Sexual dimorphism modulates metabolic and cognitive alterations under HFD nutrition and chronic stress exposure in mice. Correlation between spatial memory impairment and BDNF mRNA expression in hippocampus and spleen.

AIMS: The accumulated evidence suggests that lifestyle - specifically dietary habits and stress exposure - plays a detrimental role in health. The purpose of the present study was to analyze the interplay of stress, diet, and sex in metabolic and cognitive alterations. MAIN METHODS: For this purpose, one-month-old C57Bl/6J mice were fed with a standard diet or high-fat diet (HFD). After eight weeks, one subgroup of mice from each respective diet was exposed to 20 weeks of chronic mild stress (CMS), whilst the others were left undisturbed. KEY FINDINGS: After 28 weeks of HFD feeding, mice from both sexes were overweight, with an increase in caloric intake and abdominal and subcutaneous fat pads. Stress exposure induced a decrease in body weight, related to a decrease in caloric efficiency in both males and females. Results indicate that males are more susceptible than the females in modulating metabolic and cognitive functions under HFD and CMS. Although both sexes demonstrated HFD-induced weight gain, fat accumulation, insulin resistance, high cholesterol, only males exposed to CMS but not females have (i) impaired glucose tolerance with higher glucose level; (ii) significant prolonged latency in Barnes test, suggesting cognitive impairment; (iii) increased IFN-gamma expression in hippocampus, suggesting greater neuroinflammatory response; (iv) poorer cognitive performance related to a decrease in hippocampal and spleen BDNF mRNA expression. SIGNIFICANCE: The main finding in this study is the presence of a sexual dimorphism in modulating metabolic and cognitive functions under HFD and CMS, showing males are more susceptible than females. In addition, poorer cognitive performance was related to a decrease in hippocampal BDNF mRNA expression. Interestingly, these changes were observed in the spleen as well.

Oligonucleotide IMT504 Improves Glucose Metabolism and Controls Immune Cell Mediators in Female Diabetic NOD Mice.

Type 1 diabetes occurs as a consequence of progressive autoimmune destruction of beta cells. A potential treatment for this disease should address the immune attack on beta cells and their preservation/regeneration. The objective of this study was to elucidate whether the immunomodulatory synthetic oligonucleotide IMT504 was able to ameliorate diabetes in NOD mice and to provide further understanding of its mechanism of action. We found that IMT504 restores glucose homeostasis in a diabetes mouse model similar to human type 1 diabetes, by regulating expression of immune modulatory factors and improving beta cell function. IMT504 treatment markedly improved fasting glycemia, insulinemia, and homeostatic model assessment of beta cell function (HOMA-Beta cell) index. Moreover, this treatment increased islet number and decreased apoptosis, insulitis, and CD45+ pancreas-infiltrating leukocytes. In a long-term treatment, we observed improvement of glucose metabolism up to 9 days after IMT504 cessation and increased survival after 15 days of the last IMT504 injection. We postulate that interleukin (IL)-12B (p40), possibly acting as a homodimer, and Galectin-3 (Gal-3) may function as mediators of this immunomodulatory action. Overall, these results validate the therapeutic activity of IMT504 as a promising drug for type 1 diabetes and suggest possible downstream mediators of its immunomodulatory effect.

In vivo xenoestrogenic actions of cadmium and arsenic in anterior pituitary and uterus.

