Diabetologia (1996) 39: 1546–1553
`
`(cid:211) Springer-Verlag 1996
`
`Effects of subcutaneous glucagon-like peptide 1
`(GLP-1 [7–36 amide]) in patients with NIDDM
`
`M.A. Nauck1, D. Wollschla¨ger2, J. Werner2, J. J. Holst3, C. Ørskov3, W. Creutzfeldt4, B. Willms2
`1 Department of Medicine, Ruhr-University Bochum, Knappschafts-Krankenhaus, Bochum, Germany
`2 Fachklinik fu¨ r Diabetes und Stoffwechselkrankheiten, Bad Lauterberg, Germany
`3 Departments of Anatomy and Physiology, Panum Institute, University of Copenhagen, Denmark
`4 Division of Gastroenterology and Endocrinology, Department of Medicine, Georg-August-University, Go¨ ttingen, Germany
`
`Summary Intravenous glucagon-like peptide (GLP)-
`1 [7–36 amide] can normalize plasma glucose in non-
`insulin-dependent diabetic (NIDDM) patients. Since
`this is no form for routine therapeutic administration,
`effects of subcutaneous GLP-1 at a high dose (1.5
`nmol/kg body weight) were examined. Three groups
`of 8, 9 and 7 patients (61 – 7, 61 – 9, 50 – 11 years;
`BMI 29.5 – 2.5, 26.1 – 2.3, 28.0 – 4.2 kg/m2; HbA1 c
`11.3 – 1.5, 9.9 – 1.0, 10.6 – 0.7 %) were examined: af-
`ter a single subcutaneous injection of 1.5 nmol/kg
`GLP [7–36 amide]; after repeated subcutaneous in-
`jections (0 and 120 min) in fasting patients; after a
`single, subcutaneous injection 30 min before a liquid
`test meal (amino acids 8 %, and sucrose 50 g in
`400 ml), all compared with a placebo. Glucose (glu-
`cose oxidase), insulin, C-peptide, GLP-1 and gluca-
`gon (specific immunoassays) were measured. Gastric
`emptying was assessed with the indicator-dilution
`method and phenol red. Repeated measures ANO-
`VA was used for statistical analysis. GLP-1 injection
`led to a short-lived increment in GLP-1 concentra-
`tions (peak at 30–60 min, then return to basal levels
`after 90–120 min). Each GLP-1 injection stimulated
`insulin (insulin, C-peptide, p < 0.0001, respectively)
`and inhibited glucagon secretion (p < 0.0001). In fast-
`ing patients the repeated administration of GLP-1
`
`normalized plasma glucose (5.8 –
`0.4 mmol/l after
`240 min vs 8.2 – 0.7 mmol/l after a single dose,
`p = 0.0065). With the meal, subcutaneous GLP-1 led
`to a complete cessation of gastric emptying for 30–
`45 min (p < 0.0001 statistically different from pla-
`cebo) followed by emptying at a normal rate. As a
`consequence,
`integrated incremental glucose re-
`sponses were reduced by 40 % (p = 0.051). In conclu-
`sion, subcutaneous GLP-1 [7–36 amide] has similar
`effects in NIDDM patients as an intravenous infu-
`sion. Preparations with retarded release of GLP-1
`would appear more suitable for therapeutic purposes
`because elevation of GLP-1 concentrations for 4
`rather than 2 h (repeated doses) normalized fasting
`plasma glucose better. In the short term, there ap-
`pears to be no tachyphylaxis, since insulin stimulation
`and glucagon suppression were similar upon repeated
`administrations of GLP-1 [7–36 amide]. It may be
`easier to influence fasting hyperglycaemia by GLP-1
`than to reduce meal-related increments in glycaemia.
`[Diabetologia (1996) 39: 1546–1553]
`
`Keywords GLP-1 [7–36 amide], incretin, insulin, gluca-
`gon, pharmacokinetics.
`
`Corresponding author: Priv.-Doz. Dr. med. M. Nauck, Depart-
`ment of Medicine, Ruhr-University Bochum, Knappschafts-
`Krankenhaus, In der Schornau 23–25, D-44 892 Bochum, Ger-
`many
`Abbreviations: BMI, Body mass index; EDTA, ethylene di-
`amine tetra-acetic acid; GIP, gastric inhibitory polypeptide;
`GLP-1, glucagon-like peptide-1; RM-ANOVA, repeated mea-
`sures analysis of variance; NIDDM, non-insulin-dependent di-
`abetes mellitus; IR, immunoreactive.
