INTRODUCTION TO DIABETES AND ANTI-DIABETIC DRUG

SCREENING METHODS

Mrs. Reena Rodrigues

What is DIABETES?Diabetes is diagnosed when a person has too much glucose (sugar) in the blood. This happens because the pancreas cannot make enough insulin.

Insulin is produced in the pancreas and has two jobs in the body - the first is to transport glucose from the blood supply into fat and muscle cells, where it can be used for energy. The second is to switch off the liver once the level of glucose in the blood is high enough. Diabetes is the result of the body not creating enough insulin to keep blood glucose levels in the normal range.

Types of Diabetes Body makes little or no insulin due to an overactive immune system.

Body prevents the insulin it does make from working right.

Measurement of anti-diabetic activity

In vitro methods In vivo methods

Isolated pancreas of rat Assay for α-Amylase GLUT2 transport activity Assay for α-Glucosidase

Effect of insulin sensitizer drugs

Effect of thiazolidinediones on PPAR γ

Hypoglycemic effects Effect on liver receptor

agonist

Isolated pancreas of ratEnzymatically digesting the tissues connecting the islets to the exocrine tissue. Pancreas is excised from a euthanized animal and cut into 1–2 mm pieces.The process of collagenase digestion

Separating islets from non-islet tissue. Gradient separation of islets and pancreatic acinar tissueIslet yieldIslet culture conditionsIslet morphologyIslet viability

Culturing isolated islets in an environment that maintains cell viability.Glucose-stimulated insulin secretionGlucose-stimulated calciumIslet dissociation and cell identification

A freshly isolated islet shown among pancreatic acinar tissue on the day of isolation. b Islets purified from acinar tissue, after incubation at 37°C and 5% CO2 for 18–20 h. c Islet incubated 18–20 h with darkened, hypoxic center.

a GSCa for islets cultured in 5.5 mM glucose (solid, mean of n = 8) and 11 mM glucose (dashed, mean of n = 9).  b. Mean amplitude of the calcium response during each phase (phase 0, 1, and 2) for islets cultured in 5.5 mM glucose (solid, n = 37) or 11 mM glucose (stripes, n = 34).

Effect of insulin sensitizer drugs

Thiazolidinediones are potent activators of PPAR γ

The db/db mouse is a model of obesity, diabetes, and dyslipidaemia wherein leptin receptor activity is deficient because the mice are homozygous for a point mutation in the gene for the leptin receptor.

Various assays for measuring anti-diabetic property – Binding assay Transactivation assay Lipogenesis assay Protein digestion assay

Models for anti-diabetic screeningWhy need for animal models for diabetes?

Diabetes is a chronic disease with various complications.

These complications take years to develop. Animal models save time.

Animal Models for Type-1 & Type-2 Diabetes

Chemically induced diabetes Streptozocin (STZ) induced diabetes Alloxan induced diabetes Goldthioglucose obese diabetic mouse model Atypical antipsychotic-induced diabetic model

Surgically induced diabetes Duodenal-jejunal by pass non-obese T-2 DM Non obese partial pancreatectomized diabetic animals

Genetically induced diabetic animal model Zukker Diabetic Fatty Rat Goto-Kakizaki rat LEW.1WR1 rats NONcNZO10 mouse C57BL/6J mice Kuo Kundo mice Tsumara Suzuki Obese Diabetes mice db/db mice Obese rhesus monkey (Macaca mullata)

Streptozocin (STZ) induced diabetes Streptozocin (STZ) is a glucosamine-nitrosourea compound that has been in clinical

trial since 1967. Six adult Wistar rats weighting 250-300 grams (75-90 days old) were used for

inducing diabetes. The animals were injected by streptozotocin at the dose of 60 mg/kg of the body weight intravenously. Streptozotocin induces diabetes within 3 days by destroying the beta cells.

Diabetic animals and non-diabetic control group were kept in metabolic cages individually and separately and under feeding and metabolism control. Glucose in the blood of diabetic rats exceeded that of the non-diabetic control ones. Food consumption was measured in terms of (gr.), water consumption was measured in terms of (ml) and urine volume was measured in terms of (ml) on a daily basis while every 2- 4 weeks in 80 days the levels of C-peptide, insulin and glucose in blood serum were also measured, so that chemical diabetes was verified in rats injected with Streptozotocin.

E.g -

Representative photomicrographs of hematoxylin and eosin–stained liver (A,B), kidney (C, D), and pancreas (E, F) of normal nondiabetic rats (first column) and STZ-induced diabetic rats (second column). Original magnification ×400.

Duodenal-jejunal by pass non-obese T-2 DM

This model reverses type-2 diabetes (T-2 DM) in Goto-Kakizaki rats, a rodent model of non-obese T-2 DM.

Two weeks post-duodenal-jejunal bypass, oral glucose tolerance was measured.

After three weeks, insulin-induced signal transduction and glucose disposal was measured in skeletal muscle.

The study proved that bypassing of the proximal small intestine does not increase skeletal muscle glucose disposal.

Genetically induced C57BL/6J mice• Type-2 diabetic model by simply feeding high fat feed to non obese,

non diabetic C57BL/6J mouse strain.• It is characterized by marked obesity, hyperinsulinaemia, insulin

resistance and glucose intolerance• They exhibit marked fasting as well as basal hyperglycaemia in

contrast to normal basal glucose level seen in C57BL/6J (ob/ob) mice.• Its usefulness for drug testing has been reported in the literature as

these mice treated with orally active inhibitor of dipeptidyl peptidase-IV (LAF237) are shown to have normalized glucose tolerance in association with augmented insulin secretion.

After feeding high-fat diet

Diabetes: Symptoms & Complications

References http://

www.imedpub.com/articles/animal-models-for-biological-screening-of-antidiabetic-drugs-an-overview.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056052/

http://medind.nic.in/iaf/t07/i2/iaft07i2p60.pdf

https://www.dovepress.com/impact-of-streptozotocin-on-altering-normal-glucose-homeostasis-during-peer-reviewed-fulltext-article-DDDT

https://www.ncbi.nlm.nih.gov/pubmed/10647060

By, Mrs. Reena RodriguesMumbai 2016reena_rod13@yahoo.comsevielle@gmail.com