The insulin centennial—100 years of milestones in biochemistry

A hundred years ago, Frederick Banting and Charles Best, in the laboratory of John MacLeod at the University of Toronto, performed a historic experiment demonstrating that an extract from pancreata in which the pancreatic ducts had been ligated to allow isolation of the “internal secretion” lowered blood glucose in dogs. This experiment broke ground for a fertile field that now extends into essentially all branches of physiology, endocrinology, biochemistry, cell biology, genetics, and molecular biology. This centennial is being celebrated by the retelling of this extraordinary story and the research triumphs that mark the trajectory to the present. Several journals have already paid tribute to these events (1Sims E.K. Carr A.L.J. Oram R.A. DiMeglio L.A. Evans-Molina C. 100 years of insulin: Celebrating the past, present and future of diabetes therapy.Nat. Med. 2021; 27: 1154-1164Crossref PubMed Scopus (4) Google Scholar, 2Fralick M. Zinman B. The discovery of insulin in Toronto: Beginning a 100 year journey of research and clinical achievement.Diabetologia. 2021; 64: 947-953Crossref PubMed Scopus (8) Google Scholar, 3Lewis G.F. Brubaker P.L. The discovery of insulin revisited: Lessons for the modern era.J. Clin. Invest. 2021; 131e142239Crossref PubMed Scopus (5) Google Scholar, 4White M.F. Kahn C.R. Insulin action at a molecular level - 100 years of progress.Mol. Metab. 2021; 52: 101304Crossref PubMed Scopus (1) Google Scholar, 5Kahn C.R. 100 Years of progress in understanding insulin, its mechanism of action, and its roles in disease and diabetes therapy.Mol. Metab. 2021; 52: 101318Crossref PubMed Scopus (0) Google Scholar). We wish to highlight the role of our two ASBMB journals, The Journal of Biological Chemistry and The Journal of Lipid Research in publishing impactful biochemical studies in this field. We have selected 27 articles for republication. The choice of so few articles from a vast literature was very difficult and somewhat arbitrary, but it allows us to highlight a subset of the many great discoveries that advanced our understanding of insulin and to recognize the remarkable insights of early pioneers of this field. Many of the most important articles in the field were published in other journals and are therefore not included in this series. What comes across in reading the earlier publications in this field is how prescient were the insights and speculations of the brilliant scientists who launched and advanced this field. In threading together these stories, we have gained a new appreciation of the hard work, zealous dedication, and brilliant insights of the scientists involved in this quest. Although diabetes is principally defined by hyperglycemia, the role of lipids in diabetes has been a dominant theme in this field and we point to several highlights in this story. With the realization that a substance produced in the pancreas is capable of lowering blood glucose and is likely missing in people with diabetes, it became critical to purify and identify this substance. The first purification of insulin, from the cow pancreas, was performed by the Canadian biochemist James Collip and was used to treat the first human patient with diabetes, Leonard Thompson, in 1922. American scientists were also working on purifying insulin. The first article on insulin that was published in the JBC is a study of precipitation methods by Kimball and Murlin in 1923 (6Kimball C.P. Murlin J.R. Aqueous extracts of pancreas. 3. Some precipitation reactions of insulin.J. Biol. Chem. 1923; 58: 337-348Abstract Full Text PDF Google Scholar). They tested a series of alcohols, acetone, ether, toluene, xylene, trichloroacetic acid, and several salts. The method was to centrifuge the precipitate, resuspend in water, and immediately inject into rabbits. What is most remarkable about the article is how inconclusive it was regarding insulin’s properties. It did not promote any one precipitation method and ended by saying, “With regard to the properties of insulin as it has been observed in this laboratory, not much can be said. It is a white, amorphous powder probably insoluble in neutral water when pure. It gives no protein reactions of any kind, and the most potent that have been analyzed have had a low nitrogen content, 4 to 6% dry weight.” The real significance of that article (6Kimball C.P. Murlin J.R. Aqueous extracts of pancreas. 