Lasker basic medica l COMMENTARY research award

A tale of two hormones

Jeffrey M Friedman

“Der Mensch denkt, Gott lenkt.” (“Man equivalent to a death sentence1. The only avail- Kleiner’s story had great personal resonance proposes, God disposes.”) able treatment was a starvation diet advocated for me. Here was another Jewish scientist, the —German proverb by Frederick Madison Allen, a physician work- grandson of immigrants, working a century ing at the Rockefeller Institute for Medical earlier at the same institution as me. But on A little more than a century ago, an arc of Research (now ) and the cusp of isolating the most important hor- research began that culminated in the identi- a leading authority on diabetes1–5. Allen was mone ever discovered, he walked away from it. fication of insulin by four scientists working the first to realize that diabetes was a general How did Kleiner come to study diabetes? Why in Toronto. With astonishing speed, this land- disorder of metabolism and that acidosis and did his studies cease so abruptly? Did Kleiner mark discovery became a life-saving treatment death could be forestalled if caloric intake was and his colleagues fully understand the impli- for thousands of people with diabetes around restricted. When acidosis developed, calories cations of his research? What was the personal the world. In time, insulin was established as were further reduced, and, for many, diabetes impact of his having missed the opportunity of the most important anabolic hormone and was a race between starvation and acidosis, the a lifetime? Kleiner’s work and career also raise found its place in the pantheon of medicine’s ultimate result of either condition often being a general question: what are the elements of a greatest discoveries. death. The discovery of insulin by Frederick discovery? These were the questions I set out Fifteen years ago, leptin was identified as Banting, Charles Best, James Bertram Collip to answer. another metabolic hormone with major cata- and John James Macleod changed the treatment bolic effects. This story, however, is not about of diabetes forever by providing a hormonal Israel Kleiner’s magnificent paper leptin or insulin. Rather, it is about the path treatment from the pancreas that could rapidly In 1889, Josef von Mering and Oskar to discovery for each of them and about the restore bedridden, cachectic and moribund Minkowski observed that removing a dog’s path to discoveries in general. It is a story of children and young adults to healthy lives. pancreas resulted in polyuria, polydipsia and discoveries made and not made. It is about how One of the many scientists who tested the diabetes10. After Eugene Opie noted that the I found my way into the world of Israel Kleiner, ability of pancreatic extracts to treat diabe- islets of Langerhans in the pancreas were often who, but for the mysterious winds that shape tes before the discovery of insulin was Israel destroyed in humans with diabetes, the crucial our paths, might have been the man who dis- Kleiner (Fig. 1), a biochemist who also question became how this structure controlled covered insulin almost a century ago. worked at the Rockefeller Institute between sugar metabolism11. One possibility was that I have had the privilege of listening to numer- 1910 and 1919. In a study published in 1919, the islets of Langerhans produced a hormone, ous first-hand accounts from many great sci- two years before Banting and Best published referred to then as an ‘internal secretion’, entists describing their moments of discovery. their first paper, Kleiner conclusively showed that regulated glucose concentrations in the And over the course of my career, I, too, have that extracts of pancreas but not other tissues blood. Beginning with Georg Ludwig Zuelzer experienced this thrill. But even before the dis- could lower blood glucose in diabetic dogs6. in the early 1900s12, a number of scientists covery of leptin, I was fascinated by the expe- Referring to Kleiner’s work, Bliss wrote, “of all began to test the possibility that the pancreas riences of people who have made important the publications before the work at Toronto, it might make a hormone that controls blood discoveries. My interest in great discoveries was the most convincing”7, and later opined sugar levels, although definitive evidence of led me to The Discovery of Insulin, by Michael that “his controls had been impressive; his this was lacking. Thus, although Joseph Pratt Bliss1. This enthralling work tells the tale of the follow-up discussion was a beautiful piece of and Macleod had concluded that individuals 1922 discovery of insulin. In gripping fashion, scientific writing”7. with diabetes were missing an internal secre- it traces the progression of ideas that led to one Then, Kleiner’s work abruptly ceased. While tion