Diabetes mellitus is a metabolic disorder characterized by increased blood glucose level associated with discharge of glucose in urine. There are two major types of diabetes mellitus, that is, insulin-dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM).
SCREENING METHODS FOR ANTIDIABETIC AGENTS
Diabetes mellitus is a metabolic disorder characterized by
increased blood glucose level associated with discharge of glucose in urine.
There are two major types of diabetes mellitus, that is, insulin-dependent
diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM).
Insulin-dependent diabetes mellitus, also called type 1 diabetes, occurs due to
complete loss of pancreatic β-islet cells and hence there is
insulin deficiency. Noninsulin-dependent diabetes mellitus, also called as type
2 diabetes, is due to insulin resistance. Insulin resistance is developed due
to defects at the receptor level or insulin signalling at the postreceptor
level. This defect may be in the effector cells such as the skeletal muscle,
the adipose tissue etc., or in the β-islet cells. A large number of
drugs including herbs and minerals with suspected antidiabetic activity have
been successfully tested in the laboratory. The various animal models to screen
antidiabetic activity are listed in this section.
Models for Insulin-Dependent Diabetes Mellitus
Alloxan-induced
diabetes
Alloxan is a cyclic urea compound which induces per-manent
diabetes. It is a highly reactive molecule which produces free radical damage
to β-islet cells and causes cell death. Alloxan at a dose level
of 100 mg/kg in rats produces diabetes. In rabbits, a dose level of 150 mg/kg
infused through a marginal ear vein produces diabetes in 70% of the animals.
Albino rats of either sex weighing 150–200 g are injected
with a single dose of alloxan monohydrate (100 mg/kg body weight) dissolved in
normal saline (0.9%) by intraperitoneal route. The animals are kept for 48 h
during which food and water is allowed ad libitum. The blood glucose level shows the triphasic response with
hyperglycaemia for 1 h followed by hypoglycaemia that lasts for 6 h and stable
hyperglycaemia after 48 h. The animals showing fasting blood glucose level
above 140 mg/dl after 48 h of alloxan administration are considered diabetic.
Drug samples to be screened are administered orally for a period of six weeks.
After six weeks of treatment, blood samples are collected from 8 h fasting
animals through a caudal vein. Serum is separated by cooling centrifuge (2–4°C)
at 3000 r.p.m. for 10 min. The serum glucose level is estimated by glucose oxidase-peroxidase
method (GOD-POD kit) using an autoanalyser.
Streptozotocin-induced
diabetes
Streptozotocin is a broad-spectrum antibiotic which causes β-islet cells damage by free radical generation.
Strepto-zotocin induces diabetes in almost all species of animals excluding
rabbits and guinea pigs. The diabetogenic dose of Streptozotocin varies with
species. In mice, the dose level is 200 mg/kg through i.p. and in beagle dogs
15 mg/ kg through i.v. for three days. Adult albino rats of either sex weighing
150–200 g are injected with Streptozotocin (60 mg/kg body weight) prepared in
citrated buffer (pH 4.5) solution by i.p. route. The citrated buffer is
prepared by mixing 53.9 parts of 0.1 M citric acid and 46.1 parts of 0.2 M
disodium hydrogen orthophosphate and finally adjusted to a pH of 4.5. The blood
glucose level shows the same triphasic response as seen in alloxan-treated
animals. Animals showing fasting blood glucose level above 140 mg/ dl after 48
h of Streptozotocin administration are considered diabetic. Drug samples to be
screened are administered orally for a period of six weeks. After six weeks of
treat-ment, blood samples are collected from 8 h fasted animals through a
caudal vein. Serum is separated by cooling centrifuge (2–4°C) at 3000 r.p.m.
for 10 min. The serum glucose level is estimated by glucose oxidase-peroxidase
method (GOD-POD kit) using an autoanalyser.
Virus-induced
diabetes
Viruses are one of the etiological agents for IDDM. They
produce diabetes mellitus by infecting and destroying β-islet cells of pancreas. Various human viruses used for
inducing diabetes include: RNA picornovirus, coxsackie B4 (CB-4), and
encephalomyocarditis (EMC-D).
