Discovery, characterization, and clinical development of the -like

Daniel J. Drucker, … , Joel F. Habener, Jens Juul Holst

J Clin Invest. 2017;127(12):4217-4227. https://doi.org/10.1172/JCI97233.

Harrington Prize Essay Endocrinology Gastroenterology

The discovery, characterization, and clinical development of glucagon-like--1 (GLP-1) spans more than 30 years and includes contributions from multiple investigators, science recognized by the 2017 Harrington Award Prize for Innovation in Medicine. Herein, we provide perspectives on the historical events and key experimental findings establishing the biology of GLP-1 as an -stimulating glucoregulatory . Important attributes of GLP-1 action and enteroendocrine science are reviewed, with emphasis on mechanistic advances and clinical proof-of-concept studies. The discovery that GLP-2 promotes mucosal growth in the intestine is described, and key findings from both preclinical studies and the GLP-2 clinical development program for short bowel syndrome (SBS) are reviewed. Finally, we summarize recent progress in GLP biology, highlighting emerging concepts and scientific insights with translational relevance.

Find the latest version: https://jci.me/97233/pdf The Journal of Clinical Investigation HARRINGTON PRIZE ESSAY Discovery, characterization, and clinical development of the glucagon-like peptides

Daniel J. Drucker,1 Joel F. Habener,2 and Jens Juul Holst3 1Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, University of Toronto, Toronto, Ontario, Canada. 2Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Harvard University, Boston, Massachusetts, USA. 3Novo Nordisk Foundation Center for Basic Metabolic Research, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.

sequences of cloned recombinant cDNA copies of messenger RNAs. The Habener The discovery, characterization, and clinical development of glucagon-like- lab utilized this technology to elucidate peptide-1 (GLP-1) spans more than 30 years and includes contributions from amino acid sequences from multiple investigators, science recognized by the 2017 Harrington Award cDNAs and isolated from angler- Prize for Innovation in Medicine. Herein, we provide perspectives on the fish in the early 1980s (3–5) and the rat historical events and key experimental findings establishing the biology proglucagon cDNA and sequences of GLP-1 as an insulin-stimulating glucoregulatory hormone. Important followed shortly thereafter (refs. 6, 7, Fig- attributes of GLP-1 action and enteroendocrine science are reviewed, with ure 1, and Figure 2). Corresponding pro- emphasis on mechanistic advances and clinical proof-of-concept studies. glucagon sequences from hamster, bovine, The discovery that GLP-2 promotes mucosal growth in the intestine is and human were identified by Graeme described, and key findings from both preclinical studies and the GLP-2 Bell and others in the early 1980s (8–10). clinical development program for short bowel syndrome (SBS) are reviewed. These sequences revealed that glucagon Finally, we summarize recent progress in GLP biology, highlighting emerging and re­lated GLP sequences were encoded concepts and scientific insights with translational relevance. by larger precursors, termed pre- prohormones (Figure 2). The anglerfish preproglucagons (Figure 2A), isolated and characterized by Lund and Goodman (3–5), were interesting as there were two different The endocrine activity of the gastroin- second hormone, GLP-1. We high- cDNAs encoded by separate (nonallelic) testinal tract has been studied for more light 3 decades of science from multiple genes and they each contained a glucagon- than a century, with gut such as laboratories supporting the development related sequence, in addition to glucagon. emerging from the seminal stud- of GLP-1– and GLP-2–based therapies. The two anglerfish glucagon–related pep- ies of Bayliss and Starling (1). The concept GLP-1 is now used for the treatment of tides resembled GIP, a glucoincretin hor- that the gut also controlled pancreatic type 2 diabetes (T2D) and obesity, whereas mone released from the gut into the circu- islet secretions was supported by experi- GLP-2 is used for the therapy of short bow- lation during meals, subsequently shown ments demonstrating that administration el syndrome (SBS). by Dupre and Brown in 1973 to augment of crude gut extracts lowered blood glu- glucose-dependent insulin secretion (11). cose in animals. The development of the Discovery of GLP-1 Unlike the two anglerfish preproglucagons, insulin radioimmunoassay enabled the Although GIP was isolated through classical each of which harbored glucagon and a sin- description of the incretin effect, namely peptide purification and protein sequenc- gle glucagon-related peptide, the mammali- that glucose administered into the gut ing methodology, the discovery of the an preproglucagons all contained glucagon potentiated insulin secretion to a greater GLP-1 sequence stemmed from the appli- and two additional glucagon-related pep- extent than isoglycemic stimulation of cation of recombinant DNA approaches tides, designated GLP-1 and GLP-2 (Figure insulin secretion achieved through i.v. developed in the laboratories of Stanley 2B). Notably, the corresponding amino acid glucose administration. The first incretin Cohen, Paul Berg, and Herb Boyer in the sequences of the GLP-1s in the four mam- hormone, glucose-dependent insulinotro- early 1970s. This remarkable new techn­ malian species were identical (12), with pic polypeptide (GIP), was isolated by John ology allowed for a rapid and accurate conservation of sequence implying as yet Brown in the 1970s (2). Here, we describe prediction of the amino acid sequences of unknown but potentially important biolog- the discovery and characterization of the by the decoding of the nucleotide ical actions of GLP-1. Collectively, these findings further supported the evolving notion at the Conflict of interest: D.J. Drucker is supported by the Canada Research Chairs program and a BBDC-Novo Nordisk time that small peptide hormones are Chair in incretin biology, and has received speaking or consulting honorarium from Arisaph, Intarcia, Merck, Pfizer, synthesized in the form of larger prohor- and Novo Nordisk Inc. Mt. Sinai Hospital receives research support from GSK, Merck, and Novo Nordisk, for preclin- ical studies in the Drucker laboratory. J.J. Holst has served as a consultant or advisor to Novartis Pharmaceuticals, mones and that the final bioactive pep- Novo Nordisk, Merck Sharp & Dohme, and Roche and has received fees for lectures from Novo Nordisk, Merck Sharp tides are formed posttranslationally by & Dohme, and GlaxoSmithKline. selective enzymatic cleavages from the Reference information: J Clin Invest. 2017;127(12):4217–4227. https://doi.org/10.1172/JCI97233 prohormones (Figure 2B). Earlier studies

jci.org Volume 127 Number 12 December 2017 4217 HARRINGTON PRIZE ESSAY The Journal of Clinical Investigation

Figure 1. Historical timeline depicting seminal events in the discovery and development of gluca- gon-like peptide therapeutics.

