Volume 211, number 2, 169-174
`
`FEB 04356
`
`January 1987
`
`Volume 211, number 2, 169-174 FEB 04356 January 1987 Truncated glucagon-like peptide I, an insulin-releasing hormone from the distal gut J.J. Holst, C. 0rskov, 0. Vagn Nielsen and T.W. Schwartz
`
`Truncated glucagon-like peptide I, an insulin-releasing
`hormonefrom the distal gut
`
`J.J. Holst, C. Orskov, O. Vagn Nielsen and T.W. Schwartz
`
`Institute of Medical Physiology C, The Panurn Institute, Laboratory ofMolecular Endocrinology, Department of Clinical
`Institute of Medical Physiology C, The Panum Institute, Laboratory ofMolecular Endocrinology, Department of Clinical
`Chemistry and Department of Surgery C at Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
`Chemistry and Department of Surgery C at Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
`
`Received 23 October 1986; revised version received 20 November 1986
`
`By hydrophobic gel permeation and high pressure liquid chromatography weisolated from pig intestinal
`mucosa a peptide which corresponds to proglucagon 78-107 as suggested by chromatography and determi-
`nation ofits N-terminal sequence. Natural and synthetic proglucagon 78-107 dose dependently and potently
`increased insulin secretion from the isolated perfused pig pancreas. Proglucagon 78-107 also secreted by
`the small intestine may participate in the hormonalcontrolofinsulin secretion.
`
`Glucagon-like peptide; Incretin; Monobasic cleavage
`
`1. INTRODUCTION
`
`Received 23 October 1986; revised version received 20 November 1986 By hydrophobic gel permeation and high pressure liquid chromatography we isolated from pig intestinal mucosa a peptide which corresponds to proglucagon 78-107 as suggested by chromatography and determi- nation of its N-terminal sequence. Natural and synthetic proglucagon 78-107 dose dependently and potently increased insulin secretion from the isolated perfused pig pancreas. Proglucagon 78-107 also secreted by the small intestine may participate in the hormonal control of insulin secretion. Glucagon-like peptide; Incretin; Monobasic cleavage 1. INTRODUCTION The mammalian glucagon precursor (pro- glucagon) is a 180 amino acid peptide. Besides glucagon itself it contains two glucagon-like se- quences, originally designated ‘glucagon-like pep- tides 1 and 2’ (GLP-1 and GLP-2), which are separated by a 13 amino acid spacer sequence [ 1,2]. The glucagon-like sequences (GLP-1 shown in fig.l), which are about 50% homologous with glucagon, are flanked by pairs of basic amino acids, putative processing sites. Proglucagon ap- pears to be processed differently in the mammalian pancreas and small intestine [3-51. In the pig and rat pancreas the following peptides are produced and secreted upon appropriate stimulation: (i) glucagon; (ii) glicentin-related pancreatic peptide (GRPP) corresponding to proglucagon l-30; (iii) a large peptide that contains both the GLP-1 and the GLP-2 sequences [4-61. In the pig small intestine the major secreted products are the 69 amino acid glucagon-containing peptide, glicentin, and the Correspondence address: J.J. Hoist, Institute of Medical Physiology C, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark two glucagon-like sequences GLP-1 and GLP-2 as separate peptides, not as parts of one large peptide [4]. Gel filtration studies have shown that the glucagon-like peptides secreted from the pig small intestine have approximately the same size as syn- thetic replicas of the two glucagon-like peptides synthesized according to their structure as predicted from the proglucagon sequence [4]. The exact structure of the ileal glucagon-like peptides is not known, however. We therefore isolated the naturally occurring glucagon-like peptide 1 from acid-ethanol extracts of pig small intestinal mucosa and determined part of its sequence. In addition, we studied biological effects of the peptide as well as the effects of a peptide synthesized according to the structure of the natural peptide. 2. MATERIALS AND METHODS 2.1. Isolation of proglucagon 78-107 Ileal mucosa was excised from anaesthesized pigs and immediately frozen. Acid ethanol-extracts were prepared according to method II in [7]. In short, frozen tissue was homogenized in 4 vols acid ethanol and centrifuged. 5 vols cold diethyl ether were added to the supernatant and the aqueous Published by
`
`(pro-
`The mammalian glucagon precursor
`glucagon) is a 180 amino acid peptide. Besides
`glucagon itself it contains two glucagon-like se-
`quences, originally designated ‘glucagon-like pep-
`tides 1 and 2’
`(GLP-1 and GLP-2), which are
`separated by a 13 amino acid spacer sequence
`[1,2]. The glucagon-like sequences (GLP-1 shown
`in fig.1), which are about 50% homologous with
`glucagon, are flanked by pairs of basic amino
`acids, putative processing sites. Proglucagon ap-
`pears to be processed differently in the mammalian
`pancreas and small intestine [3—5]. In the pig and
`rat pancreas the following peptides are produced
`and secreted upon appropriate stimulation:
`(i)
`glucagon; (ii) glicentin-related pancreatic peptide
`(GRPP) corresponding to proglucagon 1—30;(iii) a
`large peptide that contains both the GLP-1 and the
`GLP-2 sequences [4-6]. In the pig small intestine
`the major secreted products are the 69 amino acid
`glucagon-containing peptide, glicentin, and the
`
`two glucagon-like sequences GLP-1 and GLP-2 as
`separate peptides, not as parts of one large peptide
`[4]. Gel filtration studies have shown that
`the
`glucagon-like peptides secreted from the pig small
`intestine have approximately the same size as syn-
`thetic replicas of the two glucagon-like peptides
`synthesized
`according
`to their
`structure
`as
`predicted from the proglucagon sequence[4]. The
`exact structure of the ileal glucagon-like peptidesis
`not known, however. We therefore isolated the
`naturally occurring glucagon-like peptide 1 from
`acid-ethanol extracts of pig small intestinal mucosa
`and determined part of its sequence. In addition,
`westudied biological effects of the peptide as well
`as the effects of a peptide synthesized according to
`the structure of the natural peptide.
