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- Sexually dimorphic effects of ancestral exposure to vinclozolin on stress reactivity in rats. [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141253.
How an individual responds to the environment depends upon both personal life history as well as inherited genetic and epigenetic factors from ancestors. Using a 'two-hit, 3 generations apart' model, we tested how F3 descendants of rats given in utero exposures to the environmental endocrine-disrupting chemical (EDC) vinclozolin reacted to stress during adolescence in their own lives, focusing on sexually dimorphic phenotypic outcomes. In adulthood, male and female F3 vinclozolin- or vehicle-lineage rats, stressed or non-stressed, were behaviorally characterized on a battery of tests, then euthanized. Serum was used for hormone assays, and brains for qPCR and transcriptome analyses. Results showed that the effects of ancestral exposure to vinclozolin converged with stress experienced during adolescence in a sexually dimorphic manner. Debilitating effects were seen at all levels of the phenotype, including physiology, behavior, brain metabolism, gene expression, and genome-wide transcriptome modifications in specific brain nuclei. Additionally, females were significantly more vulnerable than males to transgenerational effects of vinclozolin on anxiety but not sociality tests. This fundamental transformation occurs in a manner neither predicted by the ancestral exposure or the proximate effects of stress during adolescence, an interaction we refer to as synchronicity.
- Estradiol stimulates apolipoprotein A-IV gene expression in the nucleus of the solitary tract through estrogen receptor-α [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141239.
Although estrogens have been implicated in the regulation of apolipoprotein A-IV (apo A-IV) gene expression in the nucleus of the solitary tract (NTS), previous studies have not defined the molecular mechanism. The aim of this study was to examine the transcriptional mechanisms involved in regulation of apo A-IV gene expression. Using cultured primary neuronal cells from rat embryonic brainstems, we found that treatment with 10 nM E2 or PPT (an ERα agonist), but not DPN (an ERβ agonist), significantly increased apoA-IV gene expression, compared with vehicle treatment. This effect of E2 was abolished when the cells were incubated with E2 linked to bovine serum albumin, which prevents E2 from entering cells, implying that a nongenomic mechanism of E2 is not involved. Two putative estrogen response elements were identified at the 5'-upstream region of the apoA-IV gene promoter, but only one of them was able to recruit ERα, leading to increased apo A-IV gene expression, as determined by chromatin immunoprecipitation (ChIP) assay and luciferase activity analysis. A cyclic regimen of E2 or PPT treatment for 8 cycles (4 days/cycle, mimicking the ovarian cycle of female rats) in ovariectomized (OVX) female rats significantly reduced food intake and body weight gain, and increased apo A-IV gene expression in the NTS, relative to vehicle. These data collectively demonstrate that nuclear ERα is the primary mediator of E2's action on apo A-IV gene expression and suggest that increased signaling of endogenous apo A-IV may at least partially mediate E2-induced inhibitory effect on feeding.
- 5-HT obesity medication efficacy via POMC activation is maintained during aging. [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141223.
The phenomenon commonly described as "the middle-age spread" is the result of elevated adiposity accumulation throughout adulthood until late middle-age. It is a clinical imperative to gain a greater understanding of the underpinnings of age-dependent obesity and in turn, how these mechanisms may impact the efficacy of obesity treatments. In particular, both obesity and aging are associated with rewiring of a principal brain pathway modulating energy homeostasis, promoting reduced activity of satiety melanocortin pro-opiomelanocortin (POMC) neurons within the arcuate nucleus of the hypothalamus (ARC). Utilizing a selective ARC deficient POMC mouse line, here we report that former obesity medications augmenting endogenous 5-hydroxytryptamine (5-HT) activity d-fenfluramine and sibutramine require ARC POMC neurons to elicit therapeutic appetite suppressive effects. We next investigated whether age-related diminished ARC POMC activity therefore impacts the potency of 5-HT obesity pharmacotherapies, lorcaserin, d-fenfluramine and sibutramine and report that all compounds reduced food intake to a comparable extent in both chow fed young lean (3-5 months old) and middle-aged obese (12-14 months old) male and female mice. We provide a mechanism through which 5-HT anorectic potency is maintained with age, via preserved 5-HT-POMC appetitive anatomical machinery. Specifically, the abundance and signalling of the primary 5-HT receptor influencing appetite via POMC activation, the 5-HT2CR, is not perturbed with age. These data reveal that though 5-HT obesity medications require ARC POMC neurons to achieve appetitive effects, the anorectic efficacy is maintained with aging; findings of clinical significance to the global aging obese population.
