Both Subcutaneously and Intravenously Administered
`Glucagon-Like Peptide I Are Rapidly Degraded From
`the NH2-Terminus in Type II Diabetic Patients and in
`Healthy Subjects
`
`Carolyn F. Deacon, Michael A. Nauck, Maibritt Toft-Nielsen, Lone Pridal, Berend Willms,
`and Jens J. Holst
`
`The fate of exogenous glucagon-like peptide I (GLP-I)(7—
`36) amide was studied in nondiabetic and type II diabetic
`subjects using a combination of high-pressure liquid chro-
`matography (HPLC), specific radioirnmunoassays (RIAs),
`and a sensitive enzyme-linked irnmunosorbent assay
`(ELISA), whereby intact biologically active GLP-I and its
`metabolites could be determined. After GLP-I administra-
`
`tion, the intact peptide could be measured using an
`NH2-temrinally directed RIA or ELISA, while the dii1’er-
`ence in concentration between these assays and a CO0H-
`terminal-specific RIA allowed determination of NH,-
`terrninally truncated metabolites. Subcutaneous GLP-I
`was rapidly degraded in a time-dependent manner, form-
`ing a metabolite, which co-eluted on HPLC with GLP-1(9-
`36) amide and had the same immunoreactive profile.
`Thirty minutes after subcutaneous GLP-I administration
`to diabetic patients (n = 8), the metabolite accounted for
`88.5 t 1.9% of the increase in plasma irnmunoreactivity
`determined by the COOH-terminal RIA, which was higher
`than the levels measured in healthy subjects (78.4 3:
`3.2%; n = 8; P < 0.05). Intravenously infused GLP-I was
`also extensively degraded, but no significant diiferences
`were seen between the two groups. Intact GLP-I ac-
`counted for only 19.9 2 3.4% of the increase in immuno-
`reactivity measured with the COOII-terminal RIA in
`nonnal subjects (n = 8) and 25.0 1 4.8% of the increase
`in diabetic subjects (n = 8), the remainder being the
`NH,-terminally tnmcated metabolite. Diabetes 44:l126—
`1131, 1995
`
`hour the Department of Medical Physiology (C.F.D., JJ.H.), Panum Institute,
`University of Copenhagen, and the Department of Endocrinology (M.T.-N.),
`Hvidovre Hospital, Copenhagen, and the Department of Diabetes Phannacology
`(L.P.), Novo Nordisk A/S, Bagsvaerd, Denmark; and the Department of Medicine
`(M.A.N.), Ruhr University, Bochum, and Fachklinik fur Diabetes und StotTwech-
`selkrankheiten (B.W.), Bad Lauterberg, Germany.
`Address correspondence and reprint requests to Dr. C.F. Deacon, Department of
`Medical Physiology, Panum Institute, University of Copenhagen, Blegdamsvej 3,
`DK-2200 Copenhagen N, Denmark.
`Received for publication 7 March 1995 and accepted in revised fonn 18 May
`I995.
`ANOVA, analysis of variance; BMI, body mass index; ELISA, enzyrne-linked
`immunosorbent assay; GLP-I, glucagon-like peptide I; HPLC, high-pressure liquid
`clmomavography; RIA, radioimmunoassay.
`
`1126
`
`he processing and secretion of peptide hormones
`have been studied in detail, but it is only recently
`that the metabolic fate of the products has re-
`ceived attention. In many cases, degradation pro-
`ceeds rapidly, giving rise to biologically inactive fragments.
`However, there is evidence that metabolites of peptides may
`be biologically active in their own right, acting as agonists or
`antagonists of the parent hormone at its receptor.
`Tissue-specific post-translational processing of the gluca-
`gon precursor (proglucagon) within the intestinal L-cells
`gives rise to glucagon-like peptide I (GLP-l)(7-36) amide,
`which corresponds to prog1ucagon(78—107) amide (1,2).
`GLP-I(7—36) amide and the related nonamidated glycine-
`extended peptide GLP-I(7—37) amide are highly potent in
`stimulating insulin and inhibiting glucagon secretion (3,4),
`effects that are glucose-dependent (5-7). Because of these
`actions, both peptides have attracted interest as possible
`therapeutic agents in the management of diabetes (7-11).
