A COMPREHENSIVE REVIEW ON IN VITRO AND

IN VIVO MODELS USED FOR ANTIDIABETIC

ACTIVITY

1
 DECLARATION & CERTIFICATE

I, Robinpreet Kaur ,hereby declare that the matter embodied in the project entitled
"A Comprehensive Review On In vitro And In vivo Models Used For
Antidiabetic Activity'' Submitted to Global Group of Institutes,Amritsar
[Department of Pharmacy] is the result of my direct investigations and project has been
composed under supervision and guidance of Ms. Richa Arora, Assistant Professor
[Department of Pharmacy], Global Group of Institutes, Amritsar. I also declare that
nothing in part or full has been submitted for the award of any degree,diploma, or
fellowship to any other Institute or University . All the ideas and references have been
duly acknowledged. If anything wrong is detected in the future then I shall be held
responsible for the same.

Name of student: Robinpreet Kaur

Place: Amritsar

2
ACKNOWLEDGEMENT

I would like to express my sincere gratitude to several individuals and organizations for
supporting me throughout my project course. I thank the GOD, Almighty for keeping me blessed
with his spiritual support all long.

I wish word had the power to express my gratitude and homage towards my very
esteemed and reverend guide Ms. Richa Arora, Assistant professor, Global Group of Institutes ,
punjab for her masterly guidance and for helping inspiring and more over invigorate guidance,
energizing criticism, constant support, and encouragement along with perseverance towards this
project work.

I thank deeply from my heart to my beloved parents for showing their unconditional love,
enormous patience, unwavering support and overwhelming encouragement, prayers and optimism
which never let me in sedation during the course of this project.

I have no word for my friend Mehakpreet kaur for their encouragement throughout my
project work. I am highly thankful to my brother Rajanpreet singh, Gaganjot Singh, for all
sorts of helps. I will never forget the fun, joy and immense pleasure that we all have done
throughout his memorable work.

I also wish to express my sincere thanks to the Global Group of Institutes for accepting
me into the graduate program. In addition, I am deeply indebted to the department of pharmacy.

Warm and sincere thanks to all my respected teachers, family members and friends.

Robinpreet kaur

3
 CONTENTS

S.NO. TITLE Page no.

1. Introduction 6-7

1.1 Insulin Deficiencies 7-8

2. Review Of Literature 8

2.1 Insulin 9-10

2.2 Types 11-12

2.3 Invivo Models 13

2.3.1 Alloxan Induced 13

2.3.2 Streptozotocin Induced 14

2.3.3 Diathizone Induced 14

2.4 Invitro Models 14-15

2.4.1 Alpha- Amylase Inhibition 15

2.4.2 Alpha- Glucosiase Inhibition 16-18

3. Conclusion 19-20

4. References 21-28

4
ABSTRACT

Diabetes mellitus- commonly known as diabetes, is a diseaseX characterized by high blood sugar
level. This high blood sugar level is due to either the body not producing insulin in normal level or
cells not responding to the insulin produced by the body. Diabetes is currently one of the major
health issues facing the world, according to the World Health Organization (WHO). WHO
explains Diabetes is defined as "a chronic condition that results from either insufficient insulin
production by the pancreas or insufficient insulin use by the body."India has more diabetics than
any other country in the world, per IDF (International Diabetes Federation) figures. According to
data, there are currently over 62 million diabetics in the country, and by the year 2030, it is
projected that there will likely be over 100 million diabetics living in India. The many in vivo and
in vitro models that are used to assess this serious disease are highlighted in the current review
paper.The use of clinical, genetic, metabolic, and other risk factors for DM and its chronic
microvascular and macrovascular complications, as well as insights into variations in response to
and tolerance of commonly used medications, dietary changes, and exercise, will all be covered in
this review. We also introduce readers to personalized medicine as it relates to diabetes mellitus
(DM). These developments in "omic" knowledge and methods also offer hints about the
pathophysiological processes underlying DM and its complications.