`
`Glucagon-like peptide 1 (GLP-1) [7–36 amide] is an
`insulinotropic hormone secreted from enterogluca-
`gon-producing L cells in the lower gut, i. e. the ileum
`and colon/rectum [1, 2]. GLP-1 [7–36 amide], to-
`gether with gastric inhibitory polypeptide (GIP)
`from the upper gut, acts as a physiological incretin
`hormone [3, 4]. In pharmacological concentrations,
`exogenous GLP-1 [7–36 amide or 7–37] also raised
`insulin and lowered glucagon concentrations in
`
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`M. A. Nauck et al.: Subcutaneous GLP-1 [7–36 amide] effects in NIDDM
`
`1547
`
`Table 1. Characteristics of NIDDM patients studied
`Parameter
`Study A
`Fasting patients
`Single dose
`5/3
`61 – 7
`29.5 – 2.5
`9 – 3
`11.3 – 1.5
`1429 – 262
`9.6 – 2.5
`1/7
`1/7
`162 – 28
`94 – 16
`
`GLP-1 [7–36 amide] s. c.:
`Sex (male/female)
`Age (years)
`Body mass index (kg/m2)
`Diabetes duration (years)
`HbA1c (%)
`Diet (kCal/day)
`Glibenclamide (mg/day)
`Metformin (yes/no)
`Acarbose (yes/no)
`RRsystolic (mm Hg)
`RRdiastolic (mm Hg)
`Data are mean – SD
`
`Study B
`Fasting patients
`Repeated dose
`4/5
`61 – 9
`26.1 – 2.3
`11 – 7
`9.9 – 1.0
`1538 – 348
`10.5 – 0
`2/7
`0/9
`138 – 19
`72 – 13
`
`Study C
`Liquid meal
`Single dose
`3/4
`50 – 11
`28.0 – 4.2
`10 – 4
`10.6 – 0.7
`1416 – 253
`10.5 – 0
`6/1
`0/7
`136 – 12
`82 – 11
`
`Significance
`ANOVA
`(p -value)
`0.69
`0.03
`0.096
`0.59
`0.054
`0.66
`0.39
`0.007
`0.35
`0.043
`0.012
`
`non-insulin-dependent diabetic (NIDDM) patients
`[5, 6]. By these mechanisms, plasma glucose was nor-
`malized within 3–4 h by i. v. GLP-1 [7–36 amide] in
`NIDDM patients with secondary failure of sulpho-
`nylurea treatment
`[7]. Therefore, GLP-1 [7–36
`amide] or GLP-1 [7–37] (which has an identical ac-
`tion profile in rats [8], normal humans [9] and
`NIDDM patients [10]) has been suggested for use in
`the therapy of NIDDM patients [5–7, 10, 11]. How-
`ever, a mode of administration has to be sought that
`both makes use of the potential to normalize glycae-
`mia in NIDDM patients [7, 10] and that will be ac-
`ceptable when compared with other current therapy.
`One obvious way is the s. c. administration of this
`peptide hormone. Limited experience using small do-
`ses of GLP-1 [7–36 amide] have been reported by
`Gutniak et al. [12]. However, they only used it to re-
`duce postprandial glycaemic excursions. GLP-1 [7–
`36 amide] was injected shortly before the ingestion
`of a meal, and the effects noted were smaller in com-
`parison to the normalization of fasting hyperglycae-
`mia that can be achieved by i. v. GLP-1 [7–36 amide]
`in NIDDM patients [6, 10]. Since i. v. GLP-1 [7–36
`amide] profoundly influences fasting hyperglycaemia
`in NIDDM patients [7, 10], it was the aim to also ex-
`amine the effect of s. c. GLP-1 in fasting patients,
`and to compare it to a preprandial administration.
`The amount administered in the present study was
`the maximum dose that was free of severe side-effects
`in young, healthy volunteers [13]. Preliminary results
`have been communicated in abstract form [14].
`
`Subjects, materials and methods
`
`Study protocol. The study protocols were approved by the eth-
`ics committee of the medical faculty of the Georg-August Uni-
`versity, Go¨ ttingen, prior to the study. Written, informed con-
`sent was obtained from all participants.
`
`Subjects. Three groups of NIDDM patients were studied, hav-
`ing been admitted to a specialized diabetes clinic because of
`
`unsatisfactory metabolic control. Their characteristics are
`shown in Table 1. All were treated with diet and oral agents
`(sulphonylurea in all, plus acarbose or metformin in some).
`Diabetes, on average, had been diagnosed approximately
`10 years earlier. Metabolic control at the time of the study, as
`indicated by HbA1 c values of 10–11 % (normal: 4.3–6.1 %),
`was unsatisfactory. Most patients were slightly obese.
