solution, on a similar schedule). We performed a specific immunohistochemical study on each anterior-pituitary cell population, along with the morphometry of these cells and of the parietal and visceral adipose tissue, and measured the levels of serum leptin and triglycerides. The MSG animals evinced significant changes in volume density (VD), cell density (CD), and cell size (CS) in the corticotropes, thyrotropes, and LH gonadotropes, but not in the somatotropes, lactotropes, and FSH gonadotropes. The modification common to the three cell types was a hyperplasia, but with different results depending on cell size. Furthermore, in the MSG rats significant changes were also observed in the VD, CD, and CS of the adipose tissue, consisting of adipogenesis and decrease of adipocyte size in visceral fat, together with probable lipogenesis as judged by an increase in adipocyte size in the parietal fat. The serum levels of leptin and triglycerides appeared significantly higher in MSG animals. For the first time in this animal model, and at the level of three neuroendocrine axes, our results suggest changes that correlate hypothalamic damage, cellular pituitary alterations, and the response of the adipose tissue as a target organ for MSG insult.

Neonatal monosodium l-glutamate (MSG) treatment destroys hypothalamic arcuate nucleus neuronal bodies, thus inducing several metabolic abnormalities. As a result, rats develop a phenotype characterized by hyperleptinemia and by impaired NPY but normal prepro-orexin hypothalamic mRNAs expression. Thus, our study was designed to explore whether hypothalamic effects of orexin A on food intake and glucocorticoid production develop in the absence of full hypothalamic NPY-ergic activity. For this purpose we evaluated, in control and MSG-treated rats, the consequences of intracere-broventricular (icv) orexin A administration on food intake and changes in circulating levels of ACTH and glucocorticoid. Our results indicate that orexin A icv treatment stimulated hypothalamic-pituitary-adrenal (HPA) axis activity in both MSG-damaged and normal animals, with this response even more pronounced in neurotoxin-damaged rats. Conversely, food intake was only enhanced by icv orexin A injection in normal rats. Our study further supports that acute hypothalamic effects of orexin A on food intake and glucocorticoid production are due to independent neuronal systems. While intact arcuate nucleus activity is needed for the orexinergic effect induced by icv orexin A administration, conversely, orexin A-stimulated HPA axis function takes place even in the absence of full NPY-ergic activity.

Genetic, social, metabolic, endocrine and neural events participate in the physiopathological development of obesity. Because of the multifactorial background of obesity, up to now, it has been very difficult to fully understand the whole disease. In fact, the relationship between several signals, through very complex mechanisms, is only partially known. Obesity, from a neuroendocrine point of view, implies taking into account abnormalities in both hypothalamic and endocrine functions. Among altered functions in obesity, namely those involving the hypothalamo-pituitary-adrenal and adipo-insular axes activities and the neural circuitry controlling food intake and energy expenditure have been subjects of major research. This review attempts to update information about this disorder by, firstly, analyzing each of the already mentioned systems and, secondly, focusing on the normal function of integrated processes. Finally, the discussion of some altered mechanisms and the role played by each of them in the development/maintenance of the obesity phenotype are also revisited.

Up to now it is unclear whether there is a relationship between insulin resistance and circulating leptin levels (LEP) in women with polycystic ovary syndrome (PCOS). To assess the role of LEP in PCOS and to clarify the relationship between plasma LEP levels and insulin resistance (IR) in PCOS patients, we studied 49 women with PCOS and 14 normal premenopausal women. All subjects were evaluated by a 2 hours, 75 g, oral glucose tolerance test. Fasting plasma LEP, insulin, glucose, insulin sensitivity indexes and LEP:body mass index (BMI) were determined. Results were analyzed by ANOVA and the Pearson's correlation test when appropriate. The results indicate that: 1) no differences were found in basal plasma LEP levels (ng/ml) between normal (17.6 +/- 4.9) and PCOS (21.9 +/- 2.8) women; 2) in PCOS patients, a significant (P0.01) correlation between plasma LEP levels and BMI and insulin sensitivity indexes were found; and 3) seventeen PCOS patients were insulin resistant (IR) and showed higher basal plasma LEP levels (32.8 +/- 4.3, P0.01) and LEP:BMI (0.95 +/- 0.09, P0.05) than non insulin resistant (non IR) PCOS subjects (16.2 +/- 3.2 and 0.61 +/- 0.08, respectively). Our results suggest that PCOS seems to be associated with normoleptinemia, however, if IR are analyzed separately from non IR PCOS patients, there is a clear relationship between IR PCOS and hyperleptinemia, regardless of the BMI. The present study strongly supports bi-directional relationship between fat and carbohydrate metabolisms under a very particular physiopathological condition (PCOS).

Neonatal treatment of rats with monosodium l-glutamate, which destroys hypothalamic arcuate nucleus neuronal bodies, induces several metabolic abnormalities; as a result, rats develop a phenotype of pseudoobesity. This study was designed to explore, in the monosodium l-glutamate-treated female rat, the influence of chronic hyperleptinemia on adrenal cortex functionality. For this purpose, we evaluated in control and hypothalamic-damaged rats: (a) in vivo and in vitro adrenocortical function, (b) adrenal leptin receptor immunodistribution and mRNA expression, and (c) whether the inhibitory effect of leptin on adrenal function remains. Our results indicate that, compared to normal counterparts, pseudoobese animals displayed (1) hyperadiposity, despite being hypophagic and of lower body weight, (2) in vivo and in vitro enhanced adrenocortical response to ACTH stimulation, (3) an in vitro adrenal fasciculata-reticularis cell hyper-sensitivity to ACTH stimulus, (4) hyperplasia of their adrenal zona fasciculata cells, and (5) adrenal fasciculata-reticularis cell refractoriness to the inhibitory effect of leptin on ACTH-stimulated glucocorticoid production due, at least in part, to decreased adrenal leptin receptor expression. These data further support that increased hypothalamo-pituitary-adrenal axis function, in the adult neurotoxin-lesioned female rat, is mainly dependent on the development of both hyperplasia of adrenal zona fasciculata and adrenal gland refractoriness to leptin inhibitory effect. Our study supports that adrenal leptin resistance could be responsible, at least in part, for enhanced glucocorticoid circulating levels in this phenotype of obesity., Instituto Multidisciplinario de Biología Celular

