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Prolonged fasting is a major challenge for living organisms. An appropriate metabolic response to food deprivation requires the activation of the corticotropin-releasing factor-producing neurons of the hypothalamic paraventricular nucleus (PVHWe estimated the activation of the PVHWe found that food deprivation results in the activation of the PVHFood deprivation-induced activation of the PVH

White adipose tissue (WAT) browning has gained interest due to its impact in obesity. Here, we evaluated the effect of androgens on the Ucp1-dependent thermogenic process from inguinal (IAT) and retroperitoneal (RPAT) WAT. Surgically androgens depleted rats (ODX) showed basal thermogenic activation (room temperature) in both WAT depots, which expressed higher levels of Ucp1, Prdm16 and Pgc1a. WAT pads from ODX cold-exposed rats (ODX-C) expressed increased levels of Ucp1 and Pgc1a and showed high UCP1 protein content. In primary beige adipocyte cultures, testosterone decreased the mitochondrial marker Cox8b and mitochondrial content. Finally, testosterone and dihydrotestosterone (DHT) decreased the expression of Ucp1, Pcg1a and Prdm16 in forskolin-stimulated beige adipocytes, an effect that was prevented by the antiandrogen flutamide. In conclusion, androgen deficient rats developed WAT depots with enhanced basal and cold-stimulated thermogenic activity. Additionally, in vitro androgen treatments inhibited the thermogenic program, effect which was mediated by the androgen receptor pathway.

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.

Spexin (SPX) is a novel adipokine playing an emerging role in metabolic diseases due to its involvement in carbohydrate homeostasis, weight loss, appetite control, gastrointestinal movement, among others. Moreover, plasma levels are reduced in obese and type II diabetic patients. In vitro, SPX favors lipolysis in adipocytes and hepatocytes and inhibits white adipogenesis. Therefore, the aim of this study was to evaluate the role of SPX in white adipose tissue (AT) thermogenesis. C57BL/6J male mice were treated or not with SPX for ten days (ip. 29 µg/kg/day; CTR and SPX). At day 3 mice were randomly divided: a group was kept at room temperature (RT) and the other at 4°C to stimulate thermogenesis (CTR-C and SPX-C). Caloric intake and body weight was daily recorded. At the end of the protocol plasma, Brown AT (BAT), abdominal AT (Epidydimal, EAT) and subcutaneous AT (Inguinal, IAT) depots were collected for several measurements. We found that caloric intake was increased when animals were exposed to cold (P

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.

Spexin (SPX) is a novel adipokine related to many metabolic effects, such as gastrointestinal movements, insulin and glucose homeostasis, lipid metabolism and energy balance. This study evaluates the role of SPX in the improvement of the metabolic and inflammatory profile in fructose-rich-diet obese mice. Adult Swiss mice were supplemented or not with fructose (20% in tap water, FRD and CTR, respectively) for 10 weeks. The last ten days, mice were treated or not with SPX (ip. 29 μg/Kg/day, FRD-SPX and CTR-SPX, respectively). A positive correlation was observed between body weight prior to treatment and weight loss after SPX challenge. Moreover, plasma and liver triglycerides and adipose tissue (AT) features (mass, adipocyte hypertrophy, mRNA of leptin) were improved. SPX also induced a reduction in epididymal AT (EAT) expression of TNFα, IL1β and IL6 and an improvement in IL10 and CD206. M1 macrophages in EAT, principally the Ly6C− populations (M1a and M1b), were decreased. Adipocytes from FRD-SPX mice induced less macrophage activation (IL6, mRNA and secretion) than FRD after overnight co-culture with the monocyte cell line (RAW264.7) in stimulated conditions (M1 activation, LPS 100 ng/mL). Finally, in vitro, monocytes pre-incubated with SPX and stimulated with LPS showed decreased inflammatory mRNA markers compared to monocytes with LPS alone. In conclusion, SPX decreased body weight and improved the metabolic profile and adipocyte hypertrophy. Inflammatory Ly6C− macrophages decreased, together with inflammatory marker expression. In vitro studies demonstrate that SPX induced a decrease in M1 macrophage polarization directly or through mature adipocytes.

