INTRODUCTION

l.DIABETES MELLITUS

1.1 GENERAL

Diabetes mellitus, a debilitating disease is known since antiquity. The ancient

Sanskrit compendium of Charka Samhita mentioned this disorder as 'madhumeha'

(honeyed urine). This ancient treatise dating way back to Vedic period of Indian

civilization also includes the first ever reference to two types of diabetes, with

description closely matching those of the insulin dependent diabetes and non insulin

dependent diabetes mellitus (Bajaj, 1992; and Bajaj and Madan, 1993). The Egyptian

papyrus, dating back to 1550 BC found in grave in Thebes in 1862 and named after

the Egyptologist George Ebers, testifies to the long history of diabetes. This contains

the description of a polyuric state resembling diabetes mellitus. In 170 AD, Aretaeus

of Cappodocia wrote "A dreadful affliction being melting down of the flesh and limbs

in to urine, life is short, unpleasant, painful, thirst unquenchable, the viscera seem

scorched up affected by nausea restlessness death is inevitable." Aretaeus referred

this disease as 'diabetes' meaning 'to run through' or 'a siphon' in Ionian Greek and

implying the uncontrolled loss of urine leading to death (Bloom and Ireland, 1992).

In the 17ili century, Thomas Willis distinguished diabetes from other polyuric states.

In 1776, Matthew Dobson discovered the presence of sugar in both diabetic serum

and urine. The English physician John Rollo was one of the first to use the adjective

'mellitus' from the Latin and Greek roots of 'honey' used to distinguish the condition

from other polyuric disease in which glycosuria was absent (Latin, insipidus) (Pickup

and Williams, 1992). This disorder is characterized by features ranging from

asymptomatic stage to acute ketoacidosis and to chronic micro and macro vascular

complications. The term diabetes mellitus is used to describe the syndrome, a

collection of diseases and conditions that have hyperglycemia and glucose

intolerance in common, due to either insulin deficiency or impaired effectiveness of

insulin action (Harris and Zimmet, 1992)

Diabetes is now one of the most common non-communicable disease globally. It is

the fourth or fifth leading cause of death in most developed countries and there is

substantial evidence that it is epidemic in many developing and newly industrialized

nations (Amos et ai, 1997). Diabetes mellitus is a growing pandemic afflicting more

than 100 million people around the globe (2.1 % of the global population) and

expected to have reached 165 million in 2000 A.D. and 300 million in 2025 A.D.

(Eshwe' ge el ai, 1997).

1.2 CLASSIFICATION OF DIABETES MELLITUS

Diabetes and other categories of glucose intolerance have been classified by many

workers but the largely accepted classification has been developed by an

International Working group sponsored by the National Diabetes Data group

(NODG) of the National Institute of Health, U.S.A. The expert committee ofW.H.O.

on diabetes endorsed the classification given by NOOG. The classification is

presented in the Table 1.

1.2.1 INSULIN DEPENDENT DIABETES MELLITUS OR TYPE I

DIABETES

Insulin-dependent diabetes mellitus (lOOM) or Type I diabetes is an autoimmune

disease that affects 0.3 % population of the world. It is caused by auto aggressive T-

cells that infiltrate the pancreas and eventually destroy the insulin producing p-islet

cells. This results in the hypoinsulinaemia and hyperglycemic condition (Bach, J.F.,

1995). Although the pathogenesis of autoimmune diabetes or IODM has been

extensively studied, the precise mechanism involved in the initiation of p-islet cell

destruction is still not clear. The etiology of 100M appears to be a heterogeneous

category in the sense that various genetic and environmental factors are associated

with it. Inheritance is polygenic with genotype of the major histocompatibility

complex being the strongest determinant. Close to 90% 100M patients show either

HLA, OR3 or OR4 or both together and there are even closer association with OQB.

But this does not mean that 100M patients are having this particular haplotype,

2

Table: 1 Classification of Diabetes and Other Categories of Glucose Intolerance

CLASS

I. Insulin-dependent Diabetes mellitus IDDM

(Type I )

2. Noninsulin-dependent Diabetes mellitus,

NIDDM (Type II)

a) Non-obese

b) Obese

3. Malnutrition Related Diabetes mellitus (MRDM)

a) Fibrocalculous Pancreatic diabetes

b) Protein deficient pancreatic diabetes

4. Gestational Diabetes mellitus (GDM)

FORMER TERMINOLOGY

Juvenile diabetes, Juvenile onset diabetes,

JOD, ketosisprone diabetes, brittle diabetes

Adult / maturity onset diabetes, MOD,

ketosis-resistance diabetes.

Tropical diabetes, pancreatic diabetes,

ketosis resistance diabetes of young.

Gestational diabetes.

5. Other type of Diabetes including diabetes associated Secondary diabetes

with certain conditions and syndromes

a) Pancreatic disease

b) Hormonal

c) Drug or chemical induced

d) Insulin or it's receptor abnormalities

e) Certain genetic syndrome

f) Miscellaneous

6. Impaired glucose tolerance ( IGT) Asmyptomatic diabetes

a) Nonobese IGT

b) Obese IGT

(Modified from Bloom and Ireland, 1992)

rather it means these particular genetic make up are more susceptible to develop the

disease (Cudworth and Bodansky, 1982; Bloom and Ireland, 1992). However, in

monozygotic twins, the concordance rate is only 50%, indicating the importance of

the environmental factors (Tisch and McDevitt, 1996). Animal models used in the

study of IDDM, such as the Bio-Breeding (BB) rats and the non-obese diabetic

(NOD) mice, have enhanced our understanding of the pathogenic mechanism

involved in the disease. Macrophages are one of the most important immunocytes

involved in the initiation of J3-islet cell destruction. The presentation of the J3-cell

specific auto antigens by macro phages to CD4+ helper T -cells, in association with

MHC class II molecules, might be the initial steps in the development of

autoimmune IDDM. Most of the infiltrating immunocytes at the time of diagnosis of

IDDM are CD8+ T cells, suggesting that these cells play a major role in the

destruction of pancreatic J3- cells at a later stage of the autoimmune process as a final

effectors (Y oon et ai, 1998).

Role of glutamic acid decarboxylase, as single autoantigen for diabetes, is shown in

non-obese diabetic (NOD) mouse model. A single self-protein expressed by J3-islet

cells, glutamic acid decarboxylase, controls the development of IDDM in NOD mice

(Yoon et ai, 1999).

1.2.2 NON-INSULIN DEPENDENT DIABETES MELLITUS OR TYPE II

DIABETES

Non-insulin dependent diabetes mellitus (NIDDM) or Type II diabetes is the most

common type of diabetes. Majority of patient, close to 80% has this type of diabetes,

which is late onset occurring mostly after thirty-five years of age in human beings.

NIDDM is characterized by triad of (1) resistance to insulin action on glucose uptake

in peripheral tissues, especially skeletal muscle and adipocytes (2) impaired insulin

action to inhibit hepatic glucose production, and (3) dysregulated insulin secretion

(DeFronzo, 1982). In most cases Type II diabetes is polygenic disease with complex

inheritance pattern (Kahn et ai, 1996). Environmental factors, especially diet,

3

physical activity and age interact with the genetic predisposition to affect disease

prevalence. Susceptibility to both insulin resistance and insulin secretory defects

appears to be genetically determined. (Kahn et ai, 1996).

The factor most likely to enhance the problems of Type II diabetes is obesity.

Carbohydrate rich diet enhances the demand for insulin and obesity causes the

resistance in the peripheral tissues to the action of insulin. Though there are quite a

few dissimilarities between Type I and Type II diabetes, there are some similarities

as well. Both types may develop the same degenerative changes in the blood vessels,

nervous system, kidney and eyes, suggesting that the degeneration arises due to

hyperglycemia rather than any inherited etiological factor which may be responsible

for degenerative changes leading to long term complications (Bloom and Ireland,

1992).

