Ruijin Shao

Increase of SUMO‐1 expression in response to hypoxia: direct interaction with HIF‐1α in adult mouse brain and heart in vivo

The present study investigates the regulation of small ubiquitin‐related modifier‐1 (SUMO‐1) expression in response to hypoxia in adult mouse brain and heart. We observed a significant increase in SUMO‐1 mRNAs and proteins after hypoxic stimulation in vivo. Because SUMO‐1 interacts with various transcription factors, including hypoxia‐inducible factor‐1β (HIF‐1β) in vitro, we not only demonstrated that the HIF‐1α expression is increased by hypoxia in brain and heart, but also provided evidence that SUMO‐1 co‐localizes in vivo with HIF‐1α in response to hypoxia by demonstrating the co‐expression of these two proteins in neurons and cardiomyocytes. The specific interaction between SUMO‐1 and HIF‐1α was additionally demonstrated with co‐immunoprecipitation. These results indicate that the increased levels of SUMO‐1 participate in the modulation of HIF‐1α function through sumoylation in brain and heart.

Expression of Progesterone Receptor (PR) A and B Isoforms in Mouse Granulosa Cells: Stage-Dependent PR-Mediated Regulation of Apoptosis and Cell Proliferation

Abstract The intracellular progesterone receptor (PR) in the mammalian ovary is a part of the physiological pathway that facilitates ovulation. Two PR isoforms (A and B) exist, with different molecular and biological functions. Previous studies have revealed that the cellular ratio of the PR isoforms is important for progesterone-responsive tissues and is under developmental control in different species. However, the relative expression of PR isoforms in the ovary is unknown. In this study we have demonstrated first that the expression of both PR isoforms in mouse granulosa cells was rapidly up-regulated by hCG treatment and dramatically down-regulated when the granulosa cells were undergoing luteinization. The relative level of protein expression of the A and B forms was 2:1 and the highest total PR protein expression was found after hCG stimulation. Second, we demonstrated that the expression of PR protein was specific to granulosa cells of periovulatory follicles and was absent in undifferentiated granulosa cells of growing follicles. It was not detected in other cell types (i.e., corpora lutea or any stage of follicles with features of apoptosis). Third, we demonstrated that treatment with the PR antagonist RU 486 in vivo resulted in down-regulation of both isoforms in parallel with increased activation of caspase-3, a decreased level of proliferating cell nuclear antigen, and a reduced rate of ovulation. Fourth, we demonstrated, in vitro, that the PR antagonists RU 486 and Org 31710 increased internucleosomal DNA fragmentation parallel with a decrease in DNA synthesis in granulosa cells, which express PR. These results indicate that PR and its isoforms participate in regulation of ovulation, along with suppression of granulosa cell apoptosis and promotion of cell survival in the mouse ovary.

Glucagon-like peptide 1 receptor induced suppression of food intake, and body weight is mediated by central IL-1 and IL-6

Significance There is a growing interest in the gut- and hindbrain-produced hormone glucagon-like peptide 1 (GLP-1), and GLP-1–targeting drugs are in clinical trials for treatment of obesity, and already in the clinic for treatment of type 2 diabetes. Therefore, the implications of information arising from our study are clinically relevant and considerable. GLP-1 receptor stimulation decreases feeding and body weight likely via the CNS, effects of unquestioned scientific and clinical importance, considering the alarming rates of obesity. Despite this, there is scarce information about the mediators and mechanisms behind the effects of GLP-1. In this study, we found surprising evidence that two cytokines, interleukin-6 and interleukin-1, mediate antiobesity effects of GLP-1 receptor stimulation at the level of the CNS. Glucagon-like peptide 1 (GLP-1), produced in the intestine and the brain, can stimulate insulin secretion from the pancreas and alleviate type 2 diabetes. The cytokine interleukin-6 (IL-6) may enhance insulin secretion from β-cells by stimulating peripheral GLP-1 production. GLP-1 and its analogs also reduce food intake and body weight, clinically beneficial actions that are likely exerted at the level of the CNS, but otherwise are poorly understood. The cytokines IL-6 and interleukin 1β (IL-1β) may exert an anti-obesity effect in the CNS during health. Here we found that central injection of a clinically used GLP-1 receptor agonist, exendin-4, potently increased the expression of IL-6 in the hypothalamus (11-fold) and the hindbrain (4-fold) and of IL-1β in the hypothalamus, without changing the expression of other inflammation-associated genes. Furthermore, hypothalamic and hindbrain interleukin-associated intracellular signals [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and suppressor of cytokine signaling-1 (SOCS1)] were also elevated by exendin-4. Pharmacologic disruption of CNS IL-1 receptor or IL-6 biological activity attenuated anorexia and body weight loss induced by central exendin-4 administration in a rat. Simultaneous blockade of IL-1 and IL-6 activity led to a more potent attenuation of exendin-4 effects on food intake. Mice with global IL-1 receptor gene knockout or central IL-6 receptor knockdown showed attenuated decrease in food intake and body weight in response to peripheral exendin-4 treatment. GLP-1 receptor activation in the mouse neuronal Neuro2A cell line also resulted in increased IL-6 expression. These data outline a previously unidentified role of the central IL-1 and IL-6 in mediating the anorexic and body weight loss effects of GLP-1 receptor activation.