It is known that the neonatal treatment of rats with monosodium L-glutamate (MSG) induces several metabolic abnormalities, resulting in enhanced adiposity and hyperleptinemia. Our study was designed to explore the consequences of MSG-induced chronic hyperleptinemia on adrenal sensitivity to the inhibitory effect of exogenous leptin. Neonatal male rats treated with MSG or vehicle (controls, CTR) were followed during 150 days in order to study changes observed over development in body weight, food consumption as well as in vivo hypothalamo-pituitary-adrenal (HPA) axis and adipocyte functions. During adulthood, adrenal response to adrenocorticotropin (ACTH) was evaluated both in vitro and in vivo in order to determine the adrenal sensitivity to the inhibitory effect of leptin. For this purpose, sham-operated as well as CTR and MSG rats with bilateral adrenal enucleation (AE) were used. Our results indicate that: (1) between 30 and 150 days of age, MSG animals developed hypophagia, accompanied by arrest in body weight gain, and concomitant enhanced basal levels of all HPA axis components and of leptin; (2) adrenals from of 150-day- old MSG rats displayed an in vitro adrenocortical hyperresponse to ACTH stimulation as well as an adrenal refractoriness to the physiological inhibitory effect of leptin on ACTH-stimulated glucocorticoid output, and (3) bilateral AE in adult MSG-treated rats transiently reversed the MSG-induced hyperleptinemia, restoring normal leptin levels as well as a normal adrenal sensitivity to the inhibitory effect of leptin. Our data indicate that adrenal exposure to the chronically high plasma leptin levels observed in MSG rats is involved in the loss of the inhibitory regulatory effect of leptin at the adrenal level, being therefore, at least in part, responsible for the increased total and free glucocorticoid production measured in MSG adult rats. Furthermore, this study strongly suggests that the adrenal overfunction, frequently associated with different phenotypes of obesity, could be due to an adrenal resistance to the leptin-negative regulation.

It is recognized that there exists a link between hyperandrogenicity and insulin resistance.By using the neonatally androgenized female rat we explored whether this treatment modifies peripheral insulin sensitivity and visceral fat function at adulthood.On day 5 of age, female Sprague-Dawley pups were injected, sub cutaneous, with either 50 ml of sterile corn oil alone (CT) or containing 1.25 mg of testosterone propionate (TP) and further used for experimentation on day 100 of age. CT and TP rats were killed by decapitation in non-fasting condition and blood samples were kept frozen for measurement of different metabolites. Immediately after sacrifice, freshly dissected visceral fat pads were used for isolation of adipocytes, these cells were then incubated with medium alone or containing different concentrations of insulin in order to determine leptin secreted into the medium. Additionally, in vivo metabolic responses to intravenous high glucose load were performed in, 24 hour-fasting, CT and TP rats.We found that neonatal androgenization induced adult animals displaying higher visceral adiposity mass, body weight and leptinemia than CT rats. No group differences were found in basal circulating levels of several hormones and metabolic parameters. The results of the high glucose load 90-min test indicated that TP and CT rats developed similar glycemia but this accounted because of an early significantly higher peak values of circulating insulin in TP than in CT rats, regardless of similar enhancement in circulating glucocorticoid concentrations in both groups. While high glucose load significantly increased, over the baseline, circulating leptin concentrations as early as 30 min post-glucose in CT rats, in TP animals, it significantly enhanced leptinemia only by the end of the test. Finally, results of in vitro incubations of isolated visceral adipocytes indicated that cells from androgenized rats spontaneously released more leptin than control cells, although they were less responsive than CT cells to insulin-induced leptin output.Our study strongly supports the hypothesis that development of insulin resistance seems to be dependent on early hyperandrogenicity.

Neonatal L-monosodium glutamate (MSG) administration in rats induces several neuroendocrine and metabolic disruptions. Leptin, the adipocyte product, modulates several neuroendocrine systems including the hypothalamic-pituitary-gonadal (HPG) axis in mammals. The aim of the present study was to determine whether MSG-induced chronic hyperleptinemia could play any relevant role in the hypogonadism developed by male rats when examined in adulthood. We found that 120-day-old MSG male rats displayed significant hyperleptinemia, hypogonadism, and undisturbed basic testis structure and spermatogenesis. In vitro studies in purified Leydig cells from normal (CTR) and MSG-damaged rats revealed that basal and human chorionic gonadotropin (hCG)-stimulated 17-hydroxy-progesterone (17-HO-P4), Δ4-androstenedione (Δ4A) and testosterone (T) secretions were significantly lower in MSG than in CTR cells. Exposure to murine leptin (mleptin, 10–8M) significantly inhibited hCG-elicited T secretion by CTR cells after 180 min incubation. While mleptin significantly inhibited hCG-stimulated Δ4A output and the Δ4A:17-OH-P4 ratio of secretion, conversely, it failed to modify the ratio T:Δ4A release by CTR Leydig cells. Interestingly, the effects of mleptin found on CTR Leydig cells were absent in MSG Leydig cells. Finally, endogenous hyperleptinemia was associated with a significant decrease in Leydig cell expression of Ob-Rb mRNA in MSG rats. In summary, this study demonstrates that: (1) mleptin inhibited testicular steroidogenesis in CTR rats; (2) MSG-treated rats showed lower in vitro 17-OH-P4, Δ4A and T production under basal and post-hCG stimulation conditions; (3) purified Leydig cells from MSG-treated rats displayed resistance to the inhibitory action of mleptin on T release, and (4) endogenous leptin exerts a modulatory effect on Leydig cell Ob-Rb mRNA expression. The inhibitory effect of leptin on testicular function is thus abrogated in MSG-damaged rats. The testicular leptin-resistance developed by MSG rats seems to be due to early chronic exposure of Leydig cells to high leptin circulating levels, which in turn down-regulate testicular Ob-Rb expression. It remains to be determined whether the testicular dysfunction of MSG rats can be reversed after correction of hyperleptinemia or whether it is an irreversible effect of the hypothalamic lesion.

