Diabetes mellitus is chronic metabolic disorder charecterised by hyperglycemia, glycosuria, hyperlipamea,negative nitrogen balance and sometimes ketonaemia caused by absolute or relative deficiency of insulin.

Types of DiabetesType 1:Insulin dependent diabetes mellitus Or juvenile-onset diabetes, charecterised by absolute deficiency of insulin resulting from autoimmune destruction of -cells.β

Treatment:INSULIN(exogenously injected)Type 2:Non insulin dependent diabetes mellitus Or Maturity onset diabetes, caused by both insulin resistance and impaired insulin secretion by β-cells.

Treatment:Oral hypoglycemic drugs,weight reduction, exercise & dietary modification.

ANTIDIABETIC AGENTS:includes Insulin preparation, Oral hypoglycemic agents & peptide mimetics used to improve glycemic control.

Insulin:It is an endocrine hormone secreted by the β-cells of pancreas. It is 51 aminoacid protein composed of 2 polypeptide chain. An A-chain of 21 AA & B-chain of 30 AA. Two interchain disulfide bond is found between Cys-6 & Cys-11 of A chain.Insulin is derived from 86 AA precursors known as

proinsulin.

1)Human insulin are prepared from enzymatic conversion of terminal AAof procine insulin(Novolin) or by means of R-DNA technology(Humulin).

2)5 synthetic analogues-Lispro insulin(Humalog), Insulin aspart(Novolog), Insulin glulisine(Apidra), Insulin glargine(lantus) & Insulin detamir(Levamir)

Insulin preparationsSHORT ACTING: 1)Lispro insulin

2)Insulin aspart 3)Insulin glulisine 4)Regular insulinINTERMEDIATE ACTING: 1)Isophane insulin Or Neutral protamine hagedorn 2)Insulin zinc suspension Or LenteLONG ACTING: 1)Insulin detamir 2)Insulin glargine 3)Protamine Zinc insulin Oral antidiabetic agents1)Sulfonyl ureas :1st generation-Tolbutamide, Tolazamide, Acetohexamide, Chlorpropamide 2nd generation-Gliburide, Glipizide, Glibenclamide.2)Biguanides:Metformin, phenformin3)Meglitinide derivative:Repaglinide, Nateglinide4)Thiazolidinediones:Rosiglitazone, Pioglitazone5)α-glucosidase inhibitor:Acarbose, Meglitol

Peptide mimetics1)Exenatide 2)Pramlintide

MECHANISM OF ACTIONSulfonly ureas:1)Stimulates insulin release from β-cells of the pancreas by blocking ATP sensitive K+ channel resulting in depolarization, then leads to Calcium influx.2)Reduction in serum glucagon,3)Increased binding of insulin to target tissues & receptors.

Biguanides:1)Dcreasing hepatic glucose output, largely by inhibiting gluconeogenesis,2)Enhances insulin mediated glucose disposal in muscle & fat,3)Retarding intestinal absorption of glucose4)Promotes peripheral glucose utilization by enhancing anaerobic glycolysis.

Meglitinide derivative :Similar to sulfonylurease.

Thiazolidinedione:Stimulates PPAR-γ which in turn promotes transcription of genes involved in insulin signaling.

α-glucosidase inhibitor:It delays carbohydrate absorption, reducing increased blood glucose level.

Methods to induce experimental diabetes mellitus

INVIVO METHODS

Models for IDDM

1. Pancreatectomy in dogs2. Alloxan induced diabetes3. Streptozotocin induced diabetes4. Hormone induced diabetes5. insulin antibodies induced diabeted6. Virus induced Diabetes7.Genetic models- The NOD mouse

The BB rat

Models for NIDDM

Neonatal STZ Model of NIDDM (Chemically Induced Diabetes)

Genetically diabetic animals

Spontaneous Diabetic rats

1. BB rat2. WBN/KOB rat3. Cohen diabetic rat4. GOTO-KAKIZAKI rat5. Zucker Fatty rat6. OLETF rat7. Obese SHR rat

Spontaneous Diabetic mouse

8. KK mouse9. NOD mouse10. Diabetes mouse11. CBA/ CA mice12. Wellesley mouse

Other species with inherited diabetic symptoms

1. Sand rat (Psammomys obesus)2. Spiny mouse (Acomys cahirinus)3. African hamster (Mystromys Albicaudatus)4. TUCO-TUCO (Ctenomis talarum)5. Black apes (Macaca nigra)

