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CHAPTER 1

INTRODUCTION

1.1 DIABETES MELLITUS

Diabetes is a chronic metabolic disorder of carbohydrate, fat and

protein metabolism characterized by increased fasting and post-prandial blood

sugar levels. Increasing epidemic of non-insulin dependent diabetes mellitus

(NIDDM) is anticipated to rise to two-fold from the current estimate of 150

million by 2025 (Freeman et al 2009). The pathophysiological hallmarks of

NIDDM include insulin resistance, pancreatic d-cell dysfunction, and

increased endogenous glucose production. In normal conditions, insulin

regulates glucose homeostasis through suppression of hepatic glucose

production and stimulation of peripheral glucose uptake in major target tissues

such as skeletal muscle and adipocytes (Giugliano et al 2008).

1.2 TYPES OF DIABETES MELLITUS

The two most common types of diabetes are insulin-dependent

diabetes mellitus (IDDM) or (type 1) and non-insulin-dependent diabetes

mellitus (NIDDM) or (type 2).

1.2.1 Type 1 Diabetes Mellitus (IDDM)

IDDM (or Juvenile onset diabetes mellitus) results due to cell

mediated autoimmune destruction of the insulin secreting く-cells of the

pancreas leading to a deficiency of insulin in the body. Although the onset of

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the disease can occur at any age, it usually affects the younger population of

ages between 5-10 years accounting for 5-10% of all diagnosed cases. The

patients with type 1 diabetes must rely on insulin medication for survival. The

risk factors for Type 1 Diabetes include autoimmune, genetic and

environmental factors (NDFS, 2005). A reduction in insulin levels, decreased

utilization of glucose, and increased gluconeogenesis from elevated levels of

regulatory hormones including catecholamines, glucagon, and cortisol, results

in diabetic ketoacidosis.

1.2.2 Type 2 Diabetes Mellitus (NIDDM)

Two key features in the pathogenesis of type 2 diabetes mellitus (or

Adult onset diabetes mellitus) a decreased ability of insulin to stimulate

uptake of glucose in peripheral tissues, insulin resistance, and the inability of

the pancreatic く-cell to secrete insulin adequately due to く-cell failure. The

major sites of insulin resistance in type 2 diabetes are the liver, skeletal

muscle and adipose tissue (White et al 2003). Both defects such as insulin

resistance and く-cell failure are caused by a combination of genetic and

environmental factors like lifestyle habits (i.e., physical inactivity and poor

dietary intake), obesity, although the genetic factors are still poorly

understood (Holt et al 2004). Type 2 diabetes is increasingly being diagnosed

at any age nowadays and accounts for nearly 90-95% of all diagnosed cases

of diabetes. It results from majorly old age, obesity, family history of

diabetes, impaired glucose metabolism, physical inactivity and race/ethnicity

(Li et al 2004).

NIDDM is a complex disease that is currently thought to be

influenced by more than a single gene or environmental factor. Although the

contribution of genetic and environmental factors to the development of

NIDDM differs among individuals, patients generally have two common

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metabolic abnormalities: insulin resistance and defects in glucose-stimulated

insulin secretion, which leads to a disease state (Fig.1.1).

Figure 1.1 Schematic diagram of progressive pathogenesis of NIDDM

(Hee Sook Jun et al 1999).

1.2.3 Other Type of Diabetes Mellitus

Gestational diabetes is characterized by the elevation of blood

glucose level during pregnancy, which is a significant disorder of

carbohydrate metabolism due to hormonal changes during pregnancy. It is

more common among obese women and women with a family history of

diabetes. It usually resolves once the baby is born. However, after pregnancy,

5-10% of women with gestational diabetes are found to have type 2 diabetes

and 20-50% of women have a chance of developing diabetes in the next 5-10

years (NDFS, 2005).

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1. 3 INSULIN RESISTANCE

Insulin resistance is a key pathophysiological feature of type 2

diabetes. Impaired insulin secretion and free radical formation are the initial

events triggering the development of insulin resistance and its causal relations

with dysregulation of glucose and fatty acid metabolism. Even though

numerous oral hypoglycemic drugs exist alongside insulin, still there is no

promising therapy for NIDDM (Sumana et al 2001). Although some of the

drugs such as sulphonylureas, and few biguanides are valuable drugs for the

treatment of hyperglycemia in NIDDM these therapies are limited by their

poor pharmacokinetic properties, secondary failure rates and accompanying

side effects (Melander et al 1988). Hence in NIDDM patients, the decreased

ability of insulin to stimulate glucose disposal into muscle or adipose tissues

results in insulin resistance (Kahn et al 1994). Although the molecular basis

of NIDDM is poorly understood, it is well established that insulin signaling,

including the activation of insulin receptor activity, is impaired in most of the

patients with NIDDM.