Among neurodegenerative diseases, Alzheimer’s disease (AD) is a leading cause of death in elderly individuals. AD is characterized, among other clinical findings, by unexplained weight loss, cachexia and altered immune function. To explore whether any relationship between gender and circulating levels of several eating-controlling metabolites exist, we evaluated leptin, tumor necrosis factor (TNF)-α, triiodothyronine (T3), free (F) thyroxine (T4), TSH, PRL, insulin (INS), and cortisol in 15 AD-treated patients (age range 55–82 years): 9 postmenopausal females (without hormone replacement therapy) and 6 males. The results (mean ± SEM) indicated that circulating leptin levels were significantly (p < 0.05) higher in female AD (40.34 ± 11.1 ng/ml) than in male AD (6.07 ± 1.39 ng/ml) patients. The difference found in circulating leptin levels was noticed regardless of BMI (26.75 ± 1.77 and 24.55 ± 1.93 kg/m2, in females and males, respectively) and waist:hip ratios (0.91 ± 0.03 and 0.94 ± 0.02, in females and males, respectively). Moreover, serum TNF-α concentrations were also significantly (p < 0.02) higher in AD females (12.24 ± 1.47 pg/ml) than in AD males (6.62 ± 1.44 pg/ml), regardless of TNF-α:BMI ratios (0.50 ± 0.09 and 0.28 ± 0.08, in females and males, respectively; p > 0.05). Finally, no differences were observed between gender (in female and male AD patients, respectively) in circulating levels of T3 (151.33 ± 9.91 vs. 116 ± 17.04 ng/dl), FT4 (1.26 ± 0.08 vs. 1.24 ± 0.06 ng/dl), TSH (1.28 ± 0.16 vs. 2.46 ± 0.67 µIU/ml), PRL (10.53 ± 2.47 vs. 12.61 ± 2.37 ng/ml), INS (11.76 ± 1.95 vs. 8.59 ± 1.34 µIU/ml) and cortisol (15.71 ± 1.23 vs. 12.63 ± 1.47 µg/dl). These results indicate that our AD group of patients, with normal corticoadrenal and thyroid functions and normoprolactinemia, displayed a gender-related characteristic in the circulating levels of two very important anorectic signals, leptin and TNF-α, being both higher in female than in male AD patients, regardless of BMI. Our study suggests that increased circulating levels of both anorexigenic adipokines may contribute to the metabolic changes observed in AD females.

Malnutrition induces profound deleterious effects on several metabolic and neuroendocrine functions. In the present study, we examined the impact of maternal food restriction, during gestation and lactation, on the metabolic-neuroendocrine function of their male offspring at 21 and 60 d of age. Well-nourished (WN) and undernourished (UN) pregnant rats were used, during gestation and lactation, until pups were weaned. Twenty-one-day-old WN and UN male pups were studied in basal and postinsulin conditions. Additional groups of weaned (WN and UN) male rats were fed either ad libitum (WN-WN and UN-WN) or in a restricted fashion (WN-UN and UN-UN) until experimentation at age 60 d. Body weights of mothers and their male offspring were monitored. Basal and post-insulin plasma concentrations of several metabolic fuels were evaluated. Our results indicate that 21-d-old UN male rats exhibited (vs their WN counterparts), decreased body weights, similar basal and postinsulin glycemia, similar basal plasma adrenocorticotropic hormone (ACTH) and corticosterone levels but diminished ACTH response to insulin treatment, and basal hypoleptinemia and significant insulin-induced leptin release. Finally, at 60 d of age, long-term UN (WN-UN and UN-UN) rats showed lower plasma (basal and postinsulin) glucose, and basal triglyceride levels than their counterparts (WN-WN and UN-WN). Sixty-day-old rats submitted to either food restriction protocol also showed a reduced hypothalamo-pituitary-adrenalaxis response to insulin-induced hypoglycemia and basal hypoleptinemia, in spite of restoration of normal body weights. These results further indicate a clear metabolic-neuroendocrine dysfunction in male pups of UN mothers, with the abnormality partially present at weaning and deteriorated by adulthood, even after the recovery of normal body weight. Our study strongly supports the importance of the irreversibility of a deleterious allostatic state resulting from fetal and early postnatal undernutrition., Facultad de Ciencias Exactas, Instituto Multidisciplinario de Biología Celular

It is well recognized that the reciprocal interaction established between the immune and neuroendocrine systems is crucial for the homeostatic adaptation of individuals during septicemia. In the present study, using an in vivo rat model, we investigated the degree of participation of central and peripheral epinergic systems in the modulation of the hypothalamic-pituitary-adrenal and immune axes’ functions during endotoxemia. For this purpose, acute endotoxemia was induced in adult male rats pretreated intraperitoneally with either different inhibitors of phenylethanolamine-N-methyltransferase (PNMT) [which are active either peripherally (SKF 29661) or both peripherally and centrally (SKF 64139), thus lowering epinephrine (EPI) synthesis] or vehicle only (CTRL). Twelve hours after pretreatment, animals were intraperitoneally injected with vehicle alone (basal) or vehicle containing bacterial lipopolysaccharide (LPS) and sacrificed 2 h later. A significant (p < 0.05 vs. the respective basal value) hypoglycemia was found in all groups studied. No pretreatment modified basal plasma adrenocorticotropic hormone (ACTH), glucocorticoid and cytokine concentrations. Endotoxin-stimulated ACTH secretion was severalfold (p < 0.05) higher than the respective basal value in CTRL and in SKFs-pretreated rats; however, the plasma ACTH levels after LPS were significantly (p < 0.05 vs. CTRL and SKF-29661 values) reduced in SKF-64139-pretreated rats. All groups studied showed an appropriate adrenal response to endotoxin challenge. Although no differences were found in basal anterior pituitary (AP) ACTH content among groups, LPS treatment significantly (p < 0.05 versus the respective basal value) decreased AP ACTH in CTRL and SKF 29661 groups. No pretreatment modified the basal medial basal hypothalamus (MBH) corticotropin-releasing hormone (CRH) content. Conversely, SKF 64139 pretreatment significantly (p < 0.05 vs. CTRL and SKF 29661 values) reduced basal median eminence (ME) CRH content, and LPS administration significantly (p < 0.05) decreased ME CRH in CTRL and SKF-29661-pretreated rats. SKF 64139 pretreatment significantly (p < 0.05) enhanced basal MBH and ME arginine vasopressin (AVP) contents. LPS administration significantly (p < 0.05) decreased MBH AVP in CTRL and SKF-29661-pretreated rats and diminished (p < 0.05 vs. basal values) ME AVP in all groups. The plasma tumor necrosis factor alpha (TNFα) concentrations were enhanced severalfold (p < 0.05 vs. basal values) after LPS treatment in CTRL rats, but not in SKFs-treated animals. In order to explore the reduced cytokine release after LPS in PNMT-inhibited rats, additional ex vivo experiments were performed by using peripheral mononuclear cells (PMNC) from both CTRL and SKF-29661-pretreated rats. The results of these experiments confirmed an immune dysfunction after inhibition of peripheral EPI synthesis; in fact, basal and concanavalin-A-stimulated TNFα secretions were significantly (p < 0.05) lower in SKF-29661-treated than in CTRL PMNC, while, interestingly, addition of EPI (10–7 M) to the medium fully restored these effects. These data demonstrate that: (1) the mechanism whereby LPS-induced hypoglycemia was independent of epinergic activity; (2) selective central inhibition of epinergic function reduced endotoxin-stimulated ACTH secretion, an effect that could mainly be due to a decrease in CRH-ergic activity; (3) the central epinergic system positively and negatively modulates CRH- and AVPergic functions, respectively, and (4) inhibition of peripheral PNMT activity reduced immune system function in vivo and ex vivo. It is further suggested that low peripheral levels of EPI could be beneficial for the body’s defense mechanisms during endotoxic shock.

