No description available.

Background: Exposure to perfluorooctane sulfonic acid (PFOS) or to perfluorooctanoic acid (PFOA) increases mouse and human peroxisome proliferator–activated receptor alpha (PPARα) subtype activity, which influences lipid metabolism. Because cholesterol is the substrate from which testosterone is synthesized, exposure to these substances has the potential to alter testosterone concentrations. Objectives: We explored associations of total testosterone and sex hormone–binding globulin (SHBG) concentrations at age 15 years with prenatal exposures to PFOS, PFOA, perfluorohexane sulfonic acid (PFHxS), and perfluoronanoic acid (PFNA) in females. Methods: Prenatal concentrations of the perfluoroalkyl acids (PFAAs) were measured in serum collected from pregnant mothers at enrollment (1991–1992) in the Avon Longitudinal Study of Parents and Children (ALSPAC). The median gestational age when the maternal blood sample was obtained was 16 weeks (interquartile range, 11–28 weeks). Total testosterone and SHBG concentrations were measured in serum obtained from their daughters at 15 years of age. Associations between prenatal PFAAs concentrations and reproductive outcomes were estimated using linear regression models (n = 72). Results: Adjusted total testosterone concentrations were on average 0.18-nmol/L (95% CI: 0.01, 0.35) higher in daughters with prenatal PFOS in the upper concentration tertile compared with daughters with prenatal PFOS in the lower tertile. Adjusted total testosterone concentrations were also higher in daughters with prenatal concentrations of PFOA (β = 0.24; 95% CI: 0.05, 0.43) and PFHxS (β = 0.18; 95% CI: 0.00, 0.35) in the upper tertile compared with daughters with concentrations in the lower tertile. We did not find evidence of associations between PFNA and total testosterone or between any of the PFAAs and SHBG. Conclusions: Our findings were based on a small study sample and should be interpreted with caution. However, they suggest that prenatal exposure to some PFAAs may alter testosterone concentrations in females.

The glycoprotein hormones, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) are heterodimeric proteins with an identical α-subunit associated noncovalently with the hormone specific β-subunit and play important roles in reproduction and overall physiology of the organism (Pierce & Parsons, 1981). The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have a large extracellular domain (ECD)comprising of 9-10 leucine rich repeats (LRR) followed by a flexible hinge region, a seven helical transmembrane domain (TMD) and a C terminal cytoplasmic tail (Vassart et al, 2004). Despite significant sequence and structural homologies observed between the ECDs of the receptors and the specific β-subunits of the hormones, the hormone-receptor pairs exhibit exquisite specificity with very low cross-reactivity with other members of the family. Several biochemical, immunological and molecular biological tools have been employed to elucidate the structure– function relationship of the hormones and their receptors. These studies also helped in deciphering some of the regions present in both the hormones and the receptors involved in maintaining the specificity of their interaction (Fan & Hendrickson, 2005b; Fox et al, 2001; Wu et al, 1994). However, the complete understanding of the hormone-receptor contact sites and mechanism of receptor activation are still an enigma. Understanding the molecular details of these phenomena can lead to the development of novel strategies of regulating hormone action. Binding of FSH to FSHR occurs in the large extracellular NH2-terminal domain where the participation of the LRRs (amino acids 18-259) is essential to determine the ligand selectivity (Dias & Van Roey, 2001; Fan & Hendrickson, 2005a; Szkudlinski et al, 2002). In fact, mutations in these regions lead to reduction in binding of the agonist to the receptor. It is not known how the signal from the large extracellular domain liganded complex is transmitted to the TMD (amino acids 367-695). It is envisioned that hormone binding to the LRRs leads to series of conformational changes leading activation of the TMD resulting in signal transduction. The recently reported crystal structure of the single chain form of FSH in complex with the leucine rich repeats of the FSHR (amino acids 1-268) (Fan & Hendrickson, 2005b), although provides detailed understanding of the molecular interactions of the LRRs with the hormone, fails to provide any insights into mechanism of receptor activation as the information regarding critical interaction of the hormone with TMD. This structure also did not provide any information on the role of the hinge region (amino acids 259-366) that connects the LRRs to the TMD in hormone binding and activation of the receptor. In the present study an attempt has been made to understand the role of the hinge region in hormone binding and signal transduction. The overall objective of the study is to elucidate the molecular details of the hormone receptor interactions, particularly FSH-FSHR interaction. Antibodies to glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone-receptor interactions and signal transduction. While the TSH receptor antibodies and their effects on the overall physiology have been well documented (Khoo & Bahn, 2007; Rapoport & McLachlan, 2007), reports of such antibodies against FSHR or LHR and their possible effects on the reproductive functions are not available. In the present study, effects of FSHR antibodies with different specificities on FSH-FSHR interactions have been investigated. Antibodies to different regions of rat FSHR, were raised and extensively characterized and their effects of FSH-FSHR interactions and