1.4 INSULIN RESISTANCE AND OBESITY

The association of obesity with type 2 diabetes has been recognized

for decades, and the major basis for this link is insulin resistance. Insulin

resistance is a fundamental aspect of the etiology of type 2 diabetes and is

also linked to a wide array of other pathophysiologic secondary complications

such as hypertension, hyperlipidemia, atherosclerosis (i.e., the metabolic

syndrome, or syndrome X), and polycystic ovarian disease (Reaven et al

1995). Although many details of the mechanism by which the enlarged

adipose tissue mass that defines obesity, causing systemic insulin resistance

remain unknown, the past several years have witnessed an explosive increase

in the understanding of what may now be referred to as the adipo-insulin axis

(Barbara et al 2000). There are also grounds for considering the related

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possibility that insulin resistance and hyperinsulinemia, in addition to being

caused by obesity, can also contribute to the development of obesity.

In general, obesity leads to hyperglycemia, which in turn leads to

and exacerbates insulin resistance. Insulin resistance, if not treated, results in

hyperinsulinemia and eventually leads to full blown type 2 diabetes (Kahn et

al 2000). Obesity or excessive adiposity, particularly visceral adiposity,

contributes to and worsens insulin resistance (Kopelman et al 2000).

Body fat distribution is an important variable to consider in obesity

and its related metabolic complications with visceral fat playing an important

role in relating regional fat distribution and metabolic complications. Insulin

resistance and hyperinsulinemia appear to imply that development of obesity

and free fatty acid oxidation is the most important factor that triggers the

progression of diabetes (Lemieux et al 1994). Increased lipid oxidation is

concomitant with a decrease in glucose oxidation, glucose storage and

insulin-mediated inhibition of hepatic glucose production. Imbalance in the

carbohydrate metabolism and the efforts of physiological system to neutralize

the changes causes an overload in the endocrine system leading to defects in

lipid metabolism.

Most of the hypoglycemic drugs to different extents, apart from

their blood glucose lowering effect promote adipogenesis (Moller et al 2001)

Thus, these drugs treat one of the key symptoms of type 2 diabetes,

hyperglycemia, but exacerbate the condition of being overweight or obese.

Therefore, while these drugs are beneficial over short term, they are not

optimal for long term usage. An ideal anti-diabetic drug would be a

compound that exhibits hypoglycemic activity without promoting weight gain

(adiposity).

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1.5 PATHOPHYSIOLOGY AND COMPLICATIONS OF

DIABETES MELLITUS

NIDDM is known to have a strong genetic component with

contributing environmental determinants. Although the disease is genetically

heterogeneous, there appears to be a fairly consistent phenotype once the

disease is fully manifested. Whatever the pathogenic causes, the early stage of

type 2 diabetes is characterized by insulin resistance in insulin-targeting

tissues, mainly the liver, skeletal muscle, and adipocytes. Insulin resistance in

these tissues is associated with excessive glucose production by the liver and

impaired glucose utilization by peripheral tissues, especially muscle. These

events undermine metabolic homeostasis, but may not directly lead to overt

diabetes in the early stage. With increased insulin secretion to compensate the

insulin resistance, the baseline blood glucose levels can be maintained within

normal ranges, but the patients may have impaired response to post prandial

glucose loading and oral glucose tolerance tests. The chronic over-stimulation

of insulin secretion gradually diminishes and eventually exhausts the islet く-

cell reserve. A state of absolute insulin deficiency ensues and overt clinical

diabetes becomes fully blown (Seely et al 1993 and Olefsky et al 1999). The

transition from impaired glucose tolerance to type 2 diabetes can also be

influenced by ethnicity, degree of obesity, distribution of body fat, sedentary

lifestyle, aging, and other medical conditions (Clark et al 1998)

The quality of life of type 2 diabetic patients with chronic and

severe hypoglycemia is adversely affected. The characteristic symptoms are

tiredness and lethargy which can become severe and lead to poor work

performance in adults. The most common acute complications are metabolic

problems (hyperosmolar hyperglycemic nonketotic syndrome or HHNS) and

infections. The long-term complications are macrovascular (hypertension,

dyslipidemia, myocardial infarction, stroke), microvascular (retinopathy,

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nephropathy, diabetic neuropathy, diarrhea, neurogenic bladder, impaired

cardiovascular reflexes, sexual dysfunction), and diabetic foot disorders

(Davidson et al 1991).

