Insulin
I can only imagine the pain Harold Thompson felt as he carried his dying son, Leonard, into the hospital.
It was January 1922 and Leonard was still only 13 years old.
Leonard had been diagnosed just over two years before at the age of 11. His illness was terminal and there was no known cure.
weighed a frail 29 kg (65 pounds) Leonard drifted in and out of a coma. Hospital staff alikened his appearance to that of a victim of famine. With a distended abdomen, losing his hair falling out, and breath that smelled of acetone [1]
desperate to save his son‘s life Harold permitted doctors to inject Leonard with a newly discovered drug that had never before been tried on another human being.
While the first injection failed a second, 12 days later, revived the boy. [2]
Leonard’s disease was type 1 diabetes. The wonderdrug was insulin .
Insulin had first been reported in pancreatic extracts in 1921, with the original discovery of insulin from the pancreas of a dog.
having been identified by Canadian scientists Frederick G. Banting and Charles H. Best and by Romanian physiologist Nicolas C. Paulescu, who was working independently and called the substance “pancrein.” After Banting and Best isolated insulin, they began work to obtain a purified extract, which they accomplished with the help of Scottish physiologist J.J.R. Macleod and Canadian chemist James B. Collip.
Until the discovery of insulin Type 1 diabetes was fatal within months. Lives were extended with a starvation diet. Sometimes these diets were as strict as 450 calories a day, as carbohydrates could only be consumed at the bare minimum to keep the patient alive. Some patients starved to death on a diet of vegetables, fat, protein and whiskey.
Naturally, the full story is more complicated and contains as many failures as it does success. insulin and the treatment of diabetes still had a journey ahead.
The first insulin injections utilized hormone extracts from pigs, sheep, and cattle,
by the 1970s pancreata from 56 million pigs and cattle were being used each year to supply insulin for just the United States alone.
The effort is international and takes in the skills of many different disciplines such as physiologists and biochemist and chemist, it combines research from industries as diverse as that into into poisonous gases, and the clothing fibre wool.
Decades of research to characterise insulin combined with development of recombinant DNA technology together allows the treatment of diabetes mellitus using biosynthesized insulin.
This simple statement belies how long the journey was. It took until 1955 To identify all the amino acids in the two chains of insulin and the amino acids that formed disulphide bonds that join them.
by the early 1980s certain strains of bacteria had been genetically modified to produce human insulin. Today the treatment of diabetes mellitus relies primarily on a form of human insulin that is made using recombinant DNA technology. (
Britannica)
(Over the ensuing decades, however, multiple researchers sought to determine the precise identity of insulin, concluded that it was indeed a protein, identified insulin's amino acid sequence, synthesized insulin using conventional protein synthesis, showed that this “man-made” version was actually biologically active, elucidated its crystal structure; developed a technique to measure its levels in biologic specimens, cloned its cDNA, and eventually – employing recombinant DNA technology – made insulin the first recombinant therapeutic protein. [3]
Insulin was attractive as a project for two reasons. Firstly, it was one of the few pure proteins then readily available. It was easily purchased in bottles from a local pharmacy. Secondly, insulin was of major medical interest, having been used for the treatment of diabetes since the 1920s, so pharmaceutical funding could be obtained for research on the protein
Previous work by Chibnall's team indicated insulin had a much simpler composition than most proteins. Critically, it lacked two of the most commonly occurring amino acids in other proteins (tryptophan and methionine). Maurice Rees, Chibnall's chief assistant, had also discovered insulin contained a much higher content of one group of amino acids, known as alpha amino acids, than the team could account for. These amino acids appeared at just one terminal, labelled N, on the chain. Overall, the findings suggested insulin was made up of relatively short polypeptide chains. This meant the chains were potentially amenable to chemical analysis
Sanger's first task was to identify the free amino acids at the N terminal of the insulin chain. Free amino acids are single molecules that are not bound by peptide bonds to other amino acids. A number of researchers had already devised some techniques to determine end-group amino acids in proteins. However, as yet, none of these techniques had produced any reliable results. Sanger first investigated a solubility product method developed by Max Bergmann and colleagues at the Rockefeller Institute, New York. He soon rejected it, however, because it necessitated very accurate weighing of many small samples which was extremely laborious given the weighing equipment of the time
Instead, he used partition chromatography. Call Tian Inc photography to understand the composition of amino acids in wool had been developed as part of an effort to revive the wool industry in the face of competition from synthetic fibres.
Now a method was needed to mark up the amino acids and make them visible via partition chromatography. Markers or labels for the amino acids Which required heating are unsuitable because heating the nature protein and insulin is a protein.
One options is a fluoro compound. However, this is toxic.
Bit of an aside here the scientists that worked on isolating fluorine from fluorides are known as the fluorine martyrs.
fluorodinitrobenzene (FDNB) was being synthesised as part of research into the physiological effects of poisonous gases. And FDNB reacted with amino acids at room temperature.
The resulting dinitrophenyl-amino-acids also proved more stable than the peptide bonds when subjected to heat hydrolysis and appeared bright yellow when put into a solution. This was important for the next stage – partition chromatographY.
