INSTITUTE: UIPS

DEPARTMENT: PHARMACY
M. Pharmacy-Ist Sem (Pharmacology)
Subject Name and Code: Cellular and Molecular
Pharmacology– 22PHT-624

DISCOVER . LEARN . EMPOWER

Lovedeep Singh, Assistant Professor, UIPS, CU
Course Outcome
CO Number Title Level

CO1 Describe the fundamental knowledge on the structure Remember

and functions of cellular components.
CO2 Describe the molecular pathways affected by drugs. Understand

CO3 Explain the science of genomics Understand

CO4 Illustrate the applicability of molecular pharmacology Understand

and biomarkers in drug discovery process.
CO5 Analyse molecular biology techniques as applicable for Understand
pharmacology.

CO6 Appraise the cell viability assay Understand

Glucose uptake assay

Lovedeep Singh, Assistant Professor, UIPS, CU

 Glucose uptake assay
Glucose is the main source of energy in organisms, including humans and the uptake of
glucose by different tissues such as skeletal muscles and liver is an important step in
glucose utilization.
➢ Generally, glucose is transported across membranes by two different mechanisms, i.e.,
Na+ glucose co-transporter and Na+ independent glucose transporter glycoproteins
(GLUT).
➢ In mammalian cells, families of glucose transporters (GLUT) are located on the liver and
muscles and perform this function.
➢ On the liver cells, GLUT2 is predominant, while GLUT4 is more predominant on the
skeletal muscles. Glucose transporters facilitate the movement of glucose across the
plasma membrane down its chemical gradient.
Lovedeep Singh, Assistant Professor, UIPS, CU
➢ Glucose uptake assays are done using radiolabeled glucose or radiolabeled glucose
analogs such as 2-deoxyglucose (2-DG) and 3-O-methyl-D-glucose (3-MG).
➢ Insulin resistance—also called prediabetes—is characterized by elevated fasting blood
glucose levels ranging from 101 to 125mg/dL (5.6–6.9mM) or impaired glucose
tolerance (140–199mg/dL, 2 h after a 75-g oral glucose challenge)
➢ The lack of insulin prevents glucose uptake in the peripheral tissues, including adipose
tissue, and skeletal muscle, resulting in high blood glucose levels.
➢ Skeletal muscle is essential for glucose clearance and is responsible for over 80% of
glucose uptake from an oral glucose load, postprandial.
➢ Insulin resistance is caused by the desensitization of muscle to the insulin released by
the pancreas to elicit glucose uptake, leading to elevated blood glucose levels.

Lovedeep Singh, Assistant Professor, UIPS, CU

➢ Insulin resistance disrupts both the amount of glucose uptake into skeletal muscle and
the timing of that uptake.
➢ Under normal conditions, postprandial glucose uptake into muscle increases linearly
with time. However, with insulin resistance and T2D, there is a delay in insulin action
and glucose uptake, causing diminished overall glucose uptake by the skeletal muscle.
Mechanism of glucose uptake
➢ When contraction occurs via exercise (depicted by yellow lightning bolts) multiple
pathways are activated, which can all lead to GLUT4 translocation and glucose uptake.
➢ Contraction causes Rac1-GTP loading/activation, which induces PAK1 (P21-activated
kinases-1) phosphorylation/activation, and F-actin remodeling to activate the exercise
stimulated GLUT4 vesicle pool; it can also activate NOX2 (NADPH oxidase 2) which
causes reactive oxygen species (ROS) formation.
Lovedeep Singh, Assistant Professor, UIPS, CU
➢ ROS can also be stimulated directly by contraction, leading to peroxynitrite and
subsequent GLUT4 translocation, or can cause AMPK activation.
➢ Nitric oxide (NO) production can lead to S-nitrosylation of proteins that cause GLUT4
translocation.
➢ Muscle excitation can also cause calcium influx into the muscle cell (far right), which
can activate CAMKII and lead to glucose uptake.
➢ Calcium influx can also stimulate cross-bridge cycling, the process of muscle fiber
contraction, which leads to GLUT4 translocation and glucose uptake into the cell.

