Chapter II

Review of Literature

CHAPTER II

Review of Literature

Overweight and obesity are generally defined as abnormal or excess fat

accumulation that presents a risk to health. Although overweight can be caused by

accumulation of muscle, obesity is always associated with accumulation of excess fat.

Fat is a metabolic fuel and has the highest energy per gram of fat. Consequently if

excess calories have to be stored, fat is the most efficient storage form since it is energy

dense.

Metabolic fuels of the body: Carbohydrates versus Fats

The fuel reserves of a typical 70 kg man are shown in Table 1 (Cahill 1976).

Table – 1

Available Energy Reserves (Kcal)

Organ Glucose /

Glycogen

Triacylglycerol Mobilizable

Protein

Blood 60 45 0

Liver 400 450 400

Brain 8 0 0

Muscle 1200 450 24000

Adipose 80 135000 40

Triacyglycerol in adipose tissue is a far greater energy reserve than glycogen in

liver and muscle. The second largest reserve is the mobilizable protein in the muscle.

By definition an essential nutrient is one which the body is unable to synthesize.

By that definition, glucose is not an essential nutrient since it can be synthesized from

non-carbohydrate sources like lactate and amino acids. Thus there is no actual

physiological requirement for dietary carbohydrates. However, about 15 gm

carbohydrates per day can limit nitrogen loss in the body. Increasing the carbohydrate

intake to 50 gm/day makes the use of amino acids for a glucose synthesis completely

not necessary. Diets containing less than 100 gm carbohydrates per day result in

ketosis. The maximal level of carbohydrate intake would occur if all the calories

requirements of an individual are met by carbohydrates alone. This would put a limit

of about 16 gm/kg body weight. (Mc Donald, 2005).

Glucose is not the preferred fuel of muscle cells under resting metabolic

condition, or even under mild exercise. In fact cardiac muscle prefers ketones (Sato

et.al., 1995). In the evolutionary history of mankind fat and protein were the major

macro nutrients, and consumption of dietary carbohydrates was opportunistic.

Digestion Absorption Transport and Metabolism of Dietary Fats

Since most humans are omnivorous, their diet will contain a mixture of fats

from plant, animal and microbial sources. Fat is an integral part of our diet. In

addition to providing energy, it also gives flavor and taste to food, helps in the

absorption of fat soluble vitamins, is a constituent of cell membranes, functions as

signaling molecules, and finally its distribution in the body gives shape to the human

body.

Although the word “fat” is assumed to represent glycerol ester of fatty acids, fat

includes esters of sphingosine and sterols also. A comprehensive naming of lipids was

addressed by the International Union and Pure and Applied Chemistry and the

International Union and Biochemistry and Molecular Biology Commission on

Biochemical Nomenclature in 1976 (IUPAC – IUB 1977). However, in the post

genomics era, to facilitate international communication about lipids a comprehensive

classification of lipids with common platform that is compatible with informatics

requirements was developed to classify massive amount of data. Hence lipids were

divided into eight categories as follows: (Fahy et al., 2005).

1. Fatty acids

2. Glycerolipids

3. Glycerophospholipids

4. Sphingo lipids

5. Sterol lipids

6. Prenol lipids

7. Saccharo lipids

8. Polyketides

All these lipids would be part of the diet of an individual. However,

glycerolipids would predominate in the amounts consumed.

Digestion of Fats

The digestion of fat begins in the mouth where the food is mixed with a small

amount of lingual lipase secreted by Ebner’s glands on the tongue. The lingual lipase

accumulates in the stomach between meals and remains active when the gastric pH is

less than 3.0 (Fink et al.,1984).

The lipids associated with the lingual lipase in the stomach protect the enzyme

from inactivation. The enzyme activity was irreversibly lost at pH range between 1-

2.4. Thus there is a small but significant amount of fat digestion in the stomach.

Most fat digestion is initiated in the duodenum where the food from the stomach

(acid chyme) is mixed with bile. Bile emulsifies fat making it easier for the pancreatic

lipase. In response to the acid chyme, duodenum secretes secretin . Secretin stimulates

the pancreas and bile ducts to release sodium bicarbonate to neutralize the acid.

Sodium bicarbonate flows into the duodenum through the pancreatic duet. Secretin is

shut off when the pH of the dnodenal contents becomes neutral.

Cholecystokinin (CCF) is screted in the duodenum in response to partially

digested fat and protein. CCK stimulates the release of degistive enzymes of panereas

and stimulates contraction of the gall bladder to empty bile into the duodenum.