- GLP-1 analogue liraglutide enhances proinsulin processing in pancreatic β-cells via a PKA-dependent pathway. [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141218.
Hyperproinsulinemia has gained increasing attention in the development of type 2 diabetes. Clinical studies have demonstrated that glucagon-like peptide-1 (GLP-1)-based therapies significantly decrease plasma proinsulin/insulin ratio in patients with type 2 diabetes. However, the underlying mechanism remains unclear. Prohormone convertase 1/3 (PC1/3) and PC2 are primarily responsible for processing proinsulin to insulin in pancreatic β-cells. We have recently reported that Pax6 mutation downregulated PC1/3 and PC2 expression, resulting in defective proinsulin processing in Pax6 heterozygous mutant (Pax6(m/+)) mice. In this study, we investigated whether and how liraglutide, a novel GLP-1 analogue, modulated proinsulin processing. Our results showed that liraglutide significantly upregulated PC1/3 expression and decreased the proinsulin/insulin ratio in both Pax6(m/+) and db/db diabetic mice. In cultured mouse pancreatic β-cell line Min6, liraglutide stimulated PC1/3 and PC2 expression and lowered the proinsulin/insulin ratio in a dose- and time-dependent manner. Moreover, the beneficial effects of liraglutide on PC1/3 and PC2 expression and proinsulin processing were dependent on GLP-1 receptor (GLP-1R)-mediated cAMP/PKA signaling pathway. The same mechanism was recapitulated in isolated mouse islets. In conclusion, liraglutide enhanced PC1/3- and PC2-dependent proinsulin processing in pancreatic β-cells through activation of the GLP-1R/cAMP/PKA signaling pathway. Our study provides a new mechanism for improvement of pancreatic β-cell function by the GLP-1-based therapy.
- Disparate changes in kisspeptin and neurokinin B expression in the arcuate nucleus following sex steroid manipulation reveal differential regulation of the two KNDy peptides in rats. [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141200.
Kisspeptin, neurokinin B (NKB) and dynorphin A are co-expressed in a population of neurons in the arcuate nucleus (ARC), termed KNDy neurons, which were recently recognized as important elements for the generation of GnRH pulses. However, the topographic distribution of these peptides and their regulated expression by sex steroids are still not well understood. In this study, detailed examination of NKB and kisspeptin immunoreactivity in the rat ARC was carried out, including comparison between sexes, with and without sex steroid replacement. Neurons expressing kisspeptin and NKB were more prominent in the caudal ARC of females, whereas neurons expressing NKB, but not kisspeptin, were the most abundant in the male. Sex steroid manipulation revealed differential regulation of kisspeptin and NKB; while kisspeptin immunoreactive (-ir) cells increased in response to gonadectomy, NKB remained unchanged. Furthermore, the number of NKB-ir cells increased upon sex steroid replacement compared to gonadectomy, while kisspeptin did not, suggesting that sex steroids differently regulate these peptides. In addition, only in females did the density of kisspeptin- and NKB-ir fibers in the ARC increase upon sex steroid replacement in relation to sham and OVX, respectively, suggesting sex-specific regulation of release. In conclusion, our observations reveal sex differences in the number of kisspeptin- and NKB-ir cells, which are more prominent in the caudal ARC. The divergent regulation of kisspeptin and NKB peptide contents in the ARC as function of sex and steroid milieu enlarge our understanding on how these neuropeptides are post-transcriptionally regulated in KNDy neurons.
- Genetic Rescue of Glycosylation-deficient Fgf23 in the Galnt3 Knockout Mouse. [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141199.
Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence ((176)RHTR(179)↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in autosomal dominant hypophosphatemic rickets (ADHR) patients, and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23, but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by Galnt3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus in Galnt3 knockout mice.
- Ezrin is an actin binding protein that regulates Sertoli cell and spermatid adhesion during spermatogenesis* [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141163.
During spermatogenesis, the transport of spermatids and the release of sperms at spermiation, and the remodeling of the BTB in the seminiferous epithelium of rat testes require rapid re-organization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein, we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction (AJ) at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (apical ES) in the adluminal compartment, and the Sertoli cell-cell interface known as basal ES at the blood-testis barrier (BTB). Ezrin is expressed at the basal ES/BTB in all stages, except IX, of the epithelial cycle. Its knockdown by RNAi in vitro perturbs the Sertoli cell tight junction (TJ)-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (e.g., N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilaments at the ES in rat testes.
