`
`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
`
`Institute of Medical Physiology C, The Panum Institute, Laboratory of Molecular Endocrinology, Department of Clinical
`Chemistry and Department of Surgery Cat 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 we isolated from pig intestinal
`mucosa a peptide which corresponds to proglucagon 78-107 as suggested by chromatography and determi(cid:173)
`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; lncretin; Monobasic cleavage
`
`1. INTRODUCTION
`
`(pro(cid:173)
`The mammalian glucagon precursor
`glucagon) is a 180 amino acid peptide. Besides
`glucagon itself it contains two glucagon-like se(cid:173)
`quences, originally designated 'glucagon-like pep(cid:173)
`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(cid:173)
`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
`
`Institute of
`Correspondence address: J .J. Holst,
`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(cid:173)
`thetic replicas of the two glucagon-like peptides
`their structure as
`synthesized according
`to
`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 prog/ucagon 78- 107
`Heal 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 Elsevier Science Publishers B. V. (Biomedical Division)
`00145793/87/$3.50 © 1987 Federation of European Biochemical Societies
`
`169
`
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`
`isolated at - 50°C. The resulting
`phase was
`precipitate was then dissolved in distilled water
`containing 8 mol/1 urea. The OLP-1 immunoreac(cid:173)
`tive peptide was isolated in 4 consecutive steps.
`The procedure was monitored with a radioim(cid:173)
`munoassay developed for synthetic OLP-1 (pro(cid:173)
`glucagon 71-107) (4), using antiserum 1953 raised
`against synthetic OLP-1, 1251-labeled synthetic
`OLP-1, and synthetic OLP-1 (1-37 amide, code
`no.7166, Peninsula, Belmont, CA, USA) for stan(cid:173)
`dards. Determined with this radioimmunoassay ex(cid:173)
`tracts of the pig ileum mucosa contain 88 ±
`4 pmol/g of immunoreactive OLP-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 Cl 8, 40-63 µm
`(HPLC Technology, England), and eluted with a
`gradient of 20-800Jo ethanol in water containing in
`addition 0.01 OJo trifluoroacetic acid (TFA, Pierce,
`Rockford, IL). From the OLP-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 SephadexR 0-50,
`fine grade (Pharmacia, Uppsala, Sweden) and
`eluted with 0.5 M acetic acid at a flow rate of
`1 ml/min at 4°C. OLP-1 immunoreactive frac(cid:173)
`tions were then subjected to reverse-phase high(cid:173)
`pressure liquid chromatography on an 8 x 250 mm
`NucleosilR Cl8 column employing LKB pumps
`and detectors (LKB, Bromma, Sweden). The col(cid:173)
`umn was eluted with water containing 0.1 OJo TF A
`and a gradient of acetonitrile (grade S, Rathburn
`Chemicals, Ltd, Walkerburn, Scotland) from Oto
`800Jo. Finally, OLP-1 immunoreactive fractions
`were subjected to isocratic HPLC with 430Jo
`ethanol in water containing in addition 0.01 OJo
`TF A 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 lOOJo TFA in water and subjected to
`automated sequence analysis by sequential Edman
`degradation on an Applied Biosystems 470A gas(cid:173)
`phase sequenator with the 02 NV AC program
`(batch 1) or the 02RPTH program (batch 2), pro(cid:173)
`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
`
`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 µm 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 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(cid:173)
`tained from Peninsula Laboratories (San Carlos,
`CA) by custom synthesis (lot no.008802; peptide
`purity by amino acid analysis 720Jo). Before the
`peptide was used in physiological studies, it was
`purified to homogeneity by isocratic HPLC as
`described above. Sequence determination of syn(cid:173)
`thetic peptide confirmed the structure as being pro(cid:173)
`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 150Jo
`washed bovine erythrocytes, 0.1 OJo human serum
`albumin (reinst, trocken, Behringwerke Marburg,
`FRO), 5 or 7 mmol/1 glucose, 50Jo dextran T-70,
`aprotmm
`100000 KIU/1
`(TrasylolR, Bayer,
`Leverkusen, FRO), and 5 mmol/1 of a mixture of
`amino acids (Amodex Asa, Pharmacia, Uppsala,
`Sweden). The medium was gassed with 950Jo ox(cid:173)
`ygen and 50Jo CO2. Synthetic proglucagon 78-107
`was infused intra-arterially for 5 or 10 min periods
`in doses corresponding to final perfusate concen(cid:173)
`trations of 10- 10 - 10-s mol/1. Efffoent 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).
`
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`
`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 C18 TechoprepR. col(cid:173)
`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(cid:173)
`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(cid:173)
`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(cid:173)
`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 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(cid:173)
`tional processing [11).
