Anna Benrick

Measurement of a Comprehensive Sex Steroid Profile in Rodent Serum by High-Sensitive Gas Chromatography-Tandem Mass Spectrometry

Accurate measurement of sex steroid concentrations in rodent serum is essential to evaluate mouse and rat models for sex steroid-related disorders. The aim of the present study was to develop a sensitive and specific gas chromatography-tandem mass spectrometry (GC-MS/MS) method to assess a comprehensive sex steroid profile in rodent serum. A major effort was invested in reaching an exceptionally high sensitivity for measuring serum estradiol concentrations. We established a GC-MS/MS assay with a lower limit of detection for estradiol, estrone, T, DHT, progesterone, androstenedione, and dehydroepiandrosterone of 0.3, 0.5, 4.0, 1.6, 8, 4.0, and 50 pg/mL, respectively, whereas the corresponding values for the lower limit of quantification were 0.5, 0.5, 8, 2.5, 74, 12, and 400 pg/mL, respectively. Calibration curves were linear, intra- and interassay coefficients of variation were low, and accuracy was excellent for all analytes. The established assay was used to accurately measure a comprehensive sex steroid profile in female rats and mice according to estrous cycle phase. In addition, we characterized the impact of age, sex, gonadectomy, and estradiol treatment on serum concentrations of these sex hormones in mice. In conclusion, we have established a highly sensitive and specific GC-MS/MS method to assess a comprehensive sex steroid profile in rodent serum in a single run. This GC-MS/MS assay has, to the best of our knowledge, the best detectability reported for estradiol. Our method therefore represents an ideal tool to characterize sex steroid metabolism in a variety of sex steroid-related rodent models and in human samples with low estradiol levels.

Mice Chronically Fed High-Fat Diet Have Increased Mortality and Disturbed Immune Response in Sepsis

Background Sepsis is a potentially deadly disease that often is caused by gram-positive bacteria, in particular Staphylococcus aureus (S. aureus). As there are few effective therapies for sepsis, increased basic knowledge about factors predisposing is needed. Methodology/Principal Findings The purpose of this study was to study the effect of Western diet on mortality induced by intravenous S. aureus inoculation and the immune functions before and after bacterial inoculation. Here we show that C57Bl/6 mice on high-fat diet (HFD) for 8 weeks, like genetically obese Ob/Ob mice on low-fat diet (LFD), have increased mortality during S. aureus-induced sepsis compared with LFD-fed C57Bl/6 controls. Bacterial load in the kidneys 5–7 days after inoculation was increased 10-fold in HFD-fed compared with LFD-fed mice. At that time, HFD-fed mice had increased serum levels and fat mRNA expression of the immune suppressing cytokines interleukin-1 receptor antagonist (IL-1Ra) and IL-10 compared with LFD-fed mice. In addition, HFD-fed mice had increased serum levels of the pro-inflammatory IL-1β. Also, HFD-fed mice with and without infection had increased levels of macrophages in fat. The proportion and function of phagocytosing granulocytes, and the production of reactive oxygen species (ROS) by peritoneal lavage cells were decreased in HFD-fed compared with LFD-fed mice. Conclusions Our findings imply that chronic HFD disturb several innate immune functions in mice, and impairs the ability to clear S. aureus and survive sepsis.

Interleukin‐6 Gene Knockout Influences Energy Balance Regulating Peptides in the Hypothalamic Paraventricular and Supraoptic Nuclei

Interleukin (IL)‐6 is a pro‐inflammatory cytokine that also affects metabolic function because IL‐6 depleted (IL‐6−/−) mice develop late‐onset obesity. IL‐6 appears to act in the central nervous system, presumably in the hypothalamus, to increase energy expenditure that appears to involve stimulation of the sympathetic nervous system. In the present study, we explored possible central mechanisms for the effects exerted by IL‐6 on body fat. Therefore, we measured the effects of IL‐6 depletion in IL‐6−/− mice on expression of key hypothalamic peptide genes involved in energy balance by the real time polymerase chain reaction. Additionally, co‐localisation between such peptides and IL‐6 receptor α was investigated by immunohistochemistry. IL‐6 deficiency decreased the expression of several peptides found in the paraventricular nucleus (PVN), which is a nucleus that has been attributed an adipostatic function. For example, corticotrophin‐releasing hormone (CRH), which is reported to stimulate the sympathetic nervous system, was decreased by 40% in older IL‐6−/− mice. Oxytocin, which is reported to prevent obesity, was also decreased in older IL‐6−/− animals, as was arginine vasopressin (AVP). The IL‐6 receptor α was abundantly expressed in the PVN, but also in the supraoptic nucleus, and was shown to be co‐expressed to a high extent with CRH, AVP, oxytocin and thyrotrophin‐releasing hormone. These data indicate that depletion of endogenous IL‐6, a body fat suppressing cytokine, is associated with the decreased expression of CRH and oxytocin (i.e. energy balance regulating peptides) as well as AVP in the PVN. Because IL‐6 receptor α is co‐expressed with CRH, oxytocin and AVP, IL‐6 could stimulate the expression of these peptides directly.

