BIOLOGICAL EVALUATION OF HYPOGLYCEMIC AGENTS

About Authors:
Kataria Sahil, Aggarwal Ashutosh, Middha Akanksha, Sandhu Premjeet
Seth G. L. Bihani S.D. College of Technical Education,
Institute of Pharmaceutical Sciences and Drug Research,
Sri Ganganagar, Rajasthan,
INDIA

INTRODUCTION
Diabetes is a chronic (lifelong) disease marked by high levels of sugar in the blood.
There are three major types of diabetes:
• Type 1 diabetes is usually diagnosed in childhood. Many patients are diagnosed when they are older than age 20. In this disease, the body makes little or no insulin. Daily injections of insulin are needed. The exact cause is unknown. Genetics, viruses, and autoimmune problems may play a role.

• Type 2 diabetes is far more common than type 1. It makes up most of diabetes cases. It usually occurs in adulthood, but young people are increasingly being diagnosed with this disease. The pancreas does not make enough insulin to keep blood glucose levels normal, often because the body does not respond well to insulin. Many people with type 2 diabetes do not know they have it, although it is a serious condition. Type 2 diabetes is becoming more common due to increasing obesity and failure to exercise.

• Gestational diabetes is high blood glucose that develops at any time during pregnancy in a woman who does not have diabetes. Women who have gestational diabetes are at high risk of type 2 diabetes and cardiovascular disease later in life.
There are many risk factors for type 2 diabetes, including:
•Age over 45 years
• A parent, brother, or sister with diabetes
•Gestational diabetes or delivering a baby weighing more than 9 pounds
•Heart disease
•High blood cholesterol level
•Obesity
•Not getting enough exercise
•Polycystic ovary disease (in women)
•Previous impaired glucose tolerance
•Some ethnic groups (particularly African Americans, Native Americans, Asians, Pacific Islanders, and Hispanic Americans)

Reference ID: PHARMATUTOR-ART-1189

Symptoms
High blood levels of glucose can cause several problems, including:
•   Blurry vision
•   Excessive thirst
•   Fatigue
•   Frequent urination
•   Hunger
•   Weight loss
However, because type 2 diabetes develops slowly, some people with high blood sugar experience no symptoms at all.
Symptoms of type 1 diabetes:
•   Fatigue
•   Increased thirst
•   Increased urination
•   Nausea
•   Vomiting
•   Weight loss in spite of increased appetite
Patients with type 1 diabetes usually develop symptoms over a short period of time. The condition is often diagnosed in an emergency setting.
Symptoms of type 2 diabetes:
•   Blurred vision
•   Fatigue
•   Increased appetite
•   Increased thirst
•   Increased urination

Signs and tests
A urine analysis may be used to look for glucose and ketones from the breakdown of fat. However, a urine test alone does not diagnose diabetes.
The following blood tests are used to diagnose diabetes:
•   Fasting blood glucose level -- diabetes is diagnosed if higher than 126 mg/dL on two occasions. Levels between 100 and 126 mg/dL are referred to as impaired fasting glucose or prediabetes. These levels are considered to be risk factors for type 2 diabetes and its complications.
•   Hemoglobin A1c test -- this test has been used in the past to help patients monitor how well they are controlling their blood glucose levels. In 2010, the American Diabetes Association recommended that the test be used as another option for diagnosing diabetes and identifying pre-diabetes. Levels indicate:
•   Normal: Less than 5.7%
•   Pre-diabetes: Between 5.7% - 6.4%
•   Diabetes: 6.5% or higher
•   Oral glucose tolerance test -- diabetes is diagnosed if glucose level is higher than 200 mg/dL after 2 hours. (This test is used more for type 2 diabetes.)
•   Random (non-fasting) blood glucose level -- diabetes is suspected if higher than 200 mg/dL and accompanied by the classic diabetes symptoms of increased thirst, urination, and fatigue. (This test must be confirmed with a fasting blood glucose test.)
Treatment
The immediate goals are to treat diabetic ketoacidosis and high blood glucose levels. Because type 1 diabetes can start suddenly and have severe symptoms, people who are newly diagnosed may need to go to the hospital.
The long-term goals of treatment are to:
•   Prolong life
•   Reduce symptoms
•   Prevent diabetes-related complications such as blindness, heart disease, kidney failure, and amputation of limbs
These goals are accomplished through:
•   Blood pressure and cholesterol control
•   Careful self testing of blood glucose levels
•   Education
•   Exercise
•   Foot care
•   Meal planning and weight control
•   Medication or insulin use
There is no cure for diabetes. Treatment involves medicines, diet, and exercise to control blood sugar and prevent symptoms⁸

Methods to induce experimental diabetes mellitus

Pancreatectomy in dogs
Dysfunction of the visceral tract has been considered for a long time to be the cause of diabetes mellitus. Bomskov (1910) reported severe diabetic symptoms in dogs after cannulation of the ductus lymphaticus. This observation, however, could not be confirmed in later experiments . The technique was similar to that described for ligation of the thoracic duct in dogs. Von Mehring and Minkowski (1890) noted polyuria, polydipsia, polyphagia, and severe glucosuria following 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 reduced the elevated blood sugar levels in pancreatectomized dogs by injection of extracts of the pancreatic glands. The technique of complete pancreatectomy in the dog has been used by many scientists as a relevant animal model for diabetes mellitus in man¹⁸