Lovedeep Singh, Assistant Professor, UIPS, CU

Mechanism of glucose uptake

Lovedeep Singh, Assistant Professor, UIPS, CU

The different methods employed for measuring glucose uptake are as follows:
1. Measurement of glucose uptake using radiolabeled 3-o-methyl-glucose
▪ The glucose uptake activity in cultured cells is commonly analyzed using radioactive 3-
methyl glucose (3-MG).
▪ It is a non-phosphorylatable glucose analog and it accurately measures the rate of glucose
transport as there is no interference from glucose-metabolizing steps.
▪ As the cells take-up radio-labeled glucose, there is an increase in radioactivity in cells.
▪ Therefore, the appearance of radioactivity is directly proportional to glucose up taken by
cells.

Lovedeep Singh, Assistant Professor, UIPS, CU

▪ The advantages of this method include better signal-to-noise ratio, high specificity, need of
very short incubation time and no interference from glucose metabolism.
▪ However, the major limitation is use of radioactive substrate and need of special
precautions to deal with radioactive substances.
▪ Therefore, this method may not be routinely used in laboratory.
2. Measurement of glucose uptake using radio-labeled 2-deoxyglucose
▪ The measurement of 2-deoxyglucose (2-DG) in the cells is also a reliable method to
estimate the amount of glucose up-taken.
▪ The method is based on the fact that glucose and 2-deoxyglucose are taken up by the cells
in the same manner.
▪ Therefore, the amount of 2-dexoyglucose up-taken by the cells represents the amount of
glucose up-taken by the cells.
Lovedeep Singh, Assistant Professor, UIPS, CU
▪ As 2-DG enters inside the cells, it is phosphorylated to form 2-DG-6 phosphate,
which accumulates in the cells.
▪ The accumulation of 2-DG-6 phosphate takes place because it is not further metabolized
inside the cells.
▪ This assay is particularly useful to assess the extent of glucose uptake by glucose trans-
porters, GLUT1 and GLUT4.
3. Measurement of glucose uptake by detecting intensity of fluorescence
▪ To overcome the problems related to radiolabeled analogs such as high cost, requirement
of specialized equipment and training, non-radio-labeled forms of deoxyglucose
are employed.
▪ Therefore, a rapid, inexpensive, and reproducible non-radioactive microplate assay for
analyzing glucose uptake has been devised.
Lovedeep Singh, Assistant Professor, UIPS, CU
▪ The principle is that as deoxyglucose enters inside the cells, it is allowed to react with
substrate that gives fluorescence.
▪ The intensity of fluorescence is directly proportional to amount of deoxyglucose present
inside the cells.
▪ In this assay, deoxyglucose is phosphorylated to deoxyglucose-phosphate.
▪ Thereafter, enzyme glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the
conversion of deoxyglucose-6 phosphate to 2-deoxy-6-phosphogluconate and
concurrently, there is conversion of NADP+ to NADPH.
▪ Resazurin is employed as a substrate, which is acted upon by diaphorase to form
fluorescence emitting resorufin.
▪ The intensity of fluorescence is directly proportional to amount of deoxyglucose.

Lovedeep Singh, Assistant Professor, UIPS, CU

Principle involved in measuring glucose uptake by measuring the intensity
of fluorescence

Lovedeep Singh, Assistant Professor, UIPS, CU

Application
▪ The major application of glucose uptake assay is study the phenomenon of insulin
resistance.
▪ These tests are very commonly employed for screening antidiabetic agents, which
produce beneficial effects in diabetes by decreasing insulin resistance.

Lovedeep Singh, Assistant Professor, UIPS, CU

 References:
Textbooks

T1 Basic Cell Culture (Practical Approach ) by J. M. Davis (Editor)

T2 Animal Cell Culture: A Practical Approach by John R. Masters (Editor)

T3 Current porotocols in molecular biology vol I to VI edited by Frederick M.Ausuvel etla.

Reference Books

R1 The Cell, A Molecular Approach. Geoffrey M Cooper.

R2 Pharmacogenomics: The Search for Individualized Therapies. Edited by J. Licinio and M –L.Wong

R3 Handbook of Cell Signaling (Second Edition) Edited by Ralph A.et.al

R4 Molecular Pharmacology: From DNA to Drug Discovery. John Dickenson et.al

R5 Basic Cell Culture protocols by Cheril D.Helgason and Cindy L. Miller

 THANK YOU

For queries
Email: [email protected]

Lovedeep Singh, Assistant Professor, UIPS, CU