Investigating the role of adipose tissue in mobility and aging: design and methods of the Adipose Tissue ancillary to the Study of Muscle, Mobility, and Aging (SOMMA-AT)

Abstract

Background: Age-related changes in adipose tissue affect chronic medical diseases and mobility disability but mechanism remains poorly understood. The goal of this study is to define methods for phenotyping unique characteristics of adipose tissue from older adults.

Methods: Older adults enrolled in study of muscle, mobility, and aging selected for the adipose tissue ancillary (SOMMA-AT; N = 210, 52.38% women, 76.12 ± 4.37 years) were assessed for regional adiposity by whole-body magnetic resonance (AMRA) and underwent a needle-aspiration biopsy of abdominal subcutaneous adipose tissue (ASAT). ASAT biopsies were flash frozen, fixed, or processed for downstream applications and deposited at the biorepository. Biopsy yields, qualitative features, adipocyte sizes, and concentration of adipokines secreted in ASAT explant conditioned media were measured. Inter-measure Spearman correlations were determined.

Results: Regional, but not total, adiposity differed by sex: women had greater ASAT mass (8.20 ± 2.73 kg, p < .001) and biopsy yield (3.44 ± 1.81 g, p < .001) than men (ASAT = 5.95 ± 2.30 kg, biopsy = 2.30 ± 1.40 g). ASAT mass correlated with leptin (r = 0.54, p < .001) and not resistin (p = .248) and adiponectin (p = .353). Adipocyte area correlated with ASAT mass (r = 0.34, p < .001), BMI (r = 0.33, p < .001), adiponectin (r = -0.22, p = .005) and leptin (r = 0.18, p = .024) but not with resistin (p = .490).

Conclusion: In addition to the detailed ASAT biopsy processing in this report, we found that adipocyte area correlated with ASAT mass, and both measures related to some key adipokines in the explant conditioned media. These results, methods, and biological repositories underscore the potential of this unique cohort to impact the understanding of aging adipose biology on disease, disability, and other aging tissues.

Keywords: Adipokines; Adipose tissue; Human aging; SOMMA.

Figure 1.

Flowchart of SOMMA-AT participant enrollment for ASAT biopsy collection.

Figure 2.

Abdominal subcutaneous adipose tissue biopsy sample processing in order of priority. When total ASAT yield was <500 mg, A. ~100 mg ASAT were allocated toward formalin-fixed paraffin-embedded (FFPE) histological sectioning and B. Up to 4 separate pieces of ~100 mg ASAT were flash frozen in liquid nitrogen (LN₂) for future assessments. No further processing was performed. When total ASAT yield was ≥500 mg, C. pieces of ~100mg ASAT were processed for explant conditioned media (CM). This was performed in duplicate where ASAT yield was sufficient. After 3 h of incubation in a shaking water bath at 37 °C, ASAT were captured on top a 40 μm cell strainer and flash frozen in LN₂. CM were centrifuged at 500 x g for 5 min before being frozen at -80 °C. D. ≥500 mg ASAT were digested with collagenase solution in a shaking water bath at 37 °C until a “soup-like” consistency, or up to 60 min. Free-floating adipocytes were left to separate from the stromal vascular fraction (SVF) by flotation for 5–10 min. Internatant and SVF were transferred into a new conical tube via a syringe needle and centrifuged at 500 x g for 5 min. Free-floating adipocytes were washed with Hank’s Balanced Salt Solution (HBSS) before isolation with a wide-bore pipette tip and frozen at −80 °C. For the removal of red blood cells (RBCs) from the SVF, cells were resuspended in ACK lysing buffer and incubated at room temperature for 5 min, followed by centrifugation at 500 x g for 5 min. Incubation with lysing buffer was repeated up to 3 times if SVF was still red in color. SVF were resuspended in growth media and filtered through a 100 μm cell strainer. SVF were grown in a 37 °C, 5% CO₂ incubator and frozen into cryovials at −80 °C for 24 h in a cell freezing container and moved to vapor phase in LN₂ for long-term storage.

Figure 3.

Total abdominal subcutaneous adipose tissue biopsy yield correlates with total ASAT mass and age. A. Pearson’s correlations (R and p-value) of ASAT biopsy yield with the ASAT mass and B. with age.

Figure 4.

Needle-aspirated abdominal subcutaneous adipose tissue biopsies in older adults. Representative images of abdominal subcutaneous adipose tissue (ASAT) biopsy samples captured after the removal of blood, blot clots and connective tissue. A–D. Decreasing amounts of total ASAT sample yield obtained per participant. E–H. Increasing amounts of blood clots present throughout the ASAT. I–L. ASAT color ranged from yellow, pale yellow to white. ASAT were assessed by 2 independent observers at each of the clinical sites.

Figure 5.

Representative images of formalin-fixed paraffin-embedded histological sections of abdominal subcutaneous adipose tissue. Computer-aided measurements of adipocyte area were performed on Visiopharm. Intracellular space within the adipocytes were defined within the membrane borders (green). An example immunohistochemistry staining is shown in the image (red pixels) but not quantified for this report. Abdominal subcutaneous adipose tissue histology sections from a A. woman and B. man.

Figure 6.

Adipokine profiles of abdominal subcutaneous adipose tissue from older adults. Measurements of 3 ASAT-secreted factors from abdominal subcutaneous adipose tissue explants in conditioned media, adjusted for ASAT mass. Adipokine concentrations of A. adiponectin, B. leptin and C. resistin, stratified by sex. Data are mean ± SD; ANCOVA adjusted for ASAT mass measured by AMRA® magnetic resonance imaging; ^**p < .01.

Figure 7.

Spearman correlation matrix of adipocyte size, anthropometric characteristics, and adipokine concentration levels. Mean area of histology-derived adipocytes correlated to abdominal subcutaneous adipose tissue (ASAT) mass, body mass index (BMI), age, and concentration of three adipokines (adiponectin, leptin, and resistin) measured in ASAT explant conditioned media. *p < .05.