1.3 EXPERIMENTAL DIABETES

The equivalent of Type I diabetes mellitus (IDDM) in human beings is chemically

induced in animals to generate experimental diabetics to be used for laboratory

purpose. Various drugs can induce diabetes and hence are called 'diabetogenic'.

These chemicals act by generation of free radicals and alteration of endogenous

scavengers of these free radical species. Secondly, by depleting the NAD in the p-

cells by increasing the activity of poly-ADP ribose synthetase and thirdly, inhibiting

the active calcium transport and colmodulin activated protein tyrosine kinase (Y oon

et ai, 1987). Diazooxide is one such diabetogenic agent of this group and has been

used in the treatment of the hyperglycemia induced by islet cell tumors.

Streptozotocin, a nitric oxide derivative of methyl-urea complex, is used mainly to

induce diabetes in experimental animals and occasionally used in the treatment of

hypoglycemia due to an insulinoma. Streptozotocin has direct and destructive action

on the pancreatic p-cells. Alloxan is another powerful suppressant of the pancreatic

p-cells and its administration is confined to experimental animals (Bloom and Ireland,

1992). Alloxan produces cytotoxicity owing to its conversion to anionic free radicals

4

(Nukatsuka et ai, 1989). The cytotoxic action of alloxan is specific to pancreatic p-cells (Asayama et ai, 1984).

Pancreatic islet cells, treated with alloxan, exhibit multiple cellular necrosis marked

deregulation, extensive vesiculation of the endoplasmic reticulum as well as Golgi

complex, enlarged mitochondria with disrupted cristae and mitochondrial rupture

(Watkins III and Sanders, 1995). Alloxan diabetes is characterized by hyperglycemia,

ketonuria, glucosuria, polydipsia, polyuria, loss in body weight, polyphagea,

hyperlipemia, and acidosis (Yoon et ai, 1987). Alloxan induced diabetes in the

laboratory animals is equivalent to typical insulin-dependent diabetes mellitus

symptoms and complications associated with short term and long term complications

of IDDM. Alloxan induced diabetes produced in the laboratory animals has been

widely used to study the pathology, physiology, biochemistry, genetics and

therapeutics of IDDM. All the experimental work in this thesis has been also carried

out on alloxan induced experimental Wistar rats.

1.4 METABOLIC DERANGEMENTS ASSOCIATED WITH DIABETES

MELLITUS.

Patients of diabetes mellitus exhibit wide spectrum of derangement in carbohydrate

metabolism, from those having mild or asymptomatic disease without fasting

hyperglycemia to those having severe fasting hyperglycemia in fully expressed

clinical disease (Foster, 1987). Even though attention is much more focused on the

disordered carbohydrate metabolism, there are alterations in most of the pathways of

intermediary metabolism. Insulin is a major anabolic hormone in the body and

derangement of insulin secretion and function not only affects glucose metabolism

but also fat and protein metabolism. The deficiency of insulin, therefore, causes the

most severe derangement of metabolism in Type I diabetes. The assimilation of

glucose and other sugars into muscles and adipose tissues are sharply diminished or

abolished. Not only does the storage of glycogen in liver and muscle decrease, but

also the latter is depleted by glycogenolysis. Fasting hyperglycemia may reach many

5

times higher than normal and when the level of circulating glucose exceeds the renal

threshold, glycosuria ensues. The excessive glycosuria induces an osmotic diuresis

and thus polyurea, causing profound loss of water and electrolytes (Na, K, Mg, and

P). These obligatory water losses, combined with hyperosmolarity resulting from the

increased level of glucose tend to deplete the intracellular water, for example, in the

osmoreceptors of brain. In this manner, intense thirst (polydipsia) appears. Through

poorly defined pathways, increased appetite (polyphagia) develops, thus completing

the classic triad of diabetes mellitus - polyurea, polyphagia and polydipsia (Carton et

ai, 1989). With the deficiency of insulin the metabolic scale swings from insulin

promoted anabolism to catabolism of protein and fat. Proteolysis follows and

gluconeogenic amino acids are removed by liver and used as building blocks in

gluconeogenesis, worsening the deranged carbohydrate metabolism. Two acute

complications of diabetes mellitus follow diabetic ketoacidosis and nonketoic

hyperosmolar coma. Diabetic ketoacidosis occurs almost exclusively in Type I

diabetes and is stimulated by severe insulin deficiency coupled with absolute or

relative increase of Glucagon. The insulin deficiency causes excessive breakdown of

adipose stores, resulting in the increased level of free fatty acids. Oxidation of such

free fatty acids in liver through Acetyl-CoA produces ketone bodies. Glucagon is the

hormone that accelerates the breakdown of such fatty acids through oxidation. The

rate at which these ketone bodies are formed may exceed the rate at which

acetoacetic acid and beta hydroxy butyric acid can be utilized by muscles and other

tissues. Ketogenic amino acids further aggravate the lipid metabolism derangement.

Ketogenesis, thus, increases leading to ketonemia and ketoneurea. If the urinary

excretion of ketone is compromised by dehydration, the plasma hydrogen IOn

concentration increases and systemic metabolic ketoacidosis results.

In Type II diabetes, polyurea, polyphagia and polydipsia may accompany the fasting

hyperglycemia but ketoacidosis is rare. Adults, particularly the elderly diabetics,

develop nonketoic hyperosmolar coma, a syndrome engendered by severe

dehydration resulting from sustained hyperglycemic diuresis which is coupled with

the inability of these patients to drink water. The absence of ketoacidosis, and its

6

symptoms (nausea, vomiting, respiratory difficulties), delays the seeking of medical

attention (Arky, 1982). The metabolic derangement and its clinical correlates are

presented in the Table 2.

1.5 LONG TERM COMPLICATIONS OF DIABETES MELLITUS

Diabetes mellitus is no longer regarded as a single disease rather it is a group of

chronic disorders with persistent hyperglycemia resulting in several long as well as

short term complications. (Harris and Zimmet, 1992). Diabetics, in general, have a

substantially reduced life expectancy with age specific mortality rates approximately

twice to that of non-diabetic population. This rate tends to diminish with increasing

age at the onset of diabetes (Cudworth and Bodansky, 1982, Ronald, 1994).

Hyperglycemia plays an important role in the pathogenesis of all major long term

complications of diabetes including microvascular and macrovascular complications,

impaired cellular immunity, protein glycosylation, abnormality in cell growth and

cell differentiation (Brownlee and Cerami, 1981). Major microvacular complications

of diabetes include retinopathy, nephropathy and neuropathy. The pathophysiology

of the diabetic retinopathy is due to the elevated blood flow to the retina and the

intravascular pressure. Retinopathy may be apparent at the time of the diagnosis,

especially in NIDDM. Diabetes is the most common cause of adult blindness either

due to retinopathy, cataract or glaucoma. Glomerular hyperfiltration and the

excessive urinary excretion of protein lead to pathophysiology of nephropathy.

Neuropathy leads to neurological disorders. Diabetic neuropathy is characterized by

variety of morphological changes associated with decreased sensory and motor

conduction velocities including decreased number of intra-membrane particle on the

myelin surface; endoneural edema with resultant shrinkage of axon and Schwan

cells; increased permeability of nodal gap substance; basement membrane thickening

in the intra- and peri-neural vessels; axonal degeneration and segmental

demyelination. Following the biochemical changes, some changes are also observed

in nerves; changes in the composition and synthesis rate of various myelin lipid and

protein components; increased activity of polyol pathway; decreased concentration

7

Table 2: Biochemical and Clinical Correlation of Diabetes mellitus

Metabolic defects Chemical abnormalities

A. Carbohydrate metabolism '

I. Diminished uptake by tissues such as muscle,

adipose tissue and liver.