had demonstrated that insulin and para- GLP-1(7-37). Further, at the carboxyl- note, in addition to glucagon, the hormone are synthesized as pre- terminal region of the putative GLP-1 pep- contained a large peptide with immuno- prohormones. The hydrophobic amino tide resides a sequence RGRR predicting reactive determinants for both GLP-1 and terminal sequences, termed the pre- or a prohormone convertase–directed cleav- GLP-2 (but not glucagon), consistent with signal sequences, are cleaved from the age site followed by an amidation of the incompletely cleaved proglucagon (major nascent polypeptide chains during their penultimate arginine by a peptidylglycine proglucagon fragment [MPGF]) (Figure 3, translation on ribosomes, leaving the α-amidating monooxygenase (14) result- A and C, and ref. 19). In contrast to find- prohormones as the precursor of the ing in peptides of 36 and 30 amino acids, ings in pancreas, immunoreactive GLP-1 peptide hormones. The A and B chains GLP-1(1-36)amide and GLP-1(7-36)amide. peptides detected in gut extracts consisted of insulin and were The availability of the amino acid entirely of smaller peptides (refs. 15 and found to be cleaved from their respective sequences of GLP-1 and GLP-2 obtained 16, and Figure 3, B and C). These findings prohormones by the actions of specific from the decoding of the nucleotide indicated that the cleavage of proglucagon endopeptidases, prohormone conver- sequences based on the genetic code into small GLP-1–immunoreactive pep- tases, which cleave proteins at the sites allowed for the chemical synthesis of the tides was more efficient in the gut com- of two consecutive basic amino acids, predicted peptides, examination of their pared with pancreas. These observations arginine and lysine (13). These earlier biological activities, and preparation of were also consistent with the incretin con- findings of prohormone convertase spec- peptide-specific antisera. Daniel Drucker cept in which, in response to oral nutrients, ificity were applied to deduce potential joined the Habener laboratory in the sum- glucoincretin hormones such as GIP (and cleavage sites in proglucagon. mer of 1984, with the original intent of subsequently GLP-1) originate from the studying the molecular control of thyroid gut and not the pancreas. Structure-activity properties hormone biosynthesis. However, the thy- Svetlana Mojsov in the Habener lab- of GLP-1 roid group, led by Bill Chin, was decamp- oratory detected glycine-extended and Examination of the amino acid sequence ing for the Brigham and Women’s Hospi- arginine-amidated isoforms of GLP-1 — as of proglucagon initially presented a tal, and Daniel was assigned to work on the well as both the amino-terminally extended conundrum regarding the processes by proglucagon gene. To understand whether peptides GLP-1(1-37) and GLP-1(1-36)amide which potentially bioactive GLP-1 pep- proglucagon might be processed to yield and the amino-truncated peptides GLP-1 tides might be liberated from the pro- multiple proglucagon-derived peptides (7-37) and GLP-1(7-36)amide — in extracts hormone (Figure 2B). In keeping with (PGDPs), including GLP-1 and GLP-2, of pancreas (Figure 3A). Nevertheless, the the rule that bioactive peptides are Drucker transfected a proglucagon cDNA abundance of these peptides was much cleaved from prohormones at sites con- expression vector into fibroblasts, pituitary greater in the gut (20). sisting of two basic amino acids (13), cells, and islet cells (15). Although minimal several investigators initially predicted processing was observed in fibroblasts, Bioactivities of the GLP-1 that bioactive GLP-1 would be a peptide immunoreactive GLP-1 and GLP-2 was peptides of 37 amino acids beginning with histi- detected by chromatography and radio- The earliest studies of the bioactivities of dine and ending in glycine, GLP-1(1-37). immunoassays in medium and extracts GLP-1(1-37) were indecisive. One study However, further inspection of the pro- from transfected pituitary and islet cells found that the 37 amino acid sequences revealed a second (15). Application of similar chromatogra- activated adenylyl cyclase in membranes single basic amino acid followed by his- phy and radioimmunoassay techniques prepared from rat pituitary and hypothal- tidine residing 6 amino acids carboxyl- in studies of rat (16), pig (17), and human amus (21), whereas another study failed to proximal to the first histidine, predict- (18) tissues revealed distinct profiles of detect any effects of the peptide on glucose ing a GLP-1 peptide of 31 amino acids, PGDPs in pancreas and gut (Figure 3). Of and insulin in -treated rabbits

4218 jci.org Volume 127 Number 12 December 2017 The Journal of Clinical Investigation HARRINGTON PRIZE ESSAY

bioactivities for the extended forms, GLP-1

(1-37) and GLP-1(1-36)amide, have yet been determined. Furthermore, no distinctive bio­ logical activities have been attributable spe- cifically to the amidated forms of GLP-1.

The view from Denmark In Copenhagen, Jens Holst and colleagues were interested in the incretin effect and were studying the condition of postpran­ dial reactive after gastric surgery (27). This type of hypoglycemia was clearly hyperinsulinemic, yet the sig- nal for insulin secretion was unknown. Looking for possible candidates, they were inspired by Lise Heding’s work on gluca- gon and her identification of the immu- nological differences between gut and pancreatic glucagon (28). Knowing that glucagon would stimulate insulin secre- tion, they were interested in the numer- ous cells in the gut that produce immu- Figure 2. Structure and processing of anglerfish and human proglucagon. Representation of the noreactive glucagon (29). Eventually, this structures of proglucagon cDNAs from Anglerfish (A) and Human (B), with tissue-specific liberation of individual proglucagon-derived peptides in pancreas or intestine. PC1, prohormone convertase 1; PC2, work led to the identification of glicentin prohormone convertase 2; MPGF, major proglucagon fragment. Arrows in A represent sites of cleave by and (Figure 2B), which prohormone convertase enzymes. both contain the full glucagon sequence, explaining the immunoreactivity in the (22). To determine whether the shorter The demonstration that GLP-1 directly gut (30–32). GLP-1 isoforms were bioactive, Drucker increased cAMP levels provided condi- Having identified all of the molecular incubated truncated forms of GLP-1 with tional evidence for the existence of a Gs components of glicentin also in the pan- multiple cell lines, examining cell growth, protein-coupled receptor in β cells. In creas (33), they proposed that glicentin , and signal transduc- studies of insulin secretion using the iso- represents at least part of a common gut tion. Remarkably, insulin-producing islet lated perfused pancreas, Mojsov and Weir and pancreatic glucagon precursor, which cells responded to N-terminally truncated demonstrated that GLP-1(7-37) and not undergoes differential processing in the forms of GLP-1, exhibiting increased lev- GLP-1(1-37) stimulated insulin secretion two tissues, a hypothesis subsequently con- els of cyclic AMP accumulation within at concentrations as low as 50 pM (ref. 24 firmed through identification of the human minutes of exposure to 50 pM–5 nM GLP- and Figure 5, A–C). Likewise, as outlined proglucagon gene by Graeme Bell and col- 1(7-37) (Figure 4A). In contrast, neither below, Jens Holst and colleagues showed leagues (7). However, it was also clear that glucagon, GLP-1(1-37), or GLP-1(1-36)– that luminal glucose stimulated GLP-1 proglucagon was larger than glicentin, and NH2 increased cAMP accumulation at secretion from the perfused intestine (Fig- the interest focused on peptides contained these concentrations (23). GLP-1(7-37) ure 5D), and doses from 500 pM to 5 nM within the MPGF representing the remain- also increased the levels of insulin mRNA GLP-1(7-36)amide stimulated insulin secre- der of proglucagon (minus glicentin) (34). transcripts and stimulated insulin secre- tion in the perfused pig pancreas (Figure The early work decoding the anglerfish tion at 25 mM but not 5.5 mM glucose in 5E and ref. 25). Thus, it turned out that a proglucagon cDNA by Lund and Habener studies with isolated islet cell lines (Figure cleavage in proglucagon at the single basic (3) followed by elucidation of the ham- 4, B and C, and ref. 23). In contrast, nei- amino acid, arginine, and not the double ster proglucagon cDNA by Graeme Bell ther glucagon nor GLP-2 induced insulin basic amino acids, generated the active (9) supported a hypothesis that cleavage gene expression. Furthermore, GLP-1 GLP-1 peptides, GLP-1(7-37) and GLP-1 of MPGF might result in liberation of the

(7-37) had no effect on levels of angioten- (7-36)amide (Figure 5C). First in man studies GLPs. The Holst group quickly developed sin gene expression in the insulinoma cell reported in December 1987 by Kreymann radioimmunoassays for GLP-1 and GLP-2 line and did not change levels of glucagon, and Bloom (26) rapidly established the to test this hypothesis. To their excitement, , and corticotropin mRNAs in islet insulinotropic actions of GLP-1(7-36)amide they found that MPGF was indeed differ- or pituitary cell lines (23). Hence, these in human subjects. Although numerous entially processed in the gut, but not in the findings (Figure 4, A–C) published in 1987 studies have demonstrated that the amino- pancreas, to yield two GLPs (ref. 17 and (23) established that GLP-1(7-37) directly terminally truncated forms of GLP-1, GLP- Figure 3C). However, using the perfused augments glucose-dependent insulin bio- 1(7-37), and GLP-1(7-36)amide are active pancreas preparation, they soon realized synthesis and secretion from β cells. glucoregulatory hormones, no compelling that neither of the two GLPs used in these

jci.org Volume 127 Number 12 December 2017 4219 HARRINGTON PRIZE ESSAY The Journal of Clinical Investigation