Methods of screening

Assay of insulin and insulin like activity

1. Hypoglycemic effectsa. Blood sugar lowering effects in rabbitsb. Blood sugar determinations in mice

2. Insulin target cells/tissue of ratsa. Lipogenesis assayb. Insulin signaling in the liver

c. Insulin receptor bindingAssay of other glucose regulating peptide hormones

3. Bioassay of glucagon4. Receptor binding of Amylin

Blood Glucose lowering activity of antidiabetic drugs

1. Euglycemic clamp technique2. Effects of insulin sensitizer drugs

Activity on isolated organs, cells, and membranes

1. Perfusion of isolated rat pancreatic islets2. Interaction of sulfonylureas with β-cell membranes3. Isolated diaphragm4. Isolated hepatocytes

Inhibition of polysaccharide degrading enzymes

1. Inhibiton of amaylase invitro2. Evaluation of α-glucosidase inhibitors using inverted sac technique

Effects on secondary diabetic symptoms

1. Aldose reductase inhibition invitro2. Effect of nerve blood flow3. Effect of streptozotocin induced cataracts

INVITRO METHODS Assays of insulin and of insulin-like activity Insulin target cells/tissues of rats Epididymal fat pad of rats Blood glucose lowering activity of antidiabetic drugs Effects of thiazolidinediones on peroxisome proliferator-activated receptor-

Screening of Antidiabetic agentsIt can be performed by Invivo & Invitro method1)Invivo Method:

Models for IDDM1.Chemically indused diabetes:Most commonly used animal model of diabetes.Chemical agents which produce diabetes can be classified into 3 categories & include agents that A)Specifically damage β-cellB)Cause temporary inhibition of insulin productionC)Diminish the metabolic efficacy of insulin in target tissues

®Alloxan-induced diabetes®Streptozotocin-induced diabetes

2. Hormone induced diabetes3. Insulin antibodies induced diabetes4.Diabetes induced by viral agents5.Surgically induced diabetes6. Genetic model

Models for NIDDM1. Neonatal STZ model for NIDDM2. Other chemically induced3. Genetic models of NIDDMMonogenic model of obesity & NIDDMYellow mouse(Agouti mouse)Obese & diabetic mouseTubby mouseFat mouseZukker diabetic fatty ratKoletsky & JCRPolygenic models of obesity & NIDDMNew Zealand obese(NZO) mouseJapanese KK mouse Nagaya-Shibata-Yasuda(NSY) mousePBB/Ld mouseOtsuka-Long-Evans-Jokustima Fatty Rat(OLEFT)Goto-Kakisaki ratChinese HamsterDjungarain HamsterSouth African Hamster

Alloxan-Induced Diabetes :Alloxan is cyclic urea analog, was agent in this category which reported to produce permanent diabetes in animals.

Mechanism of action: it is not very clear,1)Alloxan is highly reactive molecule that is readily reduced to diuleric acid, which is then auto-oxidized back to alloxan resulting in production of free radicals. These free radicals damage DNA of β-cells & causes cell death.2)Its ability to react with protein SH groups, especially the membrane proteins like glukokinase on the β-cells, finally resulting in cell necrosis.

Body weight Dose with route resultsRabbits 2-3.5kg infused via the ear vein

with 150 mg/kg 70% of the

animals to

alloxan monohydrate (5.0 g/100 ml, pH 4.5) for 10 min

become hyperglycemic and uricosuric.

The rest of the animals either die or are only temporarily hyperglycemic

Rats(Wistar or Sprague-Dawley strain)

150–200 g injected subcutaneously with 100–175 mg/ kg alloxan

---------------

Beagle dogs 15–20 kg I. injected intravenously with 60 mg/kg alloxan

II. receive daily 1 000 ml 5% glucose solution with 10 IU Regular insulin for one week

III. canned food ad libitum.

IV. a single daily dose of 28 IU insulin is administered subcutaneously

The blood glucose level shows triphasic change 1) a rise at 2 hour2)hypoglycemic phase at 8hr 3)increase at 24hr due to depletion of β-cells of insulin.

Drawbacks:

1) High mortality in rats.2)Causes ketosis in animal due to free fattyacid generation.3)Diabetes induced is reversible.4)Some species like guinea pigs are resistant to its diabetogenic action.