1.6 INSULIN AND ITS SIGNALLING CASCADE

1.6.1 Role of Insulin

Insulin was discovered by Banting and Best in 1921 and is the first

line of remedy for type 1 diabetes, while oral anti-hyperglycemic agents exert

glycaemic control over NIDDM. Insulin is the main hormone controlling the

intermediary metabolism, having action on liver, muscle and adipocytes. Its

overall function is to conserve energy by facilitating the uptake, utilization

and storage of glucose, amino acids and fats after ingestion of food. Insulin

influences the glucose metabolism in all tissues, increasing the glucose

transport and utilization in muscle tissues and adipocytes. Insulin increases

fatty acid synthesis and triglyceride formation in adipose tissue, inhibiting

lipolysis. It also increases protein synthesis in muscle. Conversely, a fall in

circulating plasma insulin reduces cellular glucose uptake and glucose

homeostasis, affecting all the biochemical metabolic pathways and mobilizing

the fuel from the endogenic source.

1.6.2 Insulin signaling events

The insulin receptor is a hetero-tetrameric membrane protein

consisting of two identical g and く subunits. Insulin binds to the g subunit of

the insulin receptor (IR); activating the intrinsic kinase activity in the dsubunit, which results in an intramolecular trans-autophosphorylation reaction

whereby one d subunit tyrosine phosphorylates the adjacent d subunit. The

insulin receptor substrate (IRS 1-4) family of proteins (Hunter et al 1998),

specifically interacts with the phosphorylated IR through a phosphotyrosine

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binding (PTP) module, which then facilitates phosphorylation of IRS on a

number of tyrosine residues via activated IR (Sesti et al 2001). These

phosphotyrosine residues on IRS proteins provide docking sites for proteins

of Src Homology 2 (SH2) domains with p85 being the most important

regulatory subunit of the Type IA phosphatidylinositol 3' kinase (PI3K).

Figure 1.2 Insulin signaling pathway (PI3K-dependent pathways are

indicated as in skeletal muscle)

1.6.3 PI3 Kinase - Molecular switch at cellular level

Probing the molecular mechanisms of insulin signaling, it is

apparent that insulin causes the activation of a tyrosine kinase in the

intracellular domain of its receptor, which activates the Src homology-2

(SH2) domain and the multifunctional docking protein insulin receptor

substrates-1&2 (Keller et al 1994). Multi-site tyrosine phosphorylation of

IRS-1 and IRS-2 appear to recruit a number of signaling molecules through

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their src-homology-2 (SH2) domains. This includes the major down stream

signaling switch PI3 Kinase (Backer et al 1992).

The family of PI3 Kinase phosphorylates the inositol ring at the D-

3 position to give PI3-phosphate from Phosphatidylinositol, PI-4,5-

bisphosphate from PI-4-phosphate and PI-3,4,5-trisphosphate from PI-4,5-

bisphosphate, Kotani et al 1995. Hara et al 1994 demonstrated that

microinjection of dominant negative mutant p85 inhibits the translocation of

GLUT4 in 3T3-L1 cells using a plasma membrane lawn assay in addition to

inhibiting production of PI-3,4,5-trisphosphate suggesting that this lipid may

be a mediator for downstream effects on glucose transport.

Figure 1.3 Schematic diagram of insulin signaling and glycogen

synthesis pathway

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The phosphatidyl inositol 3 kinase (PI3K) pathway is involved

mainly in mediating the metabolic effects of insulin, such as glucose

transport, glycogen and protein synthesis, ions and amino acid transport, and

lipid metabolism (Saltiel et al 2001). The PI3K is a heterodimer composed of

a p110 catalytic subunit and a p85 regulatory subunit. After the SH2 domain

of the p85 regulatory subunit of PI 3-kinase attaches to IRS-1, p110 catalytic

subunit, which is responsible for the lipid kinase activity of PI3K, catalyzes

the phosphorylation of membrane-bound PIP2 to PIP3. This step has been

shown to be a critical step which ultimately leads to insulin dependent

translocation of glucose transporter 4 (GLUT4) to plasma membrane.

Increased PIP3 activates a protein kinase cascade, stimulating the

phosphoinositide dependent kinase (PDK) (Alessi et al 1997), which

phosphorylates and activates two classes of serine/threonine kinases: protein

kinase B (PKB, also known as Akt) and the atypical protein kinase C (PKC)

(isoforms こ and そ) (Le Good et al 1998). Members of the PKC family of

serine/threonine kinases have been implicated in several of insulin’s actions.

Different isoforms of PKC have been shown to undergo translocation from

the cytosol to the membrane in response to insulin stimulation in different

tissues (Tsuru et al 2002). Atypical PKCs (こ and そ) have been proposed to

play a role in insulin-dependent glucose transport (Standaert et al 1999).