Gastric inhibitory peptide (GIP) produced in the duodenum decreases the

stomach contractions and slows the emptying of the stomach into duodenum. GIP,

along with the glucagon like peptide (GLP-1), are known as incretins. Both of them

regulate gastric emptying.

After digestion, monoglycerides and fatty acids associate with bile salts and

phospholipids to from micelles. Micelles are much smaller than emulsion droplets.

The average diameter of a micelle is about 4-7 nm whereas that of a emulsion droplet

is about 1 m. Most fat absorption takes place in the jejunal mucosa by passive

diffusion. The bile salts left behind are reabsorbed further down to GI tract in the

Ileum, transported to the liver and recycled and secreted back into the digestive tract.

Physiologically important lipases are listed in table-2.

Table – 2

Lipase Location Substrate Products

Lingual Mouth Stomach TAGs with MCFA

FFA + DAG

Pancreatic Lipase +

Colipase

Small Intestine TAG with LCFA FFA + 2 MGA

Intestinal Lipase with Bile acids

Small Intestine TAG with MFCA FFA + Glycerol

PLA2 + bile acids Small Intestine PL with unsaturated

FA at Sn2

Unsaturated FA +

Lyso PL

Lipoprotein lipase Capillary Walls TAG in chylomicrons or VLDL

FFA + glycerol

Horomone sensitive

lipase

Adipose Cell TAG stored in

adipose cells

FFA + Glycerol

Absorption and Transport of Lipids

Fatty Acids

Many factors influence the transport of fatty acids. Chain length and

unsaturation influence their absorption. Thus medium and short chain fatty acids are

better absorbed them long chain FA. Secondly the positional distribution of fatty acid

in the Triglyceroide determines whether FA are absorbed as 2-manoacyl glycerol or as

free fatty acids, and hence influence the composition of chylomicrons.

In the intestinal uncosa triglyceridex are resynthesized using 2-

monoacylglycerols. The long chain polymer saturated fatty acids are present not only

on triglycerides but also on phospholipids, and this may effect their bioavailability and

final destination. (Ramirez et al., 2001).

A protein – dependent mechanism for the transport of lipids is proposed.

FAT/CD36 plays a key role in the uptake of fatty acids (Abumrad 2005). Fatty acids

are taken up from the intestinal lumen into theenterocytesand they are esterified with

glycerol to form triacylglycerols (Mansbach and Gorelick 2007).

Medium chain fatty acids and unsaturated fatty acids are more efficiently

absorbed than long chain saturated fatty acids. The medium chain fatty acids are

actually digested in the stomach itself by gastric lipase and absorbed in the stomach

(Christensen et al., 1995). They can also be absorbed in the aqueous phase of the

intestinal contents, and are bound to albumin and transported to the liver via the portal

vein (Decker 1996).

When the chain length of saturated fatty acids is increased there is an increase

in the proportion of fatty acids absorbed into the lymphatic pathway (Thomson et al.,

1989).

As early as 1890, Arnschink (Arnschink, 1890) reported that digestibility of

tristearein in dogs was 9 – 14%. Hoagland and Snider studied absorption of lauric

(12:0) myristic (14:0) palmitic (16:0) and stearic (18:0) acids and their tringlycerides in

rats fed with amounts varying from 5- 25%. At 5% of the diet lauric acnd myristic acid

were over 98% digestible. When fatty acid content in the diet was at 10%, the

digestibility of tristearin was 8% (Hoagland and Srider, 1943).

Cholesterol

Cholesterol absorption in the intestine is a multistep process. There are three

sources of intestinal cholesterol from the diet, from bile and from intestinal epithelial

sloughing. Diet provides about 300-500 mg cholesterol in a typical western diet bile

provides 800 – 1200 mg of cholesterol per day and the turnover of intestinal mucosal

epithelium provides about 300 mg of cholesterol. Although the entire small intestine is

capable of absorbing cholesterol, major absorption takes place in the duodenum and

proximal jejunum.

Cholesterol is absorbed solely as a monomer. It is rapidly exported from the

mucosal cells to the intestinal lymph (Wang and Carey 2003) upon entering the

enterocyte approximately half the cholesterol molecules move to the endoplasmic

reticulum where they are esterified by Acyl CoA : Cholesterol acyl transferase (ACAT)

before incorporation into nascent chylomicron particle. All the cholesterol that moves

from the intestinal lumen into intestinal mucosal cells is unesterfied. However,

cholesterol entering the lyneple is 70 – 80% esterifed. Cholesterol that escapes

intestinal absorption in excreted in the fees and constitutes a major route for its

elimination from the body (Wang 2003).