Alloxan has completely replaced by STZ because of these drawbacks.

Streptozotocin induced diabetesStreptozotocin (STZ, 2-deoxy-2-(3-(methyl-3- nitrosoureido)-D-glucopyranose) is synthesized by Streptomyces achromogenes and is used to induce both insulin-dependent and non-insulin-dependent diabetes mellitus (IDDM and NIDDM, respectively).

Mechanism of action

1) By process of methylation.

2)STZ is a nitric oxide (NO) donor and NO was found to bring about the destruction of pancreaticislet cells, it was proposed that this molecule contributes to STZ-induced DNA damage

3)STZ was found to generate reactive oxygen species, which also contribute to DNA fragmentation and evoke other deleterious changes in the cells.

4)NO and reactive oxygen species can act separately or form the highly toxic peroxynitrate (ONOO

.

PROCEDURE Male Wistar rats weighing 150–220 g :fed with a standard diet :- injected with 60 mg/kg

streptozotocin intravenously or i.p. Mice (175-200mg/kg i.p. or i.v.) Dogs (15mg/kg for 3 days) As with alloxan, three phases of blood glucose changes are observed.

A. Initially, blood glucose is increased, reaching values of 150–200 mg% after 3 h. B. Six–eight h after streptozotocin, the serum insulin values are increased up to 4

times, resulting in a hypoglycemic phase C. followed by persistent hyperglycemia.

After the dose of 60 mg/kg i.v., symptoms occur already after 24–48 h with hyperglycemia up to 800 mg%, glucosuria and ketonemia.

Histologically, the beta-cells are degranulated or even necrotic.

Advantage1)Greater selectivity towards β-cells2)Lower mortality rate3)Longer or irreversible diabetes induction

DisadvantageGuinea pigs & rabbits are resistant to its diabetogenic action.

Pancreatectomy in dogsGENERAL CONSIDERATIONSVon Mehring and Minkowski (1890) noted polyuria, polydipsia, polyphagia, and severe glucosuria fol-lowing removal of the pancreas in dogs. The final proof for the existence of a hormone in the pancreas was furnished by Banting and Best (1922) who could reduce the elevated blood sugar levels in pancreatectomized dogs by injection of extracts of the pancreatic glands.PURPOSE AND RATIONALEThe technique of complete pancreatectomy in the dog has been used by many scientists as a relevant animal model for diabetes mellitus in man.

PROCEDURE

Male Beagle dogs weighing 12–16 kg are used. The animal is anesthetized with an intravenous injection of 50 mg/kg pentobarbital

sodium and placed on its back. After removal of the fur and disinfection of the skin a midline incision is made from the

xyphoid process reaching well below the umbilicus. Bleeding vessels are ligated and the abdomen is entered through the linea alba. The falciform ligament is carefully removed and the vessels ligated. A self-retaining retractor is applied. By passing the right hand along the stomach to the pylorus, the duodenum with the head

of the pancreas is brought into the operating field. First, the mesentery at the unicate process is cut and the process itself is dissected free. The glandular tissue is peeled off from the inferior pancreatico-duodenal artery and vein. The vessels themselves are carefully preserved. Along a line of cleavage which exists between the pancreas, the pancreaticoduodenal

vessels and the duodenal wall, the pancreas is separated from the duodenum and from the carefully preserved pancreaticoduodenal vessels.

The small vessels to the pancreas are ligated. The dissection is carried out from both sides of the duodenum. In the area of the accessory pancreatic duct the glandular tissue being attached very

firmly has to be carefully removed in order to leave no residual pancreatic tissue behind. The surgical field is checked once more for pancreatic remnants. The concavity of the duodenum and its mesentery is approximated by a few silk stitches

and the omentum is wrapped around the duodenum. Retroperitoneal injection : 5 ml 1% procaine solution is given to prevent intussusception

of the gut. 250 000 IU penicillin G : in saline solution are instilled into the peritoneal cavity. The abdominal wall and the subcutaneous layer are closed by sutures and finally the skin

is sutured with continuous everting mattress stitches. After the operation, the animal receives:- via a jugular vein catheter for 3–4 days the

following treatment: I. 1000 ml 10% glucose solution with 10 IU human insulin Regular

II. 3 ml 24% (sulfadioxin/trimethoprim) solutionIII. 2 ml 50% metamizol and 400 IU secretin.

On the third day, the animal is offered milk. After the animal has passed the first milk feces, it is given daily dry food together with a

preparation of pancreatic enzymes Insulin is substituted with a single daily subcutaneous dose of 34 IU Retard-Insulin

Vitamin D3 is given every three months as a intramuscular injection of 1 ml Vigantol forte.