`
`2. MATERIALS AND METHODS
`
`2.1. fsolation of proglucagon 78-107
`Ileal mucosa was excised from anaesthesized
`pigs and immediately frozen. Acid ethanol-extracts
`were prepared according to method II in [7]. In
`short, frozen tissue was homogenizedin 4 vols acid
`ethanol and centrifuged. 5 vols cold diethyl ether
`were added to the supernatant and the aqueous
`
`Institute of
`J.J. Holst,
`address:
`Correspondence
`The
`Panum Institute,
`Medical
`Physiology C,
`Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
`
`Elsevier
`Published by Elsevier Science Publishers B.V. (Biomedical Division)
`Science Publishers B. V. (Biomedical Division) 00145793/87/%3.50 0 1987 Federation of European Biochemical Societies 169
`00145793/87/$3.50 © 1987 Federation of European Biochemical Societies
`169
`
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`DR. REDDY’S LABORATORIES, INC.
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`

`

`-—50°C. The resulting
`isolated at
`phase was
`precipitate was then dissolved in distilled water
`containing 8 mol/I urea. The GLP-1 immunoreac-
`tive peptide was isolated in 4 consecutive steps.
`The procedure was monitored with a radioim-
`munoassay developed for synthetic GLP-1 (pro-
`glucagon 71—107) [4], using antiserum 1953 raised
`against synthetic GLP-1, 1**I-labeled synthetic
`GLP-1, and synthetic GLP-1 (1-37 amide, code
`no.7166, Peninsula, Belmont, CA, USA) for stan-
`dards. Determined with this radioimmunoassay ex-
`tracts of the pig ileum mucosa contain 88 +
`4 pmol/g of immunoreactive GLP-1. Extract of
`1 kg mucosa correspondingto ileal mucosa from 3
`pigs of 40-45 kg was applied to a3 x 15 cm glass
`column packed with Techoprep C18, 40-63 zm
`(HPLC Technology, England), and eluted with a
`gradient of 20-80% ethanol in water containing in
`addition 0.01% trifluoroacetic acid (TFA, Pierce,
`Rockford, IL). From the GLP-1 immunoreactive
`fractions the ethanol was removed by evaporation
`and the pool was applied to a 50 x 1000 mm
`(K 50/100) column packed with Sephadex® G-S0,
`fine grade (Pharmacia, Uppsala, Sweden) and
`eluted with 0.5 M acetic acid at a flow rate of
`1 ml/min at 4°C. GLP-1 immunoreactive frac-
`tions were then subjected to reverse-phase high-
`pressure liquid chromatography on an 8 x 250 mm
`Nucleosil® C18 column employing LKB pumps
`and detectors (LKB, Bromma, Sweden). Thecol-
`umn was eluted with water containing 0.1% TFA
`and a gradient of acetonitrile (grade S, Rathburn
`Chemicals, Ltd, Walkerburn, Scotland) from 0 to
`80%. Finally, GLP-1 immunoreactive fractions
`were subjected to isocratic HPLC with 43%
`ethanol
`in water containing in addition 0.01%
`TFAas the mobile phase.
