`
`lnsulinotropin: Glucagon-like Peptide I (7-37) Co-encoded in the Glucagon Gene
`Is a Potent Stimulator of Insulin Release in the Perfused Rat Pancreas
`
`Svetlana Mojsov,* Gordon C. Weir,* and Joel F. Habener*
`*Laboratory of Molecular Endocrinology, Massachusetts General Hospital and Howard Hughes Medical Institute, Harvard Medical
`School, Boston, Massachusetts 02114; and *Joslin Diabetes Center and New England Deaconess Hospital, and Harvard Medical School,
`Boston, Massachusetts 02115
`
`Abstract
`
`Insulin secretion is controlled by a complex set of factors that
`include not only glucose but amino acids, catecholamines, and
`intestinal hormones. We report that a novel glucagon-lilce pep(cid:173)
`tide, co-encoded with glucagon in the glucagon gene is a potent
`insulinotropic factor. The glucagon gene encodes a proglucagon
`that contains in its sequence glucagon and additional glucagon(cid:173)
`like peptides (GLPs). These GLPs are liberated from proglu(cid:173)
`cagon in both the pancreas and intestines. GLP-I exists in at
`least two forms: 37 amino acids GLP-I(l-37), and 31 amino
`acids, GLP-I(7-37). We studied the effects of synthetic GLP-Is
`on insulin secretion in the isolated perfused rat pancreas. In the
`presence of 6.6 mM glucose, GLP-I(7-37) is a potent stimulator
`of insulin secretion at concentrations as low as 5 X 10-11 M (3-
`to IO-fold increases over basal). GLP-I(l-37) had no effect on
`insulin secretion even at concentrations as high as 5 X 10-7 M.
`The earlier demonstration of specific liberation of GLP-I(7-37)
`in the intestine and pancreas, and the magnitude of the insuli(cid:173)
`notropic effect at such low concentrations, suggest that GLP(cid:173)
`I(7-37) participates in the physiological regulation of insulin se(cid:173)
`cretion.
`
`Introduction
`
`Pancreatic glucagon and intestinal glicentin are synthesized in
`the form of a 180-residue protein, preproglucagon encoded in
`a single gene ( 1 ). The precursor contains in addition to glicentin
`and glucagon the sequences of two glucagon-like peptides
`(GLPs)1, GLP-1 and GLP-11, separated by an intervening peptide
`(IP-II) (2-5). The posttranslational processing of preproglucagon
`differs in pancreas and intestine (1, 6). In the pancreas the pre(cid:173)
`cursor is processed to glucagon and GLP-1, and in both large
`and small intestines glicentin, GLP-1, GLP-11 and IP-11-leucine(cid:173)
`amide are found. Both pancreas and intestine contain GLP-1 in
`at least two forms-31 and 37 residues long (I).
`The close similarity of the amino acid sequence ofGLP-Is
`and GLP-11 with glucagon and the other peptides related in
`structure to glucagon (secretin, vasoactive intestinal peptide,
`gastric inhibitory peptide, growth hormone-releasing hormone)
`
`Received for publication 28 October 1986.
`
`I. Abbreviation used in this paper: GLP, glucagon-like peptide.
`
`J. Clin. Invest.
`© The American Society for Clinical Investigation, Inc.
`0021-9738/87/02/0616/04 $1.00
`Volume 79, February 1987, 616-619
`
`616
`
`S. Mojsov, G. C. Weir, and J. F. Habener
`
`suggests that the GLPs might have a role in metabolic regulation.
