efficacy of the dipeptidyl peptidase iv inhibitor isoleucine thiazolidide (p32/98) in fatty zucker...

OR I G I N A L A R T I C L E doi: 10.1111/j.1463-1326.2007.00813.x

Efficacy of the dipeptidyl peptidase IV inhibitor isoleucine

thiazolidide (P32/98) in fatty Zucker rats with incipient and

manifest impaired glucose tolerance

P. Augstein,1 S. Berg,1 P. Heinke,1 S. Altmann,1 E. Salzsieder,1 H. U. Demuth2 and

E. J. Freyse1

1Institute of Diabetes ‘Gerhardt Katsch’, Karlsburg, Germany2Probiodrug AG, Halle/Saale, Germany

Aim: Incretin enhancers are a new class of antidiabetic drugs with promising therapeutic potential for type 2 diabetes.

Therapeutic intervention in prediabetes is an attractive strategy for preventing or delaying diabetes onset. The aim of

the present study was to investigate the therapeutic effects of incretin enhancement on incipient impaired glucose

tolerance (iIGT) and manifest IGT (mIGT) using the dipeptidyl peptidase IV (DPP-4) inhibitor P32/98- and fatty Zucker

rat (ZR, fa/fa) as a model.

Methods: ZRs were classified into groups with iIGT and mIGT (n ¼ 10 per group). P32/98 (21.61 mg/kg body weight)

was administered orally to ZR with iIGT and mIGT once daily for 6 and 3 weeks respectively. Assessments included

body weight, morning blood glucose and insulin, oral glucose tolerance test (oGTT; 2 g glucose/kg), plasma parameters

and blood glucose day–night profile (DNP). In addition, glucose responsiveness of isolated islets and islet morphology

were analysed.

Results: P32/98 decreased non-fasting morning blood glucose more effectively in ZR with iIGT than in ZR with mIGT.

Compared with study entry, P32/98 improved DNP of blood glucose in ZR with mIGT and nearly normalized DNP in

ZRwith iIGT. An acute bolus of inhibitor reduced glucose load during oGTT in rats chronically treated with placebo or

P32/98. In contrast to placebo-treated rats, rats receiving long-term treatment with P32/98 required less insulin during

oGTT. This effect was larger in rats with iIGT vs. rats with mIGT. In isolated pancreatic islets of ZR with mIGT,

treatment with P32/98 decreased pancreatic insulin content and increased glucose responsiveness, while the b-cellvolume density was unaffected. P32/98 significantly reduced triglycerides and non-esterified fatty acids. Intestinal

growth was comparable between inhibitor- and placebo-treated fatty rats.

Conclusions: Enhancement of incretin with the DPP-4 inhibitor P32/98 has therapeutic effects in hyperinsulinaemia,

hyperglycaemia and IGT in ZR with iIGT and mIGT. Apparently, administration of P32/98 in ZR with iIGT results in

more efficient b-cell function, which is associated with less need for insulin to cope with deterioration of glucose

tolerance. Importantly, P32/98 has a strong effect on dyslipidaemia in mIGT. P32/98 has no side effect on intestinal

growth. Daily intake of P32/98 is a promising strategy for treatment of glucose intolerance and has the potential to

prevent type 2 diabetes.

Keywords: dipeptidyl peptidase IV, fatty Zucker rats, glucagon-like peptide 1, ile-thiazolidide, impaired glucose tolerance, pancre-

atic islets, prediabetes, type 2 diabetes

Received 27 April 2007; revised version accepted 27 August 2007

Correspondence:

Petra Augstein, PhD, Institute of Diabetes ‘Gerhardt Katsch’, Karlsburg e.V., Greifswalder Str.11e,17495 Karlsburg, Germany.