2. Overproduction of glucose (via

glycogenolysis and gluconeogenesis) by tissues

such as liver and kidney.

B. Fat metabolism I. Increased lipolysis.

2. Decreased lipogenesis

3. Increased production oftriglycerides.

4. Decreased removal oftriglycerides.

C. Protein metabilism I. Diminished uptake of amino acids and

diminished synthesis of proteins.

L Increased proteolysis.

(Modified from Arky, 1982)

Hyperglycemia

Elevated plasma fatty acids level.

Elevated plasma glycerol level

Elevated plasma ketone

Hypertriglyceridemia

Metabolic acidosis.

Clinical correlation

Polyurea

Polydipsia

Polyphagea

Fatigue

Muscle weakens

Pruritus

Blurred vision

Loss of adipose tissues.

Nausea and vomiting.

Abdominal pain, formation

of acetone on breathing.

Exudative xanthoma

Lipemia retinalis and

pancreatitis

Hyperventilation because

of heavy breathing

Loss of muscle mass

Weakness.

and synthesis rate of myoinositol and phosphatidyl ionositol and decreased

exoplasmic transport of choline acetylase, acetylcholine esterase, norepinephrine and

several glycoproteins (West, 1982).

The macrovascular complications in diabetes include the complications of large

arteries. Macrovascular complications in diabetes may be due to the abnormalities in

plasma lipids and the changes in the composition of the arterial (cardiac and cerebral)

wall (Brownlee and Cerami, 1981). The main complication in this type, being

artherosclerosis, leads to cardiovascular and cerebral dysfunction. Other long term

complications of diabetes include the diabetic foot, connective tissue and bone joint

diseases, ulceration, skin disease and impotency in diabetic males.

2 ANTIDIABETIC AGENTS

2.1 GENERAL

Though, diabetes mellitus is a debilitating disease known since antiquity, it was only

in 1778 that Thomas Cawely postulated in the London Medical Journal about the

possible involvement of pancreas damage in the development of this disorder. The

importance of pancreas was established by Oskar Minkowski in 1889, who described

with Von Mering, how pancreatectomy made a laboratory dog urinate excessively

and he tested the urine for sugar and found large amount of sugar in it (Bloom and

Ireland, 1992). After the development of the concept of hormones by Ernst Starling in

1904, European scientists and elsewhere concentrated their attention on extraction of

insulin from the pancreas. Finally, a Canadian surgeon Fredrik Banting, working with

his student Charles Best, was successful in discovering and isolating insulin in 1921.

The discovery of insulin was promptly translated into a life saving remedy. This was

the beginning of a new era for the treatment of diabetes by insulin (Bloom and

Ireland, 1992). The success of the insulin therapy for 100M (Type I diabetes

mellitus) was overwhelming and even blunted exploring other approaches for a long

time. Existence of NIDDM (Type II Diabetes mellitus), where the insulin therapy

8

becomes ineffective because of the increased insulin resistance of the insulin

responding tissues, makes it prudent to look for alternatives for insulin therapy

(Ramasarma, 1996). Besides, were it not for the devastating effects of hypoglycemia,

Type I diabetes mellitus would be rather easy to treat. Enough insulin to lower

glucose level to the non-diabetic range would eliminate symptoms, undoubtedly

prevent the specific long term complications (retinopathy, nephropathy, and

neuropathy) and reduce the risks associated with these complications to basal line.

But the negative effects of hypoglycemia, particularly on the brain, are real (Cryer,

1999). It is now well established that glycemic control makes a difference for people

with diabetes. It prevents or delays the short term as well as specific long term

complications (Ohkubo et ai, 1993). The metabolic complications are not always

reverted back to complete normalcy (Alberti and Press, 1982; Lacy, 1995). For the

effective therapy of Type II diabetes various drugs, including the derivatives of

sulphonylurea (eg. Tolbutamide) and bigunides (eg. Phenoformins) are used which

enhances the insulin secretion by pancreatic p-cells and sensitizing the target cells of

insulin action or enhancing the binding of insulin to the receptors. Toxic effects of

these drugs limit their use (Ramasarma, 1996). There is renewed interest in finding

the alternatives for such drugs and insulin, which act possibly at the intercellular level

and are effective in both types of diabetes is now resurgent the world over. The

insulin-mimetic properties and the anti diabetogenic properties of various chemical

and natural agents have hence been studied.

Some metals are known to be capable of lowering the blood sugar level. Among

metal elements, the insulin mimetic effects of Vanadium (Meek et ai, 1971; Heyliger

et ai, 1985, Baquer, 1998), selenite (Ezakki, 1990), molybdate (Goto et ai, 1992),

tungstate (Goto et ai, 1992), zinc (Shiseva et ai, 1992), manganese (Baquer et ai,

1975; 1982) and chromium (Singh et ai, 1992) have all been demonstrated. However,

vanadium has been favored over the rest due to the promising insulin mimetic effects

it has been showing. More than a quarter of the medicine in use today comes from

plants i.e. from traditional medicine (Laughlin, 1963). Extracts of various plant

materials, capable of decreasing the blood glucose level are known in the ancient

9

system of the medicine like Ayurveda, Unani and Homeopathic medicine (Murthy,

1995). Some plant materials have been tested in the animal model system and their

hypoglycemic effects have been elucidated (Murthy, 1995). Active ingredients are

found to be present in more than one part of the plant. Infew cases the active

components are isolated and tested. A very important fact is that most of the plant

extracts used were non-toxic in nature, which make the use of such natural products

for their antidiabetic proprieties favorable. Among the plant extracts studied so far,

Alium sativum (garlic) bulbs (Sheela and Augusti, 1992), Momordica charantia

(Bitter gourd) fruit extract (Leatherdale et al 1981; Mukherjee and Chandershekher,

1992; Shibib et al 1993; Pugazhenthi and Murthy, 1996; Ahmed et ai, 1998), Ficus

bengalensis (Banyan) bark extract (Babu et ai, 1987; Shukla et ai, 1994), Ficus

carica leaves (Serrac1ara et ai, 1998); and Trigonella foenum graecum (fenugreek)

whole seed powder (Moorthy et ai, 1989) have been confirmed to possess antidiabetic

properties.

2.2 VANADIUM

In 1801, in Mexico, Andres Manuel del Rio first discovered one of the vanadium

compounds, but he erroneously thought that it was chromium. Thirty years later, the

Swedish chemist Nils Gaberial Sefstrom was credited for the discovery of the

element. He was so much dazzled with the lustre and brightness as well the variety of

colors of its salt in the solution that he named it after 'Vanadis,' the Scandinavian

counterpart of 'Venus' (Schroeder et ai, 1963). However, it was a century later, when

Marden and Rich in 1927, purified vanadium in crystalline form (Dafnis and Sabatini,

1994).

2.2.1. CHEMISTRY OF VANADIUM

Vanadium, element number 23, atomic weight 50.94, belongs to group V of periodic

table and shares common chemical characteristics with other member of the same

10

group, nitrogen, phosphorus and arsenic. It is the 21 5t most abundant element in the

Earth's crust, with an average concentration of 135 ppm (Nechay, 1984). All the

elements of group V have five valence electrons permitting a maximum oxidation

state of +5. Vanadium belongs to Vb group and has three electrons in the d-orbitals

imparting it a 3-d electron orbital configuration, which characterizes its unusual

oxidation features (Phillips and Nechay, 1983). Its multiple oxidation states range

from - 1 to +5 and the most common oxidation states are +3, +4 and +5; while the

most stable oxidation state is +4. Metallic vanadium does not occur in nature. At the

higher oxidation states, vanadium forms negatively charged oxyanions for example:

vol-, H2 V04-, Hvol- similar to phosphate compounds found in urine. Figure 1

presents some of vanadium compounds.