from the pancreas and that a pancreatic of the key achievements of the twentieth cen- history has correctly bestowed the credit for extract could be used to treat diabetes13,14, this tury and arguably the first instance in which discovering insulin on the Toronto group, Bliss view was not universal. In 1913, Allen wrote, science provided a new lifesaving medicine for wrote of Kleiner, “in 1919 he was closer to suc- “though pancreas feeding may have at a least a the morbidly ill. cess than any of them, and made no claims at digestive value in some cases of diabetes, injec- Before the discovery of insulin, a diagnosis all”8. Kleiner never publicly discussed why his tions of pancreatic preparations have proved of diabetes, particularly in children, was often research stopped so precipitously, other than both useless and harmful. The failure began writing four decades later “why we did not with Minkowski and continued to the pres- Jeffrey M. Friedman is at The Rockefeller continue and isolate the antidiabetic factor is ent without an exception”15. Forty-two years University, New York, New York, USA. a long story and has no place in the present later, in a letter to Kleiner, Allen acknowledged e-mail: [email protected] discussion”9. that it was Kleiner’s pioneering discovery that

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entered the graduate school at Yale, then ence of the pancreas the circulation rids itself called the Sheffield Scientific School, and easily of the intravenously introduced dextrose; did research in the laboratory of Lafayette but that it is unable to do it satisfactorily in the Mendel18. Mendel was one of the intellectual absence of the pancreas”26. This distinction is giants at the school and is now considered to important because, starting with the brilliant be the father of nutrition research in the US. work of Claude Bernard showing that the liver Mendel and his colleagues had developed an can synthesize glucose27,28, the prevailing view apparatus that allowed them to selectively was that diabetes primarily results from glu- deprive an animal of specific nutrients and cose overproduction. We now know that both used it to show that animals require essen- overproduction and decreased consumption tial amino acids that cannot be synthesized contribute to the disease, and Kleiner and endogenously19. Among many other contri- Meltzer’s results were the first to show that butions, he also reported the consequences of insulin regulates glucose use. vitamin A deficiency19. Although the complete data set was not Kleiner’s thesis was entitled Studies in published until 1919, Kleiner’s and Meltzer’s Intermediary Metabolism, and his research preliminary report was the first to show a ben- focused on pyrimidines, with the ultimate eficial effect of a pancreatic extract on blood goal of understanding more about the func- glucose levels in diabetic dogs6. The only prior tion of nucleic acids18. After teaching at report measuring blood glucose after injection Tulane University in New Orleans for a of pancreatic extracts showed an increase rather year, he applied to the laboratory of Samuel than a decrease in blood glucose levels29. The Meltzer at the Rockefeller Institute, express- implications of Kleiner’s and Meltzer’s findings, Figure 1 Photograph of Israel Kleiner as he ing an interest in returning to research20. By published seven years before Banting’s and neared retirement as a professor of 1910, the Rockefeller Institute had already Best’s first paper, were reported in a prominent at the New York Medical College. established a reputation as one of the leading article in the New York Times on August 19, non-university research institutes in the world 1915 under the headline “Find Diabetes Cause, pancreatic extracts lowered blood glucose that and one of the few places in the US that pro- Now Seek a Remedy: Rockefeller Institute had helped to change his opinion, as he wrote, vided funding for research21. Simon Flexner, Doctors Say Disease is Due to a Failure of the “I can corroborate Joseph H. Pratt’s statement the director, had assembled a group of highly Pancreas”26,30. that you announced a pancreatic extract with distinguished international scientists whose However, efforts to seek a remedy were sugar-reducing properties something like eight mission was to address problems of human soon interrupted. The world was at war. The years before the announcement by Banting and health. In 1904, Samuel Meltzer was the first research enterprise at Rockefeller was dis- Best”16. physiologist appointed at the Institute22. rupted and, regarding to this period, Kleiner The controversy about whether diabetes Meltzer was a highly distinguished member later wrote, “in the meantime, the first World results from a deficiency of a hormone made of the National Academy of Sciences, and his War had come about and interfered with much by the pancreas existed largely because the most notable achievement is the development of this work”9. Kleiner did not take up his stud- available tools were limited. There were two of the respirator, or ‘insufflation device’, as he ies of the effects of pancreatic extracts until key problems. First, it was possible to mea- referred to it23. 