`Proglucagon processing has been studied in detail (12),
`but little is known of the metabolic fate of GLP-l(7—36) amide
`once it has been produced. This has partly been due to a lack
`of suitable methodology, since most radioimmunoassays
`(RlAs) do not distinguish between the intact peptide and any
`metabolites. Few studies have examined GLP-I degradation,
`but the fate of pharmacological (rnicromolar) concentrations
`of the peptide in vitro has been reported (13). We have
`recently shown that physiological (picomolar) concentra-
`tions of GLP-l(7-36) amide are degraded by human plasma in
`vitro by endogenous dipeptidyl peptidase IV (EC 3.4.14.5),
`forming GLP-I(9—36) amide (14). Furthermore, we also
`showed that this NH2-terminally truncated peptide is a major
`endogenous metabolite in vivo. GLP-l(9—36) amide lacks
`eiflcacy, but it may act as an antagonist at the GLP-I receptor
`in vitro (15).
`This study has examined the fate of exogenous GLP-1(7-
`36) amide in type II diabetic and nondiabetic subjects, with a
`combination
`of
`high-pressure
`liquid
`chromatography
`(HPLC), specific RIAS, and a sensitive sandwich enzyme-
`linked immunosorbent assay (ELISA).
`
`RESEARCH DESIGN AND METHODS
`
`Subjects and study protocols. The studies were approved by the local
`ethics committees of Copenhagen and Frederiksberg municipalities and
`
`DIABETES, VOL. 44, SEPTEMBER 1995
`
`SANOFI-AVENTIS Exhibit 1057 - Page 1126
`IPR for Patent No. 8,951,962
`
`

`
`Georg-August-University, Gottingen, and written consent was obtained
`from all participants after the nature and possible risks of the study had
`been explained to them. The studies were carried out in nondiabetic
`subjects and in patients with type II diabetes. The nondiabetic subjects
`were healthy with no personal or family history of diabetes or gastro-
`intestinal disease (study 1: six men, two women, age 24 : 2 years, body
`mass index [BMI] 21.9 : 2.3 kg/mz; study 2: eight men, zero women, age
`23 I 1 years, BMI 22.2 .+. 0.5 kg/mg). The diabetic subjects were treated
`with diet and sulfonylurea compounds, and some also received met-
`formin or acarbose treatment (study 1: five men, three women, age 61 1
`2 years, BMI 29.5 I 2.5 kg/m2, duration of diabetes 9 : 3 years; study 2:
`three men, five women, age 58 I 2 years, BMI 30.0 : 1.8 kg/m2, duration
`of diabetes 12 : 2 years).
`All antidiabetic medication was continued until the evening before
`the study, which was performed the morning after an overnight fast The
`studies examined the fate of GLP-I(7—36) amide given as either a single
`subcutaneous bolus injection (study 1; 1.6 nmovkg) or a continuous
`intravenous infusion (study 2; 1.2 pmol-kg"-min"). Assay methodology
`was validated by examining in detail the fate of 4.5 nmovkg GLP-I given
`subcutaneously to nondiabetic subjects. The peptide (Saxon, Hannover,
`Germany, and Peninsula Laboratories Europe, St. Helens, U.K.) was
`dissolved in sterile 0.9% saline solution containing in addition 1% human
`serum albumin. its stability in solution was examined by HPUC analysis
`of infusion product samples, and it was verified to be intact GLP-I(7-36)
`amide and not to be degraded during the experiment. Blood samples
`were taken in the basal state and for up to 4 h after GLP-I administration,
`into chilled tubes containing EDTA (3.9 mmol/l) and aprotinin (500
`kallikrein-inhibitory units/ml), and kept on ice to prevent
`in vitro
`degradation of GLP-I(7—36) amide (14). The plasma was separated by
`centrifugation at 4°C and stored at —20°C until analysis.
