Anti Diabetic

ANTI-DIABETIC
In-vivo & In-vitro Models to Study

anti-diabetic activity of new
Substances
- PAYAL KATE
- F.Y.M.Pharm, Pharmacology
Diabetes Mellitus
 A Chronic disease, occurs when pancreas is not producing enough insulin or
the produced insulin cannot be used by the body.
 Two Types -
a) Type I(Juvenile Diabetes) : Inability of pancreas to produce enough insulin.
b) Type II : Inability of body to respond to produced insulin.
 Symptoms of Diabetes :
1) Increased thirst
2) Slow-healing cuts and sores
3) Fatigue
4) Blurred vision
5) Frequent urination
6) Unexplained weight loss
Pathophysiology of Hyperglycemia
 Hyperglycemia, in turn, contributes directly to insulin resistance and
impairs pancreatic β-cell function, effects described as glucose
toxicity. Such glucose toxicity sets up a various cycle of maladaptive
mechanisms leading to further deterioration of β-cell function and
more severe insulin resistance.
IN-VIVO & IN-VITRO STUDIES
 In-vivo studies :
Studies in which the effects of various biological entities are tested
on whole living organisms or cells, usually animals (including humans).
Examples: Development of antibiotics, development of new drugs.
 In- vitro studies :
In- vitro studies Known as “test tube” experiments. These are the
studies performed with microorganisms, cells or biological molecules
outside their normal biological context. These are performed in
labwares. Example: in-vitro fertilization techniques.
CLASSIFICATION OF ANTI-DIABETIC
Screening methods of In-Vivo models
1. Chemical induced diabetes :
(a)Alloxan induced diabetes
(b) Streptozotocin induced diabetes
(c)Monosodiuxm glutamate induced diabetes
2. Hormone induced diabetes :
(a)Growth hormone induced diabetes
(b) Corticosteroid induced diabetes
3. Insulin antibodies-induced diabetes
4. Diabetes induced by viral agents
(a) D-variant encephalomyocarditis(EMC-D)
(b) Coxsackie viruses
5. Pancreatectomy in dogs
6. Other Diabetogenic compounds
Screening methods of In-Vitro models
METHODS :
1. Isolated pancreas of rat
2. Isolated rat liver
3. Isolated target tissue: muscle cell lines
4. Isolated hepatocytes
5. Assays for insulin or insulin like substances on adipocytes
6. Assay of glucose transport in adipocytes
7. Glucose uptake by the isolated diaphragm from mice and rats
Alloxan induced diabetes
 Alloxan is a derivative of pyrimidine.
 Alloxan produces selective necrosis of beta cells.
 Alloxan is used for induction of diabetes in experimental animals such
as mice, rats, rabbits and dogs.
 The routes and dose of alloxan required may vary depending upon the
animal species.
 A First short lived hypoglycemic phase lasting for 30 min from the first
minutes of alloxan administration. The hypoglycemic stage may be due to the
stimulation of insulin release and high levels of plasma insulin levels.
 The mechanism at back of the hyperinsulinemia is due to the short term
increase of ATP availability and glucokinase inhibition.
 The second phase is the increase in the blood glucose levels one hour after
administration of alloxan, the plasma insulin concentration decreases.
 The pronounced hyperglycemia lasts for 2-4 hours is due to decrease plasma
insulin concentrations. This may be due to inhibition of insulin secretion and
beta cell toxicity.
 Alloxan treatment brings out a sudden rise in insulin secretion in the
presence and absence of glucose. The insulin release occurs until the
complete suppression of the islet response to glucose.
 Alloxan reacts with two sulfhydryl in the glucokinase resulting in disulfide
bond and inactivation of the enzyme.
Dosage-
 Alloxan monohydrate injected by IV or SC or through IP route.
 Rat – 65mg/kg (i.v) /65mg/kg (s.c) /150 mg/kg(i.p)
 Mice – 100-200 mg/kg (i.v)
 Dogs – 50 mg/kg
 Rabbit – 150 mg/kg (i.v)
Evaluation –
 Fasting blood glucose level
Streptozotocin induced diabetes
 Streptozotocin (STZ) is a naturally occurring chemical that particularly
produces toxin to the beta cells of the pancreas.
 Two hours after injection, the hyperglycemia occurs due to the decrease
in blood insulin levels.
 STZ impairs glucose oxidation and decreases insulin synthesis and release.
 STZ changes the DNA in pancreatic B cells. The B cell death is due to
alkylation of DNA by STZ.
 After injecting STZ to animal ,
after 72 hrs FBG > 200mg/dl.
Insulin Induced Diabetes
 The insulin antibodies have the affinity and capacity to

