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INSULIN

INSULIN

Submitted by,

Vignesh

Xll-A

Reg No:

INSULIN

V


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INSULIN

INSULIN

Submitted b

Vivek

Xll-A

Reg

INSULIN

,

Visweswar

o:


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Acknowledgement

It is my humble pleasure to acknowledge my deep sense of gratitude to my

Biology teachers Mrs. Priya Govindan and Mrs Indhulekha and Lab assistant

Mr. Praveen for their valuable support, constant help and guidance at each

and every stage, without which it wouldnt have been possible to complete this

project.

I also register my sense of gratitude to our Principal Rev. Fr Mathew Arekalam

for his immense encouragement that has made this project successful.

Most of all I thank the Almighty for paving the path for my successful

completion of this project.


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CHRIST NAGAR SENIOR SEC. SCHOOL

CHAVARAPURAM, THIRUVALLAM

TRIVANDRUM, KERALA -695027

CERTIFICATE

This is to certify that this project work in Biology is a bonafide record

done by Reg no

for the AISSCE practical examination during the academic year 2011-

2012

Teacher in charge Examiner

Head of Institution

School seal


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Contents

Introduction

Discovery of Insulin

Structure of Insulin

Natural synthesis

Recombinant DNA technology in te synthesis of insulin

Physiological effects

What is diabetes? What causes diabetes?

Testing For Diabetes Treatment for diabetes

Conclusion

Bibliography


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Introduction

Insulin is a hormone. It makes our bodys cells absorb glucose fromthe blood. the glucose is stored in the liver and muscle as glycogen and stops

the body from using fat as a source of energy.

When there is very little insulin in the blood, or none at all, glucose

is not taken up by most body cells. When this happens our body uses fat as a

source of energy. Insulin is also a control signal to other body systems, such as

amino acid uptake by body cells. Insulin is not identical in all animals- their

levels of strength vary.

Porcine insulin, insulin from pig, is the most similar to human

insulin. Humans can receive animal insulin. However, genetic engineering has

allowed us to synthetically produce human insulin.


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DISCOVERY OF Insulin

In 1992, Dr.Frederick Banting wanted to make pancreatic

extract, which he hoped would have anti-diabetic qualities. In 1921, at the

University of Toronto, Canada, along with medical student Charles Best, they

managed to make the pancreatic extract.

Their method involved tying a string around the pancreaticduct. When examined several weeks later, the pancreatic digestive cells had

died and been absorbed by the immune system. The process left behind

thousands islets. They isolated the extracts from the islets and produced

isletin. What they called isletin became known as insulin.

Banting and Best managed to test this extract on dogs that

had diabetes. They discovered insulin.

At this point, Professor J. Mcleod, who had placed the

laboratory at their disposal, said he wanted to see a re-run of the whole trial.

After doing so he decided to get his whole research team to work on the

production and purification of insulin.

Bantingss lab where

insulin was first isolated


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J.B. Collip joined the scientific team, which now consisted of

Banting, Best, Collip and Mcleod. They managed to produce enough insulin, in

a pure form, to be able to test it on patients.

In 1922 the insulin was tested on Leonard Thompson, a 14year old diabetes patient who lay dying at the Toronto General Hospital. He

was given an insulin injection. At first he suffered severe allergic reactions and

further injections were cancelled. The scientists worked hard on improving the

extract and then a second dose of injections were administered on Thompson.

The results were spectacular.

Collip did not get on too well with Banting and Best

apparently-and he soon left the project. Best continued trying to improve theextract and managed eventually to produce enough for the hospitals demand.

Their work was privately published. Eli Lilly Company soon got to hear about it

and offered to assist. It was not long before the Eli Lilly company managed to

produce large quantities of refined insulin

In 1923 Banting and Mcleod were awarded the Nobel

Prize in physiology or Medicine. Banting shared his prize with Best and Mcleod

shared his with Collip. The patent for insulin was sold to the University ofToronto for one dollar.

C. H. Best and F. G. Banting ca. 1924

C. H. Best and F. G. Banting


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StRuCtuRE OF Insulin

Circulating, and biologically active, insulin is monomeric.

It is composed of two polypeptide chains: chain A has 21 amino acids and chain

B has 30 amino acids (in humans). Two disulfide bridges (residues A7 to B7,

and A20 to B19) covalently tether the chains, and chain A contains an internal

disulfide bridge (residues A6 to A11). Notably, the positions of these three

disulfide bonds are invariant in mammalian forms of insulin. The amino acid

sequence of both polypeptide chains and disulfide bridge positions are shown

in panel 1. At micromolar concentrations, insulin dimerizes, and in the

presence of zinc, it further associates into hexamers.