Hormone induced diabetesGrowth hormone induced diabetes

a) the diabetogenic action of pure anterior pituitary growth hormone in cats. b) In intact adult dogs and cats the repeated administration of growth hormone induces an

intensively diabetic condition with all symptoms of diabetes including severe ketonuria and ketonemia.

c) Rats of any age subjected to a similar treatment do not become diabetic but grow faster and show striking hypertrophy of the pancreatic islets.

Example:-

Hormone induced diabetes

Principle: - Dexamethasone: is a steroid possessing immunosuppresion action, which causes an autoimmune reaction in the islets and produces type 1 diabetes.

Procedure: - Adult rats weighing 150-200 gm are injected with dexamethasone at a dose level of 2-5

mg/kg body weight i.p twice a day. Repeated injection of 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 root. Blood glucose is analysed to determine the activity

Genetically diabetic animalsinsulin secretion and increased serum lipids. Insulin secretory capacity is lost concomitant with amyloid infiltration into the islets of Langerhans. Secondary manifestations are atherosclerosis, thickened basement membranes of muscle capillaries, and cataracts. The genetic predisposition in these monkeys is exacerbated by changes in diet and environment.

General considerations Several animal species, mostly rodents, were described to exhibit spontaneously diabetes

mellitus on a hereditary basis. Up to now at least 6 genetically diabetic animal models exhibit defects in the leptin

pathway: The ob mutation in the mouse resulted in leptin deficiency.

BB RAT

OBESE SHR RAT

KK MOUSE

ZDF RAT

The db mutation in the mouse and the cp and fa mutations in the rat are different mutations of the leptin receptor gene.

The fat mutation in the mouse results in a biologically inactive carboxipeptidase E, which processes the prohormone conversion of POMC into α-MSH, which activates the hypothalamic MC4 receptor. Finally the Agouti yellow (y) mouse exhibit an ubiquitous expression of the Agouti protein which represents an antagonist of the hypothalamic MC4 receptor.

Spontaneously diabetic rats

BB RAT

The BB rat (Bio Breeding (BB) rat) is a model of spontaneous diabetes associated with insulin deficiency and insulitis due to autoimmune destruction of pancreatic beta cells. Diabetes is inherited as an autosomal recessive trait and develops with equal frequency and severity among males and females. The onset of clinical diabetes is sudden, and occurs at about 60–120 days of age. Within several days, diabetic animals are severely hyperglycemic,hypoinsulinemic,and ketotic unless insulin treatment is instituted.

WBN/KOB RAT

TUCO-TUCOCBA mice

Spontaneous hyperglycemia, glucosuria and glucose intolerance have been observed in aged males of an inbred Wistar strain, named the WBN/Kob rat

OBESE SHR RATAfter several generations of selective inbreeding, these obese SHR exhibited obesity, hypertension, and hyperlipidemia. In addition, some rats developed hyperglycemia and glucosuria associated with giant hyperplasia of pancreatic islets.

Spontaneously diabetic mice

KK MOUSEThe animals were moderately obese and showed polyphagia and polyuria. Mice at the ageof seven months or older showed glucosuria and blood sugar levels up to 320 mg%. The pancreatic insulin content was increased, but histologically degranulationof the beta-cells and hypertrophy of the islets were found. Sections of the liver showed a reduction of glycogen and an increase in lipid content.

KK-AY MOUSEcarrying the yellow obese gene (Ay). These mice develop marked adiposity and diabeticsymptoms in comparison with their littermates, black KK mice. Blood glucose and circulating insulin levels as well as HbA1c levels were increased progressively from 5 weeks of age. Degranulation and glycogen infiltration of B cells were followed by hypertrophyand central cavitation of islets. Lipogenesis by liver and adipose tissue were increased. Insulin sensitivity of adipose tissue was more remarkably reduced than in black KK mice to its complete loss at 16 weeks of age.

