Adipocyte stiffness increases with accumulation of lipid droplets

Abstract

Adipogenesis and increase in fat tissue mass are mechanosensitive processes and hence should be influenced by the mechanical properties of adipocytes. We evaluated subcellular effective stiffnesses of adipocytes using atomic force microscopy (AFM) and interferometric phase microscopy (IPM), and we verified the empirical results using finite element (FE) simulations. In the AFM studies, we found that the mean ratio of stiffnesses of the lipid droplets (LDs) over the nucleus was 0.83 ± 0.14, from which we further evaluated the ratios of LDs over cytoplasm stiffness, as being in the range of 2.5 to 8.3. These stiffness ratios, indicating that LDs are stiffer than cytoplasm, were verified by means of FE modeling, which simulated the AFM experiments, and provided good agreement between empirical and model-predicted structural behavior. In the IPM studies, we found that LDs mechanically distort their intracellular environment, which again indicated that LDs are mechanically stiffer than the surrounding cytoplasm. Combining these empirical and simulation data together, we provide in this study evidence that adipocytes stiffen with differentiation as a result of accumulation of LDs. Our results are relevant to research of adipose-related diseases, particularly overweight and obesity, from a mechanobiology and cellular mechanics perspectives.

Figure 1

The suggested structure-function relationships in fat tissues: the closed-loop coupling between mechanical loads that are being developed in weight-bearing adipose tissues and the adipogenic differentiation process. When adipocytes differentiate, their stiffness gradually changes. Hence, the distributions of strains and stresses in and around the cells change. If the process involves a large number of cells, these stiffness changes also reflect to the tissue scale. At the cell level, such stiffness changes appear to relate to the increasing contents of lipid droplets (adipogenesis) and to rearrangement of the cytoskeleton, which are regulated by activation of different mechanotransduction pathways (5–7). To see this figure in color, go online.

Figure 2

(a) The atomic force microscopy (AFM) nanoindentation experiments; and (b) a finite element (FE) model simulating these experiments. N = nucleus; LD = lipid droplet; Cy = cytoplasm. (c) Cross-sectional view of the FE mesh in the model shown in (b). To see this figure in color, go online.

Figure 3

The interferometric phase microcopy system. BS = beam splitter; R = retro-reflector for beam path adjustments; M = mirror; MO = microscope objective; L = lens. To see this figure in color, go online.

Figure 4

Experimental results of the atomic force microscopy studies. (a) Example of the repeated measurements of the effective stiffness (ES) for indentations over the lipid droplets (LDs) in five different cells; each cell is presented with a different symbol. The white and black markers represent different LDs within each cell. The ES over each LD were tested three times. (b) Comparison of ES for indentation over LDs versus over the nucleus for all cells. ^∗p < 0.05. (c) The LD over nucleus ES ratios, calculated for the same cell each time (pooled for all cells). The error bars in frames (b) and (c) are the minimum and maximum values.

Figure 5

Example force versus indentation depth relationships acquired in the atomic force microscopy experiments and calculated from the corresponding finite element simulations when the indenter was (a) above/near lipid droplets (LDs) and (b) above/near nuclei. Models Nos. 1, 2, and 3 correspond to adipocytes at days 8–9, 15–16, and 22–23 postinduction of differentiation, respectively. The diamond and square marks in panel (a) represent LDs located at the peripheries and centers of the cells, respectively. To see this figure in color, go online.

Figure 6

Finite element calculated effective stiffness (ES) versus (a) horizontal axis length, (b) vertical axis length, (c) volume, and (d) surface area of lipid droplets (LDs) in the adipocyte models. The lengths of the two semiprincipal axes of LDs that were parallel to the cell bottom surface (horizontal axes) were equal. Models Nos. 1, 2, and 3 correspond to adipocytes at days 8–9, 15–16, and 22–23 postinduction of differentiation, respectively.

Figure 7

Results of the interferometric phase microcopy studies: (a) example optical path delays of adipocytes at days 10 (right frame) and 13 (left frame) postinduction of differentiation; (b) the corresponding standard deviation maps. The scale bars are 5 and 1.5 microns-wide in the right and left frames, respectively. To see this figure in color, go online.

Figure 8

Differences of the optical path delay with respect to its temporal mean value at three different time-points (from left to right; a different lipid droplet is presented in each row). The scale bars are 1, 0.5, 0.5, 1.5, and 1.5 microns-wide in (a), (b), (c), (d), and (e), respectively. To see this figure in color, go online.

Figure 9

The state of shear deformations in the cytoplasm, in the simulation case where the stiffness of the lipid droplet was greater than the stiffness of the cytoplasm. To see this figure in color, go online.