Cadmium (Cd) and arsenic (iAs) are toxic metals ubiquitously present in the environment. Both pollutants exert nonmonotonic dose responses, being mostly cytotoxic at high concentrations but mimicking estrogen (E2) effects at low doses. Xenoestrogenic activity of Cd and iAs has been demonstrated in different hormone-dependent tumor cell lines; however, their actions in vivo remain largely unknown. Here, we investigated whether in vivo administration of low doses of Cd and iAs through drinking water would display xenoestrogenic effects in the anterior pituitary gland and uterus of ovariectomized rats. Cd (1ppm) and iAs (0.1ppm) exposure increased the wet weight of anterior pituitary gland and uterus and induced proestrus- and estrus-like vaginal smears. Both metals stimulate cell proliferation of these tissues as they increased the expression of proliferation markers. More importantly, they augmented soluble guanylyl cyclase α1 subunit expression, which has been linked to hormone-dependent tumor progression. Also, Cd and iAs modified protein levels of full-length estrogen receptor α and its truncated variants in an E2-like manner. Anterior pituitary hormone secretion was differentially affected by both metals. Luteinizing hormone synthesis and release were strongly diminished after Cd exposure and only mildly reduced by iAs. Both metals were able to increase prolactin synthesis, although only iAs augmented serum prolactin levels. This study shows for the first time that Cd and iAs exert strong xenoestrogenic effects on anterior pituitary gland at low doses. The differences between Cd and iAs E2-like behavior indicate that other Cd- and iAs-specific mechanisms could be involved. Altogether, these results contribute to the knowledge of reproductive disorders associated with Cd and iAs environmental contamination.

Proposed mechanisms for oligonucleotide IMT504 induced diabetes reversion in a mouse model of immunodependent diabetes.

Type 1 diabetes (T1D) originates from autoimmune ß-cell destruction. IMT504 is an immunomodulatory oligonucleotide that increases mesenchymal stem cell cloning capacity and reverts toxic diabetes in rats. Here, we evaluated long-term (20 doses) and short-term (2-6 doses) effects of IMT504 (20 mg·kg(-1)·day(-1) sc) in an immunodependent diabetes model: multiple low-dose streptozotocin-injected BALB/c mice (40 mg·kg(-1)·day(-1) ip for 5 consecutive days). We determined blood glucose, glucose tolerance, serum insulin, islet morphology, islet infiltration, serum cytokines, progenitor cell markers, immunomodulatory proteins, proliferation, apoptosis, and islet gene expression. IMT504 reduced glycemia, induced ß-cell recovery, and impaired islet infiltration. IMT504 induced early blood glucose decrease and infiltration inhibition, increased ß-cell proliferation and decreased apoptosis, increased islet indoleamine 2,3-dioxygenase (IDO) expression, and increased serum tumor necrosis factor and interleukin-6 (IL-6). IMT504 affected islet gene expression; preproinsulin-2, proglucagon, somatostatin, nestin, regenerating gene-1, and C-X-C motif ligand-1 cytokine (Cxcl1) increased in islets from diabetic mice and were decreased by IMT504. IMT504 downregulated platelet endothelial cell adhesion molecule-1 (Pecam1) in islets from control and diabetic mice, whereas it increased regenerating gene-2 (Reg2) in islets of diabetic mice. The IMT504-induced increase in IL-6 and islet IDO expression and decreased islet Pecam1 and Cxcl1 mRNA expression could participate in keeping leukocyte infiltration at bay, whereas upregulation of Reg2 may mediate ß-cell regeneration. We conclude that IMT504 effectively reversed immunodependent diabetes in mice. Corroboration of these effects in a model of autoimmune diabetes more similar to human T1D could provide promising results for the treatment of this disease.

In Vivo and In Vitro Arsenic Exposition Induces Oxidative Stress in Anterior Pituitary Gland.

Inorganic arsenic (iAs) is at the top of toxic metalloids. Inorganic arsenic-contaminated water consumption is one of the greatest environmental health threats worldwide. Human iAs exposure has been associated with cancers of several organs, neurological disorders, and reproductive problems. Nevertheless, there are no reports describing how iAs affects the anterior pituitary gland. The aim of this study was to investigate the mechanisms involved in iAs-mediated anterior pituitary toxicity both in vivo and in vitro. We showed that iAs administration (from 5 to 100 ppm) to male rats through drinking water increased messenger RNA expression of several oxidative stress-responsive genes in the anterior pituitary gland. Serum prolactin levels diminished, whereas luteinizing hormone (LH) levels were only affected at the higher dose tested. In anterior pituitary cells in culture, 25 µmol/L iAs significantly decreased prolactin release in a time-dependent fashion, whereas LH levels remained unaltered. Cell viability was significantly reduced mainly by apoptosis evidenced by morphological and phosphatidylserine externalization studies. This process is characterized by early depolarization of mitochondrial membrane potential and increased levels of reactive oxygen species. Expression of some key oxidative stress-responsive genes, such as heme oxygenase-1 and metallothionein-1, was also stimulated by iAs exposure. The antioxidant N-acetyl cysteine prevented iAs-induced effects on the expression of oxidative stress markers, prolactin release, and apoptosis. In summary, the present work demonstrates for the first time that iAs reduces prolactin release both in vivo and in vitro and induces apoptosis in anterior pituitary cells, possibly resulting from imbalanced cellular redox status.

