IMT504 protects beta cells against apoptosis and maintains beta cell identity, without modifying proliferation.

We have demonstrated that oligodeoxynucleotide IMT504 promotes significant improvement in the diabetic condition in diverse animal models. Based on these results, here we evaluated whether these effects observed in vivo could be due to direct effects on ß-cells. We demonstrate by immunofluorescence that IMT504 enters the cell and locates in cytoplasm where it induces GSK-3ß phosphorylation that inactivates this kinase. As GSK-3ß tags Pdx1 for proteasomal degradation, by inactivating GSK-3ß, IMT504 induces an increase in Pdx1 protein levels, demonstrated by Western blotting. Concomitantly, an increase in Ins2 and Pdx1 gene transcription was observed, with no significant increase in insulin content or secretion. Enhanced Pdx1 is promising since it is a key transcription factor for insulin synthesis and is also described as an essential factor for the maintenance ß-cell phenotype and function. Dose-dependent inhibition of H2 O2 -induced apoptosis determined by ELISA as well as decreased expression of Bax was also observed. These results were confirmed in another ß-cell line, beta-TC-6 cells, in which a cytokine mix induced apoptosis that was reversed by IMT504. In addition, an inhibitor of IMT504 entrance into cells abrogated the effect IMT504. Based on these results we conclude that the ß-cell recovery observed in vivo may include direct effects of IMT504 on ß-cells, by maintaining their identity/phenotype and protecting them from oxidative stress and cytokine-induced apoptosis. Thus, this work positions IMT504 as a promising option in the framework of the search of new therapies for type I diabetes treatment.

Early Exposure to a High-Fat Diet Impacts on Hippocampal Plasticity: Implication of Microglia-Derived Exosome-like Extracellular Vesicles.

Adolescence is a transitional period from childhood to adulthood characterized by puberty and brain maturation involving behavioral changes and environmental vulnerability. Diet is one of the factors affecting brain health, potentially leading to long-lasting effects. Hence, we studied the impact of early exposure (P21-60) to a high-fat diet (HFD) on mouse hippocampus, analyzing inflammation, adult neurogenesis, dendritic spine plasticity, and spatial memory. Glycemia and seric pro-inflammatory IL1ß were higher in HFD mice without differences on body weight. In the HFD hippocampus, neuroinflammation was evidenced by Iba1+ cells reactivity together with a higher expression of TNFα and IL1ß while the neurogenic capability in the dentate gyrus was strongly reduced. We found a predominance of immature Dil-labeled dendritic spines from CA1 neurons along with diminished levels of the scaffold protein Shank2, suggesting a defective connectivity. Moreover, the HFD group exhibited spatial memory alterations. To elucidate whether microglia could be mediating HFD-associated neuronal changes, the lipotoxic context was emulated by incubating primary microglia with palmitate, a saturated fatty acid present in HFD. Palmitate induced a pro-inflammatory profile as shown by secreted cytokine levels. The isolated exosome fraction from palmitate-stimulated microglia induced an immature dendritic spine phenotype in primary GFP+ hippocampal neurons, in line with the in vivo findings. These results provide novel data concerning microglia to neuron communication and highlight that fat excess during a short and early period of life could negatively impact on cognition and synaptic plasticity in a neuroinflammatory context, where microglia-derived exosomes could be implicated. Graphical Abstract ᅟ.

Evaluation of sodium arsenite exposure on reproductive competence in pregnant and postlactational dams and their offspring.

We investigated arsenite exposure on the reproductive axis of dams (during pregnancy and at cyclicity resumption) and their offspring. Pregnant rats were exposed to 5 (A5) or 50ppm (A50) of sodium arsenite in drinking water from gestational day 1 (GD1) until sacrifice at GD18 or two months postpartum. Offspring were exposed to the same treatment as their mothers from weaning to adulthood. A50-pregnant rats gained less weight, showed increased testosterone and estradiol but pregnancy was unaffected. After lactation, arsenic-exposed dams presented compromised cyclicity, decreased estradiol, increased follicle-stimulating hormone (FSH), less preovulatory follicles and presence of ovarian cysts, suggesting impaired reproduction. A50-offspring presented lower body weight; A50-female-offspring showed elevated gonadotropin releasing hormone (GnRH), FSH and testosterone, while A50-males showed diminished GnRH/FSH, but normal testosterone. We conclude that arsenite at the present exposure levels did not compromise pregnancy outcome while it negatively affected reproductive physiology in postpartum dams and their offspring.

Arsenite in drinking water produces glucose intolerance in pregnant rats and their female offspring.