Chronic malnutrition is one of the most important causes of several metabolic, immune and neuroendocrine dysfunctions. The aim of the present study was to determine the influence of chronic food restriction on basal neuroendocrine, immune and adipocyte functions and during the acute-phase response to endotoxic shock in female rats. The effect of refeeding of undernourished rats on the above-mentioned functions was also investigated. For these purposes, plasma total protein, glucose, triglycerides, ACTH, corticosterone, tumor necrosis factor-α (TNF) and leptin (LEP) levels were determined in basal condition and 2 h after endotoxin (LPS; 180 µg/kg body weight, i.p.) administration in 3 different groups: (1) well-nourished (WN) controls; (2) undernourished (UN) rats as a consequence of chronic food restriction, and (3) UN rats re-fed to restoration of their body weights in the WN rat range. The results indicate that UN rats, in comparison with WN controls, developed an arrest in body weight gain as well as in basal hypoglycemia, hypotriglyceridemia, hypoleptinemia, hypercorticosteronemia and enhanced adrenal glucocorticoid content; however, no changes in basal total protein, ACTH and TNF plasma levels and in anterior pituitary ACTH concentrations were found. When endotoxic shock was induced, the LPS-induced hypoglycemia developed in WN rats was abolished in UN animals, and both ACTH and TNF plasma concentrations after endotoxin, albeit significantly (p < 0.05) higher than the respective basal values, were significantly (p < 0.05) lower in UN than in WN control rats. Despite the high basal plasma corticosterone concentration in UN vs. WN rats, the LPS-induced glucocorticoid release was similar in WN and UN rats. Additionally, LPS treatment did not modify basal plasma LEP levels, regardless of the group. Interestingly, UN rats fed ad libitum for 15 days restored their body weight to WN rat range values, and the various metabolic dysfunctions seen in UN rats in both basal and post-LPS conditions were fully normalized. Our results clearly indicate that chronic undernutrition not only affects, as earlier described, reproductive function but also metabolic, neuroendocrine, immune and adipocyte functions, and that the effects induced by undernutrition can be fully reversed after recovery of normal body weight. The present study strongly supports the involvement of the metabolic status in the effectiveness of the defense mechanisms developed in patients in inflammatory stress conditions.

Cytokines secreted by bacterial endotoxin-activated immune cells are substances known to stimulate the hypothalamo-pituitary-adrenal (HPA) axis function. The present study was designed to better understand the effect of different mediators of inflammation, such as cytokines and histamine, on the acute HPA axis response induced by administration of a single dose of bacterial lipopolysaccharide (LPS) in adult, male, BALB/c mice. Two different experimental designs were set up. In the first design, mice (n = 8–11 per group) were injected i.p. with LPS (90 µg/kg body weight) and killed by decapitation 2 or 6 h after treatment. Additional groups of mice were pretreated i.p. 12 h before LPS treatment with: (a) 3–4 mg IgG/kg body weight of either an anti-tumor necrosis factor-α (TNF)-α, anti-interleukin (IL)-1β- or IL-6 serum; (b) IL-1 receptor antagonist (IL-1ra) (120 µg/kg body weight) immediately before LPS and also 3 h later (when animals were killed 6 h after LPS injection), or (c) 182 µg/kg body weight of clemastine, an antagonist of H1 histaminergic receptors, 2 h before LPS treatment; animals were killed in a similar fashion to that described for treatment with LPS alone. In the second experimental design, mice were pretreated (i.p., 10 mg/kg body weight, 30 min before administration of a similar dose of LPS) with different blockers of histaminergic pathway function such as: (a) mepyramine, another anti-H1, (b) cimetidine, an H2 receptor blocker, and (c) Rα-methylhistamine dihydrochloride, an H3 presynaptic receptor agonist which inhibits histamine synthesis and output. These animals were then killed by decapitation 40 min after endotoxin treatment. After decapitation, trunk blood was collected for further determination of plasma levels of both ACTH and corticosterone (B) by specific assays. The results indicate that plasma levels of both ACTH and B were several-fold increased over baseline, 2 and 6 h after LPS administration. Two hours, the effect of LPS on ACTH output was not modified by pretreatment with anti-IL-1β IgG, anti-IL-6 IgG, anti-TNF-α IgG nor with IL-1ra, although IL-1ra treatment was able to fully block the IL-1β (35 µg/kg body weight)-stimulated HPA axis function, 1 and 2 h after cytokine administration. Six hours after LPS administration, anti-IL-1β and anti-TNF-α IgGs were both able to significantly reduce HPA axis response to the endotoxins, whereas anti-IL6 IgG had no effect. Anti-IL-1β IgG reduced only B secretion, whereas anti-TNF-α IgG decreased both ACTH and B secretion. The blockade of histaminergic pathway functions did not impede the LPS-induced ACTH and B release regardless of the product employed. The present results indicate that TNF-α, and to a lesser extent IL-1β, are the most relevant cytokines involved in HPA axis response to endotoxin administration. Our data also suggest that, in mice, HPA axis activation after infection appeared to be independent of stimualtion of the histaminergic pathway.

The aim of the present study was to determine the role of the endogenous sex steroid environment in the hypothalamo-corticotrope (HC) function in both sham-operated (SHAM) and bilaterally adrenalectomized (ADX) rats. For this purpose adult rats of both sexes were used 3 and 6 weeks after either SHAM or ADX. The results indicate that: a) in SHAM animals, basal plasma ACTH levels were significantly higher in females than in males, and this sexual dimorphism was overridden by ADX, regardless of the time post-surgery; b) although basal anterior pituitary (AP) ACTH content was similar in SHAM animals of both sexes, 3- and 6-week ADX induced higher AP ACTH in males than in females; c) at 3- and 6-weeks, ADX rats of hoth sexes had an AVP: CRH ratio (r), in the median eminence (ME) and medial basal hypothalamus (MBH), increased several fold over the respective SHAM-value and, although no sexual dimorphism was found at week 3 post-ADX, by 6-weeks post ADX, these ratios were significantly higher in both brain tissues of females than in those of males; and d) the in vitro ME CRH and AVP output in response to high potassium concentrations (hK+; 28 and 56 mmol/l), was concentration-related, regardless of sex and surgery, and was characterized by enhanced secretion of neuropeptides by MEs from ADX in comparison to SHAM rats of both sexes, and a sexual dimorphism was found in this parameter, consisting in general, in greater neuropeptide output from tissues of female than of male animals. Our results indicate that: 1) there is a gender-dependent characteristic of the HC axis function in glucocorticoid-replete rats and 2) the absence of the glucocorticoid negative feedback mechanism is responsible for either the expression or for the override of the sexual dimorphism in different parameters, a phenomenon which dependent on the time elapsed after ADX., Instituto Multidisciplinario de Biología Celular, Facultad de Ciencias Exactas

