This document discusses experimental methods for evaluating anti-diabetic drugs. It describes various animal models of diabetes used, including those induced chemically, genetically, or surgically. Methods for measuring anti-diabetic activity include assessing glucose lowering effects in vivo in animals like rabbits, rats, mice, and dogs. The euglycemic clamp technique, a gold standard method, quantifies insulin sensitivity by infusing glucose to maintain blood sugar levels during insulin infusion in rats. A variety of genetic and transgenic animal models of type 1 and type 2 diabetes are also summarized.

1. Experimental evaluation of
anti-diabetics
Dr Kirtan Bhatt
Department of Pharmacology
KIMS, Bangalore

2. Protocol
• Introduction
• Need for animals models in diabetes mellitus
• Animals models of diabetes mellitus
For IDDM
For NIDDM
• Measurement of glucose lowering / anti-diabetic activity
In vivo methods
In vitro methods
• Conclusion

3. Introduction
“Diabetes mellitus can be defined as a heterogenous group of chronic
disorders of carbohydrate, lipid and protein metabolism characterized
by high blood glucose levels due to relative or absolute deficiency of
insulin”

4. Epidemiology of diabetes
• Prevalence in 10 countries with highest prevalence ranges from 11.6%
to 30.9%
• Worldwide prevalence has risen dramatically in last 2 decades – 30
million cases in 1985 to 285 million in 2010
• 438 million will have diabetes by 2030 (IDF)
• Asian phenotype – onset at a lower BMI and younger age

5. Need for animal models for DM
• Diabetes a chronic disease with many different complications
• Complications take years to develop
• Animal models save time

6. Animal models of diabetes
• For IDDM
Chemically induced diabetes
Hormone induced diabetes
Virus induced diabetes
Surgery induced diabetes
Insulin antibody induced diabetes
Genetic models
• For NIDDM
Neonatal STZ induced NIDDM
Other chemicals
Genetic models

7. Chemically induced diabetes
Chemicals that induce diabetes can be classified into 3 categories
 Specifically damage β cell
 Cause temporary inhibition of insulin production and/or secretion
 Diminish metabolic efficacy of insulin target tissues
1. Alloxan induced diabetes
2. Streptozocin induced diabetes

8. Alloxan induced diabetes
• A cyclic urea analog, the first agent to be introduced in this category
to induce permanent diabetes in animals
• Shows a triphasic reponse
• Species to species variation
Alloxan
Diuleric
acid
Alloxan +
free
radicals
Damages
the DNA of
β cells
Cell death

9. Rabbits:
weight 2-3.5 kg are taken
ear vein – 150 mg/kg alloxan monohydrate (5g/100 ml, pH 4.5) for 10
minutes resulting in 70% of animals becoming hyperglycemic and
uricosuric.
The rest either die or are temporarily hyperglycemic.
Rats:
Wistar or Sprague – Dawley strain
Weight 150-200 g
Inj. Alloxan 100-175 mg/kg SC
Male Beagle dogs:
weight 15-20 kg
inj. Alloxan IV 60 mg/kg

10. Drawbacks of alloxan
• High mortality in rats
• Causes ketosis in animals due to free fatty acid generation
• Diabetes induced is reversible
• Guinea pigs are resistant to alloxan

11. Streptozocin induced diabetes
Streptozocin [2-deoxy-2-methyl(3-methyl-3-nitrosourea)1-
Dglucopyranose] is a broad spectrum antibiotic produced from
Streptomyces achromogens.
Mechanism of β cell damage
By process of methylation
Free radical generation
Nitric oxide production

12. Diabetogenic doses vary with species
Rats – 50-60 mg/kg IP or IV
Mice – 175-200 mg/kg IP or IV
Dogs – 15 mg/kg for 3 days
Blood glucose levels show triphasic response as seen with alloxan
• Hyperglycemia at 1 hour
• Hypoglycaemia that lasts for 6 hours
• Stable hyperglycemia by 24-48 hours after STZ administration

13. Advantages and disadvantages: :
• Greater sensitivity towards β cells
• Lower mortality rates
• Longer and irreversible diabetes induction
• But, guinea pigs and rabbits are resistant to its diabetogenic action.

14. Growth hormone induced diabetes
• Cotes and co-workers (1949) described the diabetogenic action of
anterior pituitary growth hormone in cats.
• Repeated administration of GH induces an intensively diabetic
condition with all the symptoms of diabetes including ketonemia and
ketonuria in intact adult dog.
• Rats of any age subjected to similar treatment do not become
diabetic but grow faster and show hypertrophy of pancreatic cells.

