It is the aim of the thesis to investigate the properties of Insulin while reviewing current academic research on the application of insulin and its analogues.
Delving into the discovery of insulin, I shall discuss who isolated the compound and how it was done. Like many discoveries the true nature of the compound was not realised till scientific and clinical investigation had proved a beneficial bases of the drug to patients suffering from diabetes. Structural determination of the compound through spectroscopic analysis gave greater insight to the mechanism of action.
Like other biological systems, storage introduces a new interesting aspect to insulin, how polymerisation can affect stability and biological availability. The use of non-human insulin on patients who require treatment has been extensive, how does the variation of insulin within the animal kingdom affect its effectiveness on human patients, and how does the cost of such treatments compare to synthetically prepared insulin.
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This investigation will include an overview to the function within the pancreas, as pancreatic production of insulin is vital to maintain the healthy concentration of sugars within the blood plasma. As there are many large and complex proteins that all play parts in developing the molecule the review will focus on interesting transformations, in particular proinsulin with its disulphide bridges and cleavage from C-peptide to complete the synthesis of insulin.
An in depth study of the insulin cycle will highlight the need for insulin in the dynamic equilibration of glucose levels within the body. Liver function will be investigated as a central player in the conversion of fatty acids, the energy stores, to ketone bodies that are central to bringing energy into the muscles.
Difficulties facing patients with diabetes and the role of different insulin analogues to control sugar levels will be investigated. Drugs which have different release timelines within the body will be looked at and how different ligands and binding agents can prolong the absorption of insulin. Looking at the methods of administrating the drug will also be evaluated, and the side effects it may cause.
Delaying the onset of Diabetes related conditions can be achieved by living a balanced lifestyle, where exercise and diet are to a prolonged healthy life. These simple strategies can be underestimated by a healthy individual. Exercise and diet are essential to controlling blood sugars without direct intervention with more insulin drugs. Limited exposure to drugs of any nature is known to cause detrimental effects and the smaller the concentrations in the body the better.
The future of Diabetes is unknown. Further research into the illness continues, with the aim to target the faulty genes that can lead to the illness. Diabetes is on the rise, as a direct result of poorer lifestyles particularly in the post industrialised economies of the world, as well as an aging population further increasing the chances of contracting diabetes, especially type 2.
A drug, a protein, a hormone, and an academic mystery, from its discovery in the 1920's insulin has become an invaluable tool for treating Diabetes. Since then a greater understanding has evolved from countless papers on the role of insulin. The history of insulin dates back to 1869, when the Beta-cells of insulin were first noticed under the microscope by a medical student in Berlin. Some twenty years later, a medical researcher studying the role of the pancreas in digestion noticed that after removal of the pancreas from dogs flies had been attracted to their urine, investigating this revealed a high sugar level, establishing a link between Diabetes and the pancreas. This also established the true medical name for Diabetes, Diabetes Mellitus which means a siphon of honey. In 1901, Eugene Opie, suggested that the cause of Diabetes may be due to the destruction of the Islets of Langerhans, in which the insulin producing beta-cells are contained.
Another twenty years passed before the isolation of insulin by two research groups, one in a Toronto and the other in Bucharest. Nicolae Paulescu the Romanian researcher, was the first to isolate and prove the positive effect that insulin had on lowering the glucose levels, albeit in dogs. However controversially, the Nobel Prize for the discovery of insulin went to Banting and Best in Canada, although their discovery was after that of Paulescu. Banting and Best proved very successfully on diabetic children, who, after receiving their injection of insulin made a miraculous recovery within a few minutes to the amazement of their beside relatives (1).
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News of this incredible drug travelled fast and patients from other countries were more than eager to receive it themselves. Production and refinement of their technique was started by the medicinal company Eli Lilly in 1922, a year before Banting and Best were presented with the Nobel Prize. Since then further Nobel Prizes have been awarded to Fredrick Sanger for the resolving the Amino Acid sequence insulin in 1958, to Dorothy Crowfoot Hodgkin, 1964, for the Spatial Conformation of insulin by x-ray diffraction and crystallography. Although no Nobel Prize was awarded, another notable historical first was achieved in 1977; insulin was the first ever drug to be produced by the recombinant DNA method exploiting the E. coli virus, the drug was approved by the FDA under the brand name Humulin. At the same the Nobel Prize for medicine was awarded for the Radioimmunoassay of Insulin, a new method of investigating the levels or hormones in the blood. (2)
Banting & Best Nobel Lecture.
