Improved Glucose Tolerance in Zucker Fatty Rats
`by Oral Administration of the Dipeptidyl Peptidase
`IV Inhibitor Isoleucine Thiazolidide
`
`Raymond A. Pederson, Heather A. White, Dagmar Schlenzig, Robert P. Pauly, Christopher H.S. McIntosh,
`and Hans-Ulrich Demuth
`
`The hormones glucose-dependent insulinotropic polypep-
`tide (GIP) and glucagon-like peptide (GLP)-1 act on the
`pancreas to potentiate glucose-induced insulin secretion
`(enteroinsular axis). These hormones (incretins) are
`rapidly hydrolyzed by the circulating enzyme dipeptidyl
`peptidase IV (DP IV) into biologically inactive NH2- t e r-
`minally truncated fragments. This study describes the
`e ffect of inhibiting endogenous DP IV with a specific DP
`IV inhibitor, isoleucine thiazolidide (Ile-thiazolidide), on
`glucose tolerance and insulin secretion in the obese
`Zucker rat. In initial studies, the specificity of Ile-thiazo-
`lidide as an inhibitor of incretin degradation was deter-
`mined using matrix-assisted laser desorption/ioniza-
`tion–time of flight mass spectrometry. These results
`showed that inhibiting DP IV activity with Ile-thiazoli-
`dide blocked the formation of NH2-terminally truncated
`GIP and GLP-1. Oral administration of Ile-thiazolidide
`resulted in rapid inhibition of circulating DP IV levels by
`65% in obese and lean Zucker rats. Suppression of DP IV
`levels enhanced insulin secretion in both phenotypes with
`the most dramatic effect occurring in obese animals
`(150% increase in integrated insulin response vs. 27%
`increase in lean animals). Ile-thiazolidide treatment
`improved glucose tolerance in both phenotypes and
`restored glucose tolerance to near-normal levels in obese
`animals. This was attributed to the glucose-lowering
`actions of increasing the circulating half-lives of the
`endogenously released incretins GIP and, particularly,
`GLP-1. This study suggests that drug manipulation of
`plasma incretin activity by inhibiting the enzyme DP IV is
`a valid therapeutic approach for lowering glucose levels
`in NIDDM and other disorders involving glucose intoler-
`ance. Diabetes 47:1253–1258, 1998
`
`The term enteroinsular axis refers to the signaling
`
`pathways between the gut and pancreatic islets
`that amplify the insulin response to absorbed
`nutrients (1–3). Glucose-dependent insulinotropic
`polypeptide (GIP) and the truncated form of glucagon-like pep-
`
`From the Department of Physiology (R.A.P., H.A.W., R.P. P., C.H.S.M.), Uni-
`versity of British Columbia, Va n c o u v e r, Canada; and the Department of Drug
`Biochemistry (D.S., H.-U.D.), Hans Knoell Institute for Natural Product
`Research, Halle, Germany.
`Address correspondence and reprint requests to Dr. R.A. Pederson, Uni-
`versity of British Columbia, 2146 Health Sciences Mall, Va n c o u v e r, B.C. V6T
`1Z3, Canada. E-mail: pederson@unixg.ubc.ca.
`Received for publication 17 March 1998 and accepted in revised form 5
`May 1998.
`DP IV, dipeptidyl peptidase IV; GIP, glucose-dependent insulinotropic
`polypeptide; GLP, glucagon-like peptide; MALDI–TOF MS, matrix-assisted
`laser desorption/ionization–time of flight mass spectrometry.
`
`tide-1 (GLP-1(7-36) amide) are considered to be the most
`important insulin-releasing hormones (incretins) comprising
`the enteroinsular axis (2–4). GIP and GLP-1 are members of
`the glucagon family of peptides and share considerable NH2-
`terminal sequence identity, including alanine residues in posi-
`tion 2 from the NH2-terminus. GIP and GLP-1(7-36) have been
`shown to be substrates of the circulating exopeptidase dipep-
`tidyl peptidase IV (DP IV) (5–8), a peptidase that specific a l l y
`cleaves the first two amino acids from peptides with an NH2-
`terminal penultimate proline or alanine residue (9). The prod-
`ucts of DP IV hydrolysis, GIP(3-42) and GLP-1(9-36), have
`been shown by us and others to lack insulinotropic activity
`(10–13). Numerous studies support the view that DP IV–medi-
`ated hydrolysis of these hormones is the primary mechanism
`of their inactivation in vivo (5–8).
