4. INTRODUCTION
• Glucose is the primary source of energy for many organisms, and the 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 measuring glucose
consumption.
• Glucose uptake occurs on a rapid time scale of 10minutes or less and
specifically measures transporter activity, Whereas changes in glucose
concentration involve a multitude of pathways and typically take several
hours.
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5. DIABETES MELLITUS (DM)
• Diabetes mellitus (DM) is a group of diseases
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.
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8. TYPES OF DM
• Type 1 DM
• It is due to insulin deficiency and is formerly known as-
Type I
Insulin Dependent DM (IDDM)
Juvenile onset DM
• It is due to pancreatic islet β-cell destruction predominantly by an autoimmune
process.
• Usually develops in childhood or early adulthood.
• It accounts for up to 10% of all DM cases.
• Develops as a result of the exposure of a genetically susceptible individual to an
environmental agent 8
9. • Type 2 DM
• It is a combined insulin resistance and relative deficiency in insulin secretion and is
frequently known as-
Type II
Non insulin Dependent DM (NIDDM)
Adult onset DM
• It results from insulin resistance with a defect in compensatory insulin secretion.
• Insulin may be low, normal or high.
• About 30% of the DM 2 patients are undiagnosed (they do not know that they have
the disease) because symptoms are mild.
• It accounts for up to 90% of all DM cases
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10. MEASURING GLUCOSE UPTAKE
• Glucose metabolism is a key process in many organisms. A lack of insulin-
stimulated glucose uptake is associated with type 2 diabetes, while high
glucose uptake is a sign of the high glycolytic rates associated with cancer.
Measuring glucose uptake can determine the effects of various treatments for
diabetes and cancer.
• The gold standard method of assaying glucose uptake relies on detection of
radio-labeled glucose analogs. Alternative colorimetric and fluorometric
glucose uptake assays often lack the sensitivity and robustness needed to
reliably measure glucose uptake in cells.
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11. NON METABOLIC GLUCOSE ANALOG
2-NBDG 2-(N-(7-NITROBENZ-2-
OXA-1,3-DIAZOL-4-YL)AMINO)-2-
DEOXYGLUCOSE
• Much larger than Glucose
• Not transported in the same way as
Glucose
• May not be an accurate indicator of
Glucose Transporter Activity
2-DG 2-DEOXY GLUCOSE
• Widely Used
• Structurally Similar to Glucose
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14. • For a long time, the gold standard for measuring glucose uptake has been the radio-
labeled analog 2H-2DG and the measurement of accumulated 2H-2DG6P.
• Although the method is sensitive and works well, it requires multiple wash steps.
• Many researchers do not use this method because they want to avoid the handling
and disposal of radioactive material.
• Commercial non-radioactive methods use non-radiolabeled 2DG and measure the
accumulation of 2DG6P through the action of G6PDH(glucose-6-phosphate
dehydrogenase).
• This enzyme oxidizes 2DG6P to form NADPH, which can then be used to generate
probes for absorbance or fluorescence assays.
• Both assays are amenable to multiwell plate formats, but they require multiple
processing steps.
• Due to a cycling detection reaction, the absorbance assay is most sensitive and can
detect very low amounts of 2DG6P. 14
16. PRINCIPLE
• Glucose uptake activity was analyzed by measuring the rate of uptake of
radioactively tagged 2-deoxy 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 10μM 2-deoxy glucose
and 0.25μCi 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 plant extract on the
glucose uptake activity. 16
18. WHY MEASURE GLUCOSE UPTAKE
• Changes in glucose uptake can reflect overall changes in metabolism, but
there are many specific processes as well.
• In cancer cells, measuring glucose uptake can monitor the overexpression of
glucose transporters or identify glucose transporter inhibitors.
• With fat and muscle cells, changes in GLUTS translocation upon insulin
stimulation can be observed by measuring glucose uptake.
• In immunologically relevant cells, measuring glucose uptake can be used to
follow the transformation of certain cell types from one stage to another.