`Analytical procedures
`RIAs. Plasma aliquots were extracted by ethanol precipitation (70% v/v,
`final concentration), and assayed using three diiferent antisera raised
`against GLP-I as described below. Antiserum 2135 (16,17) is side-viewing
`and measures all molecules containing the central sequence of GLP-I(7-
`36) amide, recognizing both NH2-terminally and COOH-tenninally trun-
`cated peptides and having a cross-reactivity of 79% with GLP-l(9—36)
`amide. Antiserum 91022 (18)
`is directed toward the Ni-I2-terminal
`sequence of GLP-I(7-36) amide and cross-reacts fully with COOH-
`tenninally truncated fragments but <0.296 with the NH2-terminally
`truncated peptide GLP-I(9—36) amide. Antiserum 89390 (19) has an
`absolute requirement for the intact arnidated COOH-tenninus of GLP-
`l(7-36) amide and cross-reacts <0.0l% with GLP-l(7—34) or GLP-l(7-35)
`and ruuy with NH2-tenninally truncated peptides including GLP-I(9-36)
`amide. For all assays, standard and ml-labeled tracers were GLP-I(7w‘36)
`amide, and separation of antibody-bound peptide from antibody-free
`peptide was achieved with plasma-coated charcoal (16,19).
`ELISA. Unextracted plasma aliquots were assayed by a newly de-
`scribed sandwich ELISA (20), which uses an NH2-terminally directed
`polyclonal antibody (91022 as described above) and a COOH-terminally
`directed monoclonal antibody (GLP-I-F5), with GLP—l(7-36) amide as
`standard. The assay cross-reacts with GLP-I(7—34) and GLP-[(7-35) but
`does not recognize peptides with larger deletions at the COOH-temiinus
`and cross-reacts <1% with NH2-terminally truncated peptides. it also
`detects precursors of GLP-I(7—36) amide, cross-reacting 6596 with GLP-
`l(l—36) amide lproglucagon(72—107) amide] and 71% with major pro-
`glucagon fragment (MPGF)
`[proglucagon(72-158)]. However, under
`conditions where GLP-I(7—36) amide is administered exogenously, the
`assay can be considered as specific for biologically active GLP-I.
`HPLC. Aliquots of plasma (500 pl) from nondiabetic subjects receiving
`4.5 nmovkg GLP-I(7—36) amide subcutaneously were extracted on C1,;
`Sep-Pair cartridges (Waters-Millipore, Milford, MA) and subjected to
`analytical reverse-phase HPLC with the use of a Nucleosil 120-5 pm C3
`column (Macherey-Nagel, Diiren, Germany) eluted with gradients of
`acetonitrile in 0.1% trifluoroacetic acid, as previously described (14).
`Fractions were collected and assayed for GLP-I with antisera 2135 and
`91022 as described above. The method has an overall recovery of 41%
`and a detection limit of ~95 fmol.
`Statistical analysis. Data are expressed as means : SE and were
`analyzed by analysis of variance (ANOVA) and Student’s t test for paired
`and nonpaired data as appropriate. P < 0.05 was considered significant.
`
`RESULTS
`
`Validation of analytical determinations. HPLC analysis
`of plasma from normal subjects receiving 4.5 nmol/kg GLP-
`
`DIABETES, VOL 44, SEPTEMBER 1995
`
`C.F, DEACON AND ASSOCIATES
`
`80
`
`O O
`
`Ga" 0 ouruuoraov %
`
`40
`40
`
`80
`
`40
`
`00 O omuuoraav %
`
`[GLP-1]
`
`tmolltractlon
`
`[GLP-1]
`
`tmolltractlon
`
`1 0
`
`2 5
`
`3 0
`
`2 0
`1 5
`(min)
`Time
`FIG. 1. Plasma (500 ill) from a representative healthy subject was
`analyzed by reverse-phase HPLC on a Nucleosil C, column at 15 min (A)
`and 30 min (B) after subcutaneous administration of GLP-[(7436) amide
`(4.5 nmol/kg) and measured with sideoviewing (2135) and NH,-terminal
`(91022) RlAs for GLP-I. Concentrations have not been corrected for the
`recovery of 41%. Elution positions of the intact peptide (1) and the
`Nil,-terminally truncated metabolite (II) are indicated.