`According to our results, the cleavage of pro(cid:173)
`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 mono basic proteolytic pro(cid:173)
`cessing is found for many precursors as recently
`reviewed [12). The natural porcine GLP-1, which
`according to the present study corresponds to pro(cid:173)
`glucagon 78-107 amide, is thus very similar to
`anglerfish, catfish and salmon GLP-1 ([13-15); see
`fig. I). 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 homology of the re(cid:173)
`maining peptide with glucagon is even more pro-
`
`s:eecies
`
`Residue no. in human :eroglucagon
`72
`His Asp Glu Phe Gl u Arg ~s Ala
`
`G:u G:y e eee e V:l e S~r ~
`
`If
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`
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`
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`
`Human
`ox
`Hamster
`Rat
`Guinea pig
`Anglerfish
`Salmon
`Catfish
`
`If
`
`If
`
`If
`
`If
`
`If
`
`If
`
`+
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`
`If Asp
`If Asp
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`
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`
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`
`If Tyr
`If Tvr
`
`If
`
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`
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`
`If Asn
`
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`
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`
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`
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`
`Soecies
`
`Residue no. in human ~roglucagon
`
`G Glu Gly Gln 0 Ala
`
`,,
`
`,,
`
`,,
`
`,,
`
`''
`
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`
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`
`Glu 8 if~ Ala 0 8 Val
`
`If
`
`If
`
`If
`
`If
`
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`
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`
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`
`Lys
`
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`
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`
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`
`Human
`ox
`Hamster
`Rat
`Guinea pig
`Anglerfish
`Salmon
`Catfish
`
`If
`
`If
`
`If
`
`If
`
`If Lys Asp
`If Gln
`If Gln
`
`If
`
`If
`
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`
`If
`
`fl
`
`If
`
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`
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`
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`
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`
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`
`110
`107
`Lys Gly Arg Gly Arg Arg
`"
`"
`"
`
`If
`
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`
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`
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`
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`
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`
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`
`If Val Asp Arg
`If Val Ser
`If Thr
`
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`
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`
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`
`If Lys Ala
`If Lys Ser
`If Lys Ser
`
`If
`
`If
`
`If
`
`If Gln Val
`If Ala +
`+
`If Gln Pro Lys Pro
`
`Fig.I. Sequences from 8 species of proglucagon regions containing glucagon-like peptide 1. Glucagon-like peptide-I
`containing sequences of proglucagon from all species whose proglucagon structure has been determined
`[l,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. Veriical 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
`
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`
`nmoLl
`1000
`
`500
`
`25
`
`51
`
`31
`
`101Fr1ct1on
`numb•r
`
`0
`
`nmoVI
`~50
`
`05
`
`10Kd
`
`21
`
`41 ,,act10
`n11mb1r
`
`Isolation of porcine natural GLP-1. The
`Fig.2.
`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 (430Jo ethanol in water). The dotted line shows
`UV-absorption at 280 nm.
`
`nounced (see fig.I). Since the N-terminal part of
`the glucagon molecule is essential for its biological
`activity [16), proglucagon 78-107 might be ex(cid:173)
`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 (1-36 amide, Peninsula) on
`blood glucose or insulin levels in fasting rabbits in
`what was considered as pharmacological doses.
`Schmidt et al. [18) found a weak insulinotropic ef(cid:173)
`(1-36 amide, code
`fect of synthetic GLP-1
`no.7166, Peninsula), but not of synthetic GLP-2
`(1-34, code no.7156) on isolated rat islet at rather
`high concentrations of GLP-1 and GLP-2 and in
`the presence of 10 mmol/1 glucose. We [19) recent(cid:173)
`ly demonstrated inhibition of gastric acid secretion
`in healthy volunteers during submaximal pen-
`
`172
`
`Table 1
`
`Sequence
`cycle
`
`Amino
`acid
`
`Amino acid
`in mam(cid:173)
`malian pro(cid:173)
`glucagon
`78-96
`
`Yield of amino
`acid phenyl(cid:173)
`thiohydantoin de(cid:173)
`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
`
`His
`Ala
`Glu
`Gly
`Thr
`Phe
`X
`Ser
`Asp
`Val
`X
`X
`Tyr
`Leu
`Glu
`X
`Gin
`X
`
`37
`108
`76
`81
`43
`80
`
`22
`a
`64
`
`46
`
`18
`105
`54
`
`64
`
`32
`47
`
`24
`14
`16
`
`16
`
`a The unmethylated PT A-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
`in batch 2.