15. Glucocorticoids induced diabetes
• Ingle (1941) described hyperglycemia and glucosuria in forced fed rats with
cortisone.
• Hausberger and Ramsay in 1953 showed that experimental diabetes by
cortisone can be induced in guinea pigs and rabbits without forced feeding.
(Abelove and Paschkis,1954)
• In the rat, adrenal cortex, stimulated by corticotrophin, has the capacity to
secrete amounts of steroids which induce diabetes (Ingle et al 1946)

16. Virus induced diabetes
They may produce DM by:
• Infecting and destroying β cells in pancreas.
• A less infecting or cytologic variant producing a comparable damage by
eliciting immune auto-reactivity to β cells.
• Viruses that produce systemic effects, not directly affecting β cells.
Viruses used for inducing DM:
• Coxsackie B4
• Encephalomyocarditis (EMC-D and M variants)
• Mengo-2T
• Reoviruses
• Lymphocytic Choriomengitis Virus (LMCV) Armstrong variant

17. Surgery induced diabetes
• Induction of DM can be achieved through surgical removal of all or
part of pancreas.
• Depending on the amount intact pancreatic cells, DM may range in
duration from a few days to several months.

18. Disadvantages:
• Surgical removal of pancreas also causes loss of α and δ cells in addition to β
cells.
• There is also loss of pancreatic enzymes for digestion.
• The total resection is difficult to achieve and the severity of DM is strain
specific.

19. Insulin antibody induced diabetes
Bovine insulin, dissolved in acidified water (pH 3.0) is incorporated in water-oil emulsion
based on complete Freund’s adjuvant or a mixture of paraffin oil and lanolin.
Guinea pigs (300-400 g) are given 1 mg insulin SC divided doses and at monthly intervals.
They are bled by cardiac puncture 2 weeks after 2nd and subsequent dose.
IV injection 0.25-1.0 ml guinea pig anti-insulin serum to rats

20. Genetically diabetic animals
In recent years, various animals have been shown to exhibit diabetes
mellitus spontaneously
• The ob mutation in mice resulted in leptin deficiency
• The fat mutation in mouse results in biologically inactive carboxypeptidase E,
which processes the prohormone conversion of POMC into MSH-α, which
activates the hypothalamic MC4 receptor.
• Agouti yellow mouse exhibit ubiquitous expression of Agouti protein which
represents an antagonist of hypothalamic MC4 receptor.

21. Spontaneous rat models Spontaneous Mouse models
BB rat KK mouse
WBN/Kob rat KK-Ay mouse
Cohen diabetic rat NOD mouse
Goto Kakizaki rat Diabetic Db/Db mouse
Zucker fatty rat Welleseley mouse
Wdf/Ta-Fa rat Diabetes obesity syndrome in CBA/Ca mice

22. Transgenic animals
1. Skeletal muscle and insulin resistance/MIRKO mice model
2. Adipose tissue and insulin resistance/FIRKO mouse
3. Mouse with specific disruption of IR gene in β cells which show a selective loss of insulin
secretion in response to glucose and progressive impairment of glucose tolerance
4. The liver specific insulin receptor knock-out (LIRKO) mouse
5. CNS specific disruption of IR gene (NIRKO)
6. IRS-1 deficient mouse which show genetically determined insulin resistance
7. PPAR γ inactivation

23. STZ induced NIDDM
• Neonatal rats of Sprague-Dawley strain are taken
• Treated with STZ 80-100 mg/kg IP at birth or within 5 days of birth
which leads to severe β cell destruction, deficiency in pancreatic
insulin levels and rise in plasma glucose.
• In contrast to adults, the β cells in neonates partially regenerate.
• Following an initial spike in plasma glucose, these rats become
normoglycemic by 3 weeks.

24. Other chemical methods
• For NIDDM in rabbits - adrenaline (0.1 mg/kg SC).
• The hyperglycemia is seen at 1 hour and lasts for 4 hours. Oral
hypoglycemic agents can be screened by this method.
• Other chemicals are 8-hydroxy quinoline, biphenyl thio carbazine,
EDTA (partially depancreatized rats), thiazides, chlorthiazide,
hydrochlorthiazide, diazoxide and furosemide.