`
`Volume 211, number 2 FEBSLETTERS January 1987 phase was isolated at - 50°C. The resulting precipitate was then dissolved in distilled water containing 8 mol/l urea. The GLP-1 immunoreac- tive peptide was isolated in 4 consecutive steps. The procedure was monitored with a radioim- munoassay developed for synthetic GLP-1 (pro- glucagon 71-107) [4], using antiserum 1953 raised against synthetic GLP-1, 1251-labeled synthetic GLP-1, and synthetic GLP-1 (l-37 amide, code no.7166, Peninsula, Belmont, CA, USA) for stan- dards. Determined with this radioimmunoassay ex- tracts of the pig ileum mucosa contain 88 + 4 pmol/g of immunoreactive GLP-1. Extract of 1 kg mucosa corresponding to ileal mucosa from 3 pigs of 40-45 kg was applied to a 3 x 15 cm glass column packed with Techoprep C18, 40-63 pm (HPLC Technology, England), and eluted with a gradient of 20-80% ethanol in water containing in addition 0.01% trifluoroacetic acid (TFA, Pierce, Rockford, IL). From the GLP-1 immunoreactive fractions the ethanol was removed by evaporation and the pool was applied to a 50 x 1000 mm (K SO/lOO) column packed with SephadexR G-50, fine grade (Pharmacia, Uppsala, Sweden) and eluted with 0.5 M acetic acid at a flow rate of 1 ml/min at 4°C. GLP-1 immunoreactive frac- tions were then subjected to reverse-phase high- pressure liquid chromatography on an 8 x 250 mm NucleosilR Cl8 column employing LKB pumps and detectors (LKB, Bromma, Sweden). The col- umn was eluted with water containing 0.1% TFA and a gradient of acetonitrile (grade S, Rathburn Chemicals, Ltd, Walkerburn, Scotland) from 0 to 80%. Finally, GLP-1 immunoreactive fractions were subjected to isocratic HPLC with 43% ethanol in water containing in addition 0.01% TFA as the mobile phase. 2.2. Sequence determination Solvent was removed under vacuum from the HPLC purified peptide. It was reconstituted in 0.06 ml of 10% TFA in water and subjected to automated sequence analysis by sequential Edman degradation on an Applied Biosystems 470A gas- phase sequenator with the 02 NVAC program (batch 1) or the 02RPTH program (batch 2), pro- grammes modified from Hunkapillar et al. [S], available at Applied Biosystems, Foster City, CA, USA. All chemicals were purchased from Applied Biosystems. The phenylthiohydantoin derivatives 170 of amino acids were either (batch 1) characterized by HPLC on a Hewlett-Packard 1084 liquid chromatograph with a 0.45 x 25 cm column of CN (5 pm particles, IBM Instruments) and a sodium acetateiacetonitrile gradient elution system as described [9], or, in the case of batch 2, the samples from the sequenator were methylated before HPLC by treating the dried derivatives with acidified methanol (1 M HCl in methanol; Applied Biosystems) for 10 min at 50°C. The amino acid derivatives were then characterized on an Applied Biosystems PTH column, 2.1 x 22 cm, using the elution system recommended by the manufacturer. Aminobutyric acid was used as internal standard during the HPLC for correction of elution time and for quantifying the amino acid derivatives. 2.3. Effect of natural and synthetic GLP-1 on insulin secretion Synthetic proglucagon 78-108 amide was ob- tained from Peninsula Laboratories (San Carlos, CA) by custom synthesis (lot no.008802; peptide purity by amino acid analysis 72%). Before the peptide was used in physiological studies, it was purified to homogeneity by isocratic HPLC as described above. Sequence determination of syn- thetic peptide confirmed the structure as being pro- glucagon 78-107 amide. The biological effect of both the synthetic proglucagon 78-107 fragment and the isolated natural peptide was studied using perfused porcine pancreas, prepared and perfused as described in [lo]. The pancreas was isolated together with the supplying arteries and veins and perfused with an artificial medium consisting of Krebs-Ringer bicarbonate buffer, containing 15% washed bovine erythrocytes, 0.1% human serum albumin (reins& trocken, Behringwerke Marburg, FRG), 5 or 7 mmol/l glucose, 5% dextran T-70, aprotinin 100000 KIU/l (TrasylolR, Bayer, Leverkusen, FRG), and 5 mmol/l of a mixture of amino acids (Amodex Asa, Pharmacia, Uppsala, Sweden). The medium was gassed with 95% ox- ygen and 5% COz. Synthetic proglucagon 78-107 was infused intra-arterially for 5 or 10 min periods in doses corresponding to final perfusate concen- trations of 10-‘“-10-8 mol/l. Efflrrent fractions were collected every minute and kept on ice until centrifugation. The supernatants were stored at -20°C until assay. Insulin was measured in all fractions as described earlier [lo].