`The specific liberation of GLP-1 and GLP-11 in the intestine
`indicates that these peptides may be components of the entero(cid:173)
`insular axis (7), which comprises multiple intestinal factors in(cid:173)
`fluencing the release of hormones produced in the pancreatic
`islets. Further, they may be incretins, endocrine transmitters
`produced in the gastrointestinal tract that are released by nu(cid:173)
`trients and stimulate insulin secretion in the presence of elevated
`glucose if exogeneously infused in amounts not exceeding blood
`levels achieved after food ingestion (8). Detection of both GLP-
`1(1-37) and GLP-1(7-37) in pancreas and intestines raises the
`possibility that GLP-1(1-37) is itself a prohormone that undergoes
`a proteolytic cleavage at the single arginine residue at position
`6 to release the biologically active GLP-1(7-37). In these studies
`we used synthetic GLP-1(1-37) and GLP-1(7-37) to investigate
`their effects on insulin secretion in the perfused rat pancreas
`and find that GLP-I(7-37) has uniquely potent insulinotropic
`actions.
`
`Methods
`
`Synthesis of peptides. Glucagon and GLP-ls were synthesized by the
`stepwise solid-phase method (9). Because the assembly of the peptide
`chain proceeds in the carboxyl- to the amino-terminal direction, GLP(cid:173)
`I( 1-37) and GLP-1(7-37) were prepared in the same synthesis by separating
`the peptide resin after incorporation of a protected histidyl residue at
`position 7 and continuing the assembly of amino acids on the other
`aliquot of the peptide resin to obtain protected GLP-1(1-37) peptide resin.
`Peptides were purified by preparative reverse-phase C-18 chromatography.
`Purified peptides were shown to be homogeneous by amino acid analysis,
`preview-sequence analysis, and high performance liquid chromatography
`(HPLC) on reverse-phase C-18 and ion-exchange DEAE-52 columns.
`Radioimmunoassays. Development of the antisera and competitive
`binding radioimmunoassays for glucagon and GLP-1 are described else(cid:173)
`where ( 1 ). In brief, samples were incubated with the antisera in borate
`buffer (pH 8.1) for 24 hat 0°C, followed by addition of 1251-labeled
`peptide for an additional 24 h in a total volume of 0.5 ml. Separation
`of the antibody bound from the free peptide was accomplished with
`dextran-coated charcoal. Assay sensitivity with all three antisera was I 0
`pg/ml. The antiserum against GLP-1 was obtained by immunization
`with GLP-1(1-37) and is directed against both the amino-terminal (1-6)
`part of the molecule and to 7-37 determinants. Therefore, the amount
`ofGLP-i(7-37) may be over or underestimated with respect to GLP-1(1-
`37) in the assay. The assay for insulin ( 10) used charcoal separation and
`rat insulin standards (Novo Research Institute, Copenhagen, Denmark).
`Rat-perfused pancreas experiments. The preparation of the in situ
`isolateq rat pancreas has been described previously ( 11, 12). The perfusate
`contained bicarbonate buffer (pH 7.4) and 120 mg/di glucose, 4% dextran
`T-70, and 0.2% bovine serum albumin, and was equilibrated with 95%
`0 2 and 5% CO2 • The first 20 min of each perfusion was an equilibration
`period and is not represented in the data graphs.
`
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`After the initial 20-min equilibration period, aliquots of perfusate
`were removed every 2-4 min for additional 20 min, thus allowing the
`system to equilibrate for a total of 40 min. The perfusion, with GLP-
`1(1-37) or GLP-1(7-37), was for 6 min and samples were collected at I(cid:173)
`min intervals. The peptide perfusions were followed by equilibration
`periods of20 min, during which four samples 5 min apart were collected.
`A second 6-min perfusion followed with the same peptide as the first
`perfusion only at I 00 times higher concentration of peptide. Again, sam(cid:173)
`ples I min apart were collected. The entire perfusion time was between
`70 and 85 min.
`In each aliquot of perfusate obtained, insulin was determined by
`radioimmunoassay. In addition the efficiency of delivery of the GLP-ls
`was confirmed by radioimmunoassay of corresponding aliquots of per(cid:173)
`fusate in which insulin was measured (I).