Drinking water is the main source of arsenic exposure. Chronic exposure has been associated with metabolic disorders. Here we studied the effects of arsenic on glucose metabolism, in pregnant and post-partum of dams and their offspring. We administered 5 (A5) or 50 (A50) mg/L of sodium arsenite in drinking water to rats from gestational day 1 (GD1) until two months postpartum (2MPP), and to their offspring from weaning until 8 weeks old. Liver arsenic dose-dependently increased in arsenite-treated rats to levels similar to exposed population. Pregnant A50 rats gained less weight than controls and recovered normal weight at 2MPP. Arsenite-treated pregnant animals showed glucose intolerance on GD16-17, with impaired insulin secretion but normal insulin sensitivity; they showed dose-dependent increased pancreas insulin on GD18. All alterations reverted at 2MPP. Offspring from A50-treated mothers showed lower body weight at birth, 4 and 8 weeks of age, and glucose intolerance in adult females, probably due to insulin secretion and sensitivity alterations. Arsenic alters glucose homeostasis during pregnancy by altering beta-cell function, increasing risk of developing gestational diabetes. In pups, it induces low body weight from birth to 8 weeks of age, and glucose intolerance in females, demonstrating a sex specific response.

Juvenile exposure to a high fat diet promotes behavioral and limbic alterations in the absence of obesity.

The incidence of metabolic disorders including obesity, type 2 diabetes and metabolic syndrome have seriously increased in the last decades. These diseases - with growing impact in modern societies - constitute major risk factors for neurodegenerative disorders such as Alzheimer's disease (AD), sharing insulin resistance, inflammation and associated cognitive impairment. However, cerebral cellular and molecular pathways involved are not yet clearly understood. Thus, our aim was to study the impact of a non-severe high fat diet (HFD) that resembles western-like alimentary habits, particularly involving juvenile stages where the brain physiology and connectivity are in plain maturation. To this end, one-month-old C57BL/6J male mice were given either a control diet or HFD during 4 months. Exposure to HFD produced metabolic alterations along with changes in behavioral and central parameters, in the absence of obesity. Two-month-old HFD mice showed increased glycemia and plasmatic IL1ß but these values normalized at the end of the HFD protocol at 5 months of age, probably representing an acute response that is compensated at later stages. After four months of HFD exposure, mice presented dyslipidemia, increased Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, hepatic insulin resistance and inflammation. Alterations in the behavioral profile of the HFD group were shown by the impediment in nest building behavior, deficiencies in short and mid-term spatial memories, anxious and depressive- like behavior. Regarding the latter disruptions in emotional processing, we found an increased neural activity in the amygdala, shown by a greater number of c-Fos+ nuclei. We found that hippocampal adult neurogenesis was decreased in HFD mice, showing diminished cell proliferation measured as Ki67+ cells and neuronal differentiation in SGZ by doublecortin labeling. These phenomena were accompanied by a neuroinflammatory and insulin-resistant state in the hippocampus, depicted by a reactive phenotype in Iba1+ microglia cells (increased in number and soma size) and an impaired response to insulin given by decreased phosphorylated Akt levels and increased levels of inhibitory phosphorylation of IRS1. Our data portray a set of alterations in behavioral and neural parameters as a consequence of an early-life exposure to a quite moderate high fat diet, many of which can resemble AD-related features. These results highly emphasize the need to study how metabolic and neurodegenerative disorders are interrelated in deep, thus allowing the finding of successful preventive and therapeutic approaches.

Hyperprolactinemia induced by hCG leads to metabolic disturbances in female mice.

The metabolic syndrome is a growing epidemic; it increases the risk for diabetes, cardiovascular disease, fatty liver, and several cancers. Several reports have indicated a link between hormonal imbalances and insulin resistance or obesity. Transgenic (TG) female mice overexpressing the human chorionic gonadotropin ß-subunit (hCGß+ mice) exhibit constitutively elevated levels of hCG, increased production of testosterone, progesterone and prolactin, and obesity. The objective of this study was to investigate the influence of hCG hypersecretion on possible alterations in the glucose and lipid metabolism of adult TG females. We evaluated fasting serum insulin, glucose, and triglyceride levels in adult hCGß+ females and conducted intraperitoneal glucose and insulin tolerance tests at different ages. TG female mice showed hyperinsulinemia, hypertriglyceridemia, and dyslipidemia, as well as glucose intolerance and insulin resistance at 6 months of age. A 1-week treatment with the dopamine agonist cabergoline applied on 5-week-old hCGß+ mice, which corrected hyperprolactinemia, hyperandrogenism, and hyperprogesteronemia, effectively prevented the metabolic alterations. These data indicate a key role of the hyperprolactinemia-induced gonadal dysfunction in the metabolic disturbances of hCGß+ female mice. The findings prompt further studies on the involvement of gonadotropins and prolactin on metabolic disorders and might pave the way for the development of new therapeutic strategies.

