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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)
23
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
24
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).
25
‚ 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.
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