Recent Preprints

Microphysiological Modeling of Vascular Adipose Tissue for Multi-Throughput Applications

Struss M, Bellas E. Microphysiological Modeling of Vascular Adipose Tissue for Multi-Throughput Applications.

bioRxiv 2024.01.30.578061; doi: https://doi.org/10.1101/2024.01.30.578061

Adipose tissue (AT) is a highly vascularized endocrine organ which regulates whole-body metabolic homeostasis. Key AT functions which rely on vascularization include insulin-stimulated glucose uptake and lipolysis (lipid mobilization to supply energy). Most in vitro AT models do not include the vasculature, and while microphysiological systems (MPS) incorporate spatial organization of cells, 3D environments, and perfusion by external pumps, they are too large to fit traditional cultureware. Thus, we developed a novel miniaturized vascularized adipose tissue (µAT) platform compatible with traditional 24 well plates. Using this µAT platform, we quantified vascular permeability and adipocyte function by insulin-stimulated glucose uptake and lipolysis assays. Shear flow decreased vascular permeability and increased insulin-stimulated glucose uptake. Treatment with forskolin, an adenyl cyclase agonist, increased lipolysis, and decreased vascular permeability. This µAT platform allows for the facile screening of compounds in a physiologically relevant system where both adipocyte and vascular function can be evaluated.

Simulated Microgravity Enhances Adipocyte Maturation and Glucose Uptake via Increased Cortical Actin Remodeling

Anvari G, Struss M, Bellas E. Simulated Microgravity Enhances Adipocyte Maturation and Glucose Uptake via Increased Cortical Actin Remodeling.
bioRxiv 2024.01.30.578049; doi: https://doi.org/10.1101/2024.01.30.578049

Adipocytes are mechanically responsive cells, yet little is known about how the lack of mechanical loading may affect adipocytes and their function. To model the lack of mechanical loading, we exposed engineered adipose tissue (AT) constructs to simulated microgravity (sµg) conditions. We found sµg enhanced lipid accumulation (lipogenesis) and lipid mobilization (lipolysis). Adipocyte maturation involves a phenotypic switch from actin stress fiber disruption and cortical actin formation. Sµg exposure increased cortical actin formation through mechanoresponsive signaling pathways involving Ras homolog family member A (RhoA) and Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) downstream targets, cofilin and actin-related protein 2/3 (ARP2/3). Adipocytes cultured in sµg have increased glucose transporter type 4 (GLUT4) translocation to the cell membrane and insulin-stimulated glucose uptake, independent of the canonical Akt pathway. GLUT4 translocation to the cell membrane and insulin-stimulated glucose uptake was limited when we inhibited new formation of branched cortical actin using an ARP2/3 inhibitor, CK-666. This study demonstrated that sµg enhances adipocyte maturation via increased lipogenesis and lipolysis and cortical actin remodeling which further enhanced glucose uptake. Therefore, targeting these mechanosensitive pathways pharmacologically or simulating microgravity on earth as a non-pharmacological modality are novel approaches to improving adipocyte function and AT metabolism and possibly for treating related comorbidities such as type 2 diabetes and obesity.

Flow Cytometry Strategies for Rapidly Characterizing Heterogeneous Adipocyte Populations in 3D In Vitro Constructs

Struss M, Anvari G, Bellas E. Flow Cytometry Strategies for Rapidly Characterizing Heterogeneous Adipocyte Populations in 3D In Vitro Constructs.

bioRxiv 2024.01.30.578065; doi: https://doi.org/10.1101/2024.01.30.578065

Adipose tissue (AT) is an endocrine organ that regulates whole body metabolism and supports energy needs of other tissues. Two key adipose tissue functions are insulin-stimulated glucose uptake and lipid metabolism. As the prevalence of metabolic diseases, such as obesity, continue to rise, there is a growing need for new methods to study adipose tissue and its main cell type, adipocytes. Adipocytes are unique cells, distinguished by their large spherical shape housing large lipid droplet(s). For many in vitro models (and in tissues), adipocytes are derived from a heterogenous population of precursor cells, leading to varying degrees of adipogenesis and adipocyte maturation. Common single cell characterization methods provide data based gene and protein expression but do not account for the morphological variability in cells such as adipocytes at different stages of maturation, and are expensive to run. More traditional methods, such as microscopy or colorimetric assays, are often time consuming with intrinsic challenges due to overlapping or coincident features or lose single cell adipocyte details due to the destructive nature of the assay. Here, we show how flow cytometry can be used to characterize adipocyte populations while preserving critical details at the individual adipocyte level.

This protocol provides multiple workflows for indirect measurements of lipogenesis (lipid accumulation), protein content (branched actin formation), and adipocyte functions (insulin-stimulated glucose uptake).
The flow cytometry workflows presented in this work show the effectiveness of binning individual adipocytes based on their level of maturity and allow for comparisons within subpopulations traditional methods cannot provide.