NOD MOUSEThe NOD mouse strain was established by inbreeding diabetic CTS mice derived originally from the JCLICR strain. Like the BB rat, the NOD mouse is a model of insulin dependent diabetes mellitus and develops hypoinsulinemia secondary to autoimmune destruction of pancreatic β cells in association with insulitis and auto antibody production. NOD mice develop diabetesabruptly between 100 and 200 days of age, as well as rapid weight loss, polyuria, polydipsia, and severe glucosuria.

Chinese hamsterthe Chinesehamster (Cricetulus griseus). Blood sugar levels of diabetic hamsters were elevated from a normal of 110 mg% up to 600 mg%. Severe polyuria, glucosuria, ketonuria, and proteinuria were observed. The diabetic condition could be improved by administration of insulin, and oral antidiabetic drugs were effective in mildly diabetic hamsters.

Other species with inherited diabetic symptoms

SAND RATThe sand rat (Psammomys obesus) lives in the desert regions of North Africa and the Near East. In the laboratory the animals develop diabetic symptoms when fed Purina laboratory chow instead of an all vegetable diet The diabetic syndrome in the sand rat usually develops within 2–3 months with variations in severity between the animals.n Severely hyperglycemic animals die prematurely from ketosis. Initially, the pancreatic islets appear normal. In the intermediate stage of the disease, degranulation of pancreatic β cells is observed. This is followed by β cell degeneration and necrosis with resultant insulinopenia and ketonuria.

SPINY MOUSEThe spiny mouse (Acomys cahirinus) is a small rodent living in the semi-desert areas of the Eastern Mediterranean

MACACA NIGRAAbnormal signs include hyperglycemia, decreased clearance of glucose, in intravenous tolerance tests, reduced

ASSAY OF INSULIN AND INSULIN LIKE ACTIVITYHypoglycemic effects

Blood sugar lowering effect in rabbits

PURPOSE AND RATIONALEA biological assay of insulin preparations in comparison with a stable standard using the blood sugar lowering effect in rabbits

PROCEDURE Four groups of at least 6 randomly distributed rabbits weighing at least 1.8 kg are kept in

the laboratory and maintained on a uniform diet for not less than one week before use in the assay.

About 24 h before the test each rabbit is provided with an amount of food that will beconsumed within 6 h.

The same feeding schedule is followed before each test day. During the test all food and water is withheld until the final blood sample has been taken.

The rabbits are placed into comfortable restraining cages to avoid undue excitement. Immediately before use two solutions of the standard preparation are made, containing 1 unit and 2 units of insulin per ml, respectively, and two dilutions of the preparation being examined which, if the assumption of potency is correct, contain amounts of insulin equivalent to those in the dilutions of the standard preparation.

As diluent, a solution is used of 0.1–0.25% w/v of either m-cresol or phenol and 1.4 to 1.8 w/v of glycerol being acidified with hydrochloric acid to a pH between 2.5 and 3.5

Each of the prepared solutions is injected subcutaneously to one group of rabbits, using the same volume, which should usually be between 0.3 and 0.5 ml for each rabbit, the injections being carried out according to a randomized block design.

Preferably on the following day, but in any case not more than 1 week later, each solution is administered to a second group of rabbits following a twin crossover design.

One hour and 2.5 h after each injection a suitable blood sample is taken from the ear vein of each rabbit

Blood sugar is determined by a suitable method, preferably using glucose oxidase.

BLOOD GLUCOSE LOWERING ACTIVITY OF ANTIDIABETIC DRUGS

Blood glucose lowering activity in rabbits

PURPOSE AND RATIONALEThe rabbit has been used since many years for standardization of insulin .Therefore, it hasbeen chosen as primary screening model for screening of blood glucose lowering compounds as well as for establishing time-response curves and relative activities

PROCEDUREGroups of 4–5 mixed breed rabbits (e.g. Hoe:BASK, SPFWiga) of either sex weighing 3.0–4.5 kg are used. For insulin evaluation, food is withheld overnight. For evaluation of sulfonylureas and other blood glucose lowering agents the animals are on a normal diet (e.g. Era mixed feed 8 300) prior to the experiment. The animals are gently placed into special restraining boxes allowing free access to the rabbit’s ears. Oral blood glucose lowering substances are applied by gavage in 1 ml/kg of 0.4% starch suspension or intravenously in solution. Several doses are given to different groups. One control group receives the vehicle only. By puncture of the ear veins, blood is withdrawn immediately before and 1, 2, 3, 4, 5, 24, 48, and 72 h after treatment. For time-response curves values are also measured after 8, 12, 16, and 20 h. Blood glucoseis determined in 10 μl blood samples with the hexokinase enzyme method

EVALUATIONAverage blood sugar values are plotted versus time for each dosage. Besides the original values, 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 experimentalgroup are compared statistically with the t-test or the WILCOXON test for each time interval with those of the control group.