Mentlein in Kiel, Holst and Deacon showed that the GLP-1 molecule was cleaved by the enzyme dipeptidyl-peptidase-4(DPP-4) in vivo and that inhibitors of this enzyme could completely protect the molecule (43). In fact, the circulating half-life of GLP-1 was only 1.5–2 minutes in human subjects with diabetes, and they proposed that inhibitors of DPP-4 could maintain higher levels of intact active endogenous GLP-1 for therapeutic purposes (44). Sub- sequent studies soon demonstrated that DPP-4–resistant GLP-1 analogues were longer-acting than native GLP-1 (45). Fur- thermore, inhibitors of DPP-4 completely prevented the breakdown of GLP-1 in the circulation and amplified the insulinotro- pic actions of GLP-1 (46). This exciting development, presented in a Perspectives article in Diabetes in 1998 (47), was soon followed by the development of clinically useful inhibitors, first vildagliptin and sub- sequently sitagliptin. It remained to be understood whether Figure 3. Processing of proglucagon and glucagon-like peptides in the pancreas and intestine. GLP-1 receptor agonists would actually Detection of immunoreactive forms of GLP-1 in extracts from rat pancreas (A) and intestine (B) as adapted from ref. 16. Characterization of proglucagon-derived peptide immunoreactivity secreted be useful for clinical diabetes therapy or from perfused pig pancreas and intestine (C) using peptide-specific antisera reveal tissue-specific whether tachyphylaxis would develop posttranslational processing of the PGDPs, as outlined in ref. 17. upon chronic administration. The group in Copenhagen administered synthetic GLP-1 by constant s.c. infusion for 6 studies had any effect on pancreatic hor- stimulating activity attenuated as plasma weeks to a group of individuals with mone secretion. They therefore decided glucose levels started to fall, limiting the long-standing T2D (48). Fortunately, no to isolate the naturally occurring hormone fall to 0.5–1 mmol/l. tachyphylaxis was observed; GLP-1 ther- from porcine and human and gut extracts, At that time, the Copenhagen group apy reduced fasting and mean plasma and they found that the naturally occur- realized that GLP-1 was extremely interest- glucose by 4.3 and 5.5 mmol/l; glycated ring peptide was a truncated from of GLP-1 ing and, in further studies, demonstrated hemoglobin by 1.3 %; and body weight by representing proglucagon (aa 78-108) that it strongly inhibited gastric motility 2 kg. Moreover, insulin sensitivity and β (Figure 5C) and subsequently found to be and gastric and pancreatic exocrine secre- cell function, assessed by clamp studies, amidated, corresponding to proglucagon tion (39), consistent with an important greatly improved. Importantly, no limit-

78-107amide (35). Importantly, this hormone role for this hormone as a regulator of ing side effects were recorded­ (48), pro- was potently insulinotropic (Figure 5E and upper gastrointestinal function. They also viding proof of concept in 2002 for GLP-1 ref. 25), so they had also described a new demonstrated that infusions of GLP-1 in therapy in subjects with T2D. It was now incretin hormone in 1987; however, they humans inhibited appetite and food intake, clear that GLP-1–based therapies had tre- wondered whether GLP-1 was more inter- actions subsequently exploited in the clinic mendous potential. esting than the already known incretin to treat obesity (40). In studies published GIP, which exhibited a diminished effect in 1993 by Michael Nauck and colleagues The Toronto perspective: on insulin secretion in patients with T2D in Göttingen, i.v. infusion of GLP-1 com- the physiology of GLPs and (36). They soon found, using the perfused pletely normalized severely elevated fast- discovery of GLP-2 pancreas, that GLP-1 — in contrast to GIP — ing glucose concentrations in patients with Building on the availability of cloned pro- also powerfully inhibits glucagon secretion long-standing T2D as a consequence of the glucagon gene sequences in the Habener (37). Eventually, they demonstrated that actions of GLP-1 to stimulate insulin and lab (6), Daniel Drucker and Jacques during infusions of physiological amounts inhibit glucagon secretion (41). Although Philippe pursued the analysis of the molec- of GLP-1 into humans, insulin secretion GLP-1 clearly had therapeutic potential, ular control of islet α cell proglucagon would be stimulated and glucagon secre- s.c. injections of GLP-1 were disappoint- gene expression in the mid 1980s (49, 50). tion inhibited, resulting in a decrease in ingly ineffective (42). The explanation was Upon returning to Toronto in 1987, Druck- hepatic glucose production (38). However, an extremely rapid metabolism and inacti- er extended these studies to examine pro- the effect was self-limiting, with the insulin- vation of GLP-1. With inspiration from Rolf glucagon gene expression in the intestine

4220 jci.org Volume 127 Number 12 December 2017 The Journal of Clinical Investigation HARRINGTON PRIZE ESSAY

Figure 4. GLP-1 directly stimulates signal transduction and glucose-dependent insulin secretion in islet cells. GLP-1(7-37) directly stimulates cyclic AMP accumulation (A), insulin gene expression (B), and glucose-dependent insulin secretion (C) in a rat insulinoma cell line, as adapted from ref. 23.

and CNS. He isolated human neonatal Two unexpected observations were the Drucker lab demonstrated that native brainstem cDNAs encoding proglucagon, made during isolation of GLUTag cells. GLP-2 robustly increased small bowel which exhibited an identical sequence to First, mice harboring s.c. GLUTag cell growth in Fischer 344 rats with an inac- that described for islet proglucagon (51). tumors exhibited a marked reduction tivating mutation in the Dpp4 gene (60). Molecular cloning of the rat intestinal pro- of pancreatic islet α cell mass (57). Sec- Furthermore, a GLP-2 analogue with a sin- glucagon cDNA similarly revealed an open ond, mice with s.c. GLUTag, InR1-G9, or gle amino acid substitution [Gly2]-GLP-2 reading frame identical to that elucidated RIN1056A glucagon-producing tumors exhibited substantial resistance to DPP-4 for the rat pancreatic islet proglucagon all exhibited marked enlargement of the cleavage and robust intestinotrophic activ- cDNA (52). Moreover, in studies carried small bowel. These findings led Drucker ity in normal rats in vivo (60). Hence, the out in collaboration with Patricia Brubak- to reinvestigate the link between glucagon- importance of DPP4 for cleavage of both er, forskolin, cholera toxin, and dibutyryl producing tumors and gut growth, first GLP-1 and GLP-2 became evident very cyclic AMP increased the synthesis and reported in a human subject studied at the early in the study of the GLPs. secretion of intestinal PGDPs from primary Hammersmith hospital in 1970 by Dowl- The identification of GLP-2 as an cultures of rat intestinal cells (52). At the ing and colleagues in London (58). A series intestinal growth factor spurred a series time, there were no differentiated GLP-1– of simple experiments from the Drucker of experiments examining the actions of secreting lines suit- lab published in 1996 identified GLP-2 as GLP-2 in the context of experimental gut able for studies of intestinal proglucagon the PGDP with the most potent intestino- injury. GLP-2 administration was gener- gene expression. Accordingly, Ying Lee, a trophic activity in mice (58). Remarkably, ally associated with preservation of gut fellow in the Drucker laboratory, generated although immunoreactive GLP-2 had been mucosal structure and function in the a transgenic mouse expressing the SV40 T detected in intestinal extracts of various setting of chemical, radiation, or surgical- antigen cDNA under the control of the pro- species (refs. 16, 59, and Figure 3), no pre- ly induced intestinal injury in preclinical glucagon gene promoter. This transgenic vious biological activity had yet been iden- studies (61–63). Notably, GLP-2 also rapid- mouse reproducibly developed GLP-1– tified for GLP-2 in vivo. ly increased nutrient absorption in normal secreting enteroendocrine tumors of the The actions of GLP-2 to stimulate rodents (64) and in animals with surgical colon (53), enabling isolation of the first small bowel growth were rapid, detectable gut resection mimicking SBS (65). Excit- differentiated GLP-1–producing entero- within days, and associated with increased ingly, the findings in animals were soon endocrine L cell line in 1992, designated crypt cell proliferation (58). Surprising- extended to humans with SBS. In a pilot GLUTag cells. GLUTag cells were easily ly, when similar doses of GLP-2 were study carried out around 2000–2001, propagated ex vivo; secreted immunoreac- administered to rats, intestinal growth Jeppesen and colleagues demonstrated tive GLP-1, glicentin, oxyntomodulin, and was not significantly increased, although that native GLP-2 administered twice dai- GLP-2 in response to cyclic AMP analogues an increase in crypt plus villus height was ly for 35 days increased nutrient absorp- (54, 55); and resembled primary cultures observed (60). With hindsight, these find- tion, energy absorption, and weight gain of nonimmortalized gut endocrine cells ings reflected the importance of DPP-4 for in human subjects with SBS (66). These in regard to their response to a battery of the degradation of GLP-2, more evident findings, namely expansion of intesti- secretagogues (56). in rats than in mice. Subsequent studies in nal mucosal surface area coupled with