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 pancreatic duct is cleaned, doubly ligated and cut between the ligatures. The dissection proceeds until one encounters a small lobe containing the main pancreatic duct. The glandular tissue adheres here firmly to the duodenum. Blunt dissection and ligation of the vessels is followed by ligation of the pancreatic duct. By pulling on the pylorus and the stomach, the pyloric and the splenic parts of the pancreas are delivered into the wound. The duodenal part is placed back into the abdominal cavity. The mesentery of the body and tail of the pancreas is cut with scissors. The small vessels are doubly ligated and cut. The pancreatic tissue is bluntly dissected from the splenic vessels. The pancreatic branches of the splenic vessels are doubly ligated and cut. Working in direction from the spleen to the pylorus, the pyloric part of the pancreas is the last one to be dissected. Finally, all pancreatic tissue is removed. 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 of 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:
1000 ml 10% glucose solution with 10 IU human insulin Regular, 3 ml 24% Borgal (sulfadioxin/trimethoprim) solution, 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.¹⁴

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Alloxan induced diabetes
Hyperglycemia and glucosuria after administration of alloxan has been described in several species, such as in dogs,in rabbits,in rats and in other species .Guinea pigs have been found to be resistant. Dosage and treatment regimen have been elaborated for the most frequently used species. In most species a triphasic time course is observed: an initial rise of glucose is followed by a decrease, probably due to depletion of islets from insulin, again followed by a sustained increase of blood glucose¹⁸

PROCEDURE
Rabbits weighing 2.0 to 3.5 kg are infused via the ear vein with 150 mg/kg alloxan monohydrate (5.0 g/100 ml, pH 4.5) for 10 min resulting in 70% of the animals to become hyperglycemic and uricosuric. The rest of the animals either die or are only temporarily hyperglycaemic ³

Rats of Wistar or Sprague-Dawley strain weighing 150–200 g are injected subcutaneously with 100–175 mg/ kg alloxan ⁹

Male Beagle dogs weighing 15–20 kg are injected intravenously with 60 mg/kg alloxan. Subsequently, the animals receive daily 1 000 ml 5% glucose solution with 10 IU Regular insulin for one week and canned food ad libitum. Thereafter, a single daily dose of 28 IU insulin is administered subcutaneously ¹⁶

Streptozotocin induced diabetes
Rakieten et al. (1963) reported the diabetogenic activity of the antibiotic streptozotocin. The compound turned out to be specifically cytotoxic to beta-cells of the pancreas.

PROCEDURE
Male Wistar rats weighing 150–220 g fed with a standard diet are injected with 60 mg/kg streptozotocin intravenously. As with alloxan, three phases of blood glucose changes are observed. Initially, blood glucose is increased, reaching values of 150–200 mg% after 3 h. Six–eight hr after streptozotocin, the serum insulin values are increased up to 4 times, resulting in a hypoglycemic phase which is followed by persistent hyperglycemia. Severity and onset of diabetic symptoms depend on the dose of streptozotocin. 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. A steady state is reached after 10–14 days allowing to use the animals for pharmacological tests.¹⁵

Genetically diabetic animals
Several animal species, mostly rodents, were described to exhibit spontaneously diabetes mellitus on a hereditary basis. These findings were highly appreciated with the expectation to get more insight into the pathogenesis of diabetes in humans. During the last few years since the discovery of leptin and its downstream signal transduction cascade tremendous new insight of the genetics of diabetic and obese animal disease models derived. 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. 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 receptor¹⁸

Spontaneously diabetic rats
The occurrence of spontaneous diabetes has been reported in several strains of rats:

· BB RAT The BB rat (Bio Breeding (BB) rat) is a model of spontaneous diabetes associated with insulin deficiency andinsulitis 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 hyperglycaemic ,hypoinsulinemic, and ketotic unless insulin treatment is instituted.

· WBN/KOB RAT Spontaneous hyperglycemia, glucosuria and glucose intolerance have been observed in aged males of an inbred Wistar strain, named the WBN/Kob rat.These animals exhibit impaired glucose tolerance and glucosuria at 21 weeks of age. Obvious decreases in the number and size of islets are found already after 12 weeks of age. Fibrinous exudation and degeneration of pancreatic tissue are observed in the exocrine part, mainly around degenerated islets and pancreatic ducts in 16 weeks old males. These rats develop demyelinating, predominantly motor neuropathy, later accompanied by axonal changes

· GOTO-KAKIZAKI RAT Non-obese, insulin-resistant Goto-Kakizaki (GK) rats are a highly inbred strain of Wistar rats that spontaneously developed type II diabetes. Defects in glucose stimulated insulin secretion, peripheral insulin resistance, and hyperinsulinemia are seen as early as 2 to 4 weeks after birth. Impaired skeletal muscle glycogen synthase activation by insulin was observed, accompanied by chronic activation of diacylglycerol-sensitive protein kinase C.

· ZUCKER-FATTY RAT The Zucker-fatty rat is a classic model of hyperinsulinemic obesity. (Zucker 1965). Obesity is due to a simple autosomal recessive (fa) gene and develops at an early age. Obese Zucker rats manifest mild glucose intolerance, hyperinsulinemia, and peripheral insulin resistance similar to human NIDDM. However, their blood sugar level is usually normal throughout life ¹⁷

Spontaneously diabetic mice

· KK MOUSE Nakamura (1962, 1967) reported on a diabetic strain of the KK-mouse. The animals were moderately obese and showed polyphagia and polyuria. Mice at the age of seven months or older showed glucosuria and blood sugar levels up to 320 mg%. The pancreatic insulin content was increased, but histologically degranulation of 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 MOUSE Iwatsuka et al. (1970) reported on yellow KK mice (also named KK-Ay mice), carrying the yellow obese gene (Ay). These mice develop marked adiposity and diabetic symptoms 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 hypertrophy and 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. Renal involvement is uniquely marked by early onset and rapid development of glomerular basement membrane thickening .
KK-Ay mice can be used to demonstrate the extrapancreatic action of antidiabetic drugs, such as glimepiride, a novel sulfonylurea.

