Understanding Obesity One Adipocyte At A Time

As the rate of obesity and overweight increases worldwide at an alarming rate, the quest goes on to find new therapeutics targeting obesity-associated disorders, including type-2 diabetes, insulin resistance, fatty liver disease, and cardiovascular events.

White adipose tissue, our major nutrient-storing organ, has been shown to be at the heart of obesity-associated pathologies (Lotta et al., 2017) by releasing excess lipids to the periphery and secreting disadvantageous adipokines and pro-inflammatory proteins in the obese state. However, a major hurdle impairing the research of fat cells (adipocytes) is the inability to perform many of the most common cellular and molecular analysis methods on adipocytes.

Mature adipocytes are significantly larger than normal cells, having a diameter of 20–150 μm in mice and 40–200 μm in humans, compared to 8-15 μm in diameter for normal cells such as lymphocytes or fibroblasts. They are, moreover, lipid-filled, making them highly buoyant and preventing the cells from mixing evenly in a solution, which in many cases is required for efficient molecular analysis. Lastly, the adipocytes can lyse easily, unleashing their high lipid content to the rest of the solution, thus making adipocyte work a very sticky and sometimes messy business.

Importantly, these features have, to date, largely prevented many types of analyses of adipocytes on a single-cell level, as normal methods such as flow cytometry or serial dilutions cannot be easily performed. The use of flow cytometry, in particular, would allow for the relatively fast analysis and separation (sorting) of large numbers of adipocytes with specific features, such as expressing a specific protein or having one or many lipid droplets, from a bulk, mixed sample. Previous attempts to use flow cytometry to analyze adipocytes have produced mixed reports, sometimes reporting a spiral-like cell population identified by the flow cytometer, other times a population with high complexity features, resulting in a high so-called side-scatter (SSC) signal.

In a recent paper published in Cell Reports, we described a detailed method on how to analyze and sort freshly-isolated mouse and human adipocytes. Using a fluorescent-reporter mouse strain whose adipocytes all are stained fluorescently red, we discovered that common flow cytometry methods miss the bulk of mature adipocytes due to their large size and buoyancy. Importantly, the resource paper showed how simple adjustments of the flow cytometer, such as using in-tube stirring to acquire the cells and adding filters to lower the fluorescent signal, can help to adapt flow cytometry to adipocytes, making both analysis and sorting possible.

The research group also demonstrated the applicability of the method by sorting multilocular so-called beige adipocytes from mouse subcutaneous fat samples and demonstrated a heterogeneous expression pattern of one of the most common pro-lipolytic receptors: beta-adrenergic receptor β2, on the surface of human adipocytes. We also tested and commented on various cellular dyes commonly used in molecular research, such as nuclear and lipid staining dyes, and their usability and particularities when used in combination with flow cytometry.

Taken together, the research report improves the applicability of flow cytometry for analyzing and sorting different types of adipocytes by highlighting common pitfalls and their solutions. This will lead to a greater understanding of one of our most difficult cell types to analyze, the adipocyte, and perhaps contribute to the discovery of novel ways to improve adipocyte function and expression pattern during obesity.

The method is described in the paper Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity, recently published in the journal Cell Reports. This work was conducted by Carolina Hagberg, Qian Li, Maria Kutschke, Debajit Bhowmick, Endre Kiss, Olga Shilkova, Viviana Kozina, and Kirsty L. Spalding from the ICMC, Karolinska Institutet, Sweden, together with Irina G. Shabalina and Jan Nedergaard from Stockholm University, Sweden; Matthew J. Harms and Jeremie Boucher from AstraZeneca; and Anders Thorell from Ersta Hospital, Sweden.

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