Protonation of Vanadium takes place at the acidic solutions and below pH 3.5 it

becomes a monovalent cation (VO} In basic solution, the element occurs as vol-

again, having chemistry similar to one of the phosphate (POl} In neutral solutions,

vanadium occurs as H2 V04- (Phillips and Nechay, 1983). In the aqueous solution,

vanadium compounds undergo rapid hydrolysis and polymerization that are

concentration and pH dependent. Thus vanadium is a very labile system (Shaver et ai,

1995) and rapidly interacts with the potential ligands such as nitrogen bases and

hydroxy groups to give mixtures of complexes. The complex aqueous chemistry of

vanadium and its relevance to biochemical effects have been well studied (Crans et

ai, 1994a, 1994b). The combination of vanadium with hydrogen peroxide under

physiological conditions, generate several peroxovanadium species in equilibrium

with one another depending on the pH of the solution and the concentration of

vanadium and H20 2 (Campbell et ai, 1989). Vanadium forms complexes with many

compounds used in the buffers (eg. EDT A, tricine, citrate and phosphate) and thus

HEPES buffer is recommended as the buffer of choice (Crans, 1994 b). Vanadium

also forms water soluble neutrally charged complexes with organic compounds like

maltoI, picoline, oxalic acid and kojic acid which act as ligand precursors (Yuen et aI,

1992 a, 1992 b, 1993; Sakurai et ai, 1993; Yuen et ai, 1997). These organic

complexes are lipophilic (Finnegan et aI, 1987).

II

Figure 1 : Vanadium Compounds

A. Tetravalent vanadium (yIV) - Yanadyl sulphate (YOS04)

B. Pentavalent vanadium (yIV) - Orthovandate (YOl)

c. Pentavalent vanadium (yIV) - Metavanadate (YOl)

D. Pentavalent vanadium (yIV) -Diperoxovanadate [HOOY(02)2+1

E. Tetravalent vanadium (yIV) - Bis (maltolato) oxovanadium [YO(mahl

2.2.2 BIOLOGICAL IMPORTANCE OF VANADIUM

Vanadium has been recognized as an essential nutritional requirement m higher

animals. It seems to be a trace metal required for normal growth and development,

also being necessary for growth and survival of mammalian cell culture (Macara,

1980). Vanadium is present in a variety of foods that we commonly eat. Rich dietary

source of vanadium (> 1 ppm) includes gelatin, skimmed milk powder, grape nuts,

dried lentils, dried Navy beans, lobsters, green pea, radishes, hazel nuts, cabbage,

oats, potato, turnip greens, rye seeds, lettuce, vegetable oils (Schroeder et ai., 1963).

Relatively little vanadium is found in fruits, wheat, millet, meat, fish, butter and

cheese (Schroeder et ai., 1963). The daily intakes of vanadium in humans have been

estimated to vary from 10 Jlg to 2 mg, depending on the environmental level of the

region of study (Nielson and Uthus, 1990; Ramasarma and Crane, 1981).

Vanadium belongs to a group of microelement, long recognized for their nutritional

and medicinal values. It was widely used for therapeutic purpose at the tum of the

20th century in France for the treatment of anemia, tuberculosis, chronic rheumatism

and diabetes (Schroeder et ai., 1963). It was also recommended to increase appetite,

strength and weight in a dose of 5mg/day in form of sodium metavanadate (Schroeder

et at., 1963). In recent years, termed as "Nutritional revolution" vanadium

compounds have become popular among body builders as an addition to multivitamin

nutritional supplements (Willsky, 1990).

Vanadium is accumulated from the sea bed by tunicates by special cells called as

vanedocytes in the trivalent form (V3+) in concentration as high as 0.15 M (Smith,

1989; Smith et ai., 1991). The uptake, tissue distribution and the excretion of various

vanadium compounds in rats have been studied comprehensively (Saxena et ai.,

1992; Setyawati et ai., 1998). The distribution of vanadium, in different body tissue

of rats after oral or i.p. treatment, was concentrated in bone> kidney> liver> spleen

>heart >testes > lung> pancreas> brain in 24 hours (Saxena et ai., 1992 ; Setyawati

12

et al., 1998). In man, vanadium is found in the fat tissues and serum in very high

concentration but not found regularly in any other tissue (Schroeder et al., 1963).

2.2.3. INSULIN MIMETIC EFFECT OF VANADIUM ON DIABETES

A report in 1971 (Meek et aI., 1971) showed for the first time that there was decrease

in the expired 14C02 on oxidation of glucose-l- 14C in rats given vanadate in drinking

water. This was the first report to show the possible relationship of vanadium and

glucose metabolism. This was supported by the experiment of Tolman and co-

workers who found that addition of metavanadate or vanadyl sulphate to preparation

of adipocytes, hepatocytes and diaphragm from the rat showed the following effects:

stimulation of glycogen synthesis in liver and the diaphragm, stimulation of glucose

oxdation and transport in adipocytes and inhibition of gluconeogenesis, similar to

those obtained with insulin (Tolman et ai., 1979). This was followed by reports

further confirming the above observation of insulin mimetic action of vanadate on

isolated adipocytes (Dubyak and Kleinzeller, 1980; Shechter and Karlish, 1980).

A major breakthrough was the demonstration in 1985 that oral administration of

sodium orthovanadate solution lowered the elevated blood sugar level in

Streptozotocin induced diabetic rats (Heyliger et aI., 1985). The relative high dose of

sodium orthovanadate used earlier was subsequently lowered to an optimal dose of

0.6 mg/ml from 0.8 mg/ml in drinking water to act as hypoglycemic agent for

experimental diabetic rats (Myerovitch et aI, 1987; Paulson et ai, 1987; Saxena et ai,

1992; Valera et ai, 1993). Sodium orthovanadate and vanadyl sulfate treatment,

through intraperitoneal injections, reduced the blood glucose level in diabetic rats

(Strout et ai, 1990; Sakurai et ai, 1990). Peroxo vanadium compounds were also

found to possess hypoglycemic activity in animals (Posneret ai, 1994; Shiheva et aI,

1994; Ramasarma, 1996). Peroxides of vanadium (peroxovanadium, pervanadate) are

about 100 fold more potent than vanadate in manifesting the biological effects of

insulin. These compounds stimulate glucose transport at low concentration and

enhance insulin-binding capacity to cells. The latter was due to an apparent increase

13

in receptor affinity (Fantus et aI, 1989; Posner et aI, 1994; Yu et aI, 1996; Yuan et aI,

1997). The hypoglycemic as well as other antidiabetic properties of organic vanadium

compounds like bis (maltolato) oxovanadium (IV), bis (picolinato) oxovanadium

(IV), bis (oxalato) oxovanadium (IV) and bis (kojato) oxovanadium (IV) have been

elucidated earlier (Yuen et aI, 1992, 1993; Sakurai et aI, 1993; Kordowiak et aI, 1997;

Yuen et aI, 1997).

Insulin like actions mediated by vanadium compounds in various responsive tissues

are shown in Table 3. Most of the insulin mimetic effects of vanadium compounds

have been periodically reviewed (Shechter, 1990; Dafnis and Sabatini, 1994;

Ramasarma, 1996; Sekar et aI., 1996, Baquer, 1998, Poucheret et aI, 1998; Fantus and

Tsiani, 1998; Verma et aI, 1998 Badmaev et ai., 1999). Vanadate treatment to diabetic

animals affects glucose metabolism, in the liver and peripheral tissues in vivo

(Sakurai et aI, 1990; Saxena et aI, 1992; Gupta and Baquer, 1998). Both hepatic

glucose production and glucose disposal in experimental diabetic rats were restored to

normal levels after administration of vanadate (Blondel, 1989). Vanadate elicited an

insulin like stimulation of the rate of glucose oxidation and 2-deoxyglucose transport

in isolated rat adipocytes in vitro (Dubyak and Kleinzeller, 1980), the maximum

stimulation was dependent on both the extracellular vanadium concentration and the

period of incubation. Similarly, the in vitro incubation of isolated adipocytes and

skeletal muscle with vanadate increased glucose uptake, glycolysis and glycogen

synthesis (Tamura et aI, 1984; Clark, 1985). These in vitro studies suggested that

maximal activating concentration of the above parameters by vanadium compounds

were nearly equal to or equivalent to the maximal activation by insulin. No additional

effect was observed in the reversal pattern of rat adipocyte glycogen synthase when

insulin and vanadate were added together in vitro Tamura et aI, 1983; 1984).