· NOD MOUSE The 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 diabetes abruptly between 100 and 200 days of age, as well as rapid weight loss, polyuria, polydipsia, and severe glucosuria. Without insulin treatment, they do not survive for more than one month and usually die from ketosis. The onset of diabetes can be prevented by an immunmodulating drug or by a soluble interleukin-1 receptor. Hutchings and Cooke (1995) compared the protective effects afforded by intravenous administration of bovine or ovine insulin to young NOD mice. Bergerot et al. (1997) reported that feeding small amounts (2–20 μg) of human insulin conjugated to cholera toxin B subunit can effectively suppress β-cell destruction and clinical diabetes in adult NOD mice.
Insulin-dependent diabetes mellitus in NOD Mice is the result of a CD4+ and CD8+ T cell-dependent auto-immune process directed against the pancreatic β-cells

· DIABETES MOUSE (DB/DB) The diabetes db/db mouse strain is derived from an autosomal recessive mutation having occurred spontaneously in mice of the C57BL/KsJ strain which was identified as a mutation in the leptin receptor gene. On this basis, the diabetes mouse (C57BL/6J db/db) consistently develops a severe diabetic syndrome similar to that found in the C57BL/KsJ ob/ob mouse, characterized by early onset of hyperinsulinemia, and hyperglycemia up to 20 to 25 mmol/l. The db/db mouse, in contrast to the ob/ob mouse, develops significant nephropathy. Mutations on the leptin receptor result in an obese phenotype identical to that of ob mice. C57BL/KsJ ob/ob mice are phenotypically the same as other strains of db mice. The leptin receptor (Ob-R) gene encodes 5 alternatively spliced forms, Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd. In the C57BL/KsJ ob/ob mouse strain, the Ob-Rb transcript contains an insert with a premature stop codon as a result if abnormal splicing⁴

· TRANSGENIC ANIMALS
Transgenic Mice Schaefer et al. (1994) described a transgenic mouse model of chronic hyperglycemia. The extracellular ligand binding domain of the human insulin receptor can be expressed as a stable soluble protein that is efficiently secreted and binds insulin with high affinity. Expression under the mouse transferrin promoter of a transgene encoding a secreted derivative of the human insulin receptor in transgenic mice results in the accumulation of this high-affinity insulin-binding protein in the plasma. Alterations of glucose homeostasis are induced including post absorptive hyperglycemia concomitant with increased hepatic glucose production and hyperinsulinemia.
Palmiter et al. (1987) developed a method of deleting specific cell lineages that entailed microinjection into fertilized eggs of a chimeric gene in which a cellspecific enhancer/promoter is used to drive the expression of a toxic gene product. Microinjection of a construct in which the elastase I promoter /enhancer is fused to a gene for diphtheria A polypeptide resulted in birth of mice lacking a normal pancreas because of the expression of the toxin in pancreatic acinar cells. A small pancreatic rudiment, containing islet and ductlike cells, was observed in some of the transgenic mice.
    Aichele et al. (1994) used a synthetic peptide corresponding to an immunodominant epitope of lymphocytic choriomeningitis virus glycoprotein (LCMV GP) to prime or to tolerize CD8+ T cells in vivo. Peptidespecific tolerance was then examined in transgenic mice expressing LCMV GP in the β islet cells of the pancreas. These mice developed CD8+ T cell-mediated diabetes within 8–14 days after LCMV infection. Specific peptide-induced tolerance prevented autoimmune destruction of β islet cells and diabetes in this transgenic mouse model.
Oldstone et al. (1991) showed that virus infection triggers insulin-dependent diabetes mellitus in a transgenic mouse model.
Ablation of tolerance and induction of diabetes by virus infection in viral antigen transgenic mice was reported by Ohashi et al. (1991).
    Von Herrath et al. (1994) investigated how virus induces a rapid or slow onset insulin-dependent diabetes mellitus in two distinct transgenic mouse models.
    Von Herrath et al. (1995) evaluated the role of the costimulatory molecule B7-1 in overcoming peripheral ignorance in transgenic mice which expressed the glycoprotein or nucleoprotein of lymphocytic choriomeningitis virus as the self-antigen in pancreatic β-cells. Von Herrath and Holz (1997) reported that pathological changes in the islet milieu precede infiltration of islets and destruction in β-cells by autoreactive lymphocytes in a transgenic model of virus-induced IDDM. RIP-LCMV transgenic mice that express the viral glycoprotein or nucleoprotein from lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter (RIP) in pancreatic β-cells develop autoimmune diabetes after infection with LCMV. Upregulation of MHC class II molecules associated with the attraction/ activation of antigen presenting cells to the islets occurs as soon as 2 days after LCMV inoculation of transgenic mice, clearly before CD4+ and CD8+ lymphocytes are found entering the cells. Possibilities of treatment of virus-induced autoimmune diabetes were discussed ¹⁸
    Hebert et al. (1996) created transgenic mice to study the influence of overexpression of glutamine:fructose- 6-phosphate amidotransferase on insulin resistance.
    Moritani et al. (1996) reported the prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes as a gene therapy model for autoimmune diabetes.
Birk et al. (1996) generated transgenic NOD mice carrying a murine Hsp60 transgene driven by the H°2Eα class II promoter in order to examine the hypothesis of a pathogenic role for self-reactive cells against the stress protein Hsp60 in autoimmune destruction of pancreatic cells in the diabetes of NOD mice.
    Terauchi et al. (1997) reported development of noninsulin- dependent diabetes mellitus in double knockout mice with disruption of insulin receptor substrate-1 and β-cell glucokinase genes.
Insulin resistance and hyperinsulinemia in insulin receptor substrate-1 knockout mice was discussed by Jenkins and Storlien (1997).
Ueki et al. (2000) could restore insulin-sensitivity in IRS-1-deficient mice by adenovirus mediated gene therapy.
     Brüning et al. (1997) developed a polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles. Mice double heterozygous for null alleles in the insulin receptor and insulin receptor substrate-1 genes exhibit the expected ~50% reduction in expression of these two proteins, but a synergism at the level of insulin resistance with 5- to 50-fold elevated plasma insulin levels and comparable levels of β-cell hyperplasia.
    Withers et al. (1998) showed that disruption of IRS-2 impairs both peripheral insulin signaling and pancreatic β-cell function. IRS-2-deficient mice showed progressive deterioration of glucose homeostasis because of insulin resistance in the liver and skeletal muscle and a lack of β-cell compensation for this insulin resistance.
   Kulkarni et al. (1999) reported that in mice a tissue specific knockout of the insulin receptor in pancreatic β cells creates an insulin secretory defect similar to that in type 2 diabetes²