signaling were investigated. It was found that a polyclonal antibody against the hinge of the receptor (RF2 antiserum, amino acids 218-336), while having no significant effect on hormone binding and response could stimulate the receptor by itself bypassing the hormone. This stimulation of FSHR was very specific as this antiserum could not stimulate LHR or TSHR and could be blocked by preincubating the antibody with the antigen. Through competition experiments with different synthetic peptides of human FSHR, a stretch of hinge region corresponding to amino acids 296-331 was identified as the site recognized by the stimulatory antibody. This antibody did not interfere in hormone binding and could also bind to the pre-formed hormone-receptor complex suggesting that the binding site of the antibody may not participate directly in hormone binding. Subsequently the antibody was extensively characterized for its effect of hormone receptor interactions (Chapter 2). Previous studies considered the hinge region to be an inert linker connecting the LRRs to the TMD, a structural entity without any known functional significance (Vlaeminck-Guillem et al, 2002). However, the data with RF2 antibody suggested a direct role of the hinge region in signal transduction. Therefore, a systematic study to dissect the role to hinge region in hormone binding and signal transduction was conducted. Several truncations, deletions, activating and inactivating point mutations in the FSHR were generated to understand the mechanism of receptor activation. Firstly, these mutant receptors were characterized for their ability to translocate to the cell surface when transfected in the cultured mammalian cells. Secondly, affinity of all the mutant receptors for the hormone was determined in order to understand the effect of mutations on hormone binding. Finally, the cAMP response of these mutant receptors to the hormone and the stimulatory antibody was investigated to understand the effects of mutations on signal transduction. The results are described in Chapter 3. The hormone binding analysis and the affinity measurement of the mutant receptors showed that the LRRs are involved in high affinity hormone binding while the hinge region may not contribute to the process. This is in agreement with the crystal structure data which showed that the hormone was bound to the truncated receptor fragment representing only the LRRs (Fan & Hendrickson, 2005b). These binding data also corroborated the earlier data indicating that the antibodies against the hinge region do not interfere in hormone-receptor interactions. Further, the analysis of different N-terminally truncated receptor mutants provided strong evidence indicating that the constraining intramolecular interactions between the extracellular and the transmembrane domains are required to maintain the FSHR in an inactive conformation in the absence of an agonist. The analysis of the constitutive basal activity of the mutant receptors in absence of hormone suggested that certain regions of the extracellular domain had an attenuating effect over the TMDs that prevented constitutive activation of the receptor. This was demonstrated by a marked increase in the basal constitutive activity of the receptor upon the complete removal of its extracellular domain. Detailed analysis of the mutants suggested that LRR portion does not contribute to this attenuating effect, but it is the hinge region that perhaps interacts with the TMDs and dampens its basal constitutive activity. This attenuating effect was further narrowed down to a small stretch of 35 amino acids (296-331) within the hinge region. It was striking that the similar stretch was identified as the binding site of the stimulatory receptor antibody. In pharmacology, an ‘inverse agonist’ is an agent which binds to the receptor and reverses the constitutive activity of receptors. Thus the hinge region of the receptor could be termed as a ‘tethered inverse agonist’ of the TMD, since it is covalently associated with the TMD and their interactions dampen the basal constitutive activity of the receptor. However, careful comparison of the activities of the mutants (receptors harboring deletions and gain-of-function mutations) with maximally stimulated wild-type FSHR indicated that these mutations of the receptor resulted only in partial activation of the serpentine domain suggesting that only the ECD in complex with the hormone is the full agonist of the receptor. Moreover, the hinge region stabilizes the TMD in an inactive conformation and the activating mutations disengage the inhibitory ECD–TMD interactions bringing about partial activation of the receptor. Most interestingly, the deletion of amino acids 296-331 from hFSHR resulted in no further response to the hormone indicating that this part of the receptor is also critical for hormonal activation, perhaps playing a dual role in the attenuation of the basal activity and a direct involvement in the hormonal activation of the receptor. Progressive sequential deletions of ten amino acids from 290 to 329 yielded similar results (high basal cAMP production with concomitant loss of hormone and antibody response) clearly demonstrating that the integrity of this region is absolutely essential for hormonal activation. In conclusion, the study provides a conclusive evidence to show that the hinge region of FSHR, although not involved in primary high affinity hormone binding, plays a critical role in the modulation of the receptor activity in absence, as well as, presence of the hormone. A large array of reproductive abnormalities is associated with malfunctioning of FSHR. To explore the possibility of using the stimulatory antibodies for therapeutic purpose, three inactivating mutations of