Adipogenesis is dependent on cytoskeletal remodeling that determines and maintains cellular shape and function. Cytoskeletal proteins contribute to the filament-based network responsible for controlling the shape of adipocytes and promoting the intracellular trafficking of cellular components. Currently, the understanding of these mechanisms and their effect on differentiation and adipocyte function remains incomplete. In this study, we identified the non-muscle myosin 10 (MYH10) as a novel regulator of adipogenesis and adipocyte function through its interaction with the insulin-dependent glucose transporter 4 (GLUT4). MYH10 depletion in preadipocytes resulted in impaired adipogenesis, with knockdown cells exhibiting an absence of morphological alteration and molecular signals. MYH10 was shown in a complex with GLUT4 in adipocytes, an interaction regulated by insulin induction. The missing adipogenic capacity of MYH10 knockdown cells was restored when the cells took up GLUT4 vesicles from neighbor wildtype cells in a co-culture system. This signaling cascade is regulated by the protein kinase C ζ (PKCζ), which interacts with MYH10 to modify the localization and interaction of both GLUT4 and MYH10 in adipocytes. Overall, our study establishes MYH10 as an essential regulator of GLUT4 translocation, affecting both adipogenesis and adipocyte function, highlighting its importance in future cytoskeleton-based studies in adipocytes.

Adipose tissue is a complex organ composed of different cellular populations, including mesenchymal stem and progenitor cells, adipocytes, and immune cells such as macrophages and lymphocytes. These cellular populations alter dynamically during aging or as a response to pathophysiology such as obesity. Changes in the various inflammatory cells are associated with metabolic complications and the development of insulin resistance, indicating that immune cells crosstalk with the adipocytes. Therefore, a study of the cell populations in the adipose tissue and the extracellular matrix maintaining the tissue niche is important for the knowledge on the regulatory state of the organ. We used a combination of methods to study various parameters to identify the composition of the resident cells in the adipose tissue and evaluate their profile. We analyzed the tissue structure and cells based on histology, immune fluorescence staining, and flow cytometry of cells present in the tissue in vivo and these markers’ expression in vitro. Any shift in cells’ composition influences self-renewal of the mesenchymal progenitors, and other cells affect the functionality of adipogenesis.

Adipose tissue plays a leading role in obesity, which, in turn, can lead to Type 2 diabetes. Adipocytes (AD) respond to the biomechanical stimulation experienced in fat tissue under static stretch during prolonged sitting or lying. To investigate the effect of such chronic stimulation on adipocyte cell metabolism, we used an in vitro system to mimic the static stretch conditions. Under in vitro culture stretching, cells were analyzed at the single‐cell level and we measured an increase in the projected area of the AD and higher content of lipid droplets. A decrease in the projected area of these cells’ nucleus is associated with peroxisome proliferator‐activated receptor‐gamma expression and heterochromatin. This is the first study to reveal proteins that were altered under static stretch following a mass spectrometry analysis and main pathways that affect cell fate and metabolism. Bioinformatics analysis of the proteins indicated an increase in mitochondrial activity and associated pathways under static stretch stimulation. Quantification of the mitochondrial activity by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) assay and the ATPase related proteins specifically measured ATP5B indicated an increase in adipogenesis which points to a higher rate of cell metabolism under static stretch. In summary, our results elaborate on the metabolism of AD exposed to biomechanical stimulation, that is, associated with altered cellular protein profile and thereby influenced cell fate. The static stretch stimulation accelerated adipocyte differentiation through increased mitochondrial activity. Hence, in this study, we introduce a new perspective in understanding the molecular regulation of mechano‐transduction in adipogenesis.

Adipose tissue is a complex organ composed of various cell types and an extracellular matrix (ECM). The visceral adipose tissue (VAT) is dynamically altered in response to nutritional regimens that lead to local cues affecting the cells and ECM. The adipocytes are in conjunction with the surrounding ECM that maintains the tissue’s niche, provides a scaffold for cells and modulates their signaling. In this study, we provide a better understanding of the crosstalk between nutritional regimens and the ECM’s stiffness. Histological analyses showed that the adipocytes in mice fed a high-fat diet (HFD) were increased in size, while the ECM was also altered with changes in mass and composition. HFD-fed mice exhibited a decrease in elastin and an increase in collagenous proteins. Rheometer measurements revealed a stiffer ECM in whole tissue (nECM) and decellularized (deECM) in HFD-fed animals. These alterations in the ECM regulate cellular activity and influence their metabolic function. HFD-fed mice expressed high levels of the receptor for advanced-glycation-end-products (RAGE), indicating that AGEs might play a role in these processes. The cells also exhibited an increase in phosphoserine332 of IRS-1, a decrease in the GLUT4 transporter levels at the cells’ membrane, and a consequent reduction in insulin sensitivity. These results show how alterations in the stiffness of ECM proteins can affect the mechanical cues transferred to adipocytes and, thereby, influence the adipocytes’ functionality, leading to metabolic disorders.

Plasma membrane proteins are extremely important in cell signaling and cellular functions. Protein expression and localization alter in response to various signals in a way that is dependent on cell type and niche. Compartmental quantification of the expression of particular proteins is a very useful means of understanding their role in cellular processes. Immunofluorescence staining is frequently used to investigate the distribution of proteins of interest. Here, we present an imaging method for quantifying the membrane to cytoplasm ratio (MCR) of proteins analyzed at single-cell resolution. This technique provides a robust quantification of membrane proteins and contributes new insights into membrane expression dynamics. We have developed a protocol that uses immunostaining to assess protein expression according to the fluorescent cellular distribution and to compute the MCR. The method was applied to measure the MCR of glucose transporter 4 (GLUT4) in response to insulin in 3T3-L1 cells, an in-vitro model for adipocyte function and adipogenesis. The results revealed informative changes in the subcellular localization of GLUT4 following insulin induction. MCR analysis is a powerful imaging tool that can be generally applied to membrane proteins to provide a rapid and efficient quantitative analysis of protein distribution and sub-cellular processes in cells.