`The tripeptide Ile-Pro-Ile (diprotin A) acts as a competitive
`substrate of DP IV in vitro (14), and it has been shown to
`block DP IV–mediated incretin degradation in vitro ( 6 , 7 ) .
`Diprotin A has not been effective in inhibiting DP IV levels
`in vivo, as this tripeptide serves as a substrate for DP IV and
`high concentrations (molar range) are required to inhibit
`circulating DP IV levels in the rat (R.A.P., R.P. P., unpublished
`o b s e r v a t i o n s ) . Ile-thiazolidide is a highly specific reversible
`competitive transition-state analog inhibitor of DP IV (Ki =
`130 nmol/l) synthesized by H.-U.D. (9,15). We have recently
`demonstrated that matrix-assisted laser desorption/ion-
`ization–time of flight mass spectrometry (MALDI–TOF MS)
`is a highly sensitive and specific method to study the
`hydrolysis of GIP and GLP-1 by DP IV (8). In the present
`s t u d y, we have used this technique to investigate the effec-
`tiveness of Ile-thiazolidide as an inhibitor of DP-IV cataly-
`sis of these hormones.
`The Zucker fatty rat exhibits abnormalities in glucose
`metabolism that characterize NIDDM, i.e., insulin secretory
`defects as well as insulin resistance (16,17) leading to hyper-
`insulinemia and glucose intolerance. Based on the known
`incretin-metabolizing actions of DP IV and the specificity of
`Ile-thiazolidide as a DP IV inhibitor, it was hypothesized that
`this compound could influence glucose tolerance in vivo by
`increasing the circulating half-lives of the incretins GIP and
`GLP-1. The use of an animal model of NIDDM was deemed
`appropriate given the effectiveness of exogenous GLP-1 as
`a glucose-lowering agent in NIDDM patients (18).
`We first established that orally administered Ile-thiazoli-
`dide was effective in inhibiting circulating levels of DP IV
`in rats. We then undertook a study to determine the effect
`of DP IV inhibition by orally administered Ile-thiazolidide
`on glucose tolerance and insulin secretion in the fatty
`Zucker rat.
`
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`IMPROVED GLUCOSE TOLERANCE IN ZUCKER RATS
`
`RESEARCH DESIGN AND METHODS
`In vitro inhibition of DP IV by Ile-thiazolidide. Pooled human serum (20%)
`was incubated with GIP(1-42) (30 µmol/l) or GLP-1(7-36) (30 µmol/l) in 0.1 mmol/l
`Tricine buffer, pH 7.6, at 30°C in the presence or absence of 20 µmol/l Ile-thiazoli-
`dide. After a 21- to 24-h incubation, an equal volume of analyte and matrix (2 , 6 -
`diydroxyacetophone) was combined, crystallized, and analyzed by MALDI-TOF MS
`as described by Pauly et al. (8). All spectra represent the cumulative sum of 250 sin-
`gle laser shots. Signals were quantified as relative amounts of GIP(1-42) or GLP-1(7-
`36): the net substrate peak height divided by the sum of the net substrate and
`product peak heights. Net peak heights were defined as peak height minus baseline.
`Animals. A colony of Zucker rats was bred in the physiology department at the
`University of British Columbia. Age-matched groups (10–12 weeks) of obese
`(fatty) and lean animals of either sex were used. Fatty rats were homozygous
`(f a/f a), and lean animals were either F a/f a or F a/F a. All experiments were car-
`ried out on conscious unrestrained rats.