GABA(B) receptors in neuroendocrine regulation.

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA(A) and GABA(C) (ionotropic receptors) and GABA(B) (metabotropic receptor). Here we reviewed the participation of GABA(B) receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA(B1) knock-out mice, that lack functional GABA(B) receptors. Our general conclusion indicates that GABA(B )receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA(B) receptor activation, as demonstrated in the rat and also in the GABA(B1) knock-out mouse. In addition, hypothalamic and pituitary GABA(B) receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA(B) receptors depends on physiological conditions, being age and sex critical factors.These results indicate that patients receiving GABA(B) agonists/antagonists should be monitored for possible endocrine side effects.

Adenohypophyseal and hypothalamic GABA B receptor subunits are downregulated by estradiol in adult female rats.

Gamma-aminobutyric acid (GABA) participates in neuroendocrine regulation. Since steroid hormones have been shown to modulate the GABAergic system, here we evaluated the effect of chronic in vivo estradiol administration on GABA B receptor (GABA(B)R) expression. GABA(B1) and GABA(B2) subunits were analyzed by Western Blot and RT-PCR, in hypothalami and anterior pituitaries of adult female rats: a) treated for 1 week with estradiol-valerate (a single dose of 100 mug /kg: E1), b) implanted with a 10 mg pellet of estradiol-benzoate for 5 weeks (E5) or c) on proestrous (P), d) ovariectomized (OVX). Pituitary GABA(B)R levels were correlated to a biological effect: baclofen, a GABA(B)R agonist, action on intracellular calcium titers ([Ca(2+)](i)) in pituitary cells. E5 pituitaries showed a significant decrease in the expression of GABA(B1) and GABA(B2) mRNAs compared to P. The GABA(B1a) splice variant of GABA(B1) was always more abundant than GABA(B1b) in this tissue. Similar to the pituitary, hypothalamic GABA(B1) and GABA(B2) mRNAs decreased in E5; this was confirmed at the protein level. In the hypothalamus GABA(B1b) was the main variant expressed in P rats, and was the one significantly sensitive to estradiol-induced decrease, as determined by Western Blots. Castration did not modify GABA(B)R expression with regards to P in either tissue. In P pituitary cells baclofen induced a decrease in [Ca(2+)](i), in contrast this effect was lost in E5 cells. We conclude that chronic estradiol treatment negatively regulates the expression of the GABA(B)R subunits in the pituitary and the hypothalamus. This effect is coupled to a loss of baclofen action on intracellular calcium in pituitary cells.

Expression of gamma-aminobutyric acid B receptor subunits in hypothalamus of male and female developing rats.

GABA and its receptors show particular ontogenic distributions in different rat brain areas. Recently, GABAB receptors (GBR) have been described to assemble as heterodimers formed by a GBR1a/b and a GBR2 subunit. Here, the ontogeny of rat GBRs and the pattern of subunit expression in both sexes were determined in the hypothalamus, a critical area for homeostatic regulation. Male and female rats were sacrificed at 1, 4, 12, 20, 28, 38 days of life and at adulthood and hypothalami were removed and frozen. Western blots analysis for GBR1 and GBR2 subunits showed that both were expressed in male and female hypothalamic membranes from day 1 to adulthood. In females, both GBR1a and GBR1b were maximally expressed in newborns and decreased towards adulthood. At birth, expression of GBR1a was significantly higher than GBR1b, while at 38 days, GBR1b was more abundant. In males, GBR1a and GBR1b expression was higher in young animals and decreased gradually showing adult levels between the second and third weeks of age without differences between isoforms. Comparing GBR1 variants levels in hypothalamus between sexes, GBR1a was significantly more abundant in females at birth while at 38 days its expression was higher in males; GBR1b showed no sex differences along development. GBR2 was detected in hypothalami of females and males at all ages; maximum levels were observed at 12 days and adult levels were attained at 38 days, without sex differences. This is the first report on the ontogeny of hypothalamic GABAB receptors in male and female rats, with a particular developmental pattern of subunit and isoform expression and presenting some sex differences.

