PhD William Rostène
email: william.rostene@inserm.fr.

Pierre De Meyts

Insulin: a hundred year-old discovery with a fascinating history

Keywords: Diabetes, insulin, pancreas, discovery of insulin, Nobel Prize, patents and therapeutic developments

Diabetes has been known since antiquity. We present here a historical perspective on the concepts and ideas regarding the physiopathology of the disease, on the progressive focus on the pancreas, in particular on the islets discovered by Langerhans in 1869, leading to the iconic experiment of Minkowski and von Mering in 1889 showing that pancreatectomy in a dog induced polyuria and diabetes mellitus. Subsequently, multiple investigators searched for the active substance of the pancreas and some managed to produce extracts that lowered blood glucose and decreased polyuria in pancreatectomized dogs, but were too toxic to be administered to patients. The breakthrough came 100 years ago when the team of Frederick Banting, Charles Best and James Collip working in the Department of Physiology headed by John Macleod at the University of Toronto managed to obtain pancreatic extracts that could be used to treat patients and rescue them from the edge of death by starvation, the only treatment then available. This achievement was quickly recognized by the Nobel Prize in Physiology or Medicine to Banting and Macleod in 1923. At 32, Banting remains the youngest awardee of this Prize. Here we discuss the work that led to the discovery and its main breakthroughs, the human characters involved in an increasingly dysfunctional relationship, the controversies that followed the Nobel Prize, and the debate as to who actually "discovered" insulin. We also discuss the early commercial development and progress in insulin crystallization in the decade or so following the Nobel Prize.

In this review commemorating the 100th anniversary of the discovery of insulin, we focus on historical aspects and attempt to show how this outstanding discovery changed the direction of diabetes research and clinical care, although milestones in basic research were attained at a slower pace.

The premises

Although the description of diabetes symptoms can be found in ancient manuscripts from Egypt, China and India, there is no doubt that in all civilisations the discomfort of diabetic patients was observed in the population. A raging thirst associated with an increased urination, excessive hunger and a sweet taste of urine could not possibly have been overlooked by the eminent physicians of the ancient times. In 2019, the International Diabetes Federation reported that 463 million people in the world (one person in 11) had diabetes (2).

The oldest known manuscript in which the symptoms of diabetes were mentioned is a papyrus dating from 1550 BC found in a sarcophagus in Luxor, sold to a German professor of archaeology, George Moritz Ebers in 1872 [START_REF] Porta | Diabetes in Ancient Times: The Long and Winding Road to Insulin[END_REF]. It is now housed in the Leipzig University library. It describes various diseases and possible treatments and refers to prescriptions for A c c e p t e d M a n u s c r i p t 5 excessive urination. The Charaka's and Susruta's medical books of Hindu Medicine (1000 -500 BC) report "a sweet taste in urines" in some disorders of urination. In the Chinese medicine, it is also reported among the signs of "Xiao-Ke" or "thirst infusing into urine", a sweet urine that attracts dogs (500 BC) [START_REF] Porta | Diabetes in Ancient Times: The Long and Winding Road to Insulin[END_REF]. Until the 18 h century, attraction by sweet urine of ants, flies and bees was a biological test to detect the presence of sugar in urine and blood.

The term diabetes comes from the Greek physician Demetrius of Apamea (3 rd century BC); it refers to a large amount of water passing through the body [START_REF] Porta | Diabetes in Ancient Times: The Long and Winding Road to Insulin[END_REF]. It is also from the Greek Rufus of Ephesus (late 1 st -early 2 nd centuries AD) that the word "pancreas" was given in relation to the fleshy structure of the organ [START_REF] Porta | Diabetes in Ancient Times: The Long and Winding Road to Insulin[END_REF]. The first accurate description of the symptoms of diabetes was made by Aretaeus Cappadocis (81-138 AD) [START_REF] Porta | Diabetes in Ancient Times: The Long and Winding Road to Insulin[END_REF]. Avicenna, a famous physician in Persia (980-1037 AD) in his important medical book, the Canon, also described in detail the symptoms of diabetic patients (excessive thirst and frequent urination) associated with kidney dysfunction [START_REF] Porta | Diabetes in Ancient Times: The Long and Winding Road to Insulin[END_REF].

As a result, during the Middle Ages and until the 17 th century, diabetes was mainly seen as a disease of the kidney and bladder.

Thomas Willis (1621Willis ( -1675)), the physician of King Charles I of England, and an eminent neuropathologist, is probably the first who made a distinction between diabetes "mellitus" in relation to the sweet taste of the urine, and other forms of diabetes [START_REF] Willis | Pharmaceutice rationalis, sive diabtriba de medicamentorum operationibus in humano corpore[END_REF]. In 1776, Matthew Dobson (1732Dobson ( -1784)), an English physician and experimental physiologist, showed that the sweet taste was due to the presence of sugars capable of fermentation [START_REF] Dobson | Experiments and observations on the urine in diabetes[END_REF]. He also found that not only urine but blood contained sugar. John Rollo (1750-1809), a Scottish military surgeon in the Royal Artillery [START_REF] Marble | In: Diabetes, its medical and cultural history[END_REF], in his Notes at the end of the 18 th century, suggested the Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 6 first diet by restriction of carbohydrates to diabetic patients. He thought that diabetes was an affliction of the stomach [START_REF] Rollo | Cases of Diabetes Mellitus with the results of the trials of certain acids and other substances in the cure of the lues venerea[END_REF].

The main breakthrough: the 19 th century

In 1815, the French chemist Michel-Eugène Chevreul (1786-1889) identified the sugar in the urine as glucose [START_REF] Chevreul | Note sur le sucre du diabète[END_REF]. In 1848, the German pharmacist and chemist Hermann Christian von Fehling (1811Fehling ( -1885)), developed a method for measuring glucose, which was used almost until the present day [START_REF] Fehling | Die quantitative bestimmung von Zucker und Stärkmehl mittelst Kupfervitriol[END_REF], using a solution of copper sulfate mixed with alkali and potassium sodium tartrate (Rochelle salt), known as Fehling's solution.

In the middle of the 19 th century, a great step forward in our understanding of sugar metabolism was made by the French physiologist Claude Bernard (1813-1878) [START_REF] Young | Claude Bernard and the discovery of glycogen. A century of retrospect[END_REF][START_REF] Wise | A matter of doubt-The novel of Claude Bernard[END_REF][START_REF] Grmek | [END_REF] (Fig. 1). Among other important discoveries such as thermoregulation, effects of poisons such as curare in the neuromuscular junction, and the physiology of the blood gases, Claude Bernard was fascinated by the controversies between French scientists on the origin of sugar in the blood. In 1817, Bernard's mentor, François Magendie (1783Magendie ( -1855)), in his textbook of physiology, wrote that "it was impossible to say what is the role of the liquid of the pancreas" [START_REF] Magendie | Précis élémentaire de Physiologie[END_REF]. Bernard noticed that the effect of the pancreas on digestion begins just before birth and the pancreatic juice may cut fat into fatty acids and glycerol. However, in spite of his skilful expertise in vivisection, Claude Bernard was unable to carry out successful pancreatectomy in dogs [START_REF] Bernard | Leçons sur le diabète et la glycogénèse animale[END_REF]. He therefore decided to focus on metabolism. In 1848, the year he co-created the Société de Biologie -where he published all his major contributions to physiology -, he showed, in the famous experiment of the "washed liver", that this organ A c c e p t e d M a n u s c r i p t 7 was able to store and produce glucose, independently of the exogenous nutrition with sugar or carbohydrates. He gave the name of glycogen to the reserve form of glucose produced by the liver in 1855 [START_REF] Bernard | Leçons sur le diabète et la glycogénèse animale[END_REF][START_REF] Bernard | De l'origine du sucre dans l'économie animale[END_REF][START_REF] Bernard | Nouvelles recherches expérimentales sur les phénomènes glycogéniques du foie[END_REF]. He correctly postulated that diabetes was characterized by an overproduction of glucose by the liver [START_REF] Bernard | Nouvelles recherches expérimentales sur les phénomènes glycogéniques du foie[END_REF]. Another outstanding contribution of Claude Bernard in relation to diabetes was the "diabetic piqûre" made in 1849 [START_REF] Binet | Centenaire d'une découverte de Claude Bernard : Diabète sucré par piqûre nerveuse[END_REF][START_REF] Bernard | Chiens rendus diabétiques[END_REF]. He punctured the fourth ventricle of rabbits and dogs and, in a few minutes, observed the presence of glycosuria, thus opening a long discussion on the possible role of the central nervous system in diabetes. He was correct when he wrote that "glycemia stays constant in normal situation whatever the alimentary diet, glycosuria is just a symptom of diabetes, not the disease itself". In 1865, he published his masterpiece, Introduction à l'étude de la médecine expérimentale, still a must-read by aspirants to biomedical research [START_REF] Bernard | Introduction à l'étude de la médecine expérimentale[END_REF].

