Biological Effects and Metabolic Rates of
`Glucagonlike Peptide-1 7-36 Amide and
`Glucagonlike Peptide-1 7-37 in Healthy
`Subjects Are Indistinguishable
`
`CATHRINE 0RSKOV, ANDRE WETTERGREN, AND JENS J. HOLST
`
`The biological effects and the metabolism of the
`intestinal hormone glucagonlike peptide-1 7-36 amide
`and glucagonlike peptide-1 7-37 were studied in
`normal healthy subjects. GLP-1 7-36 amide and GLP-1
`7-37 equipotently stimulated insulin secretion
`(integrated hormone response 0-60 min, 631 ± 2 11 vs.
`483 ± 1 77 pmol/h x L " 1 ) and C-peptide secretion
`(integrated hormone response 9064 ± 1804 vs.
`9954 ± 2031 pmol/h x L " 1 ) and equipotently lowered
`plasma glucose (integrated decrease 48.3 ± 5.7 vs.
`46.2 ± 8.4 mmol/h x L ' 1 ) and plasma glucagon
`(integrated decrease 80.4 ± 24.3 vs. 156.0 ± 34.6
`pmol/h x L"1). Both GLP-1 7-36 amide and GLP-1
`7-37 lowered the plasma concentration of free fatty
`acids significantly. The plasma half-lives of GLP-1
`7-36 amide and GLP-1 7-37 were 5.3 ± 0.4 vs.
`6.1 ± 0.8 min, and the metabolic clearance rates of the
`two peptides also were similar (14.6 ± 2.4 vs.
`12.2 ± 1 .0 pmol/kg x min). In conclusion,
`COOH-terminal amidation is neither important for the
`metabolism of GLP-1 nor for its effects on the
`endocrine pancreas. Diabetes 42:658-61,1993
`
`The posttranslational processing of PG in the
`
`small intestine gives rise to GLP-1, which has
`been shown to have profound effects on the
`endocrine pancreas in many species (1-5).
`GLP-1 is secreted in humans in response to physiological
`stimuli, e.g., a mixed meal (4). The exact structure of
`
`From the Department of Clinical Biochemistry and the Department of Gastro-
`intestinal Surgery C, Rigshospitalet; and the Institute of Medical Physiology C,
`Panum Institute, Copenhagen, Denmark.
`Address correspondence and reprint requests to Dr. Cathrine 0rskov,
`Department of Medical Anatomy B, Panum Institute, Blegdamsvej 3, DK-2200
`Copenhagen N, Denmark.
`Received for publication 29 October 1992 and accepted in revised form 23
`December 1992.
`GLP-1, glucagonlike peptide-1; PG, proglucagon; FFA, free fatty acid;
`NIDDM, non-insulin-dependent diabetes mellitus; RIA, radioimmunoassay;
`ANOVA, analysis of variance.
`
`GLP-1 has been a matter of some debate. A GLP-1
`molecule corresponding to PG 78-107 amide has been
`isolated from human small intestine and sequenced (6).
`This peptide has been designated GLP-1 7-36 amide.
`As in other peptides, the COOH-terminal amide is de-
`rived from a Gly residue positioned next to the COOH-
`terminus, catalyzed by the so-called amidation enzyme,
`PAM (7,8). Because COOH-terminal Gly-extended inter-
`mediates frequently are found together with the amidated
`peptides, it is conceivable that smaller amounts of a
`Gly-extended GLP-1 corresponding to PG 78-108, also
`called GLP-1 7-37, may be found in humans. Thus, in the
`rat intestine, 66% of the intestinal GLP-1 immunoreactivity
`has been shown by chromatography to correspond to
`GLP-1 7-36 amide and 33% to GLP-1 7-37 (9).
`Both GLP-1 7-36 amide and the Gly-extended GLP-1
`7-37 have been shown previously to stimulate insulin
`secretion in humans (2,4,10-13). Furthermore, GLP-1
`7-36 amide has been shown to inhibit glucagon secre-
`tion in humans (4,13), whereas the effect of GLP-1 7-37
`on glucagon is unknown. Because of the effects on
`insulin secretion and glucagon secretion, both peptides
`have been suggested as a possible treatment for NIDDM
`(11-14).
