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Aim An adverse endogenous environment during early life predisposes to metabolic disorder development. We previously reported adverse metabolic and adipose tissue effects in adult male rats born to dams fed with a fructose-rich diet (FRD). The aim of this work was to determine the effect of a FRD consumed by the pregnant mother on the white adipose tissue (WAT) browning capacity of male offspring at adulthood. Main methods Adult S D male offspring from control (C) and FRD-fed mothers were exposed during one week to a cold stimulus. WAT browning capacity was studied through in vivo and in vitro approaches. Key findings After cold exposure, WAT browning was higher in fructose-programmed animals as evidenced by an increase in ucp-1 gene expression, protein levels, and higher UCP-1 positive foci. Moreover, pgc1-α gene expression was increased. In vitro studies showed a lower adipogenic capacity in cells of prenatally fructose-exposed animals differentiated with a white differentiation cocktail, while a higher ucp-1 expression was noted when their cells were treated with a pro-beige differentiation cocktail. Significance For the first time we demonstrate that pre-natal fructose exposure predisposes programmed male rats to a higher WAT browning-induced response, under stimulated conditions, despite an apparent lower basal thermogenic capacity. These results should be considered in future studies to generate new therapeutic approaches to deal with adverse programming malnutrition effects.

Aim Dexamethasone (DXM) is a synthetic glucocorticoid whose effects in early and terminal adipogenesis have been addressed. In this study, we evaluated if DXM affects adipocyte precursor cells (APCs), priming them for further adipogenic differentiation. For this purpose, we analyzed APCs number and competency after DXM treatment. Materials and methods Adult male rats were injected for 2 or 7 days with either DXM (30 μg/kg of weight, sc.) or vehicle. Stromal vascular fraction (SVF) cells from retroperitoneal adipose tissue (RPAT) were isolated to quantify APCs by flow cytometry (CD34+/CD45−/CD31−). Also, expression of competency markers (PPARγ2 and Zfp423) was assessed. Additionally, SVF cells from control rats were incubated with DXM (0.25 μM) alone or combined with a mineralocorticoid receptor (MR) antagonist (Spironolactone 10 μM) and/or a glucocorticoid receptor (GR) antagonist (RU486 1 μM) to assess APCs competency and adipocyte differentiation. Key findings APCs from 2 days DXM-treated rats showed increased expression of PPARγ2 and Zfp423 (competency markers), but did not affect APCs percentage by FACS analysis (CD34+/CD45−/CD31−). Additionally, we found that DXM treatment in SVF also increased APCs competency in vitro, predisposing APCs to further adipocyte differentiation. These effects on APCs were abrogated only when both, MR and GR, were blocked. Significance Overall, our results suggest that DXM primes APCs for differentiation mainly by enhancing Zfp423 and PPARγ2 expressions. Also, we showed that the inhibition of MR and GR was necessary for the complete abolishment of DXM effects.

The impact of neonatal androgen receptor (AR) stimulation/blockage, due to testosterone propionate (TP)/AR antagonist treatment, on individual anthropometry and neuroendocrine-metabolic function was evaluated in the juvenile female rat. Pups (age 5 days) were s.c. injected with TP (1.25 mg), flutamide (F; 1.75 mg), and TP + F or vehicle (control, CT) and studied on day 30 of age. Body weight (BW), parametrial adipose tissue (PMAT) mass, food intake, adipoinsular axis, and steroidogenic functions were examined. Opposite to TP-rats, F-treated rats developed hypophagia, grew slowly (BW and PMAT), and displayed heightened peripheral insulin sensitivity. These F effects were abrogated in TP + F animals. Accordingly, TP rats displayed hyperleptinemia, an effect fully prevented by F cotreatment. Finally, androgen-treated animals bore an irreversible ovarian dysfunction (reduced circulating levels of 17HOP4 and ovary 17HOP4 content and P450c17 mRNA abundance). These data indicate that early stimulation of AR enhanced energy store, blockage of AR activity resulted in some beneficial metabolic effects, and neonatally androgenized rats developed a severe ovarian dysfunction. Our study highlights the important role of AR in the early organizational programming of metabolic and neuroendocrine functions., Instituto Multidisciplinario de Biología Celular, Centro de Endocrinología Experimental y Aplicada

Pregnant women with impaired insulin sensitivity are at risk for developing pregnancy-induced hypertensive disorders (PIHD). We analyzed glucose and insulin circulating levels throughout a 2-h oral 75 g glucose tolerance test in pregnant women, and related the 2-h insulinemias to PIHD prevalence.Pregnant women (gestational week 24-28) were submitted to a glucose overload, and glucose and insulin plasma concentrations were measured throughout the test. These peripheral metabolite levels, the homeostasis model assessment (HOMA) values and the glucose to insulin ratio (G:Ir) were analyzed. Anthropometric parameters and pregnancy outcome were recorded.Women with normal fasting glycemia, insulinemia and HOMA values, G:Ir and 2 h-glycemia but whose 2 h-insulinemia was higher than 215.25 pM were at greater risk for developing late pregnancy hypertension and preeclampsia compared to women of similar characteristics but whose 2 h-insulinemias were lower than 215.25 pM.2-h insulinemias higher than 215.25 pM after a 75 g glucose overload could be highly indicative of women at increased risk of developing PIHD.