Initial tyrosine kinase phosphorylation and its downstream cellular events in

the insulin signaling cascade results in the translocation of insulin-stimulated

GLUT4 and the glucose homeostasis becomes normalized. PKB

phosphorylates and regulates the function of many cellular proteins involved

metabolism, apoptosis and proliferation (Nicholson et al 2002). Among the

targets of PKB are glycogen synthase kinase 3 (GSK3) and glycogen-

associated protein phosphatase 1 (PP1). GSK3 is phosphorylated and

inactivated in response to PKB stimulation. Decreased activity of GSK3 leads

to the dephosphorylation and activation of glycogen synthase (GS). Insulin

activates GS by promoting its dephosphorylation, through the inhibition of

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kinases such as protein kinase A or GSK3, and activation of PP1 (Brady et al

1997). Upon PPI activation downstream of PI3K, PKB transmits the insulin

signal by phosphorylation of GSK3, the forkhead transcription factors and

cAMP response element-binding protein (Chen et al 2001).

1.6.4 Glucose and Glucose transporters

Glucose is the primary source of energy for the body's cells, and

blood. Glucose is transported from the intestines or liver to body cells via the

bloodstream, and is made available for cell absorption via the hormone

insulin. Many factors affect a person's blood sugar level and the body's

homeostatic mechanism when operating normally, restores the blood sugar

level to a narrow range of about 80 to 120 mg/dL. Mostly mammalian cells

transport glucose through a family of membrane proteins known as glucose

transporters (GLUTs).

1.6.5 Impact of glucose transporters

GLUTs are integral membrane proteins which contain 12

membrane spanning helices with both the amino and carboxyl termini

exposed on the cytoplasmic side of the plasma membrane. Glucose is

transported into the cell via facilitative glucose transporters which catalyze

the transport of glucose into the target tissues. Different isoforms of GLUTs

distributed in the insulin sensitive targeted tissues are responsible for the

glucose transport (Table 1.1). Among the established functional facilitative

glucose transporter isoforms (GLUT1 to 11), GLUT5 is a fructose transporter.

GLUT1 is ubiquitously expressed with particularly high levels in human

erythrocytes and in the endothelial cells lining the blood vessels of the brain.

GLUT3 is expressed primarily in neurons and together GLUT1 and GLUT3

allow glucose to cross the blood brain barrier and enter neurons. GLUT2, a

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low affinity glucose transporter is present in liver, intestine, kidney, and

pancreatic く cells.

The GLUT4 isoform is the major insulin responsive transporter that

is predominantly restricted to skeletal muscle, adipose tissue, and cardiac

muscle. GLUT8, which is specifically expressed in the testes and GLUT11

enhance glucose transport in placenta, pancreas and kidney. In contrast to the

other GLUT isoforms, which are primarily localized to the cell surface

membrane, GLUT4 transporter proteins are sequestered into specialized

storage vesicles that remain within the cells interior under basal conditions

(Pessin et al 1999).

As the post prandial glucose levels rises, there is a subsequent

increase in the circulating insulin, which activates the intracellular signaling

cascades that ultimately result in the translocation of the GLUT4 storage

compartments to the plasma membrane through a process called exocytosis.

The overall insulin dependent shift in the cellular dynamics of GLUT4 vesicle

trafficking, results in a net increase of GLUT4 protein levels on the cell

surface, thereby increasing the rate of glucose uptake occur. So, glucose

transporters are essential for sugar transport and are responsible for energy

supply to the cell.

Diminished expressions of GLUT4 transporters constitute an

obvious mechanism of insulin resistance, since there are fewer intracellular

transporters available for the recruitment to the plasma membrane. Both the

reduced basal level and insulin stimulated glucose transport in isolated

adipocytes is accompanied by reduced expression of GLUT4 proteins and the

encoding of mRNA in type 2 diabetes (Garvey et al 1993). Given the critical

role of GLUT4 in insulin stimulated glucose transport, pre-translational

events leading to cellular GLUT4 depletion appear to be the predominant

mechanism of insulin resistance in adipocytes.