`All patients were studied on two occasions; in randomized
`order, in a single-blind fashion, placebo (0.9 % NaCl with 1 %
`human serum albumin) or GLP-1 [7–36 amide] in a dose of
`1.5 nmol/kg body weight was administered in the morning af-
`ter an overnight fast as a single or repeated dose. Anti-dia-
`betic medication (Table 1) was given until the night before
`the first experiment. Between the experiments, 1 day with a
`regular eating and treatment schedule (including all drugs)
`was allowed.
`Three protocols were compared: Study A examined the ef-
`fect of a single s. c. injection of GLP-1 [7–36 amide] (adminis-
`tered at time 0 min) in fasting NIDDM patients followed for
`240 min. Study B examined the effect of a repeated s. c. injec-
`tion of GLP-1 [7–36 amide] (administered at time 0 and
`120 min) in fasting NIDDM patients followed for 240 min.
`Study C examined effects of a preprandial single s. c. injection
`of GLP-1 [7–36 amide] (administered at time –30 min) in
`NIDDM patients. At time 0 min, a liquid mixed meal made
`up of a commercially available amino acid solution (Amino-
`steril N-Hepa 8 %; Fresenius AG, Bad Homburg, Germany)
`and sucrose (50 g per 400 ml) was administered via a nasogas-
`tric tube. Results were observed over 240 min.
`
`Peptides. Synthetic GLP-1 [7–36 amide] was purchased from
`Saxon Biochemicals GmbH, Hannover, Germany (PGAS
`242). The peptide was dissolved in 0.9 % NaCl containing 1 %
`human serum albumin (Merieux, Norderstedt, Germany), fil-
`tered through 0.2 mm nitrocellulose filters (Millipore, Bed-
`ford, Mass., USA) and stored frozen at –30(cid:176) C as previously
`described [4, 5, 7]. Net peptide content rather than gross
`weight was used for dose calculations. High performance liq-
`uid chromatography profiles (provided by the manufacturer)
`showed that the preparation was more than 99 % pure (single
`peak coeluting with appropriate standards). Samples were
`analysed for bacterial growth (standard culture techniques)
`and for pyrogens (Limulus amebocyte lysate endo-LAL,
`Chromogenix AB, Mo¨ lndal, Sweden). No bacterial contami-
`nation was detected. Endotoxin concentrations in the GLP-1
`[7–36 amide] stem solutions were always less than 0.03 endo-
`toxin units (EU)/ml.
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`1548
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`M. A. Nauck et al.: Subcutaneous GLP-1 [7–36 amide] effects in NIDDM
`
`Experimental procedures. One forearm vein was punctured
`with a teflon cannula (Moskito 123, 18 gauge; Vygon, Aachen,
`Germany), and kept patent using 0.9 % NaCl.
`In experiments with fasting NIDDM patients, after drawing
`basal blood specimens, at 0 min, GLP-1 [7–36 amide] was ad-
`ministered s. c. into the periumbilical region. The injected vol-
`ume was 1 ml per 85 kg body weight. In study B, at 120 min
`the subcutaneous injection of GLP-1 [7–36 amide] was re-
`peated.
`
`Blood specimens. Blood was drawn into heparinized tubes (im-
`munoreactive [IR] insulin and C-peptide measurements). A
`sample was stored in NaF (Microvette CB 300; Sarstedt, Nu¨ m-
`brecht, Germany) for the measurement of glucose. For gluca-
`gon and GLP-1 [7–36 amide] measurements, blood was drawn
`into tubes containing ethylene diamine tetra-acetic acid
`(EDTA) and aprotinin (Trasylol; 20 000 kallikrein inhibitor
`units/ml, 200 ml per 10 ml blood; Bayer AG, Leverkusen, Ger-
`many). After centrifugation, plasma for hormone analyses
`was kept frozen at –30(cid:176) C.
`
`Gastric emptying. In study C, before the start of the experi-
`ments, a nasogastric tube (Freka-Erna¨hrungssonde, 120 cm,
`CH 12, Fresenius AG) was placed and tape-fixed with the tip
`approximately 55 cm from the nostrils. Gastric juice was aspi-
`rated and an acid pH was ascertained using pH-sensitive lack-
`mus paper. The gastric lumen was washed with 100 ml tap wa-
`ter warmed to 37(cid:176) C. If instilled water could not be completely
`aspirated, the position of the tube was adjusted to allow a
`near-complete aspiration of instilled fluid. The patients were
`in a semi-recumbent position, with the upper body 45(cid:176) upright.