`
`2.2. Sequence determination
`Solvent was removed under vacuum from the
`HPLC purified peptide. It was reconstituted in
`0.06 ml of 10% TFA in water and subjected to
`automated sequence analysis by sequential Edman
`degradation on an Applied Biosystems 470A gas-
`phase sequenator with the 02 NVAC program
`(batch 1) or the O2RPTH program (batch 2), pro-
`grammes modified from Hunkapillar et al.
`[8],
`available at Applied Biosystems, Foster City, CA,
`USA.All chemicals were purchased from Applied
`Biosystems. The phenylthiohydantoin derivatives
`
`170
`
`Volume 211, number 2
`
`FEBS LETTERS
`
`January 1987
`
`of amino acids were either (batch 1) characterized
`by HPLC on a Hewlett-Packard 1084 liquid
`chromatograph with a 0.45 x 25 cm column of CN
`(5 «2m particles, IBM Instruments) and a sodium
`acetate/acetonitrile gradient elution system as
`described [9], or,
`in the case of batch 2,
`the
`samples from the sequenator were methylated
`before HPLC bytreating the dried derivatives with
`acidified methanol (1 M HCl in methanol; Applied
`Biosystems) for 10 min at 50°C. The amino acid
`derivatives were then characterized on an Applied
`Biosystems PTH column, 2.1 x 22 cm, using the
`elution system recommended by the manufacturer.
`Aminobutyric acid was used as internal standard
`during the HPLC for correction of elution time
`and for quantifying the amino acid derivatives.
`
`2.3. Effect of natural and synthetic GLP-1 on
`insulin secretion
`Synthetic proglucagon 78-108 amide was ob-
`tained from Peninsula Laboratories (San Carlos,
`CA) by custom synthesis (lot no.008802; peptide
`purity by amino acid analysis 72%). Before the
`peptide was used in physiological studies, it was
`purified to homogeneity by isocratic HPLC as
`described above. Sequence determination of syn-
`thetic peptide confirmed the structure as being pro-
`glucagon 78—107 amide. The biological effect of
`both the synthetic proglucagon 78—107 fragment
`and the isolated natural peptide was studied using
`perfused porcine pancreas, prepared and perfused
`as described in [10]. The pancreas was isolated
`together with the supplying arteries and veins and
`perfused with an artificial medium consisting of
`Krebs-Ringer bicarbonate buffer, containing 15%
`washed bovine erythrocytes, 0.1% human serum
`albumin (reinst, trocken, Behringwerke Marburg,
`FRG), 5 or 7 mmol/I] glucose, 5% dextran T-70,
`aprotinin
`100000 KIU/I
`(Trasylol®,
`Bayer,
`Leverkusen, FRG), and 5 mmol/1 of a mixture of
`amino acids (Amodex Asa, Pharmacia, Uppsala,
`Sweden). The medium was gassed with 95% ox-
`ygen and 5% CO>2. Synthetic proglucagon 78-107
`was infused intra-arterially for 5 or 10 min periods
`in doses corresponding to final perfusate concen-
`trations of 107'°-10-® mol/l. Efftuent fractions
`were collected every minute and kept on ice until
`centrifugation. The supernatants were stored at
`—20°C until assay. Insulin was measured in all
`fractions as described earlier [10].
`
`MPI EXHIBIT 1027 PAGE 2
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`DR. REDDY’S LABORATORIES, INC.
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`

`Volume 211, number 2
`
`FEBS LETTERS
`
`January 1987
`
`3. RESULTS AND DISCUSSION
`
`Theresults of the isolation procedure are shown
`in fig.2. A homogeneous peak of immunoreactive
`GLP-1 was eluted from the C18 Techoprep® col-
`umn, and wasfurther purified by gel filtration and
`repeated HPLC. The sequence of the first 17
`aminoacids is shownin table 1. This sequence cor-
`responds to proglucagon 78—94; in other words, a
`truncated form of GLP-1. By gel filtration on
`Sephadex G 50 columnsthe natural peptide eluted
`exactly at the position of synthetic proglucagon
`78—-107 amide. In addition, on analytical reverse-
`phase HPLC using a 24—48%acetonitrile gradient
`over 45 min the natural and the synthetic peptide
`had the sameretention time (26 min). This strong-
`ly suggests that the natural peptide corresponds to
`proglucagon 78-107. In the present study we could
`not determine whether the natural peptide has a
`free a-carboxyl group or is amidated. An amidated
`form was chosen for the synthetic peptide because
`
`of the presence in proglucagon ofa glycine residue
`after the C-terminal dibasic flanking sequences of
`GLP-1; in many propeptides this sequence gives
`rise to carboxyamidation during the posttransla-
`tional processing [11].
