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. 2014:537:93-122.
doi: 10.1016/B978-0-12-411619-1.00006-9.

Quantifying size and number of adipocytes in adipose tissue

Sebastian D Parlee  1 , Stephen I Lentz  2 , Hiroyuki Mori  1 , Ormond A MacDougald  3
Affiliations

Quantifying size and number of adipocytes in adipose tissue

Sebastian D Parlee et al. Methods Enzymol. 2014.

Abstract

White adipose tissue (WAT) is a dynamic and modifiable tissue that develops late during gestation in humans and through early postnatal development in rodents. WAT is unique in that it can account for as little as 3% of total body weight in elite athletes or as much as 70% in the morbidly obese. With the development of obesity, WAT undergoes a process of tissue remodeling in which adipocytes increase in both number (hyperplasia) and size (hypertrophy). Metabolic derangements associated with obesity, including type 2 diabetes, occur when WAT growth through hyperplasia and hypertrophy cannot keep pace with the energy storage needs associated with chronic energy excess. Accordingly, hypertrophic adipocytes become overburdened with lipids, resulting in changes in the secreted hormonal milieu. Lipids that cannot be stored in the engorged adipocytes become ectopically deposited in organs such as the liver, muscle, and pancreas. WAT remodeling therefore coincides with obesity and secondary metabolic diseases. Obesity, however, is not unique in causing WAT remodeling: changes in adiposity also occur with aging, calorie restriction, cancers, and diseases such as HIV infection. In this chapter, we describe a semiautomated method of quantitatively analyzing the histomorphometry of WAT using common laboratory equipment. With this technique, the frequency distribution of adipocyte sizes across the tissue depot and the number of total adipocytes per depot can be estimated by counting as few as 100 adipocytes per animal. In doing so, the method described herein is a useful tool for accurately quantifying WAT development, growth, and remodeling.

Keywords: Adipocytes; Cell number; Cell size; ImageJ; Metamorph; Quantitative histomorphometry.

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Figures

Figure 6.1
Figure 6.1
Expression of Adiponectin increases and secreted frizzled-related protein 1 (sFRP1) decreases in epididymal adipose tissue sampled progressively from the testicle. Twelve-week-old C57BL6/J (N=12) mice were sacrificed and epididymal adipose tissue harvested. The depot was divided into quarters and orientation to the testicle noted. Total RNA was harvested and analyzed by RT-qPCR. Results indicate an inverse relationship with adiponectin expression increasing and sFRP1 decreasing in samples more distal to the testicle. Two-way ANOVA with Bonferroni post-hoc analysis, *p<0.05 when compared to the proximal quarter (area 1).
Figure 6.2
Figure 6.2
The distribution of adipocyte sizes and the linear relationship between average adipocyte volume and epididymal WAT weight. Ten-week-old C57BL6/J mice fed a 60% high-fat diet for 6 weeks were sacrificed and epididymal WAT harvested, fixed, sectioned and ~5000 adipocytes per mouse counted using the MetaMorph-based method described herein. The distribution of adipocytes within the adipose depot is calculated using the frequency function in Excel. (A) C57BL6 mice (N=10) have a mean adipocyte area of approximately 3600.98±820 μm2 (open circle) and a median of 2970 μm2 (black square). (B) The relationship between average adipocyte volume weight of epididymal WAT is linear (r2=0.92).
Figure 6.3
Figure 6.3
Adipocytes size and average area are similar when quantified by MetaMorph or ImageJ. Ten-week-old C57BL6/J mice (N=5) fed a 60% high-fat diet for 6 weeks were sacrificed and epididymal adipose tissue depots harvested, fixed, sectioned and adipocytes counted using either the MetaMorph or ImageJ method described herein. The frequency distribution of adipocyte sizes is equivalent whether analyzed by MetaMorph or ImageJ. No significant differences were found in either the frequency distribution (A, two-way ANOVA with Bonferroni post-hoc analysis) or average adipocyte area (B, Student’s t-test).
Figure 6.4
Figure 6.4
Inguinal adipose tissue has smaller adipocytes than epididymal adipose tissue. Ten-week-old C57BL6/J mice (N=5) fed a 60% high-fat diet for 6 weeks were sacrificed and epididymal (eWAT) and inguinal (iWAT) adipose tissue depots harvested, fixed, sectioned and adipocytes counted using the MetaMorph method described herein. The distribution of adipocyte areas indicates iWAT has greater numbers of adipocytes with areas below 2000 μm2 and fewer adipocytes with areas greater than 2000 μm2 when compared to eWAT (A, B). The area-under-the-curve of this distribution quantifies this elevated number of smaller (≤2000 μm2) and decreased number of larger (≤2000 μm2) adipocyte in iWAT. The difference in adipocyte size is evident in representative H&E stained samples of both iWAT and eWAT (C). Two-way ANOVA with Bonferroni post-hoc analysis, *p<0.05 compared to respective adipocyte area increment (A and B).
Figure 6.5
Figure 6.5
The frequency of small adipocytes but not the average adipocyte area changes in epididymal adipose tissue located on the testicle. Ten-week-old C57BL6/J mice fed a 60% high-fat diet for 6 weeks were sacrificed and epididymal WAT harvested and divided into five equal sections according to the proximity to the testicle before fixing, sectioning and counting of adipocytes (~1000 per sample) using the MetaMorph method described herein. Whereas the average size of adipocytes did not differ between samples (A), there was a higher frequency of small adipocytes (1000–2000 μm2) directly adjacent to the testicle (labeled 1). Two-way ANOVA with Bonferroni post-hoc analysis, *p<0.05 compared to samples 2–5.
Figure 6.6
Figure 6.6
Counting the area of as few as 100 adipocytes provides an accurate distribution of adipocyte size in WAT. Ten-week-old (N=10) C57BL6/J mice fed a 60% high-fat diet for 6 weeks were sacrificed and epididymal WAT harvested, fixed and sectioned. For each individual animal 3, 10, 100, 300, 500 or 1000 adipocytes were randomly counted 10 times and the average adipocyte area calculated. While there was no significant difference in the mean adipocyte area (A) when counting between 3 and 1000 adipocytes, the variance around this mean is significantly greater when counting 3 or 10 adipocytes (B). The distribution of adipocyte areas, however, does not differ whether you count 100, 300, 500 or 1000 adipocytes. Accordingly counting a minimum of 100 adipocytes is sufficient to estimate mean adipocyte size, minimize variance around that mean and provide an accurate estimation of the distribution of adipocytes in WAT. One-way ANOVA with Tukey post hoc analysis, *p<0.05 versus 1000 adipocytes counted.
Figure 6.7
Figure 6.7
A minimum of five animals are required to detect a ~20–25% difference in mean adipocyte radius. C57BL6/J mice fed a chow diet (22-week old, N=10) or 60% high-fat diet (10-week old, N=10) for 6 weeks were sacrificed and epididymal WAT, fixed and sectioned. The distribution of adipocyte sizes was used to calculate the mean radius of adipocytes in the epididymal WAT of mice fed standard chow (26.7 ± 3.1 μm) or high-fat diet (33.8 ± 3.9 μm) (A). Average adipocyte volume was calculated and a linear relationship to weight of epididymal WAT observed (B). Based on the calculated variance in the radius of adipocytes from WAT of mice fed a standard chow (C) or high-fat (D) diet, a power equation (1 − β=0.8, α=0.05) indicates that a of five mice would be required to detect a 7 μm or ~20–25% difference between treatments.
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