Diabetologia (1993) 36: 741-744
`
`Diabetologia
`
`©Springer-Verlag 1993
`
`Normalization of fasting hyperglycaemia by exogenous glucagon-like
`peptide 1 (7-36 amide) in Type 2 (non-insulin-dependent)
`diabetic patients
`M.A. Nauck1, N. Kleine2, C. 0rskov3, J. J. Holst3, B. Willms2, W. Creutzfeldt1
`1 Division of Gastroenterology and Endocrinology, Department of Medicine, Georg-August-University, Gottingen, Germany
`2 Fachklinik ftir Diabetes und Stoffwechselkrankheiten, Bad Lauterberg, Germany
`3 Departments of Anatomy B and Physiology C, Pan um Institute, Copenhagen, Denmark
`
`Sunuuary. Glucagon-like peptide 1 (GLP-1) (7-36 amide) is
`a physiological incretin hormone that is released after
`nutrient intake from the lower gut and stimulates insulin
`secretion at elevated plasma glucose concentrations. Pre(cid:173)
`vious work has shown that even in Type 2 (non-insulin(cid:173)
`dependent) diabetic patients GLP-1 (7-36 amide) retains
`much of its insulinotropic action. However, it is not known
`whether the magnitude of this response is sufficient to
`normalize plasma glucose in Type 2 diabetic patients with
`poor metabolic control. Therefore, in 10 Type 2 diabetic
`patients with unsatisfactory metabolic control (HbA"
`11.6±1.7 % ) on diet and sulphonylurea therapy (in some
`patients supplemented by metformin or acarbose ), 1.2 pmol
`xkg- 1 xmin- 1 GLP-1 (7-36amide) or placebo was
`infused intravenously in the fasting state (plasma glucose
`13.1±0.6 mmol/l). In all patients, insulin (by 17.4 ± 4.7 nmol
`x 1- 1 x min; p = 0.0157) and C-peptide (by 228.0 ± 39.1
`nmol x 1- 1 x min; p = 0.0019) increased significantly over
`
`basal levels, glucagon was reduced (by -1418 ± 308 pmol
`x 1- 1 x min) and plasma glucose reached normal fasting
`concentrations (4.9±0.3mmol/l) within 4h of GLP-1
`(7-36 amide) administration, but not with placebo. When
`normal fasting plasma glucose concentrations were reached
`insulin returned towards basal levels and plasma glucose
`concentrations remained stable despite the ongoing infu(cid:173)
`sion of GLP-1 (7-36 amide). Therefore, exogenous GLP-1
`(7-36 amide) is an effective means of normalizing fasting
`plasma glucose concentrations in poorly-controlled Type 2
`diabetic patients. The glucose-dependence of insulinotropic
`actions of GLP-1 (7-36 amide) appears to be retained in
`such patients.
`
`Key words: Type 2 (non-insulin-dependent) diabetes mel(cid:173)
`litus, incretin hormones, glucagon-like peptide 1 (7-36
`amide), pancreatic glucagon, enteroinsular axis.
`
`Glucagon-like peptide 1 (7-36 amide) is an insulinotropic
`hormone secreted from enteroglucagon-producing cells
`in the lower gut, i.e. the distal jejunum, ileum and
`colon/rectum [1]. Since after meals plasma concentrations
`increase and reach the concentration range necessary to
`stimulate insulin secretion, GLP-1 (7-36 amide), together
`with gastric inhibitory polypeptide (GIP) from the upper
`gut, act as physiological incretin hormones [2, 3]. In phar(cid:173)
`macological concentrations, exogenous GLP-1 (7-36
`amide or 7-37) raised insulin and lowered glucagon con(cid:173)
`centrations also in Type 2 (non-insulin-dependent) dia(cid:173)
`betic patients [4, 5]. It also reduced meal-related insulin
`requirements in both Type 1 (insulin-dependent) and
`Type 2 diabetic patients [6]. However, after a mixed meal,
`not only influences on insulin and glucagon secretion, but
`also other effects, such as an inhibition of gastric emptying
`may have contributed to the "anti-diabetogenic effect"
`[7]. Therefore, the aim of the present study was to charac(cid:173)
`terize the endocrine pancreatic and glucose response to a
`pharmacological dose of exogenous GLP-1 (7-36 amide)
`
`in fasting Type 2 diabetic patients. The subjects were se(cid:173)
`lected to represent a group of Type 2 diabetic patients
`which could not be satisfactorily controlled by oral anti(cid:173)
`diabetic drugs. Since the expected insulinotropic response
`involves the potential risk of hypoglycaemic reactions, it
`was of special interest to evaluate the glucose-dependence
`of the insulin response to GLP-1 (7-36 amide) in these pa(cid:173)
`tients, i.e. to see whether the insulin response declines
`again when normal fasting glucose concentrations are ap(cid:173)
`proached.