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Assays of insulin and of insulin-like activity

Hypoglycemic effects

Blood sugar lowering effect in rabbits

A biological assay of insulin preparations in comparison with a stable standard using the blood sugar lowering effect in rabbits has been proposed already in 1925 by Harrison et al. The biological assay of insulin using the blood sugar lowering effect in rabbits has been until recently the official assay in several pharmacopoeias, such as European Pharmacopoeia,;; British Pharmacopoeia; United States Pharmacopoeia and The National Formulary
The rabbit blood glucose bioassay as well as the mouse convulsion assay and the mouse glucose assay were used for establishing international standards for highly purified human, porcine and bovine insulin
In several pharmacopoeias, the biological assays have been replaced by chemical methods (British Pharmacopoeia 1999; European Pharmacopoeia, 3rd Edition 1997; but the rabbit blood sugar method is still valid in the United States Pharmacopeia USP 24, 2000.

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 be consumed 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 diluents , 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 oxidase⁵

Hypoglycemic seizures in mice
The biological assay of insulin using hypoglycaemic seizures in mice has been suggested already in 1923 by Fraser. The biological standardization of insulin using the mouse convulsion method has been published in detail by the Health Organisation of the League of Nations in 1926 and has been until recently the official assay in several pharmacopoeias, such as European Pharmacopeia, British Pharmacopoeia 1988.
In most pharmacopoeias, the biological assays have been replaced by chemical methods (British Pharmacopoeia 1999; European Pharmacopoeia, 3rd Edition 1997).

PROCEDURE
Ninety-six mice of either sex (but not of mixed sexes) weighing 20 ±5 g are randomly distributed into 4 groups. The mice are deprived of food 2–20 h immediately preceding the test. Solutions of the insulin standard and of the test preparation containing 30 and 60 milli Units/ml are prepared by diluting the original solution with 0.9% NaCl solution, pH 2.5. 0.5 ml/20 g mouse of these solutions are injected subcutaneously. The mice are kept at a uniform temperature, between 29 and 35 °C, in transparent containers within an air incubator with a transparent front. The mice are observed for 1.5 h and the number of mice is recorded that are dead, convulse or lie still for more than 2 or 3 s when placed on their backs.

EVALUATION
The percentage of mice of each group showing the mentioned symptoms is calculated and the relative potency of the test solution calculated using a 2 + 2 point assay¹¹

Blood sugar determinations in mice
Eneroth and Ahlund recommended a twin crossover method for bio-assay of insulin using blood glucose levels in mice instead of hypoglycaemic seizures giving more precise results. This test was induced into the British Pharmacopoeia 1980 and continued up to 1988.

PROCEDURE
Non-fasting mice of the same strain and sex are used having body masses such that the difference between the heaviest and lightest mouse is not more than 2 g. The mice are assigned at random to four equal groups of not less than 10 animals. Two dilutions of a solution of the substance or of the preparation to be examined and 2 dilutions of the reference solution are prepared using as diluent 0.9% NaCl solution adjusted to pH 2.5 with 0.1 N hydrochloric acid and containing a suitable protein carrier. 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 injected subcutaneously to one group of mice according to a randomized block design. Not less than 2.5 h later, each solution is administered to a second group of mice following a twin crossover design. Exactly 30 min after each injection, a sample of 50 μl of blood is taken from the orbital venous sinus of each mouse. Blood glucose concentration is determined by a suitable method

EVALUATION
The potency is calculated by the usual statistical methods for the twin-cross-over assay¹⁰

Binding assays

Immunoassay
The first description of an immunoassay of endogenous plasma insulin in man has been given by Yalow and Berson. Yalow et al. providing evidence that the bioassays hitherto being used (isolated rat diaphragm, epididymal fat pad tissue) measure insulin- like activity but not true insulin levels in blood.

