Håkan Billig

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.

Comparative studies on the expression of somatostatin receptor subtypes, outcome of octreotide scintigraphy and response to octreotide treatment in patients with carcinoid tumours

We have compared the expression of somatostatin receptor (sstr) subtypes with the outcome of somatostatin receptor scintigraphy and the effect of somatostatin receptor activation in patients with disseminated carcinoid tumours. Tumour tissues from nine patients with midgut carcinoids (ileal) and three patients with foregut carcinoids (gastric, thymic) were analysed using Northern blotting. Expression of somatostatin receptors was demonstrated in all tumours (12 out of 12), with all five receptor subtypes present in 9 out of 12 tumours. Somatostatin receptor scintigraphy using [111In]DTPA-D-Phe1-octreotide visualized tumours in all patients (12 out of 12). The 111In activity concentrations in tumour tissue (T) and blood (B) were determined in three tumours 1-7 days after injection of the radionuclide. The T/B 111In activity concentration ratios ranged between 32 and 651. Clinically, treatment with the long-acting somatostatin analogue octreotide resulted in marked symptom relief accompanied by a significant reduction in tumour markers, for example urinary-5-HIAA levels (28-71% reduction). Incubation of midgut carcinoid tumours in primary culture with octreotide (10 microM) resulted in a reduction in spontaneously secreted serotonin (45-71% reduction) and 5-HIAA (41-94% reduction). The results demonstrate that carcinoid tumours possess multiple somatostatin receptor subtypes and that somatostatin analogues such as octreotide, which preferentially bind to somatostatin receptor subtype 2 and 5, can be used in the diagnosis and medical treatment of these tumours. In the future, novel somatostatin analogues with subtype specific receptor profiles may prove to be of value for individualizing the treatment of disseminated carcinoid tumour disease.

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.

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.

Progesterone Receptor-Mediated Inhibition of Apoptosis in Granulosa Cells Isolated from Rats Treated with Human Chorionic Gonadotropin

Abstract Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.

The effects of physical activity and estrogen treatment on rat fast and slow skeletal muscles following ovariectomy

Decreased estrogen production is associated with changes in the skeletal, cardiovascular and muscular systems. At the level of skeletal muscles, it has been shown that a reduction in force production occurs at menopause but the underlying mechanisms are still unknown. The aim of the study was to investigate the effects of ovariectomy on myosin heavy chain (MyHC) composition. Additionally, we studied the effects of physical activity and the combined effects of physical activity and estrogen treatment on MyHC content in ovariectomised (OX) animals. Twenty-five rats were randomly assigned to five different groups: controls, runners, OX, ovariectomised runners and ovariectomised runners receiving estrogen. Exercise consisted of voluntary running for 5 weeks. Two muscles were analysed: m. extensor digitorum longus, EDL, (fast muscle) and m. soleus (slow muscle). MyHC content was analysed on 8% gel electrophoresis. The level of running activity is reduced in OX animals and estrogen administration is associated with the normalisation of the level of physical activity. Ovariectomy induces a shift from fast to slow MyHC isoforms in both the soleus and EDL. When OX animals are allowed to run, alterations in MyHC isoforms are still observed in the EDL but not in the soleus. When physical activity is combined with estrogen treatment no alterations are observed in both muscles. In conclusion, this study shows that ovariectomy induces alterations in the contractile properties of skeletal muscles and that physical activity in combination with estrogen treatment are associated with the maintenance of slow and fast muscle characteristics.

Expression of the growth hormone-binding protein messenger RNA in the liver and extrahepatic tissues in the rat: Co-expression with the growth hormone receptor

A cDNA encoding a growth hormone-binding protein (GH-BP) was recently cloned from mouse and rat liver. The GH-BP in these species is identical to the extracellular part of the GH receptor (GH-R) with the transmembrane and intracellular domain substituted for a hydrophilic tail. In the present study the expression of the GH-BP and GH-R was studied in rat liver and extrahepatic tissues. Specific transcripts with estimated sizes of 1.2 kb (GH-BP) and 4.0 kb (GH-R) were found in the liver from both sexes. The expression of GH-BP increased with age up to puberty suggesting that it is developmentally regulated in a similar manner as GH-R. GH-BP mRNA was found in all extrahepatic tissues examined that contained GH-R mRNA. The ratio between the 1.2 kb and 4.0 kb transcripts varied between tissues indicating that GH-R and GH-BP transcripts may be separately regulated. The co-expression of GH-BP and GH-R suggests a functional role for the GH-BP in the local regulation of GH action.

Distribution and hormonal regulation of membrane progesterone receptors β and γ in ciliated epithelial cells of mouse and human fallopian tubes

BackgroundThe controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma.MethodsWestern blot and immunohistochemistry with specific antibodies were used to characterize the expression and cellular localization of the mPRs in mouse and human tissues. Taqman (Quantitative Polymerase Chain Reaction) assays were used to quantify mRNA levels in the fallopian tubes of two different mouse models after injections with different hormones and specific antagonists.ResultsIn the fallopian tubes of both mouse and human, the expression of mPR beta and mPR gamma proteins was exclusively found in the ciliated cells. Whereas mPR beta was found on the cilia, mPR gamma was localized at the base of the same ciliated cells, as previously reported. In gonadotropin-primed mice, both mPRs genes were down-regulated after an injection with progesterone. Treatment with estradiol rapidly down-regulated the level of mPR beta mRNA and protein in immature mice. The mPR gamma protein was down-regulated around the time of ovulation in cycling women, similar to the regulation observed in mice stimulated to ovulate via gonadotropin injections.ConclusionOur findings show the presence and hormonal regulation of two distinct mPRs associated with the cilia of the fallopian tubes in both mice and women. It is hypothesized that these receptors are involved in the control of ciliary movement and, thus, gamete transport in the fallopian tubes of mammals.

Sex steroid levels temporarily increase in response to acute psychosocial stress in healthy men and women.

It is well known that acute psychosocial stress activates the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). However, the effect of acute psychosocial stress on the hypothalamic-pituitary-gonadal (HPG) axis and levels of sex steroids are less known. The aim of this study was to investigate the effect of acute psychosocial stress on serum concentrations of sex steroids in healthy men and women. Twenty men and 19 women (age 30-50 years) underwent Trier Social Stress Test (TSST), a tool for investigating psychobiological stress responses in a laboratory setting. Blood samples were collected before, directly after the stress test, and after 30 min of recovery. Concentrations of androgens were measured with high specificity LC-MS/MS method; concentrations of cortisol, estradiol and sex hormone-binding globulin were determined using immunoassays. In both men and women we observed significantly elevated levels of testosterone, estradiol, androstenedione and sex hormone binding globulin along with significantly increased adrenocorticotropic hormone (ACTH), serum cortisol, heart rate, systolic blood pressure (SBP), and diastolic blood pressure (DBP) as a response to the stressor. Thus, even though the HPG axis and the production of sex steroids may be inhibited during prolonged periods of stress, the sex steroid levels may increase in the initial phase of acute psychosocial stress.

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.