Maternal androgen excess reduces placental and fetal weights, increases placental steroidogenesis, and leads to long-term health effects in their female offspring

Here, we tested the hypothesis that excess maternal androgen in late pregnancy reduces placental and fetal growth, increases placental steroidogenesis, and adversely affects glucose and lipid metabolism in adult female offspring. Pregnant Wistar rats were randomly assigned to treatment with testosterone (daily injections of 5 mg of free testosterone from gestational days 16 to 19) or vehicle alone. In experiment 1, fetal and placental weights, circulating maternal testosterone, estradiol, and corticosterone levels, and placental protein expression and distribution of estrogen receptor-α and -β, androgen receptor, and 17β-hydroxysteroid dehydrogenase 2 were determined. In experiment 2, birth weights, postnatal growth rates, circulating testosterone, estradiol, and corticosterone levels, insulin sensitivity, adipocyte size, lipid profiles, and the presence of nonalcoholic fatty liver were assessed in female adult offspring. Treatment with testosterone reduced placental and fetal weights and increased placental expression of all four proteins. The offspring of testosterone-treated dams were born with intrauterine growth restriction; however, at 6 wk of age there was no difference in body weight between the offspring of testosterone- and control-treated rats. At 10-11 wk of age, the offspring of the testosterone-treated dams had less fat mass and smaller adipocyte size than those born to control rats and had no difference in insulin sensitivity. Circulating triglyceride levels were higher in the offspring of testosterone-treated dams, and they developed nonalcoholic fatty liver as adults. We demonstrate for the first time that prenatal testosterone exposure alters placental steroidogenesis and leads to dysregulation of lipid metabolism in their adult female offspring.

Continuous Administration of a P450 Aromatase Inhibitor Induces Polycystic Ovary Syndrome with a Metabolic and Endocrine Phenotype in Female Rats at Adult Age

Studying the mechanisms for the complex pathogenesis of polycystic ovary syndrome (PCOS) requires animal models with endocrine, reproductive, and metabolic features of the syndrome. Hyperandrogenism seems to be a central factor in PCOS, leading to anovulation and insulin resistance. In female rats, continuous administration of letrozole, a nonsteroidal inhibitor of P450 aromatase, at 400 μg/d starting before puberty induces hyperandrogenemia and reproductive abnormalities similar to those in women with PCOS. However, despite high circulating testosterone levels, these rats do not develop metabolic abnormalities, perhaps because of their supraphysiological testosterone concentrations or because estrogen synthesis is completely blocked in insulin-sensitive tissues. To test the hypothesis that continuous administration of lower doses of letrozole starting before puberty would result in both metabolic and reproductive phenotypes of PCOS, we performed a 12-wk dose-response study. At 21 d of age, 46 female Wistar rats were divided into two letrozole groups (100 or 200 μg/d) and a control group (placebo). Both letrozole doses resulted in increased body weight, inguinal fat accumulation, anovulation, larger ovaries with follicular atresia and multiples cysts, endogenous hyperandrogemia, and lower estrogen levels. Moreover, rats that received 200 μg/d had insulin resistance and enlarged adipocytes in inguinal and mesenteric fat depots, increased circulating levels of LH, decreased levels of FSH, and increased ovarian expression of Cyp17a1 mRNA. Thus, continuous administration of letrozole, 200 μg/d, to female rats for 90 d starting before puberty results in a PCOS model with reproductive and metabolic features of the syndrome.

Downregulation of cilia-localized Il-6Rα by 17β-estradiol in mouse and human fallopian tubes