Postnatal development of the endocrine pancreas in mice lacking functional GABAB receptors.

Adult mice lacking functional GABAB receptors (GABAB1KO) have glucose metabolism alterations. Since GABAB receptors (GABABRs) are expressed in progenitor cells, we evaluated islet development in GABAB1KO mice. Postnatal day 4 (PND4) and adult, male and female, GABAB1KO, and wild-type littermates (WT) were weighed and euthanized, and serum insulin and glucagon was measured. Pancreatic glucagon and insulin content were assessed, and pancreas insulin, glucagon, PCNA, and GAD65/67 were determined by immunohistochemistry. RNA from PND4 pancreata and adult isolated islets was obtained, and Ins1, Ins2, Gcg, Sst, Ppy, Nes, Pdx1, and Gad1 transcription levels were determined by quantitative PCR. The main results were as follows: 1) insulin content was increased in PND4 GABAB1KO females and in both sexes in adult GABAB1KOs; 2) GABAB1KO females had more clusters (<500 µm(2)) and less islets than WT females; 3) cluster proliferation was decreased at PND4 and increased in adult GABAB1KO mice; 4) increased ß-area at the expense of the α-cell area was present in GABAB1KO islets; 5) Ins2, Sst, and Ppy transcription were decreased in PND4 GABAB1KO pancreata, adult GABAB1KO female islets showed increased Ins1, Ins2, and Sst expression, Pdx1 was increased in male and female GABAB1KO islets; and 6) GAD65/67 was increased in adult GABAB1KO pancreata. We demonstrate that several islet parameters are altered in GABAB1KO mice, further pinpointing the importance of GABABRs in islet physiology. Some changes persist from neonatal ages to adulthood (e.g., insulin content in GABAB1KO females), whereas other features are differentially regulated according to age (e.g., Ins2 was reduced in PND4, whereas it was upregulated in adult GABAB1KO females).

Neonatal xenoestrogen exposure alters growth hormone-dependent liver proteins and genes in adult female rats.

The hypothalamic-growth hormone (GH)-liver axis represents a new concept in endocrine regulation of drug toxicity. Preponderant sex differences are found in liver gene expression, mostly dependent on the sexually dimorphic pattern of GH secretion which is set during the neonatal period by gonadal steroids. We tested if GH-dependent sexually dimorphic liver enzymes and proteins was perturbed by neonatal Bisphenol A (BPA) treatment in female rats. Female rats were sc injected with BPA (50 or 500 µg/50 µl) or castor oil vehicle from postnatal day 1 to 10. At five months serum prolactin, pituitary GH, and serum and liver insulin growth factor-I (IGF-I) were measured by RIA. Major urinary proteins (MUPs) were determined by electrophoresis. Liver Cyp2c11, Cyp2c12, Adh1, Hnf6, and Prlr mRNA levels were determined by real time PCR. Pituitary GH content and liver IGF-I concentration were increased by neonatal BPA treatment, indicating partial masculinization of the GH axis in treated females. GH-dependent female predominant liver enzyme genes (Cyp2c12 and Adh1) and a transcription factor (Hnf6) were downregulated or defeminized, while there were no changes in a male predominant gene (Cyp2c11) or protein (MUP). Our findings indicate that perinatal exposure to BPA may compromise the sexually dimorphic capacity of the liver to metabolize drugs and steroids.

Participación de los receptores GABAB en la regulación del eje gonadotrófico: evaluación en ratones GABA / Participation of GABAB receptors on neuroendrocrine regulation in the regulation of the hypothalamic-pituitary-gonadal axis: evaluation in GABA mouse

