International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

_________________________________________Research Article

Invitro Models for Antidiabetic Activity Assessment

Krutika Thorat*, Leena Patil, Dnyanesh Limaye and Vilasrao Kadam
Bharatividyapeeth college of Pharmacy, Sector-8, C.B.D., Belapur, Navi Mumbai, India.
INTRODUCTION
Antidiabetic effects can be studied invivo using
animal models or invitro using a variety test
systems. Invitro tests can play a very important role
in the evaluation of antidiabetic activity of drugs as
initial screening tools where the screening of large
number of potential therapeutic candidates may be
necessary. They might provide useful information
on the mechanism of action of therapeutic agent.
Biological material used in these includes perfused
whole organs, isolated tissues, cell culture systems,
or tissue slice preparations. While in vivo
biological systems using live animals (whole
organisms) are necessary to study how such
mechanisms
behave
under
clinical
or
pathophysiological
conditions,
data
from
experiments carried out in in vitro systems can
establish mechanisms and define toxicities.
Models to study inhibition of Carbohydrate
digesting enzymes
Assay of -amylase inhibitory activity
The effect of sample on -amylase activity can be
studied using an enzyme-starch system.1 Sample is
mixed by stirring with 25 mL of 4% potato starch
in a beaker; 100 mg of -amylase is added to the
starch solution, stirred vigorously, and incubated at
37C for 60 minutes. After the incubation period
0.1 M NaOH is added, to terminate enzyme
activity. The mixture is centrifuged (3000 xg; 15
minutes) and the glucose content in the
supernatant
is
determined.
Assay of -glucosidase inhibitory activity
A crude enzyme solution of rat intestinal glucosidase and sucrase, prepared according to the
method of Dahlqvist,2 is used to assay the glucosidase and sucrase inhibitory activities,
according to the method of Honda and Hara. 3 Ten
milliliters of enzyme solution and varying
concentrations of the sample is incubated together
for 10 minutes, at 37C, and the volume was made
up to 210 L with maleate buffer, pH 6.0. The
enzyme reaction is started by adding 200 l of 2
mM p-nitrophenyl--D-glucopyranoside solution
and further incubated at 37C for 30 minutes. The
reaction is terminated by treating the mixture in a
boiling water bath for five minutes. After the
addition of 1.0 ml of 0.1 M disodium
hydrogenphosphate solution, the absorption of
liberated p-nitrophenol is read at 400 nm.

Vol. 3 (2) Apr Jun2012

Assay
of
sucrase
inhibitory
activity
The effect of sample on sucrase activity was
assayed according to the method of Honda and
Hara. 3The enzyme solution (10 l) and varying
concentrations of the sample is incubated together
for 10 minutes at 37C, and the volume is made up
to 200 L with maleate buffer (pH 6.0). The
enzyme reaction is started by adding 100 l sucrose
solution (60 mM). After 30 minutes, the reaction is
terminated by adding 200 L of 3,5dinitrosalysilic acid reagent and treating the
mixture in a boiling water bath for five minutes.
The absorbance of the solution is read at 540 nm.
The percent inhibitory activities were calculated
using the following formula

Where, Abs control is the absorbance of the control

reaction (containing all reagents except the test
sample), and the Abs sample is the absorbance of
the test sample. An untreated enzyme solution was
used as the control. All the experiments are carried
out
in
triplicate.

Models to study inhibition of intestinal glucose

uptake
The determination of insulin based on the
stimulation of glucose uptake by the isolated
diaphragm from mice and rats has been used by
many investigators to study the effects of insulin
and insulin-mimetic substances on muscle tissue.
Intact washed rat diaphragms are incubated (30
min, 37 C) in HEPES-buffered saline (25 mM
HEPES,
120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM
MgCl2, 5 mM glucose, 0.5 mM sodium pyruvate,
1.5 mM KH2PO4, pH 7.4) under constant bubbling
with 95% O2/5% CO2. The diaphragms are then
washed two times with the same buffer lacking
glucose and further incubated (30 min) in 5 ml of
glucose-free buffer in the presence of test
compounds or insulin. Glucose transport is initiated
by addition of 50
l of 10 mM 2-[13H]deoxyglucose (10 Ci/ml) in the absence or
presence of 25 M cytochalasin B (control). After
15 min, the diaphragms are rinsed four times with
icecold buffer containing 10 mM glucose, 25 M