Working in Berlin in the laboratory of Rudolf Virchow (1821-1902), a medical student, Paul Langerhans (1847-1887) (Fig. 1), characterized some small "heaps of cells" in the pancreas in his 1869 medical thesis [START_REF] Langerhans | Beiträge zur mikroskopischen Anatomie des Bauchspeicheldrüse[END_REF]. He did not work further on the pancreas and was the first to describe the dendritic cells of the epidermis [START_REF] Langerhans | Über die Nerven der menschlischen Haut[END_REF]. His scientific career and his premature death from tuberculosis have been detailed in the outstanding biography by Björn M. Hausen (1940Hausen ( -2017) ) [START_REF] Hausen | Die Inseln des Paul Langerhans. Eine Biographie in Bildern und Dokumenten[END_REF]. Gustave-Edouard Laguesse (1861Laguesse ( -1927)), who became Professor of Histology in Lille, France, called these cell clusters in 1893 the islets of Langerhans, and suggested they may be the source of the substance involved in blood glucose control [START_REF] Laguesse | Sur la formation des îlots de Langerhans dans le pancréas[END_REF][START_REF] Hoet | His demonstration of the significance of the islands of Langerhans[END_REF]. Laguesse also coined the word endocrine, which means "internally secreting" [START_REF] Fossati | Laguesse à Lille en 1893 crée le terme « endocrine » et ouvre l'ère de l'endocrinologie. Son modèle : l'îlot endocrine du pancréas et le diabète[END_REF].

The last decade of the 19 th century was characterized by an increased interest in diabetes, particularly in France and in Germany. Apollinaire Bouchardat (1806Bouchardat ( -1886)), the leading clinical diabetologist of the 19 th century, observed that weight reduction by decreasing [START_REF] Bouchardat | De la glycosurie ou diabète sucré; son traitement hygiénique avec notes et documents sur la nature et le traitement de la goutte[END_REF]. Though he was one of the first scientists to suggest that the pancreas is the main organ involved in diabetes, it was only in 1889 that two German medical researchers, Oskar Minkowski (1858-1931) and Josef von Mering (1849Mering ( -1908) ) (Fig. 1), working in the laboratory of Bernhard Naunyn in Strasbourg, made one of the most important discoveries in diabetes research [START_REF] Mering | Diabetes mellitus nach Pankreasextirpation[END_REF][START_REF] Mering | Diabetes mellitus nach Pankreasextirpation[END_REF]. They showed that removing the pancreas of a dog (performed to explore the role of pancreatic juices in lipid metabolism) unexpectedly induced incessant urination. Minkowski found sugar present in the urine. He and von Mering concluded that the pancreas plays a key role in diabetes by secreting a substance which lowers the level of blood glucose [START_REF] Mering | Diabetes mellitus nach Pankreasextirpation[END_REF][START_REF] Mering | Diabetes mellitus nach Pankreasextirpation[END_REF]. Whereas Claude Bernard said that pancreatectomy was impossible to be carried out in dogs, they successfully removed the gland and showed that it induced polyuria and glycosuria. More importantly, they grafted a piece of pancreas in a pancreatectomized animal and observed that the symptoms disappeared. The opposite experiment led to a definite conclusion: when the engrafted pancreas fragment was removed, diabetes reappeared [START_REF] Mering | Diabetes mellitus nach Pankreasextirpation[END_REF].

Since then, the number of publications reporting on the putative internal pancreatic secretion that ameliorated glycosuria and hyperglycemia increased dramatically. In 1893, the French physiologist Emmanuel Hedon (1863-1933) and Gaston Giraud (1888Giraud ( -1975) ) reproduced the experiments of Minkowski and von Mering, furthermore showing that leaving a small piece of the pancreas prevented diabetes, refuting the notion that the loss of exocrine function may be the cause of diabetes [START_REF] Hédon | La courbe de la glycémie dans les premières heures qui suivent la pancréactectomie[END_REF]. However, extraction of the internal secretion from the islets of Langerhans was challenging since they represent only a small portion of the pancreatic tissue which is otherwise mainly involved in exocrine function. In 1909, the Belgian physiologist Jean De Meyer (1878-1934) (Fig. 2), at the Free University of Brussels, investigated the actions of the internal secretion of the pancreas on the kidney, and proposed to name this internal secretion "insuline" (30). He also demonstrated that perfusion of pancreatic extract in the liver of a diabetic dog resulted in glycogen formation [START_REF] Meyer | Sur les relations entre la sécrétion interne du pancréas et la fonction glycogénique du foie[END_REF].

In 1916, the English physiologist Sir Edward Albert Schäfer (1850-1935) (Fig. 2) proposed in a compendium on The Endocrine organs [START_REF] Schäfer | The endocrine organs. An introduction to the study of internal secretions[END_REF], to name the internal secretion of the pancreas "insuline" (with French spelling), apparently unaware of De Meyer's earlier proposal.

Remarkably, Schäfer also proposed in the same paper the existence of the precursor proinsuline, half a century before its discovery by Donald F. Steiner (1930Steiner ( -2014) ) at the University of Chicago [START_REF] Steiner | Insulin biosynthesis: evidence of a precursor[END_REF]. Schäfer also introduced the term endocrine, for which he is usually credited, without mention of Laguesse's proposal 25 years earlier. In 1918, he added to his last name the name of his mentor, the eminent physiologist William Sharpey (1802-1880), becoming known as Sir Edward Sharpey-Schafer.

Several researchers at the end of the 19 th century and beginning of the 20th came very close to obtain a pancreatic extract able to improve glycemia (34-36, and references therein).

The first to claim success in decreasing glycosuria in pancreatectomized dogs with a pancreatic extract was the French physiologist and endocrinologist Marcel Eugène Emile Gley (1857Gley ( -1930)). In 1891, he described a procedure for pancreatectomy, showing it caused experimental diabetes [START_REF] Gley | Procédé de destruction du pancréas. Troubles consécutifs à cette destruction[END_REF]. In 1892, he confirmed that the atrophy of the exocrine pancreas does not cause diabetes [START_REF] Gley | Note préliminaire sur quelques effets de la destruction lente du pancréas : importance de la fonction digestive du pancréas[END_REF]. From 1892 to 1905, he tried to make pancreatic extracts from sclerosed remains of animal pancreas and obtained extracts that effectively diminished sugar in the urine of pancreatectomized dogs and improved their symptoms, in essence [START_REF] Gley | Action des extraits de pancréas sclérosé sur des chiens diabétiques[END_REF]. Bizarrely, Gley chose not to publish his results, but instead consigned them in a sealed envelope that he deposited in 1905 at the Société de Biologie in Paris with instructions for it to be opened only upon his explicit request [START_REF] Gley | Action des extraits de pancréas sclérosé sur des chiens diabétiques[END_REF]. He then stopped working on pancreatic extracts, claiming insufficient resources. In 1922, after Banting and Best went public with their discovery, Gley requested at the December 23, 1922 meeting of the Société de Biologie that the envelope be opened and read [START_REF] Gley | Action des extraits de pancréas sclérosé sur des chiens diabétiques[END_REF]. He described what he carried out on February 20, 1905 with pancreatic extracts on sclerosed dog pancreas, and suggested that: "It will be quite important to isolate the active principle of these extracts; it means the internal secretion of the pancreas and to study the mechanism of action". Gley, unlike other protagonists in the insulin discovery saga (see below), did not attempt to claim priority, and congratulated Macleod for having achieved a great simplification of his method [START_REF] Gley | Action des extraits de pancréas sclérosé sur des chiens diabétiques[END_REF]. Macleod acknowledged Gley's contribution in his book on Carbohydrate Metabolism and Insulin [START_REF] Macleod | Carbohydrate metabolism and insulin[END_REF].

Georg Ludwig Zuelzer (1870-1949) (German spelling: Zülzer) in Berlin conducted several experiments and extraction procedures to obtain potent canine pancreatic extracts to treat diabetes [START_REF] Jörgens | They got very near the goal: Zülzer, Scott and Paulescu[END_REF]. He first injected adrenal extract into rabbits and observed an increase in glycosuria. When he injected a pancreatic extract at the same time the increase in glycosuria did not appear [START_REF] Zuelzer | Experimentelle Untersuchungen über den Diabetes[END_REF]. He filed a patent in 1912, naming the active substance acomatol [START_REF] Zuelzer | Pancreas preparation suitable for the treatment of diabetes[END_REF], but he made a mistake by asserting that the substance was not a protein. He started to use the appropriate chemical extraction by alcohol, made a collaboration with Hoffmann La Roche, but the preparation was not pure enough to avoid serious side effects in animals and patients [START_REF] Jörgens | They got very near the goal: Zülzer, Scott and Paulescu[END_REF]. The First World War unfortunately put an end to his efforts toobtain insulin. In 1909, Ernest Lyman Scott (1877Scott ( -1966)), a student studying physiology at the University of Chicago Medical School, submitted a master's thesis project to his supervisor, Prof. Anton Julius Carlson (1875Carlson ( -1956)), focused on the search for a substance in the pancreas that lowers blood glucose [START_REF] Scott | On the influence of intravenous injections of an extract of the pancreas on experimental pancreatic diabetes[END_REF]. As this topic was outside of his field of research, Carlson was not enthusiastic but he agreed for Scott to carry out some experiments. In contrast to Zuelzer, and to the general belief at the time, Scott embarked on a search for a protein using an 85% alcohol extraction. In a publication in 1912 in the American Journal of Physiology, he described carefully an appropriate method of extraction [START_REF] Scott | On the influence of intravenous injections of an extract of the pancreas on experimental pancreatic diabetes[END_REF]. An anecdote is that the paper of Scott was written by Carlson without any exchange with Scott, even if the latter was the single author. In 1914 Scott published a standard test for blood glucose [START_REF] Scott | The content of sugar in the blood under common laboratory conditions[END_REF]. He became Professor of Physiology at Columbia University where he did a distinguished career having stopped working on insulin. After the publication of the Toronto research, Scott in 1923 in a correspondence to the Journal of the American Medical Association (JAMA) discussed his priority of the extraction procedure with a high percent of alcohol [START_REF] Scott | Priority in discovery of a substance derived from the pancreas, active in carbohydrate metabolism[END_REF]. Nicolas Constantin Paulesco (Romanian spelling: Nicolae Paulescu) (1869-1931), a student from Romania, came to Paris to pursue his medical studies in 1888 [START_REF] Jörgens | They got very near the goal: Zülzer, Scott and Paulescu[END_REF]. After receiving his MD in 1897, he worked with famous French physician and diabetologist Etienne Lancereaux Unfortunately, similarly to the work carried out by Zuelzer and others, his substance was not pure enough to avoid toxic side effects and was not administrable to humans. Paulesco attempted further purification and tried rectal administration in humans but was not successful. Paulesco was a fervent Christian nationalist and became involved in Romanian extreme right politics, as will be discussed later.