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19. System Model Cells Purpose
Fat & Muscle
Cells
3T3-L1, 3T3L1
MBX, L6, C2C12
Monitor GLUT4 translocation in
response to insulin
Immunologically
relevant cells
T cells and
macrophages
Monitor cell activation
Cancer cells Any cancer cell
(e.g,HCT116)
Monitor glucose transporter over
expression, identify glucose
transporter inhibitors or measure
general changes in metabolism 19
20. GLUCOSE UPTAKE ASSAY USING 3T3-L1
CELLS
• Insulin promotes glucose uptake, metabolism and storage in adipose tissue and
skeletal muscle.
• Insulin stimulates phosphorylation of insulin receptor substrates (IRS) 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 there by
favouring glucose entry into the muscle cells and adipose tissue.
• Inactivation of GSK-3 in 3T3 L1 differentiated cells[adipocytes] stimulates glucose
uptake which can be measured by incorporation of 2-deoxy- 14C-glucose and
quantified by using scintillation counter. 20
21. GLUCOSE UPTAKE ASSAY USING 3T3-L1
ADIPOCYTES
• 2-deoxy -D-[3H] glucose uptake of 3T3-L1 adipocyte is used to measure the glucose
transport system38.
• 3T3-L1 adipocyte cells cultured on 12 well microtitre plate are incubated in a
transport solution containing μCi 2- deoxy D-[3H]glucose (10 mCi/ mmol) and 0.1
mM 2-deoxy-Dglucose for 7minutes.
• Uptake of glucose is terminated by the addition of 50 mM glucose and 0.1 M
NaOH/ 0.1% PBS for disruption of cells.
• Radioactivity incorporated in the cells is determined using Scintillation counter.
• Protein is used to standardize the glucose transport values.
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22. GLUCOSE UPTAKE- GLO™ ASSAY
• We have developed a luminescent glucose uptake assay that is based on the same
principle as the a fore mentioned assays.
• The accumulation of 2DG6P and detection through the enzymatic action of
G6PDH.
• The luminescent assay has a number of advantages:»
Similar sensitivity to the radioactive assay but without any wash steps or handling
and disposal of radioactive material.
Larger signal window than the absorbance and fluorescence assays, making it easier
to detect small changes and requiring less assay optimization.
Amenable to high-throughput screening due to fewer false hits and steady glow-
type signals.
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23. • The luminescent assay can detect glucose uptake from as few as 5,000 cells
with a greater than threefold signal-to-background ratio, and the signal
remains linear up to at least 50,000 cells.
• The luminescent assay is the best combination of simplicity and sensitivity
available for a non-radioactive multiwell plate method.
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25. 2DG UPTAKE ASSAY KIT
• Measuring uptake of 2-deoxyglucose (2DG) in tissues and cells is are liable approach
method to estimate the amount of glucose uptake and thereby to explore the regulation of
glucose metabolism and mechanism of insulin resistance.
• Radioisotope-labeled 2DG is usually used to measure 2DG uptake both in vivo and in vitro.
• However a specialized RI facility with strict limitations is required to carry out the
radioisotope (RI) method and cannot be handled in ordinal laboratories.
• Furthermore, radioactive 2DG administered into cultured cells remains in extracellular space,
and therefore the results obtained must be corrected by separating the extracellular 2DG and
intracellular 2DG/2DG-6-phosphate (2DG6P) in the cells
• This kit is based on the enzymatic method for the direct measurement of amount of 2DG6P
without the use of radioisotope materials
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26. • The luminescent assay can detect glucose uptake from as few as 5,000 cells
with a greater than threefold signal-to-background ratio, and the signal
remains linear up to at least 50,000 cells.
• The luminescent assay is the best combination of simplicity and sensitivity
available for a non-radioactive multiwell plate method.
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27. A COMPARISON OF THE GLUCOSE UPTAKE-
GLO™ ASSAY WITH THE CONVENTIONAL
RADIOACTIVE METHOD
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28. APPLICATION
• Glucose uptake experiments are commonly used to measure cellular
metabolic activity and glucose transport. Glucose uptake can be studied using
radiolabeled glucose itself, or radiolabeled glucose analogs such as 2-deoxy-
D-glucose (DOG) or 3-O-methyl-D-glucose.