Amazingly the mechanism of action of Insulin is still not completely understood. There are systems, of greater size and complexity that scientific endeavour had managed unravel and gain a complete understanding of, thus it would seem quite strange that the very first protein to have its amino acid sequenced and its spatial conformation determined still can't be fully understood. Consequently there has been great academic interest in insulin. It is also of great importance as it function is crucial in the cell absorption of glucose and plays a role in many other biological systems. Every year there are around 300 papers published on different aspects of the chemistry and biological function of insulin.(3)
Insulin's basic function within the body is to enable cells to accept glucose. Glucose is required in the cell to provide energy through the "combustion" with oxygen, to give energy. When insulin binds to the insulin receptor, a change occurs within the cell, this change within the cell that enables glucose channels to open, allowing a passive transport due to lower glucose concentrations in then cells Glucose transport channels are opened in the cell wall when binding with insulin.
There are two theories of thought concerning the binding of the protein to receptor. In one case the idea is that specific interactions between atoms and groups on opposite molecules form strong attractions causing the binding to take place. The other theory of thought is more holistic, it is not just the bond forming of a few groups and atoms but it describes the interaction between the protein and receptor as the sum of many interactions which play together to form a strong bond. It also has a strong emphasis on the spatial conformation of the molecules, detailing a "lock and key" concept.
Dealing first with the idea that insulin forms specific bonding interactions with its receptor. The detail of these interactions be it, ionic forces, hydrogen bonding and hydrophobic interactions can be difficult specify and quantify. The sites of hydrophobic interactions are often difficult to identify as the interaction is not totally understood. However the use of neutron diffraction studies has been instrumental to determining where solvent interactions are present, therefore it is likely hydrophobic interactions are taking place where the solvent is absent. This hydrophobic quantification process is also helped by examining and understanding the structure of insulin, knowing the amino acid sequence, we can determine the polarity of the local environment and the likelihood of hydrophobic interactions.
The structure of insulin is well defined. Since 1964 we have known the spatial conformation of all non-hydrogen atoms with the help of x-ray diffraction, since then developments in older techniques and increasing computer power has enabled x-ray diffraction become the dominant definitive structural determination tool. The most recent innovation in diffraction techniques is that of neutron crystal diffraction, it is without doubt the most expensive technique in the science with a requirement of a nuclear reactor to produce high speed neutrons. Neutron diffraction has able to determine the hydrogen bonds in the structure, allowing an insight to hydrogen bonding, solvent interactions and non-static atoms in the structure.
Spectroscopic methods of structural determination have found greater difficulty, due to the complexity and size of the protein. Nevertheless studies by NMR, Electromass spray, Dynamic Light Scattering,________ have agreed with the evidence presented by x-ray and neutron diffraction. These studies have also shown new features in the chemistry often presenting new hypothesise for structure and the interactions therein.
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Secondary Structure- a helix's due to hydrogen bonding NH---- CO at biological pH. The two peptide chains both have alpha-helix. The secondary structure of insulin, in the form of the dimer, was for a long time considered to be the active complex of insulin. Published in many articles of that time, 1200KDa, however in _____ was discovered the real mass of insulin as a monomer was not 1200KDa but 600KDa.
What happens when insulin misfolds, do we find beta-sheets, thermodynamically accessible, kinetically accessible?? Effects on activity?
Tertiary Structure- structure stabilised due to thiol disulphide bridges, formed by oxidation of sulfhydryl on cystine. Salt bridges. Non polar interior so hydrophobic interactions, polar exterior helps with solvations, van der Waals.
Quaternary Structure, granules, 2 zinc ions in hexamer, stored in Beta cells. The size of the hexamer reduces the ability of the complex to move around the body easily, thus most of the granules are stored in the pancreas. There is no activity of the hexamer towards insulin receptor on adipose tissue or muscle.
The data from spectroscopic and crystallographic analysis has helped to determine the physical properties of insulin. Knowing these properties is crucial if new analogues of insulin are to be devised. The large size of the protein means there will be poor absorption through stomach. New innovations, see references on complexed insulin, possibility of prodrug combining within the body, current research.
Biosynthesis of Insulin, synthesis from mRNA when transcribed by ribosome, the DNA portion used is from the 11 chromosome.
Chemical Synthesis is technically very difficult, due to the number of Amino Acids involved. Retrosynthesis would suggest division of the molecule into alpha chain and beta chain helps to reduce the complication with side reactions, then other major divisions. Biologically proinsulin is the precursor one peptide chain which is divided twice to give the c-peptide and the alpha and beta chains of insulin. However should chemically synthesis be explored it is soon found to be not economically viable. The commercial rout is using yeast or E. coli, modifying their DNA, then allowing replication and subsequent removal and purification.
