Alzanati, Nadia
(2017)
Polycystic ovarian syndrome and adipose tissue: contribution of peripheral androgen synthesis to hyperandrogenism in polycystic ovarian syndrome.
PhD thesis, University of Nottingham.
Abstract
Background:
Polycystic ovarian syndrome (PCOS) is the most common endocrine, reproductive, metabolic and psychological disorder in women of childbearing age, affecting 6-10% of premenopausal women.
Hyperandrogenism is the most important biochemical feature of the syndrome, which is responsible for the clinical features of PCOS, and is frequently associated with metabolic disturbance, such as insulin resistance, dyslipidaemia, glucose intolerance and hypertension, regardless of the presence of obesity. In several studies, attention has focused on androgen as the main factor for development of metabolic disturbance, which observed in women with PCOS.
The relationship between adipose tissue and the pathophysiology of PCOS, in terms of development of hyperandrogenism and its relation to development of insulin resistance with compensatory hyperinsulinemia still not fully understood. Based on epidemiological studies, the association between circulating androgen levels and insulin resistance, as well as central obesity is a direct correlation. Although ovaries and adrenal glands are the main sources of androgens, adipose tissue is also one of the most important peripheral tissues involved in the production of androgens. Adipose tissue is not just an organ with energy storage; it also has endocrine, paracrine and autocrine functions, due to secretion of active peptides, known as adipokines, and hormones, such as androgens.
In order to understand the role of adipose tissue in the development of hyperandrogenism, we hypothesised that “Androgen metabolic pathways leading to testosterone production in subcutaneous adipose tissue are altered in women with PCOS.” The excess adipose tissue androgen synthesis plays an important role in PCOS pathogenesis.
Aims:
The main aim of this study was to analyse and compare the expression level of the two key enzymes in androgens synthesis, 17-α-hydroxylase/17.20-lyase (CYP17A1) and 17-β-hydroxysteroid dehydrogenase type 5 (AKR1C3) in adipose tissue of women with and without PCOS. These are responsible for locally synthesised sex steroid hormones, mainly androgens; CYP17A1 is responsible in the production of the precursors of androgens and AKR1C3 is responsible in the conversion of inactive androgen (androstenedione) to its active form, testosterone.
In addition, to understand the mechanism of androgen production this study employed isolated pre-adipocytes cultures and in vitro differentiated to mature adipocytes with close regulation of the impact of insulin and LH as the most two hormones which have effect in sex steroid synthesis. In doing so, we investigated androgen synthesis by activation and expression of the main steroidogenic enzymes CYP17A1 and AKR1C3 in adipocytes of non-PCOS and PCOS women. This enabled us to study the difference in the CYP17A1 mRNA and AKR1C3 mRNA expression levels, as well as the concentration of testosterone secretion across non-PCOS and PCOS cultures. This also indicated the probability of a pathway leading to localised synthesis of adipocytes and to investigate if there is a role for PI3-K signaling pathway in insulin regulation of testosterone synthesis in peripheral adipose tissue of women with and without PCOS.
Methods:
In order to achieve these aims, subcutaneous adipose tissue samples (SC) were surgically obtained during gynecological surgery from women with and without PCOS. All participants were of reproductive age (20-45) with a BMI of 20-35kg/m2. Total RNA was isolated from frozen adipose tissue samples of non-PCOS (n=8) and PCOS (n=8) after matching, using Trizol reagent method, followed by reverse transcription. Quantitative RT-PCR was performed to determine the expression of a panel of reference genes (GAPDH, ACTB, and LPR10), and target genes (CYP17A1 and AKR1C3). Data were analysed with GenEx and compared using ΔΔCt method.
AKR1C3 protein expression in non-PCOS (n=3) and PCOS (n=3) was measured and compared by using western blot (WB) technique.