The biochemist Israel Simon Kleiner (1885Kleiner ( -1966) ) did numerous experiments between 1915 and 1919 (57) while assistant in physiology at the Rockefeller Institute, demonstrating that intravenous injections of pancreatic emulsions in pancreatectomized diabetic dogs corrected hyperglycemia. There were no toxic effects if the extract was given slowly and highly diluted, suggesting a possible therapeutic application in humans. The experiments were well A c c e p t e d M a n u s c r i p t 13 controlled and carefully described, making Kleiner probably the closest of the early investigators to a therapeutic solution [START_REF] Jörgens | They got very near the goal: Zülzer, Scott and Paulescu[END_REF]. The Toronto team confirmed Kleiner's experiments [START_REF] Best | The preparation of insulin[END_REF]. Unfortunately he stopped working on this problem when he left Rockefeller in 1919.

The famed nutritionist John Raymond Murlin (1874Murlin ( -1960) ) [START_REF] Nasset | Investigator, teacher, colleague[END_REF] with Benjamin Kramer at the laboratory of Physiology at Cornell University described in 1913 pancreatic extracts that were effective in decreasing both glycosuria and hyperglycemia in pancreatectomized dogs [START_REF] Murlin | The influence of pancreatic and duodenal extracts on the glycosuria and the respiratory metabolism of depancreatectomized dogs[END_REF]. After a long gap in investigating pancreatic extracts, in 1922, Murlin, then Professor of Physiology at Rochester University, did some trials on diabetic patients with initial failure due to toxic side effects but later claimed some success [START_REF] Sutter | Three-month study of the influence of the antidiabetic substance on a case of severe diabetes[END_REF]. In 1923, he discovered glucagon [START_REF] Murlin | Aqueous extracts of the pancreas. 1. Influence on the carbohydrate metabolism of depancreatized animals[END_REF].

The Toronto story

Nobody could have predicted that the discovery of insulin would take place at the University of Toronto. It was carried out by Frederick Grant Banting, Charles Herbert Best, John James Rickard Macleod and James Bertram Collip (see graphical abstract). Surprisingly enough, except for Macleod as will be discussed later, none of them had any training in endocrinology.

Frederick Banting (1891-1941)

Banting's biography has been vividly written by Michael Bliss [START_REF] Bliss | A Biography[END_REF]. Frederick tried to obtain a position at this hospital but his application was declined. In the beginning of the summer 1920, he moved to London, Ontario, a small city where the University of Western Ontario is located, and opened a general practice. It was a convenient position as his fiancée was working as a teacher in a nearby town. He only saw a few patients until the fall when he supplemented his general practice with some orthopaedic cases. He felt depressed; he found a part-time job as a demonstrator in surgery and anatomy at the University. On November 1, 1920, he had to give a course on carbohydrate metabolism. He had worked all day the day before, reading all he could find about the anatomy of the pancreas and its function. He went to bed with the latest issue of the journal Surgery, Gynecology and Obstetrics in which he read a paper (64) from Moses Barron (1884Barron ( -1974) ) (Fig. 3 pancreas in diabetic patients suffering of obstruction of the pancreatic ducts by stones, and thus recalled in his paper the gradual atrophy of acini in contrast to the islets of Langerhans after duct blockade, as previously shown by several investigators in various animal species [START_REF] Barron | The relation of the islets of Langerhans to diabetes with special reference to cases of pancreatic lithiasis[END_REF]. Barron also reported cases in man and results by Opie and Carlson [START_REF] Opie | On the relation of the chronic intestinal pancreatitis to the islands of Langerhans and to diabetes mellitus[END_REF][START_REF] Carlson | Pancreas, its endocrine function, and relation to the sex-life of women[END_REF]. He concluded that "… the islets secrete a hormone directly into the lymph or blood streams (internal secretion), which has a controlling power over carbohydrate metabolism and which is necessary for the utilization of sugar by the tissues". Banting was fascinated by that paper [START_REF] Hengele | Insulin's centenary: the birth of an idea[END_REF], woke up with an idea at 2:00 am on October 31, 1920, and wrote a few words in his notebook : "Diabetus: ligate pancreatic ducts of dogs. Keep dogs alive till acini degenerate leaving islets. Try to isolate the internal secretion of these to relieve glycosurea". In February 1923, Barron wrote a letter to Banting (Fig. 3) saying that "he was flattered that Banting On November 8, 1920, Banting had an appointment with John Macleod. Banting outlined his idea but Macleod soon realized that Banting had no real knowledge of the pancreas or diabetes and that he did not know how to carry out the appropriate experiments in order to confirm his hypotheses.

Macleod also thought that it would be risky to venture in a field in which several eminent researchers had failed. But in spite of his lack of enthusiasm, he was curious and only warned Banting that success was unlikely. Macleod respected Banting's skills and experience as a surgeon, which could be an asset to managing surgical procedures on dogs. Macleod

The discovery of Insulin

The story of the discovery of insulin has been plentifully reported over the years and the main source remains the book of Michael Bliss [START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF], first published in 1982; a special edition was published on the 25 th anniversary of the first edition in 2007 and a new special edition for the 100 th anniversary of insulin's discovery is just being published in 2021. Here we will focus on the important steps which led to the discovery, highlighting the actual breakthrough made by the Toronto team, in particular a description of the biochemical advances made to increase the purity and yield of the internal secretion of the pancreas, making insulin available to be safely used in humans.

First experiments and discovery

On May 17, 1921, just after completing his last exam for graduation from the physiology and biochemistry courses, Charles Best started working with Banting. They had discussions about the literature. With the help of Macleod, Banting began to operate on several dogs in order to ligate the ducts to obtain gradual atrophy of the pancreas as previously described [START_REF] Hédon | La courbe de la glycémie dans les premières heures qui suivent la pancréactectomie[END_REF][START_REF] Barron | The relation of the islets of Langerhans to diabetes with special reference to cases of pancreatic lithiasis[END_REF] A c c e p t e d M a n u s c r i p t 19 or tried complete pancreatectomy [START_REF] Mering | Diabetes mellitus nach Pankreasextirpation[END_REF][START_REF] Mering | Diabetes mellitus nach Pankreasextirpation[END_REF]. In mid-June, before leaving for Scotland for three months, Macleod gave Banting and Best some advice about the experiments to carry out.

Banting was mainly interested in the surgical part and Best only had Macleod's courses on carbohydrate metabolism to refer to. The first results were erratic. The main problem was the summer heat that particular year in Toronto. It was very difficult to keep diabetic animals alive for a long period of time due to infections in bad sanitary animal facility conditions. A lot of dogs died even if Banting's surgical technique improved. Best was in charge of testing sugar in urine (Lewis-Benedict method) and blood (Myers-Bailey method) and to prepare some pancreatic extracts. The first encouraging data were obtained on July 30 when the diabetic dog n°410, depancreatized in two stages, showed a lower blood sugar level from 0.20 to 0.11-0.12 % (normal range 0.085-0.15 %) with intravenous injections of a pancreatic extract obtained from a duct-ligated dog degenerated pancreas sliced and chilled in Ringer solution, ground up and filtered. The other idea to graft pancreas in diabetic dogs was thus abandoned. They named "isletin" the extract prepared from degenerated pancreas and kept frozen. In late April/early May 1922 in the publication in the American Journal of Physiology (70) they settled with Macleod on "insulin", unaware of the previous naming by De Meyer and Schäfer (30,[START_REF] Schäfer | The endocrine organs. An introduction to the study of internal secretions[END_REF]. On Best's suggestion in the beginning of August 1921, Banting stopped the tedious Hedon method of two-step pancreatectomy [START_REF] Hédon | La courbe de la glycémie dans les premières heures qui suivent la pancréactectomie[END_REF] and it was a success, even if the dog n°408 died after four days from infection. It is the dog we can see on the famous photograph of Banting and Best on the roof of the Medical building and not, as often wrongly said, Marjorie. Best noted that the extract he obtained "can be kept active for at least four days, is destroyed by boiling, and extracts of other organs are inactive". He also quoted that the extracts were potent on cats and rabbits showing that it worked across species. On August 9, Banting and Best wrote a letter to Macleod reporting their data and Best worked hard to improve the method of purification with 0.2% hydrochloric acid in 60% ethyl ethanol. The alcohol was evaporated and the dry residue redissolved in saline. In previously pancreatectomized dog n°35, they confirmed that an extract of its own pancreas showed a blood sugar drop from 0.38 to 0.18% in four hours. The following day the same dog received an extract of whole beef pancreas and also showed a decrease in blood sugar level from 0.28 to 0.11% in four hours [START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF][START_REF] Banting | The Internal Secretion of the Pancreas[END_REF].