The hormone has a compact three-dimensional structure, consisting of three

helices and three conserved disulfide bridges (Panel 2). This basic fold is

present in all members of the insulin peptide family, despite divergent

sequences. A cluster of hydrophobic residues that form the core of the small

protein contributes to protein stability. This is further enhanced by constraintof the polypeptide backbone by the disulfide bridges. Surrounding its core, the

monomer has two extensive nonpolar surfaces. The first is flat and mostly

aromatic, and is buried upon dimer formation contributing to an antiparallel

beta sheet structure. The other surface is more extensive and is buried upon

hexamer formation. Interestingly, insulin uses the same surfaces for binding to

its cognate receptor that it does for self-assembly.


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Insulin Hexamer

Quat

Insul

ernary structure of

un


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NAtuRAL Synthesis

Insulin is produced in the pancreas and released when any of several stimuli

are detected. These stimuli include ingested protein and glucose in the blood

produced from digested food.

Insulin undergoes extensive posttranslational modification

along the production pathway. Production and secretion are largely

independent; prepared insulin is stored awaiting secretion. Both C-peptide and

mature insulin are biologically active.

In mammals, insulin is synthesized in the pancreas within the

-cells of the islets of Langerhans. One million to three million (pancreatic

islets) form the endocrine part of the pancreas, which is primarily an exocrine

gland. The endocrine portion accounts for only 2% of the total mass of the

pancreas. Within the islets of Langerhans, cells constitute 60-80% of all the

cells.

In -cells, insulin is synthesized from the proinsulin precursor

molecule by the of proteolytic enzymes, known as prohormone convertase, as

well as the exoprotease carboxypeptidase E. These modifications of proinsulin

remove the center portion of the molecule (i.e., C-peptide). The remaining

polypeptides (51 amino acids in total), the B- and A- chains, are bound

together by disulfide bonds.

Islets of Langerhans


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RECOMBINANt DNA tECHNOLOGY IN tHE

SYNtHESIS OF HuMAN INSuLIN

The nature and purpose of synthesizing human insulinAlthough bovine and porcine insulin are similar to human insulin, their

composition is slightly different, consequently, a number of patients immune

systems produce antibodies against it, neutralizing its actions and resulting in

inflammatory responses at injection sites. Added to these adverse effects of

bovine and porcine insulin, were fears o long term complications ensuring

from the regular injection of a foreign substance, as well as a projected decline

in the production animal derived insulin. These factors led researchers to

consider synthesizing Humulin by inserting the insulin gene into a suitable

vector, the E. coli bacterial cell, to produce insulin that is chemically identical

to its naturally produced counterpart. this has been achieved by Recombinant

DNA technology

Inside the Double helixThe genetic code for insulin is found in the DNA at the top

of the short arm of the eleventh chromosome. It contains 153 nitrogen bases

(63 in the A chain and 90 in the B chain)


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Insulin synthesis from the genetic codeThe double strand of the eleventh chromosome of DNA divides in

two, exposing unpaired nitrogen bases which are specific to insulin production.

Using one of the exposed DNA strand as a template,

messenger RNA forms in the process of transcription

The nitrogen bases of mRNA are grouped into threes, known as codons.

Transfer RNA (tRNA) molecules, three unpaired nitrogen bases bound to a

specific amino acid, collectively known as anti codon pair with complementary

bases on the mRNA

The reading of the mRNA by the tRNA at the ribosome is known as

translation. A specific chain of amino acids is formed by the tRNA following the

code determined by the mRNA. The base sequence of the mRNA has been

translated into an amino acid sequence which link together to form specific

proteins such as insulin.

The Vector (Gram negative E. coli).A weakened strain of the common bacterium Escherrichia

coli ( E. coli), an inhabitant of human digestive tract, is the factory used in the

genetic engineering of insulin.

When the bacterium reproduces, the insulin gene is

replicated along the plasmid, a circular section of DNA.

In E. coli, B-galactocidase is the enzyme that controls the

transcription of the genes. To make the bacteria produce the insulin, the

insulin gene needs to be tied to this enzyme.


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Inside the genetic engineers toolboxRestriction enzymes, naturally produced by the bacteria, act

like biological scalpels, only recognizing particular stretches of nucleosides,

such as the one that code for insulin.

This makes it possible to sever certain nitrogen base pairs

and remove the section of insulin coding DNA from one organisms

chromosome so that it can manufacture insulin. DNA ligase is an enzyme which

serves as a genetic glue, welding the sticky ends of exposed nucleotides

together.