`
`Results
`
`To optimally study the effects ofGLP-1(7-37) and GLP-1(1-37)
`on insulin secretion we used separate perfusions with each pep(cid:173)
`tide, perfusing twice at two different concentrations of peptides
`and allowing 20-min time intervals between the two perfusions.
`In perfusions of two separate pancreases using this protocol,
`GLP-1(7-37) was a potent stimulator of insulin secretion, giving
`about a 20-fold stimulation at 5 X 10-9 M and a sixfold stim(cid:173)
`ulation at 5 X 10-11 M (Fig. I). In comparison, GLP-1(1-37),
`also studied in two pancreases, showed no effect on insulin se(cid:173)
`cretion at either 5 X 10-9 or 5 X 10-1 M (Fig. 2). At the latter
`concentration no effect was observed even during a 15-min per(cid:173)
`fusion period (Fig. 2 B).
`Using a slightly different perfusion protocol than that dee
`scribed above (Figs. I and 2) we gave alternate 5-min infusions
`of the peptides at concentrations ranging from 5 X 10-1 to 5
`X 10- 12 M to five additional individual pancreases. We repro(cid:173)
`ducibly observed insulin release in response to GLP-I(7-37) at
`concentrations as low as 5 X 10- 11 M, and little if any insulin
`responses to GLP-1(1-37) at concentrations as high as 5 X 10-1
`M. Thus, the potent insulinotropic actions ofGLP-I(7-37) have
`been observed in studies of seven separate pancreases.
`Effects of glucagon on insulin secretion in the perfused pan(cid:173)
`creas have been established previously (13). We also compared
`the effects of glucagon to that of the GLP-Is. We used synthetic
`
`glucagon in the concentration range of I 0- 11-10-1 M and found
`it to be less potent than GLP-I(7-37).
`
`Discussion
`
`The results of these studies clearly indicate that GLP-I(7-37) has
`potent insulinotropin activity. The liberation of this peptide from
`proglucagon in the intestine, and to a lesser extent in the pancreas
`(1), raises the possibility that GLP-I(7-37) has a role in endocrine
`regulation in the entero-insular axis (7). Our data, taken together
`with earlier observations that glucagon-like immunoreactivity
`in crude gut extracts released insulin after ingestion of glucose
`;md fat, (8) suggest that GLP-I(7-37) could potentially be an
`incretin. Of all the known intestinal hormones tested for their
`insulin-releasing potency in the past, gastric inhibitory peptide
`has been considered as a possible incretin (14, 15). However,
`the concentrations of gastric inhibitory peptide required to stim(cid:173)
`ulate insulin secretion exceed the physiologic levels of the peptide
`achieved after a meal. In the rat-perfused pancreas in the presence
`of8.9 mM glucose, gastric inhibitory peptide (10-9 M) increased
`insulin secretion sixfold (16). We find a comparable increase in
`insulin secretion with GLP-1(7-37) at concentrations 100-fold
`lower than those required for an insulinotropic response to gastric
`inhibitory peptide. By radioimmunoassay we have measured
`both GLP-I(l-37) and GLP-1(7-37) levels of~ 150 pg/ml (50
`pM) in rat portal blood and 50 pg/ml ( 15 pM) in peripheral
`blood (S. Mojsov, unpublished results). Therefore, the insuli(cid:173)
`notropic effect that we have observed at concentrations ofGLP-
`1(7-37) of between 5 and 50 pM are well within the physiological
`levels ofGLP-1(7-37) found in the circulation.
`There has been considerable interest in the potential intra~
`islet relationships which might occur between A, B, and D cells,
`such that the secretory product of one cell type might influence
`the function of a neighboring cell ( 17). Interaction could take
`place via a paracrine mechanism or through a local intra-islet
`portal system. Glucagon can stimulate both insulin and soma(cid:173)
`tostatin secretion (13, 18), but because there appears to be a
`functional compartmentalization between islet cells, it is unclear
`whether glucagon can actually reach B and D cells ( 19). Taking
`into account the vascular arrangement of the rat islet, the glu-
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`Figure I. The effects of sepa-
`rate perfusions in two represen-
`tative pancreas GLP-1(7-37) at
`two concentrations, 5 X 10-11
`and 5 X 10-9 M. Solid lines,
`insulin values determined by
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`given peptide.