www.ijrpbsonline.com

730

International Journal of Research in Pharmaceutical and Biomedical Sciences

cytochalasin B, blotted with filter paper and

homogenized. Portions of the homogenate are used
for protein determination. One-ml portions of the
supernatant of a centrifugation at 10 000 g for 15
min are mixed with 10 ml scintillation cocktail and
counted for radioactivity. Specific glucose
transport (dpm/mg of protein) is calculated as the
difference
between
diaphragm-associated
radioactivity measured in the absence (total uptake)
and presence of cytochalasin B (non-specific
uptake). Under these experimental conditions,
transport is linear for 30 min.4
Models to study insulin secretion from cells of
the pancreas
Insulin secreting cell lines reported in the
literature, retain a differentiated function and
respond to secretagogues and inhibitors of insulin
secretion 5.
The insulin secreting HIT cell line was developed
by Santerre et al. 6 by isolating pancreatic islets
from the hamster, dispersing the islets into single
cells, transforming the cell isolates with the simian
virus 40 (SV40), and cloning out the insulinsecreting cell lines.
Experiments for assessment of glucose transport
activity in HIT cells and Western blot analysis for
GLUT2 in these cells after incubation with
glibenclamide and troglitazone were performed by
Masuda et al. (1995)7. Asfari et al.8 derived INS-1
cells and INS-2cells from parental RINm5f cells.
The betacyte, also called the HEP G2ins/g cell, is
a genetically engineered insulin-secreting human
liver cell line that is glucose responsive . 9,10,11
Models based on muscle as an insulin target
tissue
The effect of insulin on glucose uptake in the
soleus muscle of rats during hemorrhagic shock
was studied by Chaudry et al. (1975).
Total uptake of glucose
Adipocytes are incubated with D-[U-14C]glucose
(0.2 mM final concentration) for 20 min. Cells are
separated from the medium by centrifugation on
silicon oil, removed and counted for radioactivity.
This assay measures the total insulin-stimulated
glucose uptake (signal cascade, glucose transport,
glucose metabolism) irrespective whether the
glucose is utilized via the oxidative or nonoxidative pathway. Conversion into lipids,
glycogen or membrane-impermeable intermediary
products (glucose-6-phosphate) will be detected.
Adipocytes are incubated with trypsin (4 mg/ml)
for 15 min at 4 C. After addition of soy bean
trypsin inhibitor (8 mg/ml) the cells are washed
three times by flotationand used for determination
of total uptake of glucose. This assay measures the
total glucose uptake into cells with inactivated
insulin receptor. Thus only compounds that bypass

Vol. 3 (2) Apr Jun2012

ISSN: 2229-3701

the first step in the insulin signal transduction

cascade (binding of insulin to its receptor) will
provide positive results.
Transport of 2-deoxy-glucose
The transport of 2-deoxy-D-[1-3H] glucose
(Amersham, specific activity 2030 Ci/mmol,
aqueous solution) by isolated rat adipocytes or 3T3
adipocytes is measured as described by Gliemann
et al. (1972)12, Foley and Gliemann (198113, ,),
Mller and Wied (1993) 14. All incubations are
performed in a 25 C shaking water bath. A 50 l
adipocyte cell suspension is pipette into minisorp
tubes (Nunc, Denmark) and equilibrated at 25 C
for 30 min. Insulin (80 nM stock in KRH/5% BSA)
and substances (lyophilized, 2 mg/ml) are added in
50 l KRHB and incubated for 30 min at 25 C. To
correct
for
2-deoxyglucose
unspecifically
associated with the cell surface or entrapped in
extracellular spaces, 2 l of cytochalasin B (20 M
final conc.) from a 1 mM stock solution (in 10%
ethanol, diluted with H2O from a 10 mM stock in
ethanol at the day of use) and, as a control, 2 l of
20% ethanol were added prior to addition of
radiolabeled 2-deoxy-glucose and incubated under
identical conditions. The transport assay is started
by addition of 50
l of 0.1 mM 2-deoxy[3H]glucose (2 Ci per ml of KRHB containing 10
l /ml of 3H-stock, 0.2 mCi/ml), is added and the
incubation continued for another 10 (max. 20) min
at 25 C (total volume of the final incubation
mixture = 150 l). The assay is terminated by
centrifugation of 100 l samples on top of 250 l
dinonylphthalate (Merck, Darmstadt, FRG) in 500
l plastic tubes (Beckman) in a microfuge (10 000
g, 1 min, room temperature). The adipocytes
remain on top of the oil, while the buffer is below
the oil. The tube is cut just below the adipocyte
layer, which is transferred into 5 ml aqueous
scintillation cocktail (Zinsser Nr. 312 or Beckman
Ready Safe) and counted. Specific transport is
calculated as the difference between total cellassociated radioactivity (absence of cytochalasin B)
and
unspecifically
entrapped
radioactivity
(presence of cytochalasin B). Under the conditions
used, 2-deoxyglucose transport is linear with time
up to 20 min. The glucose transport stimulation
factor is calculated as ratio between stimulated
specific transport (presence of insulin or extract)
minus basal specific transport (absence of insulin
or extract) and basal specific transport. For insulin
(2 nM), this factor usually lies in the range between
15 and 25. This assay measures the insulinstimulated specific transport of glucose across the
plasma membrane via facilitated diffusion (glucose
carriers) including the signal cascade irrespective
of glucose utilization (as revealed by the nonmetabolizable deoxyglucose).