Adipose tissue (AT) expansion is the result of two processes: hyperplasia and hypertrophy; and both, directly or indirectly, depend on the adipogenic potential of adipocyte precursor cells (APCs). Glucocorticoids (GCs) have a potent stimulatory effect on terminal adipogenesis; while their effects on early stages of adipogenesis are largely unknown. In the present work, we study, in a model of high GC levels, the adipogenic potential of APCs from retroperitoneal AT (RPAT) and its relationship with RPAT mass expansion. We employed a model of hyper-adiposity (30- and 60-day-old rats) due to high endogenous GC levels induced by neonatal treatment with L-monosodium glutamate (MSG).We found that the RPAT APCs from 30-day-old MSG rats showed an increased adipogenic capacity, depending on the APCs’ competency, but not in their number. Analyses of RPAT adipocyte diameter revealed an increase in cell size, regardless of the rat age, indicating the prevalence of a hypertrophic process. Moreover, functional RPAT alterations worsened in 60-day-old rats, suggesting that the hyperplastic AT expansion found in 30-day-old animals might have a protective role. We conclude that GCs chronic excess affects APCs’ adipogenic capacity, modifying their competency. This change would modulate the hyperplastic/hypertrophic balance determining healthy or unhealthy RPAT expansion and, therefore, its functionality., Facultad de Ciencias Médicas

The aim of this work was to determine the effect of a fructose rich diet (FRD) consumed by the pregnant mother on the endocrine-metabolic and in vivo and in vitro adipose tissue (AT) functions of the male offspring in adulthood. At 60 days of age, rats born to FRD-fed mothers (F) showed impaired glucose tolerance after glucose overload and high circulating levels of leptin (LEP). Despite the diminished mass of retroperitoneal AT, this tissue was characterized by enhanced LEP gene expression, and hypertrophic adipocytes secreting in vitro larger amounts of LEP. Analyses of stromal vascular fraction composition by flow cytometry revealed a reduced number of adipocyte precursor cells. Additionally, 60 day-old control (C) and F male rats were subjected to control diet (CC and FC animals) or FRD (CF and FF rats) for three weeks. FF animals were heavier and consumed more calories. Their metabolic-endocrine parameters were aggravated; they developed severe hyperglycemia, hypertriglyceridemia, hyperleptinemia and augmented AT mass with hypertrophic adipocytes. Our study highlights that manipulation of maternal diet induced an offspring phenotype mainly imprinted with a severely unhealthy adipogenic process with undesirable endocrine-metabolic consequences, putting them at high risk for developing a diabetic state., Facultad de Ciencias Médicas

A sex steroid-dependent modulation of the immune function in mammals is accepted, and evidence suggests that while estrogens enhance, androgens inhibit the immune response. The aim of this study was to explore in the adult male rat the effect of either neonatal flutamide (FTM) treatment or prepubertal orchidectomy (ODX) on endocrine markers in the basal condition and peripheral tumor necrosis factor alpha (TNFα) levels during inflammatory stress. For these purposes, (1) 5-day-old male rats were subcutaneously injected with either sterile vehicle alone or containing 1.75 mg FTM, and (2) 25-day-old male rats were sham operated or had ODX. Rats were sacrificed (at 100 days of age) in the basal condition for determination of peripheral metabolite levels. Additional rats were intravenously injected with bacterial lipopolysaccharide (LPS; 25 μg/kg body weight, i.v.) and bled for up to 4 h. Data indicate that (1) ODX increased peripheral glucocorticoid levels and reduced those of testosterone, whereas FTM-treated rats displayed low circulating leptin concentrations, and (2) LPS-induced TNFα secretion in plasma was significantly enhanced in the FTM and ODX groups. Our study supports that neonatal FTM treatment affected adiposity function, and adds data maintaining that androgens have a suppressive role in proinflammatory cytokine release in plasma during inflammation. Fil: Ongaro Gambino, Luisina. 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.. Lausanne University Hospital; 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