`At –30 min, GLP-1 [7–36 amide] was administered s. c., and at
`0 min, the liquid meal (warmed to 37(cid:176) C) was instilled into the
`stomach. Previous studies had indicated peak plasma concen-
`trations around 30 min after s. c. injection of GLP-1 [7–36
`amide] [13]. The meal consisted of 32 g mixed amino acids
`(131 kCal = 40 % of total caloric content) and 50 g sucrose
`(196 kCal = 60 %). Total energy content was 327 kCal (energy
`density 0.82 kCal/ml) [15, 16].
`Gastric emptying was determined exactly as in a previous
`study testing intravenously infused GLP-1 [7–36 amide] in a
`similar group of NIDDM patients [15], by a double-sampling
`dye dilution technique using phenol red (Merck AG, Darms-
`tadt, Germany), according to George [17], with modifications
`introduced to reduce the measurement error by Hurwitz et al.
`[18]. In principle, at all time-points chosen to measure gastric
`volume, a known amount of the non-absorbable phenol red
`dye was added to the translucent test meal in a volume of 5 to
`15 ml. After thorough mixing with gastric contents for approx-
`imately 2 min, a gastric sample was drawn, and the resulting
`step-up in phenol red concentrations was determined photo-
`metrically. Increasing amounts of phenol red were used as the
`experiments proceeded to obtain clearly measurable incre-
`ments in optical density also in the presence of previously in-
`stilled phenol red [18]. In vitro, this method measured gastric
`volume with an accuracy of less than 6 % (coefficient of varia-
`tion).
`
`Laboratory determinations. Glucose was measured using a glu-
`cose oxidase method with a Glucose Analyser 2 (Beckman In-
`struments, Munich, Germany). Plasma IR-insulin and C-pep-
`tide were determined using commercial immunoassay kits. In-
`sulin was measured using an insulin microparticle enzyme im-
`munoassay (MEIA) IMx Insulin, Abbott Laboratories, Wies-
`baden, Germany, which shows a correlation coefficient 0.982
`vs RIA 100; Pharmacia, Freiburg, Germany (the assay used in
`our previous study [15]). Intra-assay coefficients of variation
`
`were less than 4.0 %. C-peptide was measured using C-peptide
`antibody-coated microtitre wells (C-peptide MTPL EIA) from
`DRG Instruments GmbH, (Marburg, Germany). Human insu-
`lin and C-peptide were used as standard.
`IR-GLP-1 was determined in ethanol-extracted plasma as
`previously described [19], using antiserum 89 390 (final dilu-
`tion 1:150 000) and synthetic GLP-1 [7–36 amide] for tracer
`preparation and as standard. Recovery of GLP-1 [7–36 amide]
`standards after alcohol extraction was 75 – 8 %. The experi-
`mental detection limit (2 standard deviations over samples
`not containing GLP-1 [7–36 amide]) was less than 5 pmol/l.
`Antiserum 89 390 binds proglucagon-derived peptides con-
`taining the amidated carboxy-terminus of GLP-1 [7–36 amide],
`thereby being relatively specific for GLP-1 [7–36 amide] [20].
`Plasma samples with expected high concentrations were di-
`luted 1:10 with assay buffer before analysis.
`Pancreatic glucagon was assayed in ethanol-extracted plas-
`ma using antibody 4305 [21]. GIP was determined using antise-
`rum R 65 and synthetic human GIP for the preparation of stan-
`dards and 125GIP tracer (purified by HPLC) as described by
`Krarup et al. [22]. GIP was not measured in the experiments
`with fasting patients, since GLP-1 [7–36 amide] does not stimu-
`late GIP release in humans [4, 5].
`Each patient’s set of plasma samples was assayed at the
`same time to avoid errors due to inter-assay variation.
`
`Symptoms. During the experiments, the volunteers were ob-
`served and frequently asked about their state of well-being.
`Their answers were recorded using an open questionnaire.
`
`Statistical analysis
`
`Subject characteristics are reported as mean – SD, experimen-
`tal results are reported as mean – SEM. Integration was car-
`ried out according to the trapezoidal rule, separately calculat-
`ing increments over and decrements below mean baseline val-
`ues. Metabolic clearance rates were calculated as the dose of
`s. c. GLP-1 [7–36 amide], divided by the integrated incremental
`response. Significances of differences were tested using re-
`peated measurement analysis of variance (RM-ANOVA;
`NCSS Version 5.01, Kaysville, Utah, USA). If a significant in-
`teraction of treatment and time was documented (p < 0.05),
`values at single time points were compared by Student’s t-test
`(paired analyses; GLP-1 [7–36 amide] vs placebo). For the
`analysis of time courses one-way ANOVA was used, followed
`by t-tests (vs mean basal values), if indicated. For contingency
`table analysis, a chi square-test was used. A two-sided p-value
`less than 0.05 was taken to indicate significant differences.