jci.org Volume 127 Number 12 December 2017 4221 HARRINGTON PRIZE ESSAY The Journal of Clinical Investigation

generation of Glp1r–/– mice. These ani- mals generated by Louise Scrocchi exhib- ited impaired oral glucose tolerance and reduced insulin levels after glucose stim- ulation, demonstrating the critical role of endogenous GLP-1 as an incretin hormone in 1996 (69). Unexpectedly, Glp1r–/– mice also exhibited fasting hyperglycemia, and impaired i.p. glucose tolerance, establish- ing the importance of the GLP-1 for β cell function beyond its original description as an incretin. Moreover, GLP-1R–deficient β cells exhibit enhanced sensitivity to apop- totic injury, whereas pharmacological acti- vation of GLP-1R signaling attenuated β cell death in mice in vivo, as well as in cul- tures of purified rat β cells studied ex vivo (70). The importance of GLP-1R signaling for the response to cellular stress was high- lighted by findings that activation of GLP- 1R signaling attenuated the development of ER stress in β cells by enhancing ATF-4 induction and accelerating recovery from translational repression via augmentation of ER stress–stimulated ATF-4 translation (71). Collectively, these findings explain how GLP-1, despite acting to simultane- ously enhance insulin biosynthesis and secretion, maintains functional β cell mass through attenuation of ER stress. The actions of GLP-1 to stimulate insu- lin and inhibit glucagon secretion, together Figure 5. Truncated forms of GLP-1 stimulate glucose- with its inhibitory effects on food intake dependent insulin secretion. GLP-1(7-37) (A) but not GLP-1(1-37) (B) stimulates insulin secretion from and gastric emptying, spurred considerable the perfused rat pancreas as adapted from ref. 24. effort in development of GLP-1–based ther- Sequences of GLP-1(1-37), GLP-1(7-37), and GLP-1 apeutics. Remarkably, the first GLP-1R ago- (7-36 ). (C) Intraluminal glucose stimulates entero- amide nist approved for clinical use was glucagon, GLP-1, and GLP-2 secretion from the perfused (synthetic exendin-4), a peptide originally pig (D), as adapted from ref. 17. Demonstration isolated from Heloderma suspectum lizard that GLP-1(7-36)amide stimulates insulin secretion from the perfused pig pancreas (E), as adapted from ref. 25. venom by John Eng in 1992 (72). Subse- quent molecular cloning studies published by the Drucker laboratory in 1997 demon- strated that the lizard genome contains two distinct proglucagon gene sequences encoding GLP-1 and a separate proexendin enhanced nutrient absorption (67), togeth- in increased fluid and energy absorption, gene, largely restricted in its expression to er with substantial preclinical data, sup- while reducing the requirements for PN, in the salivary gland (73). The original clinical ported the initiation of a drug development two separate placebo-controlled Phase 3 development program for exendin-4 uti- program to test the efficacy of GLP-2 in studies (67, 68). was approved lized twice-daily injections of the unmodi- human subjects with parenteral nutrition– for chronic therapy of subjects with fied peptide. Pivotal studies in subjects with dependent (PN-dependent) SBS. Clinical PN-dependent SBS in the US in Decem- T2D ultimately resulted in the approval studies were initiated with h[Gly2]-GLP-2, ber 2012 (67). of twice-daily exenatide, the first GLP-1R a degradation-resistant GLP-2 analogue agonist in 2005 (Figure 1). These efforts discovered in the Drucker lab (60) and sub- The physiology of GLP-1 action spurred the development of a once-weekly sequently designated teduglutide. Once Complementary studies in the Drucker lab form of exenatide in a microsphere suspen- daily teduglutide administration in human were focused on defining the physiological sion, ultimately the first once-weekly ther- subjects with PN-dependent SBS resulted importance of endogenous GLP-1 through apy approved for diabetes in 2012 (Figure

4222 jci.org Volume 127 Number 12 December 2017 The Journal of Clinical Investigation HARRINGTON PRIZE ESSAY