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Table 1.1 Types and distribution of various glucose transporters

Types of

Glucose

Transporters

Major Tissue Distribution

GLUT1 Brain, Erythrocytes, Placenta, Kidney, Colon, Adipose

Tissue, Muscle

GLUT2 Liver, Kidney, Small Intestine, く cell

GLUT3 Brain, Neurons, Placenta, Kidney, Fetal Muscle

GLUT4 Skeletal Muscle, Adipose Tissue, Cardiac myocytes

GLUT5 Small Intestine, Testes

GLUT7 Hepatic microsome, Endoplasmic Reticulum

GLUT8 Testes, Placenta, Blaostocyst

GLUT9 Spleen, Peripheral leucocytes, Brain

GLUT10 Liver, Pancreas

GLUT11 Heart, Skeletal Muscle, Placenta, Kidney

GLUT12 Skeletal Muscle, Adipose tissue, Small Intestine, Prostate

GLUT13 Brain

Altered GLUT4 expression in skeletal muscle may also contribute

to insulin sensitivity. In skeletal muscle GLUT4 protein levels are positively

correlated with insulin stimulated glucose uptake rates in vitro with

differentiated L6 myotubes (Walker et al 1989) and in vivo (Garvey et al

1994). The clear implication is that the abnormality in basal GLUT4

targeting/trafficking is a distinct defect, intrinsic to the glucose transport

system, which is mechanistically linked to impaired GLUT4 translocation.

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1.7 PHARMACOLOGICAL TREATMENT AND LIMITATIONS

1.7.1 Glucose Lowering Drugs - Oral administration

Five classes of oral hypoglycemic agents are being used for the

treatment of type 2 diabetes (Table 1.2) including sulphonylureas, biguanides,

alpha-acarbose inhibitors, insulin secretagogue and thiazolidinediones.

1.7.2 Sulfonylureas

Sulfonylureas including first generation (e.g., tolbutamide) and

second generation (e.g., glyburide) sulfonylureas, enhance insulin secretion

from the pancreatic く-cells. The main side effect of this drug is hypoglycemia

and is also usually associated with weight gain due to hyperinsulinemia,

which has been implicated as a cause of drug failure (Kelly et al 1995)

1.7.3 Biguanides

Biguanides include the drug metformin, which was originally

derived from a medicinal plant, Galega officinalis. Metformin reduces plasma

glucose via inhibition of hepatic glucose production and increase of muscle

glucose uptake. It also reduces plasma triglyceride and LDL-cholesterol

levels. Side effects include weakness, fatigue and shortness of breath, nausea,

dizziness, lactic acidosis, and kidney toxicity.

1.7.4 Alpha-glucosidase inhibitors

Alpha-glucosidase inhibitors includes the drug acarbose. This drug

decreases the postprandial glucose levels by interfering with carbohydrate

digestion and delaying gastrointestinal absorption of glucose. The major side

effects are bloating and diarrhea.

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1.7.5 Thiazolidinediones

Thiazolidinediones are represented by troglitazone, rosiglitazone

and pioglitazone. This expensive oral agent acts by improving insulin

sensitivity in muscle and, to a much lesser extent, in the liver. These drugs

decrease plasma triglyceride levels, but such decrease may be associated with

weight gain and an increase in LDL-cholesterol levels. Liver toxicity is a

concern requiring monthly monitoring of liver function. Since troglitazone

(Rezulin) was more toxic to the liver than rosiglitazone and pioglitazone

resulting in dozens of deaths from liver failure in March 2000 the FDA has

withdrawn the product from the market.

1.7.6 Meglitinides

Meglitinides drug name Repaglinide augments insulin secretion,

but side effects include weight gain, gastrointestinal disturbances, and

hypoglycemia.

Table 1.2 Summary of various limitations of current drug therapies

Anti-diabetic drugs Mechanism of action Limitations/side

effects

Sulfonylureas Insulin secretagogues

(through voltage gated Ca2+

channel

activation)

Hypoglycemia,

weight gain

Biguanides

Not clearly understood

t gluconeogenesis

r Insulin mediated GUT

tintestinal Glu absorbtion

Gastrointestinal

disturbances

c- Glucosidase

inhibitors

Inhibits c – Glucosidase, the final

enzyme in the

carbohydrate digestion

Gastrointestinal

disturbances

Thiazolidinediones Ligands for PPARi(a nuclear hormone receptor, expressed

in fat and Adipocyte and regulates

Adipocyte Differentiation)

Liver toxicity,

weight gain, high

LDL cholesterol,

high cost

Meglitinides Stimulates insulin secretion Hypoglycemia,

weight gain

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1.8 INSULIN THERAPY

Insulin is usually added to an oral agent when glycemic control is

suboptimal at maximal doses of oral medications. Some diabetologists prefer

to initiate insulin therapy in patients with newly diagnosed type 2 diabetes

(DeFronzo et al 1999). Weight gain and hypoglycemia are the common side

effects of insulin therapy (Sinha et al 1996). Prolonged insulin treatment may

also carry an increased risk of atherosclerosis.