The product of the ob/ob gene, leptin, is known to be able to exert a modulator, role on HPA axis function. The aim of the present study was to determine whether endogenous ACTH and glucocorticoids exert any regulatory effect on leptin secretion. For this purpose bilaterally adrenalectomized (ADX) or sham operated (Sham) adult male rats were implanted with an indwelling i.v. catheter. A subgroup of ADX animals received, at the same time of surgery, a s.c. corticosterone (B) pellet (75 mg) (ADX+B). All animals were subjected to experimental designs 7 days after surgery. Our results indicate, as expected, that 7-day ADX animals have several fold increased basal ACTH plasma levels and non detectable circulating B, whereas ADX+B rats showed basal plasma ACTH levels in the range of Sham values and plasma B concentrations of about 5 microg/dl. Interestingly, basal plasma leptin levels were significantly (P < 0.05) decreased by 7 days post ADX, and B replacement therapy (ADX+B) restored circulating leptin to Sham levels. Acute dexamethasone (Dxmn, 30 microg/kg body weight, i.v.) treatment induced a very rapid decrease in plasma ACTH concentrations in both Sham and ADX rats, as well as a decrease in plasma B levels in Sham rats. Interestingly, Dxm test had no effect on plasma leptin levels in Sham animals; however, in ADX rats, the synthetic glucocorticoid increased plasma leptin concentrations, restoring the levels observed in Sham rats. This effect occurred at the same time when plasma ACTH levels were decreasing toward basal Sham values. These results clearly indicate that, beside the known effects of leptin on HPA axis function, circulating ACTH and glucocorticoid are able to modulate leptin secretion in plasma. The lack of circulating glucocorticoid and/or increased plasma ACTH concentrations, are responsible for decreasing leptin output, whereas decreased plasma ACTH concentrations allow an increase of leptin secretion in blood. Our data strongly support the existence of a closed, bi-directional, circuit between HPA axis function and adipose tissue metabolism. They further indicate the physiological relevance of different types of stress associated with many phenotypes of obesity.

A bidirectional relationship between the immune and neuroendocrine systems is now widely accepted. Since it is well known that the thymus plays an important role in the regulation of the immune function, we decided to explore whether a lack of the thymic function may influence hypothalamic-pituitary-adrenal (HPA) axis activity. Eight-week-old female mice of both strains, nude and control BALB/c, were used to study: (a) the in vivo response of the HPA axis to several stress stimuli acting at either the hypothalamic (insulin administration and ether vapor inhalation), pituitary (CRH and vasopressin injections) or adrenal (ACTH treatment) level and (b) the in vitro response of hypothalamic fragments to high KCl (48 mM) stimulation. The results indicate that: (1) basal plasma ACTH and vasopressin levels were significantly (p < 0.05) higher in nude than in control BALB/c mice, whereas basal plasma corticosterone concentrations were similar in both strains of mice; (2) although no significant strain-related difference in the stress-induced ACTH secretion in plasma was found, hypothalamic stimuli were able to induce a significantly (p < 0.05) higher secretion of glucocorticoid in plasma in nude than in control BALB/c mice; (3) the pattern of in vitro hypothalamic CRH release was similar in both strains of animals; however, basal AVP output and that stimulated by 48 mM KCl were significantly (p < 0.05) higher in nude than in control hypothalamic fragments, and (4) whereas hypothalamic CRH, pituitary ACTH and adrenal glucocorticoid contents were similar in both strains, hypothalamic AVP content was significantly (p < 0.05) higher in athymic than in control mice. In summary, our results indicate that nude mice have an increased vasopressinergic function which could contribute to a normal HPA axis activity; thus, adult athymic mice of BALB/c origin could compensate, due to their increased vasopressinergic function, for a robust glucocorticoid release to protect themselves immediately after aggression. It remains to be determined whether this enhanced vasopressinergic function is a result of an early adrenal insufficiency due to congenital deficiency of thymic factors known to stimulate HPA axis function.

Immune neuroendocrine interactions are vital for the individual's survival in certain physiopathological conditions, such as sepsis and tissular injury. It is known that several animal venoms, such as those from different snakes, are potent neurotoxic compounds and that their main component is a specific phospholipase A type 2 (PLA2). It has been described recently that the venom from Crotalus durissus terrificus [snake venom (SV), in the present study] possesses some cytotoxic effect in different in vitro and in vivo animal models. In the present study, we investigated whether SV and its main component, PLA2 (obtained from the same source), are able to stimulate both immune and neuroendocrine functions in mice, thus characterizing this type of neurotoxic shock. For this purpose, several in vivo and in vitro designs were used to further determine the sites of action of SV-PLA2 on the hypothalamo-pituitary-adrenal (HPA) axis function and on the release of the pathognomonic cytokine, tumor necrosis factor alpha (TNF alpha), of different types of inflammatory stress. Our results indicate that SV (25 microg/animal) and PLA2 (5 microg/animal), from the same origin, stimulate the HPA and immune axes when administered (i.p.) to adult mice; both preparations were able to enhance plasma glucose, ACTH, corticosterone (B), and TNF alpha plasma levels in a time-related fashion. SV was found to activate CRH- and arginine vasopressin-ergic functions in vivo and, in vitro, SV and PLA2 induced a concentration-related (0.05-10 microg/ml) effect on the release of both neuropeptides. SV also was effective in changing anterior pituitary ACTH and adrenal B contents, also in a time-dependent fashion. Direct effects of SV and PLA2 on anterior pituitary ACTH secretion also were found to function in a concentration-related fashion (0.001-1 microg/ml), and the direct corticotropin-releasing activity of PLA2 was additive to those of CRH and arginine vasopressin; the corticotropin-releasing activity of both SV and PLA2 were partially reversed by the specific PLA2 inhibitor, manoalide. On the other hand, neither preparation was able to directly modify spontaneous and ACTH-stimulated adrenal B output. The stimulatory effect of SV and PLA2 on in vivo TNF alpha release was confirmed by in vitro experiments on peripheral mononuclear cells; in fact, both PLA2 (0.001-1 microg/ml) and SV (0.1-10 microg/ml), as well as concavalin A (1-100 microg/ml), were able to stimulate TNF alpha output in the incubation medium. Our results clearly indicate that PLA2-dependent mechanisms are responsible for several symptoms of inflammatory stress induced during neurotoxemia. In fact, we found that this particular PLA2-related SV is able to stimulate both HPA axis and immune functions during the acute phase response of the inflammatory processes., Instituto Multidisciplinario de Biología Celular