`A direct comparison of the biological effects and the
`metabolism of the two peptides, however, has not been
`made. We therefore studied the effects and the metabo-
`lism of GLP-1 7-36 amide and GLP-1 7-37 in healthy
`volunteer subjects.
`
`RESEARCH DESIGN AND METHODS
`The study was approved by the local ethical committee of
`Copenhagen on 19 February 1992. Six healthy volunteer
`subjects, 4 men and 2 women, participated in the study.
`They were 26 ± 2 yr of age and weighed 62 ± 2 kg
`(mean ± SE). Written informed consent was obtained
`from all subjects before the study.
`Peptides. GLP-1 7-36 amide and GLP-1 7-37 were
`purchased from Peninsula (code 7241 and 7123, Mer-
`
`658
`
`DIABETES, VOL. 42, MAY 1993
`
`MPI EXHIBIT 1052 PAGE 1
`
`MPI EXHIBIT 1052 PAGE 1
`
`

`

`seyside, St. Helens, UK). The peptides were dissolved in
`0.9% saline containing 1% human serum albumin (Novo
`Nordisk, Gentofte, Denmark), subjected to sterile filtra-
`tion, and kept at -20°C until use.
`The volunteer subjects were studied after an overnight
`fast. Cannulas (Venflon, Viggo Products, Helsingborg,
`Sweden) were placed in antecubital veins—one for
`infusion and one for blood sampling. The cannulas were
`kept patent with saline. The two peptides were infused on
`separate days at least 1 wk apart. After a 20-min basal
`period, a 60-min continuous infusion of either GLP-1
`7-36 amide or GLP-1 7-37, 1.5 pmol/kg per min, was
`started. Blood was drawn in the basal state, during the
`infusion, and up to 2 h after termination of the infusion into
`heparinized tubes, 15000 IE/L for insulin measurements
`and into tubes containing EDTA, 3.9 mM, and aprotinine
`500,000 KIU/L, for glucagon, GLP-1, C-peptide, FFAs,
`and plasma glucose measurements.
`RIAs. GLP-1 immunoreactivity was measured with either
`synthetic GLP-1 7-36 amide or GLP-1 7-37 as stan-
`dards (Peninsula), 125l-labeled GLP-1 7-36 amide, and
`antiserum 2135 as described previously (15). Pancreatic
`glucagon was measured using antiserum 4305, which
`recognizes the COOH-terminus of glucagon, purified
`porcine glucagon (Novo, Bagsvaerd, Denmark), and 1 2 5I-
`labeled glucagon (gift from Novo) as described previ-
`ously (16). Insulin was measured as described previously
`(17). C-peptide was determined with a commercial RIA
`kit (C-peptide kit, Novo Nordisk a/s, Bagsvaerd, Den-
`mark). FFAs were determined with a commercial kit (Nefa
`C kit, Wako Chemicals GmbH, Germany). Plasma glu-
`cose was determined by the glucose oxidase method.
`The plasma half-lives of the two GLPs were calculated
`after linear transformation of the elimination curves in a
`semilogarithmic system. The metabolic clearance rates
`were calculated as metabolic clearance rate = infusion
`rate (pmol/kg x min)/increase in GLP-1 concentration
`above basal (pmol/ml).
`Statistical analysis. Data are presented as means ± SE
`for n = 6 unless otherwise stated. The significance of
`differences was evaluated by Wilcoxon's matched-pairs
`signed-ranks test for paired data. P < 0.05 was consid-
`ered significant. Integrated incremental responses were
`determined by calculation of the area under the curve
`with the trapezoidal rule. ANOVA followed by Newman-
`Keul's Multiple Range test was used for evaluation of
`changes as function of time.
`
`RESULTS
`The two peptides were infused in approximately equimo-
`lar amounts as estimated by RIA of infusion samples
`TABLE 1
`Comparison of integrated
`
`C. ORSKOV. A. WETTERGREN. AND J.J. HOLST
`
`Apmol/I
`200
`
`100
`
`0 L
`
`-20 0
`
`60
`
`120
`
`180 min
`
`FIG. 1. Incremental plasma concentrations of GLP-1 during and after
`Infusion of synthetic GLP-1 7-36 amide (
`) or GLP-1 7-37
`(
`). Incremental concentrations (pM) are plotted against time
`
`(232 ± 21 [GLP-1 7-36 amide] vs. 193 ± 16 [GLP-1
`7-37] nM, P > 0.05), which resulted in similar elevations
`of the plasma concentration of the two peptides (Fig. 1).