Regulation of Cholesterol Transport

Through a variety of studies, several proteins have been found to regulate the

cholesterol transport.

(a) Niemann – Pick C1 Like (NPC1L1)

This is shown to be a cholesterol uptake transporter and was identified in 2000

(Ninomiya, 2010). It is a glycoprotein of molecular mass 170 – 200 KDa and is

predicted to contain 13 transmembrane domains. Its primary structure contains a

dileucine molif at the terminus which is believed to be the targeting sequence for

endosomes (Davies et al., 2000) The estracellular loop assists of amino acids 5 to 165

which is highly conserved between species and is shown to be the cholesterol binding

domain (Infante et al., 2008).

The cholesterol transport from gut to lymph is summarized in Figure – 1

Fig. 1. www.phmd.pl

ATP Binding Cassette Transporters

ATP binding cassette transporters have a vital role in the cholesterol transport

process. There are 51 genes in human which belong to this family of transporter

(Schmitz et al., 2001). Among the ABC G subfamily G5 and G8 were found in small

intestine and liver and are implicated in the efflux of sterols from the enterocytes back

to the intestine. ABC G5 and G8 form a betrodimer (Grf et al., 2003). In the intestine,

ABC G5/G8 secrete absorbed plant sterole back into the intestinal lumen. While on the

bile canaliculus they mediate secretion of plant sterols as well as cholesterol into bile

(Tu et al., 2002a, b).

Also ABC G5/G8 are targets for the nuclear hormone receptor LXR. Upon

activation of LXR with endogenous or exogenous legends. The observed increase in

biliary cholesterol secretion depends on functional expression of ABC G5/G8 (Yu et

al., 2003).

Although several studies have shown that ABC G5/G8 are the key components

of biliary cholesterol secretion, the molecular mechanism resulting in this transport is

not known. The currently accepted model suggests that the ABC G5/G8 lifts the

cholesterol outside the outer bilayer sufficiently high for the extraction by mixed

micells (Small, 2003).

Other ABC Proteins

ABC B4 functions are a flippase for phosphaidyl choline translocating PC from

the inner to the outer bilayer of the canalicular membrane (Oude and Groen, 2000)

ATP 8B1

ATpase class I type 8B member 1 is a P type ATPase that flips phosphatidyl

serine from the outer to the inner leaflet of the canalicular membrane resulting in the

reduction of PS content and a consequent increase in the sphinogmyclin content of the

outer canalicular bilaryer. (Paul Usma, et al., 2006).

ABC B11

It is classically referred to as the bile salt export pump and mediates biliary

secretion of bile acids (Stieger, 2009).

A summary of the cholesterol transport in the bile is shown in Figure – 2.

Figure – 2 ( ahdc.vet.cornell.edu )

Denovo Biosynthetic Pathways

Fatty Acid Synthesis

In a 1986 paper Weiss et al., concluded that under usual diet with relatody high

fat content upto 40 percent even after carbohydrate rich diet, fatty acid synthase

activity remained low and they concluded that under the dietary condition of the

developed world denovo lipogenesis in man was negligible. On the contrary in young

lean rats it was 10 to 50 fold higher (Weiss et al., 1986).

Triglyceride Synthesis

Triglycerides are synthesized by two major pathways elucidated in the 50s and

60s. They are the glycerol phosphate pathway or the kennedy pathway (Kennedy 1957)

and the monoaylgylcerol pathway (Bell and Coleman 1980). They two pathways are

shown in Figure – 3.

Glycerol Phosphate Pathway Manoacylglycerol Pathway

Glycerol 3 phosphate Monoacylglycerol

GPAG MGAT

Lysophosphatidate Diacylglycerol

AGPAT DGAT1, DGAT2

Phosphatidate Triacylglycerol

PAP

Diacylglycerol

DGAT1, DGAT2

Tracylglycerol

Fig 3.

The glycerol phosphate pathway is present in most cells where as monoacyl

glycerol pathway is found in specific cell types such as enterocytes hepatocytes and

adipocytes (Xia et al., 1993). TG synthesis occurs at the endoplasmic reticulam

(Bweiss et al., 1960).