Fructose-rich diet (FRD) has been associated with obesity development, which is characterized by adipocytes hypertrophy and chronic low-grade inflammation. Interaction of adipocytes and immune cells plays a key role in adipose tissue (AT) alterations in obesity. We assessed the metabolic and immune impairments in AT in a murine obesity model induced by FRD at different periods. Adult Swiss mice were divided into groups of 6 and 10 weeks of fructose (FRD 6wk, FRD 10wk) or water intake (CTR 6wk, CTR 10wk). FRD induced increased in body weight, epidydimal AT mass, and plasmatic and liver Tg, and impaired insulin sensitivity. Also, hypertrophic adipocytes from FRD 6wk-10wk mice showed higher IL-6 when stimulated with LPS and leptin secretion. Several of these alterations worsened in FRD 10wk. Regarding AT inflammation, FRD mice have increased TNFα, IL-6 and IL1β, and decrease in IL-10 and CD206 mRNA levels. Using CD11b, LY6C, CD11c and CD206 as macrophages markers, we identified for first time in AT M1 (M1a: Ly6C+/−CD11c+CD206− and M1b: Ly6C+/−CD11c+CD206+) and M2 subtypes (Ly6C+/−CD11c−CD206+). M1a phenotype increased from 6 weeks onward, while Ly6C+/− M1b phenotype increased only after 10 weeks. Finally, co-culture of RAW264.7 (monocytes cell line) and CTR or FRD adipocytes showed that FRD 10wk adipocytes increased IL-6 expression in non- or LPS-stimulated monocytes. Our results showed that AT dysfunction got worse as the period of fructose consumption was longer. Inflammatory macrophage subtypes increased depending on the period of FRD intake, and hypertrophic adipocytes were able to create an environment that favored M1 phenotype in vitro., Instituto Multidisciplinario de Biología Celular, Instituto de Estudios Inmunológicos y Fisiopatológicos

Rats neonatally treated withmonosodiumL-glutamate (MSG) are deeply dysfunctional in adulthood. We explored the effect of an oral low dose of metformin treatment in male MSG rats on adipoinsular axis and visceral adipose tissue (VAT) dysfunctions, in both basal (nonfasting) and endotoxemia conditions. MSG rats, treated or not treated with metformin (30 days prior to experimentation), and control litter-mates (CTR) were studied at 90 days of age. Peripheral concentrations of glucose, lipids, and hormones were determined in basal and post-lipopolysaccharide (LPS) treatment conditions. Food intake and body weight (BW) were recorded and VAT mass and leptin mRNA levels were evaluated. Data indicated that MSG rats were lighter and displayed hypercorticosteronemia, hypophagia, adipoinsular axis hyperactivity, and enhanced VAT mass associated with an increased leptin gene expression. Interestingly,metformin-treatedMSG rats corrected BWcatch-up and counteracted VAT (mass and leptinmRNA level) and adipoinsular axis (basal and post-LPS) dysfunctions. Thus metformin treatment in MSG rats is able to correct several VAT and metabolic-endocrine dysfunctions. Our study suggests that a low-dose metformin therapy is effective to correct, at least in part, adipoinsular axis dysfunction in hypertrophic obese phenotypes, such as that of the human Cushing syndrome., Facultad de Ciencias Médicas

