The verified hypothesis assumed that centrally administered neurosteroid, allopregnanolone (AL), could affect basal and/or stress-induced activity of the hypothalamic-pituitary-adrenal (HPA) axis in sheep. Four groups (n = 6 each) of luteal-phase sheep were intracerebroventricularly infused for 3 days with a vehicle without stress (control); a vehicle treated with stressful stimuli (isolation and partial movement restriction) on the third day; AL (4 × 15 µg/60 µL/30 min, at 30-min intervals) treated with stressful stimuli, and AL alone. Simultaneously, the push-pull perfusion of the infundibular nucleus/median eminence and plasma sample collection were performed. After the experiment, the sheep were killed to collect the hypothalamic and anterior pituitary (AP) tissues. Stressful stimuli evoked an increase in the expression of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA in the hypothalamic paraventricular nucleus (PVN), and AVP receptor (V1b) and proopiomelanocortin (POMC) mRNA in the AP; the concentrations of perfusate CRH, and plasma adrenocorticotropic hormone (ACTH) and cortisol compared to controls. Conversely, the expression of the CRH receptor (CRHR1) mRNA in the AP was downregulated. AL decreased the expression of CRH and AVP mRNA in the PVN, and AVPRV1b and POMC mRNA in the AP in stressed sheep, compared to only stressed ones. There was also a reduction in perfusate CRH, and plasma ACTH and cortisol concentrations. AL alone decreased the expression of CRHR1 mRNA in the AP, and plasma cortisol concentration at the beginning of the collection period compared to controls. In conclusion, AL may function centrally as a suppressor of HPA axis activity in stressed sheep.
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Tomasz Misztal, Patrycja Młotkowska, Elżbieta Marciniak and Anna Misztal
Meng Guo, Yuna Li, Yan Wang, Zhenkun Li, Xiaohong Li, Peikun Zhao, Changlong Li, Jianyi Lv, Xin Liu, Xiaoyan Du and Zhenwen Chen
Recent studies raise the possibility that eukaryotic translation elongation factor 1 alpha (eEF1A) may play a role in metabolism. One isoform, eEF1A2, is specifically expressed in skeletal muscle, heart and brain. It regulates translation elongation and signal transduction. Nonetheless, eEF1A2’s function in skeletal muscle glucose metabolism remains unclear. In the present study, suppression subtractive hybridisation showed a decrease in Eef1a2 transcripts in the skeletal muscle of diabetic Mongolian gerbils. This was confirmed at mRNA and protein levels in hyperglycaemic gerbils, and in db/db and high-fat diet-fed mice. Further, this downregulation was independent of Eef1a2 promoter methylation. Interestingly, adeno-associated virus-mediated eEF1A2 overexpression in skeletal muscle aggravated fasting hyperglycaemia, hyperinsulinaemia and glucose intolerance in male diabetic gerbils but not in female gerbil models. The overexpression of eEF1A2 in skeletal muscle also resulted in promoted serum glucose levels and insulin resistance in male db/db mice. Up- and downregulation of eEF1A2 by lentiviral vector transfection confirmed its inhibitory effect on insulin-stimulated glucose uptake and signalling transduction in C2C12 myotubes with palmitate (PA)-induced insulin resistance. Furthermore, eEF1A2 bound PKCβ and increased its activation in the cytoplasm, whereas suppression of PKCβ by an inhibitor attenuated eEF1A2-mediated impairment of insulin sensitivity in insulin-resistant myotubes. Endoplasmic reticulum (ER) stress was elevated by eEF1A2, whereas suppression of ER stress or JNK partially restored insulin sensitivity in PA-treated myotubes. Additionally, eEF1A2 inhibited lipogenesis and lipid utilisation in insulin-resistant skeletal muscle. Collectively, we demonstrated that eEF1A2 exacerbates insulin resistance in male murine skeletal muscle via PKCβ and ER stress.
