Molecular and Cellular Endocrinology

Creosote bush-derived NDGA attenuates molecular and pathological changes in a novel mouse model of non-alcoholic steatohepatitis (NASH) Publication date: 1 December 2019 Source: Molecular and Cellular Endocrinology, Volume 498 Author(s): Lu Han, Stefanie Bittner, Dachuan Dong, Yuan Cortez, Hunter Dulay, Sara Arshad, Wen-Jun Shen, Fredric B. Kraemer, Salman Azhar Abstract Creosote bush (Larrea tridentata)-derived nordihydroguaiaretic acid (NDGA) was shown to have profound effects on the core components of metabolic syndrome. This study investigated the in vivo potential of NDGA for prevention or attenuation of the pathophysiologic abnormalities of NASH. A novel dietary NASH model with feeding C57BL/6J mice with a high trans-fat, high cholesterol and high fructose (HTF) diet, was used. The HTF diet fed mice exhibited obesity, insulin resistance, hepatic steatosis, fibrosis, inflammation, ER stress, oxidative stress, and liver injury. NDGA attenuated these metabolic abnormalities as well as hepatic steatosis and fibrosis together with attenuated expression of genes encoding fibrosis, progenitor and macrophage markers with no effect on the levels of mRNAs for lipogenic enzymes. NDGA increased expression of fatty acid oxidation genes. In conclusion, NDGA exerts anti-NASH/anti-fibrotic actions and raises the therapeutic potential of NDGA for treatment of NASH patients with fibrosis and other associated complications.

Empaglifozin mitigates NAFLD in high-fat-fed mice by alleviating insulin resistance, lipogenesis and ER stress Publication date: 1 December 2019 Source: Molecular and Cellular Endocrinology, Volume 498 Author(s): Tamiris Ingrid Petito-da-Silva, Vanessa Souza-Mello, Sandra Barbosa-da-Silva Abstract Aim To evaluate the pleiotropic effects of empagliflozin in the liver through lipogenesis, beta-oxidation, and endoplasmic reticulum stress pathways. Methods Male C57Bl/6 mice, 3 months of age, received a control diet (C, 10% lipids, n = 20) or high-fat diet (HF, 50% lipids, n = 20) for 10 weeks, after that, the groups were subdivided to receive empagliflozin, during 5 weeks at a dose of 10 mg/kg/day added to the diets, totalizing four groups: C, C-EMPA, HF, and HF-EMPA. We performed biochemical analyzes, oral glucose tolerance test, homeostasis model assessment of insulin resistance (HOMA-IR), indirect calorimetry, liver stereology, western blotting, RT-qPCR for genes related to beta-oxidation, lipogenesis, and endoplasmic reticulum stress. Results After the treatment with empagliflozin, there was a 4% increase in energy expenditure, a 5% reduction in body mass, improvement in glucose tolerance and insulin sensitivity and insulin resistance. The expression of Ppar alpha was greater in the HF-EMPA group with a concomitant reduction in the expression of the lipogenic genes Fas, Srebp1c and Ppar gamma, according to protein expression. In addition, HF-EMPA showed a reduction in the genes related to endoplasmic reticulum stress Chop, Atf4, and Gadd45. Conclusion Empagliflozin mitigates the development of NAFLD, confirmed through reduced expression of the genes involved in hepatic lipogenesis and genes involved in endoplasmic reticulum stress. Thus, empagliflozin may be an important tool to treat the progression of hepatic steatosis. Graphical abstract NAFLD is associated with increase in lipogenic and inflammation pathways imbalance, with detrimental for beta-oxidation and reticulum endoplasmic function. On the other hand, empagliflozin treatment ameliorates hepatic steatosis through lipogenic pathway reduction, beta-oxidation augmentation and attenuation of endoplasmic reticulum stress and consequently inflammation markers reduction, in high fat fed C57Bl/6 mice.

Adenosine stimulates neuromedin U mRNA expression in the rat pars tuberalis Publication date: 1 October 2019 Source: Molecular and Cellular Endocrinology, Volume 496 Author(s): Sayaka Aizawa, Tingting Gu, Arisa Kaminoda, Ryuya Fujioka, Fumiya Ojima, Ichiro Sakata, Takafumi Sakai, Maho Ogoshi, Sumio Takahashi, Sakae Takeuchi Abstract Neuromedin U (NMU) shows circadian expression in the rat pars tuberalis (PT), and is known to be suppressed by melatonin. Here we examined the involvement of adenosine in the regulation of Nmu expression. We found that the rat PT expressed adenosine receptor A2b and that an adenosine receptor agonist, NECA, stimulated Nmu expression in brain slice cultures. In vitro promoter assays revealed that NECA stimulated Nmu promoter activity via a cAMP response element (CRE) in the presence of adenosine receptor A2b. NECA also increased the levels of phosphorylated CRE-binding protein. These findings suggest that adenosine stimulates Nmu expression by activating the cAMP signaling pathway through adenosine receptor A2b in the rat PT. This is the first report to demonstrate that Nmu expression in the PT is regulated by adenosine, which acts as an intravital central metabolic signal, in addition to melatonin, which acts as an external photoperiodic environmental signal.