PROCEDURE
Immunization
Semisynthetic or biosynthetic human insulin is used as immunogen and as standard. Formerly, porcine insulin has been used since Yalow and Berson (1960) and subsequently many other authors have shown that antisera raised against porcine insulin react identically with human and porcine insulin. Guinea pigs weighing 350–450 g are injected subcutaneously with 0.4 ml of an emulsion of 5 mg human insulin dissolved in 1.0 ml 0.01 N HCl and 3.0 ml complete adjuvant. For boostering, 0.2 ml of an identically prepared emulsion is injected in monthly intervals. Fourteen days after the third booster injection, the animals are slightly anesthetized and 8–10 ml blood are withdrawn by cardiac puncture. Boosting is continued at monthly intervals and the animals are bled 2 weeks following each booster injection.

Antiserum
The optimal antiserum titer for use in the radioimmunoassay is determined using conditions identical to those employed in routine immunoassays. The percentage binding of 1μU ¹²⁵I insulin is determined for dilutions of antisera ranging from 103 to 106 fold. The steepness of the antisera dilution curve is a measure of the affinity of the antiserum and therefore the potential sensitivity of the radioimmunoassay. Antisera with the steepest slopes, but not necessarily the highest titer, are selected for further study. The selected antisera dilutions are then run in an immunoassay using a full range of standards. A reduction in the percent 125I-insulin bound to antibody from 50% (in the absence of unlabeled insulin) to 45% (in the presence of unlabeled insulin) (B/Bo = 0.9) is a reasonable measure of assay sensitivity.

Preparation of ¹²⁵I-insulin
Most investigators use the “chloramine-T procedure” to iodinate insulin. The reaction is carried out in a 20 ml glass vial in an ice-bath with continuous magnetic stirring. To 2.5 ml 0.05 M phosphate buffer, pH 7.5, 2.0 mCi Na125I, and 15 μl of a 1 mg/ml insulin solution are added. Then, 0.5 ml of a chloramine T (50 mg/ml) solution is added dropwise over the course of 1 min. After 10 min, 0.7 ml of a freshly prepared sodium metabisulfite solution (50 mg/ml in 0.05 M phosphate buffer, pH 7.5) is added. One ml of this reaction mixture is transferred to 10 ml 2% bovine serum albumin for determination of specific activity. In order to absorb unreacted ¹²⁵I and damaged products 2.0 g 20–50 mesh AG 1X-8 resin are added (equilibrated in 1 ml 0.05 M phosphate buffer, pH 7.5, containing 0.1 mg/ml thiomerosal and 20 mg/ml crystalline bovine serum albumin). The reaction mixture is stirred for 10 min, decanted from the resin and diluted to a concentration of less than 25 μC/ml in a solution of 0.8 M glycine, 0.2 M NaCl, 0.05 M phosphate (pH 7.5), and 2.5 mg/ml crystalline BSA. The final solution is stored in multiple aliquots at –70 °C.

The following procedure is recommended:

· A buffer is prepared from a solution of 8.25 g boric acid and 2.70 g NaOH dissolved in 1litre water. After dissolving 5.0 g of purified bovine serum albumin, pH is adjusted with concentrated HCl to 8.0.

· In disposable plastic tubes, 10 * 75 mm, the following volumes are added:

100μl serum or standard

900 μl buffer

100 μl 1 mU 125I-insulin in assay buffer

100 μl guinea pig anti-insulin antiserum diluted in assay buffer (at a concentration to bind 50% of the ¹²⁵I-insulin in the absence of unlabeled hormone)

The mixture is incubated at 4 °C for 72 h. Then, the following solutions are added

100 μl normal guinea pig serum diluted 1 : 400 in the assay buffer

100 μl rabbit anti-guinea pig globulin serum diluted in assay buffer

The mixture is again incubated at 4 °C for 72 h and then centrifuged at 4 °C and 2 000 g for 20 min. The supernatant is decanted and radioactivity counted in the precipitate for 5 min.

Calculation
Counts in the nonspecific binding tubes are subtracted from counts in all other tubes. Data are linearized using an unweighted logit-log transformation .Micro-units insulin in a logarithmic scale are plotted against the ratio B/Bo ¹²⁵I-insulin on a logit scale. The range of B/Bo between 0.4 and 0.9 is the most suitable for determination of insulin concentration in plasma.

Insulin receptor binding
Insulin receptor binding studies have been performed with various animal tissues and isolated cells as well as with cells of human origin. Human adipocytes can be used to study simultaneously insulin receptor binding and metabolic effects of insulin .The binding tests are of value to characterize newly synthesized insulin derivatives