`
`Subjects, materials and methods
`
`Study protocol
`
`The study protocol was approved by the ethics committee of the
`medical faculty of the Georg-August-University, Gbttingen prior to
`the study. Written informed consent was obtained from all partici(cid:173)
`pants.
`
`MPI EXHIBIT 1056 PAGE 1
`
`

`

`742
`
`Table 1. Patient characteristics
`
`M.A.Naucket al.: GLP-1 (7-36 amide) in Type 2diabetes
`
`Patient
`number
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`Mean
`SD
`
`Sex
`
`(m/f)
`m
`m
`m
`m
`m
`f
`m
`m
`f
`f
`7m/3f
`
`Age
`
`Height
`
`Weight
`
`(years)
`64
`54
`48
`51
`61
`42
`42
`57
`46
`52
`53
`7
`
`(cm)
`171
`170
`177
`179
`183
`164
`180
`177
`171
`153
`173
`9
`
`(kg)
`63
`74
`103
`88
`87
`75
`94
`66
`61
`65
`78
`15
`
`Body mass
`index
`(kg/m2
`)
`21.5
`25.6
`32.9
`27.5
`26.0
`27.9
`29.0
`21.1
`20.9
`27.8
`26.0
`3.9
`
`Duration of
`diabetes
`(years)
`9
`17
`15
`8
`10
`16
`2
`7
`4
`3
`9
`5
`
`Therapy Metabolic control
`HbAlc
`(%)a
`
`D,S
`D,S,B
`D,S,B
`D,S,B
`D,S,A
`D,S
`D,S
`D,S
`D,S,A
`D,S,A
`
`12.2
`11.3
`12.2
`10.6
`10.0
`10.8
`14.8
`9.1
`13.4
`12.0
`11.6
`1.7
`
`D, Diet; S, sulphonylurea treatment (glibenclamide, 10.5 mg per day); B, biguanide treatment (metformin, 1700 mg or 2550 mg per day), A,
`acarbose treatment (300 mg per day). a normal value: 4.0--6.2 %
`
`Patients
`
`Blood specimens
`
`Ten Type 2 diabetic patients were studied (Table 1). They were all
`being treated with diet and sulphonylurea compounds, and some
`were also receiving metformin or acarbose treatment. Multiple fast(cid:173)
`ing plasma glucose concentrations (from hospital charts) were
`13.l ± 2.0 mmol/l (236 ± 36 mg/di), postprandial glucose concentra(cid:173)
`tions were 15.l ± 2.7 mmol/l (272 ± 49 mg/di). Because of unsatisfac(cid:173)
`tory metabolic control, eight of these ten patients were assigned to
`insulin treatment immediately after the study.
`The patients were studied on two occasions. All anti-diabetic
`medication was continued until the morning before the studies. A
`regular meal and drug schedule was allowed for one day between the
`experiments with GLP-1 (7-36 amide) and placebo. On the study
`days, all medication was withheld until the end of the experiments.
`
`Peptides
`
`Synthetic GLP-1 (7-36 amide) was purchased from Saxon Bio(cid:173)
`chemicals GmbH, Hannover, FRG. The same lot number was used
`as in previous studies (GLP-1 (7-36 amide): PGAS 242, LotZE 865,
`net peptide content 79.3 % ) [3, 4]. The peptide was dissolved, filtered
`through 0.2 µm nitrocellulose filters (Millipore, Bedford, Mass.,
`USA) and stored frozen at -30°C as previously described. Net pep(cid:173)
`tide content rather than gross weight was used for dose calculations.
`HPLC profiles (provided by the manufacturer) showed that the
`preparation was more than 99 % pure (single peak coeluting with ap(cid:173)
`propriate standards). Samples were analysed for bacterial growth
`(standard culture techniques) and for pyrogens (Limulus amebocyte
`lysate endo-LAL; Chromogenix AB, Molndal, Sweden). No bac(cid:173)
`terial contamination was detected. Endotoxin concentrations in
`the GLP-1 (7-36 amide) stem solutions were always less than
`0.03EU/ml.