1.9 CONVENTIONAL APPROACH OF TREATMENTS

1.9.1 Exercise

Any exercise prescription should be individualized to account for

patient’s interest, physical status, capacity, and motivation. Exercising five or

six times per week enhances weight reduction. Since, people with diabetes

have not been active, exercise should start at a low level and gradually

increased to avoid adverse effects such as injury, hypoglycemia, or cardiac

problems (Alexandria et al 1994).

In type 1 diabetic patient, the lack of physiological inhibition of

insulin secretion during exercise results in a potential risk of hypoglycemia.

On the other hand, exercise-induced activation of counter regulatory

hormones might trigger an acute metabolic derangement in severe insulin-

deficient subjects. Thus, diabetic patients, before starting exercise sessions,

must be carefully educated about the consequences of physical activity on

their blood glucose and the appropriate modifications of diet and insulin

therapy. Long-term effects of regular exercise are particularly advantageous

for type 2 diabetic patients. Regular aerobic exercise reduces visceral fat mass

and body weight without decreasing lean body mass, ameliorates insulin

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sensitivity, glucose and blood pressure control, lipid profile and reduces the

cardiovascular risk.

1.9.2 Diet

Diet also a key lifestyle choice which regulates blood glucose. Since

in the heterogeneous nature of type 2 diabetes, no single dietary approach is

appropriate for all type of diabetic patients, hence meals plan and diet

modifications are generally individualized by a registered dietitian to meet the

patients need. A typical conventional approach would recommend a diet

composed of 60-65% of carbohydrate, 25- 35% of fat and 10-20% of protein,

within a limit or no alcohol consumption (Schlichtmann et al 1997).

1.10 ALTERNATIVE APPROACHES

Alternative therapies for anti-diabetic activity have been researched

extensively, particularly in India. Ideal therapies should have a similar degree

of efficacy without the troublesome side effects associated with conventional

treatments (Sinha et al 1996).

1.11 MEDICINAL HERBS

1.11.1 The importance of medicinal plants & traditional medicines

In the last few decades there has been an exponential growth in the

field of herbal medicine and it is gaining popularity both in the developing

and developed countries because of their natural origin and nil side effects

(Grover et al 2002). Plants have always been an exemplary source of drugs

and many of the currently available drugs have been derived directly or

indirectly from them. Less than 1% of some 250,000 higher plants have been

screened in-depth for their phytochemistry or pharmacological property. The

ethnomedical approach to plant drug discovery is practical, cost-effective and

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logical. This approach seems likely to increase the possibility of discovering

new drugs for the management of NIDDM (Petlevski et al 2001).

World ethnobotanical information about medicinal plants reports

more than 800 plants used in the control of diabetes mellitus (Mohamed

Bnouham et al 2006). The plants and their products can directly stimulate

insulin secretion or action and improve insulin binding. It is a big challenge to

fully exploit medicinal biodiversity to look for phytochemicals with insulin

mimetic property. Several herbs and compounds have shown anti-diabetic

activity when assessed using presently available experimental techniques

(Sangeetha et al 2010). A wide array of plant derived active principles

representing numerous chemical compounds has demonstrated the activity

consistent with their possible use in the treatment of NIDDM (Marles et al

1995).

Compounds with different structure but same therapeutic activity

isolated from plant species act as active moieties for the treatment of various

diseases including diabetes. Some of these active principles originate from

edible plants and their inclusion in the diet would undoubtedly be of some

value because of their hypoglycemic potential. Several phytomolecules

including flavonoids, alkaloids, glycosides, saponins, glycolipids, dietary

fibres, polysaccharides, peptidoglycans, carbohydrates, amino acids,

triterpeniods, steroids, xanthone, coumarins, iridoids, alkyl disulphides,

inorganic ions and guanidines obtained from various plant sources have been

reported to have anti-diabetic activity (Jarald et al 2008). Several important

drugs such as Taxol®, camptothecin, metformin, morphine and quinine have

been isolated from plant sources (Koehn et al 2005). Their contribution to the

world market for herbal remedies is as shown in Figure 1.4

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Figure 1.4 World market for drugs from plant sources

Source: Environmental Health Perspectives

1.12 MEDICINAL PLANTS AND PLANT DERIVED

COMPOUNDS IN TYPE 2 DIABETES TREATMENTS

The plant extracts and compound play an important role in treating

and preventing many of the diseases and disorders. Pioneering studies on the

active constituents of Podophyllum peltatum followed by the discovery and

development of the anti-leukemic agents, vinblastine and vincristine from

Catharanthus roseus provide convincing evidence that plants could be

sources of novel and potential chemotherapeutic agents (Joseph Baker et al

1995). The methylhydroxychalcone compound isolated from the plant

cinnamon which was reported for insulin mimetic activity in 3T3-L1

adipocytes (Karalee et al 2001) and also insulin mimetic compound from

Lagerstroemia speciosa exhibited significant glucose uptake stimulatory

effect (Fang Liu et al 2001) has been reported.