`
`Results
`
`All patients were hyperglycaemic at the start of the
`experiments (mean plasma glucose concentrations
`over 11 mmol/l; Figs. 1 and 2).
`
`Study A. A single s. c. administration of GLP-1 [7–36
`amide] reduced plasma glucose concentrations by ap-
`proximately 2–3 mmol/l (Fig. 1A), mainly during the
`initial 120 min, but
`the effect was maintained
`throughout the 240-min period. Insulin (Fig. 1B) and
`C-peptide (Fig. 1C) were stimulated (peak 30 min af-
`ter GLP-1 [7–36 amide] administration) significantly,
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`M. A. Nauck et al.: Subcutaneous GLP-1 [7–36 amide] effects in NIDDM
`
`1549
`
`Fig. 1. A–H. Effects of single (left panels, A–D) and repeated
`(right panels, E-H) s.c administrations of GLP-1 [7–36 amide]
`(1.5 nmol/kg body weight) on plasma glucose (A, E), insulin
`(B, F) C-peptide (C, G), and glucagon (D, H) concentrations
`in NIDDM patients. Arrows indicate time point of GLP-1 [7–
`36 amide] administration. Experiments with GLP-1 [7–36
`amide] (U) or placebo (k). P-values indicate significance of in-
`teraction of experiment (GLP-1 [7–36 amide] vs placebo) and
`time. *: Differences at specific time points (t-test, p < 0.05)
`
`but transiently, and returned to baseline values after
`90–120 (insulin) and 150–180 (C-peptide) min.
`GLP-1 [7–36 amide] concentrations reached peak
`values of around 400–550 pmol/l 30–60 min after s. c.
`administrations of 1.5 nmol/kg body weight GLP-1
`[7–36 amide] (Fig. 2A) and returned to baseline val-
`ues within 120–180 min after injection.
`Glucagon concentrations were transiently, but sig-
`nificantly (p < 0.0001) suppressed during 90 min after
`GLP-1 [7–36 amide] administration (Fig. 1D).
`
`Study B. In contrast to the single s. c. administration
`of GLP-1 [7–36 amide], the repeated administration
`(two doses administered at 0 and 120 min) resulted
`in a step-wise normalization of fasting glycaemia in
`NIDDM patients (Fig. 1E; p < 0.0001 vs placebo).
`
`Fig. 2. A–C. Effects of single (A) and repeated (B) s. c. admin-
`istrations of GLP-1 [7–36 amide] (1.5 nmol/kg body weight) in
`the fasting state, and of single (C) s. c. administrations of
`GLP-1 [7–36 amide] administered before a liquid test meal on
`plasma GLP-1 [7–36 amide] concentrations in NIDDM pa-
`tients. Arrows indicate time point of GLP-1 [7–36 amide] ad-
`ministration and of the intragastric installation of the meal. Ex-
`periments with GLP-1 [7–36 amide] (U) or placebo (k). P-val-
`ues indicate significance for the interaction of experiment
`(GLP-1 [7–36 amide] vs placebo) and time. *: Differences at
`specific time points (t-test, p < 0.05)
`
`The plasma glucose reached at the end of the experi-
`ment was significantly lower (5.8 – 0.4 mmol/l) than
`after the single dose (8.2 – 0.7 mmol/l; p = 0.0065, t-
`test), while there was no significant difference in the
`integrated decremental response.
`Insulin and C-peptide showed a second increment
`after the administration of GLP-1 [7–36 amide] at
`120 min (Fig. 1F and G), which made the overall insu-
`lin and C-peptide responses greater (p < 0.05 for
`both) than in the experiment with single doses of
`GLP-1 [7–36 amide] (Fig. 1B and C). The responses
`of insulin and C-peptide to the second injection of
`GLP-1 [7–36 amide] were similar in magnitude to
`those of the first or single administration (Fig. 1F and
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`M. A. Nauck et al.: Subcutaneous GLP-1 [7–36 amide] effects in NIDDM
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`Fig. 4. Effects of single s. c. administrations of GLP-1 [7–36
`amide] (1.5 nmol/kg body weight) on gastric emptying rates af-
`ter the intragastric instillation of a liquid test meal in NIDDM
`patients. Arrow indicates time point of GLP-1 [7–36 amide] ad-
`ministration. Experiments with GLP-1 [7–36 amide] (U) or pla-
`cebo (k). P-values indicate the significance for the interaction
`of experiment (GLP-1 [7–36 amide] vs placebo) and time. *:
`Differences at specific time points (t-test, p < 0.05)
`
`concentrations were reduced significantly (p < 0.0001)
`30 min after each administration (Fig. 1H). The reduc-
`tion in glucagon was similar after the first and second
`injection (p = 0.51). However, the overall reduction
`in glucagon (integrated decremental responses) was
`not significantly different from experiments with a sin-
`gle dose of GLP-1 [7–36 amide].
