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.

Understanding mechanotransduction in adipocytes is important for research of obesity and related diseases. We cultured 3T3-L1 preadipocytes on elastic substrata and applied static tensile strains of 12% to the substrata while inducing differentiation. Using an image processing method, we monitored lipid production for a period of 3-4 wk. The ratio of %-lipid area per field of view (FOV) in the stretched over nonstretched cultures was significantly greater than unity (P < 0.05), reaching ∼1.8 on average starting from experimental day ∼10. The superior coverage of the FOV by lipids in the stretched cultures was due to significantly greater sizes of lipid droplets (LDs) with respect to nonstretched cultures, starting from experimental day ∼10 (P < 0.05), and due to significantly more LDs per cell between days ∼10 and ∼17 (P < 0.05). The statically stretched cells also differentiated significantly faster than the nonstretched cells within the first ∼10 days (P < 0.05). Adding peroxisome proliferator-activated receptor-γ (PPARγ) antagonist did not change these trends, as the %-lipid area per FOV in the stretched cultures that received this treatment was still significantly greater than in the nonstretched cultures without the PPARγ antagonist (14.44 ± 1.96% vs. 10.21 ± 3%; P < 0.05). Hence, the accelerated adipogenesis in the stretched cultures was not mediated through PPARγ. Nonetheless, inhibiting the MEK/MAPK signaling pathway reduced the extent of adipogenesis in the stretched cultures (13.53 ± 5.63%), bringing it to the baseline level of the nonstretched cultures without the MEK inhibitor (10.21 ± 3.07%). Our results hence demonstrate that differentiation of adipocytes can be enhanced by sustained stretching, which activates the MEK signaling pathway.

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Understanding the mechanoresponsiveness of adipocytes and the characteristics of the mechanical stimuli that regulate adipogenesis is critically important in establishing knowledge in regard to the long-term effects of a sedentary lifestyle (or immobility in extreme medical conditions) as well as concerning obesity and related diseases. In this study we subjected 3T3-L1 preadipocytes cultured on elastic substrata to different levels of static equiaxial tensile strains within the physiological range, up to substrate tensile strain (STS) of 12%, while inducing differentiation in the cultures. Based on prior work which revealed that adipogenesis is accelerated in cultures subjected to STS of 12% by activating the mitogen-activated protein kinase kinase signaling pathway, we were specifically interested in identifying the STS levels which trigger this process. We hence monitored the production and accumulation of lipid droplets (LDs) using a non-destructive, image-processing-based method that we have previously developed, for a period of 4 weeks. The experimental data demonstrated accelerated adipogenesis in the cultures subjected to STS levels of 6%, 9%, and 12% with respect to cultures subjected to STS of 3% and (non-stretched) control cultures. This accelerated adipogenic response to the large sustained STS manifested in significantly larger numbers and greater sizes of LDs in the cultures that were stretched to large STS levels (p < 0.05), starting at approximately day 14 following induction of differentiation. Hence, indeed, there appears to be a certain tensile strain threshold, or domain-which is found within the physiological range-above which the responsiveness of adipocytes to sustained static stretching increases and is manifested in accelerated adipogenesis.