INI

INSULIN

Submitted by, Vignesh V Xll-A Reg No:

INSULIN

Submitted by, Vivek Visweswar Xll-A Reg No:

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.

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 20112012

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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

Introduction

Insulin is a hormone. It makes our bodys cells absorb glucose from the 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.

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 pancreatic duct. 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.

Bantingss lab where insulin was first isolated

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.

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 14 year 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 the extract 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 of Toronto for one dollar.

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

C. H. Best and F. G. Banting

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 constraint of 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.

Quaternary structure of Insulun

Insulin Hexamer

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

RECOMBINANt DNA tECHNOLOGY IN tHE SYNtHESIS OF HuMAN INSuLIN

The nature and purpose of synthesizing human insulin

Although 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 helix

The 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)

Insulin synthesis from the genetic code

The 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.

Inside the genetic engineers toolbox

Restriction 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 Humulin
The 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 acid compositions of both chains have been charted. Sixty three nucleotides are

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.

The issue of hyperglycemic complications in the administration of human insulin.

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.

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

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 in an expectant mother.

Testing For Diabetes

A glucose meter (or glucometer) 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 or mmol/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 and severity 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.

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

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.

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 stores into 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.

 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 insulininduced 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.

BIBLIOGRAPHY

Encyclopedia of Science Technology Recombinant DNA, Grolier Electronic publishing Directory of Modern Biotechnology Taming the beast of Diabetes by McCall

Thank you

Synthesis of Insulin

Insulin undergoes extensive posttranslational modification along the production pathway. Production and secretion are largely independent; prepared insulin is stored awaiting secretion. Both Cpeptide and mature insulin are biologically active.

Fluctuation of Blood Sugar

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

Symptoms of Diabetes