`Oral glucose tolerance test. After an overnight fast, lean or obese animals were
`administered oral glucose by syringe and feeding tube (1 g/kg) as a 40% solution
`(wt/vol). The DP IV inhibitor Ile-thiazolidide was dissolved in saline and adminis-
`tered along with the glucose at a dose of 20 µmol/l per 300 g body wt. In control
`experiments, saline was administered along with oral glucose. Blood samples were
`collected from the tail veins of conscious unrestrained rats into heparinized capil-
`lary tubes at 0 and 5, 10, 20, 30, and 60 min after glucose (glucose + Ile-thiazolidide)
`administration. Blood samples were centrifuged at 4°C, and DP IV activity was ana-
`lyzed immediately. The remaining plasma was stored at –20°C until analysis for glu-
`cose and insulin measurement. Glucose levels were measured using the glucose oxi-
`dase procedure (Beckman glucose analyzer; Fullerton, CA). To determine whether
`Ile-thiazolidide had a direct effect on insulin secretion or fasting glucose levels (in
`the absence of glucose-stimulated incretin release), in one set of experiments, Ile-
`thiazolidide was administered orally with saline instead of glucose.
`Assays. Insulin was measured by radioimmunoassay as described by Pederson
`et al. (19), using rat insulin as standard and a guinea pig anti-human insulin serum
`(GP01). Plasma DP IV activity was measured by a colorimetric assay. Gly-Pro-4-
`nitroanilide, a chromogenic substrate of DP IV, is hydrolyzed into the dipeptide
`Gly-Pro and the yellow product 4-nitroaniline, whose rate of appearance can be
`measured spectrophotometrically. The substrate consisted of 0.26 mmol/l Gly-Pro-
`nitroanilide (Sigma, St. Louis, MO) in 0.04 mol/l HEPES buffer. The assay mixture
`consisted of 270 µl of substrate and 30 µl plasma, and assays were carried out in
`96-well microtiter plates. Optical density was measured at 0, 10, and 20 min by a
`Dynatech MRX Microplate Reader (Chantilly, VA) (wavelength 405 nm). DP IV
`activity is expressed as the change in optical density over 20 min.
`Reagents. Ile-thiazolidide was synthesized in the laboratory of H.-U.D. (chemi-
`cal structure Fig. 1A) .
`Statistical analysis. Comparisons between drug-treated and control rats were
`assessed by unpaired Student’s t tests (P < 0.05 for signific a n c e ) .
`
`R E S U LT S
`In vitro inhibition of DP IV by Ile-thiazolidide. I n c u b a t i o n
`of 30 µmol/l GIP(1-42) in 20% human serum resulted in the
`hydrolysis of 71% of the native GIP into the reaction product
`GIP(3-42), as assessed by MALDI-TOF MS (Fig. 1B). Similarly,
`incubation of 30 µmol/l GLP-1(7-36) with serum for 21 h
`resulted in hydrolysis of 89.3% of the original peptide into the
`reaction product GLP-1(9-36) (Fig. 1C). Neither GIP(3-42) n o r
`GLP-1(9-36) were detected in parallel experiments conducted
`under identical conditions but in the presence of 20 µmol/l Ile-
`t h i a z o l i d i d e .
`Oral glucose tolerance in lean and obese Zucker rats. F i g-
`ure 2A and B indicate that obese Zucker rats are hyperinsu-
`linemic, exhibit fasting hyperglycemia (obese, 9.8 ± 0.33 mmol/l;
`lean, 7.5 ± 0.16 mmol/l; P < 0.05), and are glucose intolerant com-
`pared with lean age-matched control rats (peak values: obese,
`19.2 ± 0.56 mmol/l; lean, 15.47 ± 0.32 mmol/l; P < 0.05).
`In vivo inhibition of DP IV activity by Ile-thiazolidide.
`Oral administration of Ile-thiazolidide at a concentration of 20
`µmol/l per 300 g body wt resulted in significant inhibition of
`circulating DP IV activity 5 min after oral administration (Figs.
`3A, 4A, and 5A). Maximum inhibition was observed at time 30
`min (65% suppression). Preliminary experiments indicate that
`plasma DP IV activity returns to pretreatment levels after
`
`B
`
`C
`
`A
`
`FIG. 1. MALDI–TOF MS analysis of GIP(1-42) (30 µmol/l) (B) and GLP-
`1(7-36) (30 µmol/l) (C) degradation by serum DP IV in the presence
`or absence of 20 µmol/l Ile-thiazolidide. Signals of the intact hor-
`mone peaks [GIP(1-42) and GLP-1(7-36)] and the NH2- t e r m i n a l l y
`truncated DP IV reaction products [GIP(3-42) and GLP-1(9-36)] are
`identified. A: The structure of Ile-thiazolidide.