Bidirectional communication between the immune and the endocrine systems is now widely accepted as essential for the survival of the organism. Since a classical nonresponsive period of the hypothalamic-pituitary-adrenal (HPA) axis takes place shortly after birth and because endogenous sex hormones modulate immune function, the aim of the present work was to determine whether sex steroids regulate the PHA axis response to immune (bacterial, lipopolysaccharide, LPS) and nonimmune (insulin, INS) stressors in mice during development. For this purpose 7-, 15-, 30-, 45- and 60-day-old mice of both sexes were intraperitoneally injected with either vehicle alone (basal) or containing LPS (2 mg/kg body weight) or INS (12 IU/kg body weight). The animals were then killed by decapitation, 2 h or 45 min after LPS or INS, respectively. Plasma samples were assayed to measure corticosterone concentrations. The results indicated that: (a) there was a transient increase in basal plasma corticosterone levels during development, with a peak value at the juvenile age, regardless of sex; (b) a higher basal plasma corticosterone concentration in females than in males chartacterized the adult age; (c) the infantile age is a period of the HPA axis function nonresponsive to purely neuroendocrine but not to inflammatory stimuli; (d) during the juvenile age, females showed a hyporesponsive HPA axis to neurendocrine and immune stress, whereas male mice were fully unresponsive to both challenges; (e) animals of both sexes showed a maximal HPA axis response to purely neuroendocrine stress at the prepubertal age; this response to the immune stimulus was also maximal in 30-day-old males, while it was found in females after puberty (45-day-old mice); (f) sexual dimorphism in the HPA axis response to a purely neuroendocrine stimulus was found at 30 days of age or later, while this characteristic of the response to endotoxin was not present until puberty. These data clearly suggest that these are gender-dependent characteristics of the ontogeny of the HPA and HP-gonadal axes that are responsible for the sexual dimorphism of HPA axis function in mice.

The aim of the present study was to elucidate the modulatory effect of transient changes in endogenous glucocorticoids, occurring after bilateral adrenal enucleation (ENUC), on anterior pituitary (AP) proopiomelanocortin (POMC)-derived peptides synthesis and output in rats. For this purpose, adult female rats were either bilaterally ENUC, adrenalelectomized (ADX), or sham-operated (SHAM) and killed by decapitation 2, 7, 14, and 21 days after surgery. Trunk blood was collected for measurements of ACTH, β-endorphin (β-END) and corticosterone (B) concentrations; APs were quickly dissected for the determination of ACTH, β-endorphin (β-END)-like (β-END-LI) and γ3-MSH contents and adrenal glands were removed and submitted to histological study. The results indicate that ENUC and ADX increased AP POMC-related peptides synthesis and release in association with changes in the AP processing of peptides belonging to the N-terminal (γ3-MSH), mid (ACTH) and C-terminal (β-LPH/ENDs) portions of POMC. While ADX abolished plasma B levels, ENUC induced a transient (day 2) decrease in plasma B concentrations which returned to SHAM levels at 7 days after surgery. These data tallied with the histological observations carried out, indicating a time-dependent regenerative process of the adrenal which was completed by three weeks after ENUC. There was a different pattern in plasma ACTH and β-END levels between ENUC and ADX; maximal plasma peptide levels were found 7–14 days after ENUC, then falling down to SHAM values at 21 days post ENUC. Conversely, there was a constant increment in plasma peptide levels up to 21 days after ADX. At 2 days after both ENUC and ADX all peptides measured in the AP were lower than SHAM values, thus reflecting a rapid corticotrope secretion. Thereafter, 7 or more days after surgery, AP peptide content in ADX rats increased, in a time-related fashion, up to 21 days after surgery. Only β-END-LI showed a similar AP content to that of the SHAM group, thereafter indicating a preferential cleavage of POMC to β-END long after ADX (21 days). ENUC rats showed increased AP POMC peptides content throughout the whole time, and it was significantly different from SHAM and ADX values 14 days post-surgery. Interestingly, we found an increment in AP γ3-MSH, a peptide which is preferentially synthesized in the intermediate lobe of the rat pituitary, in both ENUC and ADX situations. Our results further indicate that: 1) glucocorticoids, from regenerating adrenal origin, induce a fast negative feedback mechanism on AP secretion, and 2) there might be a delayed inhibitory action of newly synthesized corticosteroids on higher levels of the central nervous system. The lack of glucocorticoids (ADX) clearly corroborates a persistent enhancement of AP POMC-related peptides synthesis and secretion. The differences in AP processing of POMC between ENUC and ADX might be due to qualitative/quantitative changes in hypotalamic ACTH secretagogues output., Facultad de Ciencias Médicas

It has been established that in vivo administration of bacterial lipopolysaccharide (LPS) enhances hypothalamo-pituitary-adrenal (HPA) axis function by a mechanism involving endotoxin-stimulated cytokine release. Since under chronic LPS treatment a tolerance of the HPA axis response takes place, the aim of the present study was to determine whether mice submitted to repeated LPS administration could present an impairment in the HPA response to insulin (INS) administration, a pure neuroendocrine challenge. For this purpose, adult female BALB/c mice were injected with 200 microliters i.p. of sterile saline solution (VEH) containing 25 micrograms of LPS in a single or repeated (at 24-hour intervals, during 5 consecutive days) fashion. Animals were then killed at either 45 min after INS (0.3 IU/mouse, i.p.) or 2 h after LPS (25 micrograms/mouse) administration on experimental day 1 (D1; without any previous LPS injection), 3 (D3; mice having received 2 previous LPS injections) or 5 (D5; mice having received 4 previous LPS administrations). Control groups were injected a similar volume of VEH alone on experimental day 1 (D1; without any previous LPS injection), 3 (D3; mice having received 2 previous LPS injections) or 5 (D5; mice having received 4 previous LPS administrations); in each group, mice were killed at either 45 min or 2 h after VEH injection. Immediately after decapitation, trunk blood was collected. Plasma tumor necrosis factor alpha (TNF), ACTH and corticosterone (B) levels were determined by specific assays. Plasma TNF, ACTH and B levels were significantly increased 2 h after the first LPS treatment (D1). Although no significant increase in plasma TNF concentration was found 2 h after the third LPS injection (D3), the corticotrope response was still significant and induced a full effect on adrenal B output. Two hours after the fifth LPS administration (D5) TNF output was minimal and the HPA axis response was significantly diminished. Finally, the pattern of the HPA axis response to INS-induced hypoglycemia was similar to that elicited after LPS challenge although somewhat delayed. Our results indicate that: (1) TNF seems to play an important role in stimulating HPA axis function after single but not after repeated endotoxin administration; and (2) an impairment in the HPA axis response to both immuneneuroendocrine (LPS) and neuroendocrine (INS) stimuli takes place after repeated LPS administration. This study further suggests that the tolerance of the HPA axis response under recurrent endotoxemia could be, at least partially, due to an impairment in both immune (TNF output) and neuroendocrine functions.

