Type I fiber decrease and ectopic fat accumulation in skeletal muscle from women with PCOS

Background: Polycystic ovary syndrome’s (PCOS) main feature is hyperandrogenism, which is linked to a higher risk of metabolic disorders in women. Gene expression analyses in adipose tissue and skeletal muscle reveal dysregulated metabolic pathways in women with PCOS, but these differences do not necessarily lead to changes in protein levels and biological function.

Methods: To advance our understanding of the molecular alterations in PCOS, we performed global proteomic and phosphorylation site analysis using tandem mass spectrometry. Adipose tissue and skeletal muscle were collected at baseline from 10 women with and without PCOS, and in women with PCOS after 5 weeks of treatment with electrical stimulation.

Results: Perilipin-1, a protein that typically coats the surface of lipid droplets in adipocytes, was increased whereas proteins involved in muscle contraction and type I muscle fiber function were downregulated in PCOS muscle. Proteins in the thick and thin filaments had many altered phosphorylation sites, indicating differences in protein activity and function. The upregulated proteins in muscle post treatment were enriched in pathways involved in extracellular matrix organization and wound healing, which may reflect a protective adaptation to repeated contractions and tissue damage due to needling. A similar, albeit less pronounced, upregulation in extracellular matrix organization pathways was also seen in adipose tissue.

Conclusions: Our results suggest that hyperandrogenic women with PCOS have higher levels of extramyocellular lipids and fewer oxidative insulin-sensitive type I muscle fibers. These could be key factors leading insulin resistance in PCOS muscle while electric stimulation-induced tissue remodeling may be protective.

Methods
Women with polycystic ovary syndrome (PCOS) were diagnosed according to the Rotterdam criteria. Subcutaneous adipose tissue and skeletal muscle biopsies from women with PCOS and controls were included in the proteomics analysis. Immunfluorescence was used to quantify perilipin-1 and slow type I myosin fibers in skeletal muscle. Picrosirius Red staining was used to quantify fibrillar collagen in adipose and muscle tissue.

3-week-old wild-type (wt) female mice on C57Bl/6J background were purchased from Janvier Labs (C57BL/6NRj, Le Genest-Saint-Isle, France). Female skeletal muscle androgen receptor knockout mice (SkMARKO) were generated by crossing ARflox mice with B6;C3-Tg(ACTA1-rtTA,tetO-cre)102Monk/J (HSA-rtTA/TRE-Cre) mice. To induce Cre recombinase expression, at 3 weeks of age, SkMARKO mice were given a diet containing 200 mg/kg doxycycline (Specialty Feeds SF11-059) for the entire duration of the experiment. At 4 weeks of age, wt and SkMARKO female mice received a subcutaneous silastic implant containing 5-10 mg DHT (5α-Androstan-17β-ol-3-one, A8380, Sigma-Aldrich, St. Louis, USA) or empty implant (n=5-8/group). A subset of DHT-exposed wt mice received a slow-release flutamide pellet (n=5, 25 mg flutamide/pellet, 90-day release, Innovative Research of America, Sarasota). At 15-17 weeks of age, the mice were euthanized and gastrocnemius muscle tissue was dissected and snap-frozen. Western blot was used to quantify slow type I myosin fibers in skeletal muscle.