Using glycine as the first test amino acid through a red herring as politician chromatography with glycine showed two bands instead of one strong band. Had a different acid been used first this will not have happened as glycine is the exception to the rule.
By 1945 Sanger had developed a three stage method for identifying, quantitatively measuring and characterising the terminal amino acids in insulin. This involved treating the protein with FDNB, subjecting it to acid hydrolysis and then separating out the coloured compounds with chromatography.
His technique made it possible to estimate insulin had four open peptide chains. Two ended with the amino acid called phenylalanine and the other two ended with the glycine amino acid. From this it appeared that insulin possessed only two types of chains, and not 18 as Chibnall had originally hypothesised. He was able to establish that the two chains were linked together by cystine, another amino acid.
Analysing amino acids further along the two insulin chains posed a major challenge, however. Most of the available separation techniques could not fractionate a product as large as the two insulin chains.
break up the protein into mini-chains of four to five amino acids, from near the N termini allowed use of paper chromatography
If you are thinking about the Smarties experiment with wet filter paper, you’re not far wrong.
The procedure entailed putting a drop of an amino acid solution on the edge of a strip of filter paper wetted with water and then dipping that paper into a solvent. Once absorbed the solvent spread across the paper in two different directions carrying with it the mixture's components. After this the paper was dried and sprayed with ninhydrin, a colouring reagent that reacts with proteins. With the components moving at different speeds on the paper it became possible to see them as distinct and physically separate spots. Critically paper chromatography could be performed with just basic equipment and several samples could be analysed simultaneously
Within a year Tuppy had identified and determined the sequence of all 30 amino acids in chain B
chain A however had fewer unique amino acids. This made it more difficult to determine their pattern. By 1953, however, Sanger and Thompson had succeeded in sequencing all 21 amino acids in chain A
Analysis to determine the composition of the amino acids of the disulphide bridges that hold the 2 Chainz together followed.
By 1955 Sanger and his team had sequenced all 51 amino acids in the two chains of insulin and worked out the position and composition of the three disulfide bridges which joined them
It is certainly a reminder to never give up hope as even as the insulin was initially administered The hospital staff was certain he was doomed.
Today insulin is available in a number of forms allowing, close mimicry of the body’s natural process of carbohydrate control . Insulin is Synthesised In yeast and bacteria by DNA recombination.
The human insulin DNA sequence was synthesise and inserted into bacteria for insul in production.
synthesising the human engine in DNA sequence Synthesis of the gene itself and assembling it.
(Insulin is a protein composed of two chains, an A chain (with 21 amino acids) and a B chain (with 30 amino acids), which are linked together by sulfur atoms (Britannica)
Insulin chains are built from individual amino acids . There are two chains known as an and B . )
Multiple synthesis are needed for each step to protect amino and carboxyl groups and then those protections have to be removed. Finally, fragments are condensed to build the full chains .)
Once that gene has been synthesised, it must be cloned. To do this, it is inserted into a bacterium in a way that the gene can be replicated in producing unlimited quantities.
Then insul in manufacture Is initiated by coaxing the bacteria into recognising the sequence and begin producing human insulin.
During the decades that insulin biology advanced through the application of techniques of protein chemistry, protein synthesis, X-ray crystallography, and assay development, another biologic revolution was taking place that would also leave insulin's mark on modern bioscience. That revolution was the understanding of DNA biochemistry, function, and techniques to manipulate it and created an ability to synthesize insulin and other proteins in a simpler way,
the UCSF group that was taking what we would call the cDNA or natural gene approach
Additional biochemical studies of what is now known as “signal transduction” and the additional analysis that became possible following the cloning of the receptor cDNA's have transformed both our understanding of insulin actions and provided new insights into parallel signaling systems used by insulin and the IGFs and the truly massive field of signal transduction from membrane receptors to numerous cellular pathways. Once again, insulin was at the heart of this burgeoning field of study.
This also opened the field to many other important aspects of insulin and its actions, many of which are covered in more detail in other articles in this issue. These include understanding the insulin molecule in evolution, defining the complimentary relationships between insulin and IGFs, understanding insulin and IGF-1 receptor signaling systems, including the role of insulin action in tissues not previously thought to be insulin sensitive, to the development of new insulin analogs to improve treatment of type 1 diabetes, and understanding the role of insulin resistance in type 2 diabetes and metabolic syndrome.
[1] Wellington, Alexander Leonard Thompson ‘ever remembered’: The first person to receive insulin Journal of Medical Biography 2022, Vol. 30(1) 64–66 https://journals.sagepub.com/doi/pdf/10.1177/0967772020974355#:~:text=On%20July%2017%2C%201908%2C%20Leonard,near%20the%20Beaches%20in%20Toronto.
[2] American diabetes Association The History of a Wonderful Thing We Call Insulin, July 2019 https://diabetes.org/blog/history-wonderful-thing-we-call-insulin
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https://www.whatisbiotechnology.org/index.php/exhibitions/sanger/insulin
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[3] Flier and Khan, October 2021 Insulin: A pacesetter for the shape of modern biomedical science and the Nobel Prize
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8513142/
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