An adverse endogenous environment during early life predisposes the organism to develop metabolic disorders. We evaluated the impact of intake of an iso-caloric fructose rich diet (FRD) by lactating mothers (LM) on several metabolic functions of their male offspring. On postnatal d 1, ad libitum eating, lactating Sprague-Dawley rats received either 10% F (wt/vol; FRD-LM) or tap water (controls, CTR-LM) to drink throughout lactation. Weaned male offspring were fed ad libitum a normal diet, and body weight (BW) and food intake were registered until experimentation (60 d of age). Basal circulating levels of metabolic markers were evaluated. Both iv glucose tolerance and hypothalamic leptin sensitivity tests were performed. The hypothalamus was dissected for isolation of total RNA and Western blot analysis. Retroperitoneal (RP) adipose tissue was dissected and either kept frozen for gene analysis or digested to isolate adipocytes or for histological studies. FRD rats showed increased BW and decreased hypothalamic sensitivity to exogenous leptin, enhanced food intake (between 49-60 d), and decreased hypothalamic expression of several anorexigenic signals. FRD rats developed increased insulin and leptin peripheral levels and decreased adiponectinemia; although FRD rats normally tolerated glucose excess, it was associated with enhanced insulin secretion. FRD RP adipocytes were enlarged and spontaneously released high leptin, although they were less sensitive to insulin-induced leptin release. Accordingly, RP fat leptin gene expression was high in FRD rats. Excessive fructose consumption by lactating mothers resulted in deep neuroendocrine-metabolic disorders of their male offspring, probably enhancing the susceptibility to develop overweight/obesity during adult life., Facultad de Ciencias Exactas, Facultad de Ciencias Médicas

The aim of this study was to evaluate the effect of ovariectomy on the acute-phase response of inflammatory stress. Ex vivo adrenocortical, peripheral mononuclear cell (PMNC) and adipocyte activities were studied in intact and ovariectomized mice. Endotoxemia was mimicked by intraperitoneal administration of bacterial lipopolysaccharide (LPS; 25 mg per mouse) to sham-operated and 21-day ovariectomized mice. Circulating corticosterone, tumor necrosis factor-α (TNFα) and leptin concentrations were monitored before and 30–120 min after the administration of LPS. Additionally, in vitro experiments were performed with isolated corticoadrenal cells, PMNCs and omental adipocytes from sham-operated and ovariectomized mice incubated with specific secretagogues. The results indicate that while ovariectomy enhanced TNFα secretion after in vivo administration of LPS, it reduced corticoadrenal response and abrogated LPS-elicited leptin secretion into the circulation. While the corticoadrenal sensitivity to ACTH stimulation was reduced by ovariectomy, the LPS-induced PMNC response was not affected. Exogenous leptin enhanced baseline PMNC function regardless of surgery. Finally, ovariectomy drastically reduced in vitro adipocyte functionality. Our data support the notion that ovariectomy modified neuroendocrine-immune-adipocyte axis function and strongly suggest that ovarian activity could play a pivotal role in the development of an adequate immune defense mechanism after injury.

It has been proposed that ghrelin induces food intake by a mechanism due to the stimulation of hypothalamic NPY-ergic activity. It is recognized that bilateral adrenalectomy (ADX) enhances hypothalamic CRH-ergic function and reduces appetite. Thus, the aim of the present study was to test whether, icv-administered, ghrelin modulates NPY- and CRH-ergic functions after food restriction (FR) and glucocorticoid deprivation. For this purpose, 1 microg ghrelin was administered icv to ad libitum (AL) eating and to corticosterone (B)-depleted (ADX) and -replete (sham and ADX+B) male animals habituated, for 15 d, to FR. Food intake, hypothalamic function, and peripheral ghrelin, ACTH, and B concentrations were evaluated 2 h after ghrelin administration. Results indicate that while icv ghrelin treatment stimulated 2-h food intake in AL rats, it failed to do so in sham- and ADX+B-FR animals; moreover, 2-h food intake was inhibited by icv ghrelin treatment in ADX-FR rats. Regarding peripheral hormone levels: (a) basal circulating ghrelin levels, already enhanced (vs AL rats) by FR, significantly increased 2 h after icv ghrelin treatment in AL and sham-FR rats; (b) central ghrelin treatment stimulated ACTH secretion in circulation of AL and glucocorticoid-replete-FR rats; and (c) B circulating levels remained unchanged after ghrelin treatment, although they were in relation to the food intake condition of rats. Finally, hypothalamic NPY mRNA expression was enhanced by FR and, in response to icv ghrelin treatment, it decreased in ADX-FR rats only. ADX-enhanced hypothalamic CRH mRNA levels were reduced by ghrelin icv administration only when animals received B replacement therapy. Our data indicate an inhibitory effect of hypothalamic ghrelin on NPY-ergic activity in FR rats lacking endogenous glucocorticoid.

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.

Estradiol has been implicated in the regulation of food intake; however, its effect seems to be exerted in a bimodal fashion. We examined whether a single im injection of estradiol valerate (E2V), lastingly effective, could induce changes in parametrial fat function that further induce a new set point of leptin sensitivity in the female rat. E2V induced severe anorexia and loss of body weight between d 4 and 12 posttreatment. E2V rats recovered normal food intake and departing body weights on wk 2 and 3 posttreatment, respectively; however, they did not reach body weights of control rats. On d 61 posttreatment, we found that unfasting E2V, vs control, rats displayed increased E2 and leptin circulating levels; reduced plasma tumor necrosis factor-alpha(TNF-alpha) concentrations; similar circulating levels of glucose, insulin, and triglyceride; and lower parametrial fat mass containing a higher number of adipocytes that, although normal in size, in vitro released more leptin. Metabolic responses to fasting indicated that unlike control animals, E2V rats did not decrease triglyceride circulating levels, and that both groups decreased plasma glucose, leptin, and insulin, but not TNF-alpha, levels. High glucose load experiments indicated that E2V animals displayed a better insulin sensitivity than control rats; did not significantly increase circulating leptin concentrations as control rats did; and, unlike control, significantly decreased plasma triglyceride levels. Our data strongly support a potent acute anorectic effect of E2 and that, after several weeks, E2 modified parametrial fat function and insulin sensitivity, protecting the organism against future unfavorable metabolic conditions., Instituto Multidisciplinario de Biología Celular