Manufacturing Humu linThe first step is to chemically synthesize the DNA chains

that the specific nucleotide sequence characterizing the A and B polypeptide

chains of insulin.

The required DNA sequence can be determined because the amino acidcompositions of both chains have been charted. Sixty three nucleotides are


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required for synthesizing the A chain and ninety for the B chain, plus a codon

at the end of each chain, signaling the termination of protein synthesis. The

synthetic A and B chain gene for a bacterial enzyme, B-galactocidase which

carried in the vectors plasmid.

The recombinant plasmids are then introduced into E. coli

cells. Practical use of Recombinant DNA technology in the synthesis of human

insulin requires millions of copies of the bacteria whose plasmid has been

combined with the insulin gene in order to yield insulin. The insulin is

expressed as it replicates with the B-galactocidase in the cell undergoing

mitosis.

The protein which is formed consists of B-galactocidase, joined to either the A or B chain of insulin. The A and B chains are then

extracted from the B-galactocidase fragment and purified. The two chains are

mixed and reconnected in a reaction that forms the disulfide cross bridges,

resulting in pure Humulin-synthetic human insulin.

Biological implications of genetically engineered Recombinant human insulin.Human insulin is the only animal protein to have

been made in bacteria in such a way that its structure is absolutely identical to

that of the natural molecule. This reduces the possibility of complications

resulting from antibody production. In chemical and pharmacological studies,

commercially available Recombinant DNA human insulin has proven in

distinguishable from pancreatic human insulin. Initially the major difficulty

encountered was the contamination of the final product by the host cells,

increasing the risk of contamination in the fermentation broth. This danger

was eradicated by the introduction of purification process.


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The issue of hyperglycemic complications in the administration of humaninsulin.Since porcine insulin was phased out, and the majority of insulin dependent

patents are now treated with genetically engineered Recombinant human

insulin, doctors and patients have become concerned about the increase in the

hypoglycaemic episodes experienced. Although hypoglycaemia can be

expected occasionally with any type of insulin, some people with diabetes

claim that they are less cognisant of attacks of hypoglycaemia since switching

from animal derived insulin to Recombinant DNA human insulin.

Although the production of human insulin is unarguable

welcomed by the majority of insulin dependent patients, the existence of

majority of diabetics who are unhappy with the product are ignored. Although

not anew drug, the insulin derived from this new method of production must

continue to be studied and evaluated, to ensure that all its users have the

opportunity to enjoy a complication free existence.


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WHAt IS DIABEtES? WHAt CAusES

Diabetes?

Diabetes (diabetes mellitus) is classified as a metabolism

disorder. Most of what we eat is broken down into glucose. Glucose is a form

of sugar in the blood it is the principal source of fuel for our bodies.

When our food is digested the glucose makes its way into

our bloodstream. Our cells use the glucose for energy and growth. However,

glucose cannot enter our cells without insulin being present-insulin makes it

possible for our cells to take in the glucose.

A person with diabetes has a condition in which the

quantity of glucose in blood is too elevated (hyperglycemia). This is because

the body either does not produce enough insulin, produces no insulin, or has

cells that do not respond properly to the insulin the pancreas produces. This

results in too much glucose building up in the blood . this excess blood glucose

eventually passes out of the body in urine. So, even though blood has plenty of

glucose, the cells are not getting it for their essential energy and growth

requirements.

There are three main types of diabetes:

Diabetes Type 1- You produce no insulin

Diabetes Type 2- you dont produce enough insulin or your insulin is not

working properly.

Gestational Diabetes- You develop diabetes just during your pregnancy


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i

In Type 1 diabetes, according to the National Diabetes Information

Clearinghouse, the immune system attacks and destroys the insulin-producing

beta cells in the pancreas, which results in little or no production of insulin. In

type 2 diabetes, the pancreas can produce insulin. The body, however, becomes

insulin resistant, which means the insulin cannot be used properly by the body.

After a few years, the insulin production decreases. Gestational diabetes is

caused by the hormones of pregnancy or not enough insulin being produced inan expectant mother.


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Testing For Diabetes

A glucose meter (orglucometer) is a medical device for determining the

approximate concentration of glucose in the blood. It is a key element of home

blood glucose monitoring (HBGM) by people with diabetes mellitus or

hypoglycemia. A small drop of blood, obtained by pricking the skin with a

lancet, is placed on a disposable test strip that the meter reads and uses to

calculate the blood glucose level. The meter then displays the level in mg/dl ormmol/l.