E-mail:

[email protected]

850 j Diabetes, Obesity and Metabolism, 10, 2008, 850–861# 2007 The Authors

Journal Compilation # 2007 Blackwell Publishing Ltd

Introduction

Impaired glucose tolerance (IGT) and impaired fasting

glucose (IFG) are considered to be prestages of type 2 dia-

betes [1]. In 2000, 41 million Americans were diagnosed

with prediabetes [2]. Importantly, cardiovascular risk

factors such as obesity, hyperlipidaemia, hypertension

and insulin resistance are prevalent among patients

with prediabetes [3,4]. The dramatic increase in the

incidence of diabetes and its complications has promp-

ted worldwide efforts to prevent diabetes onset. Life-

style modification, including weight loss and exercise,

can reduce the incidence of diabetes in patients with

IGT [5]. Also, in high-risk subjects with IGT, drugs such

as metformin, acarbose and thiazolidinediones have

been shown to prevent type 2 diabetes [6–8].

Two new classes of antidiabetic drugs with promising

therapeutic potential for people with type 2 diabetes are

incretin mimetics and incretin enhancers [9]. The incre-

tin hormone glucagon-like peptide 1 (GLP-1) is pro-

duced in the L cells of the small- and large intestine and

released by several components of digested food [9].

GLP-1 increases postprandial insulin secretion during

conditions of increased blood glucose, inhibits glucagon

secretion and decelerates stomach emptying [9,10]. The

effects of endogenous GLP-1 are markedly reduced in

type 2 diabetes but are restored after administration of

exogenous GLP-1 [9]. In patients with diabetes, continu-

ous administration of GLP-1 lowers blood glucose to

near-normal levels in both the fasting and the post-

prandial state [9]. However, circulating intact GLP-1 is

degraded within minutes by the protease dipeptidyl

peptidase IV (DPP-4), leading to a decline in plasma lev-

els of active GLP-1 after meals [9,11]. Inactivation of

GLP-1 by DPP-4 can be prevented by application of

cleavage-resistant GLP-1 analogues (incretin mimetics)

or DPP-4 inhibitors (incretin enhancers), which result in

enhanced and prolonged GLP-1 effects [11,12]. Both

strategies have been successful in clinical trials [9,10].

Several DPP-4 inhibitors have been developed during

recent years, for example, NVP-DPP 728 [12], valine-

pyrrolidide [13] and ile-thiazolidide, also known as

P32/98 [11,14]. P32/98 improved glucose tolerance,

insulin sensitivity and b-cell responsiveness [14,15] in

preclinical studies using the fatty Zucker rat (ZR, fa/fa)

[11], an animal model for IGT [16–19]. As a result of

hyperphagia, these animals become overweight and

develop insulin resistance, hyperinsulinaemia, hyper-

glycaemia and hyperlipidaemia.

The present study investigated the therapeutic efficacy

of the incretin enhancer P32/98 in prediabetes using

glucose-intolerant fatty ZR. We investigated the acute

and chronic effects of P32/98 administration on the pro-

gression of prediabetes in rats with incipient IGT (iIGT)

and manifest IGT (mIGT). We monitored the long-term

parameters such as body weight, blood glucose, plasma

insulin and food and water intake as well as glycated

haemoglobin (gHb), triglycerides and non-esterified fatty

acids (NEFA) and evaluated the morphological and

in vitro characteristics of pancreatic islets.

Materials and Methods

Animals

Male fatty (fa/fa) ZR andmale lean ZR (�/� or fa/�) were

purchased fromCharles River (Sulzfeld, Germany) at age

4 weeks (fatty ZR: n ¼ 20; lean ZR: n ¼ 11) and 9 weeks

(fatty ZR: n ¼ 20; lean ZR: n ¼ 10). The rats were housed

singly and maintained under standardized semi-barrier

conditions with controlled temperature (22 � 2 °C) anda 12-h light/dark cycle, with light on at 06:00 hours. Ani-

mals had ad libitum access to acidified tap water and

commercial standard pelleted chow (ssniff�, Soest,

Germany).

DPP-4 Inhibitor

The DPP-4 inhibitor P32/98 {Ile-Thia*Fum; isoleucine

thiazolidide; 3-N-[(2S, 3S)-2-amino-3-methylpentanoyl]-

1, 3-thiazolidine hemifumarate} was kindly provided by

Probiodrug AG (Halle/Saale, Germany). The compound

was dissolved in 5 ml/kg 1% methylcellulose solution

in saline (methyl hydroxyethyl cellulose; Carl Roth

GmbH, Karlsruhe, Germany). It was orally administered

at a dose of 83 mM (21.61 mg/kg body weight) using a

feeding tube (15 g, 75 mm; Fine Science Tools, Heidel-

berg, Germany) at 5:00 pm. Control rats received the

same amount of placebo (5 ml/kg 1% methylcellulose

solution in saline).