25. Genetic models for NIDDM
Monogenic models of obesity and NIDDM
1. Yellow mouse (the Agouti mouse)
2. Obese and diabetic mouse
3. Tubby mouse
4. Fat mouse
5. Zucker diabetic fatty rat
6. Koletsky and JCR: LA-Corpulent rats
Polygenic models of obesity and NIDDM
1. New Zealand obese mouse
2. Japanese KK mouse
3. Nagoya – Shibata – Yasuda mouse
4. PBB/Ld mouse
5. Otsuka-Long-Evans-Tokushima Fatty rat
6. Goto-Kakisaki rat
7. Chinese Hamster
8. Djungarian (Siberian) hamster
9. South African Hamster

26. Animal models for NIDDM with unknown hereditary and
environmental component
1. Sand rat
2. Spiny mice
3. Tuco-Tuco
Polygenic animal models produced by hybrid crosses
1. BSB (C57BL/6J * Mus Spretus)
2. AKR/J * SWR/J model
3. GK crosses: GK * Fisher 344 strain, GK * non-diabetic Brown Norway

27. Transgenic and knock-out animals
Genes manipulated to cause
insulin resistance:
Genes manipulated to cause
defective insulin secretion:
Genes that increase body fat
Insulin receptor GLUT-2 Knock out of uncoupling
proteins
Insulin receptor substrate 1 and 2 Glucokinase Knock out β3 adrenergic receptors
Glucose transporters Hepatic nuclear factors
Hexokinase II Islet amyloid polypeptide
TNF-α
Fatty acid binding protein 2
RAS associated with diabetes

28. Measurement of glucose lowering / anti-diabetic activity
• In vivo methods
Hypoglycemic effects
Euglycemic clamp technique
Hypoglycemic seizures in mice
Effects of insulin sensitizer drug
Effects of thiazolidinediones on PPAR γ
Anti-diabetic effects of liver X receptor agonist
• In vitro methods
Isolated pancreas of rat
Isolated rat diaphragm

29. Blood glucose lowering effects in rabbit
Primary screening model for screening of blood glucose lowering
compounds
Animals - 4-5 mixed breed rabbits of either sex with weight 3-4.5 kg
Conditions required:
for insulin – food withheld overnight,
for sulfonylureas and other blood glucose lowering agents – animals
are on a normal diet prior to experiment.
Animals should be in a special restraining box with free access to rabbit
ears

30. Procedure
• Oral blood glucose lowering agents are applied by gavage in 1 ml/kg of
0.4% starch suspension or IV in solution.
• Several doses are given to different groups. One control group receives
vehicle only.
• Blood is withdrawn immediately before and 1,2,3,4,5,24,48 and 72 hours
after treatment.
• For time-response curves values are also measured after 8,12,16 and 20
hours. Blood glucose is determined in 10 μl blood samples with the
hexokinase enzyme method.

31. Evaluation
• Average blood sugar values are plotted versus time for each dosage.
• Percentage data related to the value before the experiment are calculated.
• Mean effects at a time interval are calculated using the trapezoidal rule.
• The values of the experimental group are compared statistically with t-test
or the Wilcoxon test for each time interval with those of control group.
• Dose dependencies and relative activities are determined by means of
linear regression analysis.

32. Blood glucose lowering effects in rats
Procedure
• Male Wistar rats of 180-240 gm are kept on a standard diet (Altromin
1324).
• Groups of 4-7 non-fasted animals are treated orally or IP with various
doses of test compounds suspended in 0.4% starch suspension. One
control group receives vehicle only.
• Blood is withdrawn from the tip of the tail immediately before and
1,2,3,5 and 24 hours after administration of test compound.

33. Evaluation
• Average blood sugar values are plotted versus time for each dosage.
• Percentage data related to the value before the experiment are
calculated.

34. Modifications:
• Studies in glucose loaded rats – in this method, glucose 1 gm/kg body
weight is given following the test compound either orally or SC
• Studies in streptozocin diabetic rats – in this method, diabetes is
induced with streptozocin which leads to fall in plasma insulin levels.
Compounds which release insulin from islets as the sole hypoglycemic
mechanism of action are not effective in these animals

35. Blood glucose lowering in mice
• Eneroth and Ahlund (1968)
Animals – non-fasting mice of the same strain and sex, having body
masses not more than ± 2 gm
Procedure:
• Assigned at random to 4 equal groups of not less than 10 animals.
• Two dilutions of a solution of the preparation to be examined and 2
dilutions of the reference solution are prepared

36. • In a preliminary experiment, concentrations of 0.02 IU and 0.10 IU are
tested.
• Each of the prepared solutions (0.1 ml/10 g body weight) is given SC
to one group of mice according to a randomized block design
• 2.5 hours later, each solution is given to a second group of mice
following a twin crossover design.
• At 30 minutes after each injection, a sample of 50 μl of blood is taken
from orbital venous sinus.