www.ijrpbsonline.com

731

International Journal of Research in Pharmaceutical and Biomedical Sciences

Models based on adipocytes as an insulin target

tissue
Insulin sensitizer drugs are reported to improve
symptoms in patients with established NIDDM
(non-insulin dependent diabetes mellitus) . In
contrast to sulfonylureas, these compounds do not
lower blood glucose in normal animals. Various
animal models resembling type II diabetes are used
for evaluation. In vitro techniques showed an
increased glucose uptake of into muscle tissue and
into adipocytes.15,16
Hep G2 cells express insulin receptors, display a
number of metabolic responses to insulin and
insulin-like growth factor I and have been used for
various studies found that troglitazone (CS-045)
increased glycogen synthase I activity in Hep G2
and BC3H-1 muscle cells.
The principal substrate for the insulin and insulinlike growth factor-1 receptors is the cytoplasmatic
protein insulin-receptor substrate-1 (IRS-1). IRS-1
undergoes multisite tyrosine phosphorylation and
mediates downstream signals by docking various
proteins that contain Src homology 2 domains.
Insulin derivatives can be characterized by
phosphorylation and dephosphorylation kinetics of
the insulin receptor, insulin receptor substrate-1
and Shc which is implicated in mitogenic signal
transduction.
urther members of the insulin
receptor substrate family are identified17.
Phosphorylation and dephosphorylation kinetics
of insulin receptor and insulin receptor
substrates is studied as follows. Rat embryo
fibroblasts (Rat-1) stable overexpressing the human
receptor isoform A are grown in Dulbeccos
modified Eagles/F12 Mix medium supplemented
with 200 nM methotrexate and 10% FCS to
confluence in 6 well culture plates and
subsequently incubated for 18 h in Dulbeccos
modified Eagles/F12 Mix medium without FCS.
For phosphorylation kinetic studies, cells are
incubated with human insulin or the insulin
derivatives for various times (0120 min),
subsequently rinsed once with ice-cold buffered
saline and solubilized in lysis buffer (50 mM
HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM
EGTA, 15 mM Na4P2O7, 100 mM NaF, 10% v/v
glycerol, 1% v/v Triton X-100, 1 200 trypsin
inhibitory units/liter aprotinin, 1 mM PMSF, 1 mM
Na3VO4, pH 7.5). For the dephosphorylation
kinetics, the cells are incubated with insulin or the
test preparations for 3 min and dephosphorylation
of the insulin receptor is initiated by dilution of the
ligand. The monolayers are then kept for various
times at 37 C. After washing with icecold buffered
saline, cells are solubilized in lysis buffer. After
centrifugation (10 min at 16 000 g) the
supernatants are diluted in Laemmli buffer (50
mMDTT) and the proteins separated by SDSpolyacrylamide gel electrophoresis.