Euglycemic clamp technique

PURPOSE AND RATIONALEThe euglycemic glucose clamp technique has provided a useful method of quantifying in vivo insulin sensitivity in humans .In this technique a variable glucose infusion is delivered to maintain euglycemia during insulin infusion. Whole-body tissue sensitivity to insulin, as determined by net glucose uptake, can be quantitated under conditions of near steady state glucose and insulin levels.

PROCEDURE Male Wistar rats weighing 150–200 g are fasted overnight and anesthetized with

pentobarbital (40 mg/kg, i.p.). Catheters are inserted into a jugular vein and a femoral vein for blood collections and insulin and glucose infusion, respectively.

To evaluate the insulin action under physiological hyperinsulinemia (steady state plasma insulin concentration during the clamp test around 100 μU/dl), and maximal hyperinsulinemia (under which maximal insulin action may appear) two insulin infusion rates, 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 the blood glucose at its basal level during the clamp test.

The final glucose infusion rate is calculated from the amount of glucose infused for the last 30 min (from 60 to 90 min after start of the clamp) in which the blood glucose levels are in a steady state.

The glucose metabolic clearance rate is obtained by dividing the glucose infusion rate by the steady state blood glucose concentration.

The steady state plasma insulin concentration is calculated from the insulin concentrations at 60 and 90 min after the start of the clamp.

At the start and end of the euglycemic clamp test, free fatty acid concentration is also determined and the free fatty acid suppression rate is calculated.

EVALUATIONAll values are analyzed by one-way ANOVA. When the steady state plasma insulin is maintained at submaximal concentration by the euglycemic clamp technique, the glucose infusion rate and glucose metabolic clearance rate value are considered to reflect the state of receptor binding levels in the peripheral tissue as an index for insulin sensitivity. Under maximal hyperinsulinemiam these values are thought to reflect the state of the enzymes and glucose transport system activated after the binding to receptors, indicating mainly insulin responsiveness.

INHIBITION OF POLYSACCHARIDE DEGRADING ENZYMES

Evaluation of alpha- glucosidase inhibitors using the everted sac technique

PURPOSE AND RATIONALEThe everted sac technique allows to study the effects of intestinal enzymes on substrates in an incubation vial.

PROCEDURE Male rats weighing 120–140 g are sacrificed and the small intestines removed by cutting

across the upper end of the duodenal junction.

The intestine is stripped of the mesentery and the entire intestinal content is rinsed with cold saline solution.

The intestine is divided into 7 to 8 cm-segments and turned inside out using a Pasteur pipette.

The everted intestine is ligated at one end with a cotton thread and a second ligature is placed loosely around the opposite end ready for tying. A 1 ml syringe with Krebs-Henseleit-buffer is introduced into the lumen sac.

The end of the sac is ligated and placed in a 25 ml Erlenmeyer flask containing 6 ml of 1% starch, dissolved in Krebs-Henseleit-buffer, with or without various concentrations of the α-amylase inhibitor. Pork α-amylase (4 000 U/g starch) is also included in the 6 ml starch solution.

Following gassing with 95% O2/5% CO2, the flask is tightly capped and incubated in a shaking bath at 37 °C for 120 min.

The reaction is terminated by the addition of 10 μl 1 N HCl. At the end of the incubation period, the sac is removed from the flask and the inner fluid is collected by cutting one end of the sac.

The final volumes of the solute in the serosal and the mucosal side and the level of glucose liberation are measured.

EVALUATIONGlucose liberated in the presence of various concentrations of the α-amylase inhibitor is expressed as percentage of glucose found without the inhibitor. Doseresponse curves can be drawn plotting percent inhibition versus concentration of the inhibitor

INVITRO METHODSISOLATED PANCREASE OF RAT:

The in vitro perfusion of isolated pancreas helps us in studying the effect of drug on insulin, glucagon & somatostatin secretion without interference from other organ changes.