On November 18, the dog n°33, named Marjorie, had her pancreas removed. She was first treated with 10cc of filtered foetal calf extracts intravenously and later with the same acidalcohol extract but of beef pancreas, easier to obtain from the abattoir and less expensive than calf pancreas [START_REF] Best | The preparation of insulin[END_REF]. Her urine was sugar-free after one hour and she was kept alive for 70 days, contrarily to the dogs that had not received the extract. With the help of a pathologist they obtained the proof that no pancreatic tissue remained in Marjorie to explain the impressive effects, overcoming an objection from Macleod.

In November 1921, when James Collip arrived in Toronto to work with Macleod in a lab across the street from where Banting and Best worked, he asked Banting if he needed help for the pancreatic extracts. Banting invited Collip to help estimate the glycogen content in the liver. Collip joined the team to work on the biochemistry of pancreatic extracts. With the help of Clark Noble, the extracts could be tested on normal rabbits, given an easy assay for their efficacy [START_REF] Collip | Some Recent Advances in Endocrinology[END_REF]. Collip also developed a new blood sugar test, the Shaffer-Hartman method, based on the reduction of copper by the reducing group of the sugar, and observed that subcutaneous injections of pancreatic extracts seemed to work.

Best had trouble obtaining a pure extract with a mixture of alcohol and acid. Collip, instead of evaporating the alcohol in a warm air current as Best did, used a vacuum. He reduced the A c c e p t e d M a n u s c r i p t 22 pancreas alcoholic solution to obtain a suspension of fine particles which was filtered in order to obtain a liquid filtrate and a residue of solid particles [START_REF] Best | The preparation of insulin[END_REF][START_REF] Collip | Some Recent Advances in Endocrinology[END_REF][START_REF] Banting | The preparation of pancreatic extracts containing insulin[END_REF]. Collip found that a solution of the solid particles gave a potent hypoglycaemic effect with a decrease of ketone bodies and a huge increase in glycogen in the liver, suggesting that the extract could restore the function which was lacking in diabetes.

After a meeting with a group of physicians at the Toronto General Hospital, Dr Joe Gilchrist, a friend from Alliston and classmate of Banting, who was diabetic and had been on the Allen starvation diet [START_REF] Allen | Studies concerning diabetes[END_REF][START_REF] Allen | Prolonged fasting in diabetes[END_REF] offered to help Banting as a test person. It was a dangerous test but Gilchrist had no alternative anyway. Banting gave him some extract orally (34) but it was unsuccessful as previously reported by Rennie and Fraser in 1907 [START_REF] Rennie | The islets of Langerhans in relation to diabetes[END_REF]. On December 30, 1921, Banting made a presentation at the American Physiological Society at Yale University in New Haven, Connecticut. Banting felt nervous, being an inexperienced and awkward speaker particularly in formal situations. The reaction to his presentation was extremely positive and several questions were raised by Frederick Allen, Anton Carlson, Elliott Joslin and George Clowes, director of research for the Eli Lilly company in Indianapolis, who wanted to know more about the extraction procedure. Perhaps because Banting was intimidated, Macleod felt the need, as his supervisor, to step in and support him, answering several of the questions using "we" as if he had been directly involved in the work. Banting felt that Macleod had taken the credit for the work he considered to be his and Best's work, which upset him. As for Best, he remained silent in respect for his professor [START_REF] Best | The Personal Story of DR Charles Best, the Codiscoverer of Insulin[END_REF].

Purification and clinical tests

At the end of December 1921, the main goal was to produce a preparation suitable for human administration. Banting insisted that the first clinical trial be made with an extract Macleod asked Collip to make extract improvements. Collip observed indeed that results were much better if not all alcohol was evaporated [START_REF] Best | The preparation of insulin[END_REF][START_REF] Collip | Some Recent Advances in Endocrinology[END_REF]. He carried out experiments to determine the alcohol concentration in which the active substance can be soluble in contrast to contaminants, and then increased the alcohol concentration to 90% in which the principle was able to precipitate. Collip obtained an extract purer than that of Best. He tested the preparation on rabbits and it worked, with no inflammation reported. Thus, on January 23, two weeks after the first test, Leonard Thompson received the new extract made by Collip subcutaneously and the results were spectacular. Glycosuria and ketonuria almost disappeared, and the blood sugar dropped from 5.2 to the normal sugar concentration of 1.2 mg/cc (77) (Fig. 4). Daily injections were made until February 4 th A strange event then happened. Collip did not write down his experimental protocol, and for several weeks was unable to obtain good extracts. Nobody knows if it was due to problems with the equipment or the products used for the insulin purification, or to a change required to obtain large scale production, but no more insulin was available when the stock from The press published many articles on insulin and Banting and Best received very touching letters of thanks from diabetic patients for the gift of insulin [START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF][START_REF] Best | The Personal Story of DR Charles Best, the Codiscoverer of Insulin[END_REF].

Recognition and awards (1923)

At the end of December 1922, Best and his friend Clark Noble presented a paper on the effects of insulin on rabbits at the meeting of the Federation of American Societies for Experimental Biology. Among the assistance was Robert Barany, an otologist from Uppsala, who had won the Nobel Prize in 1914 for his work on the physiology and pathology of the vestibular apparatus. He talked with John Macleod. He wrote to him in the beginning of January 1923, saying that "the discovery of insulin is of such importance that it should be awarded the Nobel Prize". The Nobel committee knew about the bad relationship between the members of the Toronto team. Some members of the Committee quoted that point as an argument to postpone the prize, arguing that it was a too recent discovery. However, with the pressure from the media, from some other authorities such as Henry Dale as well as from the patients themselves, it would have been incomprehensible and in disagreement to Alfred Nobel's testimony, not to award the Nobel Prize for the discovery of insulin. As we know from the Nobel records, Krogh nominated Banting and Macleod and also suggested the name of Best.

By

George W. Crile from Cleveland followed Krogh. George N. Stewart, also from Cleveland, nominated only Macleod. Finally Francis G. Benedict, from Washington, nominated only Banting (https://www.nobelprize.org/nomination/archive/). The Nobel Prize to Banting and Macleod was voted on October 25, 1923 by the committee of the Karolinska Institute. On October 26, news about it spread in Toronto. Banting drove back to Toronto from Alliston.

When he arrived in his office, the phone was ringing. It was a friend who congratulated him and told him to open the newspaper. When he knew that Macleod had won the Prize with him, Banting's anger about Macleod's inclusion was unbelievable. At the same time, Best was giving a lecture at Harvard University. The students were very impressed at such discovery made by a young student. At the end of his talk, Dr Joslin read a telegram he had just received from Banting saying "I share the Nobel Prize with Best" (Fig. 5). Macleod did the same with Collip (Fig. 5). None of them attended the official ceremony in Stockholm on