Effect of androgens on sexual differentiation of pituitary gamma-aminobutyric acid receptor subunit GABA(B) expression.

Previous work demonstrated a sexually dimorphic ontogenic expression of gamma-aminobutyric acid receptors (GABA(B)R) in rat pituitary. As sex steroids determine sex-specific expression patterns, we now studied the effect of sex hormones on pituitary GABA(B)R expression. GABA(B)R subunits, measured by Western blot and by semi-quantitative RT-PCR and luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone measured by RIA were determined in two experimental designs: First experimental design: 8- and 15-day-old females (8F, 15F); 8F and 15F treated with 100 mug testosterone propionate (TP) on day 1 of life (8F100TP, 15F100TP), 8- and 15-day-old males (8M, 15M) and 8M and 15M castrated on day 1 (8MC, 15MC). Second experimental design: 8-day-old female and male animals: 8F, 8F100TP, 8F treated with 1 mug/day TP on days 1-4 (8F1TP), 8F treated with the androgen antagonist Flutamide (Flut: 2.5 mg/100 g BW of pregnant mother on days E17-E23) (8F-Flut), 8M, 8MC, 8M treated with Flut as above (8M-Flut) and 8MC-Flut. In these animals, in addition, GABA, glutamate, aspartate and taurine were measured by HPLC in hypothalami and cortex. In the first set of experiments, GABA(B1)R mRNA/protein expression was higher in 8F than in 15F, 8M or 15M. In 8F100TP, GABA(B1)R mRNA/protein decreased to male levels. TP treatment did not alter GABA(B1)R expression in 15F. There was no difference in GABA(B1)R expression between 8M and 15M and neonatal castration did not modify its expression. In the second set of experiments, TP (1 mug) or Flut did not modify GABA(B1)R in 8F, while 100 microg TP continued to decrease GABA(B1)R expression. In 8M, Flut, alone or with castration, increased GABA(B1)R mRNA/protein expression to 8F. Hypothalamic GABA content followed the same pattern as pituitary GABA(B)R expression in 8-day-old animals, suggesting a cross-regulation. With regard to hormonal levels, 100 microg, but not 1 microg TP altered gonadotropins at 8 days, although both treatments effectively androgenized females as evidenced by lack of cycling. We conclude that androgens, acting pre- and postnatally, decrease pituitary GABA(B)R subunit expression.

Guinea pig gonadotropin-releasing hormone: expression pattern, characterization and biological activity in rodents.

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating vertebrate reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. The first form of GnRH to be identified was isolated from mammals (mGnRH) and the same form has been reported for all mammals studied, which includes marsupials and placental mammals. Later, another variant, chicken GnRH-II (cGnRH-II) was shown to be expressed together with mGnRH in the brains of all jawed vertebrates, including mammals such as rats, monkeys and humans. Our objective was to characterize a third form of GnRH that was isolated previously as mRNA from guinea pigs (gpGnRH), but has not been reported for any other mammal to date. Furthermore, the gonadotropic activity of gpGnRH has not been fully characterized. Our results, using chromatographical and immunological methods, show for the first time that gpGnRH is expressed together with mGnRH in some rodents (wild guinea pig and capybara), but not in others (mouse and hamster). Also, the gonadotropic activity of gpGnRH and mGnRH was tested in two different rat cell culture systems. Although there have been reports that the salmon(s) form of GnRH is present in mammals, we did not detect sGnRH in capybara, wild guinea pigs, hamsters, rats or mice. Taken together with previous reports, the present results support the idea that the expression of multiple GnRH variants in a single species is a common pattern in most vertebrate groups.