`
`Study C. Intragastric instillation of a liquid mixed meal
`containing 50 g sucrose, together with the administra-
`tion of placebo, raised mean plasma glucose concen-
`trations to over 15 mmol/l (Fig. 3A). However, with
`GLP-1 [7–36 amide] administered s. c. 30 min before
`the meal, plasma glucose values were lowered rather
`than elevated during the initial 30–45 min after the
`meal, with a later rise that was attenuated in compari-
`son with placebo studies (Fig. 3A). The rise in glycae-
`mia over basal values (integrated incremental re-
`sponses), however, was only reduced from 529.2 –
`54.3 mmol (cid:215) l–1 (cid:215) min (placebo) to 318.6 – 106.7 (GLP-
`1 [7–36 amide], which was only of borderline signifi-
`cance (p = 0.051). There was a short increment in insu-
`lin (Fig. 3B) and C-peptide (Fig. 3C) plasma concen-
`trations during the initial 30 min after GLP-1 [7–36
`amide] administration (i. e. before the meal was giv-
`en), but later insulin and C-peptide curves were simi-
`lar to those with the placebo. Due to the initial peak
`in insulin and C-peptide (Fig. 3B and C), the overall in-
`tegrated incremental insulin (21.6 – 4.7 vs 15.9 – 3.8
`nmol (cid:215) l–1 (cid:215) min, p = 0.0032) and C-peptide (139.9 –
`20.6 vs 97.9 – 20.1 nmol (cid:215) l–1 (cid:215) min, p = 0.0054) re-
`sponse was greater with s.c GLP-1 [7–36 amide].
`The time course of GLP-1 [7–36 amide] plasma
`concentrations (Fig. 2C) after s.c injection was similar
`to that described in fasting patients (Fig. 2 A and B,
`Table 2). There was a significant increment in GLP-1
`[7–36 amide] concentrations after the liquid meal
`
`Fig. 3. A–D. Effects of a single s. c. administration of GLP-1
`[7–36 amide] (1.5 nmol/kg body weight) on plasma glucose
`(A), insulin (B), C-peptide (C), and glucagon (D) concentra-
`tions in NIDDM patients before and after feeding a liquid
`test meal. Arrows indicate the time point of GLP-1 [7–36
`amide] administration and of the intragastric instillation of
`the meal. Experiments with GLP-1 [7–36 amide] (U) or placebo
`(k). P-values indicate significance for the interaction of experi-
`ment (GLP-1 [7–36 amide] vs placebo) and time. *: Differ-
`ences at specific time points (t-test, p < 0.05)
`
`G; p = 0.99 and 0.48 for integrated incremental insu-
`lin and C-peptide responses, respectively), although
`plasma glucose concentrations had already been low-
`ered in response to the first injection (Fig. 1E).
`GLP-1 [7–36 amide] plasma values showed a sec-
`ond peak of similar magnitude (Fig. 2B), and glucagon
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`M. A. Nauck et al.: Subcutaneous GLP-1 [7–36 amide] effects in NIDDM
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`
`Table 2. Integrated incremental responses of GLP-1 [7–36
`amide] plasma concentrations after s. c. injection (cf Fig. 2)
`into fasting NIDDM patients and calculated metabolic clear-
`ance rates
`Study
`
`Interval
`[min]
`
`Integrated
`incremental
`GLP-1
`[7–36 amide]
`[nmol (cid:215) l- 1 (cid:215) min- 1]
`39.5 – 11.7
`25.6 – 2.5
`25.4 – 2.2
`40.9 – 5.0
`0.17
`
`Metabolic
`clearance
`rate
`[ml (cid:215) kg- 1 (cid:215) min- 1]
`
`51 – 7
`62 – 5
`62 – 5
`41 – 6b
`0.039
`
`Study A
`Study B
`
`0–240
`0–120
`120–240a
`- 30–240
`
`Study C
`ANOVA
`(p -value)
`Data are mean – SEM
`a Integration was carried out over true baseline values (mea-
`sured at - 15 and 0 min); b significant difference (Student’s t -
`test; p < 0.05) to both periods of study B
`
`with placebo (ANOVA: p = 0.0002, from basal 7 – 1
`to 19 – 3 pmol/l after 30 min, p = 0.002 by t-test, also
`significant vs basal at 45, 75, and 90 min).