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30. CALCIUM ION
• Play an important role various biological functions:-
• It acts as a second messenger.
• Many enzymes requires Ca as a cofactor. E.g., Lactonase transferase.
• Extracellular calcium maintains the potential difference across excitable cell
membranes.
• Bone acting as the major mineral storage site.
• Calcium ions are released from bone into the bloodstream.
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31. INTRODUCTION
• Calcium mobilization assays is a cell based second messenger assay to measure the
calcium flux associated with Gq-protein coupled receptor or inhibition or
activation.
• The method utilizes a calcium sensitive fluorescent dye that is taken up into the
cytoplasm of most cells.
• In some cells anion transporter are particularly active. The addition of probencid,
an inhibitor of anion transporter, is required for retention of this dye in the cell.
• The dye binds the calcium released from intracellular store and its fluorescence
intensity increases.
• The change in the fluorescence intensity is directly correlated to the amount of
intracellular calcium that is released into cytoplasm in response to ligand
activation of receptor 31
34. FLOW CYTOMETRIC ANALYSIS
Flow cytometry (FC) is a technique used to detect and measure physical and
chemical characteristics of a population of cells or particles.
Stimulant Phorbol myristate acetate (PMA) and
calciumionophore ionomycin
Other Material
& Reagent
T-cells, RPM! Media, Fetal bovine
Serum, penicillin.
Equipment Centrifuge ,37 °C 5% CO2 Cell
culture incubator, Cell Counter
Software Flow Jo Software
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35. ASSAY PRINCIPLE
• The Principle of flow cytometric calcium flux measurement is based on changes in
fluorescence intensity or emission wavelength of a fluorophore following chelating of
calcium ions. This is commonly reported as a plot of the fluorescence intensity against time.
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36. PROCEDURE
1) Preparation of T-cells
2) Preparation of indicator and enhancing solution
3) Preparation of loading dye by mixing indicator and enhancing solution
4) Loading dye into cells and incubate for 1hour at 37°C in CO2 Incubator.
5) Cool down the tube 20 minutes before analysis.
The ATP dose dependent (10 μM, 1 μM or 0 μM ATP) intracellular calcium
release was measured by Flow cytometry.
Cells were incubated with 520 AM dye for 30 minutes at 37 °C before ATP was
added into the cells. The baseline was acquired and the rest of the cells were
analyzed after the addition of ATP.
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37. LUMINOMETRIC ASSAY
• It is much more sensitive than the fluorescence-based calcium assays.
• It provides an optimized assay method for monitoring the G-protein-coupled
receptors and calcium channels.
• Useful for monitoring the intracellular calcium mobilization in a specified
compartment given that recombinant apoaequorin proteins.
• More sensitive than the fluorescent calcium assays.
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38. PRINCIPLE
• Luminometric assay are preferred methods in drug discovery for screening G
protein coupled receptors (GPCR).
• It uses a highly calcium- sensitive and membrane- permeable coelenterazine-
calcium indicator for the cells that are transfected with apoaequorin gene.
• Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish
Aequorea victoria that has been used extensively as a calcium indicator in
cells
• The aequorin complex emits blue light when bound to calcium ions. The
luminescence intensity is directly proportional to the Ca concentration.
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40. FLUOROMETRIC ASSAY
• Fluorometric assay provides a simple method for detecting calcium in
physiological solutions by using a red fluorescence probe.
• Indo-1-AM Calcium Assay
• It is a fluorescence-based assay for detecting intracellular calcium
mobilization.
• It provides an optimized assay method for monitoring the G-protein-coupled
receptors and calcium channels.
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41. PRINCIPLE
• Cells expressing a GPCR of interest that signals through calcium are pre-
loaded with Indo-1-AM which can cross the cell membrane.
• Once inside the cell, the lipophilic blocking groups of Indo-1-AM are cleaved
by an esterase, resulting in a negatively charged fluorescent dye that stays
inside the cell.
• Its fluorescence is greatly enhanced upon binding to calcium. When cells are
stimulated with agonists, the receptor signals the release of intracellular
calcium, which significantly increases the fluorescence of Indo-1-AM.
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