Progesterone receptor antagonists Org 31710 and RU 486 increase apoptosis in human periovulatory granulosa cells.

OBJECTIVE To investigate if progesterone receptor (PR)-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated human luteinizing granulosa cells. DESIGN Laboratory study. SETTING Göteborg University and an in vitro fertilization laboratory of a university hospital. PATIENT(S) Women undergoing oocyte retrieval for in vitro fertilization after ovulation induction with gonadotropins. INTERVENTION(S) Luteinizing granulosa cells were isolated from follicular aspirates after oocyte removal. The cells were treated with or without RU 486 (1 microM-100 microM), Org 31710 (1 microM-100 microM), progesterone (1 nM-10 microM), dexamethasone (0.5 microM-100 microM), dihydrotestosterone (1 nM-25 microM), RU 486 (10 microM-100 microM) + dexamethasone (50 microM), and picrotoxin (1 microM-100 microM) and were cultured under serum-free conditions. MAIN OUTCOME MEASURE(S) Measurement of caspase-3 activity; detection of internucleosomal DNA fragmentation using gel electrophoresis and fluorospectrophotometry; progesterone analysis of spent medium. RESULT(S) Addition of the PR antagonists RU 486 or Org 31710 in vitro to human luteinizing granulosa cells caused an increase in caspase-3 activity and a dose-dependent increase in internucleosomal DNA fragmentation. No effect on DNA fragmentation was seen after addition of dexamethasone, dihydrotestosterone, or picrotoxin. CONCLUSION(S) Nuclear PR-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated human luteinizing granulosa cells.

Intense electroacupuncture normalizes insulin sensitivity, increases muscle GLUT4 content, and improves lipid profile in a rat model of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism and insulin resistance, possibly reflecting defects in skeletal muscle and adipocyte insulin signaling. Low-frequency (2 Hz) electroacupuncture (EA) increases insulin sensitivity in female rats with dihydrotestosterone (DHT)-induced PCOS, but the mechanism is unclear. We hypothesized that low-frequency EA regulates mediators involved in skeletal muscle glucose uptake and metabolism and alters the lipid profile in rats with DHT-induced PCOS. To test this hypothesis, we implanted in prepubescent female rats 90-day continuous-release pellets containing DHT (PCOS). At 70 days of age, the rats were randomly subdivided into two groups: one received low-frequency EA (evoking muscle twitches) for 20-25 min five times/wk for 4-5 wk; the other did not. Controls were implanted with pellets containing vehicle only. All three groups were otherwise handled similarly. Lipid profile was measured in fasting blood samples. Insulin sensitivity was determined by euglycemic hyperinsulinemic clamp, soleus muscle protein expression of glucose transporter 4 (GLUT4), and phosphorylated and nonphosphorylated Akt, and Akt substrate of 160 kDa was determined by Western blot analysis and GLUT4 location by immunofluorescence staining. PCOS EA rats had normalized insulin sensitivity, lower levels of total high-density lipoprotein and low-density lipoprotein cholesterol, and increased expression of GLUT4 in different compartments of skeletal muscle compared with PCOS rats. Total weight and body composition did not differ in the groups. Thus, in rats with DHT-induced PCOS, low-frequency EA has systemic and local effects involving intracellular signaling pathways in muscle that may, at least in part, account for the marked improved insulin sensitivity.

The role of estrogen receptor α in growth plate cartilage for longitudinal bone growth.

Estrogens enhance skeletal growth during early sexual maturation, whereas high estradiol levels during late puberty result in growth plate fusion in humans. Although the growth plates do not fuse directly after sexual maturation in rodents, a reduction in growth plate height is seen by treatment with a high dose of estradiol. It is unknown whether the effects of estrogens on skeletal growth are mediated directly via estrogen receptors (ERs) in growth plate cartilage and/or indirectly via other mechanisms such as the growth hormone/insulin‐like growth factor 1 (GH/IGF‐1) axis. To determine the role of ERα in growth plate cartilage for skeletal growth, we developed a mouse model with cartilage‐specific inactivation of ERα. Although mice with total ERα inactivation displayed affected longitudinal bone growth associated with alterations in the GH/IGF‐1 axis, the skeletal growth was normal during sexual maturation in mice with cartilage‐specific ERα inactivation. High‐dose estradiol treatment of adult mice reduced the growth plate height as a consequence of attenuated proliferation of growth plate chondrocytes in control mice but not in cartilage‐specific ERα−/− mice. Adult cartilage‐specific ERα−/− mice continued to grow after 4 months of age, whereas growth was limited in control mice, resulting in increased femur length in 1‐year‐old cartilage‐specific ERα−/− mice compared with control mice. We conclude that during early sexual maturation, ERα in growth plate cartilage is not important for skeletal growth. In contrast, it is essential for high‐dose estradiol to reduce the growth plate height in adult mice and for reduction of longitudinal bone growth in elderly mice.