`
`Experimental procedures
`
`The experiments were performed in randomized •order. The tests
`were performed in the morning after an overnight fast. Two forearm
`veins were punctured with a teflon cannula (Moskito 123, 18 gauge;
`Vygon, Aachen, FRG ), which was kept patent using 0.9 % NaCl (for
`blood sampling and for GLP-1 (7-36 amide)/placebo administra(cid:173)
`tion).
`After drawing basal blood specimens at 0 min, an intravenous in(cid:173)
`fusion of GLP-1 (7-36 amide) or placebo (0.9% NaCl containing
`1 % human serum albumin; Merieux, Norderstedt, FRG) was
`started at an infusion rate of 1.2 pmol/kg per min and continued for
`240 min. Blood was drawn at 30-min intervals and plasma glucose
`was determined immediately.
`
`Blood was drawn into heparinized tubes (immunoreactive (IR) in(cid:173)
`sulin and C-peptide measurements). A sample was stored in NaF
`(Microvette CB 300; Sarstedt, Niimbrecht, FRG) for the measure(cid:173)
`ment of glucose. For glucagon and GLP-1 (7-36 amide) measure(cid:173)
`ments blood was drawn into tubes containing EDTA and aprotinin
`(Trasylol; 20000 kallikrein inhibition units/ml, 200 µl per 10 ml
`blood; Bayer AG, Leverkusen, FRG). After centrifugation, plasma
`for hormone analyses was kept frozen at - 3Q°C.
`
`Laboratory determinations
`
`Glucose was measured using a glucose oxidase method with a Glu(cid:173)
`cose Analyser 2 (Beckman Instruments, Munich, FRG). Plasma IR(cid:173)
`insulin and C-peptide were determined using commercial radioim(cid:173)
`munoassay kits (Insulin RIA 100; Pharmacia, Freiburg, FRG; RIA(cid:173)
`mat C-peptide II, Byk-Sangtec Diagnostika, Dietzenbach, FRG),
`with human insulin and C-peptide as standard.
`IR-GLP-1 was determined in ethanol-extracted plasma as pre(cid:173)
`viously described [8], using antiserum 2135 (final dilution 1:150000)
`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 experimental detection limit (2 SD
`over samples not containing GLP-1 (7-36 amide)) was less than
`5 pmol/l. Antiserum 2135 binds, with approximately equal affinity,
`to all proglucagon-derived peptides containing the GLP-1 (7-36
`amide) sequence, regardless of amino- or carboxy-terminal exten(cid:173)
`sions. Pancreatic glucagon was assayed in ethanol-extracted plasma
`using antibody4305 [9].
`Non-esterified fatty acids were quantitated enzymatically with
`acyl-CoA oxidase from Candida tropicalis using a Hitachi 705 auto(cid:173)
`analyser. Reagents were from Wako Chemicals (N euss, FRG ).
`Each patient's set of plasma samples was assayed at the same
`time to avoid errors due to inter-assay variation.
`
`Statistical analysis
`
`Results are reported as mean± SEM. Integration was carried out ac(cid:173)
`cording to the trapezoidal rule. Significances of differences were
`tested using repeated measurement analysis of variance (RM(cid:173)
`ANOVA; NCSS Version5.01, Kaysville, Utah, USA). If a significant
`interaction of treatment and time was documented (p < 0.05), values
`at single time points were compared by Student's t-test (paired ana(cid:173)
`lyses). ?-values were corrected for the number of comparisons made
`according to Bonferroni-Holm. A corrected two-sidedp-value less
`than 0.05 was taken to indicate significant differences.
