This document describes glucose uptake assays and their applications. It discusses: 1) Glucose uptake assays measure the rate of glucose transport across cell membranes by using radioactively labeled glucose. This specifically measures transporter activity over short time periods. 2) Assays use differentiated 3T3-L1 adipocytes or isolated mouse/rat diaphragm muscle tissue. Cells are treated with insulin or other compounds before exposing them to radioactive glucose. Uptake is then measured with a scintillation counter. 3) Glucose uptake assays are used to study the effects of substances like insulin on cellular metabolic activity and glucose transport, helping understand processes involved in diabetes and insulin resistance.

1. Glucose Uptake assay
Submitted by: Avishek Chakroborty
M.Pharm(Pharmacology) 1st year
Guided By: Dr. Rajesh kr. Sharma

2. CONTENT
Introduction
Regulation of plasma glucose level
Types of diabetes mellitus
Principle
Various method of Glucose uptake Assay
Application of Glucose uptake Assay

3. Introduction
 Glucose is a primary source of energy for many organism, and uptake of
glucose is a critical process. Glucose is transported across the cell’s
membrane and trapped by being phosphorylated.
 In mammalian cells, this is performed by a family of glucose transporters
(GLUT) and a few intracellular hexose kinases.
 Measuring glucose uptake is not the same as measure glucose
consumption.
 Glucose uptake occurs on a rapid time of 10 mins or less and specifically
measure transporter activity, whereas changes in glucose concentration
involve a multitude of pathways and typically take several hours.
 Diabetes mellitus(DM) is a group of disease characterized by high levels of
blood glucose resulting from defects in insulin production, insulin action,
or both.
 The effects of diabetes mellitus include long term damage, dysfunction
and failure of various organs.
Glucose Uptake Assay 3

4. REGULATON OF PLASMA GLUCOSE LEVEL

5. Types of Diabetes mellitus
Glucose uptake Assay 5
• Due to insulin deficiency and is formerly
known as Insulin Dependent Diabetes
Mellitus (IDDM).
• Deu to pancreatic islet beta- cell destruction
predominantly by an autoimmune process.
• Usually developed on children or early
adulthood.
• It accounts for up to 10% of all DM cases.
• Develops as a results of the exposure of a
genetically individual to an environmental
agents.
• It is a combined insulin resistance and
relative deficiency in insulin secretion and is
frequently known as Noninsulin Dependent
Diabetes Mellitus(NIDDM).
• It results from insulin resistance with a defect
in compensation insulin secretion
• Insulin may be low, normal or high.
• About 30% of the type ii DM patients are
undiagnosed ( they don’t know that they
have the disease
• It accounts for up to 90% of all DM cases.

6.  Glucose uptake activity was analysed by measuring the rate of uptake of
radioactively tagged 2deoxy glucose in differentiated 3T3 L1 cells .
 After starvation the cells were treated with insulin and other plant
extracts . Then these ligands will bind to the receptors on the surface of
the cells .
 These triggered the translocation of glucose transporters to the cell
surface .
 Then we treat it with radioactive cocktail containing 10uM 2-deoxy
glucose . So the radioactively tagged glucose will enter the cell along
with the normal glucose .
 By measuring this uptake liquid scintillation counter help to analyze the
glucose uptake activity and the effect of plant extract.
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PRINCIPLE

7. GLUCOSE UPTAKE ASSAY USING 3T3 LT1
CELLS
 Insulin promotes glucose uptake , metabolism and storage in
adipose tissue and skeletal muscle .
 Insulin stimulates phosphorylation of insulin receptors substrates by
kinase which leads to activation of PI3 kinase , PKB and protein
kinase C isoforms .
 Activated PKB translocate Glut4 to the cell surface and stimulate
glucose transport in muscle and fat cells whereas it phosphorylates
and inhibits GSK -3(Glycogen synthase kinase 3).
 Inhibitor of GSK- 3 enhances insulin signalling thereby favouring
glucose entry into the muscle cells and the adipose tissue .
 Inactivation of GSK-3 in 3T3 L1 differentiated cells stimulates glucose
uptake which can be measured by incorporation of 2-deoxy-14c
glucose and quantified by using the scintillation counter .
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8. CELL CULTURE
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The cell line 3T3-L1 pre-adipocytes are calculated and made to
differentiate.
The pre-adipocytes are then treated with 0.5 mM 3- isobutyl
methaylxanthine, 5 µgM dexamethasone in 10% FBS containing DMEM
for 2 days.
Later, the cells are transferred to 10% FBS/DMEM containing only
insulin and then to 10% FBS/DMEM without insulin for next 2 days.
After ten days of differentiation the cells are used for the experiments
. Chinese hamster ovaric cells over expressing the human insulin
receptor cells are grown in Ham’s F12 supplemented with 100 U /mL
penicillin ; 10 % fatal bovine serum; 2.5 µg/ml fungizone; 0.5% G-418
and 100 mg/ml streptomycin in 5% CO2 / humidified atmosphere at
370. Cells are passed two to three times a week.
Confluent cells are used for used for the experiments.

9. GLUCOSE UPTAKE ASSAY USING 3T3-L1
ADIPOCYTES
 2-deoxy-D-[3H] glucose uptake of 3T3 –L1 adipocyte is used to
measure the glucose transport system .
 3T3 L1 adipocyte cells cultured on 12 well microtiter plate are
incubated in a transport solution containing 1microCi 2-deoxy D-[3H]
glucose and 0.1mM 2-deoxy-Dglucose for 7 minutes.
 Uptake of glucoesis terminated by the addition of 50mM glucose and
0.1MNaoH/ 0.1% PBS for disruption of the cells .
 Radioactivity incorporated in the cells is determined using
scintillation counter.
 Protein is used to standardize the glucose transport values .
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10. GLUCOSE UPTAKE BY ISOLATED DIAPHRAGM
FROM MICE AND RAT
To study the effect of insulin and insulin like substance on muscle tissue .
 To study glycogen synthesis
 To study glucose transport
 To study glycogen synthase activity
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11.  Glucose uptake activity was analysed by measuring the rate of uptake
of radioactively tagged 2-deoxy glucose in differentiated 3T3 L1 cells.
 After starvation of the cells were treated with insulin and other plant
extracts .
 Then these ligands will bind to the receptors on the surface of the cells
. These triggered the translocation of glucose transporters to the cell
surface .
 Then we treat it with radioactive cocktail containing 10microM 2-deoxy
glucose and 0.25microCi of 2-deoxy –D-(3H)- glucose .
 So the radioactively tagged glucose will enter the cell along with the
normal glucose .
 By measuring this uptake rate by using liquid scintillation counter help
to analyze the glucose uptake activity and the effect of the plant
extract on the glucose uptake activity.
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12. APPLICATION OF GLUCOSE UPTAKE ASSAY
 Glucose uptake experiments are commonly used to measure cellular
metabolic activity and the glucose transport .
 Glucose uptake can be studied using radiolabelled glucose itself , or
radiolabelled glucose analogs such as 2-deoxy-D-glucose (DOG) or 3-O-
methyl –D-glucose .

13. THANK YOU