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 CHAPTER-1

INTRODUCTION

6
INTRODUCTION

Diabetes Mellitus (DM) is chronic and one of the world's most common health disorders.One of
the most prevalent medical conditions in the world is chronic diabetes mellitus (DM). The number
of those affected by DM has increased in the last 40 years. 2015 was estimated to see 1.6 million
deaths worldwide due to diabetes mellitus. In recent years, some drugs have been discovered and
used to treat diabetes mellitus[1]. In the coming years, DM has the potential to have a major
impact on productivity, life expectancy, health, and medical costs. As a result of recent significant
advancements in a variety of DM therapies and their chronic problems, life expectancy has
increased[2,3]. Diabetes mellitus is a disorder that results in an increase in blood sugar levels
because the body is not using or producing enough insulin. The pancreas secretes the hormone
insulin, which promotes the body in utilizing glucose as fuel. As a result, it keeps blood sugar
levels under control. The signs of diabetes mellitus include increased thirst, increased hunger, and
frequent urination. Ketoacidosis and a hyperosmolar hyperglycemic state are two issues brought
on by diabetes. On the other side, long-term issues can be things like cardiovascular disease,
kidney damage, eye illness, stroke, and so on. The three types of diabetes mellitus that are most
prevalent are type-1, type-2, and gestational[4]. A gastro-intestinal and endocrine organ, the
pancreas is located in the upper abdomen behind the stomach. It secretes hormones to regulate
energy metabolism as well as digesting enzymes into the colon. The pancreas is divided into two
parts: a. The exocrine pancreas, which makes up more than 95% of the organ, contains acinar,
duct, and cells linked to connective tissues, blood vessels, and nerves.. It produces and secretes
somatostatins, glucagon, insulin, and blood pancreatic polypeptide hormones[5]. Drugs are used
primarily to save life and alleviate symptoms. Secondary aims are to prevent long-term diabetic
complications and, by eliminating various risk factors, to increase longevity. Insulin replacement
therapy is the mainstay for patients with type 1 DM while diet and lifestyle modifications are
considered the cornerstone for the treatment and management of type 2 DM[6].

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1.1. Insulin Deficiencies

1. Insulin Resistance: It is a condition caused by a normal or elevated insulin level that causes
various biological reactions[7].

2. Compensatory Hyperinsulinaemic: It is a disorder caused by peripheral insulin resistance

in muscle and adipose tissue, which requires greater beta cell secretion to maintain normal blood
glucose levels[8].

3. Diabetes mellitus: Diabetes, which is more commonly known as the disease with high blood
sugar, Cleveland exhibits recognisable signs including polydipsia (increased thirst), polyuria
(frequent urination), and polyphagia (increased hunger). The body either does not create insulin at
a regular level or cells do not react to the insulin that is produced by the body, which results in a
high blood sugar level. Depending on the underlying cause of the condition, there are many forms
of diabetes[9].

8
 CHAPTER-2

REVIEW OF LITERATURE

9
2.1 INSULIN

Proinsulin is converted to insulin and C Peptide.

Insulin is referred to as the storage hormone as it promoters anabolism and inhibits

metabolism of carbohydrates, fatty acids and protein.

In the absence of insulin , most tissues cannot use glucose and fats , proteins are broken
Down to provide energy.

2.1.1. ACTION OF INSULIN

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 2.1.2. ACTION OF INSULIN PRODUCING HYPOGLYCAEMIA

TABLE NO. 1

LIVER MUSCLE ADIPOSE TISSUE

Increases glycose uptake Increases glucose uptake and Increases glucose uptake and
and glycogen synthesis utilization storage as fat glycogen

Inhibits glucogenesis and Inhibits proteolysis and Inhibits lipolysis and release
glucose output. release of amino acids into of FFA which form substrates
blood which form the for glucogenesis in the liver.
substrate for glucogenesis in
the liver.

Inhibits gluconeogenesis from

protein and FFA

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2.1.3. ADVERSE EFFECTS OF INSULIN

● Hypoglycemia
● Allergic reactions
● Lipodystrophy
● Others includes - Seizures , coma

2.2 TYPES

1. TYPE-1 DIABETES MELLITUS

[a] Insulin-dependent diabetes mellitus [IDDM]

Diabetes mellitus that is insulin dependent is another name for it. It is characterized by an inability
to produce enough insulin as a result of beta cell necrosis caused by viral invasion, chemical
activity, or autoimmune antibodies[10]. According to studies, patients with type 1 diabetes enjoy a
better quality of life than patients with type 2 diabetes[11].