`
`2
`
`Insulinotropin
`
`617
`
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`MPI EXHIBIT 1036 PAGE 2
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`Apotex v. Novo - IPR2024-00631
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`Figure 2. The effects of separate
`perfusions in two representative
`pancreas with GLP-1(1-37) at
`two concentrations, 5 X 10-9 and
`5 X 10-7 M. Details of the exper-
`iment and explanation of sym-
`bols are described in legend to
`Fig. I.
`
`cagon-containing A cells of the mantle appear to be downstream
`from the B cells of the core, and therefore glucagon may not
`reach the B cells in high enough concentration to exert a sig(cid:173)
`nificant influence (20). The mantle of A and D cells are, however,
`adjacent and this makes the possibility of paracrine interaction
`more feasible, although experimental support for such an inter(cid:173)
`action is not available. The finding that GLP-1(7-37) is a more
`potent insulin secretagogue than glucagon raises important
`questions about its potential intra-islet role.
`Amino and carboxyl-termini of glucagon, GLP-1(7-37) and
`GLP-11 are closely related to each other in their amino acid
`sequences and to vasointestinal peptide that possibly exerts a
`neuronal stimulation of insulin secretion (21 ). A most striking
`similarity among them is the conservation of a histidine residue
`at position I . It is noteworthy that gastric inhibitory peptide,
`also closely related in its structure to the GLPs, has a tyrosine
`residue at position I instead of histidine (22). Inasmuch as a
`histidine residue at this position is essential for adenylate cyclase
`stimulation in various systems, the greater insulinotropic potency
`of GLP-1(7-37) compared with GIP may in part be accounted
`for by the histidine substitution for tyrosine (23).
`Additional evidence in support of the concept that GLP-1(7-
`37) is a potent insulinotropic peptide is provided by our recent
`observation that GLP-1(7-37), and not GLP-1(1-37) or GLP-11,
`is a potent activator of adenylate cyclase at concentrations as
`low as 5 X 10- 11 Mand also stimulates cellular levels of insulin
`mRNA and insulin release in a rat insulinoma cell line (RIN-
`38) (Drucker, D. J., J. Philippe, S. Mojsov, W. L. Chick, and
`J. F. Habener, manuscript in preparation). Further, studies by
`Schmidt and co-workers showed that in isolated precultured is(cid:173)
`lets, 10-9 to 10-s M concentrations of the peptide GLP-1(1-36
`des Gly-Arg amide) were required to release insulin (24).
`Determining whether GLP-1(7-37) is the hormone whose
`primary function is to stimulate insulin secretion in response to
`feeding, or is one of a complex group of hormones involved in
`maintaining glucose homeostasis, will require further investi(cid:173)
`gation.
`
`Acknowledgments
`
`We thank Henrietta Cooper, Deanna Deery, and Adacie Allen for their
`expert experimental assistance in carrying out the pancreatic perfusions
`
`618
`
`S. Mojsov, G. C. Weir, and J. F. Habener
`
`and radioimmun~y, respectively. We thank Esther Hoomis for typing
`the manuscript.
`The studies were supported in part by Public Health Service grants
`AM 30834 and AM 20349.
`
`Note added in proof In studies of isolated perfused pig ileum and pancreas,
`Orskov et al. (25) recently reported finding secretion of a GLP-1 peptide
`from ileum, but the pancreas secreted a large peptide with both GLP-1
`and GLP-2 immunoreactivity.
`
`References
`
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`J. F. Habener. 1986. Preproglucagon gene expression in pancreas and
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`MPI EXHIBIT 1036 PAGE 3
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