Since approximately 1980, a primary goal of the management of type 1 diabetes

and type 2 diabetes mellitus has been achieving closer-to-normal levels of

glucose in the blood for as much of the time as possible, guided by HBGM

several times a day. The benefits include a reduction in the occurrence rate andseverity of long-term complications from hyperglycemia as well as a reduction

in the short-term, potentially life-threatening complications of hypoglycemia.

Four of generations blood glucose meters.


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treatment for Diabetes

Biosynthetic "human" insulin is now

manufactured for widespread clinical use using recombinant DNA technology.

Insulin currently cannot be taken orally because, like nearly all other proteins

introduced into the gastrointestinal tract, it is reduced to fragments (even

single amino acid components), whereupon all activity is lost. There has been

some research into ways to protect insulin from the digestive tract, so that it

can be administered orally or sublingually. While experimental, several

companies now have various formulations in human clinical trials.

Initially artificial insulin was isolated from the pancreas of pigs as it resembled

human insulin. However this method was discontinued due to side effects

which arose later.

Now, using recombinant DNA technology artificial insulin has been created

which can be injected into the blood via devices such as the NOVA Pen. Insulin

is available in the form of vile and has helped in breathing life into millions of

people who would otherwise be dead without this miracle discovery

Insulin Ville by NovaRapid

Insulin Pen Injectibles


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Conclusion

Insulin, a hormone vital to the normal functioning of

our body. Most of us take if for granted, but to those suffering from the lack of

it, it imparts immense meaning. The contribution by Banting and so many

others have helped millions tackle with the effects of the plague our world

faces- Diabetes. It is only in the brink that the human mind finds the will to

change and accept things. Technology has played its part in helping millions of

Diabetics, it is like a drug, once you fall into it death is inevitable. We must play

our part in staying out of this disease. This project is dedicated to spreading

awareness of the importance of insulin and treatment for diabetes.


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Physiological effects

The actions of insulin on the global human metabolism level

include:

Control of cellular intake of certain substances, most prominently

glucose in muscle and adipose tissue.

Increase of DNA replication and protein synthesis via control of amino

acid uptake.

Modification of the activity of numerous enzymes.

The actions of insulin (indirect and direct) on cells include:

Increased glycogen synthesis insulin forces storage of glucose in liver

(and muscle) cells in the form of glycogen; lowered levels of insulin

cause liver cells to convert glycogen to glucose and excrete it into the

blood.

Increased lipid synthesis insulin forces fat cells to take in blood lipids,

which are converted to triglycerides.

Increased esterification of fatty acids forces adipose tissue to make

fats (i.e., triglycerides) from fatty acid esters.

Decreased proteolysis decreasing the breakdown of protein

Decreased lipolysis forces reduction in conversion of fat cell lipid storesinto blood fatty acids.

Decreased gluconeogenesis decreases production of glucose from

nonsugar substrates, primarily in the liver. Lack of insulin causes glucose

production from assorted substrates in the liver and elsewhere.

Decreased autophagy - decreased level of degradation of damaged

organelles.

Increased amino acid uptake forces cells to absorb circulating amino

acids.


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Increased potassium uptake forces cells to absorb serum potassium;

lack of insulin inhibits absorption. Insulin's increase in cellular potassium

uptake lowers potassium levels in blood. This possible occurs via insulin-

induced translocation of the Na+/K+-ATPase to the surface of skeletal

muscle cells.

Arterial muscle tone forces arterial wall muscle to relax, increasing

blood flow, especially in microarteries; lack of insulin reduces flow by

allowing these muscles to contract.

Increase in the secretion of hydrochloric acid by parietal cells in the

stomach.

Decreased renal sodium excretion.


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BIBLIOGRAPHY

Encyclopedia of Science Technology

Recombinant DNA, Grolier Electronic

publishing

Directory of Modern Biotechnology

Taming the beast of Diabetes by McCall

Thank you


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Synthesis o

Insulin undergoes e

production pathway

independent; prepa

peptide and mature

Insulin

tensive posttranslational modificatio

. Production and secretion are largely

red insulin is stored awaiting secretion

insulin are biologically active.

along the

. Both C-


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Fluctuation of Blood Sugar

The idealized diagram shows the fluctuation of blood sugar (red) and the sugar-

lowering hormone insulin (blue) in humans during the course of a day containing

three meals. In addition, the effect of a sugar-rich versus a starch-rich meal is

highlighted


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Symptoms of Diabetes


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Date post:	06-Apr-2018
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