Long non-coding RNA GAS5 promotes osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the miR-135a-5p/FOXO1 pathway Publication date: 1 October 2019 Source: Molecular and Cellular Endocrinology, Volume 496 Author(s): Xue Wang, Ding Zhao, Yuzhu Zhu, Ying Dong, Yijun Liu Abstract Studies have shown that promoting the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts could protect against osteoporosis. Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) participate in BMSC osteogenic differentiation. This study aimed to investigate the role and underlying mechanism of growth arrest-specific transcript 5 (GAS5) in osteogenic differentiation. The mechanism was mainly focused on miR-135a-5p/FOXO1 pathway by gain- and loss-of function tests. GAS5 and FOXO1 expression was decreased, whereas miR-135a-5p expression was increased, in the BMSCs from osteoporotic mice. Levels of GAS5 and FOXO1 were increased and miR-135a-5p expression was decreased during osteogenic differentiation of BMSCs. Overexpression of GAS5 promoted, whereas knockdown of GAS5 suppressed, BMSC osteogenic differentiation. As for the mechanism, GAS5 functioned as a competing endogenous RNA for miR-135a-5p to regulate FOXO1 expression. In conclusion, GAS5 promoted osteogenesis of BMSCs by regulating the miR-135a-5p/FOXO1 axis. This finding suggests that targeting GAS5 may be a useful therapy for treating postmenopausal osteoporosis.

Aldosterone, STX and amyloid-β1-42 peptides modulate GPER (GPR30) signalling in an embryonic mouse hippocampal cell line (mHippoE-18) Publication date: 1 October 2019 Source: Molecular and Cellular Endocrinology, Volume 496 Author(s): Peter D. Evans Abstract The GPCR, GPER, mediates many of the rapid, non-genomic actions of 17β-estradiol in multiple tissues, including the nervous system. Controversially, it has also been suggested to be activated by aldosterone, and by the non-steroidal diphenylacrylamide compound, STX, in some preparations. Here, the ability of the GPER agonist, G-1, and aldosterone in the presence of the mineralocorticoid receptor antagonist, eplerenone, to potentiate forskolin-stimulated cyclic AMP levels in the hippocampal clonal cell line, mHippoE-18, are compared. Both stimulatory effects are blocked by the GPER antagonist G36, by PTX, (suggesting the involvement of Gi/o G proteins), by BAPTA-AM, (suggesting they are calcium sensitive), by wortmannin (suggesting an involvement of PI3Kinase) and by soluble amyloid-β peptides. STX also stimulates cyclic AMP levels in mHippoE-18 cells and these effects are blocked by G36 and PTX, as well as by amyloid-β peptides. This suggests that both aldosterone and STX may modulate GPER signalling in mHippoE-18 cells.

Effect of SULT2B1 genetic polymorphisms on the sulfation of dehydroepiandrosterone and pregnenolone by SULT2B1b allozymes Publication date: 1 October 2019 Source: Molecular and Cellular Endocrinology, Volume 496 Author(s): Fatemah A. Alherz, Amal A. El Daibani, Maryam S. Abunnaja, Ahsan F. Bairam, Mohammed I. Rasool, Yoichi Sakakibara, Masahito Suiko, Katsuhisa Kurogi, Ming-Cheh Liu Abstract Pregnenolone and dehydroepiandrosterone (DHEA) are hydroxysteroids that serve as biosynthetic precursors for steroid hormones in human body. SULT2B1b has been reported to be critically involved in the sulfation of pregnenolone and DHEA, particularly in the sex steroid-responsive tissues. The current study was designed to investigate the impact of the genetic polymorphisms of SULT2B1 on the sulfation of DHEA and pregnenolone by SULT2B1b allozymes. Ten SULT2B1b allozymes previously prepared were shown to exhibit differential sulfating activities toward DHEA and pregnenolone in comparison to the wild-type enzyme. Kinetic studies revealed further significant changes in their substrate-binding affinity and catalytic activity toward DHEA and pregnenolone. Taken together, these results indicated clearly a profound effect of SULT2B1 genetic polymorphisms on the sulfating activity of SULT2B1b allozymes toward DHEA and pregnenolone, which may have implications in inter-individual variations in the homeostasis of these two important steroid precursors.