Study Design

Thetimecourseofglucose intolerancewasdefinedinapre-

liminary study with ZR aged 6–32 weeks (figure 1). The

rats were classified by age, and at least six rats per class

were included in the study. Oral glucose tolerance test

(oGTT) was performed as described below in order to cat-

egorize glucose intolerance as incipient or manifest.

Ratswith iIGT received P32/98 for 6 weeks, between 9

and 15 weeks of age; rats with mIGT were treated for

3 weeks, between 20 and 23 weeks of age. Assessments

included monitoring of body weight, blood glucose,

plasma insulin and food andwater intake. At study entry

P. Augstein et al. Incretin enhancement in prediabetes j OA

# 2007 The Authors

Journal Compilation # 2007 Blackwell Publishing LtdDiabetes, Obesity and Metabolism, 10, 2008, 850–861 j 851

and termination, oGTT and day–night profile (DNP)

were performed. Blood samples were obtained from the

cut tip of the tail for measurement of gHb, NEFA and

triglycerides.

Rats with iIGT and mIGT continued to receive P32/98

or placebo treatment until they were sacrificed for pan-

creatic histology and assessment of islet function (iIGT:

2 weeks; mIGT: 5 weeks). Therefore, effects of P32/98 on

isletmorphology and functionwere investigateduntil age

17 weeks (iIGT) or 27 weeks (mIGT).

In Vivo Assessments

Body Weight

Body weight was measured daily during inhibitor ad-

ministration using a platform balance from SCALTEC

(Heiligenstadt, Germany).

Morning Non-fasting Blood Glucose and Plasma

Insulin

Twiceweekly during inhibitor administration, bloodwas

obtained from the tail vessel at 07:30hours. Blood glucose

was measured using the glucose oxidase procedure

(Super G Glukosemeßgerat; Dr. Muller Geratebau GmbH,

Freital, Germany). Plasma insulin concentration was

evaluated using the double-antibody radioimmunoassay

(RIA) method with the rat insulin kit (Linco Research,

St. Charles, MO, USA).

Glycated Haemoglobin

gHb was assayed using the VARIANT� program, with

boronate affinity high performance liquid chromato-

graphy for separation (Bio-Rad Diagnostics Group,

Hercules, CA, USA).

Plasma Parameters

Triglycerides (BM/Hitachi 911 analysis apparatus; Labor-

system Boehringer Mannheim GmbH, Mannheim,

Germany) andNEFA (commercial kit fromNEFAC;Wako

Chemicals, Osaka, Japan) were quantified using plasma

samples and manufacturer protocols.

Oral Glucose Tolerance Test

oGTTs were performed in ZR after a 16-h fast. A placebo

(1% methylcellulose solution) or P32/98 bolus (17 mM/

kg) was administered orally 30 min before the glucose

load. At time 0, 2 g glucose/kg was administered as

a 40% solution (B. Braun Melsungen AG, Melsungen,

Germany) through a feeding tube. Mixed venous blood

samples were collected from the tail vein at 0, 10, 20,

30, 40, 60, 90 and 120 min and processed for blood glu-

cose and plasma insulin, as described above.

Day–Night Profile

Blood glucose was monitored from 5 to 2 pm with

nt ¼ 3 h.

In Vitro and In Situ Assessments

The animals were lethally anaesthetized using Ketamine�

10 (Atarost GmbH, Twistringen, Germany). The pancreas

was prepared, and biopsies of the cauda pancreatis were

obtained for histology and determination of insulin con-

tent. The remaining tissue was digested with collagenase

to isolate pancreatic islets, as described below.

Pancreatic Insulin Content

A pancreatic biopsy of approximately 30 mg wet weight

was obtained. The tissue was quickly frozen in liquid

nitrogen and stored until acid extraction and radioimmu-

nological estimation of insulin content [20].

