Combined androgen excess and Western-style diet accelerates adipose tissue dysfunction in young adult, female nonhuman primates

Abstract

Study question: What are the separate and combined effects of mild hyperandrogenemia and consumption of a high-fat Western-style diet (WSD) on white adipose tissue (WAT) morphology and function in young adult female nonhuman primates?

Summary answer: Combined exposure to mild hyperandrogenemia and WSD induces visceral omental (OM-WAT) but not subcutaneous (SC-WAT) adipocyte hypertrophy that is associated with increased uptake and reduced mobilization of free fatty acids.

What is known already: Mild hyperandrogenemia in females, principally in the context of polycystic ovary syndrome, is often associated with adipocyte hypertrophy, but the mechanisms of associated WAT dysfunction and depot specificity remain poorly understood.

Study design, size and duration: Female rhesus macaques were randomly assigned at 2.5 years of age (near menarche) to receive either cholesterol (C; n = 20) or testosterone (T; n = 20)-containing silastic implants to elevate T levels 5-fold above baseline. Half of each of these groups was then fed either a low-fat monkey chow diet or WSD, resulting in four treatment groups (C, control diet; T alone; WSD alone; T + WSD; n = 10/group) that were maintained until the current analyses were performed at 5.5 years of age (3 years of treatment, young adults).

Participants/materials, setting and methods: OM and SC-WAT biopsies were collected and analyzed longitudinally for in vivo changes in adipocyte area and blood vessel density, and ex vivo basal and insulin-stimulated fatty acid uptake and basal and isoproterenol-stimulated lipolysis.

Main results and the role of chance: In years 2 and 3 of treatment, the T + WSD group exhibited a significantly greater increase in OM adipocyte size compared to all other groups (P < 0.05), while the size of SC adipocytes measured at the end of the study was not significantly different between groups. In year 3, both WAT depots from the WSD and T + WSD groups displayed a significant reduction in local capillary length and vessel junction density (P < 0.05). In year 3, insulin-stimulated fatty acid uptake in OM-WAT was increased in the T + WSD group compared to year 2 (P < 0.05). In year 3, basal lipolysis was blunted in the T and T + WSD groups in both WAT depots (P < 0.01), while isoproterenol-stimulated lipolysis was significantly blunted in the T and T + WSD groups only in SC-WAT (P < 0.01).

Limitations, reasons for caution: At this stage of the study, subjects were still relatively young adults, so that the effects of mild hyperandrogenemia and WSD may become more apparent with increasing age.

Wider implications of the findings: The combination of mild hyperandrogenemia and WSD accelerates the development of WAT dysfunction through T-specific (suppression of lipolytic response by T), WSD-dependent (reduced capillary density) and combined T + WSD (increased fatty acid uptake) mechanisms. These data support the idea that combined hyperandrogenemia and WSD increases the risk of developing obesity in females.

Study funding/competing interest(s): Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number P50 HD071836 to C.T.R. and award number OD 011092 from the Office of the Director, National Institutes of Health, for operation of the Oregon National Primate Research Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Keywords: PCOS; Western-style diet; adipose tissue; androgen; fatty acid; hyperandrogenemia; lipolysis; nonhuman primates; obesity; testosterone.

Figure 1

Study design. Female rhesus macaques were started at 2.5 years of age and maintained for 3 years on a standard chow or Western-style diet (WSD). The study involved four experimental groups: C, control animals; T, animals with chronically elevated testosterone (T) levels; WSD and T + WSD; n = 10/group (see the ‘Results’ section for details). Visceral omental-white adipose tissue (OM-WAT) biopsies were collected in years 0, 2 and 3, and sub-cutaneous (SC)-WAT biopsies in years 2 and 3. WAT biopsies were subjected to morphological analysis for the assessment of adipocyte size (cell area) and capillary network properties (see ‘Materials and Methods’ for details), and also analyzed ex vivo to quantify basal (Bas) and insulin-stimulated (Ins) fatty acid (FFA) uptake and lipolysis (glycerol release) under basal (Bas) and isoproterenol (Iso)-stimulated conditions.

Figure 2

Adipocyte area and WAT morphology. Omental (OM) (A) and sub-cutaneous (SC) (B) adipocyte area was quantified in years 0, 2 and 3 for OM-white adipose tissue (WAT), and in year 3 for SC-WAT, as described in ‘Materials and Methods.’ Average cell area per animal was calculated for 20–50 adipocytes. (C) Representative examples of Trichrome-stained histological sections of OM-WAT (year 2). Pink color demarcates cellular membranes and cytoplasm, and blue color labels fibrotic structures (scale bar = 200 μm). (A) Uppercase letters denote differences (P < 0.05) by treatment within year, and lowercase letters indicate differences (P < 0.05) within a given treatment by year. (B) Analysis of SC-WAT area data was done for year 3 only with no difference detected by treatments (P > 0.8). Bars are means ± SEM.

Figure 3

White adipose tissue (WAT) capillary networks. Omental (OM)-WAT biopsies collected in year 3 were co-labeled with BODIPY-C12 and Isolectin GS-IB4, Alexa Fluor^TM 647 conjugate and analyzed by confocal microscopy as described in ‘Materials and Methods.’ (A–D) Representative images of OM-WAT; scale bar = 50 μm; asterisks, vessel junctions. Vessel junction density (E and G) and vessel length (F and H) were calculated for OM-WAT (E and F) and SC-WAT (G and H). Total vessel length in each WAT depot was calculated using ×10 images representing confocal slice with a 1550 × 1550 × 100-μm³ dimension. Junction density is the number of junctions populating 1 μm² of a confocal projection. Overall effects of diet are indicated in inset above graphs.

Figure 4

Free fatty acid uptake in omental white adipose tissue (OM-WAT). OM-WAT explants collected in years 2 and 3 were treated with basal or insulin-containing media, labeled with BODIPY-C12 (A) and live cell marker Calcein Red-Orange AM (B), fixed and processed for microscopy analysis as described in ‘Materials and Methods.’ (A and B) Representative confocal images of insulin-treated OM-WAT explants (scale bar = 50 μm). BODIPY-C12 is distributed to micro-lipid droplets (mLDs) and the central lipid droplet (cLD). Green fluorescence is excluded from red-negative dead adipocytes (‘D’). (C) Quantification of insulin effects on BODIPY-C12 incorporation as a ratio of insulin-stimulated vs basal fluorescence. Bars are means ± SEM, n = 6–10 animals per group (average fluorescence per animal was calculated for 20–50 adipocytes). Asterisk, pair-wise comparison shows a statistically significant difference (P < 0.05) between years 2 and 3.

Figure 5

White adipose tissue (WAT) lipolysis. Omental (OM)-WAT (A and B) and sub-cutaneous (SC)-WAT (C and D) biopsies were collected in year 2 (A and C) and year 3 (B and D), divided into 50-mg explants, and treated for 2 h with basal (Bas) or isoproterenol (Iso)-containing media, in duplicate or triplicate. Conditioned media was used to determine glycerol release as described in ‘Materials and Methods.’ Lower-case letters indicate differences (P < 0.05) between treatments at Bas, and uppercase letters indicate differences between treatments cultured with Iso (P < 0.05). Bars are means ± SEM, n = 6–10 animals per group.