The final steps of tringlyceride synthesis whether from the MG pathway or the

Kennedy pathway involves the enzyme DGAT. Two DGAT enzymes have been

identified in humans DGAT1 and DGAT2. DGAT1 is located on elesomosome 8 and

has 488 amino acids. It is found in small intestine, adipose, mammary gland, testes,

thymus, skeletal mussel, spleen, heart and skin. Whereas DGAT 2 is located on

abromosome 11 and has 388 amino acids. It is found in liver, adipose tissue, mammary

gland, testes, heart and peripheral leukocytes (Yen et al., 2008).

The understanding of the physiological role of DGAT enzymes is mainly from

studies carried out on genetically modified ice. For DGAT1 the substrates may be

other than DAG. Both DGAT1 and DGAT2 are important modulators of energy

metabolism DGAT 2 and appears to be major enzyme regulating TG levels (Stone et

al., 2004).

Cholesterol Metabolism

Major source of cholesterol in the body is from the denovo biosynthetic

pathway. Cholesterol is synthesized by the mevalonate pathway and is shown in Figure

– 4.

Fig 4. Pathway of Cholesterol Biosynthesis

The major sites for cholesterol biosynthesis are liver and intestine. The

biosynthesis occurs in the cytoplasm and the endoplasmic reticulum from two carbon

acetate group of acetyl CoA. The acetyl CoA is derived from the mitochondria.

Another source of acetate in the cytosol of cells is from the pyruvate derived

from anacrobic glycolysis of glucose. Pyruvate in the presence of oxidants can be

decarboxylated to yield acetate shown as follows in Fig 5:

O O

| | [O2] | |

CH3 C – COO– CH3 - C – OH + CO2

Fig 5.

Thus in diabetics where the is an oxidative stress the glucose catalysed lipid

synthesis is enhanced due to the formation of cytosolic antate (Raghavamenon et al.,

2009).

The pathway of conversion of acetate to cholesterol is shown in Figure – 6. The

process sof cholesterol synthesis occurs in five major steps. First, Acetyl CoA is

converted to HMG-CoA. Second, HMG-CoA is converted to mevalonate. Third,

Mevalonate is converted to isoprene molecule isopenteryl pyrophosphate. Fourth, IPP

is converted to squalene and Fifth squalene is converted to cholesterol.

HMG CoA can be formed in the mitochondria as well as in the cytosol, using

similar enzymes. But the mitochondrial HMG CoA symthesis will be used for

production of ketone bodies which are used as oxidative fuels in several non hepatic

tissues (Hegardt, 1999).(Fig 6)

Figure – 6

Regulation of Cholesterol Biosynthesis

Regulation of HMG-CoA reductase is the main mechanism of regulating

denovo cholesterol biosynthesis. This is a regulatory enzyme and is regulated by four

mechanisms.

(a) Phosphorylation – Dephosphorylation

Phosphorylation of HMG CoA reductase decreases its activity. HMG CoA

reductase is phosphorylated by AMP activated protein kenase. The overall scheme is

shown in Figure – 7.

Figure – 7 ( www.geraldinemorgan.cl )

AMPK itself is activated by phosphorylatin AMPK phosphorylation is catalysed

by at least two enzymes : LKB1 which senses increasing AMP levels and calmoduli

dependent protein kenase (CaMK)

HMG CoA reductase activity is also maintained by the phosphoprotein

phosphatase inhibitor (PPI-1), which itself is regulated by phosphorylation.

(b) Feedback Inhibition

HMG CoA reductase is regulated through a multivalent feedback mechanism

(Brown and Gold stein, 1980) shown in Figure – 8.

Figure – 8 ( ebm.rsmjournals.com )

(c) Protcolytic Degradation of HMG-CoA Reductase

When the sterol content increase, the enzyme is degraded via the proteasome.

Binding ot sterols to the sterol sensing domain of HMG CoA reductase is the signal for

its degradation (Sever et al., 2003).

(d) Control of Gene Expression

Transcription may be major mechanism for regulation of cholesterol is animals

sensitive to cholesterol like man and hamsters. Whereas it is only a minor mechanisms

in rats, which are known to be resistant to dietary cholesterol. (Ness and Chambers,

2000).

During conditions of starvation, the body is known to dyrade itself in order to

provide amino acids for synthesis of essential proteins and lipids for energy (Deter et

la., 1967, Mortimore and Poso, 1984, Mortemore et al., 1985). The lipids are found

stored as lipid droplets in cells. These lipid droplets consisted of lipid esters

surrounded by a monolayer of phospholipids and separated from the hydroplulic

cytosolic environment by a coat of structural proteins generally known as perilipins

(Greenberg et al., 2011). The dynamic nature and multifunctionality of the lipid

droplets identified in recent years have elevated these lipid droplets to a status of

intracellular organelles (Greenberg et al., 2011).