The action of interleukin-6 (IL-6) impacts female reproduction. Although IL-6 was recently shown to inhibit cilia activity in human fallopian tubes in vitro, the molecular mechanisms underlying IL-6 signaling to tubal function remain elusive. Here, we investigate the cellular localization, regulation, and possible function of two IL-6 receptors (IL-6R alpha and gp130) in mouse and human fallopian tubes in vivo. We show that IL-6R alpha is restricted to the cilia of epithelial cells in both mouse and human fallopian tubes. Exogenous 17beta-estradiol (E(2)), but not progesterone (P(4)), causes a time-dependent decrease in IL-6R alpha expression, which is blocked by the estrogen receptor (ER) antagonist ICI-182,780. Exposure of different ER-selective agonists propyl-(1H)-pyrazole-1,3,5-triyl-trisphenol or 2,3-bis-(4-hydroxyphenyl)-propionitrile demonstrated an ER subtype-specific regulation of IL-6R alpha in mouse fallopian tubes. In contrast to IL-6R alpha, gp130 was detected in tubal epithelial cells in mice but not in humans. In humans, gp130 was found in the muscle cells and was decreased in the periovulatory and luteal phases during the reproductive cycles, indicating a species-specific expression and regulation of gp130 in the fallopian tube. Expression of tubal IL-6R alpha and gp130 in IL-6 knockout mice was found to be normal; however, E(2) treatment increased IL-6R alpha, but not gp130, in IL-6 knockout mice when compared with wild-type mice. Furthermore, expression levels of IL-6R alpha, but not gp130, decreased in parallel with estrogenic accelerated oocyte-cumulus complex (OCC) transport in mouse fallopian tubes. Our findings open the possibility that cilia-specific IL-6R alpha may play a role in the regulation of OCC transport and suggest an estrogen-regulatory pathway of IL-6R alpha in the fallopian tube.

Maternal testosterone exposure increases anxiety-like behavior and impacts the limbic system in the offspring

Significance Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility characterized by excessive androgen secretion. PCOS women are at an increased risk of developing depression and anxiety disorders. Although the etiology of PCOS is unclear, it is proposed to originate during fetal development because of maternal androgen excess. We describe here, in rodent models reflecting the anxiety phenotype of PCOS, evidence for disordered androgen receptor function in the amygdala, together with changes in estrogen receptor-α, serotonergic and GABAergic genes in the amygdala, and hippocampus. These findings define a previously unknown mechanism that may be critical in understanding how maternal androgen excess has the potential to increase the risk of developing anxiety disorders in daughters and sons of PCOS mothers. During pregnancy, women with polycystic ovary syndrome (PCOS) display high circulating androgen levels that may affect the fetus and increase the risk of mood disorders in offspring. This study investigated whether maternal androgen excess causes anxiety-like behavior in offspring mimicking anxiety disorders in PCOS. The PCOS phenotype was induced in rats following prenatal androgen (PNA) exposure. PNA offspring displayed anxiety-like behavior in the elevated plus maze, which was reversed by flutamide [androgen receptor (AR) blocker] and tamoxifen [selective estrogen receptor (ER) modulator]. Circulating sex steroids did not differ between groups at adult age. The expression of serotonergic and GABAergic genes associated with emotional regulation in the amygdala was consistent with anxiety-like behavior in female, and partly in male PNA offspring. Furthermore, AR expression in amygdala was reduced in female PNA offspring and also in females exposed to testosterone in adult age. To determine whether AR activation in amygdala affects anxiety-like behavior, female rats were given testosterone microinjections into amygdala, which resulted in anxiety-like behavior. Together, these data describe the anxiety-like behavior in PNA offspring and adult females with androgen excess, an impact that seems to occur during fetal life, and is mediated via AR in amygdala, together with changes in ERα, serotonergic, and GABAergic genes in amygdala and hippocampus. The anxiety-like behavior following testosterone microinjections into amygdala demonstrates a key role for AR activation in this brain area. These results suggest that maternal androgen excess may underpin the risk of developing anxiety disorders in daughters and sons of PCOS mothers.

Interleukin‐6 mediates exercise‐induced increase in insulin sensitivity in mice

Interleukin‐6 (IL‐6) is released from working skeletal muscle during exercise. We investigated the acute and the long‐term beneficial effects of IL‐6 on exercise‐induced glucose uptake in skeletal muscle and insulin sensitivity. The acute effect on exercise‐induced glucose uptake was measured in IL‐6‐deficient (IL‐6−/−) mice and wild‐type control animals using a tracer technique. There was no difference in serum disappearance of 3[H]2‐deoxyglucose after a single bout of exercise between IL‐6−/− and wild‐type mice (13565 ± 426 versus 14343 ± 1309 d.p.m. min ml−1, P= 0.5). The glucose uptake rate in the extensor digitorum longus muscle was, however, lower in IL‐6−/− compared with wild‐type mice (398 ± 44 versus 657 ± 41 nmol g−1 min−1, P < 0.01). In a long‐term study, we monitored insulin sensitivity, serum retinol‐binding protein‐4 (RBP‐4) levels, running activity, food intake, body weight and body composition in IL‐6−/− and wild‐type mice on a high‐fat diet (HFD), with or without access to running wheels. In sedentary IL‐6−/− and wild‐type mice, the HFD decreased insulin sensitivity (glucose area under the concentration–time curve increased about 20% during an insulin tolerance test, P < 0.05 for both genotypes versus baseline) and led to a 30% increase in serum RBP‐4 levels (P < 0.01 for both genotypes versus baseline). Wild‐type mice with access to running wheels were protected against these effects of the HFD and maintained their baseline insulin sensitivity and serum RBP‐4 levels. In contrast, IL‐6−/− mice did not benefit from running to the same extent as wild‐type animals. The IL‐6−/− mice with access to running wheels had a similar decrease in insulin sensitivity to their sedentary littermates (glucose area under the concentration–time curve during an insulin tolerance test in runners versus sedentary IL‐6−/− HFD mice, 312 ± 14 versus 340 ± 22 mmol min l−1, P= 0.4) and displayed a 14% increase in serum RBP‐4 compared with baseline levels (P < 0.01). Our results indicate that endogenous IL‐6 contributes to the exercise‐induced increase in insulin sensitivity, but plays only a minor role for glucose uptake into skeletal muscle during exercise.

