The overall goal of the research of the theme Signaling and ageing is to lay a biological foundation for treatment modalities in the clinic. Emphasis is on hormone signaling through receptors on the one hand and the physiological processes that occur with aging on the other. With respect to the signaling part of the theme we focus on transmembrane signaling by members of the TGFβ family of growth and differentiation factors and receptors that belong to the large family of G protein coupled receptors GPCRs, a family of high interest for drug development. These ligands and their receptors are studied in various physiological systems. One of those systems is the ovary, an organ that changes continuously throughout life, from follicle development, via the onset of puberty to the loss of menstrual cycling after menopause. We focus on the relationship between the dysfunction of the ovary in patients with polycystic ovary syndrome (PCOS) and the dysregulation of their metabolism, often displayed as obesity. We hypothesize that factors of the TGFβ family, such as AMH and BMPs, act on peripheral tissues such as fat tissue and play a role in the development of obesity in these women. A second major object of our studies is fat tissue, both white and brown fat tissue. Here we study the physiology of obesity and we try to develop new candidates for anti-diabetic drugs or drug targets. The focus here is on ghrelin and its unacylated form, UAG. Ghrelin and most probably UAG act through GPCRs and their target tissues include β-cells of the pancreas, fat and liver, all important players in the regulation of glucose and fat metabolism. A third area of our interest lies in the target organ sensitivity for steroid hormones. Many steroid hormones are further metabolized and activated by their target tissues, and this may have impact on treatment strategies for e.g. prostate and breast cancer. Lastly, we are striving to understand the molecular details of GPCR activation using the LH receptor, a receptor involved in the regulation of steroid hormone production in the gonads, as a model.

Our major achievements were in four subjects.

1.     AMH: we have definitely established the important role of anti-müllerian hormone (AMH) in the regulation of folliculogenesis in the ovary in both mice and women. AMH is involved in polycystic ovary syndrome (PCOS), in the regulation of FSH-sensitivity and therefore E2 production by granulosa cells in the ovary. Using SNP analysis we have shown a relationship between AMH and its receptor (AMHR2) and age-at menopause in interaction with parity.  Furthermore, we have established AMH as a marker for ovarian function, which has been translated to a clinical setting. In mice, we demonstrated that serum AMH levels reflect the size of the primordial follicle pool.

2.     Acylated (AG) and unacylated ghrelin (UAG). We have established that UAG is not a simple metabolite of AG with antagonistic actions. UAG acts as an insulin-sensitizer in man and in rodents. Furthermore, UAG has beneficial effects on metabolism in rodents (glucose, insulin, FFAs). Interestingly, we have shown that UAG has direct effects on brown fat tissue: UAG increases the sensitivity of the cells to energy status, stimulates their differentiation and increases their fat burning capacity.

3.     LH receptor: we have shown that a common polymorphism of the LH receptor gene is associated with disease free survival of breast cancer patients. In addition the polymorphisms changes the posttranslational modification of the LH receptor protein and renders the protein more sensitive to LH; we have identified splice mutations in the LH receptor gene causing aberrant sex differentiation; our molecular studies have shown that the C-terminal part of the hormone binding domain of the receptor is essential for transduction of the hormone binding-signal to the interior of the cell.

4.     Factors affecting steroid levels in target tissues. These factors comprise circulating steroid levels, concentrations of transport proteins and local steroid production in the target tissue. We found that circulating levels of testosterone are highly dependent on the concentration of the transport protein SHBG in blood. Furthermore, local production conversion of testosterone to oestradiol in the brain only plays a limited role in the feedback action of oestradiol on LH production in men.

In the coming years we will further study:

• the relationship between ovarian-derived growth factors and the occurrence of obesity in PCOS patients. The approach which will be used includes the development of a rodent PCOS model, analysis of diet-induced obesity in AMH-deficient mice, and in vitro studies of white and brown fat cells and their precursors in order to study the crosstalk between androgens and growth factors of the BMP family.

We have shown that in addition to androgens also growth factors of the BMP family are enhanced in PCOS patients. Such factors may have effects of fat cell differentiation and activity, in turn ameliorating insulin sensitivity in PCOS patients, increasing insulin levels and thereby worsen the ovarian characteristics of PCOS. The relationship between PCOS and obesity (50-80% of PCOS patients are obese) may therefore be the result of a vicious circle involving both the ovary and peripheral insulin target issues such as fat, muscle and liver.

• the role of UAG in the regulation of metabolism with an emphasis on fat cell function. Again a combination of mouse models and in vitro cell models will be used.

Our findings that UAG has beneficial effects on many aspects of metabolism (see above) warrants our focus on this ligand. We must further delineate the mechanism of action of UAG, identify its receptor and signal transduction and find out how UAG interacts with the many other factors that affect metabolism.

• the development of an ACTH antagonist. The approach that is used is to develop an MC2R specific antagonist that does not interfere with the other melanocortin receptors.

The medical treatment of Cushing’s disease is to date ineffective, especially compared to other treatment modalities for other pituitary diseases; excluded the non-functioning adenoma that also lacks an effective medical treatment modality. If we succeed in developing an MC2R specific antagonist, we can ‘take over’ control of adrenal glucocorticoid overproduction and replace the endogenous cortisol produced by exogenous cortisol, which will restore diurnal rhythm and serum concentrations.

• the role of local steroid production in non-endocrine organs. We will specifically aim at local steroid production in the prostate, where androgenic steroids play an important role in tumour progression, and in the heart, where the role of gluco- and mineralocorticoids will be studied. Furthermore, we will investigate alternative regulatory factors for steroid production in normal and pathological adrenal tissue, with special emphasis on members of the TGFβ/activin family of growth and differentiation factors.

Cellular steroid levels in target tissues are determined by the steroid levels in the blood, but also by local production. This means that local expression levels of steroidogenic enzymes can influence the effects of steroid hormones in steroid responsive tissues.

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