Although lipid droplets are found in all cells they are prominent in adepose

tissue where they form a single large droplet, upto 100 m in diameter and occupy

most of the volume of cytoplasm. In other cells, the size of thelipid droplets ranges

from 0.1 m to 10 m. In the adepose tissue the droplet is formed mostly with

triglyerides whereas in other tissues the core droplet contains triglycerides as well as

cholesterol (Greenberg et al., 2011).

When fat enriched diet is provided, the liver responds to the massive influx of

lipid by upregulating the biogenesis of lipid droplets (lass et al., 2011). In order to

prevent its uncontrolled expansion, lipolysis also starts.

Hypothalamic lipid antophagy is believed to control food intake. In the fed

state PI3K/ mTOR signaling maintains autophagy at basal lower levels. Starvation

increases circulating fatty acids which activate hyper thalamic autophagcy which

results in a complex signaling resulting in increased food intake. (Singh and Cuervo,

2012, Liu and Czaja, 2013).

Oxidation of Fatty Acids

The only known mechanism resulting in enzyme yield from fatty acid oxidation

is the oxidation pathway proposed by Knoop (Knoop, 1904). This pathway occurs in

only two organelles namely the mitochondria and peroxisomes. It is the mitochondrial

oxidation that is associated with energy production.

Mitochondrial oxidation

Although every cell has it own store of fatty acids, some organs like liver

specialize in fatty acid oxidation. Free fatty acids are transported bound to albumin.

This is takes up by the cell surface fatty acid transporter FAT/CD 36. The internalized

fatty acid is esterefid with CoASH forming fatty acyl CoA. It is then transported into

the mitochondrial matrix by the help of three enzymes, earmitine palmityl transferase,

carmitine palmityl transferase 2 and carntine translocase this is shown in Figure – 9.

Figure – 9 ( www.sciencedirect.com )

The four main enzymes involved in oxidation are (1) acyl CoA

dehydrogenase, (2) enoyl – CoA hydratase, (3) hydroxyacyl CoA delydrogenase and

(4) ketoacyl CoA thilase. The scheme of reactions is shown in Figure – 10.

Figure – 10 ( www.geraldinemorgan.cl )

The enzyme of oxidation are regulated transcriptionelly and post

transcriptionally. The best known transcriptional regulators are peroxisome

proliferators – activated receptors (PPARs) and the transcriptional coactivator PGC-1

(Huss and Kelly 2004) PGC-1 modulates the activity if a number of transcription

factors that can increase the expression of enzyme of oxidation. Increasing PGC-1

has been shown to result in mitochondrial biogenesis. (Lin et al., 2005).

AMP activated protein kinase (AMPK) can also activate PGC-1 by two

mechanisms one by phosphorlyating PGC-1 on ser/thr causing an increase in the

activity (Jensen et al., 2009) second by activating Sirtuir 1. Sirtuin 1 can deacetylate

PGC-1 increasing its activity (Jensen et al., 2009)

There is one more mechanism that can activate PGC-1. That is rough high fat

induced elevation of PGC –1. 9hemcock et la., 2008).

Peroxisomal Fatty Acid Oxidation

In addition to intochondrial oxidation, peroxisonal fatty acid oxidation is also

an important fat degrading mechanism. Very long chain fatty acids are preferentially

oxidized in the peroxisomer. Peroxisomes also metabolize bole and intermediate, long

chain dicarboxylic acids produced by w-oxidation.

The peroxisomal fatty acid oxidation pathway is very similar to that of

mitochondrial oxidation but with one major difference. In the peroxisomes, the first

reaction is catalysed by acyl – CoA oxidase which is coupled to the reduction of

oxygen to H2O2. (Figure – 11).

Figure – 11.

Humans have three peroxisomal acyl CoA oxidases ACOX1, ACOX2 and

ACOX3, ACOX1 is referred to as palmitoyl CoA oxidase ACOX2 is called branched

chain acyl CoA oxidase. Gene product of ACOX3 is detected only in very low

amounts and hence its role is not known.

The hydration and oxidation step is carried out by a single bifunctional protein

unlike two district enzymes used in the mitochondria.

Human peroxisomes contain two thiolases that catalyse the terminal step in the

oxidation of fatty acids. They are ACAA1 (antyl CoA-C acyl transferase 1) and

sterol carrier protein – 213 oxoacyl CoA thiolase (SCPx)

Antiobesity Therapy

Obesity is associated with substantial increase in morbidity and mortality. It

also impairs the quality of life. Hippocrates himself had stated.