`Glucagon concentrations were elevated by the liq-
`uid meal containing amino acids (Fig. 3D) in the pla-
`cebo study, but with s. c. GLP-1 [7–36 amide] there
`was a reduction of glucagon values to below fasting
`values before and shortly after the meal was instilled
`(Fig. 3D). Later, there was a delayed peak of glucagon
`concentrations (at 90 instead of 30 min). The total
`the glucagon response (1347 – 163
`magnitude of
`l–1 (cid:215) min with GLP-1 [7–36 amide] vs 1428 –
`pmol (cid:215)
`205 with placebo), however, was not different between
`GLP-1 [7–36 amide] and placebo administration
`(p = 0.53). Plasma GIP concentrations rose in re-
`sponse to the meal (not shown). GLP-1 [7–36 amide]
`administration delayed that increment by approxi-
`mately 45–60 min, also without changing the overall
`response (3386 – 661 vs 4075 – 651 pmol (cid:215) l–1 (cid:215) min,
`p = 0.19).
`Subcutaneous GLP-1 [7–36 amide] delayed gastric
`emptying by 30–45 min, with an initial complete ces-
`sation lasting 30 min (p < 0.0001). Thereafter, the
`time course of gastric emptying was parallel to the
`placebo studies with the s. c. administration of GLP-
`1 [7–36 amide] (Fig. 4). Emptying was near-complete
`after 150 and 180 min, respectively.
`
`Side effects. In study A, one female patient experi-
`enced nausea and vertigo 20 min after the s. c. admin-
`istration of GLP-1 [7–36 amide]. In study B, nausea
`and unproductive vomiting occurred with both the
`first and the second administration of GLP-1 [7–36
`amide] in one male patient. Blood pressure and pulse
`did not change in a clinically relevant fashion during
`these episodes. In study C, no clinically relevant
`side-effects were noted; likewise the placebo caused
`no symptoms.
`
`Pharmacokinetics of GLP-1 [7–36 amide]. Based on
`integrated incremental GLP-1 [7–36 amide] re-
`sponses and the dose administered s. c., an apparent
`metabolic clearance rate of approximately 50 ml (cid:215)
`kg–1 (cid:215) min–1 could be calculated (Table 2).
`
`Discussion
`
`The present results show that, with large doses of s. c.
`GLP-1 [7–36 amide], elevated fasting plasma glucose
`concentrations in NIDDM patients can be normal-
`ized as in previous studies using continuous i. v. ad-
`ministrations of GLP-1 [7–36 amide] or [7–37] [7,
`10], provided that plasma GLP-1 [7–36 amide] con-
`centrations are elevated for a prolonged period of
`time. This was achieved by repeated s. c. injections of
`GLP-1 [7–36 amide] (Figs. 1 and 2), but it can be ex-
`trapolated that similar results can be obtained with
`continuous s. c. infusions of GLP-1 [7–36 amide] or
`with a preparation that has retarded absorption ki-
`netics. Such a preparation should elevate plasma
`GLP-1 [7–36 amide] levels into the effective concen-
`tration range (~ 100 pmol/l according to previous
`studies [5–7, 10, 11]) for a minimum of 3–4 h [7, 10].
`With such an agent, one could attempt the normaliza-
`tion of fasting glycaemia by an overnight administra-
`tion. Normal fasting glucose concentrations are an
`important determinant of overall glycaemic control
`in NIDDM patients [23, 24].
`It should be considered that not all the GLP-1-like
`material detected by current
`radioimmunoassay
`methods is biologically active GLP-1 [7–36 amide].
`Recent studies have indicated the extent to which
`GLP-1 [7–36 amide] is subject to proteolytic attack
`by exopeptidases, giving rise to GLP-1 [9–36 amide],
`which is totally devoid of biological activity [25, 26].
`This has also been shown during the present experi-
`ments (details not shown, see [27]), and is also sug-
`gested if one compares the time course of GLP-1
`[7–36 amide] concentrations (Fig. 2) as measured by
`the C-terminal- (amidation-specific) antibody 89 390
`(see methods) with effects on insulin (Fig. 1B, C, F
`and G) and glucagon secretion (Fig. 1D and H). In
`any case, the duration of elevation of GLP-1 [7–36
`amide] above 100 pmol/l (a concentration that in pre-
`vious studies led to a constant stimulation of insulin
`and the suppression of glucagon secretion [7, 10])
`was longer than the duration of effects on both insu-
`lin and glucagon. This is also evident when interpret-
`ing the time course of inhibition of gastric emptying
`by GLP-1 [7–36 amide]. Such an effect had been no-
`ted under the influence of continuous i. v. infusions
`of GLP-1 [7–36 amide] in normal subjects [28, 29]
`and in NIDDM patients [15, 28]. Furthermore, the in-
`hibition of gastric emptying at plasma levels of 100
`pmol/l was near-complete for a period of at least
`2–4 h [15, 29]. In the present experiments (Fig. 4),
`
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`M. A. Nauck et al.: Subcutaneous GLP-1 [7–36 amide] effects in NIDDM
`
`gastric emptying was completely stopped for approxi-
`mately 30 min (i. e. until 60 min after the s. c. adminis-
`tration of GLP-1 [7–36 amide]), but proceeded at a
`normal rate (as with placebo administration) shortly
`thereafter, although GLP-1 [7–36 amide] plasma val-
`ues remained elevated much longer (Fig. 2C). In in-
`terpreting the gastric emptying data (Fig. 4), the
`somewhat unphysiological composition of the liquid
`meal studied should be kept in mind.