The aftermath of the discovery

The Nobel Committee received letters of protests from Zuelzer and Paulesco claiming priority, which were ignored. Paulesco became a kind of "cause célèbre" for the Romanian scientific (and political) community which still today considers that the Nobel was stolen from him by the Canadians. The fact that Banting misinterpreted and misquoted Paulesco's French publication did not help the Romanian's feeling that the Canadians had cheated. The controversy has been largely discussed over the years (see refs. 1,34,63,69). There is no doubt that Paulesco was a gifted scientist and he had his supporters both within and outside Romania [START_REF] Pavel | The priority of N.C. Paulescu in the discovery of insulin[END_REF][START_REF] Ionescu-Tirgoviste | The rediscovery of insulin[END_REF][START_REF] Murray | Paulesco and the isolation of insulin[END_REF]. The problem with attempts at his rehabilitation has been the increasing awareness of his militant fascist activities [START_REF] Jörgens | They got very near the goal: Zülzer, Scott and Paulescu[END_REF][START_REF] Laron | Paulescu : scientist and politician[END_REF][START_REF] Leiva-Pérez | From pancreatic extracts to artificial pancreas: history, science and controversies about the discovery of the pancreatic antidiabetic hormone. VI. Nicolae C. Paulescu: light and darkness[END_REF] At the same time, Best arrived in London with Margaret Mahon. He was to work on blood pressure with Henry Dale after completing his degree in medicine. In Dale's laboratory, Best identified two depressor substances present in liver extracts, histamine and choline [START_REF] Best | The nature of the vaso-dilator constituents of certain tissue extracts[END_REF]. He also described an enzyme, histaminase, able to cause the disappearance of histamine from degeneration of the exocrine but not the endocrine pancreas. Banting surmised that destruction of insulin by pancreatic enzymes may be the problem in getting an active extract, and, inspired by the fact that ligation is a surgical procedure, sketched a protocol (in which he could not even spell diabetes) based on this procedure. As persistent as he was naïve, he persuaded Prof. Macleod to let him go ahead with the help of an equally untrained medical student, Charles Best. They managed, despite all odds, to get to the stage where several had been before, rescuing pancreatectomized dogs. It turned out that the duct ligation was useless, the pancreatic enzymes being in an inactive proenzyme form, and could be omitted entirely. It is when Collip became involved that significant progress in purification was achieved. Thus, this "discovery of insulin" was not a paradigm shift as defined by Thomas Kuhn (145) but rather a paradigmatic incremental progress in purifying what was not even known yet to be a protein. The true paradigm shift had been Minkowski and von Mering's 1889 experiment, a serendipitous result that was a game changer. The successful purification of insulin was unavoidable given the progress already accomplished. It is likely that the motivation for the Nobel award was not just the purification of a pancreatic extract, but the demonstration that it saved the life of dying diabetic patients, which no other precursor had achieved. This is what struck August Krogh (and his diabetic wife) when they visited Toronto Banting and Best spent considerable energy in the rest of their life, quite successfully, to perpetuate the myth that insulin was discovered by Banting and Best, and to demean the role of their colleagues [START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF][START_REF] Best | The Personal Story of DR Charles Best, the Codiscoverer of Insulin[END_REF]146,147). Collip maintained a dignified silence on the issue. It is the great merit of historian Michael Bliss to have carefully investigated and documented the respective contributions of the four team members, and to have rehabilitated the reputation of John Macleod in his 1982 book on "The discovery of insulin" and successive editions [START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF] and his subsequent Banting biography [START_REF] Bliss | A Biography[END_REF], as well as in many scholarly publications. As Llewellys Barker said at a tribute at the University of Toronto in November 1923: "There is in insulin glory enough for all". This inspired a remarkable TV film produced by Gordon Hinch on the insulin discovery entitled Glory enough for all, based on Michael To conclude on a sobering note, as mentioned by Banting in his Nobel lecture, "insulin was not a cure for diabetes; it is a treatment". Despite a century of research, we seem nowhere near being able to prevent the autoimmune aggression that kills beta cells in type 1 diabetes, or to replace them by stem cell therapy. We seem impotent in the face of the raging worldwide epidemics of type 2 diabetes and obesity. In addition, while the insulin pioneers refused to make profit from insulin sales, the current business model of insulin distribution and sales, and health care insurance, has resulted in a situation where even in some rich countries many people cannot afford insulin and have to ration their treatment (101,148). Some new breakthroughs are badly needed. Data availability: Data sharing is not applicable to this article as no data were generated or analyzed during the current study.

Essential points

-Diabetes mellitus is an ancient disease that was fatal until insulin became available. Today there are an estimated 463 million people with diabetes worldwide.

-The discovery of a substance which regulates blood glucose levels was a long scientific process. Minkowski and von Mering in 1889 demonstrated its pancreatic origin.

-Over the following 20 years, animal pancreatic extracts that lowered blood glucose in pancreatectomized dogs were obtained by a number of investigators, but none of these extracts were suitable for human administration.

-The purification of insulin was improved in 1921 by Frederick Banting, John Macleod, Charles Best and James Collip at the University of Toronto, and the first patients were successfully treated in 1922.

-The Nobel Prize in Physiology or Medicine was awarded to Frederick Banting and John Macleod in 1923 "for the discovery of insulin"; its rapid clinical application was clearly a determining factor in the award.

-Obtaining large quantities of purified insulin, while challenging, proceeded rapidly for industrial development.

-The availability of insulin paved the way to considerable developments in basic research, but this progress was much slower than the clinical applications.

A c c e p t e d M a n u s c r i p t 4 As

 4 reported by Viktor Jörgens and Massimo Porta in the preface of their excellent book Unveiling Diabetes -Historical Milestones in Diabetology (1), two of the major advances in diabetes research to date were the discovery of the key role of the pancreas in diabetes by Oskar Minkowski and Josef von Mering in 1889 in Strasbourg and the purification of insulin and the early initiation of its clinical use by Frederick Grant Banting, Charles Herbert Best, James Bertram Collip and John James Rickard Macleod in Toronto in 1921 to 1922.

A c c e p t e d M a n u s c r i p t 10 preceding

 10 Banting and Best's result by about 25 years

  Zuelzer fled from Nazi Germany in 1934 and established a successful internal medicine practice in New York City. He died in 1949.

(

  . Lancereaux was one of the first to recognize in 1877 the likely pancreatic origin of diabetes, and he distinguished two clinical forms of diabetes, lean diabetes and fat diabetes[START_REF] Lancereaux | Traité des maladies du foie et du pancréas[END_REF][START_REF] Wright Jr | Misread and mistaken. Etienne Lancereaux's enduring legacy in the classification of diabetes mellitus[END_REF]. The physiologist Albert Dastre (1844-1917), a former student of Claude Bernard, suggested that Paulesco work on the isolation of the substance involved in the internal secretion of the pancreas. Paulesco returned to Bucharest in 1900 and became Professor of Physiology in 1905. Lancereaux and Paulesco published together in 1912 a monumental medical textbook[START_REF] Lancereaux | Traité de médecine[END_REF]. In it they argued that the internal secretion of the

  only the metabolism of carbohydrates but also lipids and proteins.Paulesco conducted a series of critical experiments on pancreatectomized dogs to isolate the internal secretion of the pancreas in an aqueous extract at the end of 1916, before his lab had to close because of the occupation of Romania by Germany in World War 1. This also delayed the publication of his results. He published a comprehensive Textbook of Medical Physiology[START_REF] Paulesco | Traité de physiologie médicale[END_REF]. In this textbook he described his yet unpublished experiments showing that intravenous injection of his pancreatic extract induced the disappearance of diabetes symptoms in pancreatectomized dogs.Paulesco's early results were finally published in French in 1921 in a series of short articles in the Comptes Rendus des Séances de la Société de Biologie[START_REF] Paulesco | Action de l'extrait pancréatique injecté dans le sang chez un animal diabétique[END_REF][START_REF] Paulesco | Influence du laps de temps écoulé depuis l'injection intraveineuse de l'extrait pancréatique chez un animal diabétique[END_REF][START_REF] Paulesco | Influence de la quantité de pancréas employée pour préparer l'extrait injecté dans le sang chez un animal diabétique[END_REF][START_REF] Paulesco | Action de l'extrait pancréatique injecté dans le sang chez un animal normal[END_REF] and in more extensive articles in the Archives Internationales de Physiologie[START_REF] Paulesco | Recherche sur le rôle du pancréas dans l'assimilation nutritive[END_REF][START_REF] Paulesco | Quelques réactions chimiques et physiques, appliquées à l'extrait aqueux du pancréas, pour le débarrasser des substances protéiques en excès[END_REF]. They were convincing and similar to what the group of Toronto published in February 1922 (see below). Paulesco named his extract "pancreine" and obtained a Romanian patent for it onApril 12, 1922.

  Banting was born on November 14, 1891 and grew up on a farm in Alliston, Ontario, a small village 90 kms northwest of Toronto. In 1912, he started medical school. His classmates included A c c e p t e d M a n u s c r i p t 14 Joseph Gilchrist and Frederick Hipwell from Alliston who played key roles in the discovery period. He set about training in the Canadian army medical service in 1915 and graduated in December 1916. As a lieutenant, he sailed from Halifax to Britain in March 1917. He was assigned to the Granville hospital at Ramsgate, in the department of orthopaedics. He stayed there for 13 months. In June 1918, he was transferred to the front, to a Canadian hospital near Cambrai in the north of France. In September 1918, as he was trying to rescue some soldiers, he was severely wounded in his right arm by a piece of shrapnel and barely escaped amputation. He stayed in Great Britain to recover until the spring of 1919, when he sailed back to Canada. He was awarded the military cross. He completed his speciality in orthopaedic surgery at the Hospital for Sick Children in Toronto but without any salary. He

  ), a pathologist who was Clinical Professor of Medicine in the Department of Pathology at the University of Minnesota in Minneapolis. The article was a survey of the literature on the histology of the pancreas, the ligation of the pancreatic Wirsung's ducts, the effects of the excision of the pancreas in animals. Barron also provided figures showing lesions of Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 15

  recalled his paper published several years before: In this article, I did not have the faintest idea or hope that it would at any time or in any way be sufficiently suggestive to start such an epoch-making investigation as you have undertaken. I feel it an honor to be in any way mentioned in connection with this work of yours and I wish that I had actually had some real part in the investigation". In 1964, Barron received the Banting Medal of the American Diabetes Association for his contribution to the discovery of insulin. The next morning Banting outlined his idea to Prof. Frederick R. Miller, who was in charge of the department of Physiology at London's Western University and asked permission to work on it in his laboratory. Banting thought that the reason for the failure to produce effective pancreatic extract was due to a destructive effect of trypsin on the hypothetical hormone. It would be possibly successful if the cells producing trypsin were destroyed by ligation of the duct of the gland and the remaining part of the gland could then be used as the original Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 16 material. It had previously been observed in some works, as mentioned above, that the ligation of the duct induced the atrophy of the acini but not of the islets. Miller did not have either facilities or competence in that field and suggested Banting consult a specialist of carbohydrate physiology, Prof. John Macleod, at the University of Toronto. John Macleod (1876 -1935) Macleod was born in Aberdeen, Scotland, in 1876. He graduated with a PhD in medicine in 1898. He moved to Leipzig and worked in the same laboratory where Paul Langerhans had spent a few weeks, years before. In 1903, he obtained a permanent position at Western Reserve University in Cleveland, Ohio. He developed an interest in carbohydrates, determination of blood glucose and liver glycogen and in experimental diabetes in dogs. He gained an international reputation on metabolism of various substances in particular in the field of carbohydrate metabolism with several publications and a textbook (68) and was appointed in 1918 to the Chair of Physiology at the University of Toronto.