Six- to eight-week-old mice are inoculated by 0.1 ml of 1:50
dilution of D-variant encephalomyocarditis (EMC) through i.p. route. The 0.1 ml
of the above dilution contains 50 PFU (plaque-forming units) of EMC virus.
Mortality due to this concentration of virus is approximately 10–20%. A
less-infecting variant produces a comparable damage by eliciting autoimmune
reactivity to the β-islet cells. Infected animals are
considered hyperglycaemic if their nonfasting levels exceed by 250 mg/dl the
levels of uninfected animals of the same strain. Drug samples to be screened
are adminis-tered orally for a period of six weeks. After six weeks of drug
treatment, blood glucose estimation is done to determine the antidiabetic
activity.
Insulin
antibodies-induced diabetes
A transient diabetic syndrome can be induced by inject-ing
guinea pigs with antiinsulin serum. It neutralises the endogenous insulin with
insulin antibodies. Diabetes per-sists as long as the antibodies are capable of
reacting with the insulin remaining in circulation.
Preparation of Antibody: Bovine insulin, dissolved in acidified water (pH 3.0) at a dose
of 1 mg is injected to guinea pigs weighing 300–400 g. Antiinsulin sera is
col-lected after two weeks of antigenic challenge.
Adult albino rats are injected with 0.25–1.0 ml of guinea
pig antiinsulin serum. Insulin antibodies induce a dose-dependent increase of
blood glucose level up to 300 mg/ dl. Slow rate intravenous infusion or an
intra-peritoneal injection prolongs the effect for more than a few hours.
However, large doses and prolonged administration are accompanied by ketonemia.
The drug sample to be screened is administered by a suitable route, and blood
glucose level is analysed to determine the activity.
Hormone-induced
diabetes
Dexamethasone: Dexamethasone is a steroid
possessing immunosupression
action which causes an autoimmune reaction in the islets and produces type 1
diabetes.
Adult rats weighing 150–200 g are injected with
dex-amethasone at a dose level of 2 5 mg/kg body weight by i.p. twice a day.
The repeated injection of the same dose level is carried out for a period of
20–30 days resulting in IDDM. The sample to be screened is administered through
a suitable route, and blood glucose level is analysed to determine the
activity.
Genetic Models
Nonobese
diabetic mouse
Nonobese diabetic mouse (NOD) is a model of IDDM.
Hypoinsulinemia is developed which is caused by autoim-mune destruction of
pancreatic β-islet cells in association with
autoantibody production.
Mice are breed at the laboratory by sib-mating over 20
generations. After 20 generations of sib-mating, spontane-ous development of
IDDM in mice is obtained. Diabetes develops abruptly between 100 and 200 days
of age. Weight loss, polyurea, and severe glucosuria are common. The animals
are treated with the drug sample to be screened. Blood sample is analysed for
glucose level to determine activity.
Bio-breeding
rat
Diabetes is inherited as an autosomal recessive trait and
develops with equal frequency and severity among males and females. Insulin
deficiency and insulitis are due to autoimmune destruction of pancreatic β-islet cells. Spon-taneous diabetes is diagnosed in a
noninbreed but closed out-breed colony of rats at bio-breeding laboratories.
Rats are breed at the laboratory by sib-mating over 20
generations. After 20 generations of sib-mating, spontaneous development of
IDDM in rats is obtained. The onset of clinical diabetes is sudden and occurs
at about 60–20 days of age. The clinical presentation of diabetes in the
bio-breeding
A rat is similar to that of its human counterpart. Marked
hyperglycaemia, glycosuria, and weight loss occur within a day of onset and are
associated with decreased plasma insulin, that if untreated will result in
ketoacidosis. The animals are treated with the drug sample to be screened for a
required period of time. The blood sample is analysed for glucose level to
determine activity.
Models for NIDDM
Streptozotocin-induced
neonatal model for NIDDM
Streptozotocin causes severe pancreatic β-cells destruction, accompanied by a decrease in pancreatic
insulin stores and a rise in plasma glucose level. In contrast to adult rats,
the treated neonates partially regenerate and become normo-glycaemic by three
weeks of age. In the next few weeks, the β-cell number increase, mainly from
the proliferation of cells derived from ducts, leads to hyperinsulinemia, and
shows symptoms similar to insulin resistance.