“Sudden death is more common in those who are naturally fat than in the lean”

(Lavie and Milani, 2003).

The major morbidities include Type 2 diabetes, metabolic syndrome,

hypertension, dyslipidemia, myocardial infarction, stroke, and son on (Flegal et al.,

2007).

In order to have an effective therapy to treat the large number of obese people,

several strategies have been employed which can be easily delivered to those in need of

them. Since it is now obvious that obesity is a result of excess energy in put over

energy expenditure, therapeutic agents have been used to target consumption of food,

digestion / absorption and increased energy expenditure. (Cooke and Bloom, 2006,

Sargent and Morore, 2009). However, these strategies though in use for over half a

centure have not shown success many of the so called “magic bullets” come with draws

because of their side effects (Rodgers et al., 2010).

The first recorded attempt at producing weight loss was by a Greek physician

Soranus Ephesus in the 2nd century AD. He pocescribed laxatives and purgatives as

well as heat, massage and exercise. These were practiced for over a thousand years. It

was in 1920s and 1930s that other treatments began to appears. Thyroid hormone was

the first to be tried but gave symptoms of hyperthyroidism. In 1933, 2,4 dimirophenol

was introduced. It was an coupler of oxidative phosphorylation. The side effects

included hypertheremia and was withdrawn in 1938 (Parascandola, 1974).

Amphetamines became popular weight loss drugs in the late 1930s. They also

caused by supporting appetite. The also caused increased alertness. However, a

combination of these drugs with other drugs resulted in mortality and hence they was

withdrawn in 1979 (Pool, 2001) Fen – Phen was introduced in 1992 but was but was

taken off in 1997 (Pool, 2001).

Orlistat was introduced in 2007. However, by 2011 there were 13 cases of

sever liver damage reported. (Aronne et la., 2011) Rimnobant is a cannabinoid receipts

antagonist (CBI) and ats on the brain reducing appetite (Akbas et al., 2009). Exenabds

is a GLP-1 analogue. It delays gastric emptying and promotes feeling of satiety (de

Luis et al., 2007) pramlintide is a synthetic analog of hormone amylin, which is

secreted by the pancreas in response to eating. Amylin delays gastric emptying and

promotes feeling of satiety.

Leptis is a hormone secreted primarily from the adepose tissue. It directly

stmulates the anorexigenic proopiomelano cortin nuerous inhibits the orexigenic

neuropeptidey neurons of the hypothalanues promoting satiety and weight loss (Ahima

and Flier, 2000). A combined therapy of analogue of panereatic peptide amylin and

lepten analog metreleptin were shown to reduce weight in a 20 week trial period. (Roth

et al., 2008).

Gastrointestinal and Pancreatic peptides that regulate food intake

The peptide hormones released from the gastrointestinal tract communicate

information about the current state of energy balance to the brain. These hormones

regulate appetite and energy expenditure by acting on key regions of the

hypothalaimnes and brain stem (Sam et al., 2012).

The GI tract is the largest endocrine organ in the body. In addition to its major

function as a digestive and absorptive organ, it regulates energy homeostasis by

regulating food intake. For this purpose, it secretes a variety of hormones which signal

satiety and hunger and can thus regulate food intake.

Cholecystokinin (CCK)

CCK is the first get hormone that has been shown to have anorexigenic action

(Gibbs et al., 1973). It regulates total amount of food intake per day. CCK is secreted

from entero endocrine cells in the duodenum and small intestine into intestinal camina

where it binds to CCK receptors on the vageusnerve tirmunal and transfer satiety

signals to the hypothalances vao the brain stem. (Lidde et al., 1985).

Pancreatic Polypeptide (PP)

Ideal intake induces PP secretion from panereatic islet PP cells via a vagal

mediated mechanism. PP levels increase in circulation and the amount of proportional

to the calorific load. The signal lasts for upto 6 hrs (Adrian et al., 1976). Plasma PP

levels in obese people has been shown to be lower (Lassmann et al., 1980). A PP

dependent hyperphagia in obese subjects has been implicated (Zipf et al., 1983).

Peptide Tyrosine – Tyrosine (PYY)

PYY is secreated from the ileum post prandially as a 36 AA peptide (1-36)

(Adrian et al., 1985). This is claved by dipephidyl peptidase (DPP-4) in circulation to

PYY (3-36) circulating PYY (3-36) binds to ZYZ receptorson presynaptic terminals on

hypothalamic neurous with high affinity (Michel et al., 1998) which results in the

induction anorexia.