1918, with Meltzer writing in his January 1919 sure sugar in the urine, but not the plasma, of In Meltzer’s laboratory, Kleiner initiated report to Flexner31, “Kleiner took up again animals with diabetes, and the imprecision of studies of the disposition of glucose after our former studies of the neutralizing effect measuring urinary glucose made it difficult for intravenous injection into normal and diabetic of intravenous injection of pancreas extracts investigators to conclusively ascertain whether (pancreatectomized) dogs24,25. In 1914, Kleiner upon the hyperglycemia and glycosuria pro- the pancreas extract was improving glucose and Meltzer reported that 1.5 hours after the duced by depancreatization.” metabolism9. Second, the extracts were quite injection of a glucose bolus, the plasma glu- By that time, research at the Institute had crude and had side effects, including fever cose concentration in a diabetic dog was more largely returned to normal32, but not for and inflammation, making them unsuitable than three times that found in a normal dog24. Kleiner. Meltzer had diabetes and decided to for human use17. These difficulties impeded In another study, Kleiner and Meltzer also retire, writing at one point to Flexner, “there is Zuelzer’s efforts to treat patients with diabetes. reported that extracts of pancreas could pre- not much difference in the fate of an old fash- Banting, Best, Macleod and, particularly, Collip vent the abnormal rise of plasma glucose after ioned and a modern diabetic. Confusion and solved the second problem. Israel Kleiner con- glucose injections into pancreatectomized dogs humbug reigned then and reign still”33. With firmed the first. and that these extracts could also correct the Meltzer’s retirement, a decision was made to Israel Kleiner was born in New Haven, hyperglycemia of diabetic dogs at baseline26. In disband Meltzer’s Laboratory of Pharmacology Connecticut in 1885. His grandfather emi- retrospect, these studies were the first to show and Physiology, and his laboratory members grated from Frankfurt, his grandmother from that insulin can correct the abnormal glucose needed to find new positions. The only posi- Alsace. His father was a tailor and had hopes tolerance of a diabetic animal. tion Kleiner could find was at the New York that his son would follow in his path. Israel had Kleiner and Meltzer concluded that glyce- Homeopathic College, and he moved there other plans (R. Glanz, A. Glanz and K. Glanz mia after a pancreatectomy was not due to an in September 1919 (ref. 34). However, before (Kleiner’s daughter and grandsons, respec- overproduction of sugar and raised the possi- moving, Kleiner completed his studies on dia- tively), personal communication). bility that “the removal of the pancreas causes a betes and published a paper, “The Action of After completing his undergraduate stud- decrease of consumption of glucose by some of Intravenous Injections of Pancreas Emulsions ies at in New Haven, Kleiner the body tissues”24, later writing, “in the pres- in Experimental Diabetes,” of which he was

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the sole author. The paper was submitted in Pancreatic September 1919 and published two months later6, approximately two and a half years Submandibular before Banting and Best’s first publication35. The paper is a masterpiece. In it, Kleiner 0.4 presented a comprehensive data set comparing the effects of injections of pancreatic and sub- 0.3

mandibular gland extracts on plasma glucose els (%) v levels in pancreatectomized dogs. The choice 0.2 of submandibular gland extract is notewor- thy; because the glands are also an exocrine 0.1

organ with high levels of digestive enzymes, Glucose le the extract is a perfect control for a pancreas 0.0 extract. In addition, to minimize immune reac- Baseline End infusion 95 min after infusion tions, Kleiner prepared the extracts from the organs of the same dog that would later receive Figure 2 Kleiner’s key experiment. The graph shows the effect of pancreatic versus submandibular it. extracts on glucose levels in pancreatectomized dogs. *P = 0.008, **P = 0.005. Data reanalyzed from The studies were all performed under ref. 6. precisely the same conditions and were well controlled, and the results were dramatic and “the fact that these pancreas emulsions lower to how the extracts were made (acid or base unequivocal even for modern standards of