`
`12–14 h in both lean and obese animals (data not shown).
`E ffect of Ile-thiazolidide on glucose tolerance in lean
`and obese Zucker rats. Figures 3 and 4 show the glucose
`and insulin responses to an oral glucose challenge in lean and
`obese Zucker rats, respectively, in the presence or absence of
`oral Ile-thiazolidide. Figures 3A and 4A show plasma DP IV
`activity in the presence or absence of oral Ile-thiazolidide. Fig-
`ures 3–5 insets show integrated insulin and glucose
`responses to an oral glucose challenge. In both lean and
`obese animals, suppression of DP IV levels enhanced the
`insulin response to oral glucose and improved glucose tol-
`erance. The insulin secretory response to oral glucose was
`greater in the presence of Ile-thiazolidide in both lean and
`obese rats. The increase in integrated insulin response result-
`ing from inhibition of circulating DP IV was greater in obese
`than in lean animals (Figs. 3B and 4B insets). The integrated
`insulin response to only glucose in the presence of Ile-thia-
`zolidide in obese rats was 150% greater than that in control
`rats compared with a 27% increase in lean animals. The
`improvement in glucose tolerance was also more dramatic in
`obese compared with lean animals after oral Ile-thiazolidide
`treatment, with a 39% decrease in integrated glucose com-
`pared with a 22% reduction after Ile-thiazolidide treatment of
`lean animals (Figs. 3C and 4C). This was most evident at
`time 60 min when glucose levels were 35% lower in obese Ile-
`thiazolidide–treated animals compared with nontreated
`
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`
`A
`
`B
`
`FIG. 2. Insulin (A) and glucose (B) responses to 1 g/kg oral glucose
`in lean (n = 6) and obese (n = 6) Zucker rats. *Significance to at least
`the 0.05 level.
`
`obese control rats (19.0 ± 0.5 vs. 12.5 ± 0.37 mmol/l), whereas
`plasma glucose levels in treated versus nontreated lean ani-
`mals were not significantly different at this time period (10.7
`± 0.4 vs. 11.9 ± 0.3 mmol/l) (Fig. 2C) .
`E ffect of Ile-thiazolidide on fasting glucose and insulin
`levels in obese Zucker rats. Ile-thiazolidide was adminis-
`tered in the absence of glucose to determine if the improve-
`ment in glucose tolerance in the obese Zucker rat was due to
`a direct glucose-lowering action of the drug. Figure 5 indicates
`that oral Ile-thiazolidide did not alter fasting glucose or
`insulin levels in the absence of endogenous incretin release.
`
`D I S C U S S I O N
`The circulating enzyme DP IV inactivates the circulating
`incretins GIP and GLP-1 by cleaving the NH2-terminal dipep-
`tide from both molecules. It has been shown by us and oth-
`ers that this occurs very rapidly in plasma and undoubtedly
`plays a regulatory role in the enteroinsular axis (5–8).
`We have previously demonstrated that circulating DP IV
`rapidly metabolizes GIP(1-42) and GLP-1(7-36) to the trun-
`cated forms GIP(3-42) and GLP-1(9-36) in vivo (6). Because
`N H2-terminal truncation destroys the insulin-releasing
`actions of both incretins (10–13), it was hypothesized that inhi-
`bition of plasma DP IV would result in improved glucose tol-
`erance by an incretin-mediated mechanism (prolonging the
`circulating half-life of intact biologically active GIP and GLP-
`1). Because GLP-1 has gained considerable importance as a
`
`R.A. PEDERSON AND ASSOCIATES
`
`glucose-lowering drug in NIDDM (18,20,21), it was of inter-
`est to determine the effectiveness of altering the circulating
`half-life of this hormone in an animal model of NIDDM, the
`obese Zucker rat. That obese Zucker rats from our colony ful-
`fill the criteria of insulin resistance, as well as fasting hyper-
`glycemia and glucose intolerance, is indicated in Fig. 2. The
`aims of the current study were twofold: 1) to determine the
`effectiveness of orally administered Ile-thiazolidide as an
`inhibitor of circulating DP IV activity and 2) to assess the
`effect of DP IV inhibition on the enteroinsular axis in the
`fatty Zucker rat.