Renal hypertrophy during diabetes, which was closely linked with highly active

polyol pathway, was effectively normalized by vanadate treatment (Saxena et aI,

1993;). The liver glycogen. level improved significantly in vanadate treated

streptozotocin induced diabetic rats, vanadate treated db/db mice, vanadate treated

14

/

Table: 3 Insulin Like Action Mediated by Vanadium Compounds in Various Responsive Tissues

Activity Effect Target tissue

Hexose transport Stimulated Skeletal muscle, adipocytes, brain, liver

Glucose oxidation Stimulated Adipocytes, diaphragm, liver

Glycolysis Stimulated Liver,heart, skeletal muscle Inhibited Kidney and brain

Gluconeogenesis Inhibited Liver and kidney

Glycogenolysis Inhibited Skeletal muscle, liver

Glycogenesis Stimulated Skeletal muscle, adipocytes, liver

Lipogenesis Stimulated Adipocytes

Lipolysis Inhibited Adipocytes

Ketogenesis Inhibited Liver

Urea cycle enzymes Inhibited Liver

Mitogenic activity Stimulated Various cultured cells

Translocation of IGF-II Stimulated Adipocytes

K+ uptake Stimulated Cardiac muscle cells

Ca2+ - Mg2+ ATPase Inhibited Adipocytes

Ca2+ influx Stimulated Adipose tissue

Intracellular pH Stimulated A-431 cell lines

Phospho tyrosine phosphatase Inhibited Liver

Cytoplasmic protein tyrosine Stimulated Adipocytes Kinase

Transmembrane protein tyrosine Stimulated Adipocytes kinase

Glyoxalase system Stimulated Liver, heart, skeletal muscle Inhibited Kidney, brain

(Modified from Shechter, 1990)

high sucrose fed rats and vanadate treated fa/fa obese Zuckar rats (Khandelwal and

Pugazhenthi, 1995). In both streptozotocin induced diabetic rats and db/db mice oral

administration of vanadate improves blood glucose level without increasing serum

insulin levels (Heyliger et aI., 1985 ; Meyerovitch et aI., 1987), thus indicating that

the primary site of action is on insulin target tissues. Vanadate has profound effect on

elevation of glucose-6-phosphate levels and increase in lipogenic capacity in starved

rat adipocytes in vivo and in vitro (Sekar et aI., 1998). The mechanism involved is the

combined action of vanadate in enhancing glucose entry and inhibiting

dephosphorylation of endogenously formed glucose-6-phosphate. The latter effect is

not exerted by insulin (Sekar et aI., 1998).

The various biochemical and biological manifestations of diabetes mellitus have been

accorded to change that occur primarily at the transcriptional level. The increased

mRNA levels of glucokinase and pyruvate kinase in diabetic rat liver was restored to

the normal levels after vanadate treatment suggesting that vanadate therapy could also

contribute to increased glucose utilization through hepatic glycolysis (Brichard et aI.,

1993; Valera et aI., 1993). Vanadate administration to diabetic rats reversed the

altered gluconeogenic enzymes, phosphoenol puruvate carboxykinase and tyrosine

aminotransferase transcript levels to control level in both liver and kidney tissues.

These results suggest that vanadate may decrease the excessive glucose production

through gluconeogenesis (Brichard et aI., 1993; Valera et aI., 1993). Vanadate

treatment also reversed the two-fold increase in the liver glucose transporters (GLUT-

2) gene expression in diabetic rats to normalcy. Earlier reports suggested that the

increase in the GLUT-2 mRNA level could be attributed to high glucose

concentrations rather than insulin deficiency (Asano et aI., 1992; Brichard et aI.,

1993; Valera et aI., 1993). Urea cycle enzyme arginase also showed an increase in the

mRNA level of experimental diabetic rats, which was normalized by vanadate

treatment (Salimuddin et aI., 1999). By decreasing the GLUT-2 transcripts in diabetic

rat liver, vanadium was able to restore normal liver glucose utilization.

15

2.2.4. MECHANISM OF ACTION OF VANADATE AS AN INSULIN

MIMETIC AGENT

Two alternative working hypotheses for vanadium signaling have been suggested,

firstly, at the insulin receptor site; and secondly at post-insulin receptor sites. The

proposed mechanism of insulin mimetic action of vanadium compounds is presented

in Figure (2).

At the insulin receptor site, vanadium was thought to stimulate phosphorylation of the

insulin receptor either directly or by activation of tyrosine kinase present in the p-subunit of the insulin receptor, or through its inhibitory effect on phosphotyrosine

protein phosphatase (PTPase) (Swarup et al., 1982). Quercetin, a cell permeable

inhibitor of insulin receptor tyrosine kinase, not only effectively blocked the effect of

insulin but also actually enhanced the same effects triggered by ¥anadate (Shisheva

and Shechter, 1992). The results suggest that the vanadium bypasses the step of

phosphorylation of tyrosine kinase associated with insulin receptor (Shechter et al.,

1995)

The major substrates of insulin receptor kinase is insulin receptor substrate-I (IRS-I)

which is closely linked to the phosphatidylinositol 3-kinase, src homology-2

phosphotyrosine protein phosphatase-2 (SGPTP-2), serine/threonine kinase like

mitogen activated protein (MAP kinase) and S6 kinase and and protein phosphatase-

lA (Flier, 1992; Hei et at., 1995; Kahn, 1995). Insulin activates PI3-kinase following

IRS-I phosphorylation and PI3-kinase association with two phosphotyrosyl-

methionyl-pronile-methionyl (PMPM) motif (Giorgetti et at., 1993; Skolmik et at.,

1993). The activation of PI3-kinase appears to be essential for promoting the

metabolic effects of insulin (Fukui and Hanafasa, 1989; Isakoff et at., 1995; Heller et

aI., 1996). The post receptor sites of vanadium action have also been explored.

Insulin like effects were obtained with vanadate in adipocytes depleted of insulin

receptors suggesting that vanadate acts through an insulin receptor independent

mechanism (Green, 1986). The basal activities of post receptor kinase, such as MAP

kinase (D'Onfrio et at., 1994) and non receptor protein tyrosine kinase (Elberg et at.,

16

Cytosoli.c ProteiJl Tyrosine Kinase

Insulin Receptor

PI.....-~ SyadIIesiI LipWs S~ ... Gale Kqwe ••••

GlueeleTr .........

,s~".~;':-:: --:· : " . -' --' 7-~:

·i:_'· ~. _~: .. ~.:~ ' ~ . ~~~.~~ ~ ... ;-!, 'i'~J

Figure 2: Schematic representation of the effect of vanadium compounds in the cells (Modified from Me Neill, 1994)

1994) can be increased by vanadate in diabetes. It is suggested that vanadium

enhances the intrinsic activities of these post receptor enzymes, perhaps by inhibiting

the phosphatases that would in turn inhibit the post receptor kinases (Goldfine, 1995;

Poucheret et at., 1998).

Vanadate activates PI-3 kinase (Li et at., 1997). However, vanadate does not activate

the insulin receptor neither does it induce IRS-I phosphorylation (Elberg et at., 1997).