`
`I(7—36) amide subcutaneously revealed a time-dependent
`formation of an NH2-terminally truncated peptide eluting in
`the position of GLP-I(9-36) amide (Fig. 1). Analysis of the
`same samples by RIA and ELISA gave concentrations of
`GLP—l irnmunoreactivity that were significantly higher when
`measured with the COOH-terminally directed RIA than when
`measured with the Ni-12-terminally directed RIA or the ELISA
`(Fig. 2). The results are expressed as the absolute increase
`above basal values (ie, after subtraction of endogenous
`concentrations) for clarity, since both the NH2-temiinal RIA
`and the ELISA also measure NH2- and COOH-terminally
`extended GLP-I peptides, resulting in concentrations in the
`basal state that are higher than those measured with the
`COOH-terminal RIA (72 t 3 pmol/l, NH2-terminal RIA; 46 1-
`12 pmol/1, ELISA; 11 : 2 pmol/l, COOH-terminal RIA).
`Concentrations of the intact peptide, determined by HPLC in
`combination with RIA (after correction for recovery), did not
`
`1127
`
`SANOFI-AVENTIS Exhibit 1057 - Page 1127
`IPR for Patent No. 8,951,962
`
`

`
`GLP-I DEGRAD/\T|ON IN HUMANS
`
`2000
`
`.—.a
`v7.0
`:1.
`§%% 1500
`u'°EQ
`E
`“moo
`W0
`2:»
`gen
`2
`
`500
`
`0 E
`
` 79:
`
`concentrations of the intact peptide were consistently lower
`in both groups compared with the immunoreactivity mea-
`sured with the COOH-terminal RIA (Fig. 4). Before the start
`of the infusion, the basal plasma concentrations were 71 : 2
`pmol/1 (NH;-terminal RIA) and 16 1 1 pmol/1 (CO0H-termi-
`nal RIA) in nondiabetic subjects and 67 t 3 pmol/I (NH2-
`temiinal RIA) and 10 I 1 pmol/1 (COOH-terminal RIA) in
`type 11 diabetic patients.
`
`DISCUSSION
`
`In this study, we have shown that in both normal nondiabetic
`subjects and patients with type II diabetes, exogenously
`administered GLP—I, whether by subcutaneous or intrave-
`nous routes, is rapidly degraded, primarily from the NH2
`terminus. Chromatographic analysis of the degradation prod-
`ucts and comparison with the results of the assays alone
`support the use of the ELISA or NH2-terminal RIA to measure
`the biologically active peptide. The assumption that the NH2-
`tenninally truncated metabolite, GLP-I(9-36) amide, accounts
`for the ditference between the concentrations measured by
`these assays and those determined by the COOH-temunal
`RIA is supported by the HPLC data and is valid, at least under
`conditions where GLP-I is administered exogenously. How-
`ever, the NH2-terminal antibody used in both the ELISA and
`the RIA also cross-reacts with NH2-terminally extended
`precursors, which precludes its use for measurement of
`endogenous GLP-I levels. Despite repeated attempts in this
`laboratory, we have been unable to raise an antibody that
`does not exhibit this cross-reactivity. However, since GLP—
`I(9w36) amide is a major endogenous metabolite (14), it is
`clearly preferable to use assays with some degree of speci-
`ficity for the NH2-terminus for determination of increases in
`biologically active GLP-I after exogenous administration.
