Obesity has become one of the leading pathophysiologic disorders in recent years. Adipose tissue is the main tissue related to obesity and is known to play a role in various physiological complications, including type 2 diabetes. To better understand how the fat tissue develops, we used an in vitro live cell imaging system to quantify the adipogenesis by means of nondestructive digital imaging to monitor the accumulation of intracellular lipid droplets (LDs), a hallmark of adipogenesis, from the macro- to the micro-scale. Analyzing the cells' shape at the single-cell level allows to quantify the cells' shape change from a fibroblast to spherical morphology, indicating the start of adipogenesis. To reveal the molecular alterations, we applied a proteomic approach using high-resolution mass spectrometry of the proliferation, confluent fibroblasts and of adipocytes. During this process, we noted the reorganization of the cells' extracellular matrix (ECM) network microenvironment from fibrillary collagen types I, III and V to collagens IV and VI, which affected the cells niche. The changes in ECM are translated for cytoskeleton remodeling according to cell fate-determining mechanisms. We quantified the cytoskeleton rearrangement of long oriented actin fibers or short cortical and disorganized fibers, associated with LDs accumulation in adipocytes. Developing in vitro models and analytical methods enable us to study differentiation into adipocytes that will advance our understanding regarding the niche conditions that affect adipogenesis. Consequently, this will enable the development of new modalities to prevent obesity and its deleterious outcomes and to develop potential treatments to battle pathophysiology-related diseases.

Lumbar spinal canal stenosis (LSCS) is a degenerative disease observed by hypertrophy of the ligamentum flavum (LF) that cause compression of the lumbar neural content. Diabetes mellitus (DM) is a risk factor for the disease and we have shown previously that DM increases the fibrosis and elastic fiber loss in patients with LSCS. The purpose of this study was to find the proteins that play a role in the development of this clinical pathogenesis and the effect of DM on protein expression. LF tissue retrieved from patients diagnosed with LSCS, some were also diagnosed with DM, were compared with LF from patients diagnosed with herniated nucleus pulposus (HNP). The tissues were analyzed by mass spectrometry for proteins profile alteration. We found that LF of LSCS/DM patients exhibited significantly higher levels of proteoglycan proteins and latent transforming growth factor β-binding protein (LTBP2 and LTBP4). Additionally, an increase of HTRA serine protease 1 and insulin-like growth factor binding protein-5 were noted. The higher fibrosis was also associated with proteins related to inflammation and slower tissue repair. Collagen 6 and transforming growth factor inhibitor are related to activation of the anti-inflammatory M2 pathway that is associated with tissue repair. The decrease of these proteins expression in LSCS/DM is associated with increased levels and activation of M1 pro-inflammatory pathways. Interestingly, C3 and C4b members of the complement complex and mannose receptorlike protein (CLEC18) paralogous proteins were detectable solely at the LSCS/DM patients' samples. Histology analysis shows that inflammatory was induced by the hyperglycemic conditions in diabetic patients involve in altering the matrix compositions. Thus, the protein profiles associated with inflammatory pathways affecting the LF suggested increasing susceptibility of developing the degeneration under hyperglycemic conditions. K E Y W O R D S diabetes mellitus, fibrosis, ligamentum flavum, mass spectrometry, spinal stenosis J Cell Biochem. 2019;120:11716-11725. wileyonlinelibrary.com/journal/jcb 11716 |

Mechanotransduction plays a role in adipose tissues by transducing the environmental mechanical signals. It is recognized that dynamic or cyclic mechanical strains suppress adipogenesis, but static strains activate the adipogenic signaling pathways. This phenomenon needs to be investigated further, given its potential use in tissue engineering of fat. We used in vitro cultures as model systems for studying differentiation and function of adipocytes. Additionally, using the finite element method, we developed here sets of multiscale models (MSM), which represent single or multiple adipocytes embedded in scaffolds, stimulated mechanically in a static regime. Based on in vitro adipocyte culture work, these models were employed to study the hypothesis that the loading state of the plasma membrane (PM) in adipocytes is influenced by neighboring cells, which could reflect positive feedback loops of en mass adipose cell differentiation. We demonstrate that under static loading, tensile strains at the PM increase with the stage of cell maturation. Furthermore, when the cell density was sufficient (above 19 cells per 100 μm(3)), progressive differentiation in some of the cells caused higher magnitudes of tensile strains in the PMs of other nearby cells. MSM are currently the only feasible means to correlate continuum (macrolevel) construct deformations to subcellular-level PM stretches in distorted cells. These macro-to-micro mechanobiology relationships, revealed through MSM, point to stimulations that promote the formation of lipid droplet accumulations and the increase of adipogenesis. Such models are a cost-effective useful platform for achieving better understanding of these deformation-driven cell processes toward optimized design of tissue-engineered fat constructs.

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 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.