37. Evaluation
The potency is calculated by statistical methods for twin crossover
studies.

38. Blood glucose lowering effects in dogs
Animals - male Beagle dogs 15-20 kg are kept on standard diet (Erka
mixed feed 8500) and food withdrawn 18 hours prior to administration
of test compound.
Procedure
• Test compound is given orally or IV at various doses. Control animals
receive vehicle only.
• Blood is collected at different time intervals upto 48 hours.
• Blood glucose is estimated with hexokinase enzyme method
(Glucoquant test kit) and insulin with an immunological method
(Riagnost kit).

39. Evaluation
• Average blood sugar values are plotted versus time for each dosage.
• Percentage data related to value before the experiment are
calculated.
• Mean effects over a time period are calculated using the trapezoidal
rule. Other statistical evaluation is similar to how it is done for
rabbits.
• Plasma insulin levels are also plotted versus time and compared with
control values.

40. Modifications:
1. Studies in pancreatectomized dogs
 Dogs are pancreatectomized 2-3 years prior to the study and kept on dry feed
and pancreatic enzymes.
 A day before the study, a shorter acting insulin is given.
 The test drug is given as oral suspension in tap water, whereas the control
animals receive only tap water.
 Blood glucose is estimated before and upto 6 hours after treatment at hourly
intervals.
2. Studies in Alloxan diabetic dogs

41. In other species
• Male guinea pigs (250-380 gm) can also be used. Blood is withdrawn
from puncture of ear veins before and 1,3,5 hours after test
compound administration.
• Genetically obese and diabetic yellow KK mice have also been used by
some for evaluation of hypoglycemic activity of potential anti-diabetic
drugs.

42. Euglycemic clamp technique
“gold standard”
Purpose and rationale:
• This is a useful method of quantifying in vivo insulin sensitivity in
humans (DeFronzo et al 1979).
• Variable glucose infusion is given to maintain euglycemia during
insulin infusion.

43. Animals: male Wistar rats weighing 150-200 gm
Procedure:
catheters inserted into a jugular vein – for blood collection
femoral vein – insulin and glucose infusion
Physiological hyperinsulinemia - 6 mU/kg/min
Maximal hyperinsulinemia - 30 mU/kg/min
The blood glucose concentrations are estimated from samples
collected at 5 minutes intervals during 90 minute clamp test.

44. • The glucose infusion rate is adjusted so as to maintain blood glucose at
basal level during the test.
• The final glucose infusion rate is calculated from the amount of glucose
infused for the last 30 minutes (from minute 60-90 from the start of the
test) in which the blood glucose is maintained in a steady state.
• The steady state plasma insulin concentration is calculated from the insulin
concentration at 60 and 90 minutes after the start of the clamp.
• Free fatty acid concentrations are also measured at the start and end of the
clamp to measure the FFA suppression rate.

45. Evaluation
• When steady state plasma insulin is maintained at submaximal
concentration by this technique, the glucose infusion rate and glucose
metabolic clearance rate value are considered to reflect the state of
receptor binding levels in peripheral tissue as an index of insulin
sensitivity.
• Under maximal hyperinsulinemia, these values are thought to reflect
the state of enzymes and glucose transport system activated after
binding to receptors, indicating mainly insulin responsiveness.

46. Hypoglycemic seizures in mice
The biological assay of insulin using hypoglycemic seizures in mice has
been suggested in 1923 by Fraser.
In most Pharmacopoeias, the biological assays have been replaced by
chemical methods now.
Animals – 96 mice of either sex weighing 20 ± 5 gm randomly
distributed into 4 groups

47. Procedure:
The mice are deprived of food 2-20 hours immediately preceding the test.
Insulin solutions standard and test are prepared by diluting 30 and 60
mIU/ml in NS with pH 2.5 and 0.5 ml/20 g injected SC.
The mice are kept at a uniform temperature, between 29-35⁰C in transparent
containers within an air incubator with a transparent front.
The mice are observed for 1.5 hours and no of mice that are dead, convulse
or lie still for more than 2-3s when placed on backs are noted.

