Diabetes mellitus Known since ages as “Madhumeha” i.e. sweetness of disease urine in Ayurveda by ‘Sushruta’
- During first century Greek physician ‘Aeretaus’ gives term Diabetes as “to flow through”
- In 1755 by ‘Dobson’ demonstrated Diabetes as by presence of sugar in urine.
- In 1909 by ‘De Mayer’ gives statement about insulin is the non-digestive part of pancreas & islet cell was responsible for pancreatic diabetes.
- It is chronic metabolic disorder, resulting from insulin deficiency characterized by hyperglycemia, altered metabolism of carbohydrate, protein , lipid & increases the risk of vascular complications.
- Diabetes is not a single disease, it is heterogeneous group of syndrome, characterized by an evaluation of Blood Glucose caused by a relative deficiency of Insulin.
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| Glucose mechanism |
Classification of Oral Hypoglycemic Agents OR classification of anti-diabetic Drugs
- Insulin secretagogus – they promote the insulin release from β-cell of pancreas.
e.g.. Sulfonylurea :- Tolbutamide, Acetohexamide
- It increases insulin uptake & utilization by target tissue
e.g.Thiazolidinediones-Rosiglitazone, Pioglitazone
- It reduces hepatic glucose output, largely by inhibiting hepatic gluconeogenesis
e.g. Biguanides - Metfomin, Phetformin
- α-Glycosidase inhibitor – they act by delaying the digestion of carbohydrate thereby decreasing glucose absorption
e.g. Acarbose, Miglitol
How to Oral hypoglycemic agents work
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| Mechanism of anti-diabetic agents |
Screening models of anti-diabetics
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| Screening methods for anti-diabetics |
Models for insulin dependence diabetic mellitus
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| Screening model for insulin dependence diabetes mellitus |
Chemical used in insulin dependence diabetes mellitus
IRREVERSIBLE Chemical
- Alloxan
- Streptozotocin
- Diphenyl thiocarbazine
- Oxine-9- Hydroxyquinolone
- Vacor
- Somatostatins
- Catecholamines
- Glucocorticoids
REVERSIBLE Chemical
- 6- amino l-asparginase
- Azide
- Cyproheptadine
- Malonates
- Glucagoan
Alloxan induce Diabetes
Alloxan Induces permanent diabetes.
Mechanism of action of alloxan
- Directly toxic to beta cells.
- Interacts with sulfhydral enzyme and inhibits it.
- Hexokinase activity.
- Protein kinase activity.
- Induces mitochondrial abnormalities.
- Damages the DNA (fragmentation).
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| Type of animal used in Screening model for insulin dependence diabetes mellitus |
Glucose Estimation Method
Glucose Oxidase method - Glucose is oxidized in Gluconic Acid by glucose oxidize. The hydrogen peroxide liberated is reduced by peroxides & Oxygen transferred to an accepter, which is color less in the reduced form but colored in oxidized form.
Urine analysis - Qualitative,Cheap,Convenient. Benedict Reagent are used but Diagnosis can’t based- may show false positive or false negative.
Hexokinase enzyme method
The procedure is a fluorometric rate method measuring the formation of NADPH catalyzed by immobilized glucose-6-phosphate dehydrogenase and hexokinase held within a tiny stirrer. The enzyme stirrer is stable for at least two months and can be used over eight-hundred assays without any loss of activity.
Glycosylated Haemoglobin (HbA1c)
Measurement of blood glucose level in diabetic suffer from variation due to dietary intake of previous day. Long term objective assessment degree of diabetic control is better done by measurement of glycosylated hemoglobin HbA1c,a minor hemoglobin component present in normal person. This is because non-enzymatic glycosylation of hemoglobin takes place over 120 day’s. This gives an estimated of diabetic control for the Preceding 6-10 day’s.
Glucose Tolerance Test (GTT)
Fasting Blood glucose level is first determined & then blood glucose level are sampled minutes to hour after an oral dose of glucose(75gm in 300 ml water) . Blood and urine sample are collected at half hourly interval.
Procedure of alloxan induced diabetes mellitus
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| Produce for alloxan induce diabetes mellitus |
Limitations of alloxan induced diabetes mellitus
- High mortality
- Ketosis
- Some species are resistant to alloxan e.g. guinea pig
- Streptozocin has almost completely replaced alloxan
Streptozotocin induced diabetic mellitus
Broad spectrum anti biotic – 200 mg/kg i.p.
Mechanism of action of streptozotocin
- β cell damage by free radical injury
- Fragmentation of DNA
- Nitric oxide generation
- Induces diabetes in all species
Procedure of streptozotocin induced diabetic mellitus
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| Procedure of streptozotocin induced diabetic mellitus |
Modifications in streptozotocin induce diabetes mellitus
- Multiple low dose over three weeks
- Cyclosporin A given with streptozotocin
Advantages of streptozotocin induced diabetic mellitus
- Greater selectivity towards beta cells
- Low mortality
- Longer and irreversible diabetes
- Guinea pigs and rabbits are resistant
Hormone induced diabetes mellitus
Dexamethasone is a long acting glucocorticoid possessing immunosuppressant action in the islets and produces type 1 diabetes.
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| Hormone induced diabetes mellitus |
Limitations for Hormone induced diabetes mellitus
Viral induced diabetes mellitus
Following virus are involve in developing diabetes mellitus -
- RNA picorna virus
- Coxsackie virus
- Encephalomyocarditis
- Mengo-2t
- Renovirus
- Lymphocytic choriomeningitis
Procedure of viral induced diabetes mellitus
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| Viral induced diabetes mellitus |
Surgically induced diabetes mellitus methods
Surgical removal of pancreas result in insulin dependent form of diabetes mellitus state.