DNA recombinant synthesis, yeast and E. coli synthesis. Advantage over chemical synthesis, the use of chemical synthesis for first implanting or modifying DNA. Production scale and cost of using these methods of insulin manufacture. The possibility of changing the product is the process flexible. Purification of product, problem of using a bacterium to produce medicine, overcome how. Possibility of using yeast as a similar method of producing Insulin.
Insulin from Bovine and Porcine sources has for a long time been a standard insulin product for diabetics. What is the modern method of isolation, who were the pioneers. Organon
Differences between insulin and its analogues - insulin determir and insulin glargine. Biological Significance of crucial AA in the sequence, disulphide bond, retention through species. Isoelectric point measurements of different drugs, changing the AA sequence to change the solubility and the biological pH.
In depth investigation on the action of insulin, binding to cell wall, possible outcomes for insulin, and the cells manipulation of insulin. Changes in the cell, allowing glucose channels to open, consequences if not functioning. The importance of insulin in cell repair and function, the use of endocytosis, how this is achieved. How are the products of endocytosis further modified to become useful for the cell?
The degradation of insulin is an essential part of the insulin cycle and is achieved through a number of routes. After the release of insulin into circulation much of the non-cell-bound insulin is degraded in five minutes on the first pass of the hepatic and renal systems, this short half life leads to efficient blood glucose management. Degradation of the insulin also takes place while adsorbed on the cell surface, or after being absorbed into the cell. Another route is via the insulin degrading enzyme (IDE) which also reduces concentration levels within the body. (3)
Extra-Cell Degradation, what happens, why is degraded, binding constant, half life adsorption. Strength of disulphide bonds.
Intra-Cell Degradation, what enzymes catalyse the destruction, where do the after products go. Function in the cell. The importance of the products of destruction. Essential to cell growth, the link between diseases and cancers and non-intracellular destruction.
Insulin as a major contributor to protein synthesis. Cell growth. Usage by body builders as an aid to muscle regeneration.
IDE, function, how often does it interact with insulin, at active site, or in blood stream, or bound to incorrect tissues?? All insulin active cells have IDE. Major player in endocytosis, what are the products of the proteolysis.
Kidney Degradation, bound to the kidneys, major player in flushing out glucose in undiagnosed diabetes, any connection. How do the kidneys bind and remove sugars from the blood stream.
Liver Degradation of insulin, role of liver, fatty acid conversion to ketone bodies, glucose to glycogen, amino acids from protein breakdown in muscle gluconeogenisis to form glucose, i.e. starving people and loss of muscle mass, body builders many meals a day to prevent protein breakdown.
Cycle restart?? What are the starting materials in the manufacture of insulin, how and where are they obtained from. How does ribosome use these building blocks to construct insulin? What are the building blocks and steps to complete the synthesis; we have from mature preproinsulin, what is before? Can we establish a clear synthesis route?
Pancreatic Function - the building blocks of insulin, mature preproinsulin mRNA, preproinsulin, proinsulin, insulin + c peptide, see notes
The synthesis of insulin begins with DNA, the 11th chromosome of human DNA contains information for the coding of DNA. During the protein synthesis of insulin the 11th chromosome unravels from the alpha helix into a single strand, the mRNA. This single strand, mRNA, is passed through the ribosome, allowing the unpaired nitrogen bases to pair with the tRNA, generating the insulin peptide sequence. Upon completion of the synthesis a peptide of mature preproinsulin is formed, mature preproinsulin is folded into preproinsulin, then between the cystine residues disulphide bonds form intramolecularly, forming proinsulin. The route to insulin is now through either two intermediates, during which amino acids are broken from the continuous peptide chain to release the c-peptide, and the insulin molecule. The insulin molecule now consists of the alpha and beta chain connected through two disulphide bridges.
The difference between type 1 and type 2 diabetes on pancreatic function. Type 2, insulin not effective, type one beta cells destroyed by autoimmune response. Can we protect or foresee autoimmune destruction? Possibility of vaccination, virus attacks Islets, autoimmune response destroys B-cells. Could this be a cure for type 1? Can type 2 be tackled in the same way?
Function of insulin within the healthy, other functions of insulin, repair of tissue, skin. Related statistically, related absolutely
Abuse of insulin, i.e. bodybuilders, Increase to metabolism, repair of damaged tissues? Detectable when under investigation?
Kidney and Liver function without normal insulin production, compensation?? Review literature
After eating, digestion of food breaks down ingested ingredients from proteins and peptides to amino acids and sugars which are absorbed through the stomach wall. This begins the glucose cycle, from here immediate response from the passing of glucose in the beta cells of the pancreas will release insulin to allow cell uptake of sugars.