PROCEDURE1)Animals used are male wistar rats weighing 200 to 250g. the pancreas is removed under pentobarbital(50mg/kg i.p.)anaesthesia.2)Once the pancreas is removed, through portal vein cannula, Krebs-Ringer bicarbonate with 2% bovine albumin and 5.5mmol/L glucose is preferred at a rate of 1.75ml/min.3)The temperature of the prefusion fluid is kept at 37.5°C and the pressure at which it is perfused is about 100mmHg.The perfusate is collected every minute for 30 minutes.4)After the first 5 min of perfusion, test compound is added till the 15 th min (conc. of test compound being 0.05-.05mM). From 16th min till 30th min, glucose of 5.5mM and 16.6mM is perfused.5)The sample is collected and stored at ₋20°C. Hormones insulin, glucagons and somatostatin are estimated radioimmunologically. At least 3 experiments per concentration are performed.6)The effect of test compound whether it increases or decreases the secreted hormones of pancreas in response to elevated glucose level is compared with the control.

Insulin target cells/tissues of ratsEpididymal fat pad of rats

Insulin-like activity can be measured by the uptake of glucose into fat cells. Adipose tissue from the epididymal fat pad of rats has been found to be very suitable.

Early studies determined the difference of glucose concentration in the medium after incubation of pieces of epididymal rat adipose tissue or measured oxygen consumption in

In experiments with radiolabelled 14C glucose, the 14CO2 is trapped and counted The principle used was measurement of 14CO2-production from 1-14C-labelled glucose by

epi-didymal fat pads from the rat. The presence of factors other than insulin in this test was proven by the persistence of

serum insulin- like activity after pancreatectomy used the manometric measurement of the net gas exchange of rat adipose tissue to quantitate small amounts of insulin.

tested insulin-like activity on adipose tissue of various species, rat, mouse and guinea pig. The epididymal fat pad assay, originally developed as bioassay of insulin-like activity in

serum samples, can be used for measuring activity of synthetic insulin derivatives as well as for evaluation of peripheral insulin- like effects of compounds such as sulfonylureas.

Several modifications (isolated fat cells, primary cultured adipocytes, 3T3 adipocytes) broadened the value of this assay.

UNIVERSITY QUESTIONS1)Enumerate the different methods used to screen anti-diabetic activity. Describe in detail STZ induced diabetes. -20M2)Write the different assay used to screen the anti diabetic drugs. -20M

REFERENCES:

Gerhard Vogel H, Wolfgang HV, Bernward AS, Jurgen S, Gunter M, Wolfgang EV.Drug discovery and evaluation. 2nd ed.Berlin,Germany: Springer;2002. p.947-1051

Drug screening method, 1st edition by S.K. Gupta

Rang HP, Dale MM ,Ritter JM. Pharmacology. 6th ed. Edinburgh (England):Harcourt Publishers Limited ;2008.p380-392.

Bertram.G.Katzung. Basic & Clinical Pharmacology.10th ed. Lange medical book: McGraw-Hill Comp;2007.p.683-705.

www.google.images/diabetics

nsulin-like activity can be measured by the uptake of glucose into fat cells. Adipose tissue from the epididymal fat pad of rats has been found to be very suitable.

Early studies determined the difference of glucose concentration in the medium after incubation of pieces of epididymal rat adipose tissue or measured oxygen consumption in

In experiments with radiolabelled glucose, the 14CO

The principle used was measurement of 14CO2-production from glucose by epi-didymal fat pads from the rat.

The presence of factors other than insulin in this test was proven by the persistence of serum insulin- like activity after pancreatectomy used the manometric measurement of the net gas exchange of

rat adipose tissue to quantitate small amounts of insulin.

tested insulin-like activity on adipose tissue of various species, rat, mouse and guinea pig.

The epididymal fat pad assay, originally developed as bioassay of insulin-like activity in serum samples, can be used for measuring activity of synthetic insulin derivatives as well as for evaluation of peripheral insulin- like effects of compounds such as sulfonylureas.

Several modifications (isolated fat cells, primary cultured adipocytes, 3T3 adipocytes) broadened the value of this assay.

Leptin is produced by interacts with six types of receptor (LepRa–LepRf). LepRb is the only receptor isoform that contains active intracellular signaling domains. This receptor is present in a number of hypothalamic nucleiventromedial nucleus

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