blood sugar in experimental diabetes without extraction), whether whole pancreas or pan- analysis. Pancreatic, but not submandibular, causing marked toxic effects indicates a possible creas after pancreatic duct ligation was used extracts lowered blood glucose levels. Papers therapeutic application to human beings.” He and how long the time interval was between of this era were generally presented in a narra- wondered whether an “emulsion of the pan- the preparation of the extract and its injection. tive form, describing each experiment that was creas from another species” would have the Owing to the highly variable protocols and the done in sequence, and the data in this paper same effect, foreshadowing the use of extracts fact that rigorous quantitative data are not pre- were in this format. Statistical analyses were prepared from animals to treat humans. He sented, it is not possible to perform statistics not typically performed in this and other con- closed by stating that the “effective agent or on the data or even draw a firm conclusion on temporaneous papers. Nevertheless, Kleiner agents, their purification, concentration and whether their pancreatic extract had an effect. summarized his data in a table in a clear and identification are suggested as promising fields In short, the results of this study, published precise manner, making it straightforward to for further work.” seven years after Kleiner’s and Meltzer’s origi- now graph these data and perform statistical Indeed they were, but not by him, as there nal report and nearly three years after Kleiner’s analyses. (In the paper, Kleiner only reported were no opportunities for Kleiner to do research definitive paper, are inconclusive. the raw data for each dog.) at the New York Homeopathic College (D. Fortunately, this paper was only the begin- The reanalyzed data (Fig. 2) show that Lehr (Professor of Pharmacology at New York ning of their group’s work. Macleod next sug- baseline glucose levels (mg dP–1) were nearly Medical College from 1941 to 1980), personal gested that Banting and Best begin working identical (pancreatic extract (PE): 0.28 ± communication). The institution did not have with Collip, a biochemist, to purify the factor. 0.012, submandibular extract (SE): 0.31 ± animal facilities at that time and, when Kleiner First, by alcohol precipitation and later iso- 0.033; P = 0.38). By contrast, glucose levels eventually resumed his research career, he had electric focusing, they prepared an extract free at the end of the infusion (PE: 0.21 ± 0.014, to conduct his studies on the effect of insulin of toxins and, less than ten months from the SE: 0.31 ± 0.022) and 95 minutes after the on glucose transit from the bloodstream at the publication of Banting’s and Best’s initial paper, infusion (PE: 0.18 ± 0.013, SE: 0.34 ± 0.029) Cold Spring Harbor Laboratory (R. Glanz, A. the first injection of insulin was administered were significantly different for pancreatic Glanz and K. Glanz, personal communica- to a human subject with diabetes41,42. Within versus submandibular extracts (P = 0.008 tion)39,40. two years, insulin was the new standard of and P = 0.005, respectively). Comparisons of Insulin was discovered three years later. In the care for diabetes. In August of 1923, Banting blood glucose in diabetic dogs before, during summer of 1921, Banting and Best, working in and Macleod were awarded the Nobel Prize in and after infusion of a pancreatic extract were the laboratory of Macleod, began their studies Physiology or Medicine. (Controversy followed even more significant. These plasma glucose on the effect of a pancreatic extract on diabetic when Banting announced that he would share levels were baseline 0.28 ± 0.012 SE versus dogs. In their first paper on this topic, published his award with Best and Macleod announced 0.21 ± 0.014 SE at the end of the infusion, P in February 1922, they reported on 71 injec- he would share his with Collip.) < 0.005, and baseline 0.28 ± 0.012 SE versus tions of pancreatic extracts into six dogs35. (In In short, although the credit for the discov- 0.18 ± 0.013 SE 95 minutes after the end of the the discussion of their paper, Banting and Best ery of insulin and its development as a treat- infusion, P = 0.0005. refer to 75 injections into 10 dogs, but the results ment for human disease belongs to Banting, Kleiner was fully aware of the implications section only shows the above data.) There were Best, Collip and Macleod, they were not the of these findings (as were others, in time)36–38. only two trials using extracts from other tissues, first to prove the existence of a pancreatic hor- The introduction of the paper stated that the one from liver and one from spleen. mone that regulates glucose—this achievement demonstration of a beneficial effect of a pan- Of the trials with interpretable data, 37 of is Kleiner’s. Even Banting and Best acknowl- creas preparation when given to a diabetic dog 63 injections were suggested to have an effect edged Kleiner’s primacy in their own paper, would “support the internal secretion hypoth- on blood glucose, although, in many cases, where they wrote that injections of pancreatic esis of diabetes” and “suggest a possible thera- the effect was small. In these studies, 25 sets extracts lower blood glucose, “thus confirm- peutic application.” In the discussion he wrote, of conditions were used, differing with respect ing Kleiner”32. Kleiner’s research predates by