`According to our results, the cleavage of pro-
`glucagon to release GLP-1 does not occur at the
`expected site of the pair of basic amino acids (69
`and 70) but after the single basic amino acid at
`position 77. Similar monobasic proteolytic pro-
`cessing is found for many precursors as recently
`reviewed [12]. The natural porcine GLP-1, which
`according to the present study correspondsto pro-
`glucagon 78-107 amide,
`is thus very similar to
`anglerfish, catfish and salmon GLP-1 ({13—15]; see
`fig.1). In fact,
`the six amino acids between the
`classical dibasic cleavage and the monobasic
`cleavage site are not encoded for by the anglerfish
`gene [13]. After removal of the 6 N-terminal amino
`acids of GLP-1 the sequence homologyof the re-
`maining peptide with glucagon is even more pro-
`
`proglucagon
`in human
`Residue no.
`
`72
`His Asp Glu Phe Glu Arg
`t
`"
`"
`"
`'
`tt
`"
`"
`1
`"
`"
`t
`"
`"
`"
`"
`tt
`"t
`
`"
`"
`
`'
`"
`
`Ala Glu Gly
`tw
`"
`wu
`
`"
`
`oy
`
`"
`
`"
`
`"
`
`'
`
`"
`
`"
`
`©) 9©) 6) G vor GS} ser
`
`Species
`Species
`
`Human
`ox
`Hamster
`Rat
`
`Guinea pig
`Anglerfish
`Salmon
`Catfish
`
`Species
`
`Human
`Ox
`Hamster
`Rat
`
`Guinea pig
`Anglerfish
`Salmon
`Catfish
`
`Volume 211, number 2 FEBS LETTERS January 1987 3. RESULTS AND DISCUSSION The results of the isolation procedure are shown in fig.2. A homogeneous peak of immunoreactive GLP-1 was eluted from the Cl8 TechoprepR col- umn, and was further purified by gel filtration and repeated HPLC. The sequence of the first 17 amino acids is shown in table 1. This sequence cor- responds to proglucagon 78-94; in other words, a truncated form of GLP-1. By gel filtration on Sephadex G 50 columns the natural peptide eluted exactly at the position of synthetic proglucagon 78-107 amide. In addition, on analytical reverse- phase HPLC using a 24-48% acetonitrile gradient over 45 min the natural and the synthetic peptide had the same retention time (26 min). This strong- ly suggests that the natural peptide corresponds to proglucagon 78-107. In the present study we could not determine whether the natural peptide has a free cu-carboxyl group or is amidated. An amidated form was chosen for the synthetic peptide because
`
`Human Cx Hamster Rat Guinea pig Anglerfish Salmon Catfish Suecies Human ox Hamster Rat Guinea pig Anglerfish Salmon Catfish of the presence in proglucagon of a glycine residue after the C-terminal dibasic flanking sequences of GLP-1; in many propeptides this sequence gives rise to carboxyamidation during the posttransla- tional processing [ 111. According to our results, the cleavage of pro- glucagon to release GLP-1 does not occur at the expected site of the pair of basic amino acids (69 and 70) but after the single basic amino acid at position 77. Similar monobasic proteolytic pro- cessing is found for many precursors as recently reviewed [12]. The natural porcine GLP-1, which according to the present study corresponds to pro- glucagon 78-107 amide, is thus very similar to anglerfish, catfish and salmon GLP-1 ([13-151; see fig.1). In fact, the six amino acids between the classical dibasic cleavage and the monobasic cleavage site are not encoded for by the anglerfish gene [ 131. After removal of the 6 N-terminal amino acids of GLP-1 the sequence homology of the re- maining peptide with glucagon is even more pro- Residue no. in human proglucagon Residue no. in human nroglucagon 6 eu Glu Gly Gln 1 Ala Lys Glu Ph Ile Ala Val Lys Gly Arg Gly Arg Arg I, 1, I, 0 I, I, I, I, 0 1, loo I, ,I @@ II II II II 11 107 11 11 I, 110 1, II 11 II 11 II II 11 II 11 11 11 II (1 II II 11 11 II II II 11 II I, II II II II 11 71 II I, It II 11 11 II I, I, I, I, II II II 11 I, 11 11 9, 1, 1, 11 ,, 11 1, 11 I, (1 I, 1, II " Lys Asp " " Ile " Asp 'I Val Asp Arg " Lys Ala 11 Gin Val " " II Gin 11 II 1, ,, v Asp t4 Val Ser w w Lys Ser U v Ala + + II Gin 11 II It 11 " Asn " II Thr I1 )t Lys Ser N Gln Pro Lys Pro Fig.1. Sequences from 8 species of proglucagon regions containing glucagon-like peptide 1. Glucagon-like peptide-l containing sequences of proglucagon from all species whose proglucagon structure has been determined [ 1,2,13-15,23,24]. The peptide which was originally designated GLP-1 (of hamster proglucagon) corresponds to proglucagon 72-108. Amino acids which occupy the same position in the glucagon molecule are encircled. Amino acids 72-77 are not coded for in the anglerfish gene. (” ” ) Same amino acid as in human proglucagon; ( + + ) gene structure not known [14,15]; (- - ) amino acids not encoded for in anglerfish gene [13]. Circle around amino acids indicates amino acids in same positions as human glucagon. Vertical line between amino acids 77 and 78 indicates position of N-terminal amino acid in glucagon-like peptide 1 from pig, anglerfish, salmon and catfish. 171
`
`roglucagon
`in human
`Residue no.