Natural products in therapeutic research continue to provide many

lead structures, which are used as templates for the development of new

drugs. Even though there are numerous plants with medicinal properties and

have been in use in siddha medicines, only 10-15% of the plant diversity has

been explored for their pharmaceutical purpose. In recent years, plants play an

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important role in drug discovery and development and there are numerous

plant derived compounds in clinical trials. More than 26 plant-based drugs

were approved/launched during 2000–2006, which also include novel

molecule-based drugs like Galanthamine HBr (Reminyl), Miglustat (Zavesca)

and Nitisinone (Orfadin) (Saklani et al 2008).

Mevinolin, a fungal product and a competitive inhibitor of く-

hydoxy-く-methyl glutaryl CoA reductase that is used for treating

hypercholesterolemia served as a template for a host of related drugs, statins

(Wang et al 1999). Plant based drugs have been a useful source for generation

of anti-diabetic drugs like miglitazone. PMI-5011 from Artemisia

dracunculus L. is in phase II clinical trial for type 2 diabetes.

1.13 INSULIN-MIMETIC COMPOUND

Gino et al 2001 reported over 50,000 samples of natural plant

extracts to isolate compounds which will mimic insulin activity. They

recently discovered a small non-peptidyl molecule (L-783,281) from a fungal

(Pseudomassaria sp) extract and have been reported as an insulin agonist in

diabetic animal models (Qureshi et al 2000). Purification of the active

compound revealed that demethylasterriquinone B1 (known as L-783,281)

structurally belong to quinone-like structure of natural product. L-783,281

seems to bind directly to the intracellular く-subunit of the insulin receptor

containing the insulin receptor tyrosine kinase activity. The binding leads to a

conformational change resulting in activation of the kinase and induction of

the insulin signaling cascade downstream of the receptor at micromolar

concentrations. L-783,281 leads to phosphorylation of a number of proteins of

the insulin signaling pathway including the く-subunit of the insulin receptor,

the insulin receptor substrate-1 and the Akt kinase (or protein kinase B). In

addition, it stimulates phosphoinositol 3-kinase. L-783,281 was also shown to

increase glucose uptake in primary adipocytes and muscle cells.

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1.14 IN VITRO - CULTURE MODEL FOR HYPERGLYCEMIC

ACTIVITY

Anandarajan et al 2006 have reported Aegles marmelos and

Syzygium cumini exhibiting glucose uptake activity via PI3K and PPARけ on

L6 myotubes using in vitro bio screen. Proanthocyanidins isolated from an

extract of grape seeds stimulated glucose uptake in insulin sensitive cells and

were also observed to exhibit significant insulinomimetic activity (Pinent et al

2004). An in vitro study suggested that amide compounds, derived from

ferulic acid appear to possess stimulatory effects on insulin secretion in rat

pancreatic RIN-5F cell (Nomura et al 2003).

The aim of the current study was to investigate the anti diabetic and

anti adipogenic properties of Cassia fistula flowers. Glucose uptake and

adipogenesis inhibitory efficacies of the crude methanolic extract of Cassia

fistula (CFME) and the bioactive pure compound isolated from it Aloe

emodin glycosides (AEG) were evaluated using L6 myotubes and 3T3-L1

adipocytes. Skeletal muscles account for approximately 75% of glucose

absorption under insulin-stimulated conditions and a reduction in insulin-

stimulated glucose uptake in skeletal muscles of type 2 diabetic patients has

been observed both in vitro (Dohm et al 1988) and in vivo (DeFronzo et al

1992). Since skeletal muscle is the primary tissue for insulin-stimulated

glucose uptake and disposal, it is considered as an important therapeutic target

tissue in type 2 diabetes. Insulin stimulates glucose uptake with high

sensitivity and maximal responsiveness only in differentiated L6 myotubes

and the expression of GLUT4 parallels the acquisition of these characteristics

as the L6 cells differentiate. L6 myotubes is therefore the best model for

studying glucose uptake (Klip et al 1990). The 3T3-L1 cell line used in the

study was selected because it plays an important role in lipid storage and

glucose homeostasis. 3T3-L1 adipocytes have been used extensively to study

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the regulation of glucose transporters, cell proliferation and insulin signaling.

During differentiation conditions, pre-adipocytes differentiate into mature

adipocytes exhibiting many of the morphological, biochemical and insulin

responsive features of normal rodent adipocyte (Zeigerer et al 2008).