It is known that in vivo administration of bacterial endotoxin activates immune cells to release cytokines, these substances in turn enhancing hypothalamo-pituitary-adrenal (HPA) axis function; additional evidence supports the existence of an immune-neuroendocrine sexual dimorphism. In the present study, we investigated: (1) the in vivo response of both the HPA and the immune systems to single and repeated endotoxin administrations in mice, and (2) whether testosterone possesses a modulatory effect on neuroendocrine-immune function under endotoxemia. For these purposes, adult male BALB/c mice were orchidectomized (Odx) or sham-operated and injected s.c, on alternate days, with either corn oil alone (Odx and Sham) or containing 20 µg of testosterone (Odx+T) until animals were killed. One week after surgery, different groups of mice were treated i.p. with bacterial lipopolysaccharide (LPS; 25 µg per mouse) in a single (day 1 D1) or repeated (at 24-hour intervals for 5 consecutive days) form. Animals were decapitated (on Dl, D3 and D5 of the treatment) 2 h after the last injection of either vehicle alone or containing LPS (the two groups were run in parellel). Trunk blood was collected and the whole medial basal hypothalamus (wMBH), the anterior pituitary (AP) and adrenal glands were dissected. Plasma tumor necrosis factor-alpha (TNFα), ACTH and corticosterone (B) concentrations as well as wMBH CRH, AP ACTH and adrenal B contents were determined by specific assays. Our results indicate: (1) a significant decrease in mice body weight after repeated LPS injections, regardless of the group; (2) a sex steroid environment-independent increase in plasma ACTH, B and TNFα levels 2 h after a single LPS injection; (3) that all these responses decreased after repeated LPS administration; (4) that a significant rise in adrenal B content occurred 2 h after the first, third and fifth LPS treatments and that such an effect was significantly enhanced by Odx and fully reversed by Odx followed by T therapy, and (5) that while hypothalamic CRH and AP ACTH were not modified by endogenous sex steroid environment or endotoxin administration, Odx significantly enhanced the LPS-induced ACTH release only 2 h after a single LPS treatment. Our findings suggest that endogenous TNFα plays a mediatory role in the acute activation of HPA axis function after LPS and that under persisting endotoxemia both immune and HPA functions are decreased. Finally, testosterone has an inhibitory role on adrenal glucocorticoidogenesis during endotoxic shock.

Various evidence suggests a bidirectional circuit between the immune and neuroendocrine systems. Because of the well-known role of the thymus in the regulation of the immune function, we designed this study to determine whether the lack of thymus may affect hypothalamo-pituitary-adrenal (HPA) axis activity by using both in vivo and in vitro paradigms in Swiss nude (athymic) and BALB/c (normal) mice. Eight-week-old female mice of both strains were used to study: (a) the in vivo response of the HPA axis to various stress stimuli acting at either hypothalamic (ether vapor inhalation, insulin administration), pituitary (CRH injection) or adrenal (ACTH treatment) level and (b) the in vitro response of pituitary and adrenal cells to CRH and ACTH stimulation, respectively. The results indicate that: (1) basal plasma ACTH levels were significantly (p0.05) higher in Swiss nude than in BALB/c mice, whereas basal plasma corticosterone (B) concentrations were similar in both strains of mice; (2) the stress-induced release of ACTH and B in plasma were significantly (p0.05) lower in Swiss nude than in BALB/c mice, regardless of the stimulus applied; (3) the in vitro pituitary response to CRH and the adrenal response to ACTH were significantly (p0.05) lower in Swiss nude than in BALB/c mice, whereas (4) hypothalamic CRH and pituitary ACTH contents were similar in both strains, adrenal B concentration was significantly (p0.05) lower in athymic mice; in addition, the nude mice adrenal glands were larger than those of BALB/c animals, due to marked hypertrophy of the zona fasciculata.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present investigation, we examined the influence of both genetic background and sex factors in the rat hypothalamo-pituitary-adrenal (HPA) axis function under both basal and post adrenalectomy (ADX) conditions. For these purposes adult female and male rats, from Sprague-Dawley (S-D), Fischer (F344/N), Lewis (LEW/N) and Buffalo (BUF) strains, were decapitated in basal condition or several (2, 7 and 14) days after ADX. Plasma stress hormones levels and adrenal corticosterone (B) concentration as well as peptide (ACTH, CRH and vasopressin, AVP) content in different tissues (anterior pituitary, AP; medial basal hypothalamus, MBH), were then evaluated by specific assays. Our results indicate that: a) despite no sex- and strain-related differences in AP ACTH and MBH ACTH secretagogues in basal condition, there exits a clear sexual dimorphism in plasma ACTH levels as well as in both plasma and adrenal B concentrations, with values significantly higher in females than in males, regardless the strain; b) ADX abolished plasma B levels and increased AP ACTH output in a time-dependent fashion up to the 14th day post surgery; c) AP ACTH content decreased 2 days after ADX, except in BUF female rats, thereafter tending to either recover or increase sham values by two weeks post ADX; d) ADX decreased MBH CRH at all periods studied, except in BUF female animals on day 14; e) ADX clearly diminished MBH AVP only in S-D rats, and f) a sexual dimorphism was also found in AP ACTH in 7-day-ADX S-D rats and in 14-day-ADX S-D and F344/N animals; also, a dimorphic pattern in MBH CRH was found in 7-day-ADX S-D as well as in 14-day-ADX F344/N and LEW/N rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-activated immune cells acutely release cytokines which, besides their effects on the immune system, increase hypothalamopituitary-adrenocortical (HPA) function to counteract the inflammatory process. The present study was designed to test, using in vitro paradigms, whether there exists a hypothalamic and/or a median eminence site of action, whereby different substances derived from the immune system could stimulate the CRH and/or the arginine-vasopressin (AVP) neuronal pathway. For this purpose, whole medial basal hypothalamus (containing the median eminence) were dissected from female rats and incubated in vitro with several concentrations of interleukin-1 (IL-1)beta, interleukin-6 (IL-6), tumor necrosis factor (TNF)-alpha, thymosin fraction 5 (TF5) or bacterial lipopolysaccharide (LPS). After a 40-min incubation period, the amounts of CRH and AVP released into the incubation medium were measured by specific radioimmunoassays (RIAs). Additional experiments were carried out by superfusing isolated rat median eminence fragments with the different test substances; CRH and AVP released into the medium were also measured by RIAs. The results indicated that IL-1 beta (10(-11) to 10(-7) M), IL-6 (0.06 x 10(-10) to 0.4 x 10(-10) M), TNF-alpha (6 x 10(-9) to 6 x 10(-7) M) and TF5 (5-500 micrograms/ml) but not LPS (1-100 ng/ml) significantly enhanced hypothalamic CRH secretion above baseline in a concentration-related fashion. Additionally, superfusion experiments demonstrated that, among all test substances, only IL-6 possesses a direct and dose-dependent CRH-releasing activity at the median eminence level. Conversely, no preparation enhanced basal AVP release in either in vitro design.(ABSTRACT TRUNCATED AT 250 WORDS)