bind insulin.
 Insulin deficiency mechanism may cause greater
postprandial hyperglycemia because antibody-bound
insulin is unavailable to tissues.
Virus Induced Diabetes
a) D-Variant Encephalomyocarditis (EMC- D)

 Viruses produce diabetes mellitus by destroying and infecting
pancreatic beta cells.
 EMC- D virus can infect and destroy pancreatic beta cells in mice and
produce insulin dependent hyperglycemia.
 EMC-D virus known as NDK25. Intraperitoneal injection of NDK25
develops noninsulin dependent diabetes mellitus.
Virus Induced Diabetes
b) Coxsackie viruses
 Coxsackie viruses cause diabetes in mice; it can infect and destroy
pancreatic acinar cells.
 Acinar cells are responsible for synthesis, storage and release of
various pancreatic digestive enzymes.
 Coxsackie B4 virus is strongly associated with the development of
insulin-dependent diabetes mellitus in humans.
Hormone Induced Diabetes
a) Growth hormone induced diabetes -

 Repeated administration of growth hormone in higher experimental
animals induces diabetes with ketonuria and ketonemia (indicating
that an alternate source of energy is used especially fatty acids) .
 Prolonged administration of growth hormone produced permanent
diabetes.
Hormone Induced Diabetes
b) Corticosteroid induced diabetes -

 Corticosteroid induces diabetes, which is called steroid diabetes.
 The prednisolone and dexamethasone, cause steroid diabetes.
 Glucocorticoids stimulate gluconeogenesis, in the liver, resulting in
increase in hepatic glucose and induce insulin resistance and
hyperglycemia.
Other diabetogenic compounds
 Dithizone injection causes a triphasic glycemic reaction in rabbits.

 A phase of initial hyperglycemia is detected after 2 h, followed by a
normoglycemic phase after 8 h and a secondary permanent
hyperglycemic phase after 24–72 h.
 Histologically, complete and partial degranulation of beta cells is
observed.
INVITRO METHODS
1. Isolated pancreas of rat
PROCEDURE
 Animals : Male Wistar rats weighing 200-250g.
 Pancreas is removed
 Pentobarbital (50mg/kg IP) given for anaesthesia.
 Through a portal vein cannula, Krebs-Ringer bicarbonate buffer with 2%
bovine albumin and 5.5mmol/l glucose is perfused at a rate of 1.75ml/min.
 Temperature : 37.5 0C
 Pressure : 100mmHg .
 Perfusate is collected every minute for 30min.
 After the first 5 min of perfusion, test compound is added.
 Glucose of 5.5Mm is perfused.
 The samples collected are stored at -200C.
INVITRO METHODS
2.Glucose uptake by the isolated diaphragm from mice
and rats
PROCEDURE
 Animal – male Sprague Dawley(70-100gm).
 Diaphragm are carefully removed and divided into two equal parts.
 Hemi-diaphragm is incubated in Krebs buffer solution with 5Mm
glucose, insulin compound to be test.
 After 30 min, hemi-diaphragm is blotted on tissue and frozen in liquid
nitrogen.
 After 4 hr at -200C, sample centrifuged for 10 min.
INVITRO METHODS
3. Isolated Hepatocytes
PROCEDURE
 Animal-male Wister (200-250gm) are selected and anaethetised with
hexobarbitol 15ml/kg
 Isolate and wash with 100ml heparinized saline solution
 Oxygenated air tube connected to portal vein.
 The sample for analysis are withdrawn by catheter and are evaluated for net
glucose production.
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ANTI-DIABETIC

In-vivo & In-vitro Models to Study

 The insulin antibodies have the affinity and capacity to

a) D-Variant Encephalomyocarditis (EMC- D)

a) Growth hormone induced diabetes -

b) Corticosteroid induced diabetes -

 Dithizone injection causes a triphasic glycemic reaction in rabbits.

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