We explored whether testosterone influences circulating leptin turnover in rats. Sham-operated male and female rats and 21-d gonadectomized rats treated or not treated with testosterone propionate were used. Anesthetized rats were implanted with an iv catheter, and then blood samples were drawn before and throughout a 60-min period following systemic leptin administration. Plasma testosterone, estradiol, and leptin concentrations were monitored. The results indicated that while gonadectomy blunted circulating concentrations of the homologous sex steroid, testosterone therapy, in gonadectomized rats, restored plasma testosterone concentrations to values found in normal male rats. Pharmacokinetic parameters evaluated during the test indicated the following: First, in the overall pharmacokinetic analyses, testosterone therapy in gonadectomized rats induced a more rapid disappearance of leptin from the circulation. Second, orchidectomy significantly enhanced the area under the curve (AUC) of circulating leptin, an effect fully reversed by testosterone treatment. Third, testosterone treatment in ovariectomized rats significantly decreased the AUC of leptin concentrations. Fourth, while gonadectomy alone did not modify circulating leptin half-life, conversely, testosterone therapy in gonadectomized rats decreased leptin half-life in the circulation. Finally, while orchidectomy reduced leptin body clearance, this parameter was increased by androgen therapy in gonadectomized rats. Our results strongly support that testosterone could play a main role in plasma leptin turnover by increasing leptin clearance rate and shortening plasma leptin half-life.

The present study was designed to assess the effect of maternal undernutrition, during gestation and lactation, on the neuroendocrine [hypothalamo-pituitary-adrenal (HPA)]-immune axis response to endotoxin (LPS) administration. For this purpose, 21-day-old male rats from both well-nourished (WN) and undernourished (UN) mothers were examined 2 h after injection (i.p.) of vehicle alone (VEH) or containing LPS (130 µg/kg BW). Circulating levels of glucose (GLU), ACTH, corticosterone (B), tumor necrosis factor-alpha (TNFα) and leptin were explored. The results indicate that: (a) mother body weight was significantly (p < 0.05) reduced, as a consequence of UN, at the second and third weeks of pregnancy; (b) no differences in basal glycemia were found in the two groups of pups, and LPS treatment did not induce hypoglycemia, in either group; (c) basal plasma ACTH, B and TNFα levels were similar in the two groups, and LPS-induced ACTH, B and TNFα secretions, although severalfold higher than respective VEH values (p < 0.05) in pups from WN mothers, were fully (ACTH and B) and partially (TNFα) abolished in products from UN mothers; (d) both mean body weights and basal plasma leptin levels were significantly (p < 0.05) lower in pups from UN than from WN mothers, and LPS administration did not modify plasma leptin values in products from both groups. In addition, results of dispersed total adrenal cells incubated in vitro indicate that: (a) both basal and ACTH (22 pM)-induced B secretion were significantly (p < 0.05) lower in cells from UN than WN pups, and (b) leptin (100 nM) was able to inhibit partially ACTH-stimulated B output by adrenal gland (AG) cells from WN pups; however, it failed to inhibit ACTH-stimulated glucocorticoid release by AG cells from UN pups. The present results indicate that undernutrition in mothers, during the very critical periods of gestation and lactation, induces in their male pups at weaning: (a) reduced circulating leptin levels and body weight values; (b) metabolic adaptation to normal carbohydrate metabolism; (c) hyporesponsiveness of the HPA and immune (TNFα) axes during endotoxemia, and (d) leptin resistance at the adrenocortical level. This study strongly supports that undernutrition of mothers results in neuroendocrine immune dysfunction of their pups; however, adrenal resistance to the inhibitory effect of leptin on glucocorticoid output is developed, probably as an adaptive mechanism to counteract unfavorable metabolic conditions.