Pre-adipocytes were isolated from fresh adipose tissue samples by enzyme digestion (collagenase) method and in vitro differentiated to mature adipocytes, which were cultured in FCS-free medium, Recombinant insulin +/-LH+/-PI3-k inhibitor (LY294002) was added to the cell culture at different concentrations, in preparation for investigating any change in the expression of steroidogenic enzymes (CYP17A1, AKR1C3) by RT-PCR, after extraction of total RNA
The supernatant was collected for testosterone measurement before and after treatment using enzyme-linked immunosorbent assay (ELISA).
Results:
Of the reference genes testes, GABDH, ACTB, and LRP10 were shown to be consistently expressed across the PCOS and non-PCOS women. The mean± SEM relative expression level of AKR1C3 mRNA in PCOS adipose tissue was 15.1± 2.0, which was significantly (P=0.0003) greater than that (3.3±1.1) of non-PCOS women. However, the expression level of CYP17A1 mRNA was not significantly (p=0.56) different between the two groups. AKR1C3 protein expression level was less expressed in PCOS and there was no significant (P>0.05) difference in the protein expression between two groups.
CYP17A1, AKR1C3 and testosterone were significantly higher in PCOS (n=5) versus the non-PCOS (n=5) in treated and un-treated cultures. Insulin did not alter CYP17A1 or AKR1C3 mRNA expression in PCOS group. In the non-PCOS, AKR1C3 significantly increased with gradual increase in insulin concentrations, 1nM/l (P=0.001), 10 nM/l (P=0.004), and 100 nM/l (P=0.0003). Insulin up regulates AKR1C3 mRNA expression (no treatment (0), 1, 10,100) (0.96±0.21) (1.59±0.84) (2.39±1.23) and (7.42±0.85) respectively. LH± insulin did not alter the expression of either of the enzymes in PCOS
Insulin increased testosterone concentration in non-PCOS but not in the PCOS, testosterone concentration in the supernatant of untreated cultured PCOS (n=5) adipocytes (mean± SEM, 129.3±2.5 pg /ml) was significantly higher (P<0.0001) than that (33.7±4.6 pg /ml) of non-PCOS (n=5) adipocytes.
Insulin addition in different concentrations (1nM/l, 10nM/l, 100nM/l) resulted in a significant increase in testosterone concentrations (94.1±7.1; 118.2±18.2, 200.0±7.3 pg/ml, respectively) in the supernatant of cultured non-PCOS adipocytes, but not in the PCOS adipocytes (118.1±1.8, 90.5±6.4, 89.3±7.6 pg/ml, respectively). The increase of testosterone levels in the non-PCOS adipocyte culture supernatant followed a dose dependent fashion. The magnitude of increase in testosterone concentrations in non-PCOS adipocyte culture supernatant was markedly increased when LH was added to insulin. Adding PI3-K inhibitor (LY294002, 10ng/ml) to insulin did not change the magnitude of insulin effects on testosterone concentrations in non-PCOS adipocyte culture supernatant.
Conclusion:
The data obtained suggests that adipose tissue has the ability to produce its own steroid hormone after detection of the main key enzymes of androgen biosynthesis, and it revealed a 5-fold increase in the expression level of AKR1C3 mRNA in subcutaneous adipose tissue of PCOS women. It is therefore possible to postulate that peripheral adipose tissue plays an important role as a source of excess androgen production in women with PCOS. This could potentially pave the way for the development of innovative therapeutic targets for the management of this very common syndrome.
In addition, we show a markedly higher CYP17A1, AKR1C3 and testosterone in peripheral adipose tissue of PCOS vs. non-PCOS women. This supports the hypothesis that peripheral adipose tissue plays an important role in the pathogenesis of hyperandrogenaemia and PCOS. Insulin and LH seem to play a role in the increased androgen synthesis in adipose tissue of PCOS women, but not through the PI3-K signaling pathway. PI3-K signaling pathway does not seem to be involved in insulin regulation of testosterone synthesis in peripheral adipose tissue of women with or without PCOS.
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