xiv volume 16 | number 10 | october 2010 nature medicine commentary several years similar studies by Nicolae Paulesco, who would nonetheless make stri- dent claims that it was he who discovered insulin1,17,43,44. Kleiner was the scientist who, building on decades of research by others, established that the pancreas contains an internal secretion Hypothalamusuus Food that can be used to treat diabetes. On the occa- intake sion of Kleiner’s seventieth birthday, Donald Van Slyke, Kleiner’s former colleague at the Energy Rockefeller Institute and one of the fathers Leptin expended of clinical chemistry wrote, “I recall the early days at the Rockefeller Institute when you demonstrated the effect of pancreas extracts Fat tissue in decreasing blood glucose concentration and urinary glucose excretion and pointed out clearly the probable therapeutic application of Figure 3 Leptin and the regulation of energy balance. Leptin is the afferent signal in a negative pancreatic extract in human diabetes. After feedback loop that maintains homeostatic control of adipose mass. It circulates in the blood and your paper in the 1919 Journal of Biological acts on the brain to regulate food intake. When fat mass falls, plasma leptin concentrations fall too, Chemistry, one may say that the next steps, in stimulating appetite and suppressing energy expenditure until fat mass is restored. When fat mass separating insulin from the extract and apply- increases, leptin levels increase, suppressing appetite until weight is lost. This system maintains homeostatic control of adipose tissue mass. Leptin acts on a receptor localized in the hypothalamus, ing it to treatment of diabetes, were inevitable. and elsewhere in brain, to regulate energy balance and other systems64,65. Leptin thus conveys A call to a teaching position interrupted your nutritional information to specific neural populations in the brain, which in turn regulate most, and own studies, and the next step was soon made perhaps all, other physiological systems. This homeostatic system enables mammalian organisms to in Toronto, but the honor of clearly showing maintain optimal levels of stored energy (fat) under a wide range of environmental conditions. the way remains yours”45. prepared mind is favored by chance.” The path to discovery the case of Banting, Best, Collip and Macleod, For many discoveries, Pasteur’s famous apho- the technical advance was the development of The discovery of leptin rism applies, that “in the fields of observa- biochemical methods, together with Collip’s The same elements played a part in the dis- tion, chance favors only the prepared mind.” competence as a biochemist, that made pos- covery of leptin in my laboratory. A set of rel- However, many other discoveries are not the sible their purification of insulin. evant hypotheses and questions were in the air. result of serendipitous observation and rather The third element is good fortune, or at least Decades before our work, in the 1950s, Gordon are ‘in the air’. Discoveries of this sort are often the absence of ill fortune, which provides a sci- Kennedy proposed that adipose tissue mass is made nearly simultaneously and referred to as entist in the right place at the right time with regulated by an endocrine system52. Building ‘multiples’46,47. With reference to this frequent the opportunity to complete his or her work. on Albert Hetherington’s and Stephen Ranson’s concurrence of important discoveries, Robert Although it can never be known whether studies from the 1930s showing that lesions in Merton paraphrased Francis Bacon, writing, Kleiner would have succeeded in purifying the hypothalamus can cause obesity in rats, “once the right path is followed, discoveries in insulin or even if he would have embarked on G.R. Hervey used parabiosis to suggest that limitless number will arise from the growing this course, he clearly lacked a suitable place to an endocrine factor that acts on the hypo- stock of human knowledge”47. Such discover- continue his studies. In 1919, there were few thalamus controls this putative homeostatic ies can often be characterized in the following institutions other than the Rockefeller Institute system53,54. way. where