Islet Histology and Morphometry

Pancreatic biopsies were fixed in Bouin’s solution, dehy-

drated and processed by standard histological procedures.

Fig. 1 Time course of impaired glucose tolerance (IGT)

showing an increase in the area under the glucose curve

(G-AUC). The absolute G-AUC for 2-h oral glucose toler-

ance test was estimated over time in Zucker rat (dark

columns) and lean controls (white columns) to define

incipient IGT (age 10–18 weeks) and manifest IGT

(age >18 weeks). Mean � s.d. are shown. Rats were

classified according to their age (6 weeks, n ¼ 7; 8 weeks,

n ¼ 7; 10 weeks, n ¼ 7; 12 weeks, n ¼ 6; 16 weeks, n ¼19; 18 weeks, n ¼ 18; 22 weeks, n ¼ 10 and 32 weeks,

n ¼ 10). *p < 0.05 vs. week 8; #p < 0.05 vs. week 10.

OA j Incretin enhancement in prediabetes P. Augstein et al.



Each biopsy was evaluated using sections from seven tis-

sue levels, at least 200 mm apart.

To determine the relative islet cell volumedensity (%of

total pancreatic tissue), 5-mm sections were stained with

haematoxylin/eosin (HE) and semi-quantified by mor-

phometry using the point-counting method [21,22]. A

225-point grid (Carl Zeiss, Jena, Germany) was randomly

placed 10 times on each section at a final magnification

of 200�. The number of intercepts over islet cells and

exocrine pancreatic tissue was counted in 70 non-

overlapping, randomly chosen fields (i.e. 13 500 points)

per biopsy. The number of intercepts over islet cells was

divided by the number of intercepts over total pancreatic

tissue, and the product was multiplied by 100.

The relative b-cell volumedensity (%of total pancreatic

tissue) was determined using sections stained with

a guinea pig insulin antibody (Dako, Hamburg, Germany)

diluted 1 : 750 in phosphate-buffered saline for 1 h at

room temperature.Antibodybindingwas visualizedusing

a secondary immunoperoxidase-labelled antibody (Dako)

and the chromogenic reagent 3-amino-9-ethylcarbazole

(Dako). All sections were counterstained with haematox-

ylin and examined by morphometry. The intercepts over

insulin-positive cells and exocrine pancreatic tissue were

counted in70non-overlapping fields.Thenumberof inter-

cepts over b cells was divided by the total number of inter-

cepts, and the product was multiplied by 100.

Formeasurement of islet size, a calibrated eyepiecewas

applied on HE-stained sections to obtain the minimum

(d1) and maximum (d2) diameter of an islet. With these

values and the formula p(d1 þ d2)2 / 16, the islet area

(mm2) could be calculated. The islets were then arbi-

trarily classified into small (<0.005 mm2), medium

(0.005–0.020 mm2) and large (>0.020 mm2) sizes, and

the relative islet distribution (%) was calculated.

Islet Function In Vitro

Pancreatic islets were isolated under aseptic conditions

by in situ collagenase digestion (SIGMA-ALDRICH).

The abdominal cavity was opened, and prewarmed

(37 °C) collagenase solution (1 mg/ml) was infused into

the bile duct after clamping the papilla vateri. The pan-

creas, expanded by the infused collagenase solution,

was removed and transferred to a tube; the tissue was

then exposed to collagenase digestion for 13 min. Diges-

tion was stopped by ice-cold Hanks balanced salt solu-

tion. After washing, the single islets were selected

under a stereomicroscope. Each experiment used islets

from one animal.

The glucose responsiveness of isolated islets was

assessed by static incubation [23] and defined as the

amount of islet insulin secretion at 20 and 2 mmol/l glu-

cose. The stimulation factor describes the glucose re-

sponsiveness and was calculated as quotient of islet

insulin secretion at 20 and 2 mM glucose.

Freshly prepared islets were preincubated in Gey-and-

Geybuffercontaining5 mmol/lglucose for 30 minat37 °Cin a humidified atmosphere (95% air and 5% CO2). Islets

were washed, and groups of 3 � 5 islets were incubated

in 400 ml Gey-and-Gey buffer with 2, 10 or 20 mmol/l

glucose for 2 h at 37 °C. Duplicate aliquots of the incuba-

tion medium were removed for RIA [24]. Groups of 4 � 5

freshly isolated islets were sonicated for estimation of the

insulin content. Duplicate aliquots of each homogenate

were used for insulin determination by RIA.