PROCEDURE
Subcutaneous adipose tissue (about 4–5 g) is obtained from the abdomen of patients undergoing gastroenterological surgery. Patients suffering from any endocrine or metabolic disorder or taking drugs known to affect metabolism have to be excluded. Other exclusion criteria are impaired glucose tolerance measured by determination of fasting blood glucose and the 2 h value after a 75 g oral glucose load. The adipose tissue is finely chopped and incubated for 90 min at 37 °C in a HEPES buffer (pH 7.4), containing human serum albumin (25 g/l) and collagenase (0.5 g/l). The isolated adipocytes are subsequently washed five times in a HEPES buffer containing 50 g/l human albumin. The diameters of adipocytes are measured at 200-fold magnification using an eyepiece micrometer. Surface and volume are calculated for every cell diameter.
Insulin receptor binding studies with isolated human adipocytes are performed in a 300 μl cell suspension containing about 1 * 10⁵ cells/ml in a HEPES buffer (10 mmol/l HEPES, 50 g/l human serum albumin, (pH 7.4) at 37 °C. The iodine labelled ligand ([125I] TyrA14-monoiodinated insulin, specific activity about 350 mCi/mg) in a final concentration of 20 pmol/l is incubated with increasing amounts of unlabeled human insulin and the insulin derivative to be tested. The reaction is stopped by adding 10 ml of chilled 0.154 mol/l NaCl and subsequent centrifugation with silicone oil
Non-specific binding is measured by incubating tracer in the presence of a large excess of unlabelled insulin. For association studies the ¹²⁵I-labelled ligand is incubated for various times (1 to 240 min) and the reaction is terminated as described above. At each time point, the non-specific binding is measured and subsequently subtracted from the corresponding data for total binding.
Dissociation rates are determined by first incubating isolated human adipocytes at 37 °C with either [¹²⁵I] TyrA14-insulin or the test compound labelled in the same position for 90 min to achieve steady-state binding conditions. Each incubation mixture is then centrifuged for 60s The adipocytes are rapidly washed twice by diluting with buffer to the original volume at 4 °C and the centrifugations and aspirations are repeated. After the third aspiration, the cells are diluted to the original volume with buffer alone or native insulin or the insulin derivative to be tested at a final concentration of 0.2 μmol/l at 22 °C. At this hormone concentration a maximal effect of ¹²⁵I-insulin dissociation is reported. The reaction is stopped at various times between 10 and 180 min and cell-associated radioactivity is determined.

EVALUATION
Results are expressed as percentage specific binding per 10 cm² plasma membrane surface area ¹⁸

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5. Assays of other glucose regulating peptide hormones

5.1 Bioassay of glucagon

Glucagon is a 29-amino-acid, single-chain polypeptide which is secreted by the α cells in the islets of Langerhans. It is synthesized from preproglucagon, a 180-amino-acid precursor Proglucagon is processed in islet and intestinal cell lines. Glucagon interacts with a 60-kDa glycoprotein receptor on the plasma membrane of target cells
A biological assay of glucagon is described in the British Pharmacopoeia 1988. The potency of glucagon is estimated by comparing its hyperglycemic activity with that of the standard preparation of glucagon using the rabbit blood sugar assay as performed for insulin determinations¹⁸

PROCEDURE
Rabbits of either sex, weighing 1.8 to 2.8 kg are maintained under uniform conditions and an adequate uniform diet for at least one week. Forty-eight hours before the beginning of the test, each rabbit is injected with 1 ml of cortisone acetate injection. The animals are deprived of food, but not water, from 16 h before each test day until the withdrawal of the last blood sample on that day. The rabbits are randomly distributed into 4 groups of at least 6 animals.
                  The standard preparation to be used is the 1st International Standard for Glucagon, porcine, established in 1973, consisting of freeze-dried porcine glucagon with lactose and sodium chloride (supplied in ampoules containing 1.49 Units), or another suitable preparation the potency of which has been determined in relation to the International Standard.
                    The entire contents of one ampoule of the standard preparation are reconstituted with 2 ml of saline solution, acidified to pH 3.0 with hydrochloric acid and diluted with the same solvent to a convenient concentration, for example 100 milli Units per ml. Two dilutions are made containing 24 and 6 milliUnits per ml, respectively, and at the same time two dilutions are made of the preparation being examined. The rabbits are injected subcutaneously with doses of 1 ml of each of the four solutions, giving the doses in random order following a twin cross-over design on two consecutive days at the same time each day. At 20 and 60 min after injection, a blood sample is taken from a marginal ear vein of each rabbit.
                   Blood glucose concentrations are determined using a suitable method, such as the glucose-oxidase procedure.

EVALUATION
The result of the assay is calculated by standard statistical methods using the means of the two blood glucose levels found for each rabbit⁵

Receptor binding and in vitro activity of glucagon
The binding of glucagon to its receptor is assayed with rat liver plasma membranes Displacement of 125I-labeled glucagon is measured for synthetic glucagon analogues in comparison to natural glucagon. Cyclic AMP formation as the first step in glucagon action on liver is measured as a bioassay in liver plasma membranes.

PROCEDURE
Preparation of membranes
Male Sprague-Dawley rats weighing 160–200 g are decapitated and the livers rapidly removed and trimmed of fat and connective tissue. The pooled livers are placed on a pre-chilled glass plate and chopped finely with a stainless steel blade. Ten-gram portions of the mincate are suspended in 35 ml of ice-cold 1 mM NaHCO3 and homogenized in a loose fitting Dounce homogenizer. Two homogenates are combined, brought to 500 ml with ice-cold medium, stirred magnetically for 5 min, and finally filtered once through two layers of cheesecloth. The filtrate is centrifuged at 1 500 g for 10 min. Supernatant fluid is aspirated to waste using a serum pipette attached to a water pump. Homogenizations and centrifugations are continued until all of the tissue has been processed. The pooled pellets are again homogenized. The suspension is adjusted to 44 ±0.1% (w/w) sucrose solution by the addition of 70% sucrose solution. Sucrose gradients are prepared in 1* 3.5-in. tubes by pipetting 26 ml of the tissue suspension followed by an overlay of 13 ml of 42.3 ±0.1% sucrose. Centrifugation is carried out at 95 100 g for 2 h. The float containing the plasma membranes can be removed by pinching the tube slightly above the float, then lifting off the float with a spoon-shaped Teflon coated spatula. These floats are transferred to a preweighed plastic centrifuge tube (50 ml), and the well mixed suspension centrifuged at 40000 g for 30 min. Following aspiration of the supernatant to waste, the tubes are reweighed in order to estimate the yield of plasma membranes. After addition of an equal volume of medium, the pellet is aspirated repeatedly through a 20-gauge needle fitted to a syringe. The plasma membrane suspension is distributed in 0.2 or 1.0 ml aliquots to screw cap plastic vessels for storage in liquid nitrogen.

