The adipose cell lineage is not intrinsically insulin resistant in polycystic ovary syndrome

Abstract

Selective resistance to the effects of insulin on glucose metabolism in skeletal muscle and adipose tissue is a key feature of polycystic ovary syndrome (PCOS). The pathogenesis of insulin resistance in skeletal muscle in PCOS involves interaction of in vivo environmental factors with intrinsic defects in insulin signaling. We aimed to determine whether (1) intrinsic defects in insulin action/signaling and cytokine secretion were present in adipose cells in PCOS and (2) insulin resistance can be induced in control adipose cells by culture in medium conditioned by insulin-resistant PCOS fibroblasts. Subcutaneous abdominal preadipocytes from obese women with PCOS (n = 7) and age- and body mass index-matched controls (n = 5) were cultured for several generations in vitro. Basal and insulin-stimulated glycogen synthesis and basal glucose transport did not differ in the preadipocytes from women with PCOS and controls. Abundance of insulin receptor (IR) beta subunit, insulin receptor substrate (IRS) 1 and 2, p85 subunit of phosphatidylinositol 3-kinase, and extracellular signal-regulated kinase (ERK)1/2 activation did not differ. Secretion of tumor necrosis factor alpha and interleukin 6 did not differ. Insulin action on glycogen synthesis in control preadipocytes was not altered by coculture with or growth in media conditioned by PCOS skin fibroblasts with constitutive serine phosphorylation of IRbeta subunit (IR ser+), indicating that IR ser+ cells do not secrete an insulin resistance-inducing factor. We conclude that in contrast to skeletal muscle and skin fibroblasts, there is no evidence for intrinsic defects in insulin signaling in the PCOS adipose cell lineage, indicating that insulin resistance in these cells is likely due to factors in the in vivo environment.

Figure 1

Basal glucose uptake and insulin-stimulated glycogen synthesis in cultured preadipocytes of obese PCOS women and age- and BMI-matched controls. Glucose uptake (n = 7 PCOS, n = 5 controls) was assessed at baseline (a). Glycogen synthesis (n = 6 PCOS, n = 5 controls) was measured at baseline and following incubation with insulin (1 – 100 nmol/l) (b). Filled circles represent PCOS and open circles represent controls. Filled bars represent PCOS and open bars represent controls. Data are presented as mean ± SEM.

Figure 2

Abundance of proximal insulin signaling proteins in cultured preadipocytes from obese women with PCOS and age- and BMI-matched controls. Abundance of IRβ (a), IRS-1 (b), p85 subunit of PI3-kinase (c) and IRS-2 (d) (n = 6 PCOS, n = 5 controls) determined by immunoblot analysis of preadipocytes from women with PCOS (P) and controls (C). Basal lysates (50 μg) were separated by SDS-PAGE (7.5% gels) and representative immunoblots are shown. Filled bars represent PCOS and open bars represent controls. Data are presented as mean ± SEM.

Figure 3

Phospho- and total- ERK1/2 in cultured preadipocytes from obese women with PCOS and age- and BMI-matched controls. Cultured preadipocytes (n = 6 PCOS, n = 5 controls) were incubated with (+) or without (-) insulin (100 nmol/l) for 10 min and lysates (50 μg) separated by SDS-PAGE (7.5% gels). Blots were probed with anti-phospho-ERK1/2, (a) then stripped and reprobed with anti-total ERK1/2 (b). Representative immunoblots are shown. Filled bars represent PCOS and open bars represent controls. Data are presented as mean ± SEM.

Figure 4

Secretion of cytokines by cultured preadipocytes from obese women with PCOS and age- and BMI-matched controls. Serum-free media from confluent preadipocytes (n = 7 PCOS, n = 5 controls) were collected after 48 h incubation and assayed for TNFα (a) and IL-6 (b) using ELISA. Filled bars represent PCOS and open bars represent controls. Results are expressed as pg/ml of media, and adjusted for protein content of each dish. Insulin-stimulated glycogen synthesis was measured in control preadipocytes cultured with media that had been conditioned by control cultured skin fibroblasts (open circles), by PCOS fibroblasts with constitutive increased serine phosphorylation of IRβ (IR ser+) (filled circles) or non-conditioned media (open diamonds) (c). Data are presented as mean ± SEM.