GPL-1

GLP-1 is produced from a large precursor peptide pre-progleagon is L cells of

ileum and colon. GLP-1 is released into circulation where it is rapidly inactivated by

DPP-4 (Mentlein et al., 1993) GLP-1 exhibits anocexigenic effects through binding to

the GLP-1 receptor which widely distributed in the brain, GI tract and pancereas

(Yamato et al., 1997).

Ghrelin

Ghrelin is a guthormone which is orexigenic, Ghrelin administration stimulated

food intake and body weight gain (Kojima et al., 1999 ) plasm ghrelin levels were

elevated during fasting. Ghrelin follows a circadian rhythm. It increases before a meal

and falls soon after eating.

Endocannabinoid System of the GI tract

Endocannabinoid receptors CB1 and CB2 are epresed in the GI tract (Sanger,

2007) Administration of CBI agonists increased food intake and reduced gastric

mobility. Whereas administrating CBI antagonists suppressed food intake and weight

gain in obese animals (Fride et al., 2005).

Satiation Peptides

Enterostatin and apolipoprotein A-IV are stimulated by fat ingestein. Although

administration of entrostatin in animals decrease dietary fat intake, it did not show any

affect in humans. (Okada et al., 1992, Kovacs et al., 2003). Apolipoprotein AIV is

synthesized primarily in the jejunum and also in duodenum and ileum acts as a satietary

peptide. It was effective in reducing meal size, food intake, and weight gain reduction

in rats, its therapeutic effects in humans is not known (fujimoto et al., 1992, Fujimoto et

al., 1993).

Amylin

It is a 37 amino acid nuroendocrime peptide hormone secreted post pradially

with insulin by pancreatic cells. It is anoxigenic (Lutz et al., 1998) Amylin exerts its

action in a vagus – independent mechanism by binding to anylin receptors in the

hindbrain area. This is in conteast to poperipheral mechanisms of most other get

derived peptodes (Rushing et al., 200, Lutz et al., 1995).

Obesity and the Gut Microbes

A more recent factors associated with obesity is the gut microbes. Whether this

is a cause of obesity or consequence of obesity is debated (Conterno et al., 2011).

Human gut contains a very large number of microorganisms which are

predominantly anaerobic bacteria and constitute 500 – 1000 species (Cani and

Delzenne, 2007) Numerically it has more number of cells than the liver which is the

largest organ in the body and contributes to our will bring (Bocci, 1992).

Studies from germ free animals have shown that they have lesser body fat than

their counter parts grown under normal conditions. Even though the normal animals

consumed less food than the germ free animals they put on more weight (Backhed et

al., 2004) coloizing the germ free animals with microbiota from obese animals resulted

in a significant greater increase in total body fat than when colonized with microbiota

from lean animals (Turnbaugh et al., 2006).

Three gut microbiota studies assigned 98% of 16Sr DNA sequence to only four

bacterial phyla : Fermicules (64%) Bacteroxidetes (23%) protiobactria (8%) and activo

bacteria (3%). The remaining 2% consisted of Verrucomicrobia F usobacteria and the

TM7 phylum (Angelakis et al., 2012).

The gut microflora was found to be different in lean and obese individuals. A

list of source major microbes associated with the gut of lean and obese individuals is

shown in Table – 3.

Table – 3

Gut Microflora in Lean and Obese Individuals

Lean Individuals Obese Individuals

Methanobrevibacter Corio bacteriaceae

Treponema Lactobacillus

Xylanibacter Enterococeus

Bacteroides Faecalibactericum Prausnitzii

Bitifobaeterium Prevotella

Clostridium

Eubacterium

Roseburia

Staphylocoeus

Escherichiacoli

(From Angelakis et al., 2012)

Thus the gut microbiota can influence the extraction of more calories from

foods that are not normally digestible, resulting in obesity (De Baise et al., 2008).

Alternate Therapies for Obesity – Traditional Medicinal Plants

Traditional medicine has addressed the problem of obesity. For example in

Indian traditional medicine, Ayurveda, obesity is one of the eight undesirable

constitutions of the body. (Chandrasekaran et al., 2012). Consequently herbal

remedies are recommended in the various texts over 70 of these plants were listed in a

recent review (Chandrasekarn et al., 2012).

Google search using “Antiobesity botanicals” revealed over 50000 sites, these

describe whole plant or a fraction.

The natural botanicals that induce weight loss can be classified under the major

mechanisms by which they act. These are summarized in Table – 4.