a scientist, particularly a Jewish scientist, The identification and characterization of First, as a result of a set of observations, a had access to the facilities and funding neces- obese (ob/ob) mice by George Snell and his question is posed. In the case of insulin, the sary to do research. Although formal quotas colleagues provided further support for the key observation was Minkowski’s finding in for Jewish students—numerus clausus—did not hypothesis that body weight is under physio- 1889 that diabetes occurs after the removal of begin until the 1920s, there were few positions logical control; they showed that an autosomal the pancreas. In this instance, chance favored available for Jews on a university faculty then, recessive mutation on mouse chromosome Minkowski’s prepared mind. The question then and for decades after (D. Lehr, personal com- 6 resulted in massive obesity and hyper- became whether there was a hormone made by munication; J. Darnell (Rockefeller University), phagia55,56. In the 1970s, Doug Coleman at the the pancreas that lowers blood glucose. personal communication)50. At the Sheffield Jackson Laboratory used parabiosis to charac- The second element is a technical advance School (the forerunner of Yale Medical School) terize ob/ob mice and diabetic (db/db) mice—a (in the hands of a scientist with the techni- the only Jew on the faculty was Lafayette second mutant model that Coleman identified cal expertise to implement it) that provides Mendel. And, despite his great achievements, that also developed severe obesity. On the basis a means for addressing the question in a new even Mendel was becoming less comfortable in of these studies, Coleman predicted that ob/ way. In Kleiner’s case, this was the development his position during same period in which the ob mice lack a blood-borne factor that regu- of methods that made it practical to measure Kleiner story unfolded51. lates body weight and that db/db mice lack its glucose in small blood samples9,48,49. It was Thus, with regard to this final requirement receptor57. the limitation of measuring urine glucose that for making key scientific discoveries, one might Thus, studies from multiple laboratories made it so difficult to establish the importance consider inverting Pasteur’s quote by suggest- over the course of several decades suggested of a pancreatic factor before Kleiner’s study. In ing that, for those making discoveries, “the that body weight is regulated by an endocrine

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Figure 4 Savin Rock, by Israel Kleiner. Savin Rock was an amusement park close to Kleiner’s childhood home. loop, that the ob gene encodes the key hormone methods available at that time were not suitable odology that we employed when in 1986 we set in this pathway and that this hormone acts on for this purpose. For those who were interested out to clone the ob gene. Eight years later, my a receptor, encoded by the db locus, located in in identifiying the ob and preceded us, laboratory reported the identification of ob in hypothalamic centers that are known to regu- and for reasons that became fully evident only mouse and human61. late weight. after the genes were cloned, the relevant fac- The ob gene encodes leptin, an adipocyte This was the working hypothesis of my lab- tors are present in amounts too small to allow hormone that functions as an afferent signal oratory when we set out to clone ob and db. their identification by a conventional feeding in a negative feedback loop that regulates food However, similarly to Allen’s skepticism about bioassay58. In addition, we now know that the intake and body weight58,62–65. This homeo- the existence of an antidiabetic hormone in the leptin does not act acutely to suppress feeding, static system enables mammals to maintain pancreas, the view that an endocrine system therefore requiring longer periods of monitor- optimal levels of stored energy (fat) under a might regulate body weight was not widely ing and possibly even multiple doses to observe wide range of environmental conditions (Fig. shared (D. Coleman (Jackson Laboratory), a robust effect58. 