`Both subcutaneous and intravenous administration of
`
`GLP-I to nondiabetic and type 11 diabetic subjects produced
`plasma concentrations of the intact peptide that were lower
`than the concentrations measured by the COOH-temiinally
`directed RIA. Indeed, within 1 h of subcutaneous adminis-
`tration to the diabetic patients, nearly all of the measured
`immunoreactivity detemuned with the COOH-terminal assay
`was accounted for by the NH2—terminally tnmcated metabo-
`lite. This metabolite constituted a greater proportion of the
`GLP-I irrununoreactivity in the diabetic subjects compared
`with healthy control subjects after subcutaneous administra-
`tion, but no such difference was seen after intravenous
`infusion of the peptide. These dilferences in metabolism
`between the two groups of subjects may be due to factors
`such as age, blood flow, or degree of obesity, but it cannot be
`excluded that they could be a result of the diabetic state per
`
`0
`
`0
`
`15
`
`30
`
`45
`
`60
`
`75
`
`TIme(rnln)
`FIG. 2. Increase in plasma concentrations of GLP-I, after subtraction of
`endogenous levels, after subcutaneous administration of GLP-l(7—36)
`amide (4.5 nmol/kg) in healthy subjects (n = 3) measured with s
`C0011-terminal amide-specific RIA (0; 89390), an NH,-terminally
`directed RIA (G, 91022), and an ELISA (A) specific for the biologically
`active peptide. Concentrations measured by the N112-terminal RIA or
`ELISA were not significantly dilferent from each other but were
`significantly lower than those measured by the C0011-terminal RIA
`(ANOVA and t test; ‘P < 0.05; “P < 0.01). Data are means 3: SE.
`
`diifer significantly from those measured by the NH2-terminal
`RIA or the ELISA, indicating that after GLP-I administration,
`these assays measure predominantly nondegraded peptide
`(Table 1). The concentrations of the NH2-terminally trun-
`cated metabolite, calculated by subtracting the ELISA or
`NH2-terminal RIA results from the COOH-terminal RIA re-
`sults, gave values that were not significantly dilferent from
`those determined by HPLC in combination with RIA (Table
`1).
`In light of these results and the fact that the sensitivity of
`the l-lPLC—RlA method precludes determination of lower
`concentrations, all subsequent measurements were made
`using either the ELISA or the NH2-terminal RIA (as a
`measure of the intact, biologically active peptide), and the
`COOI-I-terminal RIA. Metabolite concentrations were calcu-
`
`lated as the dilference between the two assays.
`GLP-I administration. After subcutaneous administration
`
`of GLP-I(7-36) amide to nondiabetic and type II diabetic
`subjects, the concentrations of the biologically active pep-
`tide reached significantly lower levels and disappeared more
`rapidly than those measured with the more conventional
`COOH-terminal RIA (Fig. 3). The concentrations in the basal
`state were 34 1' 7 pmol/1 (ELISA) and 5 I I pmol/1 (COOH-
`temtinal RIA) in nondiabetic subjects and 60 t 9 pmol/1
`(ELISA) and 11 1* 2 pmol/1 (COOH-terminal RIA) in type II
`diabetic patients.
`During intravenous infusion of GLP-I(7—36) amide,
`
`the
`
`TABLE 1
`Concentrations of GLP-I peptides (pmol/I) after subcutaneous GLP—I(7-36) amide (4.5 nmol/kg) in healthy subjects
`
`GLP—I(7456) amide
`
`GLP-I(9 -36) amide
`
`Time (min)
`
`15
`30
`45
`
`HPLC/RIA
`
`534 2 115
`306 : 86
`117*
`
`ELISA
`
`512 : 24
`394 x 48
`210 : 17
`
`91022
`
`516 : 57
`373 1 4o
`276 1 39
`
`HPLC/RIA
`
`89390-ELISA
`
`89390-91022
`
`1,207 1- 215
`1,229 1' 227
`899 1 284
`
`1,059 1 248
`1,168 i 506
`954 t 293
`
`1,055 : 216
`1,188 1 483
`878 : 261
`
`Data are means : SE. Plasma (n = 3) was measured using an NH2—terminally directed (91022) and a COOH-temiinal amide-specific (89390)
`RIA and an ELISA specific for biologically active GLP-I. Concentrations of the intact peptide and the NH2-tenninally truncated metabolite
`were determined by a combination of HPLC and RIA There were no significant dilferences between the concentrations obtained by any
`of the methods of determination either of intact GLP-I or of its metabolite (ANOVA; P > 0.05). Results for HPLC/RIA were corrected for
`recovery of 4196. *Single detemiination.