`Glu Gly Gln )
`"
`1
`"
`t
`"
`i
`tT
`
`Ala Lys Glu
`"
`tt
`"
`
`w
`wt
`
`wt
`t
`
`w
`
`u"
`
`"
`w
`"
`Lys Asp
`"
`Glin
`"
`Gln w
`
`"
`"
`"
`"
`
`wt
`
`tt
`tt
`
`w
`
`tt
`
`"
`"
`
`u
`wt
`
`w"
`"
`"
`"
`
`"
`wt
`
`1" Tyr
`ww Tyr
`
`"
`"
`
`tt Asn
`1
`A
`
`"
`"
`
`tt Thr
`"
`
`Ls
`il
`
`100
`107
`110
`Tle Ala Grp Ge) Val Lys Gly Arg Gly Arg Arg
`tt
`tt
`t
`t
`it
`Li
`1
`w
`"
`iy
`"
`w
`nt
`t
`"
`"
`"
`wt
`"
`u
`1
`tt
`"
`t
`t
`t
`"
`"
`"
`it
`"
`"
`Ui
`1
`"
`u
`"
`tt
`"
`t
`
`"
`t
`
`Val Asp Arg
`Val Ser
`"
`"Thr
`"
`
`"
`"
`"
`
`Lys Ala
`Lys Ser
`Lys Ser
`
`"
`"
`" Gin Val
`+
`"
`"Ala +
`" Gin Pro Lys Pro
`
`Fig.1. Sequences from 8 species of proglucagon regions containing glucagon-like peptide 1. Glucagon-like peptide-1
`containing sequences of proglucagon from all
`species whose proglucagon structure has been determined
`[1,2,13-15,23,24]. The peptide which was originally designated GLP-1 (of hamster proglucagon) corresponds to
`proglucagon 72-108. Amino acids which occupy the sameposition in the glucagon molecule are encircled. Amino acids
`72-77 are not coded for in the anglerfish gene. (’’ ’’) Same aminoacid as in human proglucagon; (+ +) gene structure
`not known [14,15]; (— —) amino acids not encoded for in anglerfish gene [13]. Circle around aminoacidsindicates
`amino acids in same positions as human glucagon. Vertical line between amino acids 77 and 78 indicates position of
`N-terminal amino acid in glucagon-like peptide 1 from pig, anglerfish, salmon and catfish.
`
`171
`
`MPI EXHIBIT 1027 PAGE 3
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`MPI EXHIBIT 1027 PAGE 3
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`MPI EXHIBIT 1027 PAGE 3
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1027, p. 3 of 6
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`

`Volume 211, number 2
`
`FEBS LETTERS
`
`/
`
`Lo
`
`“,
`
`1
`51
`
`nmol
`100 age
`EtOH
`
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`
`
`Volume 211, number 2 FEBS LETTERS January 1987 Fig.2. Isolation of porcine natural GLP-1. The concentration of natural GLP-1 was monitored by radioimmunoassay. Upper left part shows the results of hydrophobic chromatography. Extract of 1 kg mucosa was applied to the column and eluted with a gradient of ethanol in water (right ordinate scale). Upper right part shows gel filtration of peak fractions from the first step. Lower left part shows reverse-phase HPLC of peak fractions from the gel filtration. The column was eluted with a gradient of acetonitrile in water (right ordinate scale). The lower right part shows the results of isocratic HPLC (43% ethanol in water). The dotted line shows UV-absorption at 280 nm. nounced (see fig.1). Since the N-terminal part of the glucagon molecule is essential for its biological activity [16], proglucagon 78-107 might be ex- pected to show glucagon-like effects. So far, only few studies on the biological effects of the glucagon-like peptides GLP-1 and GLP-2 have been reported. Ghiglione et al. [17] found no effect of synthetic GLP-1 (l-36 amide, Peninsula) on blood glucose or insulin levels in fasting rabbits in what was considered as pharmacological doses. Schmidt et al. [ 181 found a weak insulinotropic ef- fect of synthetic GLP-1 (l-36 amide, code no.7166, Peninsula), but not of synthetic GLP-2 (l-34, code no.7156) on isolated rat islet at rather high concentrations of GLP-1 and GLP-2 and in the presence of 10 mmol/l glucose. We [19] recent- ly demonstrated inhibition of gastric acid secretion in healthy volunteers during submaximal pen- Table 1 Amino acid Sequence Amino Yield of amino in mam- cycle acid acid phenyl- malian pro- thiohydantoin de- glucagon rivative (pmol) 78-96 Batch 1 Batch 2 His Ala Glu Gly Thr Phe