48. Evaluation:
The percentage of mice of each group showing the above mentioned
symptoms is calculated and relative potency of test solution is
calculated using a 2+2 point assay.
Modifications:
Suggested by Young and Lewis in 1947, also done by Vogel in 1964
The rotating drum method

49. Effects of insulin sensitizer drugs
• These compounds do not lower blood glucose in normal animals
In vivo studies
• Various animals are used viz. ob/ob mice, db/db mice, Chinese hamsters,
Zucker fatty rat, yellow KK mice, obese Beagle dogs, streptozocin diabetic
rats.
In vitro studies
• Reversal of cAMP induced post insulin receptor resistance in Rat
adipocytes,
• Cultured hepatoma cells – increase glycogen synthase I activity,
• Stimulation of fructose-2,6-BP in rat hepatocytes,
• Prevention of glucose induced insulin resistance of insulin receptor in rat 1
fibroblasts,

50. Effects of thiazolidinediones on PPAR γ
• Thiazolidinediones are potent activators of PPAR γ.
• Thiazolidinediones were shown to reduce the circulating resistin
levels and increase adiponectin in mice.
• Berger et al in 1996 found a correlation of anti-diabetic actions of
thiazolidinediones in db/db mice with the conformational change in
PPAR γ.

51. Various methods in demonstrating anti-diabetic property of
thiazolidinediones are,
1. Binding assay
2. Transactivation assay
3. Lipogenesis assay
4. Protein digestion assay

52. Anti-diabetic effects of liver X receptor agonists
• Nuclear receptors LXRα and LXRβ are sensors of cholesterol
metabolism and lipid biosynthesis.
• LXRα is a target gene of PPAR γ.
• Insulin also induces LXRα in hepatocytes and increases the expression
of lipogenic enzymes and suppresses key gluconeogenesis enzymes.
• Treatment of diabetic rodents with LXRα agonist was shown to reduce
plasma glucose.

53. Procedure:
• Obese insulin-resistant female Zucker (fa/fa) rats, 10 weeks of age,
are orally gavaged for 9 days with either vehicle or the LXRα agonist.
• Eight hours after the last dose, animals are fasted overnight and on
the following morning subjected to an oral GTT.
• Blood was obtained via the tail vein at time 0 and times 15, 30, 60,
and 120 min after an oral glucose challenge.
• Plasma glucose and insulin levels are analyzed on all samples, and the
results are expressed as the product of glucose AUC and insulin AUC.

54. Isolated rat pancreas
Purpose
Used for studying the effect of drug on Insulin, glucagon, somatostatin
secretion.
Procedure
• Adult Wistar rat (150-200 gm) are fed ad libitum .
• Pancreas are removed under Pentobarbital (50 mg/kg IP)
• Through a portal vein canula Krebs-ringer bicarbonate buffer with 2%
bovine albumin & 5.5 mmol/l glucose is perfuse at rate of 1.75 ml/min. at
pressure 100mmHg.
• Perfusate is collected every min. for 30 min. after first 5 min. test drug
added till 15 min. next 16-30 min glucose is perfused.

55. Evaluation
Insulin, Glycogen, Somatostatin are estimated using
Radioimmunoassay.
The effect of test drug on hormone secretion of pancreas in response
to elevated glucose level is compared with the control.

56. Isolated rat diaphragm
Purpose:
Determination of Insulin based on the stimulation of glucose uptake by
the isolated diaphragm from rat .
Procedure:
Animal - Male Sprague Dawley (70-100 gm)
Animal sacrificed during anesthesia and diaphragm are carefully removed,
spread out and divided into two equal pieces
Hemi diaphragm are incubated in Krebs buffer solution with carbogen with
5µM glucose, insulin or compound to be tested.

57. After 30 min hemidiaphragm are blotted on tissue, grounded on porcelin
mortar pestle chilled with liquid nitrogen
After 4 hour at -200C Sample centrifuged for 10 min.
Evaluation
The concentration dependent of glucose uptake and conversion into
glycogen and concentration of insulin or insulin mimetic compound are
determined.

58. Conclusion
• For an animal model to have relevance to study of type 2 DM in
humans, either the characteristics of the animal models should
mimick the pathophysiology and natural history of diabetes or it
should develop complications of diabetes similar to that in humans.
• No single animal model encompasses all these characteristics and
there are various models in use that mimick various conditions as
seen in humans.