A delayed glucose response from digestion occurs from the amino acid conversion to sugars through a complex mechanism of the liver. The liver can also use this process to convert fatty acids to glucose in times of fasting. The storing of energy is also facilitated from the liver by the opposite reaction, conversion of sugars to fatty acids, which are then easily stored. These fat cells are very efficient energy sources weight for weight.
Drug administration, the common insulin analogues, type 2 and 1 differences. Insulin Pumps, continuous insulin administration, much better control of blood glucose; however, if NHS criteria are not met the cost of funding this type of healthcare can be as much as £5000 per annum.
Diabetics and other health problems associated with the disease. What can this tell us about the role of insulin? Cardiovascular, macrovascular, microvascular problems leading to amputations, renal failure, neuropathy (nerve failure), retinopathy (vision loss),
Balanced lifestyle and controlled diet, glycaemia control, introduce SSB article. Normally newly diagnosed type two diabetes patients are advised to control their condition through diet and exercise. Exercise reduces glucose in the blood, and reducing the consumption of sugars or sugary carbohydrates in favour of starchy foods and proteins is often advised. Exercise removes glucose from the blood and the dietary regime prevents rapid glucose uptake, allowing the already stressed pancreas beta cells to relax from very high levels of production. Using this regime one can delay the onset of further medical conditions and medications. However, normally due to the stress on the beta cells, reduction in the production of insulin will occur creating a need for extra insulin.
Diabetes and genes, who are vulnerable, what can be done?
Future predictions in the rise of diabetes, who and where are most vulnerable. Government expenditure on drugs and health care, UN predictions vs. Diabetes association predictions, what is the true picture emerging.
Other disulphides, biological role, problem with oral administration. Digestion, high acid content, affect on the disulphides. Cystine residues normally sites of disulphides? New gel-type insulin analogue, see research papers, the new type of testing the stomach absorption.
Monomer, active compound, quick acting injections for diabetics, diffuse easily into blood. Bolus insulin, before meals to supplement the basal insulin taken once a day.
Dimer, is it used, are similar compounds used. Retain conformation, two alpha, two beta, 2L or 2R, RL. The dimer being a product of the separation of the hexamer into a more kinetic product, although not active conformation of insulin it is easily transported through the body. How using the secondary structure we can predict the activity of the dimer on interactions upon cells, still being inactive due to a hydrophobic regions being protected within the centre of the oligomer. Also diffuse easily into blood
Hexamer, long storage within the pancreas released upon the heightening of blood glucose levels, then breakdown into the dimer, how similar is long acting insulin, what variations are present. Granules present in the pancreas, released when higher glucose levels present in the blood, trigger??
New types, zinc stapled insulin, see notes, other medicinal innovation. Long acting insulin, medicinal strategy for diabetics. Modification of Human Insulin, effectiveness and use in which type of patient. What are these long acting insulins.
Diabetes, differences in needs of type 1 and type 2 strategies for medicating. Type 2 patients more likely to receive less intensive therapy, more glucose lowering medications.
What are the cost and availability of current drugs and future medicinal innovations to the developed and developing economies of the world, and what implications might this imply. Eli Lilly, first to produce, money spent on new drugs. The problems with the pharmaceutical industry, capital cost, research costs, long term investment before returns.
Can a simple organic medicinal drug be formulated to behave as insulin, like penicillin compounds, problems? Are the interactions too complex for simple organic, need for a metal centre, how does the centre behave, Lewis acid or Lewis base, something else?
What are the variations on Insulin in the animal kingdom? What are the modifications in porcine, bovine, others, how does this reflect the effectiveness in humans? Drug formulations, DNA recombinement in E.Coli synthesis of Insulin
Is enough being done to identify people and prevent the disease? Risk factors...
A drug, a protein, a hormone, and an academic mystery, explain why insulin is so important, how has it met the criteria to become a protein and a hormone, why is it not fully understood.
Is insulin a wonder drug? Receptor vs. cheap organic, can it been done, are the zinc sites too important to lose. The importance of key insulin sites for receptor binding.
Who benefits the most from diabetes treatments, are drug companies working hard to find cures, or are treatments more profitable. Is our information reliable if most research from these companies or institutions funded by these companies? Public perceptions of diabetes and Pharmacompanies.
What is definite, people will die without Insulin, it' is on the rise and better ways of absorbing insulin are needed as injections are unfavourable method of administering the compound. Are oral medications in the pipeline?