Ser Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala 1 His 2 Ala 3 Glu 4 Gly 5 Thr 6 Phe 7 X 8 Ser 9 Asp 10 Val 11 X 12 X 13 T yr 14 Leu 15 Glu 16 X 17 Gin 18 X 37 - 108 18 76 105 81 54 43 _ 80 64 _ _ 22 - _a 32 64 47 _ _ _ - 46 24 _ 14 16 _ 16 - a The unmethylated PTA-Asp could be identified close to the injection artifact but was not quantified Sequence determination of the GLP-1 immunoreactive peptide isolated from porcine intestinal mucosa. The results of Edman degradation of two independent batches of peptide are shown. Identification and quantitation of the phenylthiohydantoin derivatives were performed on a Hewlett-Packard 1084 (1) or 1090A (2) chromatograph. The amino acid derivatives were methylated before identification in batch 2. (-) Uncertain identification tagastrin stimulation when GLP-1 (l-36 amide) was infused at a rate of 400 ng/kg x h. This infu- sion rate increased the plasma levels of im- munoreactive GLP-1 from 90 to about 600 pmol/l. We found no effect of synthetic GLP-1 (code 7166) or GLP-2 (code 7156; both from Peninsula) on the isolated perfused pancreas in concentrations up to lo- M either on the ex- ocrine or the endocrine secretion [4]. By contrast, natural GLP-1, isolated from pig intestinal mucosa strongly increased insulin secretion from the same preparations. At a concentration of lo-” mol/l of natural GLP-1 (determined by radioimmunoassay) 172
`
`nounced(see fig.1). Since the N-terminal part of
`the glucagon moleculeis essential for its biological
`activity [16], proglucagon 78-107 might be ex-
`pected to show glucagon-like effects. So far, only
`few studies on the biological effects of
`the
`glucagon-like peptides GLP-1 and GLP-2 have
`tagastrin stimulation when GLP-1 (1—36 amide)
`been reported. Ghiglioneet al. [17] found no effect
`was infused at a rate of 400 ng/kg x h. This infu-
`of synthetic GLP-1 (1-36 amide, Peninsula) on
`sion rate increased the plasma levels of im-
`
`munoreactive GLP-1 to_aboutfrom 90
`
`blood glucoseor insulin levels in fasting rabbits in
`what was considered as pharmacological doses.
`600 pmol/l. We found no effect of synthetic
`Schmidtet al. [18] found a weak insulinotropic ef-
`GLP-1 (code 7166) or GLP-2 (code 7156; both
`fect of
`synthetic GLP-!
`(1-36 amide, code
`from Peninsula) on the isolated perfused pancreas
`in concentrations up to 107° M either on the ex-
`no.7166, Peninsula), but not of synthetic GLP-2
`(1-34, code no.7156) onisolated rat islet at rather
`ocrine or the endocrine secretion [4]. By contrast,
`high concentrations of GLP-1 and GLP-2 and in
`natural GLP-1, isolated from pig intestinal mucosa
`the presence of 10 mmol/I glucose. We[19] recent-
`strongly increased insulin secretion from the same
`preparations. At a concentration of 107!° mol/l of
`ly demonstrated inhibition of gastric acid secretion
`in healthy volunteers during submaximal pen-
`natural GLP-1 (determined by radioimmunoassay)
`
`Table 1
`
`Sequence Amino
`cycle
`acid
`
`Amino acid
`in mam-
`malian pro-
`glucagon
`78-96
`
`January 1987
`
`Yield of amino
`acid phenyl-
`thiohydantoin de-
`rivative (pmol)
`
`Batch 1 Batch 2
`
`His
`Ala
`Glu
`Gly
`Thr
`Phe
`Ser
`Ser
`Asp
`Val
`Ser
`Ser
`Tyr
`Leu
`Glu
`Gly
`Gin
`Ala
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`
`37
`108
`76
`81
`43
`80
`—
`22
`-*
`64
`—
`-
`46
`-
`
`His
`Ala
`Glu
`Gly
`Thr
`Phe
`Xx
`Ser
`Asp
`Val
`xX
`xX
`Tyr
`Leu
`Glu
`x
`Gin
`Xx
`
`-
`18
`105
`54
`-
`64
`—
`-
`32
`47
`—
`~
`24
`14
`16
`-
`16
`~
`
`* The unmethylated PTA-Asp could be identified close
`to the injection artifact but was not quantified
`
`Sequence determination of the GLP-I immunoreactive
`peptide isolated from porcine intestinal mucosa. The
`results of Edman degradation of two independent
`batches of peptide are shown.
