The Influence Of Androgens On The Development Of Insulin Resistance In Polycystic Ovary Syndrome And Its Molecular Mechanisms

Objectives The pathogenesis of Polycystic Ovary Syndrome (PCOS) is complex, both androgens and insulin resistance(IR) play a role in the onset of PCOS. The objective is to investigate the dose and time effects of androgens on the development of IR in PCOS and the molecular mechanisms by which testosterone induces insulin resistance in 3T3-L1 adipocytes and C2C12 myotubes.Part I : The effect of testosterone propionate on the development of PCO and IR in SD ratsMethods Female Spragye-Dawley rats 21 days of age were injected daily(s.c) with different doses of testosterone propionate(0.5,0.75 or1.0 mg/100 g body weight per day)dissolved in vegetable oil(experimental group A, B, C) for 2,3, 5 ,7up to 8 weeks, Control animals were injected with vehical or nomal saline only (group VC and NSC). The changes of body weight, and length between head and tail and the waist were detected. Successive vaginal cell smear was performed at 2 and 10 weeks of age for up to 10 days. Oral glucose tolerance tests were performed during 2 and 8 weeks' treatment and peripheral blood was taken to detect insulin and glucose changes. After 3, 5 and 7 weeks treatment, every 6 rats were sacrificed after 16 hours fasting, serum was detached instantly and been kept in -70 â–¡ for future analysis of glucose and lipid metabolism, including fasting insulin(FINS), total cholesterol (TCHO), triglyceride (TG), high density lipoprotein (HDL) , low density lipoprotein (LDL) and free fatty acid (FFA) but fasting glucose was examined within 2 hours after detachment; and general morphology of ovaries were analyzed and scored on hematoxylin—eosin stained sections and visceral adipose tissue was weighted.Results (1)3 weeks of injection with testosterone (T) induced the appearance of large cystic follicles and derangement of estrous cycles, the area ratio of corpus luteum to ovary , the number ratio of corpus luteums to total follicles and atretic follicle to total follicles all displayed significance between experimental groups and control groups, P<0.05; No significance was detected between experimental groups,P>0.05; (2) T induced IR with time and dose effects, 3 weeks treatment at group C lowered insulin sensitive index compared with group VC, and group B and A induced IR with longer treatment; (3) At 39 days of age puberty in experimental animals the weight increased compared with control group, and VAT in experimental groups B and C increased after 5 weeks treatment, P<0.05;(4) Dyslipidemia appears after 3 weeks treatment at B and C groups, with TCHO and LDL increased and followed by FFA and TG abnormal. The most obvious change were seen in experimental group C, P<0.05.Conclusions It is suggested that T injection to immature female rats can induce polycystic ovaries, block ovulation, and lead to insulin resistance, whlie increase of VAT and dyslipidemia may result from the interactions of elevated testosterone and IR.Part II : The molecular mechanisms of testosterone effect on insulin resistance in 3T3-L1 adipocytes and C2C12 myotubesMethods 3T3-L1 preadipocyes and C2C12 myoblasts was differentiated into matured cells and treated with different doses of testosterone(10^-12～10^-5M)and for different hours ranging from 4 to 48 hours. The expression of Insulin Receptor Substrate-1 (IRS-1); PY₉₄₁IRS-1 and Glucose Transporter 4(GLUT4) was examined by Western Blotting; And the expression of Insulin Receptor(InsR) and the releasing of Tumor necrosis factorα(TNFα) was detected by ELISA.Results (1) the expression of IRS-1 and GLUT4 in 3T3-L1 adipocytes and C2C12 myotubes increased from 4 hours of 10^-9mol/l testosterone treatment, and the highest IRS-1 expression gained at 12 hours' treatment,while GLUT4 gained at 24hours. The highest expression of IRS-1 is 1.42±0.42 and 1.53±0.14 times and GLUT4 expression is 3.22±0.10 and 5.17±1.06 times of Ohour control in 3T3-L1 adipocytes and C2C12 myotubes respectively, P<0.05. (2) the expression of InsR, IRS-1, PY941IRS-I in 3T3-L1 adipocytes was dose-dependent, increased with testosterone doses from 10^-12M to 10^-9M, and decreased from 10^-8M to 10^-5M testosterone treatment(P<0.01, <0.05, <0.05 respectively). While GLUT4 expression in 3T3-L1 adipocytes was highest when treated with 10^-11M testosterone, which is 2.58±0.02 times of blank control(P<0.05), and it decreased with higher testosterone doses. (3) in C2C12myotubes, the expression of InsR didn't change with testosterone doses(P>0.05), and the tendency of IRS-1 change with testosterone doses likes that of 3T3-L1 adipocytes, the maximal expression at 10^-9mol/l testosterone treatment was 3.16±0.15 times higher than that of blank control, P<0.05, and GLUT4 expression increased with doses' increase(the highest expression of 10^-7mol/l is 2.99±0.15 times of blank control, P<0.05), but when the doses was 10^-7mol/l higher, the expression didn't increase any more; (4) TNFα releasing reached highest with 10^-7M testosterone treatment in 3T3-L1 adipocytes(P<0.01).Conclusions Testosterone has direct effects on the cellular expression of insulin transductional moleculars, and it is bidirectional dose- and time-dependent, which increase with low dose and short time testosterone treatment ,while decrease with higher and longer time treatment.