Validation of extracellular glucose depletion as a mock measurement of its uptake in cells and tissues ex vivo

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2022-03

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Abstract

The ongoing worldwide epidemic of diabetes increases the demand for identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis is based on the assumption that metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. Here we describe a method for measuring extracellular FD-glucose depletion and validate its correlation with intracellular FD glucose uptake in cells and tissues ex vivo. Our protocol describes two phases: intracellular and extracellular, measurements of FD-glucose uptake in 3T3-L1 cells. Cells were cultured in 96-well format, for forty-eight hours and then stimulated with FD-glucose to observe uptake in cells and depletion in extracellular medium. Likewise, we executed the ex vivo measurement of extracellular uptake of FD glucose in tissues dissected from ob/ob mice. Prior to tissues harvest, animals were treated with a canonic mediator of glucose uptake, such as insulin, an amino acid compound 2 (AAC2). AAC2 is an experimental nanomaterial that increased glucose uptake in peripheral and brain cell cultures as well as in mouse models of type 1 and type 2 diabetes. The direct comparison of extracellular FD-glucose depletion with normalized intracellular glucose uptake in cells culture showed a high correlation, suggesting that extracellular glucose depletion could be a surrogate measurement for glucose uptake assessment. Thus, Extracellular glucose depletion may be potentially applicable for high throughput kinetic and dose-dependent studies, identifying compounds with glycemic activity and their tissue-specific effects.

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Health Under the Microscope (The Ohio State University Denman Undergraduate Research Forum)

Keywords

Glucose uptake, Diabetes, Insulin resistance

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