Hypothalamic Neuroendocrine Functions in Rats with Dihydrotestosterone-Induced Polycystic Ovary Syndrome: Effects of Low-Frequency Electro-Acupuncture

Adult female rats continuously exposed to androgens from prepuberty have reproductive and metabolic features of polycystic ovary syndrome (PCOS). We investigated whether such exposure adversely affects estrous cyclicity and the expression and distribution of gonadotropin-releasing hormone (GnRH), GnRH receptors, and corticotrophin-releasing hormone (CRH) in the hypothalamus and whether the effects are mediated by the androgen receptor (AR). We also assessed the effect of low-frequency electro-acupuncture (EA) on those variables. At 21 days of age, rats were randomly divided into three groups (control, PCOS, and PCOS EA; n = 12/group) and implanted subcutaneously with 90-day continuous-release pellets containing vehicle or 5α-dihydrostestosterone (DHT). From age 70 days, PCOS EA rats received 2-Hz EA (evoking muscle twitches) five times/week for 4–5 weeks. Hypothalamic protein expression was measured by immunohistochemistry and western blot. DHT-treated rats were acyclic, but controls had regular estrous cycles. In PCOS rats, hypothalamic medial preoptic AR protein expression and the number of AR- and GnRH-immunoreactive cells were increased, but CRH was not affected; however, GnRH receptor expression was decreased in both the pituitary and hypothalamus. Low-frequency EA restored estrous cyclicity within 1 week and reduced the elevated hypothalamic GnRH and AR expression levels. EA did not affect GnRH receptor or CRH expression. Interestingly, nuclear AR co-localized with GnRH in the hypothalamus. Thus, rats with DHT-induced PCOS have disrupted estrous cyclicity and an increased number of hypothalamic cells expressing GnRH, most likely mediated by AR activation. Repeated low-frequency EA normalized estrous cyclicity and restored GnRH and AR protein expression. These results may help explain the beneficial neuroendocrine effects of low-frequency EA in women with PCOS.

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.

Understanding the mechanisms of human tubal ectopic pregnancies: new evidence from knockout mouse models

Ectopic pregnancy, a worldwide health problem, is potentially life-threatening and occurs in approximately 1.5–2% of all pregnancies in the western world; however, the precise mechanisms underlying the initiation and development of tubal ectopic pregnancy are unknown. Tubal abnormalities and dysfunction, such as altered contractility or abnormal ciliary activity, have been speculated to lead to tubal ectopic pregnancy. To elucidate the cellular and molecular mechanisms of the tubal transport process, several knockout (KO) mouse models have been developed. This review summarizes what has been learned from studies of the Fallopian tube in caspase-1, cannabinoid receptor and Dicer1 KO mice. Our understanding of the mechanisms which contribute to tubal ectopic pregnancy in humans may be enhanced through further study of these KO mouse models.

Progesterone-Receptor Antagonists and Statins Decrease De Novo Cholesterol Synthesis and Increase Apoptosis in Rat and Human Periovulatory Granulosa Cells In Vitro

Abstract Progesterone-receptor (PR) stimulation promotes survival in rat and human periovulatory granulosa cells. To investigate the mechanisms involved, periovulatory rat granulosa cells were incubated in vitro with or without the PR-antagonist Org 31710. Org 31710 caused the expected increase in apoptosis, and expression profiling using cDNA microarray analysis revealed regulation of several groups of genes with functional and/or metabolic connections. This regulation included decreased expression of genes involved in follicular rupture, increased stress responses, decreased angiogenesis, and decreased cholesterol synthesis. A decreased cholesterol synthesis was verified in experiments with both rat and human periovulatory granulosa cells treated with the PR-antagonists Org 31710 or RU 486 by measuring incorporation of [14C]acetate into cholesterol, cholesterol ester, and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory rat granulosa cells using 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (lovastatin, mevastatin, or simvastatin) increased apoptosis, measured as DNA fragmentation and caspase-3/7 activity. The increase in apoptosis caused by simvastatin was reversed by addition of the cholesterol synthesis-intermediary mevalonic acid. These results show that PR antagonists reduce cholesterol synthesis in periovulatory granulosa cells and that cholesterol synthesis is important for granulosa cell survival.