`In an initial study to characterize the activity of Ile-thiazo-
`lidide on incretin metabolism, MALDI-TOF MS was used to
`investigate the effect of DP IV inhibitor Ile-thiazolidide on the
`in vitro degradation of GIP(1-42) and GLP-1(7-36) after incu-
`bation in human serum. Results presented in Fig. 1 indicate
`that DP IV is the principal serum protease responsible for the
`degradation of GIP(1-42) and GLP-1(7-36) into the inactive
`polypeptides GIP(3-42) and GLP-1(9-36), since the presence
`of Ile-thiazolidide, a highly specific inhibitor of DP IV, was able
`to completely block the formation of the DP IV reaction
`products during the 21- to 24-h incubation.
`Oral administration of Ile-thiazolidide resulted in prompt
`(within 5 min) inhibition of circulating DP IV activity with
`maximum suppression (65%) occurring 30 min after ingestion
`(Figs. 3A, 4A, and 5A). When administered with oral glucose,
`Ile-thiazolidide resulted in a significantly greater insulin
`response and attendant improvement in glucose tolerance in
`both lean and obese Zucker rats (Figs. 3 and 4). The degree
`of enhancement of the integrated insulin response to oral glu-
`cose resulting from DP IV inhibition was greater in obese than
`in lean animals (Figs. 3B and 4B), and the pattern of insulin
`secretion after DP IV inhibition differed in fat compared with
`lean animals. The greatest difference in insulin secretion
`between treated and untreated lean animals occurred 10 min
`after oral glucose in the presence of Ile-thiazolidide. The fin d-
`ing that insulin levels do not remain elevated in the DP
`IV–inhibited lean rats, despite an increase in the half-life of
`endogenously released incretins, implies the existence of a
`mechanism that prevents the secretion of inappropriate
`amounts of insulin (even in the presence of elevated levels of
`intact GIP and GLP-1). An explanation for falling insulin lev-
`els in the presence of elevated incretin concentrations would
`undoubtedly involve the concomitant reduction in plasma glu-
`cose, as both incretins stimulate insulin in a glucose-depen-
`dent manner (2–4). In the case of obese animals, the
`enhanced insulin response to Ile-thiazolidide occurred
`throughout the 60-min sampling period. A possible explana-
`tion for this observation is the impaired islet function in these
`animals; however, a contributing factor may also be the lack
`of a glucose threshold for the insulinotropic actions of both
`GIP and GLP-1 in the f a/f a rat (22,23). Long-acting incretins
`may exert a more prolonged insulinotropic action in animals
`lacking the normal self-regulating glucose threshold pos-
`sessed by lean (normal) animals. The glucose-lowering
`actions of DP IV suppression are more dramatic in obese
`compared with lean rats (Figs. 3C and 4C), as one would
`predict from the greater insulin response in drug-treated
`obese animals. In Ile-thiazolidide–treated obese rats, the glu-
`cose tolerance curve resembled that of the lean phenotype.
`At the 60-min interval, untreated obese animals exhibited
`n e a r-peak glucose values (19 mmol/l) compared with a 35%
`
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`IMPROVED GLUCOSE TOLERANCE IN ZUCKER RATS
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`A
`
`B
`
`C
`
`A
`
`B
`
`C
`
`FIG. 3. The effect of oral administration of Ile-thiazolidide on plasma
`DP IV activity (A) and the insulin (B) and glucose (C) responses to
`oral glucose in lean Zucker rats (n = 6 for each group). Insets repre-
`sent integrated responses. *Significance to at least the 0.05 level.