. Autoimmune Type 1 diabetes mellitus: 5%–10% of people with diabetes have this type
of the disease[12].The majority of diabetes in children and teens (80%–90%) is type 1
diabetes[13].The primary causes of type 1 diabetes are an autoimmune destruction of the pancreas
cells caused by an inflammatory reaction (insulitis) mediated by T cells and a humoral (B cell)
response[14].

. Idiopathic Type 1 diabetes mellitus: It has been reported that there is a rare form of

12
type 1 diabetes (idiopathic), which is not caused by autoimmunity and is less severe than
autoimmune type 1 diabetes. The majority of individuals with this kind are of African or Asian
heritage and experience episodic ketoacidosis and insulin insufficiency to varied degrees[15].

. Fulminant Type 1 diabetes mellitus: First discovered in 2000, this unique

The form of type 1 diabetes shares certain characteristics with idiopathic type 1 diabetes, including
being non-immune mediated[16]. Through an increased immune response with no discernible
autoantibodies against pancreatic beta cells, an antiviral immune response may cause the loss of
pancreatic beta cells. Pregnancy and fulminant type 1 diabetes have also been linked, according to
reports[17].

2. TYPE-2 DIABETES MELLITUS

[b] Non Insulin-dependent diabetes mellitus[NIDDM]

Target organ insulin resistance, which results in responsiveness to both endogenous and
exogenous insulin, is also named as non-insulin dependent diabetes mellitus[18,19]. Insulin
demand in insulin-target tissues rises in type 2 diabetes patients with insulin resistance. In addition
to insulin resistance, the pancreatic beta cells' due to defect in the function prevented them from
producing enough insulin to meet the increasing demand[20].

. Insulin Resistance and signalling: The liver's inability to transport and dispose of

glucose, its inability to control glucose production through gluconeogenesis, its inability to store
glucose as glycogen, de novo lipogenesis, and hypertriglyceridemia are just a few of the complex
cellular metabolic changes caused by this condition in a variety of tissues, primarily the liver and
muscles[21].

13
GJJTABLE NO. 2

Feature Type 1 diabetes Type 2 diabetes

Onset Sudden Gradual

Age at onset Mostly in Children Mostly in adults

Body size Thin or normal Often obese

ketoacidosis Common Rare

Autoantibodies Usually present Absent

Endogenous insulin low or absent Normal, decreased or

increased

Concordance in identical 50% 90%

twins

Prevalence ~10% ~90%

2.3. IN VIVO ANIMAL MODEL OF DIABETES

2.3.1 ALLOXAN INDUCED DIABETES

Alloxan causes a pancreatic beta cells necrosis that is selective.intravenous , intraperitoneal ,

subcutaneous methods are used to give alloxan in experimental animals including mice , rats
,rabbits and dogs , alloxan is used to induce diabetes depending on the animal type the routes and
doses of alloxan requires may differ from the 1st minutes of alloxan administration , a short – lived
hypoglycaemia phase lasts for 30 minutes. The activation of insulin secretion may contribute to
the hypoglycaemia state. The short term increase in ATP availability and glucokinase inhibition

14
[22,23]. The mechanism at the root of hyperinsulinemia [24,25]. The second phase is an increase
in blood glucose levels 1 hr after alloxan injection, followed by a reduction in plasma insulin
concentration. The drop in plasma insulin concentrations causes significant hyperglycemia that
lasts for 2-4 hrs . This could be due to insulin secretion suppression and beta cell damage [26,27].
The third phase is the hypoglycaemia phase, which lasts 4 to 8 hrs after alloxan administration
[28,29]. In the presence and absence of glucose , alloxan administration causes an abrupt increase
in insulin secretion. insulin is released until the islet response to glucose is completely suppressed.
Alloxan reacts with two sulfhydryl in the glucokinase resulting in disulfide bond and inactivation
of the enzyme . The alloxan is reduced by GSH. Ferric ions are liberated from ferritin and reduced
to ferrous ions by superoxide radicals [30,31]. The fragmentation of DNA in beta cells has also
been identified as a mechanism exposed to alloxan .The disruption of intracellular calcium levels
also contribute to the diabetogenic action of alloxan[32,33].