- Cell-mass structures expressing the aromatase gene Cyp19a1 lead to ovarian cavities in Xenopus laevis. [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141096.
The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determination system. We previously reported that a W-linked gene, Dm-W, can determine development as a female. However, the mechanisms of early sex differentiation remain unclear. We used microarrays to screen for genes with sexually dimorphic expression in ZZ and ZW gonads during early sex differentiation in X. laevis, and found several steroidogenic genes. Importantly, the steroid 17α-hydroxylase gene Cyp17a1 and the aromatase gene Cyp19a1 were highly expressed in ZZ and ZW gonads, respectively, just after sex determination. At this stage, we found that Cyp17a1, Cyp19a1, or both were expressed in the ZZ and ZW gonads in a unique 'mass-in-line' structure, in which several masses of cells, each surrounded by basement membrane, were aligned along the anteroposterior axis. In fact, during sex differentiation, ovarian cavities formed inside each mass of Cyp17a1- and Cyp19a1-positive cells in the ZW gonads. However, the 'mass-in-line' structure disappeared during testicular development in the ZZ testes. These results suggested that the 'mass-in-line' structure found in both ZZ and ZW gonads just after sex determination might be formed in advance to produce ovarian cavities, and then oocytes. Consequently, we propose a view that the default sex may be female in the morphological aspect of gonads in X. laevis.
- Estrogen receptor (ER) agonists differentially regulate neuroangiogenesis in peritoneal endometriosis via the repellent factor SLIT3. [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141086.
Endometriosis is an estrogen-dependent neurovascular disorder characterised by growth of endometrial tissue (lesions) outside the uterine cavity. Patients suffer chronic pelvic pain and it has been proposed that co-recruitment of nerves/blood vessels (neuroangiogenesis) into the lesions is fundamental to the development of painful symptoms. We hypothesized that estrogen-dependent regulation of axonal guidance molecules of the SLIT/ROBO family could play a role in neuroangiogenesis occurring in endometriosis lesions found on the peritoneal wall. In tissue samples from human patients and a mouse model of endometriosis concentrations of mRNA encoded by SLIT3 were significantly higher in lesions than normal peritoneum. Estrogen regulation of SLIT3 was investigated using estradiol (E2) and selective agonists for each subtype of estrogen receptor (ERα agonist, PPT; ERβ agonist DPN). In mice, DPN (EC50 0.85) increased Slit3 mRNA concentrations compared to hormone depleted and E2-treated (EC50 0.1) animals and decreased the density of nerves but not vessels in endometriosis lesions. SLIT3 mRNA concentrations were increased in DPN-treated human endothelial cells (EC) and in PPT-treated (EC50:200) rat dorsal root ganglia neurons. Functional assays (neurite outgrowth, network formation) revealed that SLIT3 promotes angiogenesis but decreases neurogenesis. In conclusion, these data suggest estrogen-dependent expression of SLIT3 may play a key role in regulating nerve-vessel interactions within the complex microenvironment of endometriosis lesions.
- Placenta passage of the thyroid hormone analogue DITPA to male wild-type and Mct8 deficient mice. [JOURNAL ARTICLE]
- Endocrinology 2014 Jul 22.:en20141085.
After depletion of the endogenous TH, Mct8 heterozygous pregnant dams, carrying both wild-type (Wt) and Mct8 deficient (Mct8KO) male embryos were given DITPA. Effects were compared to those treated with L-T4. With DITPA treatment, serum DITPA concentration was not different in the two genotypes, which produced equal effect on serum TSH levels in both groups of pups. In contrast, with L-T4 treatment, TSH did not normalize in Mct8KO pups while it did in the Wt littermates and dams despite higher concentration of serum T4. Finally, both treatments similarly modulated the expression of the TH-dependent genes Shh, Klf9 and Aldh1a3 in brain. Thus, the ability of DITPA to cross the placenta, its thyromimetic action on the expression of TH dependent genes in brain and its better accessibility to the pituitary than L-T4, as assessed by serum TSH, make DITPA a candidate for the prenatal treatment of MCT8 deficiency.