Vol. 3 (2) Apr Jun2012

ISSN: 2229-3701

CONCLUSION
Thus this review provides the information of
various invitro studies used in antidiabetic
assessment, which can establish mechanisms for
the antidiabetic activity of the drug.
REFERENCES
1. Ou S, Kwok K, Li Y and Fu L. In vitro
study of possible role of dietary fiber in
lowering postprandial serum glucose. J
Agric Food Chem 2001;49:1026-9.
2. Dahlqvist A. Method for assay of
intestinal disaccharides. Anal Biochem
1964;7:18-25.
3. Honda M and Hara Y. Inhibition of rat
small intestinal sucrase and -glucosidase
activities by tea polyphenols. Biosci
Biotechnol Biochem 1993;57:123-4.
4. Adolfsson S, Arvill A and Ahren K.
Stimulation by insulin of accumulation
and incorporation of L-[3H]proline in the
intact levator ani muscle from the rat.
Biochem Biophys Acta. 1967;135:176
178
5. Poitout V, Olson LK and Robertson RP.
Insulin-secreting cell lines: Classification,
characteristics and potential applications.
Diabet Metabol (Paris). 1996; 22:714
6. Santerre RF, Cook RA, Crisek RMD,
Sharp JD, Schmidt RJ, William DC,
Wilson CP. Insulin synthesis in a clonal
cell line of simian virus 40-transformed
hamster pancreaticbeta cells. Proc Natl
Acad Sci USA. 1981;78:43394343
7. Masuda K, Okamoto Y, Tuura Y, Kato S,
Miura T, Tsuda K, Horikoshi H, Ishida H
and Seino Y. Effects of troglitazone (CS045) on insulin secretion in isolated rat
pancreatic islets and HIT cells: an
insulinotropic mechanism distinct from
glibenclamide. Diabetologia. 1995; 38:24
30
8. Asfari M, Janjic D, Meda P, Li G, Halban
PA and Wollheim CN. Establishment of
2-mercaptoethanol-dependent
differentiated insulin-secreting cell lines.
Endocrinology. 1992; 130: 167178
9. Simpson AM, Tuch BE, Swan MA, Tu J,
Marshall GM. Functional expression of
the human insulin gene in a human
hepatoma cell line (HEP G2). Gene
Therapy. 1995;2:223231
10. Tuch BE, Beynon S, Tabiin MT, Sassoon
R, Goodman RJ and Simpson AM. Effect
of -cell toxins on genetically engineered
insulin-secreting cells. J Autoimmun.
1997;10:239244
11. Chaudry IH, Sayeed MM and Baue AE
The effect of insulin on glucose uptake in

www.ijrpbsonline.com

732

International Journal of Research in Pharmaceutical and Biomedical Sciences

12.

13.

14.

15.

16.

17.

18.

ISSN: 2229-3701

soleus muscle during hemorrhagic shock.

Can J Physiol Pharmacol. 1975; 53:6773
Gliemann J, sterlind K, Vinten J and
Gammeltoft S. A procedure for
measurement of distribution spaces in
isolated fat cells. Biochim Biophys Acta
1972;286:19
Foley JE and Gliemann J. Accumulation
of
2-deoxyglucose
against
its
concentration gradient in rat adipocytes.
Biochim Biophys Acta. 1982; 648:100
106
Mller G and Wied S. The sulfonylurea
drug, glimepiride, stimulates glucose
transport,
glucose
transporter
translocation, and dephosphorylation in
insulin-resistant rat adipocytes in vitro.
Diabetes. 1982; 42:18521867
Colca JR. Insulin sensitiser drugs in
development for the treatment in diabetes.
Expert Opin Invest Drugs. 1995; 4:2729
Kuehnle HF. New therapeutic agents for
the treatment of NIDDM. Exp Clin
Endocrinol Diabetes. 1996;104:93
Fantin VR, Sparling JD, Slot JW, Keller
SR, Lienhard GE and Lavan BE .
Characterization of insulin receptor
substrate 4 in human embryonic kidney
293 sells. J Biol Chem. 1988; 273: 10726
10732
Ciaraldi TP, Gilmore A, Olefsky JM,
Goldberg M and Heidenreich KA. In vitro
studies on the action of CS-045, a new
antidiabetic
agent.
Metabolism.
1998;39:10561062.

Vol. 3 (2) Apr Jun2012

www.ijrpbsonline.com

733