Table – 4

Mechanisms of Antiobesity Botanicals

Antiobesity Mechanism Antiobesity Plant/ preparation

Reference

Pancreatic lipase inhibition Chitosan Bondiolotti et al., 2007,

Jun et al., 2010

Levaur Kang et al., 2006

Mate tea Martier et al., 2009

Oolong tea Kazemipoor et al., 2012

Jasmine tea Kazemipoor et al., 2012

Green tea Koo and Noh, 2007

Enhancing Thrumogresis Sece weed Maeda et al., 2007, Maeda et al., 2005

Bitter orange Haaz et al., 2006

Preventing adipocyte Turmeric Ahn et al., 2010

differentiation Capsicum Hsu and Yan 2007

Banana leaf Bai et al., 2008

Garlic Ambati et la., 2009

Flax seed Udani et al., 2004

Black soybean Kim et al., 2007

Enhancing lipid metabolism

Herbal Teas Kazemipoor et la., 2012

Cincramon Smyth and Heron, 2006

Decreasing appetite Pine nut Pasman et al., 2008

Pomegranate leaf Kazemipoor et al., 2012

Ginseng Kim et al., 2005

Hoodia gordonu Van Heerden, 2008

Since more than 70% of Indians still depends on traditional remedies for their

ailments, plant based therapies hold key to battling the obesity epidemic.

Aim and Scope of the Present Investigations

Obesity is no longer the problem of the developed countries since it can coexist

with malnourishment in the developing countries as well. Obesity is the underlying

cause for morbidity and mortality. Excess body weight demands increased work from

vital organs like the heart, until it is over burdened and stops working obesity has also

been associated with diabetes mellitus and insulin resistance. Inflammatory disorders

are also associated with obesity.

In addition to the obvious health benefits of losing excess body weight, the

concepts of beauty in the present day world demand a slimmer body shape both in men

and in women. Consequently there is an urgency in sculpting the body to acceptable

standards of health and beauty.

However, time required for exercise, the one factor that can reduce body

weight, is a luxury most obese people are unable to spare or are not willing to spare.

Hence there is a demand for “exercise-in-a-pill”.

The concept of ‘exercise- in-a-pill’ is being investigated from the point of view

of providing the benefits of exercise through a pill.

Exercise is a human invention, triggered by the natural instinet and

physiological need to be active. In the non hunter – gatherer environment, this need for

physical activity would survive only if there is a ‘reward’ associated with exercise.

There is a sense of ‘feed good’ associated with exercise. The ‘feel-good’ effect has

been identified as a result of certain hormones.

Serotonim is called happeness hormone

Endrophus reduces anxiety

Dopamine increase mental alertness

Phenyl ethylamine results in a feeling of falling in love Ghrelin reduces stress.

These hormones are released during exercise. In addition, erythropoietin has

been shown to increase the desired to exercise in experimental anaimals.

Nevertheless, exercise has been proven beyond doubt to strengthen the immune

system, cardio vascular, musculo – skeletal, gastro intestined and nuro-hormonal

systems. Coupled with this, good diet, stress management, abstinence from smoking

and such other behaviorual modification would be required for a healthy life.

In this study an attempt is made to validate traditional knowledge regarding a

botanical which is believed to lower lipids and act as an anti obesity remedy.

Artocarpus lokoocha Roxb belongs to the family moraceae. It is a large tree

which groups upto 14 mt in height. (Fig 12)

Fig 12. Artocarpus lakoocha tree and fruit

It bears nearly round or irregular fruit, dull yellow in colour. The fruit is called “vate

huli” in Kannada and is used as a souring agent in some parts of Karnataka. It is

believed to reduce body weight and control blood lipids. This study was designed to

investigate the rationale of this belief using experimental animals and a small clinical

trial.

Objectives of the Present Study

The overall objective of the study was to demonstrate lipid lowering and

antiobesity properties of the whole fruit. A. lakoocha in nutritionally obese rats and on

humans. The specific objectives are as follows

1. To prepare an ethanolic extract of the whole fruit A locoocha and to determine

its antioxidant properties.

2. To test in vitro on the ability of the extract to inhibit key enzymes in the

biosynthetic pathways of lipids namely fatty acid synthesis, cholesterol

synthesis, triacylglycerol synthesis and on enzymes of lipid mobilization like

lipoprotein lipase.

3. To test lipid lowering and body weight decrease in rats fed high fat d iet.

4. To test the antiobesity and lipid lowering effects of the whole fruit on human

volunteers.

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