Association with a cytosolic nonreceptor protein tyrosine kinase activates the PI3-

kinase after its phosphorylation on tyrosine motif (Shisheva and Shechter, 1992 a;

1999 b; 1993). Rat adipocytes contain an additional vanadate activated nonreceptor

memberanous protein tyrosine kinase. This membranous protein tyrosine kinase is

activated by autophosphorylation and interacts with PI3-kinase without involving

receptor activation and IRS-I phosphorylation (Elberg et ai., 1997). Altered pathways,

other than insulin stimulated cascade, that leads to an increase in glucose transport

activity require Ca2+ ions and Ca2+-binding protein. Vanadate increases the level of

cytoplasmic Ca2+ and cytoplasmic Ca2+ -binding protein (Shechter, 1990). The insulin

mimetic action of vanadium is further understood by its participation in physiological

reaction of oxidation converting vanadyl to vanadate that may generate H202

(Ramasarma et at., 1990). Hydrogen peroxide has been shown to mimic several of the

metabolic actions of insulin, including enhanced glucose transport, glucose oxidation

and inhibit lipolysis. Thus hydrogen peroxide is also referred to as "second

messenger" for insulin action (Mukherjee, 1980). Further interaction of vanadium

with H20 2 showed the synergistic effect in activating the insulin receptor in vitro

(Fantus et ai., 1989). As a result of reaction of vanadate and H202, peroxides of

vanadate are produced that have been found to be approximately 100 times more

potent than vanadate in mimicking the insulin like actions in vitro (Ramasarma,

1996). Nevertheless, use of peroxide of vanadium as insulin mimetic action is limited

because of their oxidizing nature, as they can deplete the antioxidant reservoir of

body, especially the glutathione system (Li et al.J995). Additional in vivo and in vitro

studies are required to understand further the mechanism of action of these

compounds.

17

2.2.5. VANADIUM IN A SAFER FORM WITH ENHANCED

BIOA V AILABILITY

The biological potential of vanadium is hampered by its toxicity. The toxicological

profile of vanadium compounds has been well documented (Dafnis and Sabatini,

1994; Domingo et a/., 1995). Effects of vanadium compounds on the gastrointestinal

system have been reported, green tongue being the most striking manifestation.

Diarrhea, vomiting, hepatomegaly and other liver abnonnalities are characteristic of

vanadium toxicity. Excessive vanadium level causing arrhythmia, bradycardia and

coronary spasm affects the cardiac system. Anemia, neutropenia and basophilic

granulation of leukocytes are results of toxic effects of vanadium on hematopoiesis

(Zaporowska and Wasilewski, 1989; 1992; 1993). Abnonnal body level of vanadium

also affects the central nervous system. Studies on the acute oral toxicity of vanadium

compounds in rats and mice showed that both vanadate and vanadyl are moderately

toxic (at dose of 0.2 to 1.0 mg/ml), the severity increases with the increasing valence

(Uobet and Domingo, 1984). The chronic response to various vanadium compounds,

following streptozotocin diabetes induction in both Wistar and Sprague-Dawely rats

has been studied extensively (Becker et a/., 1994; Yao et al., 1997).

To exploit the potential of vanadium compound and to enhance the bioavailability by

reducing its toxicity, the attempts are being made to reduce the dose of vanadium

required for therapeutic effectiveness (McNeill et al., 1994). The goal is to provide

vanadium with better gastrointestinal absorption and in a fonn that is best able to

produce the desired biological effects. It has been found that vanadium in fonn of

vanadate permeate cells and is converted intracellularly by glutathione to vanadyl

(Robinson, 1981). Both vanadate and vanadyl are biologically active. Theoretically,

providing vanadyl as opposed to vanadate would save the mammalian cell the trouble

of converting vanadate to intracellular vanadyl. Vanadyl, however, is poorly absorbed

from the gastrointestinal tract and also has the poor bioavailability to the body cells

(Robinson, 1981).

18

Vanadium being a member of transitional element family has neutral capability to

form complexes with organic compounds. Based on this property, organic chelators

of vanadium were used to bind vanadyl and surround it, so that it could be carried via

the hydrophobic surface of the biological membrane. Increased potency of vanadium,

using organic ligands, leads to the synthesis of many organic compounds of vanadium

(Cam et al. 1993; McNeil, 1992; McNeil et ai., 1992, Nandhini et ai., 1993, Sakurai

et ai., 1990). One of the best studied form of the vanadium has been organic

compound of vanadium bis (maltolato) oxovanadium or BMOV, a complex of

vanadyl with the common food additive maltol in 1:2 ratio (McNeil, 1992; McNeil et

ai., 1992). The effective therapeutic dose of BMOV was calculated to be 0.45 mg/kg

body weight from 0.65 mg for vanadyl sulphate (Ramanadham et ai., 1990).

Studies with inorganic forms of vanadium indicated that the administration of

vanadium to diabetic animals reduced food and fluid intake, causing polyphagia and

polydipsia the two characteristic symptoms of diabetes (Brichard et ai., 1988). A

similar effect was observed with the organic forms of vanadium therapy. However,

there was no incidence of diarrhoea that had previously been attributed to the

gastrointestinal toxicity of vanadium therapy (Yuen et ai., 1993). Other organic

compounds of vanadium besides BMOV that had been evaluted in the experimental

model of the diabetes includes bis (cysteinamide N-octyl) oxovanadium (IV) also

known as Naglivan (Cam et ai., 1993), bis (pyrolidine N-carbodithioato)

oxovanadium (IV) (Naki and Sakurai, 1994), and vanadyl cysteine methyl ester

(Sakurai et ai., 1990) .

In general, treatment of STZ diabetic rats with orgamc forms of vanadium is

considered more effective and advantageous, particularly from the safety point of

view compared to the inorganic vanadium treated animals (Nandhini et aI., 1993).

19

2.3 TRIGON ELLA FOENUM GRAECUM LINN. AN ANTIDIABETIC

AGENT

Trigonella foenum graecum is a leguminous, herbal annual plant belonging to family

Fabaceae. It is commonly called fenugreek and is widely used in the Indian

subcontinent. Its tender leaves are used as the green vegetable and seeds are used as

the condiment. Various medicinal properties have been attributed to Trigonella

earlier (Jain, 1991) and its use in traditional Indian medicine is known. The

hypoglycemic effect of Trigonella seed was first described by Fournier in 1958

(Shani-Mishkinsky et ai., 1974). The hypoglycemic properties of Trigonella seed

have been studied by various groups in various model systems as summarized in the

Table 4. Besides blood glucose lowering properties Trigonella also possesses

hypocholesterolemic properties as reported in the animal model system (Valette et

aI., 1984; Sharma, 1984; Petit et ai., 1993, 1995; Stark and Madar, 1993; Puri et aI.,

1989). The hypocholesterolemic effect of Trigonella seed powder was also

confirmed from the studies involving diabetic patients (Sharma, 1986 b; Sharma et

ai., 1990; Sharma and Raghuram, 1990). The Trigonella seed powder treatment of

patients with coronary heart disease combined with Type II diabetes (NIDDM)

showed significant decrease in the blood lipids (total cholesterol and triglycerides)

without affecting level of HDL (Bordia, 1997).