  A c c e p t e d M a n u s c r i p t 17 accepted to take Banting for a summer project, to provide a summer undergraduate student to help him and some dogs to start with. Two students were actually ready to accept the summer job, Clark Noble and Charles Best. The story says that they decided to split the job and tossed a coin to see who should start first. Best won the toss. Charles Best (1899 -1978) Charles Best was born in 1899 in a small town, Pembroke, in Maine, thirty miles from the Canadian border. His father was the local physician. He went through both public and high school in Pembroke. When time came for him to decide what he wanted to do, he chose to become a physician like his father. He opted for the University of Toronto. The same year, 1917, as he entered the undergraduate BA physiology and biochemistry course at the University of Toronto, one of his aunts died of diabetes. She had been followed by Elliot Joslin in Boston. Her illness and death deeply influenced Best's orientation. Like Banting, Best was enrolled in the army after being rejected twice. He finally joined the 70 th battery of the Horse Artillery in spring 1918. He was sent to England in October 1918. He returned home in December without having fought in France. He completed his second academic year in Arts and Medicine. Best worked with his close university friend Clark Noble. At a party, in February 1919, Best met Margaret Mahon. They spent their whole life together and she was a great support for him during the period before and following the discovery of insulin. Charles Best's son wrote a personal biography of his parents (69). James Bertram Collip (1892 -1965) Collip was born in Belleville, Ontario, on November 20, 1892. In 1907, he took physiology and biochemistry at the University of Toronto and received a BA in 1912. He then undertook studies in biochemistry with the influential professor of physiology then chair of Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 18 biochemistry Archibald Macallum (1858 -1934), whom Collip would succeed at McGill University in 1928. Collip obtained an MA in 1913 and a PhD in 1917 at McGIll. He moved to the University of Alberta in Edmonton where he became professor of biochemistry. In the autumn of 1921, with a Rockefeller Travelling Fellowship, he moved for a one-year sabbatical leave to Toronto with his family and started working in John Macleod's lab. In December, his skills as a biochemist enabled him to greatly contribute to the project on insulin.

  Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 20 they mentioned the problem of infection. On August 23, Macleod answered that "if you can prove that such extracts may reduce blood sugar and have controls, you can respond to potential criticisms. Your results are definitively positive but not absolutely certain. I will do all in my power to help you". Macleod also gave advice to Best to carry on with the experiments.In September, when Macleod came back to Toronto, he found that they had made the experiments he had required but he made some criticisms. He did not give any financial support to Banting. Banting was furious and wanted to leave. But the Head of the Department of Pharmacology, Velyien Henderson and Clarence Starr, the chief surgeon at the Hospital for Sick Children with whom Banting had worked in England and who had followed the work during the summer, convinced Macleod to give them more space. The first presentation of the data was given at a Journal Club on November 14, 1921.A suggestion made after the Journal Club presentation led to the idea to carry out longevity experiments in order to see how long a diabetic dog can survive with the extracts. But they were out of duct-ligated dog extracts. Knowing that the islets developed early in pancreas development and that the external secretion is not needed until digestion begins after birth, Banting thought that foetal pancreas could be used as a source of internal secretion. They went to the local slaughterhouse to obtain some foetal calf pancreas and it worked. It was the first important breakthrough for the future experiments which confirmed that the internal secretion worked across species. Furthermore, Best changed the extraction procedure by using alcohol instead of a Ringer solution on Macleod's advice and observed that extraction of whole dog pancreas with alcohol also worked. Thus, no more degenerated pancreas was needed and adult tissue extracts could be used.

  and Best. Best made a batch from beef pancreas and tested it on Marjorie. It was a filtered alcoholic solution evaporated off in a vacuum still, washed with toluene and the remaining watery solution sterilized on a filter[START_REF] Best | The preparation of insulin[END_REF].OnJanuary 11, 1922, the same extract was administered to a 13 year-old diabetic boy, Leonard Thompson at the Toronto General Hospital in the diabetes department of Dr Walter Campbell under the supervision of Pr Duncan Graham. It produced only modest effects (25% reduction) and the injection led to an abscess at the site of injection due to impurities.

  fight between them took a more dramatic turn. Macleod and Collip decided not to patent

  Collip ended in the spring of 1922. Collip returned to the University of Alberta in early May 1922 at the end of his sabbatical year. Best took over leadership from Collip of Connaught's insulin production, completed an MA degree before entering Medical School in the fall of Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 25 1922. After several attempts, Best and his colleagues at Connaught succeeded in finding a way of providing sufficient quantities of insulin by means of a new approach using acetone with slight acidification and pH adjustments at a temperature lower than 35°C. Eli Lilly introduced a benzoic acid method of purification and a rotary high vacuum pump procedure which made possible a large scale production of insulin. Their main goal was to increase the purity and yields of insulin preparations using various chemical approaches (58). Banting opened his own practice in May 1922 on Bloor Street and was able to treat diabetic patients at the Christie Street Military Hospital. By late August 1922, a larger diabetes clinic was established at Toronto General Hospital and Banting was given access to its patients. John Macleod went to a Marine station in New Brunswick trying to isolate insulin from fish. The relation between "the discoverers" remained difficult: jealousy, greed, paranoia, intermingled with hard work in dreadful conditions: a good description of the situation as reported by Barbara E. Hazlett (79). Each of them had a personal interpretation about their respective part in the discovery. It is under this tense situation that the Medical Research Council in Great Britain asked famous pharmacologist Sir Henry Dale (1875-1968) (who would in 1936 get the Nobel Prize in Physiology or Medicine for his work on acetylcholine), director at the National Institute of Medical Research, and a biochemist colleague Harold Dudley, to go to Toronto. They arrived in late September 1922 and were very impressed by the fact that extracts of pancreas were able to treat patients suffering from diabetes who until then had been starved to death. Dale was introduced to some of the first juvenile patients treated, Teddy Ryder and Elizabeth Hughes, the 15-year-old daughter of Charles Evans Hugues, the US Secretary of State, later Chief Justice of the Supreme Court. Another important visitor came to Toronto that autumn, at the end of November 1922: Prof. August Krogh from Copenhagen, a recent Nobel Prize

A

  c c e p t e d M a n u s c r i p t 26 winner whose wife was diabetic. At that time, neither Banting nor Best were aware of the real significance of Krogh's visit (see below).

  February 1923, 250 physicians, 60 clinics and 1000 patients in Canada and USA were receiving insulin. Insulin was sent to many other countries during the whole year 1923. Together with the press, politicians entered the insulin story. The Canadian Prime Minister made a recommendation to Parliament recognition for an annuity to Banting. The story of the Nobel Prize to Banting and Macleod was written many times (34,63,69,79). It was the first time a Nobel Prize was awarded for such a recent discovery. Banting is still until now the youngest recipient of the Nobel Prize in Physiology or Medicine and was the first Canadian to receive a Nobel Prize. Banting was featured on the cover of Time magazine on August 27, 1923. However, it was an embarrassing choice for the Nobel Committee. In his Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 27 testimony, Alfred Nobel had not given any indication for the selection of the candidates. It only mentioned that the discovery must have an enormous impact and should be of very great practical significance. Exactly what the discovery of insulin was. The awardees had to be alive and could not be more than 3 at the same time. Unfortunately there were four of them in the Toronto team.

28 December 10 .

 2810 Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t It was the first time that happened for the attribution of the Nobel Prize in Physiology or Medicine.

  . Paulesco mixed his science with religion and extreme right politics. He died in 1931. The following years for the Toronto investigators were honours, awards, receptions, lectures between work and writing papers. During a visit to Europe in 1934, Banting was knighted by King George VI of UK. It is only in 1925 that Macleod and Banting delivered their Nobel Lecture in Stockholm (85,86).

  Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 29 lung tissue[START_REF] Best | The inactivation of histamine[END_REF] and made important contributions on heparin, an anticoagulant drug[START_REF] Best | Heparin and the formation of white thrombi[END_REF].Best was nominated several times for the Nobel Prize in relation with these discoveries.In 1928, Macleod, whose health had been deteriorating with arthritis, decided to leave Toronto and return to Aberdeen. Best was strongly supported to replace Macleod as professor and head of the department of Physiology at the University of Toronto in 1929 after a year of co-operation with Macleod on the grounds of his youth: he was only thirty years old. In September 1930, a new lab facilities building was officially opened at 100 College Street in honor of Frederick Banting, the Banting Institute. Next to it, the Best Institute was opened in 1950. Charles Best published around 200 papers of high quality. He also set up a blood donor service which was very efficient, particularly at the beginning of World War II, when they prepared millions of dried serum and shipped them to England (90). The Connaught Laboratories produced large quantities of heparin for the prevention of blood clotting during transfusion. Frederick Banting was not as successful as Best following the discovery of insulin (34). He mainly wrote clinical papers. He became professor of Medical Research in the department of Pharmacology. He set up his own clinic for diabetic patients with some appointments from the General Hospital. His friends from Alliston, Joe Gilchrist and Fred Hipwell, took care of the clinic at 160 Bloor St in Toronto. Banting moved to cancer research. He married his second wife Henrietta, "Lady Banting" in 1939. His best achievement during the years after the discovery of insulin was his work at the National Research Council of Canada (NRC). He developed medical research in Canada, its organization, pushing to establish competitive laboratories with grants and stipends for young talented Canadian researchers. He also worked on biological warfare issues. Interestingly, two of the committee's members were Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 33 that Banting was added to the inventors to avoid further challenge on the basis of Collip andBest not being the sole inventors. Banting's name was also added to the British and Canadian patents. The three men promptly assigned their patent rights to the Board of Governors of the University of Toronto in exchange of one dollar each.The major players The Connaught Anti-Toxin Laboratories in TorontoAs described by Michael Bliss[START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF], the Connaught Laboratories played a critical role in the early production and distribution of insulin in Canada and abroad. Yet, as pointed out by Bliss's history student Christopher J. Rutty (103), the book provides only limited detail about the Laboratories and their history, because at the time Bliss did his research for the book published in 1982, the Connaught's archives had not been collected and catalogued. A 1968 book written by Connaught's second director Robert D. Defries on the first 40 years of the laboratories is cited in Bliss's book (104). The Connaught laboratories were the brainchild of an entrepreneurial, restless, enthusiastic and ambitious young physician, John Gerald FitzGerald, born on December 1882 in a rural Ontario village (103). He suffered from a manic-depressive condition and committed suicide in 1940. Yet, his vision and determination completely changed the landscape of Public Health in Canada as well as the role of the University of Toronto's research in fighting major diseases. The Connaught labs, besides their role in the insulin saga, made major contributions to the research development and large-scale production of a large range of biological products, including diphteria toxoid, pertussis vaccine, heparin, penicillin, combined vaccines, the Salk and Sabin polio vaccine, and smallpox vaccine (103,104).

A c c e p t e d M a n u s c r i p t 34 In

 34 early 1911, FitzGerald established a Laboratory of serum diagnosis in the Department of Pathology and Bacteriology at the University of Toronto. In the fall of 1913, he built a small stable and laboratory behind a private house in West Toronto hosting four horses, which enabled him to produce large doses of diphteria antitoxin for the Provincial Board of Health. In 1914 FitzGerald persuaded the University of Toronto to assume responsibility for his antitoxin production. The Antitoxin Laboratory was established in the Department of Hygiene on May 1 st , 1914, in the basement of the Medical Building. It produced tetanus antitoxin for the Canadian troops fighting in Europe. To support the war effort, Colonel Albert E. Gooderham, a local distiller and member of the university board of directors, as well as chairman of the Ontario Red Cross Society, funded the purchase of farmland 12 miles north of Toronto in York township, and the erection of new buildings. The facility was handed to the University of Toronto on October 25, 1917. It was named "Connaught Antitoxin Laboratories and University Farm", after the Duke of Connaught, patron of the Canadian Public Health Association and a friend of Gooderham. In 1946 it became "Connaught Medical Research Laboratories". It remained a self-sustaining part of the University of Toronto until 1972, when the University sold it to the Canadian Development Corporation, owned by the Canadian Government. In 1989, the Connaught labs through a variety of mergers became part of Sanofi Aventis. Its vaccine business became Sanofi Pasteur, of which the former Connaught is the Canadian component. Connaught produced insulin until 1983. From 1984 until 1993Connaught supplied Canada with insulin from Novo, later Novo Nordisk after the 1989 merger, to whom it had leased its production facility.

A c c e p t e d M a n u s c r i p t 35 Charles

 35 Best became director of the Connaught Laboratories Insulin Division from 1922 to 1925, then Assistant Director from 1925 to 1931 and Associate Director from 1931 to 1941.He led the development of the anti -coagulant heparin[START_REF] Best | Heparin and the formation of white thrombi[END_REF].David AylmerScott (1892Scott ( -1971)), who was part of the heparin team, also played a major role in standardizing the conditions for successful crystallization of insulin (see below).A detailed history of the Connaught Laboratories written by Christopher Rutty (103) as well as a rich iconography can be found on the Web Site of the Connaught Fund: Connaught.research.utoronto.ca/history.Eli Lilly and Company in IndianapolisEli Lilly and Company, in Indianapolis, Indiana, USA, was founded in 1876 by, and named after, Colonel Eli Lilly (1838-1898), a pharmaceutical chemist and veteran of the American Civil War (on the Union side) (105). It started as a small, two-story building near the main business street of Indianapolis. In 1890, Colonel Lilly turned over the management of the company to his son Joshuah Kirby Lilly Sr (1861-1948), who ran the company for 34 years and oversaw a major development. A critical step was when Joshuah Lilly hired British-born and trained biochemist George Henry Alexander Clowes (1877-1958) in the summer of 1919 as Director of Biochemical Research. Clowes transformed Eli Lilly and Company into a major science-based pharmaceutical company, and put a lot of emphasis in fostering collaboration with external academic scientists (106,107). A fateful encounter happened when Clowes attended the presentation by Macleod, Banting and s Best at the December 30, 1921 session of the American Physiological Society conference at Yale University in New Haven. Clowes in the end managed, as narrated above, to secure the rights for Eli Lilly and Company to produce and sell insulin to American patients. There were some fights between Eli Lilly and Co and the Toronto researchers on the brand name for Lilly insulin. It ended up as Iletin with A c c e p t e d M a n u s c r i p t 36 insulin as a subtitle. By the end of 1923, nearly 25.000 Americans were receiving insulin. The company's profits soared. Clowes got the Banting Medal of the American Diabetes Association in 1947 for his role in making insulin widely available. A lively biography of Clowes has been written by his grandson (107). The initial insulin success was the first step in Eli Lilly and Co becoming the market leader in insulin and diabetes therapy for decades. They would again make history in 1982 by launching DNA recombinant human insulin under the name Humulin, the first recombinant protein therapeutic (108-110). Nordisk Insulin laboratorium and Novo Therapeutisk Laboratorium in Denmark A leading figure in setting up the production and commercialization of insulin in Europe was the Danish zoologist Shack August Steinberg Krogh (1874-1949), known as August Krogh (100,111,112) (Fig. 6). Krogh became professor at the Department of Zoophysiology at the University of Copenhagen from 1916 to 1945. His wife Marie (Fig. 6) was a physician and his lifetime close collaborator. A biography of August and Marie Krogh was written by their daughter Bodil Schmidt-Nielsen in 1995 (113). August Krogh obtained the Nobel Prize in Physiology or Medicine in 1920 for discovering a mechanism that opens and closes blood capillaries according to the tissues need for oxygen. He received invitations to give lectures in prestigious place in both Europe and the USA. However, Krogh had to cancel his travels because his wife had fallen ill in the summer of 1920. She consulted a young physician that Krogh knew since 1919, Hans-Christian Hagedorn (1888-1971) (Fig. 6). He was already well-known for having designed, together with Birger Norman Jensen (1889-1946), a new and very accurate micromethod for measuring blood glucose that did not require puncturing the veins (114). Hagedorn had just passed his dissertation in the Faculty of Medicine. He found out that Marie had diabetes. Neither insulin nor oral hypoglycemic agents were available in 1922, so Hagedorn put her on a strict Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnab020/6323380 by guest on 19 July 2021 A c c e p t e d M a n u s c r i p t 44 the effect of pancreatic duct ligation on exocrine and endocrine pancreas, showing

  in 1922, leading him to nominate Banting and Macleod for the Nobel Prize. Had the Nobel Committee been wise in specifying this fact, and not just "discovery of insulin", much controversy would have been avoided.

A c c e p t e d M a n u s c r i p t 45 The

 45 controversy in limiting the award to Banting and Macleod, however, would continue, as discussed in the last chapter of Michael Bliss's 25 th anniversary edition of his book[START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF] and in refs. 146 and 147. Sadly, the relationship between the members of the Toronto quatuor, already bad before the award, steadily deteriorated afterwards in large part due to Banting's paranoid dislike of Macleod. Jesse Roth and colleagues speculated that he may have suffered of posttraumatic stress disorder after his combat experience in WWI where a shrapnel wound to his right arm almost ended in amputation (147). Macleod went back home to Aberdeen in 1928 disenchanted by his Toronto experience and did not comment on it afterwards. Banting did not achieve any notable scientific breakthroughs until his tragic death in 1941. Best and Collip pursued distinguished academic and scientific careers.

  Bliss's book, released by the Canadian Broadcasting Corporation on June 28, 1988. It won nine Gemini Awards in 1989. The above considerations in no way diminish the merits of the Toronto team as a whole nor the miracle of being able to immediately resuscitate patients dying of diabetes. Some of A c c e p t e d M a n u s c r i p t 46 them survived the insulin discoverers by many years. The isolation and purification of insulin in Toronto in 1921 undoubtedly represents one of the major medical breakthroughs of the early 20 th century.

  William Rostène, PhD, Institut de la Vision, Sorbonne Université, INSERM U968, 17 rue Moreau, 75012 Paris, France. E-mail: william.rostene@inserm.fr. Pierre De Meyts, MD, PhD. Avenue Reine Astrid 42, B-1950 Kraainem, Belgium. E-mail : pierre.demeyts@gmail.com Disclosure summary : PDM is an unpaid external consultant for Novo Nordisk A/S.