`
`MPI EXHIBIT 1056 PAGE 2
`
`

`

`M.A.Nauck et al.: GLP-1 (7-36 amide) in Type 2 diabetes
`
`743
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`o.o~~~~~~~~~~
`-30 0
`30 60 90 120 150 180 210 240
`Time [min]
`Fig.1. Plasma glucose (upper panel), insulin (middle panel) and C(cid:173)
`peptide (lower panel) responses to the intravenous administration
`of glucagon-like peptide 1 (GLP-1) (7-36 amide) (1.2 pmoVkg per
`min) or placebo in 10 Type 2 diabetic patients. Mean± SEM are
`given. e: experiments with GLP-1 (7-36 amide). 0: experiments
`with placebo. The dotted bar indicates the duration of exogenous ad(cid:173)
`ministration of GLP-1 (7-36 amide) or placebo. RM-ANOVA indi(cid:173)
`cated significant interactions of treatment (GLP-1 (7-36 amide) vs
`placebo) and time (p <0.0001) regarding glucose, insulin, and C(cid:173)
`peptide measurements. Asterisks indicate significant differences at
`single time points (p < 0.05)
`
`-30 0
`
`30 60 90 120 150 180 210 240
`Time [min]
`Fig.2. Plasmaglucagon-likepeptide 1 (GLP-1) (7-36 amide) (upper
`panel), pancreatic glucagon (antibody4305; middle panel) and non(cid:173)
`esterified fatty acid (NEFA; lower panel) responses to the intraven(cid:173)
`ous administration of GLP-1 (7-36 amide) (1.2 pmol/kg per min) or
`placebo in 10 Type 2 diabetic patients. Mean± SEM are given. e: ex(cid:173)
`periments with GLP-1 (7-36 amide). 0: experiments with placebo.
`The dotted bar indicates the duration of exogenous administration of
`GLP-1 (7-36 amide) or placebo. RM-ANOVA indicated significant
`interactions of treatment (GLP-1 (7-36 amide) vs placebo) and time
`regarding GLP-1 (7-36 amide) (p < 0.0001), pancreatic glucagon
`(p = 0.0009), and NEFA (p < 0.0001) measurements. Asterisks indi(cid:173)
`cate significant differences at single time points (p < 0.05)
`
`Results
`
`The mean fasting plasma glucose concentrations before
`the exogenous administration of GLP-1 (7-36 amide)
`was 12.7 ± 0.6 mmol/l. GLP-1 (7-36 amide) lowered plas(cid:173)
`ma glucose in all 10 Type 2 diabetic patients (Fig. l).
`After 240 min, "normal" basal plasma concentrations
`were reached ( 4.9 ± 0.3 mmol/l), whereas with placebo,
`only a slight reduction in plasma glucose concentrations
`(10.4 ± 0.7 mmol/l; p = 0.0025 by RM(cid:173)
`was noted
`ANOVA). Both insulin and C-peptide increased signifi(cid:173)
`cantly during the intravenous infusion of GLP-1 (7-
`36 amide). The integrated incremental response of in(cid:173)
`(p =
`to 17.4 ± 4. 7 nmol x I - 1 x min
`sulin amounted
`0.0157), and of C-peptide to 228.0 ± 39.1nmolx1- 1 x min
`(p = 0.0019).
`When plasma glucose values approached normal basal
`plasma glucose values, insulin and C-peptide values de(cid:173)
`creased again. Plasma glucose did not fall further. In a sub(cid:173)
`group of five patients, who had reached a normal basal
`plasma glucose concentration by 180 min, a stable plasma
`glucose concentration of 4.1 ± 0.2 mmol/l was maintained
`over the last hour of the experiment despite an ongoing
`GLP-1 (7-36 amide) infusion (details not shown). The
`
`lowest plasma glucose recorded at any time point was
`3.5 mmol/l.
`The steady-state concentration of GLP-1 (7-36 amide)
`was 123 ± 10 pmol/l with exogenous GLP-1 (7-36 amide).
`With placebo, the values remained in the basal range
`(8 ± 1 pmol/l; Fig. 2, upper panel).
`Pancreatic glucagon was significantly lowered with
`exogenous GLP-1 (7-36 amide) by -1418 ± 308 pmol
`x l - 1 x min; Fig. 2, middle panel) despite a considerable
`fall in plasma glucose (Fig.1, upper panel). When plasma
`glucose approached normal fasting levels, glucagon re(cid:173)
`turned to baseline values.
`During the exogenous administration of GLP-1 (7-36
`amide), plasma non-esterified fatty acids significantly de(cid:173)
`creased (by -26.3±3.1 mmolxI- 1 xmin) during the
`period characterized by elevated insulin and reduced glu(cid:173)
`cagon concentrations (Fig. 2, lower panel; p = 0.028).