The isolation and effect of the active hypoglycemic principle from the seed of

Trigonella was first studied by Moorthy et al. (1989). Petit et al. (1995) and

Yoshikawa et al. (1997) reported the isolation of furostanol saponins called

trigoneosides la, Ib, IIa, lIb, lIla, IIIb; Glycoside D and Trifoenoside A. These

steroid saponins are the active principles owing to their hypoglycemic effect. Petit et

aI., (1995) demonstrated that these steroid saponins administrated mixed with diet

(12.5 mg/day/300g body weight) to normal and streptozotocin induced experimental

diabetic rats, lead to significant increase in the food intake by enhancing the

motivation to eat in normal rat while modifying the circadian rhythm of feeding

behaviour. It also stabilized the food consumption in diabetic rats, which was

20

Table 4: Antidiabetic Effect of Trigonellafoenum graecum (Fenugreek) Seeds:

Antidiabetic effect by Fraction! extract used

Hypoglycemic Effect

Seed extract

Seeds Defatted seeds Seeds

Whole seed powder Whole seed powder Debitterized seed powder Seed powder Whole seed powder

Whole seed powder

Hypocholesterolemic Effect

Ethanol extract Defatted seeds! Lipid extract Defatted seeds Ethanol! water extract Ethanol! water extract Ethanol extract Purified principle Whole seed powder Whole seed powder

Whole seed powder

Hyperinsulinomic Effect

Defatted Seeds Ethanol! water extract

Model System

Alloxan diabetic rats

Alloxan diabetic rats Alloxan diabetic dogs Streptozotocin diabetic mIce Alloxan diabetic rats Type I diabetic patients Type I diabetic patients Type II diabetic patients Type II diabetic patients

Type II diabetic patients

Alloxan diabetic dogs Alloxan diabetic rats

Alloxan diabetic rats Normal rats Normal rats Hypocholestrolemic rats Hypocholestrolemic rabits Type I diabetic patients Type I diabetic patients

Type I diabetic patients

Alloxan diabetic dogs Normal rats

References

Shani (Mishkinsky) et al.,1974 Ghafghazi et al., 1977 Ribes et al., 1986 Swanston- Flatt et al., 1989

Khosla et al ., 1995 Sharma, !986 a. Sharma et al ., 1990 Madar et al., 1988 Sharma and Raghuram, 1990 Sharma et al., 1996

Valette et af., 1984 Sharma, 1984

Sharma, 1986b Petit et af., 1993 Petit et al., 1995 Stark and Madar, 1993 Puri et af., 1994 Sharma, 1986 b Sharma and Raghuram, 1990

Ribs et ai, 1984 Petit et al., 1993

accompanied by a progressive stabilization of body weight, and decrease in the

cholesterol levels. The effect of fenugreek seed powder on glyoxylase I, creatine

kinase and some gluconeogenic enzymes has been investigated on alloxan diabetic

rats (Raju et ai., 1999; Genet et ai., 1999 and Gupta et ai., 1999).

3 ENZYMIC CHANGES IN LIVER AND KIDNEY IN EXPERIMENTAL

DIABETES.

3.1 GLUCOSE-6-PHOSPHATASE (D-Glucose-6-phosphate phosphohydrolase,

EC 3.1.3.9)

Glucose-6-posphatase is an enzyme essential for the regulation of blood glucose

homeostasis. It catalyzes the hydrolysis of glucose-6-phosphate to glucose and

inorganic phosphate (Pi) (Hefferen and Howell, 1977). This is the last biochemical

reaction common to gluconeogenesis and glycogenolysis. The hepatic cells are

permeable to glucose, but are impermeable to charged esters of glucose, glucose-6-

posphate. After phosphorylation glucose is captured effectively and the release of

glucose from hepatocytes requires the hydrolysis of glucose-6-phosphate. The

enzyme is widely distributed among the mammalian tissues and is localized in

endoplasmic reticulum (Nordlie, 1976; Kaur et ai., 1983), however, it is expressed in

significant amount in the liver and kidney cortex conferring these two gluconeogenic

tissues the capacity to release glucose into blood (Wirthension et ai., 1986). Inherited

deficiency of glucose-6-phosphatase leads to glycogen storage disease characterized

by hypoglycemia while on the other hand the increased activity of glucose-6-

phosphatse further complicates the hyperglycemic state in diabetes mellitus

(Mithieux, 1997). There is 4-5 fold increase in the mRNA level in both liver and

kidney of streptozotocin induced experimental diabetic rat (Minassian et ai., 1996).

Besides liver and kidney, the glucose-6-phosphatase expression is enhanced up to

eight and six fold in duodenum and jejunum of streptozotocin induced experimental

diabetic animals (Rajas et ai., 1999) leading to enhanced enzyme activity and further

complicating the hyperglycemic state. Glucose-6-phosphatase is a crucial determinant

21

of glucose production in liver and kidney, the main gluconeogenic organs, playing

crucial role in the glucose homeostasis which is deranged in both Type I as well as

Type II diabetes.

3.2 FRUCTOSE-l,6-BISPHOSPHATASE

(D-Fructose-l,6-bisphosphate I phosphohydrolase: EC 3.1.3.11)

Fructose-l,6-bisphosphatase catalyzes the hydrolysis of fructose-l ,6-bisphosphate to

fructose-6-phosphate and inorganic phosphate (Pi). The irreversible removal of the

phosphate group at carbon one reverses the direction of the carbon flux of glycolysis,

assigning Fructose-l,6-bisphosphatase a major role in gluconeogenesis. The enzyme

has been extensively studied in herbivorous animals (Horecker et aI., 1975). Besides

the liver and kidney the presence of this enzyme in the brain was established by

Phillips and Coxan (1975) and the localization in different regions of rat brain by

Kaur (1983). Moser et aI., (1982) studied the purification and properties of fructose-

1,6-bisphosphatase from liver (Jimenez-Jativa et al., 1992). The activity of enzyme in

rat serum requires optimum alkaline pH and the activity is inhibited by cAMP. In rat

liver, fructose-l,6-bisphosphatase activity is increased in state of insulin lack and

restored to normal by insulin (Weber et aI., 1966). The diabetic rat serum showed an

increase in the activity of fructose-I ,6-bisphosphatase (Jimenez-Jativa et al.,1992).

The effect of streptozotocin induced experimental diabetes on the activity of fructose-

1,6-bisphosphatase showed that there was increase in the activity in rat liver (Shibib

et aI., 1993). To understand the glucose flux and gluconeogenesis during experimental

diabetes fructose-l ,6-bisphosphatase presents a pivotal role.

3.3 HEXOKINASE (ATP: D-Glucose-6-phospho transferase, EC : 2.7.1.2)

Hexokinase is the first enzyme in the Embeden- Meyerhof pathway which catalyzes

the phosphorylation of glucose by ATP, resulting in formation of glucose-6-

phosphate. Hexokinase is also the first regulatory enzyme in the glycolytic pathway

that regulates the entry of glucose by its phosphotransferase action, thus, controlling

22

the glucose flux inside the cell. Meyerhof (1927) first used the term "Hexokinase" for

yeast autolysate, catalyzing the phosphorylation of glucose by ATP. Later Long

(1952) worked extensively on the hexokinase of rat tissues.

Kunitz and McDonald (1946) first reported the heterogeneity of hexokinase in the

yeast. Katzen and Shimke (1965) describe four types of hexokinase based on their

electrophoretic mobility. The isoenzymes of hexokinase are assigned as type II, III,

IV and I. Hexokinase type I, II, III have a low Km for glucose «0.1) relative to its

concentration in the blood (- 5mM) and are strongly inhibited by the product of the

reaction glucose-6-phosphate. The latter is the important regulatory feature because it

prevents hexokinase from utilizing the entire Pi of the cell in the form of the

phosphorylated hexose. The low Km forms of hexokinase allow phosphorylation of

glucose even when the concentration of glucose is very low in blood and tissues

(Katzen, 1967; Sols, 1968; Harris, 1994). The type IV hexokinase, also designated as

glucokinase and uniquely confined to the liver cells, has much higher Km (- 10mM).

Glucokinase exhibits strikingly different kinetic properties from the other isoenzymes

(Katzen, 1967; Harris, 1997). This isoenzyme catalyzes an ATP-dependent

phosphorylation of glucose and is not subject to the end product inhibition

(We inhouse, 1976).