3). The identification of leptin confirmed that personal communication). Indeed, many Fortunately for my group, a methodology body weight and feeding behavior are regulated researchers at the time questioned the existence was developed in the early 1980s that allowed by a robust physiological system and not, as of a physiological system that regulates body investigators to identify mutant genes based on many believed, controlled primarily by will- weight and the relevance of the ob and db genes their position on a genetic map59,60. This task power. These findings also established adi- for human physiology, arguing that many of the was intrinsically difficult owing to the limita- pose tissue as an endocrine organ. Numerous features of the mutant mice were different from tions of available methodologies for work- reviews of this subject are available to those those of obese humans. ing with large segments of DNA that were who wish to delve deeper66–68. Determining who was correct required that megabases in length. It was not until more than Discovering a previously unknown hor- the endocrine factor proposed by Kennedy a decade later that this task became routine. mone and establishing its physiological func- and Hervey be identified and that the ob and While difficult, the task of positional cloning tion was the greatest professional pleasure that db genes be cloned. However, the biochemical did not seem impossible, and it was this meth- I have known. At 5 a.m. one morning in May

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1994, I developed an X-ray film that showed the grounding in research of either Macleod and K. Glanz, personal communication). alterations in the expression of a fat-specific or Collip, without whose expertise he would This raises one final question. If you were gene in the two available mouse lines carrying not have gotten very far, he attacked his objec- forced to make a choice between a contented the ob mutation (Fig. 3a in ref. 61). This single tive to find a pancreatic factor with a fierceness life and the thrill of having made a great dis- experiment confirmed that we had indeed born of desperation and, as much as anyone, covery, which would you choose? Many people cloned the ob gene and that it was under feed- was responsible for driving an idea that was I ask choose the former. I know what I would back control, a finding that strongly suggested decades old into a new therapy for desperately choose. that Coleman’s hypothesis that ob encodes ill patients in less than two years70. a novel hormone was correct. The elation of In contrast, even though Kleiner and ACKNOWLEDGMENTS I dedicate this article to R. Glantz, Kleiner’s daughter, peering into the depths of nature and being Meltzer were convinced that the pancreas syn- for her generosity in sharing her family history. I had the first to see something new is impossible to thesized an antidiabetic factor in 1915, there is the privilege to meet her before her death. I would describe. This article and the award that led no evidence between 1916 and 1919 that they also like to thank A. Glantz, K. Glantz and P. Kleiner me to write it are an echo of the jubilation of had any plans to purify the factor. Instead, for sharing their recollections of their grandfather and generously providing me with family documents. that moment. they worked on a set of tangentially relevant D. Lehr generously shared his memories of Kleiner Fifteen years after the discovery of leptin, projects: the effect of morphine and magne- and the environment of the New York Medical I remain mindful of the good fortune that sium sulfate on glucose metabolism and the College during the years that he and Kleiner were on made possible its discovery in my laboratory. mechanism by which the factor stimulated the its faculty. L. Hiltzik and T. Rosenbaum guided me Unlike Kleiner, I had an opportunity to con- transit of glucose from the blood. The extent through the resources of the Rockefeller Archives Center. Their help was indispensable, as were the duct my research in an environment that was to which the onset of the war or other factors resources of the American Philosophic Society. The optimal for this purpose. As an investigator shaped their focus is not known. But it is not New York Medical College generously shared its of the Howard Hughes Medical Institute at until Kleiner’s paper of 1919 that it is clear resources. G. Ceccarini performed the statistical Rockefeller University and with support from that he fully understood both the basic and analyses of the Kleiner paper and reviewed the original Banting and Best paper with great care and the US National Institutes of Health, I had ade- potential clinical importance of his findings. precision. Finally, I would like to thank J. Darnell, quate resources. And, furthermore, I benefited In 1918, Meltzer wrote to Flexner, “Kleiner A. Mark, S. Strickland, G. Ceccarini, Z. Knight, from the talents of numerous colleagues and has to pay the penalty for being a gentleman T. Rink, M. Tessier-Lavigne, J. Goldstein, T. De Lange laboratory members. and of a modest retired disposition”71. This and P.