Hasta el momento, los estudios realizados sobre la participación de los receptores GABAB (REGABAB) en la regulación neuroendocrina habían sido llevados a cabo a través de abordajes farmacológicos, mediante la utilización de agonistas y antagonistas específicos. En el presente trabajo utilizamos el modelo de ratón GABA para analizar las consecuencias endocrinas de la falta constitutiva de los RGABAB en la unidad hipotálamo-hipófiso-gonadal. No observamos diferencias en los contenidos hipofisarios ni en los niveles séricos de LH y FSH entre los genotipos en ningún sexo. Sin embargo, nuestros estudios in vitro, demostraron la existencia de alteraciones de la fisiología de los gonadotropos provenientes de hembras GABA, con una secreción basal aumentada de gonadotropinas y una menor respuesta el estímulo con GnRH. Al analizar más específicamente la funcionalidad del eje en esos ratones, encontramos alteraciones en el aumento de LH postcastración en las hembras, confirmando la participación de los RGABAB en este fenómeno. Por otro lado, en la hemras GABA adultas demostramos la presencia de alteraciones en el contenido hipotalámico de GnRH, el cual estaba francamente disminuido, y su secreción pulsátil, en la que se observa un aumento significativo de la frecuencia de los pulsos de GnRH. También observamos un aumento en los contenidos hipotalámicos de neurotransmisores aminoacídicos que podrían afectar la liberación de GnRH... (AU)

Participación de los receptores GABAB en la regulación del eje gonadotrófico: evaluación en ratones GABA / Participation of GABAB receptors on neuroendrocrine regulation in the regulation of the hypothalamic-pituitary-gonadal axis: evaluation in GABA mouse

Hasta el momento, los estudios realizados sobre la participación de los receptores GABAB (REGABAB) en la regulación neuroendocrina habían sido llevados a cabo a través de abordajes farmacológicos, mediante la utilización de agonistas y antagonistas específicos. En el presente trabajo utilizamos el modelo de ratón GABA para analizar las consecuencias endocrinas de la falta constitutiva de los RGABAB en la unidad hipotálamo-hipófiso-gonadal. No observamos diferencias en los contenidos hipofisarios ni en los niveles séricos de LH y FSH entre los genotipos en ningún sexo. Sin embargo, nuestros estudios in vitro, demostraron la existencia de alteraciones de la fisiología de los gonadotropos provenientes de hembras GABA, con una secreción basal aumentada de gonadotropinas y una menor respuesta el estímulo con GnRH. Al analizar más específicamente la funcionalidad del eje en esos ratones, encontramos alteraciones en el aumento de LH postcastración en las hembras, confirmando la participación de los RGABAB en este fenómeno. Por otro lado, en la hemras GABA adultas demostramos la presencia de alteraciones en el contenido hipotalámico de GnRH, el cual estaba francamente disminuido, y su secreción pulsátil, en la que se observa un aumento significativo de la frecuencia de los pulsos de GnRH. También observamos un aumento en los contenidos hipotalámicos de neurotransmisores aminoacídicos que podrían afectar la liberación de GnRH...

GABA(B1) knockout mice reveal alterations in prolactin levels, gonadotropic axis, and reproductive function.

gamma-Aminobutyric acid (GABA) has been implicated in the control of hypophyseal functions. We evaluated whether the constitutive loss of functional GABA(B) receptors in GABA(B1) knockout (GABA(B1)(-/-)) mice alters hormonal levels, under basal and stimulated conditions, and reproductive function. The serum hormone levels were measured by radioimmunoassay, the estrous cyclicity was evaluated by vaginal lavages, and the mating behavior was determined by the presence of vaginal plugs. A moderate hyperprolactinemic condition was observed, in which prolactin increase and thyroid-stimulating hormone decrease were similar between genotypes. Basal luteinizing hormone (LH), follicle-stimulating hormone, thyroid-stimulating hormone, and growth hormone levels were similar between genotypes in each sex. Analysis of the gonadotropin axis revealed no differences in puberty onset between female genotypes. In con trast, the estrous cyclicity was significantly disrupted in GABA(B1)(-/-) female mice, showing significantly extended periods in estrus and shortened periods in proestrus. Reproduction was significantly compromised in GABA(B1)(-/-) females, with a significantly lower proportion of mice (37.5%) getting pregnant during the first 30 days of mating as compared with wild-type controls (87.5%). Moreover, only 14% of vaginal plug positive GABA(B1)(-/-) females had successful pregnancies as compared with 75% in the controls. In addition, the postovariectomy LH rise was significantly advanced in GABA(B1)(-/-) mice, while the response to estradiol feedback was similar in both genotypes. In conclusion, our endocrine analysis of GABA(B1)(-/-) mice reveals that GABA(B) receptors are involved in the regulation of basal prolactin titers. Moreover, the hypothalamic-hypophyseal-ovarian axis is seriously disturbed, with alterations in cyclicity, postcastration LH increase, and fertility indexes. The molecular mechanism underlying these hormonal disturbances remains to be addressed.