Neonatal rats are treated with Streptozotocin (90 mg/kg body
weight) prepared in citrated buffer (pH 4.5) by i.p. at birth or within the
first 5 days following birth. After six weeks, the rats develop symptoms
similar to NIDDM. Rats showing fasting blood glucose level above 140 mg/dl are
considered diabetic. Further steps are similar to that of the alloxan-induced
model. The drug sample to be screened is administered by a suitable route, and blood
glucose level is analysed to determine the activity.
Other Chemically Induced NIDDM Models
Adrenaline-induced
acute hyperglycaemia
Adrenaline is a counter-regulatory hormone to insulin. It
increases the rate of glycogenolysis and glucose level in blood causing acute
hyperglycaemia.
Adult albino rats are injected at a dose level of 0.1 mg/ kg
through s.c. route. The dose produces peak hyperglycaemic effect at 1 h and
lasts up to 4 h. The drug sample to be analysed is administered through a
suitable route, and blood glucose level is determined. Oral hypoglycaemic
agents can be screened by this method.
Chelating Agents
Dithizone-induced
diabetes
Organic agents react with zinc in the islets of Langerhans
causing the destruction of β-islet cells, producing diabetes.
Severe necrosis and disintegration of β-cells (insulin-producing cells)
were observed, while α-cells (cells which produce
glucoagon which maintains the glucose level in the blood) remain unaltered.
Compounds such as dithizone, EDTA, 8-hydroxy quinoline are used to induce
spontane-ous type 2 diabetes in experimental animals. Dithizone at a dose level
of 40–100 mg/kg (i.v.) produces type 2 diabetes in mice, cats, rabbits, and
golden hamsters.
Adult rabbits weighing 1.8–2 kg are divided into two groups
of six animals each. An exactly weighed amount of dithizone is dissolved in
dilute ammoniacal solution (0.2 to 0.5%). The solution is warmed to 60 -70°C
for 10 min to aid solubility of dithizone. Dithizone injection at a dose level
of 50–200 mg/kg produces triphasic glycaemic reaction. Initial hyperglycaemia
is observed after 2 h and normoglycaemia after 8 h, which persists for up to 24
h. Permanent hyperglycaemia is observed after 24–72 h. The drug sample to be
analysed is administered through a suit-able route, and the blood glucose level
is determined.
Models for Insulin Sensitivity and Insulin-like
Activity
Euglycaemic
clamp technique
This method has proved to be a useful technique of
quantifying in vivo insulin sensitivity. A variable glucose infusion is
delivered to maintain euglycemia during insulin infusion. The net glucose
uptake is quantified, and the sensitivity of the body tissue to insulin is
determined.
Adult albino rats weighing 150–200 g are fasted over-night
and anaesthetized with pentobarbital (40 mg/kg i.p.). Catheters are inserted
into a jugular vein and a femoral vein for blood collection and insulin and
glucose infusion, respectively. To evaluate the insulin action under
physiological hyperinsulinemia (steady-state plasma insulin concentration
during the clamp test is around 100 μU/dl) and maximal hyperinsulinemia,
two insulin infusion rates, that is, 6 and 30 mU/kg/min are used. The blood
glucose concentrations are determined from samples collected at 5 min intervals
during the 90-min clamp test. The glucose infusion rate is adjusted so as to
maintain basal level. The glucose metabolic clearance rate is calculated by
dividing the glucose infusion rate by the steady-state blood glucose
concentration. The drug sample to be analysed is administered through a
suitable route, and the blood glucose level is determined.
Assay
for insulin and insulin-like activity
This assay involves comparing two standard solutions of
insulin with the test drug for its insulin-like activity.
Four groups of six rabbits weighing at least 1.8 kg are
used. Two standard solutions of insulin containing one unit and two units
respectively and two dilutions of sample whose potency is being examined are
prepared. As diluent, a solution of 0.1 to 0.25% w/v of either m-cresol or
phenol and 1.4 to 1.8 w/v of glycerol acidified with hydrochloric acid to a pH
between 2.5 and 3.5 is used. Each of the prepared solution (0.5 ml) is
injected subcutaneously. After 1 h and 2.5 h of each injection, a suitable blood
sample is taken from the ear vein of each rabbit, and the blood sugar level is
determined preferably by glucose oxidase method.
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