Rukmani Pandey, Pallavi Shukla, Baby Anjum, Himanshu Pawankumar Gupta, Subhashis Pal, Nidhi Arjaria, Keerti Gupta, Naibedya Chattopadhyay, Rohit A Sinha and Sanghamitra Bandyopadhyay
Estrogen deficiency reduces estrogen receptor-alpha (ERα) and promotes apoptosis in the hippocampus, inducing learning-memory deficits; however, underlying mechanisms remain less understood. Here, we explored the molecular mechanism in an ovariectomized (OVX) rat model, hypothesizing participation of autophagy and growth factor signaling that relate with apoptosis. We observed enhanced hippocampal autophagy in OVX rats, characterized by increased levels of autophagy proteins, presence of autophagosomes and inhibition of AKT-mTOR signaling. Investigating upstream effectors of reduced AKT-mTOR signaling revealed a decrease in hippocampal heparin-binding epidermal growth factor (HB-EGF) and p-EGFR. Moreover, 17β-estradiol and HB-EGF treatments restored hippocampal EGFR activation and alleviated downstream autophagy process and neuronal loss in OVX rats. In vitro studies using estrogen receptor (ERα)-silenced primary hippocampal neurons further corroborated the in vivo observations. Additionally, in vivo and in vitro studies suggested the participation of an attenuated hippocampal neuronal HB-EGF and enhanced autophagy in apoptosis of hippocampal neurons in estrogen- and ERα-deficient conditions. Subsequently, we found evidence of mitochondrial loss and mitophagy in hippocampal neurons of OVX rats and ERα-silenced cells. The ERα-silenced cells also showed a reduction in ATP production and an HB-EGF-mediated restoration. Finally in concordance with molecular studies, inhibition of autophagy and treatment with HB-EGF in OVX rats restored cognitive performances, assessed through Y-Maze and passive avoidance tasks. Overall, our study, for the first time, links neuronal HB-EGF/EGFR signaling and autophagy with ERα and memory performance, disrupted in estrogen-deficient condition.
Ángela Sánchez, Constanza Contreras-Jurado, Diego Rodríguez, Javier Regadera, Susana Alemany and Ana Aranda
Hypothyroidism is often associated with anemia and immunological disorders. Similar defects are found in patients and in mice with a mutated dominant-negative thyroid hormone receptor α (TRα) and in knockout mice devoid of this receptor, suggesting that this isoform is responsible for the effects of the thyroid hormones in hematopoiesis. However, the hematological phenotype of mice lacking also TRβ has not yet been examined. We show here that TRα1/TRβ-knockout female mice, lacking all known thyroid hormone receptors with capacity to bind thyroid hormones, do not have overt anemia and in contrast with hypothyroid mice do not present reduced Gata1 or Hif1 gene expression. Similar to that found in hypothyroidism or TRα deficiency during the juvenile period, the B-cell population is reduced in the spleen and bone marrow of ageing TRα1/TRβ-knockout mice, suggesting that TRβ does not play a major role in B-cell development. However, splenic hypotrophy is more marked in hypothyroid mice than in TRα1/TRβ-knockout mice and the splenic population of T-lymphocytes is not significantly impaired in these mice in contrast with the reduction found in hypothyroidism. Our results show that the overall hematopoietic phenotype of the TRα1/TRβ-knockout mice is milder than that found in the absence of hormone. Although other mechanism/s cannot be ruled out, our results suggest that the unoccupied TRs could have a negative effect on hematopoiesis, likely secondary to repression of hematopoietic gene expression.
Chirine Toufaily, Gauthier Schang, Xiang Zhou, Philipp Wartenberg, Ulrich Boehm, John P Lydon, Ferdinand Roelfsema and Daniel J Bernard
The progesterone receptor (PR, encoded by Pgr) plays essential roles in reproduction. Female mice lacking the PR are infertile, due to the loss of the protein’s functions in the brain, ovary, and uterus. PR is also expressed in pituitary gonadotrope cells, but its specific role therein has not been assessed in vivo. We therefore generated gonadotrope-specific Pgr conditional knockout mice (cKO) using the Cre-LoxP system. Overall, both female and male cKO mice appeared phenotypically normal. cKO females displayed regular estrous cycles (vaginal cytology) and normal fertility (litter size and frequency). Reproductive organ weights were comparable between wild-type and cKO mice of both sexes, as were production and secretion of the gonadotropins, LH and FSH, with one exception. On the afternoon of proestrus, the amplitude of the LH surge was blunted in cKO females relative to controls. Contrary to predictions of earlier models, this did not appear to derive from impaired GnRH self-priming. Collectively, these data indicate that PR function in gonadotropes may be limited to regulation of LH surge amplitude in female mice via a currently unknown mechanism.