-O. Berggren for their helpful comments. I am also mindful of the absence of ill for- description was still relevant in 1955 when COMPETING FINANCIAL INTERESTS tune that could have led to a different outcome. Elliot Joslin, founder of the Joslin Clinic, on The author declares no competing financial interests. During the eight years my laboratory was inch- the occasion of Kleiner’s seventieth birthday ing its way down the chromosome toward ob wrote of how much he appreciated Kleiner’s 1. Bliss, M. The Discovery of Insulin (The Press, Chicago, 1982). at a glacial speed, hundreds of investigators “reticence in never calling attention in public 2. Allen, F. Studies Concerning Glycosuria and Diabetes. around the world were using a set of newly to (his) close association with the discovery (WM Leonard, Boston, 1913). available tools to place newly cloned genes on of insulin”72. 3. Stillman, E. & Fitz, R. Total Dietary Regulation in the Treatment of Diabetes. (Rockefellar Institute for the mouse genetic map. If a gene happened There is one final irony. Whereas Kleiner Medical Research, New York, 1919). to map to a site on the chromosome near the by all accounts lived a happy, calm and com- 4. Anonymous. Radical new method of treating diabetes. position of a mutation, it was straightforward New York Times (13 February 1916). fortable life (R. Glanz, A. Glanz and K. Glanz, 5. Cole, R. & Flexner, S. Quarterly Report. (Rockefeller to establish whether that gene was causal by personal communication), Banting did not. Archives Center, New York, 1914–1918). sequencing mutant and wild-type DNA. In Banting’s combativeness, fierceness and 6. Kleiner, I. The action of intravenous injections of pancreas emulsions in experimental diabetes. J. Biol. several cases, mutant genes were cloned in acquisitiveness appeared not to diminish with Chem. 40, 153–170 (1919). 73 this manner, rendering for naught the years age . Following his untimely death in an air- 7. Bliss, M. The Discovery of Insulin, 39 (The University of effort by other investigators who had taken plane accident in 1941, Time titled his obitu- of Chicago Press, Chicago, 1982). 69 8. Bliss, M. The Discovery of Insulin, 238 (The University a positional-cloning approach . Throughout ary “Spark-Plug Man” and described him as “a of Chicago Press, Chicago, 1982). the seemingly endless period during which stubborn man of strong feelings, sudden tem- 9. Kleiner, I. Hypoglycaemic agents: past and present. we were searching for the ob gene, I lived in per, trenchant speech”74. Banting was never Clin. Chem. 5, 79–99 (1959). 10. von Mering J. & Minkowski, O. Diabetes mellitus nach constant fear that I would one day receive a satisfied. In September 1923, only weeks after Pankreasextirpation. Archiv. Exp. Pathol. Pharmacol. phone call informing me that someone else his having received the Nobel Prize, the fol- 26, 371–387 (1890). 11. Opie, E. On the relation of chronic interstitial pancrea- had gotten to ob first. Were this to happen, I lowing item appeared in Time with the head- titis to the islands of Langerhans and to diabetes mel- often wondered, would I have been a less able line “Greater than Insulin”: “Dr. F. G. Banting, litus. J. Exp. Med. 5, 419–428 (1900). or insightful scientist for having embarked on discoverer of insulin, would shortly announce 12. Zuelzer, G. Ueber versuche einer specifischen Fermenttherapie de Diabetes. Zeitschrift fur experimen- the course I had chosen? a new discovery of even greater importance telle Pathologie und Therapie 5, 307–318 (1908). than the world-famed diabetes treatment,” add- 13. Murlin, J.R. & Kramer, B. A quest for the anti-diabetic One last ingredient ing “Dr. Banting had something so good we hormone. J. Hist. Med. Allied Sci. 11, 288–298 (1956). There is one additional element that, although couldn’t believe it.” There is no further record 14. Pratt, J. The relation of the pancreas to diabetes. J. Am. not guaranteeing success, often has a promi- of this “amazing” discovery75. Med. Assoc. 55, 2112 (1910). 15. Allen, F. Studies Concerning Glycosuria and Diabetes. nent role in the path to discovery. This is the Kleiner grew old surrounded by his wife, 815 (WM Leonard, Boston, 1913). fierceness of one’s determination—the single- two children and five grandchildren. In addi- 16. Frederick Allen to Israel Kleiner, 24 March 1955, per- minded insistence that a body of work be seen tion to the pride and satisfaction he took from sonal letters of the Kleiner family. 17. Bliss, M. The Discovery of Insulin, 29–31 (The through to its logical conclusion. It is worth his early work on diabetes, teaching and writ- University of Chicago Press, Chicago, 1982). considering the extent to which this is an ing a well-reviewed biochemistry textbook, he 18. Kleiner, I. Studies in Intermediary Metabolism. PhD essential (though not sufficient) trait. Banting painted (Fig. 4) and, to the great amusement of thesis, Yale University (1909). 19. Chittenden, R. Biographical Memoir of Lafayette had this attribute. Although he did not have his family, wrote doggerel (R. Glanz, A. Glanz Benedict Mendel 1872–1935. in Biographical Memoirs

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