1.15 IN VIVO - MODEL FOR HYPOGLYCEMIC ACTIVITY

Several medicinal plants, in particular Indian botanicals have been

reported for potential hypoglycemic activity in the Indian system of medicines

(Ivorra et al 1989 and Saxena et al 2004). Various plant species from India

having potent hyperglycemic activity have been reported using in vivo studies

and are described in the following section listed in Table 1.3.

Table 1.3 List of traditional anti-diabetic medicinal plants

Name of the plant/

Family

Reported mechanism of action of the plant

Aegle marmelos (L.)

[Family: Rutaceae]

Increases utilization of glucose; either by direct

stimulation of glucose uptake or via the mediation of

enhanced insulin secretion (Sachdewa et al 2001a) and

also decreases the elevated glucose and glycosylated

hemoglobin levels (Kamalakkanan et al 2003)

Aloe vera (L.) Burm.f.

Common name: Aloe

[Family: Aloaceae]

Maintains glucose homeostasis by controlling the

carbohydrate metabolizing enzymes (Rajasekaran et al

2004)

Annona squamosa L.

[Family: Annonaceae]

Lowers blood glucose level (Shirwaikar et al 2004)

Caesalpinia bonducella

(L.)

[Family: Caesalpiniaceae]

Increases the release of insulin from pancreatic cells

(Sharma et al 1997)

Helicteres isora L.

[Family: Sterculiaceae]

Acts through insulin-sensitizing activity (Chakrabarti

et al 2002)

Mangifera indica L.

[Family: Anacardiaceae]

Possibly acts through intestinal reduction of the

absorption of glucose (Aderibigbe et al 1999) as well

as pancreatic and extrapancreatic mechanisms

(Muruganandan et al 2005)

Punica granatum L.

Family: Punicaceae

Inhibits intestinal alpha-glucosidase activity, leading

to antihyperglycemic property (Li et al 2005)

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1.16 MEDICINAL PROPERTIES OF CASSIA FISTULA

Cassia fistula has been widely used in folk remedies and ayurvedic

medicine for hundreds of years. It has astringent, laxative, purgative, and

vermifuge properties. This plant has been used extensively to soothe and heal

burns, cancer, constipation, convulsions, delirium, diarrhea, dysuria, epilepsy,

gravel, hematuria, pimples, and glandular tumors.

Cassia fistula Linn. belongs to the family of Caesalpinaceae and is

widely cultivated throughout India and is commonly called Sarakonrai in

Tamil. The whole plant possesses medicinal properties which are useful in the

treatment of skin diseases, inflammatory diseases, rheumatism, anorexia and

jaundice (Kirtikar et al 1991). Phytochemical investigation of this plant

revealed the presence of long-chain hydrocarbons, triglycerides, sterols,

chromones, flavanoid, anthraquinones, sugar, diterpenoid, and triterpenoids

from its leaves, flowers, seeds, pods, and fruits (Kuo et al 2002). In the

present study, the anti-diabetic and anti-adipogenic activity of Cassia fistula

flowers has been evaluated using in vitro and in vivo model.

Figure 1.5 Flowers of Cassia fistula

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Table 1.4 Taxonomical information of Cassia fistula

Genus Cassia

Species Fistula

Family Caesalpinaceae

Tribe Cassieae

Subtribe Cassiinae

Class Magnoliopsida

Division Magnoliophyta

Kingdom Plantae

1.17 OBJECTIVE OF THE STUDY

The objective of this study integrates traditional medicine and the

concept-based approach of modern sciences, with the interplay of structural

chemistry and biology. In brief, the present study was to evaluate the insulin

mimetic activity of Aloe emodin glycosides from Cassia fistula on L6

myotubes and 3T3-L1 adipocytes. To achieve the objective, four major

objectives were named.

‚ To investigate the anti-diabetic and anti-adipogenic potential

of Cassia fistula extracts on L6 myotubes and 3T3-L1

adipocytes respectively using in vitro bioassays.

‚ Isolation of the active compound showing both anti-diabetic and

anti-adipogenic activity by bioassay guided fractionation and the

structural characterization of the isolated active compound by

UV, Mass spectroscopy,1H-NMR and

13C-NMR spectral

studies. High performance thin layer chromatography

(HPTLC) method development to assay the content of the

active molecule in C. fistula methanolic extract (CFME).

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‚ Comparative assessment of the effect of CFME and the isolated

pure compound on various molecular targets in the insulin

signaling cascade in vitro to postulate the possible mechanism of

action.

‚ Evaluating the effect of CFME and the isolated pure

compound in an in vivo model (i.e. diabetic rat model) on the

carbohydrate metabolism and oxidative stress by studying the

effect of various key enzymes involved in the process to

validate the anti-diabetic effect observed from the in vitro

study.