`
`FIG. 4. The effect of oral administration of Ile-thiazolidide on plasma
`DP IV activity (A) and the insulin (B) and glucose (C) responses to
`oral glucose in obese Zucker rats (n = 6 for each group). Insets rep-
`resent integrated responses. *Significance to at least the 0.05 level.
`
`decrease in Ile-thiazolidide–treated f a/fa animals (12.5
`mmol/l). The glucose-lowering effects of suppressing circu-
`lating DP IV with Ile-thiazolidide may stem from the insulin-
`independent glucose-lowering actions of intact circulating
`GLP-1 as well as enhancing the insulin-releasing actions of GIP
`
`and GLP-1 (24–28). The effectiveness of GLP-1 as a glucose-
`lowering agent in NIDDM patients has been attributed to the
`potent suppression of glucagon secretion and inhibition of
`gastric emptying as well as enhanced insulin secretion. These
`factors, as well as increased insulin secretion, may con-
`
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`A
`
`B
`
`C
`
`FIG. 5. The effect of oral administration of Ile-thiazolidide on plasma
`DP IV activity (A) and fasting insulin (B) and glucose (C) levels in
`obese Zucker rats (n = 6 for each group). Insets represent integrated
`r e s p o n s e s .
`
`tribute to the greater glucose-lowering action of DP IV inhi-
`bition in obese compared with lean rats, considering that
`glucagon levels are exaggerated in obese Zucker rats (29). Dis-
`advantages of incretin therapy are the rapid metabolism of
`exogenously administered native peptides in the circulation
`and ineffectiveness of oral administration. Inhibition of the
`incretin-inactivating enzyme DP IV by an oral drug over-
`comes both these problems. To determine whether the DP IV
`
`R.A. PEDERSON AND ASSOCIATES
`
`inhibitor Ile-thiazolidide had direct glucose-lowering actions,
`it was administered orally without glucose to fasted obese
`rats. Results presented in Fig. 5 indicate that Ile-thiazolidide
`neither lowered fasting glucose nor enhanced insulin levels
`in obese rats, in the absence of glucose-stimulated incretin
`release. This lends support to the hypothesis that this drug
`increases insulin secretion and improves oral glucose toler-
`ance by inhibiting the degradation of GIP and GLP-1 by the
`circulating enzyme DP IV, i.e., by an incretin-mediated mech-
`anism. DP IV also plays a role in the inactivation of regulatory
`peptides (other than the incretins) that possess proline or ala-
`nine residues in the penultimate NH2-terminal position.
`Examples are growth hormone–releasing hormone, neu-
`ropeptide Y, peptide YY, and prolactin (9). The effect of short-
`term inhibition of circulating DP IV activity on the actions of
`these peptides is as yet unknown. Regarding the possible
`toxicity of Ile-thiazolidide, no deleterious effects have been
`noted on long-term cell culture (9) or after 5 days of oral treat-
`ment in rats (H.A.W., R.A.P., unpublished observations).
`In summary, inhibition of circulating DP IV enhanced
`insulin secretion and improved glucose tolerance in response
`to an oral glucose challenge in lean and obese fatty (f a/f a) rats.
`The enhanced incretin response was greater in obese than in
`lean animals, with a more profound improvement in glucose
`tolerance by Ile-thiazolidide. This was attributed to disruption
`of DP IV inactivation of GIP and GLP-1, resulting in amplifi-
`cation of the enteroinsular axis. These data support a thera-
`peutic approach of drug manipulation of plasma incretin
`activity for lowering glucose levels in NIDDM and other dis-
`orders involving glucose intolerance.
`
`A C K N O W L E D G M E N T S
`We are grateful for financial support from the Medical
`Research Council of Canada, the Canadian Diabetes Associ-
`ation, and the British Columbia Health Research Foundation.
`
`R E F E R E N C E S
`1 . Unger RH, Eisentraut AM: Entero-insular axis. Arch Intern Med 1 2 3 : 2 6 1 – 2 6 6 ,
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`2 . Pederson RA: GIP. In Gut Peptides. Walsh J, Dockray G, Eds. New Yo r k ,
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`3 . Habener JF: The incretin concept and its relevance to diabetes. E n d o c r i n o l
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