Hexokinase isoenzymes are differentially distributed in sub-cellular fractions in

different tissues. In most tissues, except liver, hexokinase is present in both soluble

(cytosolic) and particulate (mitochondrial) fractions of the cells and later contributing

as much as the 30-75% of the total enzyme activity (Baquer et aI., 1975; Wilson,

1968; Sochor et aI., 1985). The equilibrium of hexokinase between the soluble and

particulate fractions may be important factor in the control of glucose

phosphorylation (Wilson, 1968). The activity of hexokinase type II and glucokinase

are greatly diminished in the absence of insulin (Sochor et aI., 1985; Tylor and Agius,

1988). The activity of these isoenzymes are restored to normal values after treatment

with insulin and vanadium treatment in liver of diabetic animals (Sochor et al., 1985;

Saxena et aI., 1992).

23

3.4 PHOSPHOFRUCTOKINASE (PFK)

(ATP: D- Fructose-6-phosphate 1- phosphotranferase, EC 2.7.1.11)

Phosphofructokinase catalyzes the phosphorylation of fructose-6-phosphate. The role

of phosphofructokinase in the regulation of glycolysis was first suggested by C.F.

Cori in the course of his classical investigation on muscle metabolism during work

(Cori, 1942). Lardy and Parks (1952) observed that ATP strongly inhibited muscle

phosphofructokinase and suggested that this inhibition may playa crucial role in the

regulation of the carbohydrate metabolism (Bloxham and Lardy, 1973).

Phosphofructokinase phosphorylates a number of sugar phosphates including

fructose-I-phosphate and glucose-I-phosphate at the rate somewhat lower than that

obtained with fructose-6-phosphate. The enzyme lacks specificity for nucleotide

triphosphate although pyrimidine nucleotides are found to be better substrates

(Uyede, 1979).

Like all kinase reactions, phosphofructokinase requires Mg2+ for its activity and A TP-

bivalent cations as the active substrate (Muntz, 1953). Phosphofructokinase exists in

different isozymes forms- isozyme A, B, and C. Isozyme A occurs in skeletal muscle

and heart, isozyme B occurs in both liver and erythrocytes, isozyme C with A form

occur in brain and in rest of the tissues it exists in hybrid form of isozyme A and B

(Tsai and Kemp, 1973). A TP, citrate and phosphocreatine are the most studied

modulators of phosphofructokinase (Mansour, 1963; Garland el al., 1963 and Kemp,

1974). Fasting or the diabetic condition reduces the amount of phosphofructokinase in

liver (Weberl974; Donofrio el al., 1984) in alloxan diabetic rats. The kinetic

properties of phosphofructokinase in kidney is different from that in liver, in the

sense that the binding of substrate fructose-6-phosphate is not co-operative in kidney

(Sola et ai., 1991). Fructose-2,6-diphosphate is strong allosteric activator of

phosphofructokinase in liver and muscle (Uyeda el al.,1981). The regulation of its

concentration by insulin and glucagon is held to be an important factor in the control

of glycolysis and gluconeogenesis (Hers and Schaftingen, 1982). The levels of

24

fructose-2,6-bisphosphate have been measured in diabetic tissues (Sochor et aI.,

1984) and the results suggest that a relationship exists between the direction of

change of glycolysis and fructose-2,6-bisphosphate content in a range of tissues in

diabetes, in accord with the proposed role of this sugar phosphate in the regulation of

glycolysis.

3.5 GLUCOSE-6-PHOSPHATE DEHYDROGENASE

(D- Glucose-6-phosphate: NADP+ l-oxidoredutase, EC 1.1.1.49)

Glucose-6-phosphate dehydrogenase is the first enzyme of the oxidative segment of

the pentose phosphate pathway (PPP). It catalyzes the committed step of the PPP

shunt i.e. irreversible conversion of glucose-6-phosphate to 6- phosphoglucono-D-

lactone, which is very unstable and is rapidly hydrolyzed to 6-phosphogluconic acid

by a lactonase. Although, irreversible under physiological conditions the reaction is

thermodynamically reversible (Beutler and Khul, 1986). Glucose-6-phosphate

dehydrogenase is highly specific for NADP+, the Kmfor NAD+ is about thousand fold

more than that for NADP+. Hence, NADP+ is the natural coenzyme in vivo. Glucose-

6-phosphate dehydrogenase catalyzes in sequential manner in which NADP+ binds

first and released last e.g. glucose-6-phosphate dehydrogenase from human platelets,

rat liver and pig liver (Kosow, 1974; Kanji et al., 1976; Thompson at al., 1976).

Glucose-6-phosphate is substrate for glucose-6-phosphate dehydrogenase and gives

the highest Vmax and the lowest Km for the enzyme. Glucose-6-phosphate

dehydrogenase is rate limiting enzyme of PPP-shunt, responding to variety of

allosteric regulators. The enzyme is competitively inhibited by NADPH with respect

to NADP+ (Levy, 1979; Askar et aI., 1996) and for the regulation of the enzyme

activity by NADPHINADP+ ratio provides a fine control. Besides NADPH, Glucose-

6-phosphate dehydrogenase is also inhibited by many other metabolites, most

important being ATP and ADP. ATP inhibits the enzymes competitively while ADP

inhibition is non-competitive (Levy, 1979, Askar et aI., 1996). In experimental

diabetic condition the activity of glucose-6-phosphate dehydrogenase is reduced

(Sochor et aI., 1985; Pugazhenthi et aI., 1991; Saxena et aI., 1992).

25

3.6 MALIC ENZYME

(L-Malate: NADP+ oxidoreductase (oxaloacetate decarboxylase), EC 1.1.1.40)

Malic enzyme catalyzes the conversion of L-malate to pyruvate. The enzyme is well

studied in several mammalian tissues like liver (Hsu, 1970; Saito and Tomita, 1973),

kidney (Saito and Tomita, 1973), muscle (Taroni et aI., 1987), erythrocytes (Shows et

al., 1970) and brain (Salganicoff and Koeppe, 1967). Malic enzyme is an important

source ofNADPH for lipogenesis, which is a minor pathway in kidney, compared to

liver and therefore there is difference in the levels of this enzyme in both the tissues.

In liver the activity of malic enzyme is 4-5 fold higher than in kidney (Sochor et al.,

1985). A correlation between malic enzyme activity and lipid synthesis is suggested

(Luine and Kuffman, 1971) and it was shown that malic enzyme activity paralleled

change in the lipid content.

Malic enzyme activity has been shown to be affected by many hormones in several

tissues. The regulation of malic enzyme by thyroid hormone has been shown. The

increase in malic enzyme activity in liver of thyroidectomized animals after thyroid

hormone treatment is due to increase in enzyme synthesis (Li et al., 1975; Saito et ai.,

1971). The other nonhepatic tissue showing induction by thyroid hormone are kidney

and heart, though the response is lower than that in liver (Dozin et ai., 1985,1986).

Malic enzyme activity in liver is also regulated by insulin. In diabetes, a decrease in

the malic enzyme activity is observed which is reversed by the administration of

insulin (Belfoire et aI., 1974; Sohal, 1988; Pugazhenthi et aI., 1991; Saxena et al.,

1992,). Increase in the enzyme activity following the insulin treatment has been

because of increase in both enzyme quantities as well as specific activity (Drake et

aI., 1983).

26

Glycogen

Phosphorylase

G-6-Pase Glucose-I-Phosphate Pi __ - __ -_..

Glucose Glucose-6-Phosphate

UDP-Glycogen synthase

NADPH

G6PD .... 6-Phosphogluconate

6-PG Decarboxylase

ATP ADP,....----......... -I-------, ... _~ Fructose-6-Phosphate

ATP

Phosphofructokinase

ADP

· · · · · · · •

Ribulose-5-phosphate + CO2

Fr-l,6-bisPasr

Lactate .... ~ .... __ Pyruvate ••••••••• ~ TCA cycle

Figure 3 : Gluconeogeneic Shuttle , its control and interrelationship with glycolysis and HMP-shunt.