`
`1128
`
`DIABETES, VOL. 44, SEPTEMBER 1995
`
`SANOFI-AVENTIS Exhibit 1057 - Page 1128
`IPR for Patent No. 8,951,962
`
`

`
`CF.DEACONANDASSOCmTES
`
`concentrations of the metabolite may be. It has recently been
`shown that the vasoactive peptide angiotensin H is further
`metabolized to a truncated peptide, angiotensin (1-7), which
`antagonizes angiotensin H at the AT, receptor (26). A similar
`situation may also exist for GLP-I, since it has been shown in
`vitro that GLP-I(9—36) amide is an antagonist of the intact
`peptide at the GLP-I receptor (15). However, whether the
`plasma or local concentrations of the metabolite reach
`sufliciently high levels to functionally antagonize the biolog-
`ically active peptide remains to be seen.
`Elevated fasting levels of GLP-I immunoreactivity have
`been reported in type II diabetic patients (17). Moreover,
`both intravenous arginine and oral glucose caused a hyper-
`secretion of GLP-I in these patients compared with healthy
`control subjects, and this was accompanied by an impaired
`insulin response. The elevated concentrations in the fasting
`state and during arginine infusion were partly explained by
`an increase in the major proglucagon fragment [progluca-
`gon(72—l58)], although after oral glucose the main form was
`GLP-I(7—36) amide. A reduced incretin effect
`in type II
`
`{I
`Q
`—.G
`v.-.9n.
`.l¢.l
`23am
`«.935
`E
`O.
`at
`3
`E
`
`20
`
` = 60
`
`0
`
`0
`
`30
`
`60
`
`90
`
`120
`
`150
`
`Tlme(m|n)
`
` O
`
`135
`
`180
`
`225
`
`270
`
`45
`
`90
`
`ion
`I—'
`vl-21:.
`‘J0
`23%
`«.35
`g
`E
`28
`ea
`
`8E
`
`Tlme (min)
`FIG. 4. Increase in plasma concentrations of GLP-I, after subtraction of
`endogenous levels, during intravenous infusion of GU’-l(7-36) amide
`(1.2 pmol - kg" -min“) in healthy subjects (n = 8;A ) and type 11
`diabetic patients (n = 8; 8) measured with a COOH-terminal
`amide-specific BIA (0; 89390) and an N11,-terrninally directed MA (I;
`91022). Comparison between assays: ‘P < 0.05; "P < 0.01; paired t test
`(points enclosed by the square bracket are all significant at P < 0.01).
`Data are means 3: SE.
`
`1129
`
`SANOFI-AVENTIS Exhibit 1057 - Page 1129
`IPR for Patent No. 8,951,962
`
`A
`2 800
`
`
`
`G 2
`
`'-'f'.nA
`_,|0
`egé
`an
`133
`eg0
`E
`
`0
`
`30
`
`60
`
`90
`
`120 150
`
`180
`
`T|ms(mln)
`
`B
`A 400
`'8
`2
`?.n
`5»;
`23%
`an E zoo
`5”“00
`20
`=§0
`E
`
`300
`
`1oo
`
`,.
`
`
`
`-100
`
`0
`
`60
`
`120
`
`180
`
`240
`
`T|me(m|n)
`FIG. 3. Increase in plasma concentrations of GLP-I, after subtraction of
`endogenous levels. after subcutaneous administration 0! GLP-i(7—36)
`amide (1.6 nmol/kg) in healthy subjects (n = 8;A) and type II diabetic
`patients (n = 8; B) measured with a COOB-terminal amide-specific 111A
`(0; 89390) and an ELISA (A) specific for the biologically active peptide.
`Comparison between assays: ‘P < 0.05; "P < 0.01; paired t test. Data
`are means : SE.
`
`se. Further studies using age- and weight-matched subjects
`are required to clarify this point Thus, actual plasma levels
`of the biologically active peptide achieved after exogenous
`GLP—I are much lower than has previously been thought, as
`was also found for endogenous GLP-I (14). Given these
`findings, it is likely that many if not most of the published
`values of GLP-I concentrations have been overestimated in
`
`terms of the biologically active peptide, since all assays
`described thus far also measure the metabolite.
