Investigating Glucose Uptake in Cultured Cells

The transport process of glucose from the outside of cells into cells across the cell membrane is called glucose uptake. It is one of the important regulatory steps of glucose metabolism. Analysis of glucose uptake offers vital information to gain insights into glucose metabolism and its regulation in normal and disease development.

This article describes the development of an innovative glucose uptake detection method based on direct measurement of glucose levels in cultured cells. This method involves utilizing the inhibitor, 3-bromopyruvate (3BP), to temporarily inhibit hexokinases, the first enzyme that metabolizes glucose in cells. Hence, glucose metabolism was hindered, which results in glucose accumulation instead of its rapid metabolism inside the cells. Using Glucose Oxidase/HRP/OxiRedTM enzyme-based method, glucose uptake was then detected through the direct measurement of the glucose levels inside the cells. When compared to the existing methods that are commonly used, this direct glucose uptake detection method has the following advantages:

  1. Glucose uptake directly detected glucose without using structurally modified glucose derivatives
  2. No radioisotope materials are employed.
  3. Glucose from culture medium is transported inside of cells, and then detected with minimum culture condition changes and washing steps.

This sensitive, simple, and direct glucose uptake detection method offers a powerful tool for examining the process of glucose uptake and its regulation, as well as for screening and characterization of drugs that control glucose uptake during normal and disease development.

Experimental Flow Chart

Investigating Glucose Uptake in Cultured Cells

3BP Inhibits Hexokinase Enzyme Activity

Different doses of 3PB were added into purified human hexokinase enzyme solution. The hexokinase activity was measured using Hexokinase Colorimetric Assay Kit (BioVision Cat# K789-100) following the kit protocols.

Figure 1. Different doses of 3PB were added into purified human hexokinase enzyme solution. The hexokinase activity was measured using Hexokinase Colorimetric Assay Kit (BioVision Cat# K789-100) following the kit protocols.

Detection of Glucose Uptake in the Presence of 3BP in Jurkat Cells Stimulated by Serum

Jurkat cells were serum starved for 5 hours in culture medium (with glucose). 0.5 mM 3BP and 10% fetal bovine serum (FBS) was then added into the culture and incubated for different times. Glucose levels were measured following the standard procedure using Glucose Assay Kit (BioVision Inc. Cat# K606-100).

Figure 2. Jurkat cells were serum starved for 5 hours in culture medium (with glucose). 0.5 mM 3BP and 10% fetal bovine serum (FBS) was then added into the culture and incubated for different times. Glucose levels were measured following the standard procedure using Glucose Assay Kit (BioVision Inc. Cat# K606-100).

Detection Of Glucose Uptake of 3T3-L1 Cells Stimulated by Insulin

3T3-L1 cells were plated overnight, then serum starved for 24 hours. 0.5 mM 3BP and 10 or 20 ng/ml insulin were then added to stimulate glucose uptake for 30 minutes. Glucose in cells was detected using the standard procedure described above.

Figure 3. 3T3-L1 cells were plated overnight, then serum starved for 24 hours. 0.5 mM 3BP and 10 or 20 ng/ml insulin were then added to stimulate glucose uptake for 30 minutes. Glucose in cells was detected using the standard procedure described above.

Glucose Transporter Inhibitor Inhibits Insulin-Stimulated Glucose Uptake in 3T3-L1 Cells

Differentiated 3T3-L1 cells were starved for 24 hours and treated with Insulin (20 ng/ml) and Phloretin for 30 minutes. Glucose levels were measured following the standard procedure described above.

Figure 4. Differentiated 3T3-L1 cells were starved for 24 hours and treated with Insulin (20 ng/ml) and Phloretin for 30 minutes. Glucose levels were measured following the standard procedure described above.

Discussions

  1. When 3BP is absent, glucose is quickly metabolized by cytosolic enzymes during sample preparation and assay processes, and therefore it is not possible to accurately detect glucose uptake in cells (data not shown). In this article, an extensively utilized hexokinase inhibitor, 3-Bromopyruvate (3BP), was used to inhibit hexokinase activities to block glucose metabolism in cells, so that glucose uptake can be measured accurately.
  2. However, a few other glucose uptake assay methods have been developed and are commercially available, such as florescent dye-labeled glucose, isotope-labeled glucose, or 2-deoxy-D-glucose (2DG) based methods. The isotope method involves tiresome isotope labeling, incurs expensive isotope and waste costs, and is a biohazard. Other methods involved the use of structurally altered glucose derivatives; therefore, it is uncertain whether the structurally altered glucose derivatives truly represent native glucose behaviors during glucose uptake processes. Moreover, it is necessary to remove glucose in normal culture medium, which leads to another alteration of cell culture condition that may change glucose uptake behaviors of cells. In the method developed here, glucose in culture medium is directly used, without glucose structural alteration; therefore, it demonstrates true glucose behaviors for the study of glucose uptake in cells.
  3. In the new method, 3BP is used to block glucose metabolism only for a short period (30 minutes). Yet, currently, it has not been ruled out whether 3BP has an impact on other cellular functions, thus changing glucose uptake behavior of cells.

Applications

  1. Study cell signaling of cytokines, growth factors, or other regulatory factors that regulate glucose uptake in different cell types
  2. Screen and characterize drugs that inhibit or stimulate glucose uptake
  3. Study the fundamental mechanism and regulation of glucose uptake during normal and disease development processes

Advantages

  1. Simple, convenient, and easy to adapt to high-throughput (HT) assay for screening a huge number of samples
  2. Use and detect native glucose directly, no glucose structural alteration, no isotope, no fluorescence dye labeling
  3. No need to remove glucose from cell culture condition

Conclusions

An innovative glucose uptake detection method has been devised, which serves as a very useful tool for the study of glucose uptake, metabolic diseases, as well as the study of cell signaling that regulates glucose uptakes and screening drugs that inhibit or stimulate glucose uptake.

BioVision Incorporated

BioVision, Inc., is a privately held Life Science company headquartered in the beautiful San Francisco Bay Area.

BioVision develops and offers a wide variety of products including assay kits, antibodies, recombinant proteins & enzymes, and other innovative research tools for studying Apoptosis, Metabolism, Cell Proliferation, Cellular Stress, Cell Damage and Repair, Diabetes, Obesity and Metabolic Syndrome, Stem Cell Biology, Gene Regulation, Signal Transduction, etc. BioVision's products are currently being sold in more than 60 countries worldwide.


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Last updated: Feb 21, 2020 at 9:30 AM

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