The present study was designed to determine whether the glucocorticoid inhibitory feedback mechanism plays a role in the well-known tolerance of the neuroendocrine-immune axis response to repeated endotoxemia. Adult male rats underwent adrenalectomy (ADX) and were implanted with a subcutaneous corticosterone (compound B, CB, 75 mg) pellet, or sham operated and implanted with a placebo pellet. On the morning of day 8 after surgery (experimental day, D1), all rats received an intravenous injection of lipopolysaccharide (LPS) (25 µg/kg body weight) which was repeated daily until D5. Blood was drawn via intravenous indwelling catheters before (sample time zero) as well as 1, 2, 3 and 4 h after LPS treatment on D1, 3 and 5 for measurements of corticotropin (ACTH), CB, tumor necrosis factor-α (TNF-α) and leptin. In sham animals, tolerance to repeated LPS administration was complete by D5 for the corticotrope axis and the immune response. In addition, LPS was found to stimulate leptin secretion on day 1 in intact rats, an effect that also disappeared thereafter. ADX + CB rats showed only a partial tolerance of the corticotrope axis on D5, whereas tolerance of the immune response was similar to that found in sham animals. Interestingly, the acute stimulation of leptin secretion by LPS in ADX + CB rats was qualitatively similar to that of intact controls on D1, but plasma leptin levels were significantly reduced on D3 and 5 compared to controls. Our results demonstrate that the adrenal response tolerance of the hypothalamo-pituitary-adrenal axis to repeated endotoxemia. In addition, our finding that TNF-α secretion follows the same pattern in sham-operated and in adrenalectomized animals suggests that unlike the corticotrope axis, tolerance of the immune response does not depend upon stimulated CB levels. The decrease in circulating levels of leptin following ADX is consistent with the stimulatory effects of glucocorticoids on leptin secretion. However, our finding of an acute stimulation of leptin secretion by LPS in ADX + CB animals demonstrates that this effect of endotoxemia is at least partially glucocorticoid independent.

Although the pro-adipogenic effect of glucocorticoid (GC) on adipose tissue (AT) precursor cell differentiation is openly accepted, the effect of chronically high peripheral levels of GC on AT mass expansion is not fully understood. In the present study, we aim to assess the in vitro adipogenic capacity of AT precursor cells isolated from retroperitoneal (RP) AT pads of the hypercorticosteronaemic, adult neonatally treated monosodium L-glutamate (MSG) male rat. To ascertain this issue, we explored the in vitro adipogenic process of stromal-vascular fraction (SVF) cells isolated from RPAT pads of 60-day-old MSG rats. The data recorded indicated that RPAT-SVF cells from hypercorticosteronaemic MSG rats, although displaying an enhanced proliferation capacity, differentiated slower than normal cells. This dysfunction was associated with a reduction in key parameters indicative of precursor cell commitment, differentiation capacity and the percentage of fully differentiated adipocytes, with a retarded maturation process. The distorted adipogenic capacity was highly conditioned by RPAT-SVF cells displaying a low committed population and both excessive and reduced expression of anti- (Pref-1 and Wnt-10b) and pro-adipogenic (mineralocorticoid receptor) signals respectively. Notably, the normalization of peripheral corticosterone levels in MSG rats, as a result of bilateral adrenalectomy combined with GC replacement therapy, fully prevented reduced RPAT precursor cell commitment and overall impaired adipogenesis. Our study strongly supports that the impaired adipogenic process observed in the adult hypertrophic obese MSG male rat is a GC-dependent mechanism, thus explaining the unhealthy RPAT expansion observed in human hypertrophic obese phenotypes, such as in the Cushing's syndrome., Facultad de Ciencias Exactas, Instituto Multidisciplinario de Biología Celular, Centro de Endocrinología Experimental y Aplicada

We studied the effect of feeding normal adult male rats with a commercial diet (CD) supplemented with fructose added to the drinking water (10% wt/vol; fructose-rich diet, FRD) on the adipogenic capacity of stromal-vascular fraction (SVF) cells isolated from visceral adipose tissue (VAT) pads. Animals received either the CD or FRD ad libitum for 3 weeks; thereafter, we evaluated the in vitro proliferative and adipogenic capacities of their VAT SVF cells. FRD significantly increased plasma insulin, triglyceride and leptin levels, VAT mass/cell size, and the in vitro adipogenic capacity of SVF cells. Flow cytometry studies indicated that VAT precursor cell population number did not differ between groups; however, the accelerated adipogenic process could result from an imbalance between endogenous pro- and anti-adipogenic SVF cell signals, clearly shifted towards the former. The increased insulin milieu and its intracellular mediator (insulin receptor substrate-1) in VAT pads, and the enhanced SVF cell expression of Zpf423 and PPAR-γ2 (all pro-adipogenic modulators), together with a decreased SVF cell concentration of anti-adipogenic factors (preadipocyte factor-1 and wingless-type MMTV-10b) strongly support this assumption. We hypothesize that the VAT mass expansion recorded in FRD rats results from the combination of initial accelerated adipogenesis and final cell hypertrophy. It remains to be determined whether FRD administration over longer periods of time could perpetuate both processes, or whether cell hypertrophy itself remains responsible for a further VAT mass expansion, as observed in advanced/morbid obesity. This article is protected by copyright. All rights reserved. Fil: Zubiría, María Guillermina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto Multidisciplinario de Biología Celular (i); Argentina Fil: Fariña, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico La Plata. Centro de Endocrinologia Experimental y Aplicada (i); Argentina Fil: Moreno, Griselda Noemí. Universidad Nacional de la Plata. Facultad de Cs.exactas. Departamento de Cs.biologicas. Laboratorio de Invest.del Sistema Inmune; Argentina Fil: Gagliardino, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico La Plata. Centro de Endocrinologia Experimental y Aplicada (i); Argentina Fil: Spinedi, Eduardo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico La Plata. Centro de Endocrinologia Experimental y Aplicada (i); Argentina Fil: Giovambattista, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto Multidisciplinario de Biología Celular (i); Argentina