Wet Weight of the Small- and Large Intestine

The small- and large intestineswere obtained from anaes-

thetized rats. After cleaning away the chymus with tap

water, the wet weight of the intestine was measured on

a balance scale (OHAUS Corporation, Florham Park, NJ,

USA).

Statistical Methods

Statistical evaluations and graphics were performed

using Excel 2003 and the STATISTICAL PACKAGE FOR

THE SOCIAL SCIENCES Version 12.0 (SPSS, Chicago, IL,

USA). All parameters were analysed with descriptive

statistics, including the mean and s.e.m. The results of

oGTT were obtained by determining the area under the

curve (AUC) from 0 to 120 min for blood glucose (G-

AUC) and insulin (I-AUC). AUCs were calculated using

the linear trapezoidal formula. For the reactive AUC, the

baseline value was 0 min. For the DNP, the mean of all

measured blood glucose values, the time with blood glu-

cose elevation above 6.5 mmol/l and the difference be-

tween maximal and minimal blood glucose values (nBG)

were calculated. A two-tailed t-test was used to compare

treatment groups. A paired t-test was used to test within-

group differences. Significance was set at p < 0.05.

Results

Time Course of Glucose Intolerance

TheG-AUC for ZR at 6 and 8 weeks of agewas comparable

with that for lean control rats (figure 1). The lean control

rats remained glucose tolerant throughout the study,

whereas in the ZR, there was a significant increase in the

G-AUC between 8 and 10 weeks of age (775 � 24


# 2007 The Authors


vs. 1159 � 55 mmol min/l; p < 0.001), and the G-AUC

further increased until 18 weeks of age (1497 � 69 mmol

min/l; p < 0.01 vs. week 10). Therefore, the period

between 10 and 18 weeks of age was defined as a phase

of iIGT. Between 18 and 32 weeks of age, the G-AUC for

ZR remained high (p ¼ 0.72 vs. week 18). By definition,

ZRs older than 18 weeks were mIGT. Thus, we investi-

gated the efficacy of DPP-4 inhibition in 9-week-old ZR

with iIGT and 20-week-old ZR with mIGT (figure 1).

P32/98 Inhibition of DPP-4 in ZR with iIGT

Effects of Chronic Administration

Comparedwithplacebo, P32/98 reducedmean food intake

(data not shown) by 13% (25.6 � 1.1 vs. 22.2 � 1.7 g/day;

p ¼ 0.10) and bodyweight gain by 5% inZR (163 � 10 vs.

131 � 16 g; p ¼ 0.13) (figure 2). However, daily water

intake was increased by 26% with P32/98 (30.6 � 2.1 vs.

38.6 � 4.0 ml/day; p ¼ 0.08; data not shown).

During the study period, the non-fasting blood glucose

of placebo-treated ZR increased significantly (p < 0.01

vs. t0; table 1), whereas the non-fasting blood glucose of

P32/98-treated ZR remained at the initial level (p ¼ 0.25

vs. t0; table 1). Non-fasting plasma insulin tended to

increase in placebo-treated ZR (p ¼ 0.12 vs. t0; table 1),

whereas it declined significantly during the 6 weeks of

P32/98 treatment (p < 0.05 vs. t0; table 1).

DNPs confirmed the blood glucose lowering effect of

P32/98 (table 1 and figure 3a, b), demonstrating a signifi-

cant reduction of mean blood glucose and fasting blood

glucose (p < 0.01 vs. t0). There was also a trend towards

reduction of nBG compared with placebo (p ¼ 0.11;

table 1).

Importantly, P32/98 reduced the age-dependent

increase in gHb compared with placebo in iIGT ZR

(P32/98: þ0.32 � 0.14%, p ¼ 0.07 vs. t0; placebo: þ0.60

� 0.11%, p < 0.001 vs. t0; table 1). In addition, while

placebo-treated ZR demonstrated an age-related increase

in NEFA (p < 0.05 vs. t0), NEFA remained stable in ZR

treated with P32/98 (p ¼ 0.52 vs. t0; table 1). There was

a trend towards a reduction in plasma triglycerides with

P32/98 vs. placebo (p ¼ 0.09; table 1). Daily P32/98

administration did not affect the weight of the small or

large intestine (data not shown).