1). Exenatide once weekly was more effec- classes used for treatment of T2D, the GLP- clusions surrounding receptor expression tive than the twice-daily preparation, with 1R agonists and SGLT-2 inhibitors, produce and localization. greater reduction of glycemia and compa- weight loss, effective reduction of A1c, a low Elucidation of the physiological roles rable control of body weight (74). Today, incidence of hypoglycemia, and reductions of endogenous GLP-1 and GLP-2 in multiple GLP-1R agonists (small peptides in rates of major adverse cardiovascular humans has been limited in part due to lim- and high molecular weight proteins) are events and cardiovascular death (75, 87). itations associated with the available antag- approved for the treatment of T2D, and a onists. Exendin(9-39) has been widely single drug, , was approved for Challenges in studies of GLP-1 used as a GLP-1 receptor antagonist, yet the treatment of obesity in 2014 (Figure 1). and in development of GLP-1R only in short-term human studies. It seems The widespread distribution of GLP-1 agonists likely that more selective antagonists with receptors in extrapancreatic tissues stimu- In the era of rapid daily progress in bio- optimized pharmacokinetic profiles would lated considerable research into the nong- medical science, it is surprising that it advance our understanding of the impor- lycemic actions of GLP-1. Indeed, we now took almost 20 years from identification tance of endogenous GLP-1 action. Similar- understand that GLP-1 controls inflam- of GLP-1 to successful approval of the ly, GLP-2(3-33), often employed as a GLP-2 mation (75), reduces experimental world’s first clinically utilized GLP-1R ago- receptor antagonist, is both a weak antago- injury (76), and like GLP-2, acts as a potent nist. The rapid renal clearance of many nist and a partial agonist, and little data is intestinal growth factor through mech- small peptides like GLP-1, together with available to date that illuminates the phys- anisms including stimulation of crypt its inactivation by DPP4, required devel- iological importance of endogenous GLP-2 fission (ref. 77 and Figure 6). Among the opment of longer-acting peptides with in human subjects. actions of GLP-1 that have engendered the greater stability and enhanced resistance Analysis of proglucagon synthesis and most interest are its effects in the cardio- to DPP-4. The development of nausea secretion in rare enteroendocrine L cell vascular system. Native GLP-1 increases and vomiting, encountered in every GLP-1 populations in the small and large bowel flow-mediated vasodilation, enhances development program, often limited dose is also technically challenging. Transgenic rate (HR) and cardiac output, and escalation and likely delayed introduc- technologies have enabled isolation of pri- is cardioprotective in preclinical studies, tion of GLP-1R agonists, utilized at higher mary L cell populations suitable for secre- most notably in animals with ischemic doses, with greater efficacy. Indeed, it has tion and electrophysiological analyses cardiac injury (78). Although degradation- now become apparent that rates of nau- (91), and stem cell–derived gut organoids, resistant GLP-1R agonists have minimal sea and vomiting can be greatly reduced including those of human origin, herald effects on blood vessels (79), they increase with much slower titration regimens, as considerable potential for generation of HR and reduce cardiac inflammation has been the case in development of titrat- diverse enteroendocrine cell populations and infarct size in animals with experi- able GLP-1–insulin combination therapies. from different sites along the gastrointes- mental myocardial infarction (75, 80). Indeed, the development of liraglutide for tinal tract (92). Remarkably, despite the Understanding the mechanisms through obesity using a 3-mg daily dosing regimen theoretical potential of enhancing L cell which GLP-1 exerts cardioprotection is further illustrates that much greater toler- secretion for the treatment of metabolic challenging, since the GLP-1R is largely ability to GLP-1 can be achieved using pro- disorders (1), there has been scant progress expressed in atrial and not ventricular car- gressive stepwise titration regimens. in clinical development of GLP-1 secret- diomyocytes (81). Furthermore, the GLP- Studies of GLP-1 synthesis, secretion, agogues exhibiting sustained efficacy in 1R–dependent control of inflammation in and action have also been hampered by both preclinical and clinical studies. heart and blood vessels is complex, as the lack of suitable reagents and experimental predominant site of GLP-1R expression in challenges. Circulating GLP-1 levels are GLPs: current use and future the (murine) immune system is within the very low, and measurement of GLP-1 in considerations intestinal intraepithelial lymphocyte (82). the circulation is technically challenging; What does the second decade of GLP-1– Several cardiovascular outcome stud- indeed, not all commercial assays pro- based therapies portend? Currently, multi- ies have documented the safety of GLP-1R duce consistent or accurate results, and ple GLP-1R agonists are indicated for the agonists in human subjects with T2D and many exhibit problematic sensitivity and treatment of T2D, and liraglutide is also preexisting cardiovascular disease (83, 84). specificity (88). Similar challenges sur- approved for obesity; however, indica- Excitingly, reduction of major adverse car- round the quantitative detection of GLP-2 tions such as nonalcoholic steatohepatitis, diovascular events was reported in human (89), which is challenging to measure with Parkinson’s disease, and Alzheimer’s dis- subjects treated with long-acting liraglutide available commercial assays. Comple- ease are being explored. Indeed, several and (85, 86). These findings, mentary problems have been identified randomized controlled clinical trials have revealed more than a decade after the first with the antisera used to detect the GLP-1 demonstrated significant clinical improve- approval of twice-daily exenatide in 2005, and GLP-2 receptors, with problematic ment in human subjects with Parkinson’s have rekindled enthusiasm for the use of antisera utilized in dozens of publications. disease (93). Despite the appeal of cur- GLP-1R agonists, alone or in combination, The majority of commercially available rent GLP-1R agonists for the treatment of in the treatment of T2D and obesity, most antisera used to detect GLP-1R and GLP- T2D, market penetration for many GLP-1R compellingly in subjects at high risk for car- 2R exhibit impaired sensitivity and speci- agonists remains disappointing, raising diovascular disease. Today, only two drug ficity (67, 90), challenging published con- questions about the potential clinical

jci.org Volume 127 Number 12 December 2017 4223 HARRINGTON PRIZE ESSAY The Journal of Clinical Investigation

therapy of bariatric surgery–associated hypoglycemia (98). Whether exendin(9-39) will exhibit sustained efficacy and safety and be similarly useful for treatment of genetic forms of hyperinsulinemic hypo­ glycemia requires further study. The elevat- ed levels of GLP-1 detected in most subjects after bariatric surgery are widely viewed as contributing to the enhanced β cell function and reduced appetite evident in these sub- jects (99). Nevertheless, insulin sensitivity is also rapidly improved after bariatric sur- gery, and some studies suggest that GLP-1 (9-36) and its degradation products, which circulate at elevated levels after bariatric surgery, may enhance insulin sensitivity in peripheral tissues. Further research into the potential contribution of these PGDPs in the context of bariatric surgery may illu- minate the role(s) of these peptides in this unique clinical context. The cardiovascular safety and benefits (Figure 6) associated with the use of GLP- 1R agonists (75) raises further clinically relevant questions. Might nondiabetic subjects with obesity expect comparable Figure 6. The pleiotropic local and systemic actions of GLP-1 and GLP-2 secreted in response to cardioprotection with sustained GLP-1R nutrients, bacterial metabolites, or inflammatory toxins and cytokines, from enteroendocrine L agonism? Alternatively, can we identify cells of the small and . the cardioprotective mechanisms of GLP-1 action and extend the therapeutic possi- appeal, expense, and commercial success current expense of and unfamiliarity with bilities of GLP-1 therapy to nondiabetic of newer formulations. Ongoing attempts these new drugs may limit their appeal subjects at high risk for cardiovascular dis- to improve the therapeutic efficacy of GLP- in some markets. Whether longer-acting ease? Similarly, will human subjects with 1R agonists, exemplified by once-weekly versions, perhaps weekly insulin–GLP-1 prediabetes, or those at low risk for cardio- semaglutide for diabetes or once-daily combinations, are feasible and efficacious vascular disease, be suitable candidates semaglutide for obesity, are under active requires additional investigation. More- for GLP-1R agonism without long-term investigation. ITCA-650 is a long-acting over, GLP-1 may yet emerge as an ideal data establishing durability and reduction version of exenatide enabling sustained partner for peptide combinations that of complications? Additional clinical trials peptide delivery with implantation of a enable greater weight loss. Unimolecular will be required to answer these questions. small s.c. osmotic minipump every 3–6 coagonists and triagonists exhibit substan- The translational success of GLP-1 from months. Semaglutide is also being stud- tial preclinical efficacy (95) and are under bench to bedside reflects important contri- ied as a once-daily oral formulation (94), active clinical investigation. Nevertheless, butions from dozens of colleagues studying potentially extending the appeal of GLP- it may be challenging to predict the ideal GLP-1 over 3 decades, to whom we extend 1R agonists to a subset of patients that may ratio(s) of 2–3 peptide epitopes combined our thanks and appreciation. The history of traditionally eschew injectable therapies. in a single therapy that produces enhanced GLP-1 science provides instructive lessons Considerable effort is being expended control of metabolic disease, while main- relevant to modern-day experimentation to combine the established efficacy of taining an acceptable safety profile (96). and drug development. First, the field was GLP-1R agonism with additional thera- The observations that GLP-1R signal- built slowly with a series of nonflashy yet pies, enabling greater therapeutic efficacy ing in β cells is essential for control of insu- solid studies examining the physiology, delivered through a single injection. Two lin secretion, together with data suggesting pharmacology, and pleiotropic actions of insulin–GLP-1 combination therapies that exendin(9-39) may attenuate insulin native GLP-1 and multiple GLP-1R agonists. have been approved, -insulin secretion through reduction of β cell cyclic From the onset, the availability of the native glargine and liraglutide-, AMP (97), has supported investigational peptide enabled rapid proof of concept in enabling enhanced control of glucose use of exendin(9-39) for the treatment of human subjects, quickly validating discov- without weight gain. These fixed-ratio severe hyperinsulinemic hypoglycemia. eries made in preclinical studies. Most of combination therapies represent attractive Preliminary short-term data supports the the early, and the majority of subsequent, options for some patients; however, the feasibility of using exendin(9-39) in the GLP-1 studies were published in tradition-

