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2-NBDG

As we know that all cells required a molecule from which they acquired energy with the help of metabolic processes. Almost most living cells require glucose molecules to fulfill their energy requirements and regulate the level of glucose within the body with the help of homeostasis. Meanwhile, when this regulation or homeostasis of glucose within the body or cell gets disturbed, there will be an increase in the incidence of various kinds of disorders like diabetes mellitus. Diabetes Mellitus is one of the prevalent or serious metabolic disorders that is the major cause of increase  mortality not only in humans but also in different animals. This disorder causes the failure of the process that carries out the uptake of glucose by the tissue or cells from the circulation due to the response of the insulin hormone. This in return causes an increase in the level of glucose within the circulation due to the shunting of glucose uptake by the cells or tissue. So, it is necessary to detect or determine the level of glucose uptake by the cells for management of the diabetes mellitus. Scientists develop various kinds of assays or techniques that can measure the level of glucose uptake by the cell. These include the use of radioactive tracers or molecules like 2-deoxy-d-[14C] glucose. But the disadvantage of using these radioactive tracers is that there is the hazard for the proper clean-up of these molecules or problems associated with the disposal of radioactive materials. So, it is necessary to develop a method or fluorescent molecule which is non-radioactive, for the measurement of the glucose uptake by the cells. Different Studies suggest the use of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) fluorescent marker for glucose uptake measurement. This is because it is a type of fluorescent-based derivative or analog of a glucose molecule that can be used for monitoring the uptake of glucose by living cells. It is also non-radioactive, so there is no need for proper disposal management. Also, it can be easily uptake via glucose transporters into the cells. Similarly, it can not be utilized by the cell for the process of glycolysis because of its analog nature. So, it accumulates into the cells. As the cell increases the uptake of glucose analog, there will be an increase in the accumulation of the glucose analog which gives the fluorescence when it is measured with the help of fluorescent microscopy or with the help of flow cytometry. For further correction of the results, scientist use phloretin within the kit based on 2-NBDG. This phenol derivative causes the inhibition of glucose uptake by the cell. So, all is that only glucose analog is got entry into the cells via glucose transporters and give fluorescence upon flow cytometry.

structural representation of 2-NBDG

Figure 1:: structural representation of 2-NBDG

 In this study, scientists used HepG2 cells which are the human hepatocarcinoma cells and L6-rat skeletal muscle cells, and incubate them with different concentrations of 2-NBDG for different incubation times. They also stated that the D-glucose and glucose analog 2-NBDG were competing for the glucose transporters but the L-glucose does not have the competition potential for glucose transporter as compared to glucose analog. During the research, they incubated these cells at different concentrations of 0, 5, 10, 20, and 40 mM with varying rates of incubation. They suggested that the 10mM concentration is good enough to get reliable results along with the incubation time of 60min to get the effects of drugs on the cells.

Graphical representation

Figure 2:: Graphical representation of the changes in the fluorescence relative to the incubation period ranges from 0 to 180 minutes for 10mM 2-NBDG concentration for a) hepG2 hepatocarcinoma cells & b) L6-rat skeletal muscle cells.

Similarly, this fluorescent glucose analog marker can be used to monitor the changes in the process of glycolysis after the exposure of cells to endocrine and anticancer therapies in breast cancer cells. The study suggests that when it is used as compared to the fluoro-deoxyglucose ( a radioactive tracers fluorescent marker) it gives reliable results. This fluoro-deoxyglucose (FDG) is relatively expensive as compared to the 2-NBDG and is a complex method. On the other 2-NBDG has a similar mechanism of entry as D-glucose. It attaches to the glucose transporters and then it gets phosphorylated with the help of hexokinase enzymes I and II. After that, it remains in the cytoplasm of the cell until it gets decomposed into the nonfluorescent form. As we know that cancer cells continue to grow without checks. This will cause an increased requirement of glucose by the tumor cells. So, the glycolysis process, transporters for the glucose uptake, and hexokinase enzymes undergo upregulation in tumor cells. Meanwhile, as it gets entry into the cell, it gives fluorescence at 540nm and gets excited at 488nm with the help of confocal fluorescent microscopy. During this process, it exhibits the changes in the glycolysis process and gives the efficacy of the drugs for endocrine and anticancer therapies.

 One of the studies conducted on the 2-NBDG also suggested that this glucose analog can also be used for optically monitoring the localized epileptic foci. In this study, researchers first induce epileptic seizures in rats by intracortical injection of 4-aminopyridine. After that, they continuously inject 2-NBDG via the tail vein of the rat. Then, they monitor the metabolism of the brain cells with the help of fluorescent microscopy of 2-NBDG. They find that the uptake of the 2-NBDG at the intracortical injection site is greater than at the other control injection sites. This increase in the uptake is because of epileptic seizures, which cause the glial cells or neurons to upregulate the transporter receptors. So the hyperactive metabolism due to the seizures causes an increase in the uptake of glucose analog markers, which can help in the detection of localized epileptic foci.

Similarly, it can also be used for monitoring the uptake of glucose in myoblast cells (cultured myotubes). It is necessary for the management of diabetes type 2. It is because, in this type of diabetes, skeletal muscles get affected and develop insulin resistance. Various assays are in practice for the monitoring of glucose uptake in the myotubes culture cells but they require greater time for the pre-incubation procedure and starvation conditions that will lead to the alterations in the myotubes cells and their metabolism. While 2-NBDG does not change the metabolism of the myotubes as well as the morphological features of the cells. Furthermore, it is safe to be used in the inflammatory conditions of the muscle cells as well as for insulin resistance which is induced by oxidative stress.

 



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