`The biological effects of the peptides also were extremely
`similar (Fig. 2, Table 1). Both GLP-1 7-36 and GLP-1
`7-37 significantly stimulated insulin and C-peptide se-
`cretion and significantly decreased plasma glucose and
`the glucagon concentrations (Fig. 2). Neither the inte-
`grated insulin responses nor the integrated C-peptide
`responses (Table 1) to the two different synthetic GLP-1
`molecules differed significantly. Likewise, the effect on
`plasma glucose and glucagon (Table 1) was not statis-
`tically different. Both GLP-1 7-36 amide and GLP-1 7-37
`significantly lowered the concentrations of FFAs (Fig. 3).
`The half-lives of GLP-1 7-36 amide and GLP-1 7-37
`were 5.3 ± 0.4 vs. 6.1 ± 0.8 min (P> 0.05). The meta-
`bolic clearance rates of GLP-1 7-36 amide and GLP-1
`7-37 were 14.6 ± 2.4 and 12.2 ± 1.0 pmol/(kg x min
`(P>0.05).
`
`DISCUSSION
`In this study we found that both the metabolic rate and
`the biological effects of GLP-1 7-36 amide and GLP-1
`7-37 in normal subjects are identical. The question of
`whether human intestinal GLP-1 is amidated or not has
`
`incremental hormone responses (time 0 to 60 min)
`
`to GLP-1 7-36 NH2 and GLP-1 7-37
`
`Glucose (mmol/h x 1~1)
`Insulin (pmol/h x 1~1)
`C-peptide (pmol/h x 1~1)
`Glucagon (pmol/h x 1~1)
`
`Data are means ± SE. n = 6.
`*P>0.05.
`
`DIABETES, VOL. 42, MAY 1993
`
`GLP-1 7-36 NH2
`
`48.3 ± 5.7
`631 ±211
`9064 ± 1804
`80.4 ± 24.3
`
`GLP-1 7-37
`
`46.2 ± 8.4
`483 ± 177
`9954 ± 2031
`156.0 ±34.6
`
`P values*
`
`NS
`NS
`NS
`NS
`
`659
`
`MPI EXHIBIT 1052 PAGE 2
`
`MPI EXHIBIT 1052 PAGE 2
`
`

`

`mmol/l
`
`100
`
`50
`
`60
`
`120
`
`180 min
`
`FIG. 3. Plasma concentrations of FFAs before, during, and after
`intravenous Infusion of either GLP-1 7-36 amide (
`) or GLP-1
`7-37 (
`). Mean concentrations (mM ± SE) are plotted against
`time (n = 6).
`
`been a matter of debate. The background for this is the
`structure of the GLP-1 precursor, PG. In PG, the GLP-1
`sequence is flanked at its COOH-terminus by a pair of
`basic amino acids, potential posttranslational cleavage
`sites (1). However, the dibasic amino acids are preceded
`by the amino acid, Gly, a known amidation signal (18). It
`has been shown previously that amidation of peptides
`proceeds via Gly-extended precursors (7). Furthermore,
`enzymes capable of converting Gly-extended peptides
`to amidated products have been identified (8,18,19). For
`most peptide systems in which amidation occurs, small
`amounts of Gly-extended precursors remain unproc-
`essed (20). For instance, 5-10% of the gastrin molecules
`stored in the porcine antrum have been shown to be Gly
`extended (21). Mojsov et al. (3) reported that up to 33%
`of the GLP-1 immunoreactivity in a rat small intestine
`chromatographically coeluted with Gly-extended GLP-1.
`It is not yet known, however, how much Gly-extended
`GLP-1 is produced in the human small intestine. Presum-
`ably, both peptides are present with a preponderance of
`the amidated form.