Caterina Luana Pitasi, Junjun Liu, Blandine Gausserès, Gaëlle Pommier, Etienne Delangre, Mathieu Armanet, Pierre Cattan, Bruno Mégarbane, Anne-Sophie Hanak, Kamel Maouche, Danielle Bailbé, Bernard Portha and Jamileh Movassat
Islet inflammation is associated with defective β cell function and mass in type 2 diabetes (T2D). Glycogen synthase kinase 3 (GSK3) has been identified as an important regulator of inflammation in different diseased conditions. However, the role of GSK3 in islet inflammation in the context of diabetes remains unexplored. In this study, we investigated the direct implication of GSK3 in islet inflammation in vitro and tested the impact of GSK3 inhibition in vivo, on the reduction of islet inflammation, and the improvement of glucose metabolism in the Goto-Kakizaki (GK) rat, a spontaneous model of T2D. GK rats were chronically treated with infra-therapeutic doses of lithium, a widely used inhibitor of GSK3. We analyzed parameters of glucose homeostasis as well as islet inflammation and fibrosis in the endocrine pancreas. Ex vivo, we tested the impact of GSK3 inhibition on the autonomous inflammatory response of non-diabetic rat and human islets, exposed to a mix of pro-inflammatory cytokines to mimic an inflammatory environment. Treatment of young GK rats with lithium prevented the development of overt diabetes. Lithium treatment resulted in reduced expression of pro-inflammatory cytokines in the islets. It decreased islet fibrosis and partially restored the glucose-induced insulin secretion in GK rats. Studies in non-diabetic human and rat islets exposed to inflammatory environment revealed the direct implication of GSK3 in the islet autonomous inflammatory response. We show for the first time, the implication of GSK3 in islet inflammation and suggest this enzyme as a viable target to treat diabetes-associated inflammation.
Hongyu Su, Xueyi Chen, Yueming Zhang, Linglu Qi, Yun He, Juanxiu Lv, Yingying Zhang, Xiang Li, Jiaqi Tang and Zhice Xu
Cerebral circulation is important in fetal brain development, and angiotensin II (Ang II) plays vital roles in regulation of adult cerebral circulation. However, functions of Ang II in fetal cerebral vasculature and influences of in utero hypoxia on Ang II-mediated fetal cerebral vascular responses are largely unknown. This study investigated the effects and mechanisms of in utero hypoxia on fetal middle cerebral arteries (MCA) via Ang II. Near-term ovine fetuses were exposed to in utero hypoxia, and fetal MCA responses to Ang II were tested for vascular tension, calcium transient, and molecular analysis. Ang II caused significant dose-dependent contraction in control fetal MCA. Ang II-induced MCA constriction was decreased significantly in hypoxic fetuses. Neither losartan (AT1R antagonist, 10−5 mol/L) nor PD123,319 (AT2R antagonist, 10−5 mol/L) altered Ang II-mediated contraction in fetal MCA. Phenylephrine-mediated constriction was also significantly weaker in hypoxic fetuses. Bay K8644 caused similar contractions between the two groups. Protein expression of L-type voltage-dependent calcium channels was unchanged. There were no differences in caffeine-mediated vascular tension or calcium transients. Contraction induced by PDBu (PKC agonist) was obviously weaker in hypoxic MCA. Protein expression of PKCβ was reduced in the hypoxic compared with the control, along with no differences in phosphorylation levels. The results showed that fetal MCA was functionally responsive to Ang II near term. Intrauterine hypoxia reduced the vascular agonist-mediated contraction in fetal MCA, probably via decreasing PKCβ and its phosphorylation, which might play protective effects on fetal cerebral circulation against transient hypoxia.