3. Some precipitation reactions of insulin.J. Biol. Chem. 1923; 58: 337-348Abstract Full Text PDF Google Scholar) by Kimball and Murlin, however, was the discovery of glucagon, which they described as a “hyperglycemic substance” in the pancreatic extracts used to purify insulin. Patients with diabetes went on to be treated with insulin purified from cattle or pigs. However, with the development of a radioimmunoassay by Rosalyn Yalow and Solomon Berson (7Yalow R.S. Berson S.A. Reaction of fish insulins with human insulin antiserums. Potential value in the treatment of insulin resistance.N. Engl. J. Med. 1964; 270: 1171-1178Crossref PubMed Google Scholar), it became clear that many patients developed antibodies against bovine and porcine insulin, making it less effective with time. Human insulin was not to become available until late in the 1970s, when the first genetically engineered insulin was produced in Escherichia coli by scientists at Genentech (8Goeddel D.V. Kleid D.G. Bolivar F. Heyneker H.L. Yansura D.G. Crea R. Hirose T. Kraszewski A. Itakura K. Riggs A.D. Expression in Escherichia coli of chemically synthesized genes for human insulin.Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 106-110Crossref PubMed Scopus (532) Google Scholar). Rosalyn Yalow became the second woman to receive the Nobel Prize in Physiology or Medicine for her work. The first was Gerty Cori (see below). Berson and Yalow quickly realized that patients with type 2 diabetes were most often not insulin-deficient and in fact many had hyperinsulinemia. They anticipated the intense focus on insulin resistance in this field with this comment in their famous publication describing the insulin radioimmunoassay (9Yalow R.S. Berson S.A. Immunoassay of endogenous plasma insulin in man.J. Clin. Invest. 1960; 39: 1157-1175Crossref PubMed Google Scholar): “appreciation of the lack of responsiveness of blood sugar, in the face of apparently adequate amounts of insulin secreted by early maturity-onset diabetic subjects, is obviously of importance in the interpretation of the pathogenesis of this type of diabetes. However, the data at hand can only indicate that absolute insulin deficiency per se is not the cause of the hyperglycemia and suggest other possibilities that merit investigation, namely (1) abnormal tissues with a high threshold for the action of insulin; (2) an abnormal insulin that acts poorly with respect to hormonal activity in vivo but reacts well immunologically in vitro; (3) an abnormally rapid inactivation of hormonally active sites … but not of immunologically active sites on the insulin molecules; and (4) the presence of insulin antagonists. The last suggestion has been made many times by previous workers. A joint attack on the problem, utilizing both the specific immunoassay for plasma insulin and an assay method that measures the net biological effect of insulin and its inhibitors would seem to be indicated.” A pioneer in the study of post-translational processing of insulin was Don Steiner. He narrates this story in a lovingly written JBC article, published in 2011 (10Steiner D.F. Adventures with insulin in the islets of Langerhans.J. Biol. Chem. 2011; 286: 17399-17421Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The determination of the sequence and structure of insulin by Fred Sanger in 1955 (11Ryle A.P. Sanger F. Smith L.F. Kitai R. The disulphide bonds of insulin.Biochem. J. 1955; 60: 541-556Crossref PubMed Google Scholar) (who received two Nobel Prizes for his work on protein and DNA sequencing) begged the question, are the two chains of the insulin molecule derived from a common precursor? In 1967, Don Steiner published his seminal discovery, through protein purification and pulse-chase experiments in isolated islets, of a precursor to insulin, proinsulin (12Steiner D.F. Cunningham D. Spigelman L. Aten B. Insulin biosynthesis: Evidence for a precursor.Science. 1967; 157: 697-700Crossref PubMed Google Scholar). Proinsulin consists of the insulin A and B chains connected by a peptide (C-peptide), which is cleaved off in the secretory granules before secretion of mature insulin. Steiner also recognized the utility of the C-peptide as a quantifiable marker of insulin production and β-cell function, and developed its assay in 1970 (13Melani F. Rubenstein A.H. Oyer P.E. Steiner D.F. Identification of proinsulin and C-peptide in human serum by a specific immunoassay.Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 148-155Crossref PubMed Google Scholar). Steiner went on to discover several of the enzymes that process proinsulin to mature insulin. This work has had a profound impact on cell biology and endocrinology because many protein hormones, clotting factors, growth factors, receptors, and even serum albumin are synthesized as a precursor with a propeptide that is removed during its transport through the secretory pathway. The 3D structure of mature porcine insulin was solved by Dorothy Hodgkin (14Blundell T.L. Cutfield J.F. Cutfield S.M. Dodson E.J. Dodson G.G. Hodgkin D.C. Mercola D.A. Vijayan M. Atomic positions in rhombohedral 2-zinc insulin crystals.Nature. 1971; 231: 506-511Crossref PubMed Scopus (263) Google Scholar), who had applied X-ray crystallography to reveal 3D structures of several other molecules before that. She was awarded the Nobel Prize in Chemistry in 1964 for her discoveries. Not surprisingly, the 3D structure of insulin is critical for its biological activities. Graeme Bell reported in 2007 that mutations in the insulin gene that cause abnormalities in insulin maturation and folding cause diabetes (15Stoy J. Edghill E.L. Flanagan S.E. Ye H. Paz V.P. Pluzhnikov A. Below J.E. Hayes M.G. Cox N.J. Lipkind G.M. Lipton R.B. Greeley S.A. Patch A.M. Ellard S. Steiner D.F. et al.Insulin gene mutations as a cause of permanent neonatal diabetes.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15040-15044Crossref PubMed Scopus (403) Google Scholar). Protein misfolding activates a transcriptional program, the unfolded protein response, which can ultimately lead to cell death and is now recognized as a major cause of diseases caused by missense mutations in proteins, ranging from neurological diseases to metabolic diseases. Jeremy Thorner’s discovery of the yeast protease that processes the yeast mating factor, Kex2p, led to the discovery of the convertases that process proinsulin and proglucagon (16Smeekens S.P. Avruch A.S. LaMendola J. Chan S.J. Steiner D.F. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 340-344Crossref PubMed Scopus (388) Google Scholar) and then to an entire family of proprotein convertases (17Julius D. Brake A. Blair L. Kunisawa R. Thorner J. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor.Cell. 1984; 37: 1075-1089Abstract Full Text PDF PubMed Scopus (480) Google Scholar). Many endocrine disorders and some obesity syndromes are caused by mutations in these enzymes. Thus, the discovery of processing of proinsulin to insulin spurred a number of seminal subsequent discoveries. The pathways that link glucose sensing with insulin secretion have been at the heart of islet biology for decades. A milestone in the field was the 1984 discovery by Frances and Stephen Ashcroft at the University of Oxford that ATP-sensitive potassium (KATP) channels link ATP generation to insulin secretion (18Ashcroft F.M. Harrison D.E. Ashcroft S.J. Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells.Nature. 1984; 312: 446-448Crossref PubMed Google Scholar). Metabolism of glucose in the β-cell leads to a rise in ATP, the closure of the KATP channel, and the activation of voltage-gated Ca2+ channels, leading to Ca2+ influx and insulin secretion. The KATP channel is an octamer consisting of two proteins, Kir6.1 (KCNJ11) or Kir6.2 and the sulfonylurea receptor, SUR1 (ABCC8) or SUR2. Loss-of-function mutations in SUR1 or less commonly, KCNJ11, lead to hyperinsulinism (19Huopio H. Reimann F. Ashfield R. Komulainen J. Lenko H.L. Rahier J. Vauhkonen I. Kere J. Laakso M. Ashcroft F. Otonkoski T. Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1.J. Clin. Invest. 2000; 106: 897-906Crossref PubMed Google Scholar), whereas mutations in KCNJ11 that decrease ATP inhibition of KATP cause neonatal permanent neonatal diabetes (20Gloyn A.L. Pearson E.R. Antcliff J.F. Proks P. Bruining G.J. Slingerland A.S. Howard N. Srinivasan S. Silva J.M. Molnes J. Edghill E.L. Frayling T.M. Temple I.K. Mackay D. Shield J.P. et al.Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes.N. Engl. J. Med. 2004; 350: 1838-1849Crossref PubMed Scopus (932) Google Scholar). Glucokinase plays a role in glucose sensing in the liver and β-cells. In the liver, glucokinase expression is regulated by insulin, and its abundance determines the capacity of the liver to metabolize glucose, which, unlike the muscle and adipose tissue, is rate-limiting for its uptake. Matschinsky and Ellerman discovered in 1968 that glucokinase is present in β-cells (21Matschinsky F.M. Ellerman J.E. Metabolism of glucose in the islets of Langerhans.J. Biol. Chem. 1968; 243: 2730-2736Abstract Full Text PDF PubMed Google Scholar). The discovery of genes causing monogenic dominantly inherited diabetes syndromes, termed maturity-onset diabetes of the young, provided valuable mechanistic information about β-cell biology (22Fajans S.S. Bell G.I. MODY: History, genetics, pathophysiology, and clinical decision making.Diabetes Care. 2011; 34: 1878-1884Crossref PubMed Scopus (185) Google Scholar). Specifically, loss-of-function mutations in glucokinase lead to diabetes (23Gloyn A.L. Odili S. Zelent D. Buettger C. Castleden H.A. Steele A.M. Stride A. Shiota C. Magnuson M.A. Lorini R. d'Annunzio G. Stanley C.A. Kwagh J. van Schaftingen E. Veiga-da-Cunha M. et al.Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young.J. Biol. Chem. 2005; 280: 14105-14113Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), whereas the rarer gain-of-function mutants cause hyperinsulinism (24Beer N.L. van de Bunt M. Colclough K. Lukacs C. Arundel P. Chik C.L. Grimsby J. Ellard S. Gloyn A.L. Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing hyperinsulinemic hypoglycemia in humans.J. Biol. Chem. 2011; 286: 19118-19126Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). This supports the role of glucokinase as a β-cell glucose sensor that determines the capacity of the β-cell to take up glucose and oxidize it in the glycolytic pathway and led to the development of glucokinase activators as a potential treatment for diabetes (25Matschinsky F.M. GKAs for diabetes therapy: Why no clinically useful drug after two decades of trying?.Trends Pharmacol. Sci. 2013; 34: 90-99Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Other maturity-onset diabetes of the young genes include transcription factors that play roles in β-cell development and in the function of the adult β-cell. Although glucose is the best-studied insulin secretagogue, amino acids can also stimulate insulin secretion. Gain-of-function mutations in glutamate dehydrogenase cause excessive amino acid–induced insulin secretion (26Li C. Najafi H. Daikhin Y. Nissim I.B. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. Regulation of leucine-stimulated insulin secretion and glutamine metabolism in isolated rat islets.J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The product of glutamate dehydrogenase is mitochondrial α-ketoglutarate, raising the question how this metabolite could signal insulin secretion. An important clue came from the discovery that β-cells have an abundance of mitochondrial phosphoenolpyruvate carboxykinase, encoded by the PCK2 gene. Deletion of the gene in mice leads to a severe defect in insulin secretion (27Stark R. Pasquel F. Turcu A. Pongratz R.L. Roden M. Cline G.W. Shulman G.I. Kibbey R.G. Phosphoenolpyruvate cycling via mitochondrial phosphoenolpyruvate carboxykinase links anaplerosis and mitochondrial GTP with insulin secretion.J. Biol. Chem. 2009; 284: 26578-26590Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) as shown by Richard Kibbey’s laboratory at Yale University. Subsequent studies have proposed that formation of phosphoenolpyruvate promotes cycling through the pyruvate kinase reaction and this is intimately connected with the closure of the ATP channel (28Abulizi A. Cardone R.L. Stark R. Lewandowski S.L. Zhao X. Hillion J. Ma L. Sehgal R. Alves T.C. Thomas C. Kung C. Wang B. Siebel S. Andrews Z.B. Mason G.F. et al.Multi-tissue acceleration of the mitochondrial phosphoenolpyruvate cycle improves whole-body metabolic health.Cell Metab. 2020; 32: 751-766.e11Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 29Lewandowski S.L. Cardone R.L. Foster H.R. Ho T. Potapenko E. Poudel C. VanDeusen H.R. Sdao S.M. Alves T.C. Zhao X. Capozzi M.E. de Souza A.H. Jahan I. Thomas C.J. 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Full Text PDF PubMed Google This is as the first that insulin into many scientists that insulin by cells and with enzymes to their activity and glucose and used as a to its after of insulin into a an in the of from the they working by these data can be as Insulin acts the cell of tissues in a that the of from the into the cell is The of the the of metabolic for their In the of no In the of glucose, and to are by the of its into the In Charles reported in the JBC that the rate-limiting in the by insulin of glucose by the heart is transport across the plasma E. C.R. Regulation of glucose in and of insulin and in heart muscle from diabetic Biol. Chem. Full Text PDF PubMed Google Scholar). also discovered that glucose the work on and glucose into The mechanism by which insulin glucose into cells was in the late when the glucose was by a led by Graeme Bell and at the University of H. T. Y. Bell G.I. S. and characterization of the major glucose in human muscle and other Biol. Chem. 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