In the present study, we tested the effect of OS (oxidative stress) inhibition in rats fed on an FRD [fructose-rich diet; 10% (w/v) in drinking water] for 3 weeks. Normal adult male rats received a standard CD (commercial diet) or an FRD without or with an inhibitor of NADPH oxidase, APO (apocynin; 5 mM in drinking water; CD-APO and FRD-APO). We thereafter measured plasma OS and metabolic-endocrine markers, AAT (abdominal adipose tissue) mass and cell size, FA (fatty acid) composition (content and release), OS status, LEP (leptin) and IRS (insulin receptor substrate)-1/IRS-2 mRNAs, ROS (reactive oxygen species) production, NADPH oxidase activity and LEP release by isolated AAT adipocytes. FRD-fed rats had larger AAT mass without changes in body weight, and higher plasma levels of TAG (triacylglycerol), FAs, TBARS (thiobarbituric acid-reactive substance) and LEP. Although no significant changes in glucose and insulin plasma levels were observed in these animals, their HOMA-IR (homoeostasis model assessment of insulin resistance) values were significantly higher than those of CD. The AAT from FRD-fed rats had larger adipocytes, higher saturated FA content, higher NADPH oxidase activity, greater ROS production, a distorted FA content/release pattern, lower insulin sensitivity together with higher and lower mRNA content of LEP and IRS-1-/2 respectively, and released a larger amount of LEP. The development of all the clinical, OS, metabolic, endocrine and molecular changes induced by the FRD were significantly prevented by APO co-administration. The fact that APO treatment prevented both changes in NADPH oxidase activity and the development of all the FRD-induced AAT dysfunctions in normal rats strongly suggests that OS plays an important role in the FRD-induced MS (metabolic syndrome) phenotype. Fil: Fariña, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico La Plata. Centro de Endocrinologia Experimental y Aplicada (i); Argentina Fil: Garcia, Maria Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico La Plata. Centro de Endocrinologia Experimental y Aplicada (i); Argentina Fil: Alzamendi, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto Multidisciplinario de Biología Celular (i); Argentina Fil: Giovambattista, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto Multidisciplinario de Biología Celular (i); Argentina Fil: Marra, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de La Plata; Argentina Fil: Spinedi, Eduardo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto Multidisciplinario de Biología Celular (i); Argentina Fil: Gagliardino, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico La Plata. Centro de Endocrinologia Experimental y Aplicada (i); Argentina

We have previously addressed that fructose rich diet (FRD) intake for three weeks increases the adipogenic potential of stromal vascular fraction cells from the retroperitoneal adipose tissue (RPAT). We have now evaluated the effect of prolonged FRD intake (eight weeks) on metabolic parameters, number of adipocyte precursor cells (APCs) and in vitro adipogenic potential from control (CTR) and FRD adult male rats. Additionally, we have examined the direct fructose effects on the adipogenic capacity of normal APCs. FRD fed rats had increased plasma levels of insulin, triglyceride and leptin, and RPAT mass and adipocyte size. FACS studies showed higher APCs number and adipogenic potential in FRD RPAT pads; data is supported by high mRNA levels of competency markers: PPARγ2 and Zfp423. Complementary in vitro experiments indicate that fructose-exposed normal APCs displayed an overall increased adipogenic capacity. We conclude that the RPAT mass expansion observed in eight week-FRD fed rats depends on combined accelerated adipogenesis and adipocyte hypertrophy, partially due to a direct effect of fructose on APCs., Facultad de Ciencias Médicas

Aim. To test the potential role of PPARγ in the endocrine abdominal tissue dysfunction induced by feeding normal rats with a fructose rich diet (FRD) during three weeks. Methodology. Adult normal male rats received a standard commercial diet (CD) or FRD, (10% in drinking water) without or with pioglitazone (PIO) (i.p. 0.25mg/Kg BW/day; CD-PIO and FRD-PIO). Thereafter, we measured circulating metabolic, endocrine, and oxidative stress (OS) markers, abdominal adipose tissue (AAT) mass, leptin (LEP) and plasminogen activator inhibitor-1 (PAI-1) tissue content/expression, and leptin release by isolated adipocytes incubated with different concentrations of insulin. Results. Plasma glucose, insulin, triglyceride, TBARS, LEP, and PAI-1 levels were higher in FRD rats; PIO coadministration fully prevented all these increments. AAT adipocytes from FRD rats were larger, secreted a higher amount of LEP, and displayed decreased sensitivity to insulin stimulation; these effects were significantly ameliorated by PIO. Whereas AAT LEP and PAI-1 (mRNA) concentrations increased significantly in FRD rats, those of insulin-receptor-substrate- (IRS-) 1 and IRS-2 were reduced. PIO coadministration prevented FRD effects on LEP, PAI-1, and IRS-2 (fully) and IRS-1 (partially) mRNAs in AAT. Conclusion. PPARγ would play a relevant role in the development of the FRD-induced metabolicendocrine dysfunction., Facultad de Ciencias Médicas