`Identification and
`quantitation of the phenylthiohydantoin derivatives
`were performed on a Hewlett-Packard 1084 (1) or 1090A
`(2) chromatograph. The amino acid derivatives were
`methylated before identification in batch 2.
`(—)
`Uncertain identification
`
`amolA
`1000-
`
`500+
`
`o
`1
`nmol;
`50, ft
`
`
`
`
`
`
`25}
`
`430
`
`25+
`
`46.EtOH
`
`Ong |
`
`"
`
`ane
`
`Isolation of porcine natural GLP-1. The
`Fig.2.
`concentration of natural GLP-1 was monitored by
`radioimmunoassay. Upper left part showsthe results of
`hydrophobic chromatography. Extract of 1 kg mucosa
`was applied to the columnand eluted with a gradient of
`ethanol in water (right ordinate scale). Upper right part
`showsgel filtration of peak fractions from the first step.
`Lower left part shows reverse-phase HPLC of peak
`fractions from the gelfiltration. The column waseluted
`with a gradient of acetonitrile in water (right ordinate
`scale). The lowerright part showsthe results of isocratic
`HPLC (43%ethanol in water). The dotted line shows
`UV-absorption at 280 nm.
`
`172
`
`MPI EXHIBIT 1027 PAGE 4
`
`MPI EXHIBIT 1027 PAGE 4
`
`MPI EXHIBIT 1027 PAGE 4
`
`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1027, p. 4 of 6
`
`

`

`Volume 211, number 2
`
`FEBS LETTERS
`
`January 1987
`
`potent intestinal insulinotropic hormone [20]. At
`5.0 mmol/l glucose in the perfusate the in-
`sulinotropic effect was less conspicuous; but the
`relative increase in insulin secretion was approx-
`imately the same (not shown).
`On this background it might be suggested that
`the reported effects of GLP-1 in high doses could
`be due to enzymatic conversion of GLP-1 to pro-
`glucagon 78—107 in the medium. Trypsin-like en-
`zymatic activity, which maybedifficult to avoid in
`pancreatic tissue incubation studies, might be
`responsible for
`such conversion, due to the
`presence of a basic amino acid residue at position
`76.
`
`pmol /\
`
`10°'9mM
`10-10
`I
`
`I
`
`5x10 OMC7
`
`5x10-'Ok4
`I
`I
`
`10-9 M
`|
`
`5109 M
`rT
`
`Proglucagon 78 - 107
`Proglucagon 76 -107
`
`and 7 mmol/l glucose in the perfusate insulin
`secretion increased from 21.9 + 2.6 (average + SE
`of 5 min basal secretion in 2 preparations) to
`31.6 + 1.1 pmol/min (average of 5 min stimulated
`secretion); and at 107° mol/I insulin secretion in-
`creased from 32.0 + 1.1 to 73.6 + 2.9 pmol/min.
`A similar increase in insulin secretion was observed
`after administration of
`synthetic proglucagon
`78-107 amide(figs 3,4). After arterial infusion of
`synthetic proglucagon 78-107 at 10-?° mol/I in-
`sulin secretion approximately doubled and increas-
`ed more than 4-fold after 10-° mol/I. Thusthis
`peptideis at least as potent and effective as gastric
`inhibitory polypeptide (GIP), hitherto the most
`
`Volume 211, number 2 FEBS LETTERS January 1987 and 7 mmol/l glucose in the perfusate insulin secretion increased from 21.9 f 2.6 (average f SE of 5 min basal secretion in 2 preparations) to 3 1.6 + 1.1 pmol/min (average of 5 min stimulated secretion); and at low9 mol/l insulin secretion in- creased from 32.0 + 1.1 to 73.6 + 2.9 pmol/min. A similar increase in insulin secretion was observed after administration of synthetic proglucagon 78-107 amide (figs 3,4). After arterial infusion of synthetic proglucagon 78-107 at lo-” mol/l in- sulin secretion approximately doubled and increas- ed more than 4-fold after lob9 mol/l. Thus this peptide is at least as potent and effective as gastric inhibitory polypeptide (GIP), hitherto