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| Surgery induced diabetes mellitus |
Surgically induced animals
- Partial pancreatectomized animals
e.g. dog, primate, pig & rats
Advantage of surgically induced diabetes mellitus method
- Avoids cytotoxic effects of chemical diabetogens on other body organs.
- Resembles human type 2 diabetes due to reduced islet beta cell mass.
Disadvantage of surgically induced diabetes mellitus method
- cumbersome technical and post operative procedure.
- digestive problems due to excision of exocrine portion of the pancreas.
- loss of counter regulatory response to hyperglycemia.
- high mortality
Insulin antibody induced diabetes mellitus
A transient diabetic syndrome can be induced by injecting guinea pigs with anti insulin serum.
Preparation of antibody for inducing diabetes mellitus
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| Preparation of antibody for inducing diabetes mellitus |
Procedure of antibody induced diabetes mellitus
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| Procedure of antibody induced diabetes mellitus |
Limitations for antibody induced diabetes mellitus
- Effect persist as long as antibodies remain in the circulation.
- Large doses and prolonged administration- ketonemia, ketonuria, glycosuria and acidosis are fatal to animals.
Genetic models for induced diabetes mellitus
- Autoimmune destruction of pancreatic β cells in association with autoantibody production.
- Shares many characteristics with human insulin dependence diabetes mellitus.
- Spontaneously diabetes mellitus on hereditary basis.
Animal for genetic model induced diabetes mellitus
- May provide more insight into pathogenesis of diabetes in humans
- Discovered in 1974 by Drs Reignald and Clifford Chappel in a commercial rodent breeding company (Bio-breeding laboratories Ltd.) in Ottawa hypoinsulinemia is developed which is caused by autoimmune destruction of pancreatic beta cells in association with autoantibody production.
Procedure for genetic models induced diabetes mellitus
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| Procedure for genetic models induced diabetes mellitus |
Limitation for genetic models induced diabetes mellitus
- Ketosis
- Glycosuria
- Weight loss
- High mortality
Models for non-insulin dependence diabetes mellitus
- Neonatal streptozotocin model
- Genetic model
Neonatal streptozotocin model for non-insulin dependence diabetes mellitus
- Pancreatic beta cells destruction
- Decrease in pancreatic insulin stores
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| procedure for neonatal streptozotocin model for non-insulin dependence diabetes mellitus |
Genetic models for non-insulin dependence diabetes mellitus
Three type of genetic models are involve in developing non-insulin dependence diabetes mellitus.
- Monogenic models of obesity and NIDDM
- Polygenic models of obesity and NIDDM
- Animal models of NIDDM with unknown hereditary and environmental component
Monogenic models of obesity and non-insulin dependence diabetes mellitus
- Obesity
- Hyperinsulinemia
- Hyperglycemia
- Hyperlipidemia
Polygenic models of obesity and non-insulin dependence diabetes mellitus
- No single gene implicated.
- Interaction between environment and several genetic defects.
- Polygenic animal model represents human condition more closely.
Animal model of non-inslin dependence diabetes mellitus with unknown hereditary & environment component
Animals taken from natural environment developed DM when fed normal laboratory diet
Diet and nutrition induced non-insulin dependence diabetes mellitus
They remain normal in its natural habitat but develop obesity and diabetes in captivity when fed on standard laboratory chow (high energy diet) instead of its usual low energy vegetable diet.
Sand rats are used extensively in the drug testing such as protein tyrosine phosphatase inhibitor 65 and glucogan
like peptide-1 (GLP-1) analogues.
Transgenic or knockout animals
- Genes – insulin resistance
Insulin receptor
Glucose tranporters
Hexokinase II
Tumour necrosis factor α
- Genes –defective insulin secretion
GLUT-2
Glucokinase
Islet amyloid polypeptide
- Genes- increases body fat
β3 receptors knockout mouse
Uncoupling protein knockout mouse
Transgenic technique
Single gene is identified in embryonic stem cell, Female rat/mouse is allowed to mate. Next day single cell zygote is collected and maintained in culture for few hours. The desired gene is then injected into pro-nucleus of zygote and the construct integrates itself into the genome of the zygote.
The progeny has the desired characters.
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| Transgenic technique |
Advantage and disadvantage of transgenic technique
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| Advantage and disadvantage of transgenic technique |
Normoglycemic animal models
- Rabbit Model
- Rat Model
- Dog Model
Normoglycemic rabbit model
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| Normoglycemic rabbit model |
Normoglycemic Rat model
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| Normoglycemic rat model |
Normoglycemic dog model
- Beagle dog 15-20 kg
- Food is stopped 18 hours prior to administration of test compound
- Blood is collected up to 48 hours
Modification for norglycemic dog model
- Dogs are pancreatectomized 2-3 years prior
- Given pancreatic enzymes orally
- Test compound is given with an oral suspension
Insulin Assays
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| Insulin assay |
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| insulin assay |
In vitro methods on isolated organs and cells
To study the effect of the drug on insulin, glucagon and somatostatin secretion without interference from other organs
In Vitro Methods
- Assays of insulin &of insulin like activity
- Isolated organs, cell and membranes
- Insulin receptor Binding assay
- Assays of other glucose regulating peptide hormones
- Inhibition of polysaccharide degrading enzyme
- Effect on secondary diabetes symptoms
- Isolated pancreas of rat
- Isolated pancreatic islets of rat
- Isolated rat liver
- Isolated hepatocytes of rat
- Fructose 2,6-bi phosphate in rat hepatocytes
- Isolated target tissue Perfused hind limb in rats
- Muscle cell lines
- Assays for insulin or insulin like substances on adipocytes
- Assays for lipid synthesis
- Assays for glucose transport
- Glucose uptake by the isolated diaphragm from mice and rats
- Insulin receptor binding assays
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