hFSHR were analyzed. In corroboration with the earlier reports (Doherty et al, 2002; Touraine et al, 1999), the mutants A419T and L601V are incapable of transducing the signal, despite having adequate cell surface expression and wild type affinities for the hormone, mainly because of defective TMD. The RF2 antibody failed to elicit any response from these mutants suggesting that its ability to activate the receptor depends on the status of the TMD. Interestingly, the activating mutant D576G, which showed very high basal cAMP production, could be stimulated by both antibody and the hormone to the nearly wild type levels suggesting that in this mutant the interactions between the hinge region and TMD are similar to that of wild type and higher basal cAMP production could be due to different interactions of the TMD with the G-Proteins. Structure-function studies of glycoprotein hormones and their receptors have been hampered due to low levels of expression of the properly folded proteins in heterologous systems (Chazenbalk & Rapoport, 1995; Hong et al, 1999b; Peterson et al, 2000; Sharma & Catterall, 1995; Thomas & Segaloff, 1994). Previous studies from the laboratory have shown that the Pichiapastoris,which blends the advantages of both bacterial and mammalian expression systems, can be used to hyper-express biologically active hormones (Blanchard et al, 2008; Gadkari et al, 2003; Samaddar et al, 1997). In addition, the same expression system has been used to produce single chain hormone analogs (Roy et al, 2007; Setlur & Dighe, 2007). Further, methodologies for Pichiafermentation and purification of recombinant hormones from the fermentation media have been wellestablished in the laboratory. Chapter 4 describes the work carried out to express, purify and characterize a fully functional hFSHR extracellular domain. Thus a stage is now set to attempt structural studies with the receptor. The results are discussed at the end of each of these chapters and future directions have been discussed at the end of this thesis.

The main purpose of this thesis was to examine the relationship of pre-diagnostic circulating levels of sex-steroids (androgens and estrogens), sex hormone binding globuline (SHBG), insulin-like growth factor-I (IGF-I), IGF binding proteins (BP) and C-peptide (as a marker of pancreatic insulin secretion) with risk of ovarian and endometrial cancer. Additionally, the interrelationships of body mass index (BMI), sex-steroids, IGF-I and IGFBP-3 were examined. Two case-control studies were nested within 3 prospective cohort studies centered in New York (USA), Umeå (Sweden) and Milan (Italy). The ovarian study included 132 cancer cases. The endometrial study included 166 cancer cases in the IGF-I and C-peptide component and 124 postmenopausal cases in the sex-steroids component. For each case, two controls matching the case for cohort, age, menopausal status and date at recruitment were selected. In total 286 and 315 controls were included in the ovarian and endometrial cancer studies, respectively. Odds ratios (OR) and their 95% confidence intervals (CI) for cancer risk associated with increasing hormone concentrations were estimated by conditional logistic regression. The cross-sectional analysis was based on anthropometric and hormonal data from 620 controls selected for the two nested case-control studies. There was no association of prediagnostic androstenedione, testosterone, DHEAS, SHBG or estrone with ovarian cancer risk in the whole study population or in women who were pre- or postmenopausal at blood donation. In the premenopausal group, risk appeared to increase with increasing androstenedione (OR (95% CI) for the highest tertile: 2.35 (0.81-6.82), p=0.12). There was no association of IGF-I, IGFBP-1, 2, 3 or C-peptide concentrations with risk of ovarian cancer risk in the study group as a whole. In analyses restricted to subjects who had developed ovarian cancer at an early age (<55), circulating IGF-I was directly and strongly associated with risk (OR (95% CI): 4.74 (1.20-18.7), p<0.05 for the highest IGF-I tertile). In the endometrial study, previous observations were confimed that elevated circulating estrogens and androgens and decreased SHBG increase risk of developing endometrial malignancy after menopause. Multivariate ORs (95% CI) for endometrial cancer for quartiles with the highest hormone levels were: 4.13 (1.76-9.72), p<0.001 for estradiol; 3.67 (1.71-7.88), p=0.001 for estrone; 2.15 (1.05-4.40), p<0.04 for androstenedione; 1.74 (0.88-3.46), p=0.06 for testosterone; 2.90 (1.42-5.90), p<0.01 for DHEAS and 0.46 (0.20-1.05), p<0.01 for SHBG. Prediagnostic IGF-I, IGFBP-1, -2 and –3 were not related to risk of endometrial cancer in the whole study population. In postmenopausal women, levels of IGFBP-1 were inversely related to risk with an OR for the highest quartile of 0.36 (0.13-0.95), p<0.05. Endometrial cancer risk increased with increasing levels of C-peptide (p<0.01), up to an OR of 4.40 (1.65-11.7) for the highest quintile after adjustment for BMI and other confounders. The cross-sectional analyses showed that in both pre- and postmenopausal women SHBG decreased with increasing BMI. In the postmenopausal group, estrogens, testosterone and androstenedione increased with BMI, while the association with IGF-I was non-linear, the highest mean IGF-I concentration being observed in women with BMI between 24 and 25. In postmenopausal women, IGF-I was positively related to androgens, inversely correlated with SHBG, and was not correlated with estrogens. In conclusion, elevated pre-diagnostic sex-steroids, IGF-I or C-peptide increase risk of developing ovarian and endometrial cancer. BMI influences the circulating levels of these hormones, especially after menopause.