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	carbohydrates in obese patients improved diabetes. He introduced the use of physical
	training to decrease blood sugar in medical practice
	M a n u s c r i p t
	A c c e p t e d
	8

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Acknowledgments

WR thanks the Thomas Fisher Rare Books Library at the University of Toronto (UNESCO Heritage); Sanofi Pasteur Canada; Heffel Fine Art Auction House; BIU Sante Paris, Dundurn Press for the permissions to use documents; Bob and Jim Banting (Website www.bantinglegacy.ca) for the audios and unpublished documents on Frederick Banting; Janna Best for the book of her husband Henry BM Best, son of Charles Best; Tomas Hökfelt, Juleen Zierath and Thomas Perlmann for his help in the Swedish connexion and Helene Rostène for language corrections in the text. All references from the Comptes Rendus de la Société de Biologie are available on the website from the French National Library (www.gallica.bnf.fr). PDM thanks Prof. Pierre J. Lefèbvre for bringing to his attention the book in ref. 1. We would like to acknowledge the great job done by the referees whose insightful comments helped us to improve the manuscript. We dedicate this review to the memory of Michael Bliss, whom both the authors knew. In 2007, he wrote in the preface of his book The discovery of insulin (25 th anniversary edition, ref 34): "I look forward to rewriting this preface on the centennial of insulin's discovery in 2021-22, and to celebrating the occasion, once again, with my dear wife Elizabeth and our children and grandchildren". He unfortunately passed away in 2017. We hope our review, for which he was an inspiration, goes in a modest way towards fulfilling his wish for an update on the 100 th anniversary.

PDM is an unpaid external consultant for Novo Nordisk A/S.

Unfortunately, he never reached England. He died in an airplane crash on February 21, 1941 at the age of 50. His death in a Hudson bomber over Newfoundland remains an enigma. Was it sabotage or a pilot error due to bad weather or a technical problem? [START_REF] Callahan | The Banting Enigma[END_REF]. Banting had official funerals.

John Macleod had returned to Scotland in 1928. He had a position as professor of physiology at the University of Aberdeen. He then worked on the observation of Claude Bernard on the hyperglycemia due to puncture of the 4 th ventricle. He found that gluconeogenesis in rabbit liver was controlled by parasympathetic innervation. He died in Aberdeen on March 16, 1935 at the age of 59.

When James Bertram Collip definitively returned to the University of Alberta in 1922, as a full professor and head of the department of biochemistry, he moved away from general research in the field of insulin and focused on the identification and isolation of hormones of therapeutic value. Collip was a leading researcher in endocrine science during the 1920s and 1930s. His most important contribution in the first years after the insulin discovery was, in 1924, the isolation of the parathyroid hormone [START_REF] Collip | The Internal Secretion of the Parathyroid Glands[END_REF]. In 1928, Collip was appointed to the chair of biochemistry at McGill University, succeeding his mentor Archibald Macallum.

Among his prestigious graduate students was Hans Selye, "the father of stress", with whom, in 1933, in collaboration with Anderson and Thompson, he purified an adrenocorticotropic [START_REF] Collip | Demonstration of an orally active medullotrophic principle in a primary extract of pituitary tissue[END_REF][START_REF] Rostène | The Nobel Maze: from the discovery of insulin to that of stress[END_REF]. Collip also worked on the identification of thyroidstimulating hormone (TSH) [START_REF] Anderson | Studies on the physiology of the thyreotropic hormone of the anterior pituitary[END_REF]. In 1930, in collaboration with a small Montreal pharmaceutical firm, Ayerst, Collip isolated what he called Emmenin, a form of estriol from the placenta that was commercially significant because it could be administered orally [START_REF] Collip | Placental hormones[END_REF].

Later, the Ayerst researchers, on Collip's advice and using his standardization procedures, developed a different orally active estrogen from pregnant mares' urine called Premarin. Washington [START_REF] Collip | Science and War[END_REF]. In 1946, he became the first director of the Division of Medical Research [START_REF] Jb | Collip and the Development of Medical Research in Canada[END_REF]. Because of Canada's limited population, Collip argued against building national laboratories but instead promoted grants-in-aid for research at universities and teaching hospitals. In 1947, he became dean of medicine at the University of Western Ontario (99). Collip received many honors during his career. He died of a stroke in 1965 at the age of 72. He found the bitter conflict associated with the discovery of insulin absolutely distasteful. Known for his integrity, modesty, and quiet nature, Collip always refused to discuss the insulin days, only saying that "he was sure the historical record would speak for itself" as reported by Alison Li in her excellent book on Collip [START_REF] Jb | Collip and the Development of Medical Research in Canada[END_REF].

Commercialization and globalization of insulin therapy: the early days

The early developments of the pharmaceutical production of insulin have been discussed in several books and reviews [START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF][START_REF] Vecchio | The discovery of insulin: an important milestone in the history of medicine[END_REF]100,101). Later and contemporary developments in insulin therapy have been thoroughly reviewed recently in this journal by Irl Hirsch and colleagues (102). In this review, we will briefly summarize here the early developments.

Patent issues

Initially, the Toronto team started the production of insulin in collaboration with the Connaught Anti-Toxin Laboratories, founded in 1914 by a professor of hygiene, John G. FitzGerald (1883-1940) [START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF] (see below). The agreement, dated January 25, 1922, precluded that Banting, Best and Collip get into any patenting agreement with any commercial firm. Eli Lilly had already expressed interest (see below). Faced with difficulties in large scale production, the Toronto team and FitzGerald decided on May 22, 1922 to accept the offer of collaboration from Eli Lilly and Co, and to apply for an American patent. Lilly would retain the territorial rights to the United States, Central and South America. The US patent application ran into initial trouble when the chief American patent examiner became aware of the 1912 US patent granted to George Zuelzer. The patent was also contested by John R.

Murlin and reluctantly by Ernest L. Scott [START_REF] Bliss | The discovery of insulin. 25th anniversary edition[END_REF]. After support from a number of eminent scientists and also Charles Evans Hughes, the patent was finally granted under the condition Describing his remarkable biography is outside the scope of this review, for details see ref. John Abel turned his attention to insulin recently discovered in Toronto in 1924. It was recognized that the Toronto preparation contained impurities, and that it would be desirable to isolate the hormone in pure form. There was also disagreement about its chemical nature (123). At the end of 1925, Abel first obtained insulin crystals (124-126). This was a major step in obtaining a pure insulin preparation. The crystals were also used to confirm that insulin is a protein. Vincent du Vigneaud (1901Vigneaud ( -1978)) It is only in the early 30's that it was found that the key to a stable crystallization of insulin was the presence of metal ions such as zinc. David A. Scott of the Connaught laboratories while on sabbatical in London, UK, tried to crystallize insulin using Abel's method, without success (Fig. 8). Back at Connaught in January 1930, Scott realized that pancreatic tissue contains appreciable amounts of zinc, cobalt and nickel. He discovered that the addition of small amounts of zinc chloride to a buffered solution of insulin resulted in the precipitation of insulin crystals. He showed that by refining this process large quantities of highly purified insulin could be consistently produced (132,133). This was a major breakthrough in the early history of the insulin saga since it opened the way to the development of ultra-pure insulins and insulins with different PK/PD properties (102), as well as paving the way to solving the three-dimensional structure of insulin (134), although this would take another 35 years.

A brief glimpse of later milestones

The sudden availability of insulin from early 1922 was truly a miracle for the young patients who were slowly dying of starvation on Allen's diet [START_REF] Allen | Prolonged fasting in diabetes[END_REF], and for the parents who witnessed their resurrection. Further milestones in insulin therapy and diabetes treatment have been recently reviewed [START_REF] Jörgens | Unveiling Diabetes-Historical Milestones in Diabetology[END_REF]102,135,136). Insulin was also a miracle for scientists interested in basic research on proteins. Many of the major technical advances in studying proteins were made using insulin first. It was the first protein to be sequenced in 1951-1955 (137), earning Fred Sanger his first Nobel Prize in 1958, the first protein to be crystallized as narrated above, the first protein to be synthesized in the early 60's in the United States (138), Germany (139) and China (140), the first protein hormone to have its plasma level measured by radioimmunoassay in 1960 by Solomon A. Berson and Rosalyn S. Yalow (141), earning Yalow the 1977 Nobel Prize in Physiology or Medicine after Berson's death in 1972 (142), and the first protein therapeutics made from recombinant DNA to be approved by the FDA in 1982 (Humulin Lilly) (108-110). Since the scope of our review is limited to the periods preceding and immediately following the 1921 discovery, we will not elaborate further here on subsequent milestones (reviewed elsewhere [START_REF] Vecchio | The discovery of insulin: an important milestone in the history of medicine[END_REF]143,144).

Summary and conclusions

The triumph of the Toronto team in producing an efficient insulin extract that was suitable for injections in humans started as an unlikely story. Many experienced researchers had tried unsuccessfully to tackle this problem. Their preparations lowered blood glucose in pancreatectomized dogs but were too toxic for treating humans. A 29-year-old surgeon with no research experience and no expertise in diabetes or endocrinology, Frederick Banting, had an epiphany upon reading in bed a paper from a Minneapolis pathologist who described