Many reports indicate that serotonin plays a role in the regulation of the hypothalamo-pituitary-adrenocortical axis. The present study was designed to elucidate whether the activation of the central serotonergic pathway enhances adrenocorticotropin and corticosterone secretion, and if so, whether the CRH and vasopressin neuronal systems could be mediating this effect. Intraperitoneal administration of a low dose of L-5-hydroxytryptophan (an aromatic L-amino acid precursor of serotonin synthesis; 20 mg/kg bw, 30 minutes before the sacrifice) in rats pretreated with pargyline (a brain mononoamine oxidase inhibitor, which enhances monoamine activity; 75 mg/Kg bw, 16 hours before the sacrifice) and carbidopa (a peripheral active inhibitor of the decarboxylation of aromatic L-amino acids, which would permit more monoamine precursor to be available to the brain; 50 mg/Kg bw, 90 minutes before the sacrifice) increased ACTH and corticosterone secretion in plasma. Such an effect was partially blocked by metergoline (a serotonin type-1 and -2 receptor blocker; 1 mg/Kg bw, 90 minutes before the sacrifice), but not by spiperone (a serotonin type-2 and dopamine receptor antagonist; 0.5 mg/Kg bw, 90 minutes before the sacrifice). The activation of the central serotonergic system enhanced the CRH content in the median eminence, whereas it decreased the content of this neuropeptide in the medial basal hypothalamus. These effects were fully abolished by metergoline, but not by spiperone pretreatment. The activation of the serotonergic pathway did not influence the vaso-pressinergic neuronal system. In vitro experiments using hypothalamic-median eminence fragments incubated with serotonin solutions indicate that this monoamine possesses a CRH releasing effect at concentrations of 1 μM or more. The CRH secreta-gogue activity of serotonin (1 μM) was completely blocked by metergoline (1 μM) but not by spiperone (1 μM). Conversely, serotonin did not induce a significant release of vasopressin at any of the concentrations assayed. These studies strongly suggest that the enhanced pituitary-adrenocortical function after the activation of the central serotonergic pathway could be due, at least in part, through the stimulation of the CRH but not the vasopressinergic neuronal system.

Bilateral adrenalectomy (ADX) leads to increased ACTH synthesis and secretion. It is thought that endogenous glucocorticoids exert a feedback mechanism at both pituitary and brain levels. The present study has been performed in order to determine the effect of ADX on the release of hypothalamic neuropeptides with corticotropin-releasing activity (CRA) and if there exists a median eminence site of glucocorticoid action to regulate hypothalamic-pituitary-adrenal (HPA) function. Adrenalectomized and sham-operated male rats were killed at different periods after surgery (2, 5, 7 and 14 days) and trunk blood was collected for ACTH and corticosterone (B) concentrations measurement. Brain (median eminence, ME; and medial basal hypothalamus, MBH) and pituitary (anterior lobe, AP; and neurointermediate lobe, NIL) tissues were dissected in order to evaluate either peptide content or in vitro hormone release. The results indicate that ADX blunted plasma B levels and increased AP ACTH content and secretion in a time-related fashion up to the 14th day. ADX significantly decreased both CRF and CRA contents in the ME at all periods studied; ME arginine-vasopressin (AVP) increased 7 and 14 days after ADX. MBH CRF decreased after ADX, but returned to sham value 2 weeks later; similarly, MBH AVP decreased at all periods after ADX. Removal of endogenous glucocorticoids did not vary neither oxytocin (OXY) content in the ME and MBH nor AVP and OXY contents in the NIL. In our superfusion experiments, we found that ADX increased basal AVP release and did not change spontaneous CRF secretion from ME terminals. Dexamethasone (Dxm, 10 nM) diminished AVP but not CRF output by ME tissues from adrenalectomized rats. A direct relationship was found between ME CRF and 28 mM KCl (hK+)-induced CRF release by MEs from adrenalectomized rats. ME fragments from adrenalectomized rats were hyperresponsive to kH+ stimulation of AVP release. Dxm (10 nM) decreased the hK(+)-evoked CRF and AVP release by MEs from adrenalectomized rats. ADX and dexamethasone treatment did not influence basal and hK(+)-elicited ME OXY release. Additionally, a rapid glucocorticoid inhibitory effect on ACTH secretion by isolated AP cells from both sham and adrenalectomized rats was found, and an in vitro corticotrope hyporesponse to 0.63 nM CRF and 9.25 nM AVP stimulation during several days after ADX.(ABSTRACT TRUNCATED AT 400 WORDS)

The administration of testosterone propionate (TP) in the female rat at the neonatal age has been used for several yr as a model to study anovulation during adulthood. The present work was designed in order to see whether some neuroendocrine parameters vary with age in this animal model. Hypothalamic LHRH content and LH-FSH anterior pituitary (AP) content and plasma levels were evaluated in samples taken from both neonatally-androgenized and littermate control female rats at different ages (15 to 100 days old). Additionally, we have studied pulsatile LH-FSH released in plasma and in vivo AP response to LHRH in both neonatally-androgenized and control female rats during adulthood. The results indicate that the neonatal TP treatment did not induce any change in hypothalamic LHRH content over development. Neonatally androgenized rats have decreased both LH-FSH AP content and plasma levels at the infantile age (15-day old). LH-FSH AP content remained reduced in samples taken up to the 30th day of age. Plasma LH-FSH levels on the day 30 of age were similar in both groups. TP-treated rats studied on the 100th day of age had: a) an altered pulsatile rhythm of gonadotropin release in plasma due to the decreased LH-FSH trough and average mean values, and to the diminished FSH peak amplitude values, as well as an increased LH:FSH ratio; and b) an impaired in vivo LHRH-induced LH-FSH release.(ABSTRACT TRUNCATED AT 250 WORDS)