Modern lifestyle and diets have been associated with metabolic disorders and an imbalance in the normal gut microbiota. Probiotics are widely known for their health beneficial properties targeting the gut microbial ecosystem. The aim of our study was to evaluate the preventive effect of Lactobacillus kefiri (L. kefiri) administration in a fructose-rich diet (FRD) mice model. Mice were provided with tap water or fructose-added (20% w/v) drinking water supplemented or not with L. kefiri. Results showed that probiotic administration prevented weight gain and epidydimal adipose tissue (EAT) expansion, with partial reversion of the adipocyte hypertrophy developed by FRD. Moreover, the probiotic prevented the increase of plasma triglycerides and leptin, together with the liver triglyceride content. Leptin adipocyte secretion was also improved by L. kefiri, being able to respond to an insulin stimulus. Glucose intolerance was partially prevented by L. kefiri treatment (GTT) and local inflammation (TNFα; IL1β; IL6 and INFγ) was completely inhibited in EAT. L. kefiri supplementation generated an impact on gut microbiota composition, changing Bacteroidetes and Firmicutes profiles. Overall, our results indicate that the administration of probiotics prevents the deleterious effects of FRD intake and should therefore be promoted to improve metabolic disorders., Facultad de Ciencias Exactas

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

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

Metabolic functions are synchronized by the circadian clock setting daily patterns of food intake, nutrient delivery, and behavioral activity. Here, we study the impact of chronic jet-lag (CJL) on metabolism, and test manipulations aimed to overcome potential alterations. We recorded weight gain in C57Bl/6 mice under chronic 6 h advances or delays of the light-dark cycle every 2 days (ChrA and ChrD, respectively). We have previously reported ChrA, but not ChrD, to induce forced desynchronization of locomotor activity rhythms in mice (Casiraghi et al. ). Body weight was rapidly increased under ChrA, with animals tripling the mean weight gain observed in controls by day 10, and doubling it by day 30 (6% vs. 2%, and 15% vs. 7%, respectively). Significant increases in retroperitoneal and epidydimal adipose tissue masses (172% and 61%, respectively), adipocytes size (28%), and circulating triglycerides (39%) were also detected. Daily patterns of food and water intake were abolished under ChrA. In contrast, ChrD had no effect on body weight. Wheel-running, housing of animals in groups, and restriction of food availability to hours of darkness prevented abnormal increase in body weight under ChrA. Our findings suggest that the observed alterations under ChrA may arise either from a direct effect of circadian disruption on metabolism, from desynchronization between feeding and metabolic rhythms, or both. Direction of shifts, timing of feeding episodes, and other reinforcing signals deeply affect the outcome of metabolic function under CJL. Such features should be taken into account in further studies of shift working schedules in humans., Instituto Multidisciplinario de Biología Celular

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.

Aim: Androgen excess predisposes the organism to develop metabolic–endocrine and reproductive dysfunctions, among them the development of a phenotype resembling that of human Polycystic Ovary Syndrome (PCOS). Methods: We analyzed the impact of a single neonatal (5 day-old) testosterone propionate (TP; s.c. 1.25 mg/female pup) dose on: a) several metabolic–endocrine activities and b) ovarian steroidogenic and granulosa cell (GC) functions and also follicular population in juvenile and adult TP and control (CT) rats. Key findings: Compared to CT rats, TP animals were characterized by: a) accelerated growth, hyperadiposity and hyperleptinemia, b) very early (pre-weaning age) vaginal opening, c) hyperinsulinemia in adult life, d) dysfunctional ovarian steroidogenesis, e) conserved GC functionality in both juveniles (in vitro) and adults (in vivo), and f) estrous cycles arrested at estrus. Finally, histological studies of the ovaries indicated that in TP (vs. CT) rats: i) primary and antral follicle frequencies were 3- and 15-fold higher and lower, respectively, in juveniles and ii) secondary and atretic follicle frequencies were 3- and 5-fold lower and higher, respectively, in adults. Large cystic images without corpus luteum were observed in the ovaries from adult TP rats only. Significance: Our results strongly suggest that transient neonatal hyperandrogenemia induced early misprogramming of metabolic–endocrine and ovarian (steroidogenesis/folliculogenesis) functions. Conversely, TP rats preserved their ovary GC endocrine function. Our results further support the high risk of developing ovarian hyperstimulation syndrome for infertile women with transient/chronic hyperandrogenemia (PCOS) subjected to assisted reproductive technologies. Fil: Ongaro Gambino, Luisina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto Multidisciplinario de Biología Celular (i); Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Salvetti, Natalia Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Giovambattista, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto Multidisciplinario de Biología Celular (i); Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; 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. Universidad Nacional de La Plata; Argentina Fil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Ciencias Veterinarias del Litoral; Argentina. Universidad Nacional del Litoral; Argentina

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.