Estradiol and selective estrogen receptor agonists differentially affect brain monoamines and amino acids levels in transitional and surgical menopausal rat models Publication date: 1 October 2019 Source: Molecular and Cellular Endocrinology, Volume 496 Author(s): Tao Long, Jeffrey K. Yao, Junyi Li, Ziv Z. Kirshner, Doug Nelson, George G. Dougherty, Robert B. Gibbs Abstract Estrogens have many beneficial effects in the brain. Previously, we evaluated the effects of two models of menopause (surgical vs. transitional) on multiple monoaminergic endpoints in different regions of the adult rat brain in comparison with levels in gonadally intact rats. Here we evaluated the effects of estrogen receptor (ER) agonist treatments in these same two models of menopause. Neurochemical endpoints were evaluated in the hippocampus (HPC), frontal cortex (FCX), and striatum (STR) of adult ovariectomized (OVX) rats and in rats that underwent selective and gradual ovarian follicle depletion by daily injection of 4-vinylcyclohexene-diepoxide (VCD), after 1- and 6-weeks treatment with 17β-estradiol (E2), or with selective ERα (PPT), ERβ (DPN), or GPR30 (G-1) agonists. Endpoints included serotonin (5-HT) and 5-Hydroxyindoleacetic acid, dopamine (DA), 3,4-Dihydroxyphenylacetic acid and homovanillic acid, norepinephrine (NE) and epinephrine, as well as the amino acids tryptophan (TRP) and tyrosine (TYR). Significant differences between the models were detected. OVX rats were much more sensitive to ER agonist treatments than VCD-treated rats. Significant differences between brain regions also were detected. Within OVX rats, more agonist effects were detected in the HPC than in any other region. One interesting finding was the substantial decrease in TRP and TYR detected in the HPC and FCX in response to agonist treatments, particularly in OVX rats. This is on top of the substantial decreases in TRP and TYR previously reported one week after OVX or VCD-treatments in comparison with gonadally intact controls. Other interesting findings included increases in the levels of 5-HT, DA, and NE in the HPC of OVX rats treated with DPN, increases in DA detected in the FCX of OVX rats treated with any of the ER agonists, and increases in 5-HT and DA detected in the STR of OVX rats treated with E2. Many effects that were observed after 1-week of treatment were no longer observed after 6-weeks of treatment, demonstrating that effects were temporary despite continued agonist treatment. Collectively, the results demonstrate significant differences in the effects of ER agonists on monoaminergic endpoints in OVX vs. VCD-treated rats that also were brain region-specific and time dependent. The fact that agonist treatments had lesser effects in VCD treated rats than in OVX rats may help to explain reports of lesser effects of estrogen replacement on cognitive performance in women that have undergone transitional vs. surgical menopause.

GH3 and RC-4BC cell lines are not suitable as in vitro models to study prolactin modulation and AHR responsiveness in rat pituitary Publication date: 1 October 2019 Source: Molecular and Cellular Endocrinology, Volume 496 Author(s): A.D. van den Brand, E. Rubinstein, M. van den Berg, M.B.M. van Duursen Abstract Some environmental contaminants and pharmaceuticals increase the incidence of uterine tumors in toxicological studies with rats. These tumors can result from a hormonal imbalance due to rat-specific disrupted pituitary prolactin regulation, and are therefore of questionable relevance for humans. In this study we compared in vitro prolactin regulation in rat primary pituitary cells to that in pituitary cell lines, GH3 and RC-4BC. Moreover, we assessed the potential effects of aryl hydrocarbon receptor (AHR) activation on prolactin regulation by using two different AHR agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and DELAQ, the N-deethylated minor metabolite of the pharmaceutical laquinimod. In rat primary pituitary cells, known prolactin stimulant thyrotropin-releasing hormone (TRH) marginally increased prolactin secretion (1.2-fold) and gene expression (1.3-fold). In contrast, synthetic dopamine receptor agonist quinpirole, a known inhibitor of prolactin release, significantly inhibited prolactin secretion (2.6-fold) and gene expression (3.6-fold). In GH3 cells, TRH strongly increased prolactin secretion (6.8-fold) and gene expression (30.8-fold), whereas quinpirole did not affect prolactin secretion nor gene expression. In RC-4BC cells, both TRH and quinpirole did not modulate prolactin secretion nor gene expression. Prolactin secretion and gene expression did not significantly change upon exposure to TCDD or DELAQ. However, DELAQ, but not TCDD, attenuated quinpirole-inhibited prolactin gene expression by 51% in primary pituitary cells. This study shows that pituitary prolactin regulation in rat primary pituitary cells in vitro is distinctly different from rat pituitary cell lines GH3 and RC-4BC. Therefore, effects on pituitary prolactin regulation in vitro should best be performed using rat primary pituitary cells. Additionally, AHR ligands may interact with rat pituitary prolactin regulation, but this appears to depend on the ligand and constitutive prolactin secretion. However, interpretation of the in vitro results with respect to occurrence of uterine tumors in rats should take the complex regulation of prolactin release in the pituitary into account as well as the in vivo hypothalamus-pituitary-gonadal (HPG) axis and its feedback loops. Graphical abstract