Acute and Chronic Effects in the oGTT

The chronic and acute effects of DPP-4 inhibition on IGT

were evaluatedbybolus administrationofplaceboorP32/

98 30 min before oGTT in rats treated chronically with

placebo or P32/98.

At study entry, the G-AUCwas high, as expected for ZR

of this age (table 1 and figure 1). After 6 weeks of treat-

ment, placebo bolus administration produced similar

glucose tolerance curves in placebo- and P32/98-treated

ZR (figure 4a). Indeed, the G-AUC was higher at week 6

than at week 0 for both the placebo (p < 0.05 vs. t0) and

the P32/98 (p ¼ 0.08) groups (table 1). In placebo-

treated control rats, basal insulin release was slightly

higher and glucose-stimulated insulin release was sub-

stantially higher during the oGTT compared with P32/

98-treated ZR (figure 4c). In contrast, basal insulin

release and glucose-stimulated insulin release were

lower after oral glucose loading in the P32/98 group

compared with the control (p < 0.05) (figure 4c). As

a consequence, the I-AUC was lower in the P32/98

group vs. the placebo-treated group (p < 0.05; table 1).

Acute P32/98 bolus before oGTT improved the glucose

tolerance curve (figure 4b) and decreased the G-AUC for

chronic placebo-treated ZR (p < 0.05 vs. oGTT with

placebo bolus). A similar improvement in the glucose

tolerance curve (figure 4b) as well as a significant reduc-

tion in the G-AUC was achieved following acute P32/98

administration in the chronic P32/98 group (p < 0.05

vs. oGTT with placebo bolus). Thus, acute P32/98 bolus

before oGTT improved glucose tolerance in ZR rats

chronically treated with placebo or P32/98 (table 1).

In the oGTT, following acute P32/98 bolus, basal

plasma insulin was lower in the chronic P32/98 group

compared with the placebo group (p < 0.05 vs. placebo;

figure 4d). However, in chronic placebo-treated ZR, the

insulin response during the oGTT was threefold higher

than basal insulin release (figure 4d). The insulin

response was smaller in the P32/98 group vs. the pla-

cebo group (figure 4d); accordingly, the I-AUC was also

Fig. 2 Body weight gain of Zucker rat with incipient

impaired glucose tolerance treated once daily with P32/98

(d) or placebo (s). Data are the mean � s.e.m. (n ¼ 8 for

each group).




smaller in the P32/98 group (p < 0.05 compared with

placebo group; table 1).

Effects on Islet Morphology and Function

There was no difference between placebo- and P32/98

animals in islet size (p ¼ 0.55; data not shown), relative

islet distribution (p ¼ 0.52; data not shown), relative islet

cell volume density (p ¼ 0.49; data not shown) and b-cellvolume density (p ¼ 0.66; figure 5a). P32/98 also did not

affect insulin content of the islet (p ¼ 0.72; data not

shown) or pancreas (p ¼ 0.68; figure 5b).

To investigate islet function in vitro, islets were isolated

and stimulated with different glucose concentrations.

Inhibitor treatment did not affect glucose-stimulated

(10 and 20 mM glucose) insulin secretion (p ¼ 0.70 and

Table 1 Summary of results in P32/98- and placebo-treated rats with iIGT and mIGT

Placebo P32/98

Treatment (weeks) 0 6 0 6

iIGT

% gHb 2.44 � 0.05 3.04 � 0.12yy 2.59 � 0.12 2.76 � 0.05

Morning non-fasting

Blood glucose (mmol/l) 4.72 � 0.14 5.83 � 0.31yy 5.10 � 0.19 4.89 � 0.20*

Insulin (ng/ml) 17.1 � 3.9 29.5 � 6.1 23.6 � 3.7 13.8 � 2.7*,yDNP

Mean BG (mmol/l) 6.53 � 0.17 6.60 � 0.42 6.87 � 0.21 5.62 � 0.10yyDelta BG (mmol/l) 2.78 � 0.27 3.41 � 0.65 3.29 � 0.53 2.02 � 0.22