`
`These results are of considerable clinical relevance, given
`that GLP-I has been proposed as a therapeutic agent in the
`management of diabetes (7-1 1). The plasma half-life of the
`intact peptide is likely to be much shorter than the 3-11 min
`found in various in vivo studies, since these have relied on
`COOH-temiinally (21) or centrally directed antisera (4,5,22,
`23) or on following the disappearance of radiolabeled pep-
`tide (24), methods that will also measure the NH2-terminally
`truncated metabolite. However, these findings also indicate
`that GLP-l(7—36) amide may be more potent than previously
`thought, since biological effects are being observed despite
`the true concentrations of the intact peptide being lower.
`It is not clear what the physiological relevance of the high
`
`DIABETES, VOL 44, SEPTEMBER l995
`
`

`
`GLP-I DEGRADATION IN HUMANS
`
`diabetic subjects is well known (26) and has been explained
`as being caused by a diminished sensitivity of the B-cell to
`glucoincretin action rather than as a result of their reduced
`secretion (11). However, pharmacological doses of GLP-I are
`still able to stimulate insulin secretion in these patients
`(7,9,27). In the light of the findings of the present study, this
`apparent paradox may be partly explained by elevated
`concentrations of the metabolite GLP-[(9-36) amide in dia-
`betic subjects. The chromatographic method used by Qfirskov
`et al. (17) would be unlikely to differentiate between the
`metabolite, truncated only by two amino acids, and the intact
`peptide, while the side-viewing GLP-I RIA used would mea-
`sure both peptides, suggesting a hypersecretion. Further-
`more, since GLP-l(9—36) amide has no eflicacy of its own but
`could additionally antagonize the action of any residual
`intact peptide (15), this could explain the reduced insulin
`secretion and apparent diminished B—cell sensitivity to GLP-I
`seen in diabetes. Exogenously administered GLP-I(7—36)
`amide would still elicit an insulin response by transiently
`raising plasma concentrations of the intact biologically ac-
`tive peptide.
`The metabolism of GLP-I(7-36) amide cannot be ac-
`counted for entirely by the action of plasma dipeptidyl
`peptidase IV, since the measured in vitro half—life in plasma
`of 20 min (14,28) greatly exceeds the in vivo half-life of 3-11
`min (4,5,2l—24). Tissues other than the plasma must there-
`fore be important sites of GLP-I metabolism, but the enzymes
`involved have not been studied. However,
`inhibition of
`dipeptidyl peptidase IV may prove a useful adjunct in the
`management of type H diabetes, as has been the case for the
`development of angiotensin-converting enzyme inhibitors to
`treat hypertension (29) and the suggested use of neutral
`endopeptidase inhibitors to enhance endogenous atrial na-
`triuretic peptide activity in the treatment of heart failure
`(30). Inhibition of GLP-I(7-36) amide degradation would not
`only increase the availability of the biologically active pep-
`tide but would also reduce the effect of feedback antagonism
`at the level of the receptor. The amount of degradation of
`GLP-I(7-36) amide also indicates that GLP-I analogues resis-
`tant to NH2-terminal metabolism could prove useful in the
`treatment of diabetes.
`
`In summary, this study has demonstrated that GLP-I(7-36)
`amide is rapidly degraded when administered by subcutane-
`ous or intravenous routes to diabetic and nondiabetic sub-
`
`jects. Since the product of the metabolism, an NH2-terminally
`truncated peptide which lacks eificacy, cross-reacts in CO0H-
`terminally or centrally directed assays for GLP-I, the use of
`assays directed toward the NH2-terminus is preferable for
`accurate measurement of changes in the concentrations of
`the biologically active peptide after its exogenous adminis-
`tration. The relevance of the metabolite in terms of whether
`
`it is a functional antagonist in vivo requires further study.
`
`ACKNOWLEDGMENTS
`
`This work was supported by grants from the Danish Medical
`Research Council, Novo Nordisk Foundation, and Deutsche
`Forschungsgemeinschaft, Bonn 2 (Bad Godesberg) (Grant
`203/2-2).
`The technical assistance of Lise Rabenhoj, Lene Albaek,
`Annette Feldt, and Mette Winther is gratefully acknowl-
`edged. We thank Robert Ritzel, Jens Werner, and Dirk
`Wollschlinger for help with performing the experiments and
`
`1130
`
`Richard Carr for useful discussion during the manuscript
`preparation.
`
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