2.3.2 STREPTOZOTOCIN INDUCED DIABETES

Streptozotocin [STZ] is a naturally occurring substance that is a particularly toxic pancreatic beta
cell. It is employed as an animal model for hyperglycemia in medical studies [34,35]. STZ affects
insulin and glucose level in the blood. hyperglycemia occurs 2 hrs after injection due to a drop in
blood insulin levels. hypoglycemia develops 6 hrs later as a result of elevated blood insulin levels.
Finally, hyperglycemia sets in, and blood insulin levels plummet . STZ reduces insulin production
and release while glucose impairs oxidation [36,37]. STZ was found to suppress the beta cell
response to glucose at first . STZ inhibits the expression of GLUT2 in pancreatic beta cells, STZ
alter the DNA [38]. The alkylation of DNA by STZ causes beta cell death. polyADP ribosylation
is activated by STZ induced DNA damage [39,40]. The activation of polyADP ribosylation is
more important than the formation of free radicals or DNA damage diabetogenicity of STZ.
Calcium , which has the potential to cause necrosis [41,42].

2.3.3. DIATHIZONE INDUCED DIABETES

Diathizone is a sulfur containing organo sulfur chemicals with chelating properties in

experimental animals dithizone is used to induce diabetes .Zinc ,iron and potassium levels in the

15
blood of diathizonized diabetic rats were found to be greater than normal [43]. Diathizone
penetrates membranes and binds to zinc inside lysosomes,causing protons to be released which
increases diabetogenicity[44].

2.4. IN VITRO MODELS FOR ANTIDIABETIC

Biomaterials like perfused entire organs, isolated tissues, cell culture systems, or tissue slice
preparations are employed in invitro investigations to identify specific mechanisms and toxicities.
In vivo testing of antihyperglycemic effects is possible. Utilizing various test techniques in vitro
or on animals, such as inhibiting intestinal glucose uptake or carbohydrate-digesting enzymes,
etc[45].

MODELS TO STUDY INHIBITION

2.4.1. ALPHA- AMYLASE INHIBITION ASSAY

The experiment was carried out using a slightly modified version of a previously published
method (Sangeetha et al.,2012). The test sample was made with dimethylformamide.1 g/mL-1
stock solution of sulfoxide and the 0.5 mg/mL -amylase was added to the sample.solution for 10
minutes at ambient temperature. A 1.0 percent starch solution (500L) was added, and the mixture
was incubated for 10 minutes at room temperature. Following that, 1 mL of dinitrosalicylic acid
was added to the mixture .Heated the reaction mixture in a pot of boiling water 5 minutes in the
bath. Samples were evaluated after they had cooled. 10 mL distilled water was used to dilute the
samples. At 540 nm, the absorbance was measured. The enzyme inhibition activity as a proportion
of total enzyme inhibition activity The bioactive fractions were determined with the help of the
following equation's formula [46].

Abs Sample - Abs Control

Inhibition activity (%) = -------------------------------------- X100 ---------- (1)

Abs Sample

16
The absorbance of test samples is Abs Sample, and the absorbance of control responses is Abs
Control (contains all reagents except the test sample). All of the experiments were repeated three
times.

2.4.2. ALPHA- GLUCOSIDASE INHIBITION ASSAYDifferent sample concentrations

were combined with ten milliliters of the enzyme solution, incubated for ten minutes at 37°C, and
then 210 liters of maleate buffer pH 6 were added to the mixture. The enzymatic process is
initiated by adding 200 l of a 2 mM p-nitrophenyl-D-glucopyranoside solution, and it is then
incubated at 37 °C for 30 min. By placing the mixture in a bath of hot water for five minutes, the
reaction is halted. The absorbance of the released p-nitrophenol is measured at 400 nm after the
addition of 1 ml of the 0.1 M disodium hydrogen phosphate solution[47].

Abs Sample - Abs Control

Inhibition activity (%) = --------------------------------- X100 ---------- (2)

Abs sample

Where Abs Sample denotes the absorbance of test samples and Abs Control denotes the
absorbance of control reactions (which include all reagents except the test sample). The entire
experiment took place in triplicates.