Radioiodination of glucagon
Three nmol glucagon are allowed to react with a 1.0 nmol sample of carrier-free Na¹²⁵I (2.0 mC) in the presence of 1.5 nmol chloramine-T, added at a regular interval of 30 s (0.5 nmol each time). Reaction is terminated by addition of 0.5% sodium metabisulfite solution. By chromatographic purification fractions of 2 ml are collected, and the monoiodinated glucagon as determined by reverse phase HPLC is stored at –20 °C for receptor binding assays.

Receptor binding
Membrane suspensions adjusted to 50 μg protein in 400 μl of Tris-HCl buffer (25 mM, pH 7.5, with 1% BSA) are incubated for 10 min with 50 μl peptide solution (Tris-HCl buffer) and 50 μl of [¹²⁵I] glucagon (1 000 000 cpm, 25 fmol). The samples are then filtered through Oxoid filters and washed three times with 1 ml of Tris-HCl buffer. The radioactivity retained in the filters is counted by a gamma-counter.

Adenylate cyclase assay
The assay is carried out with a membrane suspension containing 25–30 μg protein in a volume of 0.1 ml of Tris-HCl buffer 25 mM, pH 7.5 containing BSA 1%, ATP 1 mM, MgCl₂ 5 mM, cAMP 1 nM, (containing 10 000 cpm of [3H]cAMP), GTP 10 mM, phosphocreatine 20 mM, with 4 * 10⁶ cpm [α³²P] ATP and 0.72 mg/ml creatinine phosphokinase (100 U/ml). Assays are run in triplicate.

EVALUATION
Results are expressed as the percentage inhibition of [¹²⁵I]glucagon specific binding for receptor binding assays. For adenylate cyclase assays the results are expressed as percent potency relative to the maximal stimulation by glucagon which is defined as 100%.¹³

Glucagon-like peptide I
Several intestinal peptides have been described to have insulinotropic or incretin activity, e.g. GIP (gastric inhibitory peptide, glucose-dependent insulin releasing peptide). More recently, glucagon-like peptide-1 (7-37) or the (7-36)amide is described as a new incretin. The insulinotropic activity has been confirmed in diabetic and non diabetic subjects. The insulin stimulatory effect of glucagon-like peptide-1(7-36)amide is glucosedependent. The peptide does not only stimulate insulin release but also inhibits glucagon secretion. Cloning and functional expression of the rat and human glucagon-like peptide1 (GLP-1) receptor has been achieved. The sequence of glucagonlike peptide-1 (7-36)amide is completely conserved in all mammalian species studied, implying that it plays a critical physiological role. Intra cerebroventricular administration of GLP-1 powerfully inhibits feeding in fasted rats, which is blocked by the GLP-1-receptor antagonist exendin(9-39)
Peptides isolated from reptile venoms, like exendin-4, were found to have similar activity as glucagon-like peptide-1(7-36)amide. Analogues and antagonists of glucagon-like peptide-1(7-36)-amide have been synthesized and evaluated in pharmacological experiments Besides insulin release from perfused pancreas , the receptor on rat insulinoma-derived cells (RINm5F cells), the cAMP formation and the insulin release were studied

PROCEDURE
Binding studies with RINm5F cells
The RINm5F cell line is derived from a radiation-induced insulin-producing rat tumor from the surface of the bottles before the experiment using phosphate buffered saline (136 mmol NaCl/l, 2.7 mmol KCl/l, 8.1 mmol Na2HPO4/l, 1.5 mmol KH2PO4/l, pH 7.3) containing 0.7 mmol EDTA/l, and centrifuged at 100 g for 5 min. The pelleted cells are resuspended in an incubation buffer (2.5 mmol Tris-HCl/l, 120 mmol NaCl/l, 1.2 mmol MgSO4/l, 1.5 mmol KCl/l, and 15 mmol CH3COONa/l, pH 7.4) containing 1% human serum albumin, 0.1% bacitracin and 1 mmol EDTA/l. Approximately 3 *10⁶ cells/tube are incubated for 5 min at 37 °C, followed by the addition of unlabelled peptide (final concentration range from 10 pmol to 1 mmol) and radiolabelled tracer (approximately 40 000 c.p.m.). Iodination of the glucagon-like peptide-1(7-36)amide is carried out using the lactoperoxidase method. The total volume of incubation is 0.3 ml. After incubation for 60 min, aliquots (200 μl) of the cell suspensions are centrifuged (11 500 g for 2 min) through an oil layer (dibutylphthalate: dinonylphthalate; 10:4, v/v). Cell surface associated radioactivity in the pellet is counted using a gamma-counter

Determination of cAMP production from RINm5F cells
Approximately 1 *10⁶ cells in 0.45 ml buffer (113 mmol NaCl/l, 4.7 mmol KCl/l, 1.2 mmol KH2PO4/l, 10 mmol HEPES/l, 2.5 mmol CaCl2/l and 1.2 mmol MgSO4/l; pH 7.4) containing 1% human serum albumin are preincubated for 10 min at 37 °C, and then incubated for 10 min after the addition of 2 μl 3-isobutyl-1-methylxanthine (50 mmol/l) in order to prevent the breakdown of cAMP. The reaction is then started by the addition of 50 μl of a peptide solution dissolved in the above buffer (final concentration range from 10 pmol/l to 1 μmol/l). After incubation for 10 min at 37 °C, the reaction is stopped by the addition of 200 μl 12% trichloroacetic acid. The reaction mixture is sonicated for 30 s at 25 W (e.g., Heat system, Ultrasonics) and centrifuged (11 500 g for 2 min). HCl (25 μl; 1 mol/l) is added to 0.5 ml supernatant. Trichloroacetic acid dissolved in the supernatant is removed by diethyl ether (3 * 1 ml) and the resulting supernatant is stored at –40 °C until cAMP assays being performed by use of a RIA kit.