4224 jci.org Volume 127 Number 12 December 2017 The Journal of Clinical Investigation HARRINGTON PRIZE ESSAY

al physiology or endocrinology subspe- sequence. Four of six exons encode separate 23. Drucker DJ, Philippe J, Mojsov S, Chick WL, cialty journals and might be described as functional domains of rat pre-proglucagon. J Biol Habener JF. Glucagon-like peptide I stimulates Chem. 1984;259(22):14082–14087. insulin gene expression and increases cyclic “descriptive” or “incremental” by many col- 7. Heinrich G, Gros P, Lund PK, Bentley RC, AMP levels in a rat islet cell line. Proc Natl Acad leagues. The elusive distribution of GLP-1 Habener JF. Pre-proglucagon messenger ribo- Sci U S A. 1987;84(10):3434–3438. receptors, often expressed in small numbers nucleic acid: nucleotide and encoded amino 24. Mojsov S, Weir GC, Habener JF. Insulinotropin: of endocrine cells, CNS or enteric neurons, acid sequences of the rat pancreatic comple- glucagon-like peptide I (7-37) co-encoded in the or rare immune cells, makes it challenging mentary deoxyribonucleic acid. Endocrinology. glucagon gene is a potent stimulator of insulin 1984;115(6):2176–2181. release in the perfused rat pancreas. J Clin Invest. to decisively identify cells, pathways, and 8. Bell GI, Sanchez-Pescador R, Laybourn PJ, 1987;79(2):616–619. reductionist mechanisms linking recep- Najarian RC. Exon duplication and divergence 25. Holst JJ, Orskov C, Nielsen OV, Schwartz TW. tor engagement to physiological actions in the human preproglucagon gene. Nature. Truncated glucagon-like peptide I, an insulin-re- and therapeutic efficacy. Nevertheless, 1983;304(5924):368–371. leasing hormone from the distal gut. FEBS Lett. the multiple actions of GLP-1, described 9. Bell GI, Santerre RF, Mullenbach GT. Ham- 1987;211(2):169–174. ster preproglucagon contains the sequence 26. Kreymann B, Williams G, Ghatei MA, in dozens of laboratories, have largely of glucagon and two related peptides. Nature. Bloom SR. Glucagon-like peptide-1 7-36: been reproducible, and occasional ques- 1983;302(5910):716–718. a physiological incretin in man. Lancet. tionable findings and controversies have 10. Lopez LC, Frazier ML, Su CJ, Kumar A, Saunders 1987;2(8571):1300–1304. been extensively studied and subsequently GF. Mammalian pancreatic preproglucagon con- 27. Holst JJ, Hending LG, Rehfeld JF. Gut glu- discarded by more careful investigation tains three glucagon-related peptides. Proc Natl cagon and reactive hypoglycaemia. Lancet. Acad Sci U S A. 1983;80(18):5485–5489. 1973;1(7810):1008. (100). Taken together, the story of the dis- 11. Dupre J, Ross SA, Watson D, Brown JC. Stimu- 28. Heding LG. Radioimmunological determination covery and characterization of the GLPs lation of insulin secretion by gastric inhibitory of pancreatic and gut glucagon in plasma. Diabe- highlights how old-fashioned biochemistry, polypeptide in man. J Clin Endocrinol Metab. tologia. 1971;7(1):10–19. physiology, molecular biology, and tradi- 1973;37(5):826–828. 29. Holst JJ. Extrapancreatic . . tional analyses of hormone action provides 12. Kieffer TJ, Habener JF. The glucagon-like pep- 1978;17(2):168–190. tides. Endocr Rev. 1999;20(6):876–913. 30. Holst JJ. Gut glucagon, , gut glu- a firm scientific foundation for the devel- 13. Steiner DF. The proprotein convertases. Curr cagonlike immunoreactivity, glicentin — current opment of multiple novel therapeutics for Opin Chem Biol. 1998;2(1):31–39. status. Gastroenterology. 1983;84(6):1602–1613. the treatment of obesity, diabetes, and 14. Kumar D, Mains RE, Eipper BA. 60 YEARS 31. Holst JJ. Evidence that glicentin contains intestinal disorders. OF POMC: From POMC and α-MSH to PAM, the entire sequence of glucagon. Biochem J. molecular oxygen, copper, and vitamin C. J Mol 1980;187(2):337–343. Endocrinol. 2016;56(4):T63–T76. 32. Holst JJ. Evidence that enteroglucagon (II) is identi- Acknowledgments 15. Drucker DJ, Mojsov S, Habener JF. Cell-specific cal with the C-terminal sequence (residues 33-69) The authors thank the members of their post-translational processing of preproglucagon of glicentin. Biochem J. 1982;207(3):381–388. laboratories and the hundreds of collabo- expressed from a metallothionein-glucagon fusion 33. Moody AJ, Holst JJ, Thim L, Jensen SL. Rela- rative colleagues who have made key con- gene. J Biol Chem. 1986;261(21):9637–9643. tionship of glicentin to proglucagon and tributions to our collective understanding 16. Mojsov S, Heinrich G, Wilson IB, Ravazzola glucagon in the porcine pancreas. Nature. M, Orci L, Habener JF. Preproglucagon gene 1981;289(5797):514–516. of glucagon-like peptide biology. expression in pancreas and intestine diversifies 34. Patzelt C, Schug G. The major proglucagon frag- at the level of post-translational processing. ment: an abundant islet protein and secretory Address correspondence to: Daniel J. J Biol Chem. 1986;261(25):11880–11889. product. FEBS Lett. 1981;129(1):127–130. Drucker, LTRI, Mt. Sinai Hospital, 600 17. Orskov C, Holst JJ, Knuhtsen S, Baldissera FG, 35. Orskov C, Bersani M, Johnsen AH, Højrup P, University Ave., Mailbox 39, TCP5-1004, Poulsen SS, Nielsen OV. Glucagon-like peptides Holst JJ. Complete sequences of glucagon-like GLP-1 and GLP-2, predicted products of the peptide-1 from human and pig small intestine. Toronto, Ontario, Canada M5G 1X5. glucagon gene, are secreted separately from pig J Biol Chem. 1989;264(22):12826–12829. Phone: 416.361.2661; Email: drucker@ small intestine but not pancreas. Endocrinology. 36. Krarup T, Saurbrey N, Moody AJ, Kühl C, lunenfeld.ca. 1986;119(4):1467–1475. Madsbad S. Effect of porcine gastric inhibitory 18. Orskov C, Holst JJ, Poulsen SS, Kirkegaard P. polypeptide on beta-cell function in type I 1. Drucker DJ. Evolving concepts and translational Pancreatic and intestinal processing of progluca- and type II diabetes mellitus. Metab Clin Exp. relevance of enteroendocrine cell biology. J Clin gon in man. Diabetologia. 1987;30(11):874–881. 1987;36(7):677–682. Endocrinol Metab. 2016;101(3):778–786. 19. Patzelt C, Schiltz E. Conversion of proglucagon 37. Orskov C, Holst JJ, Nielsen OV. Effect of truncat- 2. Brown JC, Dryburgh JR. A gastric inhibitory in pancreatic alpha cells: the major endproducts ed glucagon-like peptide-1 [proglucagon-(78-107) polypeptide. Can J Biochem. 1971;49(8):867–872. are glucagon and a single peptide, the major amide] on endocrine secretion from pig pancreas, 3. Lund PK, Goodman RH, Dee PC, HabenerJF. proglucagon fragment, that contains two glu- antrum, and nonantral . Endocrinology. Pancreatic preproglucagon cDNA contains cagon-like sequences. Proc Natl Acad Sci U S A. 1988;123(4):2009–2013. two glucagon-related coding sequences 1984;81(16):5007–5011. 38. Hvidberg A, Nielsen MT, Hilsted J, Orskov arranged in tandem. Proc Natl Acad Sci U S A. 20. Mojsov S, Kopczynski MG, Habener JF. Both C, Holst JJ. Effect of glucagon-like peptide-1 1982;79(2):345–349. amidated and nonamidated forms of glu- (proglucagon 78-107amide) on hepatic glucose 4. Lund PK, Goodman RH, Habener JF. Pancreatic cagon-like peptide I are synthesized in the production in healthy man. Metab Clin Exp. pre-proglucagons are encoded by two separate rat intestine and the pancreas. J Biol Chem. 1994;43(1):104–108. mRNAs. J Biol Chem. 1981;256(13):6515–6518. 1990;265(14):8001–8008. 39. Wettergren A, Schjoldager B, Mortensen PE, 5. Lund PK, Goodman RH, Montminy MR, Dee PC, 21. Hoosein NM, Gurd RS. Human glucagon-like Myhre J, Christiansen J, Holst JJ. Truncated Habener JF. Anglerfish islet pre-proglucagon peptides 1 and 2 activate rat brain adenylate GLP-1 (proglucagon 78-107-amide) inhibits gas- II. Nucleotide and corresponding amino cyclase. FEBS Lett. 1984;178(1):83–86. tric and pancreatic functions in man. Dig Dis Sci. acid sequence of the cDNA. J Biol Chem. 22. Ghiglione M, Uttenthal LO, George SK, Bloom 1993;38(4):665–673. 1983;258(5):3280–3284. SR. How glucagon-like is glucagon-like peptide-1? 40. Pi-Sunyer X, et al. A randomized, controlled trial 6. Heinrich G, Gros P, Habener JF. Glucagon gene Diabetologia. 1984;27(6):599–600. of 3.0 mg of liraglutide in weight management.

