A novel genetically-obese rat model with elevated 11beta-hydroxysteroid dehydrogenase type 1 activity in subcutaneous adipose tissue

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the conversion of inactive glucocorticoids to active glucocorticoids and plays an important role in the development of obesity and metabolic syndrome. 11β-HSD1 activity is lower in liver and higher in omental adipose tissue of obese rodent models like obese zucker rats, Ob/Ob and db/db mice. Here, we report the 11β-HSD1 activity in liver and adipose tissue of lean and obese rats of WNIN/Ob strain, a new genetic rat model of obesity. 11β-HSD1 activity in liver, omental and subcutaneous adipose tissues of 3 month-old male WNIN/Ob lean and obese rats was assayed. As observed in other rodent models, 11β-HSD1 activity was lower in liver and higher in omental adipose tissue. In contrast to other rodent obese models, WNIN/Ob obese rats had elevated 11β-HSD1 activity in subcutaneous adipose tissue, which is in line with the observation in human obesity. Here, we conclude that dysregulation of 11β-HSD1 in WNIN/Ob obese rat model is identical to human obesity, which makes it an excellent model for studying the effect of 11β-HSD1 inhibitors in ameliorating obesity and metabolic syndrome

Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancyResearch in context

Background: Circulating estrogen (E2) levels are high throughout pregnancy and increase towards term, however its local tissue specific actions vary across gestation. For example, myometrial E2 regulated uterotonic action is disabled until term, whereas it's proliferative function is maintained in the breast. We have identified gestationally regulated splicing events, mediated by hnRNPG and modulated by E2 that generate alternatively spliced estrogen receptor alpha (ERα) variants (ERΔ7 and ERα46) in the myometrium. These variants allow for differential, gestationally regulated, modulation of the uterotonic action of E2. Methods: Human myometrium isolated from preterm and term non-laboring and laboring pregnant women were analyzed for ERα isoforms and splice factor levels. Lentiviral mediated shRNA knockdown of hnRNPG and overexpression of ERΔ7 were performed in human myometrial (hTERT-HM) cells. Functional 3D collagen contraction assays were executed. Findings: ERΔ7 acts as a dominant negative repressor of the uterotonic action of ERα66 and ERα46 isoforms through the regulation of the myometrial gap junction protein GJA1. Elimination of hnRNPG inhibits the generation of ERΔ7 while overexpression of ERΔ7 inhibited GJA1 expression. Moreover in vivo human myometrial hnRNPG levels decline at term in an E2 dependent manner resulting in a withdrawal of ERΔ7 levels and its tocolytic action at term. Interpretation: Our findings implicate the unique role of ERΔ7 as a modulator of myometrial quiescence and define the mechanism of ERΔ7 generation, through hormonally regulated splicing events. Fund: This study was supported by NIH OPRU U01 supplement (HD047905), University of Pittsburgh and Wayne State University Perinatal Research Initiative (USA). Keywords: Estrogen receptor alpha, Alternative splicing, hnRNPG, Myometrium, Parturition, GJA

Variable Cre Recombination Efficiency in Placentas of Cyp19-Cre ROSA^mT/mG Transgenic Mice

The aromatase-Cre recombinase (Cyp19-Cre) transgenic mouse model has been extensively used for placenta-specific gene inactivation. In a pilot study, we observed unexpected phenotypes using this mouse strain, which prompted an extensive characterization of Cyp19-Cre placental phenotypes using ROSAmT/mG transgenic reporter mice. The two strains were mated to generate bi-transgenic Cyp19-Cre;ROSAmT/mG mice following a standard transgenic breeding scheme, and placental and fetal tissues were analyzed on embryonic day 17.5. Both maternal and paternal Cre inheritance were analyzed by mating the respective Cyp19-Cre and ROSAmT/mG males and females. The genotype results showed the expected percentage of Cyp19-Cre;ROSAmT/mG fetuses (73%) and Cre mRNA was expressed in all of the Cyp19-Cre placentas. However, surprisingly, only about 50% of the Cyp19-Cre;ROSAmT/mG placentas showed Cre-mediated recombinase activity as demonstrated by placental enhanced green fluorescent protein (EGFP) expression. Further genetic excision analysis of the placentas revealed consistent results showing the absence of excision of the tdTomato in all of the Cyp19-Cre;ROSAmT/mG placentas lacking EGFP expression. Moreover, among the EGFP-expressing placentas, there was wide variability in recombination efficiency, even in placentas from the same litter, leading to a mosaic pattern of EGFP expression in different zones and cell types of the placentas. In addition, we observed a significantly higher percentage of Cre recombination activity in placentas with maternal Cre inheritance. Our results show frequent mosaicism, inconsistent recombination activity, and parent-of-origin effects in placentas from Cyp19-Cre;ROSAmT/mG mice, suggesting that tail-biopsy genotype results may not necessarily indicate the excision of floxed genes in Cyp19-Cre positive placentas. Thus, placenta-specific mutagenesis studies using the Cyp19-Cre model require extensive characterization and careful interpretation of the placental phenotypes for each floxed allele

Characterization of three BACs containing parts of the AR gene.

<p>(A) Top: a map of the AR gene and flanking region is shown. Below: maps of the BACs mAR BAC-196 (RP23-102011), mAR BAC-192 (RP24-352D1) and mAR BAC-185 (RP23-316P7) are shown. AR exons are denoted by rectangles on the lines. <i>SalI</i> restiction sites are denoted with an S. Boxes above exons 1 and 8 denote the probes used for Southern blotting. The BACs contained either extended 5’ sequence, the complete AR gene or extended 3’ sequence, respectively. The BACs were end sequenced to determine their insertion sites. (B) Restriction digests followed by PFGE and Southern blotting were performed to ensure that preperations of the BACs were correct. The <i>SalI</i> digested BACs were fractionated by PFGE and subjected to Southern blotting using ³²P random primed labelled AR exon 1 and AR exon 8 probes. mAR BAC-192 was positive for both probes while mAR BAC-196 only probed positive for exon 8 and mAR BAC-185 probed positive only for exon1.</p

EGFP is expressed in the testes of mice containing the AR-EGFP transgene.

<p>Testis tissue sections from mice containing the AR-GFP transgene (A, C) and a mouse lacking the transgene (B) were probed with secondary antisera alone (A) or with antiserum against GFP and secondary antisera (B, C). Strong EGFP immunoreactivity (brown stain) was observed only in testes containing the AR-GFP transgene probed with EGFP antiserum (C). EGFP immunoreactivity was present in the cytoplasm of Sertoli cells including concentrations of EGFP in the Sertoli cell cytoplasm surrounding germ cells (arrows) and in the cytoplasm of Leydig cells (L, long arrows). Nuclei are stained blue with hematoxylin. Bar = 100 μm, magnification: 40x objective.</p

Expression of the AR-EGFP transgene in founder mice.

<p>A) Testis whole cell extracts from 6 founder mice having germ line transmission of the AR-GFP transgene and extracts from Cos7 cells infected with an adenovirus expressing EGFP (AdEGFP) by western blot. B) Whole cell extracts from the 2460 founder strain containing the AR-EGFP transgene were assayed for EGFP expression by western blot.</p