`
`Discussion
`
`The results of the present study indicate that the preserved
`incretin activity of GLP-1 (7-36 amide) may be utilized to
`normalize fasting plasma glucose concentrations in Type 2
`
`MPI EXHIBIT 1056 PAGE 3
`
`

`

`744
`
`diabetic patients. This is an extension of previous observa(cid:173)
`tions where, in mildly Type 2 diabetic patients, GLP-1 (7-
`36 amide) as aninsulinotropic agent was found to be almost
`equally as effective as in age-and weight-matched normal
`subjects [ 4]. Therefore, the insulinotropic effect of GLP-1
`(7-36 amide) is of sufficient magnitude to reduce elevated
`plasma glucose concentrations into the normal fasting
`range even in patients who no longer respond well to oral
`anti-diabetic drugs. The concentrations of GLP-1 (7-
`36 amide) reached with an infusion rate of 1.2 pmol x kg- 1
`x min - 1 were approximately two- to three-fold higher than
`peak values measured after oral glucose when using the
`same radioimmunoassay [3, 4). Therefore, the actions of
`GLP-1 (7-36 amide) observed in the present study are
`pharmacological effects, although the difference in circu(cid:173)
`lating concentrations to those encountered under physio(cid:173)
`logical conditions was not great.
`The amount of insulin secreted in response to GLP-1
`(7-36 amide) can be estimated to be approximately
`11.9 ± 2.3 U [10]. The glucagon-lowering effect of GLP-1
`(7-36 amide) [4, 11-13] may contribute to the normal(cid:173)
`ization of plasma glucose concentrations. However,
`when considering the different glucose concentration cur(cid:173)
`ves in the GLP-1 (7-36 amide) and placebo experiments,
`the influence of GLP-1 (7-36 amide) on pancreatic alpha
`cell secretion cannot be judged without accounting for the
`influence of glucose itself. The elevation in insulin concen(cid:173)
`trations may have contributed to the reduction in pancre(cid:173)
`atic glucagon. However, in some patients insulin and
`C-peptide increased very little. Nevertheless, glucagon
`concentrations were lowered and glucose concentrations
`were normalized (details not shown). This indicates that
`alpha-cell inhibition contributes to the effect on plasma
`glucose concentrations. Additional experiments will have
`to weigh the relative contributions of increased beta-cell
`and reduced alpha-cell secretion.
`In agreement with the strict glucose-dependence ofin(cid:173)
`sulinotropic actions of G LP-1 (7-36 amide) reported in
`normal subjects [2, 3, 12], when the Type 2 diabetic pa(cid:173)
`tients were hyperglycaemic, insulin and C-peptide con(cid:173)
`centrations were stimulated, with a return towards basal
`levels when glucose concentrations approached normal
`fasting levels. This shows that even in poorly-controlled
`diabetic patients, whose plasma glucose concentrations
`have not been normal over a prolonged period, a similar
`glucose threshold exists for the insulinotropic action of
`GLP-1 (7-36 amide) as seen in normal subjects. This
`should limit the risk of hypoglycaemic responses when
`GLP-1 (7-36 amide) or similarly acting analogues are
`used as therapeutic agents.
`After meals, other actions of GLP-1 (7-36 amide) may
`help to reduce the postprandial increment in plasma glu(cid:173)
`cose. GLP-1 (7-36 amide) slows the velocity of gastric
`emptying and thereby reduces the availability of nutrients
`for absorption [7]. In addition, the rise in glucose and glu(cid:173)
`cagon concentrations and in meal-related insulin require(cid:173)
`ments are decreased after a test meal in Type 1 and Type 2
`diabetic patients [6].
`In conclusion, in the fasting state, exogenous GLP-1
`(7-36 amide) stimulates insulin and reduces glucagon se(cid:173)
`cretion to a degree that leads to a normalization of plasma
`glucose concentrations even in poorly-controlled Type 2
`
`M.A.Naucket al.: GLP-1 (7-36 amide) in Type 2 diabetes
`
`diabetic patients with secondary failure after sulphonyl(cid:173)
`urea treatment. Furthermore, the strict glucose-depend(cid:173)
`ence of insulinotropic effects limits the amount of insulin
`secreted when high doses of GLP-1 (7-36 amide) are ad(cid:173)
`ministered at near-normal fasting plasma glucose concen(cid:173)
`trations.
`
`Acknowledgements. The excellent technical assistance of Ms.