jci.org Volume 127 Number 12 December 2017 4225 HARRINGTON PRIZE ESSAY The Journal of Clinical Investigation

N Engl J Med. 2015;373(1):11–22. tion by protein kinase-A in a novel mouse ing induction of endoplasmic reticulum stress. 41. Nauck MA, Kleine N, Orskov C, Holst JJ, Willms B, enteroendocrine cell line. Mol Endocrinol. Cell Metab. 2006;4(5):391–406. Creutzfeldt W. Normalization of fasting hypergly- 1994;8(12):1646–1655. 72. Eng J, Kleinman WA, Singh L, Singh G, Raufman caemia by exogenous glucagon-like peptide 1 (7-36 56. Brubaker PL, Schloos J, Drucker DJ. Regulation JP. Isolation and characterization of exendin-4, amide) in type 2 (non-insulin-dependent) diabetic of glucagon-like peptide-1 synthesis and secre- an exendin-3 analogue, from Heloderma sus- patients. Diabetologia. 1993;36(8):741–744. tion in the GLUTag enteroendocrine cell line. pectum venom. Further evidence for an exendin 42. Ritzel R, Orskov C, Holst JJ, Nauck MA. Pharma- Endocrinology. 1998;139(10):4108–4114. receptor on dispersed acini from guinea pig pan- cokinetic, insulinotropic, and glucagonostatic 57. Ehrlich P, Tucker D, Asa SL, Brubaker PL, Druck- creas. J Biol Chem. 1992;267(11):7402–7405. properties of GLP-1 [7-36 amide] after subcuta- er DJ. Inhibition of pancreatic proglucagon gene 73. Chen YE, Drucker DJ. Tissue-specific expression neous injection in healthy volunteers. Diabetolo- expression in mice bearing subcutaneous endo- of unique mRNAs that encode proglucagon- gia. 1995;38(6):720–725. crine tumors. Am J Physiol. 1994; derived peptides or exendin 4 in the lizard. J Biol 43. Deacon CF, Johnsen AH, Holst JJ. Degradation 267(5 pt 1):E662–E671. Chem. 1997;272(7):4108–4115. of glucagon-like peptide-1 by human plasma in 58. Drucker DJ, Erlich P, Asa SL, Brubaker PL. 74. Drucker DJ, et al. Exenatide once weekly versus vitro yields an N-terminally truncated peptide Induction of intestinal epithelial proliferation by twice daily for the treatment of type 2 diabetes: that is a major endogenous metabolite in vivo. glucagon-like peptide 2. Proc Natl Acad Sci U S A. a randomised, open-label, non-inferiority study. J Clin Endocrinol Metab. 1995;80(3):952–957. 1996;93(15):7911–7916. Lancet. 2008;372(9645):1240–1250. 44. Deacon CF, Nauck MA, Toft-Nielsen M, Pridal 59. Buhl T, Thim L, Kofod H, Orskov C, Harling H, 75. Drucker DJ. The cardiovascular biology of gluca- L, Willms B, Holst JJ. Both subcutaneously and Holst JJ. Naturally occurring products of proglu- gon-like peptide-1. Cell Metab. 2016;24(1):15–30. intravenously administered glucagon-like peptide cagon 111-160 in the porcine and human small 76. Fujita H, et al. The protective roles of GLP-1R 1 are rapidly degraded from the NH2-terminus in intestine. J Biol Chem. 1988;263(18):8621–8624. signaling in diabetic nephropathy: possible type II diabetic patients and in healthy subjects. 60. Drucker DJ, et al. Regulation of the biological mechanism and therapeutic potential. Kidney Diabetes. 1995;44(9):1126–1131. activity of glucagon-like peptide 2 in vivo Int. 2014;85(3):579–589. 45. Deacon CF, Knudsen LB, Madsen K, Wiberg by dipeptidyl peptidase IV. Nat Biotechnol. 77. Koehler JA, et al. GLP-1R agonists promote FC, Jacobsen O, Holst JJ. Dipeptidyl peptidase 1997;15(7):673–677. normal and neoplastic intestinal growth IV resistant analogues of glucagon-like 61. Boushey RP, Yusta B, Drucker DJ. Glucagon-like through mechanisms requiring Fgf7. Cell Metab. peptide-1 which have extended metabolic peptide (GLP)-2 reduces chemotherapy-associ- 2015;21(3):379–391. stability and improved biological activity. Dia- ated mortality and enhances cell survival in cells 78. Ban K, Noyan-Ashraf MH, Hoefer J, Bolz SS, betologia. 1998;41(3):271–278. expressing a transfected GLP-2 receptor. Cancer Drucker DJ, Husain M. Cardioprotective and 46. Deacon CF, Hughes TE, Holst JJ. Dipeptidyl pep- Res. 2001;61(2):687–693. vasodilatory actions of glucagon-like peptide 1 tidase IV inhibition potentiates the insulinotropic 62. Drucker DJ, Yusta B, Boushey RP, DeForest L, receptor are mediated through both glucagon-like effect of glucagon-like peptide 1 in the anesthe- Brubaker PL. Human [Gly2]GLP-2 reduces the peptide 1 receptor-dependent and -independent tized pig. Diabetes. 1998;47(5):764–769. severity of colonic injury in a murine model of pathways. Circulation. 2008;117(18):2340–2350. 47. Holst JJ, Deacon CF. Inhibition of the activity of experimental colitis. Am J Physiol. 1999; 79. Pujadas G, Drucker DJ. Vascular biology of dipeptidyl-peptidase IV as a treatment for type 2 276(1 pt 1):G79–G91. superfamily peptides: diabetes. Diabetes. 1998;47(11):1663–1670. 63. Boushey RP, Yusta B, Drucker DJ. Glucagon-like mechanistic and clinical relevance. Endocr Rev. 48. Zander M, Madsbad S, Madsen JL, Holst JJ. Effect peptide 2 decreases mortality and reduces the 2016;37(6):554–583. of 6-week course of glucagon-like peptide 1 on severity of indomethacin-induced murine enteri- 80. Noyan-Ashraf MH, et al. GLP-1R agonist lira- glycaemic control, insulin sensitivity, and beta- tis. Am J Physiol. 1999;277(5 pt 1):E937–E947. glutide activates cytoprotective pathways and cell function in type 2 diabetes: a parallel-group 64. Brubaker PL, Izzo A, Hill M, Drucker DJ. Intes- improves outcomes after experimental myocardial study. Lancet. 2002;359(9309):824–830. tinal function in mice with small bowel growth infarction in mice. Diabetes. 2009;58(4):975–983. 49. Drucker DJ, Philippe J, Jepeal L, Habener JF. induced by glucagon-like peptide-2. Am J Physiol. 81. Kim M, et al. GLP-1 receptor activation and Glucagon gene 5’-flanking sequences promote 1997;272(6 pt 1):E1050–E1058. Epac2 link atrial secre- islet cell-specific gene transcription. J Biol Chem. 65. Scott RB, Kirk D, MacNaughton WK, Meddings tion to control of blood pressure. Nat Med. 1987;262(32):15659–15665. JB. GLP-2 augments the adaptive response to 2013;19(5):567–575. 50. Philippe J, Drucker DJ, Knepel W, Jepeal L, Misulo- massive intestinal resection in rat. Am J Physiol. 82. Yusta B, et al. GLP-1 receptor (GLP-1R) agonists vin Z, Habener JF. Alpha-cell-specific expression of 1998;275(5 pt 1):G911–G921. modulate enteric immune responses through the glucagon gene is conferred to the glucagon pro- 66. Jeppesen PB, et al. Glucagon-like peptide 2 the intestinal intraepithelial lymphocyte (IEL) moter element by the interactions of DNA-binding improves nutrient absorption and nutritional GLP-1R. Diabetes. 2015;64(7):2537–2549. proteins. Mol Cell Biol. 1988;8(11):4877–4888. status in short-bowel patients with no colon. 83. Pfeffer MA, et al. Lixisenatide in patients with 51. Drucker DJ, Asa S. Glucagon gene expres- Gastroenterology. 2001;120(4):806–815. type 2 diabetes and acute coronary syndrome. sion in vertebrate brain. J Biol Chem. 67. Drucker DJ, Yusta B. Physiology and phar- N Engl J Med. 2015;373(23):2247–2257. 1988;263(27):13475–13478. macology of the enteroendocrine hormone 84. Holman RR, et al. Effects of once-weekly exen- 52. Drucker DJ, Brubaker PL. Proglucagon gene glucagon-like peptide-2. Annu Rev Physiol. atide on cardiovascular outcomes in type 2 expression is regulated by a cyclic AMP-dependent 2014;76:561–583. diabetes. N Engl J Med. 2017;377(13):1228–1239. pathway in rat intestine. Proc Natl Acad Sci U S A. 68. Jeppesen PB, et al. Teduglutide reduces need for 85. Marso SP, et al. Semaglutide and cardiovascular 1989;86(11):3953–3957. parenteral support among patients with short outcomes in patients with type 2 diabetes. N Engl 53. Lee YC, Asa SL, Drucker DJ. Glucagon gene bowel syndrome with intestinal failure. Gastro- J Med. 2016;375(19):1834–1844. 5′-flanking sequences direct expression of SV40 enterology. 2012;143(6):1473–1481.e3. 86. Marso SP, et al. Liraglutide and cardiovascular large T antigen to the intestine producing carci- 69. Scrocchi LA, et al. Glucose intolerance but nor- outcomes in type 2 diabetes. N Engl J Med. noma of the large bowel in transgenic mice. mal satiety in mice with a null mutation in the 2016;375(4):311–322. J Biol Chem. 1992;267(15):10705–10708. glucagon-like peptide 1 receptor gene. Nat Med. 87. Kaul S. Mitigating cardiovascular risk in type 2 dia- 54. Drucker DJ, Lee YC, Asa SL, Brubaker PL. Inhi- 1996;2(11):1254–1258. betes with antidiabetes drugs: a review of princi- bition of pancreatic glucagon gene expression in 70. Li Y, Hansotia T, Yusta B, Ris F, Halban PA, pal cardiovascular outcome results of EMPA-REG mice bearing a subcutaneous glucagon-producing Drucker DJ. Glucagon-like peptide-1 receptor OUTCOME, LEADER, and SUSTAIN-6 Trials. GLUTag transplantable tumor. Mol Endocrinol. signaling modulates β cell apoptosis. J Biol Chem. Diabetes Care. 2017;40(7):821–831. 1992;6(12):2175–2184. 2003;278(1):471–478. 88. Bak MJ, et al. Specificity and sensitivity of com- 55. Drucker DJ, Jin T, Asa SL, Young TA, Brubaker 71. Yusta B, et al. GLP-1 receptor activation mercially available assays for glucagon-like pep- PL. Activation of proglucagon gene transcrip- improves beta cell function and survival follow- tide-1 (GLP-1): implications for GLP-1 measure-

