Endocrine Abstracts

Introduction: Adipocytes have been shown to exhibit considerable heterogeneity. In humans, they vary in size from <20 to 300 µm in diameter, which equates to a several hundred-fold difference in cell volume within the same tissue. Population based studies indicate larger adipocytes are associated with metabolic diseases, but how cell size is related to metabolism is poorly understood. Recent studies measured glucose levels in individual cells in culture and showed larger fat cells were less insulin sensitive than smaller cells. However, this technique is limited in its applications and required genetic manipulation of the cells, rendering it of limited use in primary tissues. Hence, there is an unmet need to study metabolism non-invasively at the single cell level in living tissues.

Results: Using Stimulated Raman Scattering (SRS) microscopy, we have shown that we can identify and quantify heterogeneity in a label free manner using 3T3-L1 adipocytes for proof-of-concept. Furthermore, by utilizing deuterated glucose-d7, we identify heterogeneity in glucose incorporation into lipid droplets within individual cells, and individual lipid droplets. Combining this approach with spectral phasor analysis, we have begun to develop methodology to quantify cell size, lipid droplet size and number, and quantifying specific metabolic steps all within the same sample set.

Future work: We will also outline the use of a ratiometric Raman sensor that incorporates into the mitochondria (‘mitokyne’). This mitokyne is able to determine real time pH changes within mitochondria during treatment conditions; our goal is to develop a system which captures this information from a single field of cells.

Conclusion: Here, we have developed and validated a workflow of experimental systems to quantify distinct aspects of adipocyte biology which can be applied across a range of experimental research and cell types.