`S. Bierkamp, S. Ogilvie, A. Bernhard, W. Kopp, K. Illmer, M. Stix,
`and L.Rabenh¢j is gratefully acknowledged. We thank Priv.-Doz.
`Dr. V. Armstrong for the NEPA-measurements. This study was sup(cid:173)
`ported by the Deutsche Forschungsgemeinschaft, Bonn 2 (Bad Go(cid:173)
`desberg), grant Na 203/2-2, by the Danish Medical Research Coun(cid:173)
`cil, and by the Novo Nordisk Foundation.
`
`References
`
`1. Eissele R, Goke R, Willemer Set al. (1992) Glucagon-like pep(cid:173)
`tide 1 cells in the gastrointestinal tract and pancreas of rat, pig
`and man. Eur J Clin Invest 22: 283-291
`2. Kreymann B, Ghatei MA, Williams G, Bloom SR (1987) Gluca(cid:173)
`gon-like peptide 1 7-36: a physiological incretin in man. Lancet
`II: 1300-1304
`3. Nauck M, Bartels E, 0rskov C, Ebert R, Creutzfeldt W (1993)
`Additive insulinotropic effects of exogenous synthetic human
`gastric inhibitory polypeptide and glucagon-like peptide 1 (7-
`36 amide) infused at near-physiological insulinotropic hormone
`and glucose concentrations. J ClinEndocrinol Metab 76: 912-917
`4. Nauck MA, Heimesaat MM, 0rskov C, Holst JJ, Ebert R,
`Creutzfeldt W (1993) Preserved incretin activity of glucagon-like
`peptide 1 (GLP-1) (7-36 amide) but not of synthetic human gas(cid:173)
`tric inhibitory polypeptide (GIP) in patients with Type 2 diabetes
`mellitus. J Clin Invest 91: 301-307
`5. Nathan DM, Schreiber E, Fogel H, Mojsov S, Habener JF (1992)
`Insulinotropic action of glucagon-like peptide-I-(7-36) in dia(cid:173)
`betic and nondiabetic subjects. Diabetes Care 15: 270-276
`6. Gutniak M, 0rskov C, Holst JJ, Ahr en B, Efendic S (1992) Anti(cid:173)
`diabetogenic effect of glucagon-like peptide-1 (7-36)amide in
`normal subjects and patients with diabetes. N Engl J Med 326:
`1316-1322
`7. Wettergen A, Scholdager B, Mortensen PE, Myhre J, Christian(cid:173)
`sen J, Holst JJ (1990) Truncated GLP-1 (proglucagon 87-107
`amide) from distal gut: role in regulation of gastric and pancre(cid:173)
`atic functions. Digestion 46 [Suppl 1]: 120 (Abstract)
`8. 0rskov C, Holst JJ (1987) Radio-immunoassays for glucagon(cid:173)
`like peptides 1and2 (GLP-1 and GLP-2). ScandJ Clin Lab In(cid:173)
`vest 47: 165-174
`9. Holst JJ (1982) Evidence that peak II GLI or enteroglucagon is
`identical to the C-terminal sequence (residues 33-69) of glicen(cid:173)
`tin. Biochem J 207: 381-388
`10. Polansky KS, Given BD, Hirsch LJ et al. (1988) Abnormal pat(cid:173)
`terns of insulin secretion in noninsulin-dependent diabetes mel(cid:173)
`litus. N Eng!J Med 318: 1231-1239
`11. 0rskov C, Holst JJ, Nielsen OV (1988) Effect of glucagon-like
`peptide 1 (proglucagon (78-107) amide) on endocrine secretion
`from pig pancreas, antrum and nonantral stomach. Endocrino(cid:173)
`logy 123: 2009-2013
`12. Komatsu R, Matsuyama T, Namba Met al. (1989) Glucagono(cid:173)
`static and insulinotropic action of glucagonlike peptide 1-(7-36)(cid:173)
`amide. Diabetes 38: 902-905
`13. 0rskov C (1992) Glucagon-like peptide-1, anew hormone of the
`entero-insular axis. Diabetologia 35: 701-711
`
`Received: 30 November l 992
`and in revised form: 19March1993
`
`Dr.M.Nauck
`Medizinische Klinik der Ruhr-Universitat
`im Kna ppschafts-Krankenha us
`In der Schornau 23-25
`D-44892 Boch um
`Germany
`
`MPI EXHIBIT 1056 PAGE 4
`
`