The Use of Cell Painting and Fluorescence Imaging to Assess Cell Physiological Responses to Glucose Stress

Cells of organisms encounter varying conditions, including daily and potentially seasonal fluctuations in available glucose. These fluctuations correspond primarily to feeding status and nutritional intake, with fed and fasted values ranging from ~4-7 mmol/L in humans. Some mammals tolerate a wider range of intracellular conditions while others do not. For example, fruit eating bats experience a blood glucose ranging from ~3-24 mmol/L, while hibernating ground squirrels stabilize blood glucose throughout the winter fast with little variation. We investigated the responses of dermal fibroblasts obtained from skin biopsies from different mammalian species to altered in vitro glucose treatments. Fibroblasts from five species (human, rat, 13-lined ground squirrel, little brown bat, and Egyptian rousette) were cultured at baseline (8mM glucose), then subjected to 24-h of either hypoglycemic (2.5mM) or hyperglycemic (30mM) treatment. To study cellular phenotypes, we employed Cell Painting, utilizing fluorescent stains to label cellular structures. Hoechst 34580 stained DNA, providing detailed measurements of nuclear quantity and size to assess proliferation rate. The endoplasmic reticulum was stained with Alexa Fluor 488 Concanavalin A conjugate to identify its distribution and connections within the cell. The cytoskeleton was stained with Alexa Fluor 555 Phalloidin conjugate, which binds to F-actin, providing insights into structural organization within a cell. Images were analyzed through CellProfiler software, which uses a machine learning approach to extract approximately ~1400 morphological features, including area, size, fluorescent intensity, and texture variance - offering a comprehensive quantification of subtle phenotypic changes under glycolytic stress. We hypothesized that cell morphology changes would track organismal dietary preferences. Intensities of the nuclei, actin cytoskeleton and ER were the most responsive morphological changes with glucose treatment, with the lowest intensities occurring at baseline (8mM conditions) in all species. Morphology responses in 13-lined ground squirrel cells were unique in their significant increase in both actin and ER area (both pFDR=0.0001) under hypoglycemic conditions. Despite differing life histories and diets, we detected few differences in the morphological responses of the insectivorous little brown bat and the fructivorous Egyptian rousette. We also assayed cell physiology via glycolytic rate and glycolytic reserve for each species under baseline conditions using a Seahorse XFp analyzer, and conducted RNAseq for each glucose treatment condition. Little brown bats were the most striking, using very little glycolysis (0.009±0.002 mPh/min/ug protein) at baseline, which was lowest among all species (lme species effect F3,4=9.1, P=0.03). Rats had a 65% lower glycolytic reserve than any other species. Phenotypic differences across mammals, evaluated using microscopy and in the context of their organismal phenotype, will provide insights into mechanisms of cell function in response to environmental stress. Funding by NSF #2022046 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.