The effect of progesterone (P4) on fructose rich diet (FRD) intake-induced metabolic, endocrine and parametrial adipose tissue (PMAT) dysfunctions was studied in the adult female rat. Sixty day-old rats were i.m. treated with oil alone (control, CT) or containing P4 (12 mg/kg). Rats ate Purina chow-diet ad libitum throughout the entire experiment and, between 100 and 120 days of age drank ad libitum tap water alone (normal diet; CT-ND and P4-ND) or containing fructose (10% w/v; CT-FRD and P4-FRD). At age 120 days, animals were subjected to a glucose tolerance test or decapitated. Plasma concentrations of various biomarkers and PMAT gene abundance were monitored. P4-ND (vs. CT-ND) rats showed elevated circulating levels of lipids. CT-FRD rats displayed high (vs. CT-ND) plasma concentrations of lipids, leptin, adiponectin and plasminogen activator inhibitor-1 (PAI-1). Lipidemia and adiponectinemia were high (vs. P4-ND) in P4-FRD rats. Although P4 failed to prevent FRD-induced hyperleptinemia, it was fully protective on FRD-enhanced plasma PAI-1 levels. PMAT leptin and adiponectin mRNAs were high in CT-FRD and P4-FRD rats. While FRD enhanced PMAT PAI-1 mRNA abundance in CT rats, this effect was absent in P4 rats. Our study supports that a preceding P4-enriched milieu prevented the enhanced prothrombotic risk induced by FRD-elicited high PAI-1 production., Instituto Multidisciplinario de Biología Celular, Facultad de Ciencias Médicas, Facultad de Ciencias Exactas

The intake of a fructose-rich diet (FRD) in the normal female rat induces features similar to those observed in the human metabolic syndrome phenotype. We studied the impact of FRD administration to mothers on pregnancy outcome. On gestational day (Gd) zero rats were assigned to either group: ad libitum drinking tap water alone (normal diet, ND) or containing fructose (10% w/vol; FRD) through pregnancy; all rats were fed a Purina chow diet ad libitum ND and FRD rats were daily cotreated or not with metformin (60 mg/Kg/day oral; ND + MF and FRD + MF) and submitted to a high glucose load test on Gd 14. Additionally, placentas from different groups were studied on Gd 20. Data indicated that: (1) although FRD rats well tolerated glucose overload, their circulating levels of insulin were significantly higher than in ND rats; (2) the mesometrial triangle blood vessel area was significantly lower in placentas from FRD than ND dams; (3) the detrimental effects of FRD administration to mothers were ameliorated by metformin cotreatment. Our study suggests that excessive intake of fructose during pregnancy enhanced the risk for developing gestational diabetes and subsequent preeclampsia, and that metformin prevented the poor pregnancy outcome induced by FRD., Instituto Multidisciplinario de Biología Celular, Centro de Endocrinología Experimental y Aplicada

Susceptibility to inflammatory disease in infantile Lewis (LEW/N) female rats seems to be related to their impaired hypothalamo-pituitary-adrenal (HPA) axis response to different inflammatory stimuli, while the relative resistance to this type of disease in Fischer (F344/N) female rats is apparently due to their potent HPA axis response to the same stimuli. In the present study, we attempted to elucidate whether there is an impairment in the HPA axis response in the juvenile female LEW/N rat to inflammatory and noninflammatory stimuli, and also to determine whether the endogenous sex-steroid environment influences the HPA axis function in both strains of rats. For these purposes, juvenile F344/N and LEW/N rats of both sexes were submitted to different treatments: (a) inhalation of normal atmosphere or ether vapors for 1 min (Ether); (b) i.p. injection of vehicle alone or containing CRH (0.5 microgram/rat), arginine vasopressin (AVP; 5 micrograms/rat, angiotensin II (AII; 5 micrograms/rat), insulin (INS; 0.3 IU/rat), bacterial lipopolysaccharide (LPS; 100 micrograms/rat) or snake venom (SV; 100 micrograms/rat). Rats were then killed at different time intervals (in min) after treatments: 20 for Ether, AVP and CRH, 30 for AII, 45 for INS, 60 for SV and 120 for LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