Expression of nitric oxide synthase (NOS) in Anabas testudineus ovary and participation of nitric oxide-cyclic GMP cascade in maintenance of meiotic arrest Publication date: 1 October 2019 Source: Molecular and Cellular Endocrinology, Volume 496 Author(s): Poulomi Nath, Urmi Mukherjee, Subhasri Biswas, Soumojit Pal, Sriparna Das, Soumyajyoti Ghosh, Anwesha Samanta, Sudipta Maitra Abstract Participation of cyclic nucleotide-mediated signaling in nitric oxide/soluble guanylate cyclase (NO/sGC) regulation of oocyte maturation (OM) in perch (Anabas testudineus) follicle-enclosed oocytes has been investigated. Congruent with sharp decline in follicular cyclic GMP (cGMP) level, nitric oxide synthase (NOS)-inhibitor (L-NAME) attenuates protein kinase A (PKA) phosphorylation but promotes p-ERK1/2 and p-p34Cdc2 (Thr-161) in maturing oocytes. Conversely, NO donor (SNP) prevents OM, potentially through elevated cGMP synthesis. Expression and localization of Nos2 and Nos3 immunoreactivity in perch ovary varied considerably at progressively higher stages of folliculogenesis. While sGC inhibitor (ODQ) alone could induce OM, 8-bromo-cGMP attenuates 17,20β-P-induced OM indicating functional significance of NO/sGC/cGMP in perch ovary. Interestingly, high NO/cGMP inhibition of OM shows positive relation with elevated cAMP level. MIS induced OM is more susceptible to the oocyte-specific phosphodiesterase (PDE) 3 than PDE4 inhibition. Collectively, high NO/cGMP attenuation of OM potentially involves PDE3 inhibition, cAMP accumulation and PKA activation. Graphical abstract

Effects of adropin on proliferation and differentiation of 3T3-L1 cells and rat primary preadipocytes Publication date: 1 October 2019 Source: Molecular and Cellular Endocrinology, Volume 496 Author(s): Mariami Jasaszwili, Tatiana Wojciechowicz, Maria Billert, Mathias Z. Strowski, Krzysztof W. Nowak, Marek Skrzypski Abstract Adropin is a protein encoded by Energy Homeostasis Associated (Enho) gene which is expressed mainly in the liver and brain. There is evidence that biological effects of adropin are mediated via GPR19 activation. Animal studies showed that adropin modulates adiposity as well as lipid and glucose homeostasis. Adropin deficient animals have a phenotype closely resembling that of human metabolic syndrome with are obesity dyslipidemia and impaired glucose production. Animals treated with exogenous adropin lose weight, in addition to having reduced expression of lipogenic genes in the liver and fat tissue. While it was shown that adropin may contribute to energy homeostasis and body weight regulation, the role of this protein in controlling fat tissue formation is largely unknown. Thus, in the present study we investigated the effects of adropin on adipogenesis using 3T3-L1 cells and rat primary preadipocytes. We found a low Enho mRNA expression in 3T3-L1 cells and rat primary preadipocytes. Adropin stimulated proliferation of 3T3-L1 cells and rat primary preadipocytes. Stimulation of 3T3-L1 cell proliferation was mediated via ERK1/2 and AKT. Adropin reduced lipid accumulation as well as expression of proadipogenic genes in 3T3-L1 cells and rat preadipocytes, suggesting that this protein attenuates differentiation of preadipocytes into mature fat cells. In summary, these results show that adropin modulates proliferation and differentiation of preadipocytes.