`In comparison to previous studies using s. c. GLP-1
`[7–36 amide] in NIDDM patients, the dose adminis-
`tered was larger in the present examination (approxi-
`mately fivefold in comparison to Gutniak et al. [12]).
`The effects, as expected, were larger and of longer du-
`ration. From the integrated increments in GLP-1 [7–
`36 amide] concentrations (Fig. 2) after s. c. adminis-
`tration (Table 2) and the dose administered (1.5
`nmol/kg), the apparent metabolic clearance rate (ap-
`proximately 50 ml (cid:215) kg–1 (cid:215) min–1) can be calculated, in
`line with previous results in normal subjects [13].
`Since, at plasma concentrations between 100 and 300
`pmol/l, the metabolic clearance rate of GLP-1 [7–36
`amide] infused i. v. was approximately 14 ml (cid:215) kg–1
`(cid:215) min–1 [3–5], and because there is no reason to be-
`lieve that the elimination of GLP-1 [7–36 amide] ab-
`sorbed from s. c. depots should follow different kinet-
`ics compared to that of GLP-1 [7–36 amide] adminis-
`tered directly into the bloodstream, the higher meta-
`bolic clearance rate after s. c. administration (by ap-
`proximately 3.5-fold) probably indicates a reduced
`bioavailability, which can be estimated to be in the or-
`der of 25–30 %. The small differences in integrated
`GLP-1 [7–36 amide] responses between experiments
`(Table 2) most likely reflect differences in the inte-
`gration period.
`Serious side-effects were not noted; however, nau-
`sea and vomiting occurred in 2 of 24 patients studied.
`In line with our previous study in normal subjects
`[13], the dose- and concentration range used in the
`present experiments probably comes close to the
`maximum to be used in future clinical trials. Hope-
`fully with a slower release of GLP-1 [7–36 amide]
`from s. c. depots, peak values will be lower and side-
`effects should be less likely.
`As in previous studies [6, 12], the fall in plasma
`GLP-1 [7–36 amide] or [7–37] concentrations after
`administration before a meal resulted in a rebound
`increment of plasma glucose (Fig. 3A),
`insulin
`(Fig. 3B) and C-peptide (Fig. 3C), when the stomach
`started to empty again (Fig. 4). Therefore, the reduc-
`tion in glycaemia was seen only transiently, and the
`overall effect on the integrated incremental glucose
`concentrations was of borderline significance only
`(p = 0.051). This may be due to the fact that the ab-
`sorption of meal components is only slightly post-
`poned by the administration of GLP-1 [7–36 amide],
`as also suggested by the glucagon (Fig. 3D) and
`GIP responses, which remained similar in overall
`
`magnitude with the s. c. administration of GLP-1 [7–
`36 amide], although their time course was consider-
`ably different
`in comparison to placebo studies
`(Fig. 3D). If, in fact, glucose concentrations can be
`normalized in hyperglycaemic NIDDM patients, as
`has been shown with i. v. GLP-1 [7–36 amide] adminis-
`trations [7, 10], the effects on gastric emptying of
`GLP-1 [7–36 amide] administered with meals may be-
`come the most predominant effect, since the actions
`on insulin and glucagon secretion are glucose-depen-
`dent [7, 10, 30] and will be of less magnitude at de-
`creasing blood glucose levels. Along this line, the
`self-limited stimulation of insulin secretion during
`the i. v. administration of GLP-1 [7–36 amide] [7, 10]
`probably was, the consequence of the glucose-depen-
`dence of this effect. In study B, the second injection
`of GLP-1 [7–36 amide] was still able to stimulate insu-
`lin secretion, because normoglycaemia had not been
`reached with the first dose administered 120 min ear-
`lier. An alternative explanation is that the beta cells
`had been “primed” by the first injection, which may
`even potentiate secretory responses to a second stim-
`ulation, as has been shown in animal experiments [31].
`Based on these considerations, one may speculate
`that better use can be made of GLP-1 [7–36 amide]
`in the fasting state than in association with meal in-
`gestion,