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

Obesity arises on defective neuroendocrine pathways that increase energy intake and reduce mitochondrial metabolism. In the metabolic syndrome, mitochondrial dysfunction accomplishes defects in fatty acid oxidation and reciprocal increase in triglyceride content with insulin resistance and hyperglycemia. Mitochondrial inhibition is attributed to reduced biogenesis, excessive fission, and low adipokine-AMP-activated protein kinase (AMPK) level, but lateness of the respiratory chain contributes to perturbations. Considering that nitric oxide (NO) binds cytochrome oxidase and inhibits respiration, we explored NO as a direct effector of mitochondrial dysfunction in the leptin-deficient ob/ob mice.A remarkable three- to fourfold increase in neuronal nitric oxide synthase (nNOS) expression and activity was detected by western blot, citrulline assay, electronic and confocal microscopy, flow cytometry, and NO electrode sensor in mitochondria from ob/ob mice. High NO reduced oxygen uptake in ob/ob mitochondria by inhibition of complex IV and nitration of complex I. Low metabolic status restricted β-oxidation in obese mitochondria and displaced acetyl-CoA to fat synthesis; instead, small interference RNA nNOS caused a phenotype change with fat reduction in ob/ob adipocytes.We evidenced that leptin increases mitochondrial respiration and fat utilization by potentially inhibiting NO release. Accordingly, leptin administration to ob/ob mice prevented nNOS overexpression and mitochondrial dysfunction in vivo and rescued leptin-dependent effects by matrix NO reduction, whereas leptin-Ob-Rb disruption increased the formation of mitochondrial NO in control adipocytes. We demonstrated that in ob/ob, hypoleptinemia is associated with critically low mitochondrial p-AMPK and that, oppositely to p-Akt2, p-AMPK is a negative modulator of nNOS.Thereby, defective leptin-AMPK pathway links mitochondrial NO to obesity with complex I syndrome and dysfunctional mitochondria.

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.

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

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

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

It is known that ghrelin and des‐N‐octanoyl (desacyl) ghrelin modulate food intake and adipogenesis in vivo . However, desacyl ghrelin represents the majority of ghrelin forms found in the circulation. The present study explored whether ghrelin gene‐derived compounds could modulate, in vitro , adipocyte endocrine function and preadipocyte differentiation. Retroperitoneal (RP) adipocytes were cultured in the absence or presence of either ghrelin or desacyl ghrelin and in combination with either inhibitors of protein synthesis, insulin, dexamethasone (DXM), or GHSR1a antagonist. The results indicate that both ghrelin forms possess a direct leptin‐releasing activity (LRA) on RP adipocytes and significantly enhanced adipocyte ob mRNA expression. These activities were related and unrelated to the activation of GHSR1a after coincubation with ghrelin and desacyl ghrelin, respectively. Moreover, desacyl ghrelin facilitated RP preadipocyte differentiation. Desacyl ghrelin enhanced cell lipid content, and PPARγ2, and LPL mRNAs expression. The LRAs developed by different substances tested followed a rank order: ghrelin > desacyl ghrelin = insulin ≥ DXM. Additionally, desacyl ghrelin was able to enhance medium glucose consumption by mature adipocytes in culture. These data strongly support that adipogenesis and adipocyte function are processes directly and positively modulated by ghrelin gene‐derived peptides, thus further indicating that, besides their effects on food intake, ghrelin gene‐derived peptides could play an important role on adiposity for maintaining homeostasis.

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

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

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

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

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

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

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

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.