Abstract Cardiovascular diseases are the leading cause of death worldwide and type 2 diabetes is reaching a global epidemic. Epidemiological studies have identified numerous risk factors and pharmacotherapies in relation to these cardiometabolic diseases. However, the detailed molecular mechanisms of these risk factors and drug therapies generally remain incompletely understood. Elucidating the underlying molecular effects would be essential for better understanding of the disease pathogenesis and also for discovering new therapeutic targets. Quantitative serum metabolomics, which allows for simultaneous quantification of multiple circulating metabolic measures, provides a hypothesis-free approach to systematically inspect the metabolic changes in response to endogenous and exogenous stimuli. Metabolomics thus presents a valuable tool to study the detailed molecular effects of disease risk factors and drug therapies. However, current metabolomics studies are mostly conducted in small cross-sectional studies and the causal relations of the risk factors on the metabolic measures are generally unclear, providing limited public health impact. The present thesis serves as a proof-of-concept to illustrate that well-designed observational studies can be used to infer causality. With the exemplars of assessing molecular effects of two risk factors (body mass index and sex hormone-binding globulin) and two drug therapies (statins and oral contraceptives), the thesis demonstrates that an improved causal inference can be achieved in observational studies via the combination of multiple study designs, including cross-sectional, longitudinal and Mendelian randomization analysis. This robust study design approach together with metabolomics data can be also extended to study the molecular effects of other risk factors and drug therapies. With an improved molecular understanding of a wide range of risk factors and therapies, better understanding of disease pathogenesis is ensured. / Tiivistelmä Sydän- ja verisuonitaudit ovat johtava kuolinsyy maailmassa ja tyypin 2 diabetes on saavuttamassa globaalin epidemian mittasuhteet. Epidemiologiset tutkimukset ovat löytäneet useita riskitekijöitä ja lääkehoitoja edellä mainituille yleisille taudeille. Tyypin 2 diabetekseen ja sydän- ja verisuonitauteihin liittyvät yksityiskohtaiset molekylaariset mekanismit ymmärretään kuitenkin puutteellisesti. Molekylaaristen yksityiskohtien tarkempi ymmärtäminen olisi siten erittäin merkittävää sekä tautiprosessien ymmärtämiseksi että lääkehoitojen kehittämiseksi. Seerumin kvantitatiivinen metabolomiikka mahdollistaa useiden metabolisten suureiden samanaikaisen määrittämisen verenkierrosta ja tarjoaa siten hypoteesittoman lähestymistavan sekä sisäisten että ulkoisten ärsykkeiden aiheuttamien metabolisten muutosten systemaattiseen tutkimukseen. Metabolomiikka on siten arvokas työkalu yksityiskohtaisten molekylaaristen mekanismien tutkimuksessa, olipa kyseessä taudin riskitekijät tai lääkehoito. Metabolomiikkatutkimuksia on kuitenkin pääasiassa tehty pienissä poikittaistutkimuksissa ja riskitekijöihin liittyvien metabolisten suureiden syy- ja seuraussuhteet ovat yleisesti epäselviä, josta johtuen metabolisten suureiden kansanterveydellinen sovellettavuus on ollut heikkoa. Tämä väitöskirja esittelee tutkimuskonseptin hyvin suunniteltujen havaintotutkimuksien soveltamiseksi syy- ja seuraussuhteiden arvioinnissa. Työ sisältää esimerkit kahden riskitekijän (painoindeksi ja sukupuolihormoneja sitova globuliini) ja kahden lääkehoidon (statiinit ja ehkäisypillerit) molekylaaristen vaikutusten kausaalisista tutkimuksista. Tulokset havainnollistavat, että kausaalisten johtopäätösten luotettavuutta voidaan parantaa yhdistämällä useita tutkimusasetelmia, kuten poikittais- ja pitkittäistutkimuksia sekä Mendelististä satunnaistamista. Esitettyjä luotettavia tutkimusasetelmia, yhdessä metabolomiikkadatan kanssa, voidaan laajentaa muiden riskitekijöiden ja lääkehoitojen molekylaaristen vaikutusten tutkimuksiin. Parantunut molekyylitason ymmärrys useista riskitekijöistä ja lääkehoidoista johtaa myös parempaan tautiprosessien ymmärtämiseen.