Principle and applications of glucose uptake assay and calcium influx assay Diabetes Mellitus and its types Calcium regulation Glucose regulation and how it is released in cells.

1. Principle and applications of
glucose uptake and calcium influx
assay
BY
Vivek Sharma and Naveen Singh
M. Pharmacy (1st SEM)
Pharmacology

2. Glucose uptake assay

3. 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 10
minutes or less and specifically measures transporter
activity, whereas changes in glucose concentration
involve a multitude of pathways and typically take
several hours.

4. Diabetes mellitus:
 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.

5. Regulation of
plasma glucose
level

6. Insulin reduce
plasma glucose
level

7. TYPESOF 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 upto 10% of all DM cases
 Develops as a result of the exposure of a genetically
susceptible individual to an environmental agent

8. Type 2 DM:
 It is a combined insulin resistance and relative deficiency in insulin secretion and
is frequently known as.
 Type II
 Noninsulin 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 Type 2 DM 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

9. 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 containing10µM 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

10. 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 over
expression of glucose transporters or identify glucose transporter
inhibitors.
 With fat and muscle cells, changes in GLUT4 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.

12. 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.
Inhibitor of GSK-3 enhances insulin signalling thereby
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.

13. Cell culture
•The cell line 3T3-L1 pre- adipocytes are cultured and made to
differentiate.
• The pre- adipocytes are then treated with 0.5 mM 3- isobutyl
methylxanthine, 5 μg/mL insulin and 0.25 μM 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 (CHO-IR) 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 37°C. Cells are passed two to
three times a week.
• Confluent cells are used for the experiments.

14. 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-L1adipocyte cells cultured on 12 well microtitre plate are
incubated in a transport solution containing 1 μCi 2- deoxy D-[3H]
glucose (10 mCi/ mmol) and 0.1 mM 2- deoxy-Dglucose for 7
minutes.
• Uptake of glucoseis terminated by the addition of 50 mM glucose
and 0.1MNaOH/ 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

15. Glucose
uptake
by isolated
diaphragm
from
mice &rat
 To study the effect of Insulin & Insulin like substance on muscle
tissue
To study glycogen synthesis.
To study glucose transport.
To study glycogen synthase activity.

16. Count.
 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 containing10µM 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.

17. Application of
glucose
uptake assay
 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 .

18.  Introduction
CALCIUM
INFLUX
ASSAY
Based on second messenger assay which is used to measure the
calcium flux associatedGq-protein coupled receptor (inhibition or
activation).
Done by using calcium sensitive fluorescent dye and is mostly
taken up into cytoplasm.
For anion transporter cells probencid (inhibitor of anion
transporter) used for retention of florescent dye in cell
Dye binds to the released calcium and fluorescence intensity
increases.
Change in fluorescence intensity directly proportional to amount
of calcium released.

19. CALCIUM
INDICATORS
Types Of Dye
I. Luminescent Dye:- eg. Aequrin and obelin
II. Fluorescent Dye :- eg.Calcein8,
Chlortetracycline,Fluo-3,Fluo-8,Rhod-4
III. Calcium Senstive Dye:-eg. Tetra-
methylmurexide TM,arsenazo IIP,anti-
pyrylazo III and chlorphophonazo LiP.

20. Detection
techniques
I. Flow Cytometric Analysis:
II. ColorimetricAssay
III. Luminometric Assay
IV. Fluorometric Assay
V. Fluo-8Calcium Assay
VI. Rhod 4 calciumAssay
VII. Cal-520,AM Assay
VIII. Ratiometric Analysis

21. i) 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.
• Phorbol myristate acetate and
calcium ioophoe ionomocinStimulant
• T-Cells, RPMI media, Fetal
bovine Serum, peniccilin
Other Material
and reagent
• Centrifuge,37 C 5% CO2 Cell
Culture incubator,Cell counterEquipment
• Flow Jo Software
Software

22. Assay Principle

23. Procedure
T- Cell preparation
Preparation of Indicator and Enhencing solution
Loading dye preparation by mixing indicator and enhancing solution
Loading dye into cells and incubate for 1 hour in 37C CO2 incubator
Cool down the tube 20 minutes before analysis.

24. Cont.
ATP dose dependent (10µM,1µM or 0µM ATP) Intracellular calcium
measured by flow cytometry
Cells were incubated with 520AM dye for 30 min at 37C before ATP was
Added into the cells.
The baseline was acquired and the rest of cells were analyzed after ATP
addition

25. II)Luminometric
Assay
More sensitive then fluorescence based calcium assay
Provides an optimized assay method for monitoring the
GPC 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

26. Principle
and
Procedure

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 Ca2+
concentration. 23
Cells transfected with apoaequorin .The growth medium was removed and the cells were incubated with 100 µL of dye- loading
solution using for 3 hours at RT and protected from light. ATP (25 µL/well) was added to achieve the final indicated concentrations.
The EC50 of ATP is about 0.8 µM.

27. III) Fluo-8
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
Principle:- Cells expressing a GPCR of interest that signals
through calcium are pre-loaded with Fluo-8 which can cross the
cell membrane.
Once inside the cell, the lipophilic blocking groups of Fluo-8 are
cleaved by an esterase, resulting in a negatively charged
fluorescent dye that stays inside the cell.

28. Cont.
 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 Fluo-8.
Cells incubated with 100 µL of dye loading solution using
Fluo-4 and Fluo-8 for 1 hour at room temperature. Carbachol
(50 µL/well) was added to achieve the final concentrations
indicated.

29. IV)Colorimetric
assay
 colorimetric calcium assays use reagents that
measurable color change in the presence of an
analyte .
 Example :o-cresolphthalein reacts with calcium
in an alkaline environment to produce a violet
colored complex. And color intensity is
proportional to calcium concentration in the
sample .
 Colorimetric assay is the quick and easy assay
can be used to determined the calcium
concentration.(physiological range 0.4-
110mg/dl.

30. Applications
 Calcium detection assays used to dignose
Bone diseases-
osteoporosis
Kidney disease
hypercaluria
Thyroid disease
Hyperparathyroidism
Hypertension
Alzheimer’s disease

31. Refrences
 Monitoring receptor mediated changes in [Ca2+] i using single- and dual-
wavelength indicators on the FlexStation 3 reader | Molecular Devices
 https://www.slideshare.net/DipanjaliKamthe/calcium-influx-
assays
 https://www.slideshare.net/sanjukaladharan/glucose-uptake-
assay
 Glucose UptakeAssay Ppt | DiabetesTalk.Net
 Yamamoto, Norio, Manabu Ueda‐Wakagi, Takuya Sato,
Kengo Kawasaki, Keisuke Sawada, Kyuichi Kawabata,
Mitsugu Akagawa, and Hitoshi Ashida. "Measurement of
glucose uptake in cultured cells." Current protocols in
pharmacology 71, no. 1 (2015): 12-14.

32. THANK YOU