`( - )
`methylated before
`identification
`Uncertain identification
`
`tagastrin stimulation when GLP-1 (1-36 amide)
`was infused at a rate of 400 ng/kg x h. This infu(cid:173)
`sion rate increased the plasma levels of im(cid:173)
`90
`to
`about
`munoreactive GLP-1
`from
`600 pmol/1. 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 10-s M either on the ex(cid:173)
`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 10- 10 mol/1 of
`natural GLP-1 (determined by radioimmunoassay)
`
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`and 7 mmol/1 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 10-9 mol/1 insulin secretion in(cid:173)
`creased from 32.0 ± I.I 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- 10 mol/1 in(cid:173)
`sulin secretion approximately doubled and increas(cid:173)
`ed more than 4-fold after 10-9 mol/1. Thus this
`peptide is at least as potent and effective as gastric
`inhibitory polypeptide (GIP), hitherto the most
`
`potent intestinal insulinotropic hormone [20]. At
`the perfusate the in(cid:173)
`in
`5.0 mmol/1 glucose
`sulinotropic effect was less conspicuous; but the
`relative increase in insulin secretion was approx(cid:173)
`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(cid:173)
`glucagon 78-107 in the medium. Trypsin-like en(cid:173)
`zymatic activity, which may be difficult to avoid in
`pancreatic tissue incubation studies, might be
`to the
`responsible for such conversion, due
`presence of a basic amino acid residue at position
`76.
`
`Proglucogon 78 - 107
`
`pmol / l
`
`3000
`
`2000
`
`~
`IIIJ
`
`0
`
`1 3 5
`minutes
`1 3 5
`Fig.3. Insulin secretion of the isolated perfused pig pancreas in response to infusion of synthetic proglucagon 78-107
`amide. The ordinate shows the concentration of insulin in the effluent. The final perfusate concentrations of
`proglucagon 78-107 amide are indicated in boxes. Results of a single representative experiment. Perfusate glucose
`concentration was 7 .0 mmol/1.
`
`173
`
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`
`(1983)
`
`(1981) Acta
`
`[6] Moody, A.J., Holst, J.J., Thim, L. and Jensen,
`S.L. (1981) Nature 289, 514-516.
`[7] Newgard, C. and Holst, J.J.
`Endocrinol. (Kbh.) 98, 564-572.
`[8] Hunkapillar, M.W., Hewick, R.M., Dreyer, W.J.
`and Hood, L.M. (1983) Methods Enzymol. 91,
`399-413.
`[9] Hunkapillar, M.W. and Hood, L.E.
`Methods Enzymol. 91, 493-496.
`[10) Jensen, S.L., Fahrenkrug, J., Holst, J.J., Kuhl,
`C., Nielsen, O.V. and Schaffalitzky de Muckadell,
`O.B. (1978) Am. J. Physiol. 235, E381-E386.
`(11] Bradbury, A.F., Finnie, M.D.A. and Smyth, D.G.
`(1982) Nature 298, 686-688.
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`
`pmol/MiN
`200
`
`100
`
`0
`
`5
`
`10
`
`5
`
`10
`
`Minutes
`
`Fig.4. Insulin secretion (pmol/min) of the isolated
`perfused pig pancreas in response to infusion of
`synthetic proglucagon 78-107 amide at 10- 10 and
`10-9 moll!. Glucose concentration in perfusate was
`7 mmol/1. Mean ± SE, n = 4.
`
`Previously, we found that the concentration of
`immunoreactive GLP-1 in plasma rose in response
`to a mixed meal in human volunteers (21].
`Lauritsen et al. (22] have shown that the lower in(cid:173)
`testine contributes
`significantly
`to
`the
`in(cid:173)
`sulinotropic effect of luminal versus intravenous
`glucose administration (the incretin effect). Our
`results suggest that proglucagon 78-107 may be
`the incretin of the lower gut.
`
`REFERENCES
`
`(1) Bell, G.I., Santerre, R.F. and Mullenbach, G.T.
`(1983) Nature 302, 716-718.
`(2) Heinrich, G., Gros, P., Lund, P.K. and Habener,
`J.F. (1984) J. Biol. Chem. 259, 14082-14087.
`(3) Holst, J.J. (1983) Gastroenterology 84, 1602-1613.
`(4) 0rskov, A.C., Holst,
`J.J., Knuhtsen, S.,
`Baldissera, F.G.A., Poulsen, S.S. and Nielsen,
`O.V. (1986) Endocrinology 119, 1467-1474.
`[5) Patzelt, C. and Schiltz, E. (1984) Proc. Natl. Acad.
`Sci. USA 81, 5007-5011.
`
`174
`
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