4226 jci.org Volume 127 Number 12 December 2017 The Journal of Clinical Investigation HARRINGTON PRIZE ESSAY

ments in clinical studies. Diabetes Obes Metab. 2014;63(2):410–420. cy for treatment of diabetes and obesity. Cell 2014;16(11):1155–1164. 93. Athauda D, et al. Exenatide once weekly versus Metab. 2016;24(1):51–62. 89. Hartmann B, Johnsen AH, Orskov C, Adelhorst placebo in Parkinson’s disease: a randomised, 97. Serre V, Dolci W, Scrocchi LA, Drucker DJ, Efrat K, Thim L, Holst JJ. Structure, measurement, double-blind, placebo-controlled trial [pub- S, Thorens B. Exendin-(9-39) as an inverse ago- and secretion of human glucagon-like peptide-2. lished online ahead of print August 3, 2017]. nist of the murine GLP-1 receptor. Endocrinolo- Peptides. 2000;21(1):73–80. Lancet. https://doi.org/10.1016/S0140- gy. 1998;139(11):4448–4454. 90. Panjwani N, et al. GLP-1 receptor activation indi- 6736(17)31585-4. 98. Salehi M, Gastaldelli A, D’Alessio DA. Blockade rectly reduces hepatic lipid accumulation but 94. Davies M, Pieber TR, Hartoft-Nielsen ML, of glucagon-like peptide 1 receptor corrects does not attenuate development of atherosclero- Hansen OKH, Jabbour S, Rosenstock J. Effect postprandial hypoglycemia after gastric bypass. sis in diabetic male ApoE(–/–) mice. Endocrinol- of oral semaglutide compared with placebo and Gastroenterology. 2014;146(3):669–680.e2. ogy. 2013;154(1):127–139. subcutaneous semaglutide on glycemic control 99. Madsbad S, Dirksen C, Holst JJ. Mechanisms of 91. Reimann F, Habib AM, Tolhurst G, Parker HE, in patients with type 2 diabetes: a randomized changes in glucose metabolism and bodyweight Rogers GJ, Gribble FM. Glucose sensing clinical trial. JAMA. 2017;318(15):1460–1470. after bariatric surgery. Lancet Diabetes Endocri- in L cells: a primary cell study. Cell Metab. 95. Finan B, et al. A rationally designed monomeric nol. 2014;2(2):152–164. 2008;8(6):532–539. peptide triagonist corrects obesity and diabetes 100. Drucker DJ. Incretin action in the pancreas: 92. Petersen N, et al. Generation of L cells in mouse in rodents. Nat Med. 2015;21(1):27–36. potential promise, possible perils, and pathologi- and human small intestine organoids. Diabetes. 96. Tschöp MH, et al. Unimolecular polypharma- cal pitfalls. Diabetes. 2013;62(10):3316–3323.

jci.org Volume 127 Number 12 December 2017 4227