`The amounts of GLP-1 infused in the healthy subjects
`caused an increase in the plasma concentration of —150
`pM. This increase is three- to fourfold larger than the
`increase in plasma concentration seen after a mixed
`meal in normal subjects (4,12). However, we chose to
`infuse this slightly supraphysiological dose to facilitate
`the determination of the plasma half-lives and the meta-
`bolic clearance rates of the two peptides.
`The effects of GLP-1 7-36 amide and GLP-1 7-37 on
`
`C-peptide
`pmol/l
`
`Insulin
`pmol/l
`
`Glucagon
`pmol/l
`
`EFFECTS OF GLP-1 IN HUMANS
`
`6 5 4 3
`
`B
`
`800
`
`400
`
`60
`
`30
`
`0 L
`
`32 r
`
`16
`
`-20 0
`
`60
`
`120
`
`180 min
`
`FIG. 2. Plasma concentrations of glucose (A), C-peptide (B), insulin
`(C), and glucagon (0) before, during, and after Intravenous Infusion of
`either GLP-1 7-36 amide (
`) or GLP-1 7-37 (
`). Mean
`concentrations (mM ± SE or pM ± SE) are plotted against time (n = 6).
`The framed area shows the infusion period.
`
`660
`
`DIABETES, VOL. 42, MAY 1993
`
`MPI EXHIBIT 1052 PAGE 3
`
`MPI EXHIBIT 1052 PAGE 3
`
`

`

`insulin secretion, C-peptide, and glucagon were identi-
`cal. The inhibitory effect of GLP-1 7-37 on glucagon
`secretion has not been reported previously in humans.
`Both GLP-1 7-36 amide and GLP-1 7-37 decreased
`the plasma glucose concentration significantly and with
`equal strength. We noted that the plasma glucose did not
`drop below an average of 3.3 mM in spite of continued
`infusion of supraphysiological amounts. The reason for
`this seems to be that the effect of GLP-1 on the secretion
`of pancreatic glucoregulatory hormones is glucose de-
`pendent. The glucose dependency of GLP-1 7-37 in
`humans has been described previously by Andersen et
`al. (22) and Nathan et al. (11). Thus, the insulinotropic
`effect of GLP-1 is potentiated at increasing blood glucose
`levels, whereas the glucagon inhibitory effect is more
`marked at lower blood glucose levels. If GLP-1 is con-
`sidered as a possible future treatment for diabetes, this is
`interesting, because it suggests that the risk that GLP-1
`administration leads to dangerous hypoglycemia is likely
`to be extremely small (10-13).
`Both GLP-1 7-36 amide and GLP-1 7-37 decreased
`FFA levels significantly. This has not been reported
`previously. The basal levels of FFAs, however, were
`somewhat different on the two study days, making it
`difficult to directly compare the responses with the two
`GLP-1 molecules. Faulkner et al. (23) have shown previ-
`ously that GLP-1 7-36 amide lowered FFA concentra-
`tions in fasted sheep. Furthermore, Merida et al. (24)
`have reported the presence of specific GLP-1 7-36
`amide receptors on isolated human fat cell membranes.
`In this study, we found that the half-life of GLP-1 7-36
`amide was 5.3 ± 0.4 min, which is similar to the half-life
`of 4 min reported previously by Kreymann et al.(4) and
`similar to the half-life of the related hormone glucagon.
`The half-life of GLP-1 7-37 was 6.1 ± 0.8 min, which is
`very similar to that of GLP-1 7-36 amide. The half-life of
`GLP-1 7-37 in humans has not been reported previously.
`The metabolic clearance rate of GLP-1 7-36 amide was
`14.6 ± 2.4 ml/(kg x min), which is consistent with the
`value of 12 ml/(kg x min) reported previously by Krey-
`mann et al. (4) and with the value of 13 ± 3 ml/(kg x min
`by Scholdager et al. (25). A similar clearance rate for
`GLP-1 7-37 was found in this study (12.2 ± 1.0 ml/
`(kg x min). The clearance rate of GLP-1 7-37 has not
`been reported previously.
`In conclusion, the COOH-terminal amidation of GLP-1
`7-36 amide is neither crucial for the effect nor for the
`metabolism of GLP-1 in humans.
`
`ACKNOWLEDGMENTS
`This study was supported by the Danish Medical Re-
`search Council.
`We thank Lisbet Mardrup for excellent technical assis-
`tance.
`
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