2.4.2.1. GLUCOSE UPTAKE ASSAY

A. GLUCOSE UPTAKE ASSAY YEAST CELL MODEL

The Standard concept and procedure were used to conduct a glucose absorption experiment in
yeast cells.Saccharomyces spp. is a type of yeast.The suspension in distilled water was put
through its paces.Centrifugation (3000 g, 5 min) was repeated until the liquid was clear.
Supernatant fluids were collected, and distilled water was used to prepare 10% (v/v) of the
suspension.A variety of test sample concentrations were applied.1 mL glucose solution (5 mM)
was added and incubated.at 37°C for ten minutes. The chain of events began with100 liters of cell

17
suspension, followed byFurther incubation at 37 °C for 60 minutes after vortexing.The tubes were
then centrifuged for 5 minutes at 2500 g.The amount of glucose in the blood was
calculated.supernatant. The proportion of inhibitory activity that exists was calculated by applying
the formula described in a formula [48].

Abs Sample - Abs Control

Inhibition activity (%) = -------------------------------------- X100 ---------- (3)

Abs sample

Where Abs Sample denotes the absorbance of test samples and Abs Control denotes the
absorbance of control reactions (which include all reagents except the test sample). All of the
experiments were conducted out in a laboratory setting.

B. GLUCOSE UPTAKE ASSAY INSULIN TARGET

The emergence of lipotoxicity serves as a catalyst for the development of obesity and type 2
diabetes; the fundamental factor connecting the two is adipose tissue, or cell damage brought on
by increased intracellular lipid concentrations and insulin resistance. Insulin resistance either at
adipocytes or skeletal muscle results in hyperglycemia[49].

C. TOTAL UPTAKE OF GLUCOSE

D-[U-14C] glucose is treated with adipocytes for 20 minutes at a final concentration of 0.2 mM.
The separation of the cells from the media by centrifugation in silicon oil is used to measure
radioactivity. This experiment was used to determine the complete insulin-stimulated glucose
uptake (signal cascade, glucose transport, and glucose metabolism), regardless of whether the
glucose was utilized via an oxidative or non-oxidative mechanism.Trypsin (4 mg/ml) is incubated
with adipocytes for 15 minutes at 4 °C. After adding soybean trypsin inhibitor (8 mg/ml), the cells
are floated three times before being utilized to test the overall uptake of glucose[50].

D. TRANSPORT OF 2- DEOXY - GLUCOSE

Isolated rat adipocytes or murine 3T3-L1 cells and rat L6 muscle modified to over-express
GLUT4 were used to evaluate the transport of 2-deoxy-D-[1-3H] glucose (Amersham, specific

18
activity 20-30 Ci/mmol, aqueous solution), as described by Gliemann et al (1972)[51].

E.GLUCOSE UPTAKE ASSAY BY ADIPOCYTES CELL LINE

Obesity and Type 2 diabetes, as well as higher intracellular lipid concentrations and insulin
resistance, are thought to be linked to adipose tissue. Insulin resistance can be found in both
adipocytes and non-adipocytes.or skeletal muscle levels play a part in hyperglycemia.
Insulin-related pathwaysAdipocyte cell lines can be used to study resistance.such as 3T3-L1 cells
and transgenic rat L6 muscleGLUT4 should be overexpressed[52,53].

19
CHAPTER-3
CONCLUSION

20
CONCLUSION

We reviewed in vitro and in vivo models utilized in diabetes research in this review. Each model
is an important tool for studying human diabetes' endocrine, metabolisms, genetic, and underlying
mechanisms. In order to create a novel medicine for the treatment of diabetes, animal models and
in vitro approaches are required. For advanced diabetes research, more animal models, software,
and advanced methodologies must be developed, and it is envisaged that more therapeutic choices
will become available as diabetes research improves.

This updated review paper can be very useful for the researchers who are presently involved in the
research and development of a new antidiabetic drug.

The current updated review tries to cover the various in vitro And in vivo models that are used for
screening the antidiabetic activity ofa test drug. This updated review paper can be much useful for
the researchers who arepresently involved in the research and development of a new antidiabetic
drug.

21
CHAPTER-4
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