The hypothalamic damage induced by neonatal treatment with monosodium l-glutamate (MSG) induces several metabolic abnormalities, resulting in a rat hyperleptinemic–hyperadipose phenotype. This study was conducted to explore the impact of the neonatal MSG treatment, in the adult (120 days old) female rat on: (a) the in vivo and in vitro mineralocorticoid responses to ACTH and angiotensin II (AII); (b) the effect of leptin on ACTH- and AII-stimulated mineralocorticoid secretions by isolated corticoadrenal cells; and (c) abdominal adiposity characteristics. Our data indicate that, compared with age-matched controls, MSG rats displayed: (1) enhanced and reduced mineralocorticoid responses to ACTH and AII treatments, respectively, effects observed in both in vivo and in vitro conditions; (2) adrenal refractoriness to the inhibitory effect of exogenous leptin on ACTH-stimulated aldosterone output by isolated adrenocortical cells; and (3) distorted omental adiposity morphology and function. This study supports that the adult hyperleptinemic MSG female rat is characterized by enhanced ACTH-driven mineralocorticoid function, impaired adrenal leptin sensitivity, and disrupted abdominal adiposity function. MSG rats could counteract undesirable effects of glucocorticoid excess, by developing a reduced AII-driven mineralocorticoid function. Thus, chronic hyperleptinemia could play a protective role against ACTH-mediated allostatic loads in the adrenal leptin resistant, MSG female rat phenotype., Facultad de Ciencias Médicas, Comisión de Investigaciones Científicas de la provincia de Buenos Aires

Hyperandrogenemia predisposes an organism toward developing impaired insulin sensitivity. The aim of our study was to evaluate endocrine and metabolic effects during early allostasis induced by a fructose-rich diet (FRD) in normal (control; CT) and neonatal-androgenized (testosterone propionate; TP) female adult rats. CT and TP rats were fed either a normal diet (ND) or an FRD for 3 weeks immediately before the day of study, which was at age 100 days. Energy intake, body weight (BW), parametrial (PM) fat characteristics, and endocrine/metabolic biomarkers were then evaluated. Daily energy intake was similar in CT and TP rats regardless of the differences in diet. When compared with CT-ND rats, the TP-ND rats were heavier, had larger PM fat, and were characterized by basal hypoadiponectinemia and enhanced plasma levels of non-esterified fatty acid (NEFA), plasminogen activator inhibitor-1 (PAI-1), and leptin. FRD-fed CT rats, when compared with CT-ND rats, had high plasma levels of NEFA, triglyceride (TG), PAI-1, leptin, and adiponectin. The TP-FRD rats, when compared with TP-ND rats, displayed enhanced leptinemia and triglyceridemia, and were hyperinsulinemic, with glucose intolerance. The PM fat taken from TP rats displayed increase in the size of adipocytes, decrease in adiponectin (protein/gene), and a greater abundance of the leptin gene. PM adipocyte response to insulin was impaired in CT-FRD, TP-ND, and TP-FRD rats. A very short duration of isocaloric FRD intake in TP rats induced severe metabolic dysfunction at the reproductive age. Our study supports the hypothesis that the early-androgenized female rat phenotype is highly susceptible to developing endocrine/metabolic dysfunction. In turn, these abnormalities enhance the risk of metabolic syndrome, obesity, type 2 diabetes, and cardiovascular disease.

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

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

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.

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.

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

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 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.

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)

Neonatal treatment with monosodium glutamate (MSG) induces severe neuronal damage in selected brain areas, which in turn results in a number of neuroendocrine abnormalities during adult life. The present study was designed to determine what effects this partial and selective denervation of the hypothalamic-pituitary axis may have on the sensitivity of two key components of that axis, the median eminence (ME) and the anterior pituitary (AP). In order to test any changes in response that may occur after MSG treatment, the release of several peptide hormones from either the ME or the AP was evaluated in vitro, employing specific or general secretagogues. Male newborn pups of the Holtzman strain were injected with MSG every other day for 5 days, starting on day 2 of life; littermate controls received an injection of 10% NaCl. All animals were used when adult, at about 5-7 months of age. After decapitation, ME and AP tissues from control and MSG-treated rats were dissected out and incubated in vitro. Release of LHRH, SRIF, arginine vasopressin, and oxytocin from the ME was measured by direct RIA, under basal conditions and after stimulation with high K+ medium (28 mM). The results clearly indicate a marked hyperresponse of the release of each of the four neuropeptides by ME fragments from MSG-lesioned animals. The altered release was not attributable to changes in ME peptide content. In the case of the AP, the release of ACTH, LH, PRL, and GH in response to high K+ or, in some cases, to specific releasing factors, was evaluated in a dispersed cell preparation. The release of all four protein hormones was increased by high K+, and again the MSG-lesioned rats showed a pronounced hyperresponse. Corticotropin-releasing factor, at concentrations of 10(-9) and 10(-8) M enhanced ACTH release from control and MSG-lesioned rats, but the latter presented a marked hyperresponse. A similar observation on LH release was seen after stimulation with LHRH (10(-9) M). The results indicate that the neuronal damage induced by neonatal MSG treatment results in a generalized hyperresponsiveness in vitro to either specific or general secretagogues by ME or AP tissues, which may suggest the development of a denervation-type supersensitivity in the hypothalamic-pituitary axis. Since the release of these peptide hormones is sluggish in MSG-lesioned rats under in vivo conditions, it seems plausible to conclude that the major defect after the neurotoxin damage may reside in the loss of neurotransmitter systems normally innervating and regulating the activity of the peptidergic neurons.