Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The demand for measuring glucose uptake rises together with the critical need to address an epidemic increase in a multitude of diseases dependent on glucose metabolism. Underlying mechanisms of degenerative metabolic diseases, neurological and cognitive disorders¹, inflammatory² and infectious diseases³, cancer^4,5, as well as aging⁶, depend on glucose metabolism for energy and its storage, anabolic processes, protein, and gene modification, signaling, regulation of genes, and nucleic acids synthesis and replication^7,8,9. Diabetes mellitus (DM) is directly related to malfunction of glucose uptake regulation. DM is a spectrum of chronic diseases such as type-1, -2, and -3 diabetes mellitus, gestational diabetes, maturity-onset diabetes of the young, and other types of this disease induced by environmental and/or genetic factors. In 2016, the first WHO Global report on diabetes demonstrated that the number of adults living with the most widespread DM has almost quadrupled since 1980 to 422 million adults¹⁰, and this number of DM patients has been rising exponentially for the last few decades. In 2019 alone, an estimate of 1.5 million deaths was directly caused by DM¹⁰. This dramatic upsurge is due to the rise in type-2 DM and the conditions driving it, including overweight and obesity¹⁰. The COVID-19 pandemic revealed a two-fold increase in mortality in patients with DM compared to the general population, suggesting the profound yet poorly understood role of glucose metabolism in the immune defense³. Prevention, early diagnosis, and treatment of DM, obesity, and other diseases require optimization of measurements of glucose uptake by different tissues, and identification of environmental¹¹, nutritional¹², endocrine¹³, genetic¹⁴, and epigenetic¹⁵ factors affecting glucose uptake.

In research, intracellular and/or tissue uptake of glucose is commonly measured by fluorescently-labeled glucose (FD-glucose) in vitro^16,17,18 and in vivo¹⁹. FD-glucose became a preferred method compared to more precise methods using radioactively-labeled glucose²⁰, analytical mass spectroscopy analysis²¹, metabolomics²², nuclear magnetic resonance methods²³, and positron emission tomography/computed tomography (PET/CT)^5,24. Unlike FD-glucose uptake, analytical methods requiring more biological material may involve a multi-step sample preparation, expensive instruments, and complex data analysis. Effective and inexpensive measurements of FD-glucose uptake in cell cultures have been utilized in proof-of-concept experiments and may require validation by other methods.

The basis of FD-glucose application for glucose uptake studies is the reduced metabolism of FD-glucose compared to endogenous glucose²⁵. Nonetheless, both endogenous glucose and FD-glucose are dynamically distributed among all cellular compartments for use in anabolic, catabolic, and signaling processes. The compartmentalization and time-dependent processing²⁵ of FD-glucose interfere with the fluorescence measurements, and represent the major limiting factors for the use of this assay in high-throughput screening experiments, kinetic analysis, 3D cell culture, co-cultures, and tissue explant experiments. Here, we provide data demonstrating a high correlation between the extracellular depletion of FD-glucose and its intracellular uptake, suggesting the extracellular depletion of FD-glucose as a surrogate measurement for intracellular glucose uptake. The measurement of extracellular depletion of glucose was applied to validate tissue-specific differences in glucose uptake in mice treated with insulin and an experimental drug¹⁸ to provide a proof-of-principle of this method.

The current protocol describes intracellular and extracellular (Figure 1) measurements of FD-glucose uptake in 3T3-L1 cells. Protocol sections 1-7 explain the culture and growth of cells for 48 h; cell starvation, stimulation, and baseline extracellular measurements; and post-stimulation measurements of extracellular FD-glucose and intracellular measurements of FD-glucose and protein. Protocol section 8 describes the ex vivo measurement of extracellular uptake of FD-glucose in tissues dissected from ob/ob mice in the presence and absence of insulin and amino acid compound 2 (AAC2) described elsewhere¹⁸.