Determination of insulin release from RINm5F cells
One day prior to the experiment, approximately 1 *10⁶ cells are seeded into 24-well test plates. At the time of the experiment, the culture medium is aspirated, and the cells are washed with 1 ml of a modified Krebs- Ringer bicarbonate buffer containing 10 mmol HEPES/l, 5 mmol NaHCO3/l, 0.5% BSA, and 8.3 mmol glucose/l (pH 7.4). After pre-incubation for 30 min at 37 °C with the above buffer, glucagon-like peptide-1(7-36) amide and its analogues dissolved in the above buffer, are added (final concentration range from 10 pmol/l to 1 μmol/l) and incubated for 30 min at 37 °C. The incubation is stopped by aspiration of the buffer which is stored at –40 °C until measurement of insulin by radioimmunoassay. After the aspiration, the cells are washed with the phosphate-buffered saline used in the binding study, and dissolved in 0.5 ml NaOH (0.1 mol/l). After overnight incubation, the resulting solution is collected for assay of cellular protein content.

EVALUATION
Displacement curves of ¹²⁵I-labelled glucagon-like peptide-1(7-36)amide are established for various concentrations of glucagon-like peptide-1(7-36)amide and its analogues as well as dose-response curves for cAMP production and insulin release. The data are analyzed by analysis of variance followed by group comparisons (Duncan’s multiple comparisons).¹

Blood glucose lowering activity of antidiabetic drugs

Blood glucose lowering activity in rabbits
The rabbit has been used since many years for standardization of insulin Therefore, it has been chosen as primary screening model for screening of blood glucose lowering compounds as well as for establishing time-response curves and relative activities

PROCEDURE
Groups of 4–5 mixed breed rabbits 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 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 glucose is determined in 10 μl blood samples with the hexokinase enzyme method

MODIFICATIONS OF THE METHOD
For special purposes the effect of blood sugar lowering agents is studied in glucose loaded animals. Rabbits of either sex weighing 3.0–4.5 kg are treated either once (0.5 h after test compound) or twice (0.5 and 2.5 h after test compound) orally with 2 g glucose/kg body weight in 50% solution.

EVALUATION
Average 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 experimental group are compared statistically with the t-test or the WILCOXON test for each time interval with those of the control group. Differences between several treated groups and the control group are tested using a simultaneous comparison according to Dunnett or Nemenyi/Dunnett (1966). Dose dependencies and relative activities are determined by means of linear regression analysis All data for statistical comparisons have to be tested for homogeneity of variances and for normal distribution. In the case of regression analyses, the lines are additionally tested for parallelism) and for linearity. The level of significance for all procedures is chosen as 5%.¹²

Blood glucose lowering activity in rats
Rats are used for screening as well as for quantitative evaluation of blood glucose lowering agents.

PROCEDURE
Male Wistar rats weighing 180–240 g are kept on standard diet. Groups of 4–7 non-fasted animals are treated orally or intraperitoneally with various doses of the test compounds suspended in 0.4% starch suspension. One control group receives the vehicle only. Blood is withdrawn from the tip of the tail immediately before, and 1, 2, 3, 5, and 24 h after administration of the test compound. Blood glucose is determined in 10 μl blood samples with the hexokinase enzyme method

MODIFICATIONS OF THE METHOD

Studies in glucose loaded rats
For special purposes the effect of blood sugar lowering agents is studied in glucose loaded animals. One g glucose/kg body weight is given in a 50% solution either orally 5 min after oral administration or subcutaneously 5 min after intraperitoneal administration of the test compound.¹⁸

Blood glucose lowering activity in dogs
Besides experiments in rats and rabbits studies in dogs are necessary to predict the effect of a new compound in man due to differences in species related metabolism.

PROCEDURE
Male Beagle dogs weighing 15–20 kg are kept on standard diet Food is withdrawn 18 h prior to the administration of the test compound which is given either orally or intravenously in various doses. Control animals receive the vehicle only. Blood is collected at different time intervals up to 48 h. Blood glucose is determined with the hexokinase enzyme method and plasma insulin with an immunological method

MODIFICATIONS OF THE METHOD

Studies in pancreatectomized dogs
The animals are pancreatectomized up to 2–3 years prior to the study. They are kept on dry feed together with 2–3 g pancreatic enzymes Insulin is substituted with a single daily subcutaneous dose of 32 IU Insulin. For substitution of vitamin D an intramuscular dose of 1 ml is given every 3 months. On the day before the study, the animals receive 32 IU of the shorter acting insulin. This insulin is administered at the same time when food and test compound are given in the morning. The test drug is applied as oral suspension in tap water. Blood glucose is determined before and up to 6 h after treatment in hourly intervals. Control animals receive tap water only¹⁸

Euglycemic clamp technique
The 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.

EVALUATION
All 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 hyperinsulinemia these values are thought to reflect the state of the enzymes and glucose transport system activated after the binding to receptors, indicating mainly insulin responsiveness⁷

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