Inter-relation between Interleukin (IL)-1, IL-6 and Body Fat Regulating Circuits of the Hypothalamic Arcuate Nucleus

Interleukin (IL)‐1 and IL‐6 are immune modulating cytokines that also affect metabolic function because both IL‐1 receptor I deficient (IL‐1RI−/−) and IL‐6 deficient (IL‐6−/−) mice develop late‐onset obesity and leptin resistance. Both IL‐1 and IL‐6 appear to target the central nervous system (CNS) to increase energy expenditure. The hypothalamic arcuate nucleus (ARC) is a major relay between the periphery and CNS in body fat regulation (e.g. by being a target of leptin). The present study aimed to investigate the possible mechanisms responsible for the effects exerted by endogenous IL‐1 and IL‐6 on body fat at the level of the ARC, as well as possible interactions between IL‐1 and IL‐6. Therefore, we measured the gene expression of neuropeptides of the ARC involved in energy balance in IL‐1RI−/− and IL‐6−/− mice. We also investigated the interactions between expression of IL‐1 and IL‐6 in these mice, and mapped IL‐6 receptor α (IL‐6Rα) in the ARC. The expression of the obesity promoting peptide neuropeptide Y (NPY), found in the ARC, was increased in IL‐1RI−/− mice. The expression of NPY and agouti‐related peptide (AgRP), known to be co‐expressed with NPY in ARC neurones, was increased in cold exposed IL‐6−/− mice. IL‐6Rα immunoreactivity was densely localised in the ARC, especially in the medial part, and was partly found in NPY positive cell bodies and also α‐melanocyte‐stimulating hormone positive cell bodies. The expression of hypothalamic IL‐6 was decreased in IL‐1RI−/− mice, whereas IL‐1ß expression was increased in IL‐6−/− mice. The results of the present study indicate that depletion of the activity of the fat suppressing cytokines IL‐1 and IL‐6 in knockout mice can increase the expression of the obesity promoting neuropeptide NPY in the ARC. Depletion of IL‐1 activity suppresses IL‐6 expression, and IL‐6Rα‐like immunoreactivity is present in neurones in the medial ARC, including neurones containing NPY. Therefore, IL‐6, IL‐1 and NPY/AgRP could interact at the level of the hypothalamic ARC in the regulation of body fat.

Gene expression in subcutaneous adipose tissue differs in women with polycystic ovary syndrome and controls matched pair-wise for age, body weight, and body mass index.

Adipose tissue dysfunction may be a central factor in the pathogenesis of insulin resistance in women with polycystic ovary syndrome (PCOS). Gene expression in subcutaneous adipose tissue in PCOS and its relation to metabolic and endocrine features of the syndrome have been fragmentarily investigated. The aim was to assess in subcutaneous adipose tissue the expression of genes potentially associated with adipose tissue dysfunction and to explore their relation to features of the syndrome. Twenty-one women with PCOS (body mass index [BMI] 18.2–33.4 kg/m2) and 21 controls (BMI 19.2–31.7 kg/m2) were matched pair-wise for age, body weight, and BMI. Tissue biopsies were obtained to measure mRNA expression of 44 genes (TaqMan Low Density Array). Differential expression levels were correlated with BMI, glucose infusion rate (GIR), sex hormone binding globulin (SHBG), and sex steroids. In PCOS, expression of adiponectin receptor 2 (ADIPOR2), LPL, and twist-related protein 1 (TWIST1) was decreased, while expression of chemokine (C-C motif) ligand 2 (CCL2) and heme oxygenase (decycling 1) (HMOX1) was increased. TWIST1 and HMOX1, both novel adipokines, correlated with BMI and GIR. After BMI adjustment, LPL and ADIPOR2 expression correlated with plasma estradiol, and CCL2 expression correlated with GIR, in all women. We conclude that adipose tissue mRNA expression differed in PCOS women and controls and that two novel adipokines, TWIST1 and HMOX1, together with adiponectin, LPL, and CCL2, and their downstream pathways merit further investigation.