Jian Ma, Xin He, Yan Cao, Kienan O’Dwyer, Katherine M Szigety, Yuan Wu, Buddha Gurung, Zijie Feng, Bryson W Katona and Xianxin Hua
Protein arginine methyltransferase 5 (PRMT5), a symmetric arginine methyltransferase, regulates cell functions by influencing gene transcription through posttranslational modification of histones and non-histone proteins. PRMT5 interacts with multiple partners including menin, which controls beta cell homeostasis. However, the role of Prmt5 in pancreatic islets, particularly in beta cells, remains unclear. A mouse model with an islet-specific knockout (KO) of the Prmt5 gene was generated, and the influence of the Prmt5 excision on beta cells was investigated via morphologic and functional studies. Beta cell function was evaluated by glucose tolerance test (GTT) and glucose-stimulated insulin secretion (GSIS) test. Beta cell proliferation was evaluated by immunostaining. Gene expression change was determined by real-time qPCR. Molecular mechanisms were investigated in beta cells in vitro and in vivo in Prmt5 KO mice. The results show that islet-specific KO of Prmt5 reduced expression of the insulin gene and impaired glucose tolerance and GSIS in vivo. The mechanistic study indicated that PRMT5 is involved in the regulation of insulin gene transcription, likely via histone methylation-related chromatin remodeling. The reduced expression of insulin in beta cells in the Prmt5 KO mice may contribute to impaired glucose tolerance (IGT) and deficient GSIS in the mouse model. These results will provide new insights into exploring novel strategies to treat diabetes caused by insulin insufficiency.
Md Nurul Islam, Yuichiro Mita, Keisuke Maruyama, Ryota Tanida, Weidong Zhang, Hideyuki Sakoda and Masamitsu Nakazato
Ghrelin, a stomach-derived peptide, promotes feeding and growth hormone (GH) secretion. A recent study identified liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous inhibitor of ghrelin-induced GH secretion, but the effect of LEAP2 in the brain remained unknown. In this study, we showed that intracerebroventricular (i.c.v.) administration of LEAP2 to rats suppressed central ghrelin functions including Fos expression in the hypothalamic nuclei, promotion of food intake, blood glucose elevation, and body temperature reduction. LEAP2 did not inhibit neuropeptide Y (NPY)-induced food intake or des-acyl ghrelin-induced reduction in body temperature, indicating that the inhibitory effects of LEAP2 were specific for GHSR. Plasma LEAP2 levels varied according to feeding status and seemed to be dependent on the hepatic Leap2 expression. Furthermore, ghrelin suppressed the expression of hepatic Leap2 via AMPK activation. Together, these results reveal that LEAP2 inhibits central ghrelin functions and crosstalk between liver and stomach.
Olivier Dumortier, Gaia Fabris, Didier F Pisani, Virginie Casamento, Nadine Gautier, Charlotte Hinault, Patricia Lebrun, Christophe Duranton, Michel Tauc, Stéphane Dalle, Julie Kerr-Conte, François Pattou, Marc Prentki and Emmanuel Van Obberghen
Enhanced beta cell glycolytic and oxidative metabolism are necessary for glucose-induced insulin secretion. While several microRNAs modulate beta cell homeostasis, miR-375 stands out as it is highly expressed in beta cells where it regulates beta cell function, proliferation and differentiation. As glucose metabolism is central in all aspects of beta cell functioning, we investigated the role of miR-375 in this process using human and rat islets; the latter being an appropriate model for in-depth investigation. We used forced expression and repression of mR-375 in rat and human primary islet cells followed by analysis of insulin secretion and metabolism. Additionally, miR-375 expression and glucose-induced insulin secretion were compared in islets from rats at different developmental ages. We found that overexpressing of miR-375 in rat and human islet cells blunted insulin secretion in response to glucose but not to α-ketoisocaproate or KCl. Further, miR-375 reduced O2 consumption related to glycolysis and pyruvate metabolism, but not in response to α-ketoisocaproate. Concomitantly, lactate production was augmented suggesting that glucose-derived pyruvate is shifted away from mitochondria. Forced miR-375 expression in rat or human islets increased mRNA levels of pyruvate dehydrogenase kinase-4, but decreased those of pyruvate carboxylase and malate dehydrogenase1. Finally, reduced miR-375 expression was associated with maturation of fetal rat beta cells and acquisition of glucose-induced insulin secretion function. Altogether our findings identify miR-375 as an efficacious regulator of beta cell glucose metabolism and of insulin secretion, and could be determinant to functional beta cell developmental maturation.