Fasting BG (mmol/l) 6.15 � 0.23 6.85 � 0.57 7.08 � 0.30* 5.33 � 0.22yyoGTT

Placebo bolus

G-AUC (mmol min/l) 1215 � 49 1461 � 90y 1238 � 76 1447 � 123

I-AUC (ng min/ml) n.d. 2841 � 386 n.d. 1802 � 268*

P32/98 bolus

G-AUC (mmol min/l) n.d. 1184 � 78# n.d. 1280 � 122#

I-AUC (ng min/ml) n.d. 4948 � 1072 n.d. 1921 � 496*

Hyperlipidaemia

NEFA (mmol/l) 1.87 � 0.16 2.65 � 0.24y 2.70 � 0.59 2.32 � 0.27

Triglycerides (mmol/l) 7.42 � 1.07 8.92 � 1.35 7.97 � 0.88 5.90 � 0.81

0 3 0 3

mIGT

% gHb 4.18 � 0.30 3.83 � 0.29y 3.95 � 0.20 3.09 � 0.11*,yyMorning non-fasting

Blood glucose (mmol/l) 6.29 � 0.44 6.23 � 0.28 5.69 � 0.30 5.70 � 0.14

Insulin (ng/ml) 9.8 � 2.5 20.0 � 2.8yy 17.5 � 4.5 13.1 � 5.5

DNP

Mean BG (mmol/l) 8.57 � 0.58 7.19 � 0.48 9.13 � 0.71 6.31 � 0.15yyDelta BG (mmol/l) 7.38 � 1.41 4.18 � 0.80 7.78 � 1.00 2.79 � 0.47yyFasting BG (mmol/l) 7.04 � 0.50 6.34 � 0.40 11.17 � 1.03** 5.32 � 0.27yy

oGTT

Placebo bolus

G-AUC (mmol min/l) 1435 � 34 1623 � 122 1420 � 54 1518 � 100

I-AUC (ng min/ml) 1193 � 185 1297 � 144 1582 � 249 1251 � 248

P32/98 bolus

G-AUC (mmol min/l) 1116 � 49## 1234 � 65y,## 1186 � 66## 1204 � 61#

I-AUC (ng min/ml) 2710 � 434# 2087 � 340 1895 � 358## 1639 � 397

Hyperlipidaemia

NEFA (mmol/l) 2.16 � 0.17 2.08 � 0.12 1.96 � 0.13 1.60 � 0.09**,yTriglycerides (mmol/l) 7.41 � 0.63 9.01 � 0.65y 5.04 � 0.49** 6.04 � 0.73**

BG, blood glucose; DNP, day–night profile; G-AUC, areaunder the glucose curve; gHb, glycatedhaemoglobin; I-AUC, areaunder the insulin curve;

iIGT, incipient impaired glucose tolerance;mIGT,manifest impaired glucose tolerance; n.d., notdetected;NEFA,non-esterified fatty acids; oGTT,

oral glucose tolerance test.Data are the mean � s.e.m.

*p < 0.05, **p < 0.01 vs. age-matched, placebo-treated Zucker rat.

yp < 0.05, yyp < 0.01 vs. first measurement.#p < 0.05, ##p < 0.01 vs. placebo bolus.


# 2007 The Authors


p ¼ 0.80, respectively; data not shown) or the stimula-

tion factor (p ¼ 0.50; figure 5c).

P32/98 Inhibition of DPP-4 in ZR with mIGT

Effects of Chronic Administration

In ZR with mIGT, chronic treatment with P32/98 (daily

treatment for 3 weeks) did not affect weight gain com-

pared with placebo (39 � 6 vs. 33 � 7 g; p ¼ 0.49).

Chronic P32/98 treatment also did not affect these rats’

intake of food (p ¼ 0.92 compared with placebo group;

data not shown) or water (p ¼ 0.52 compared with pla-

cebo group; data not shown). Daily P32/98 intake had no

significant effect on morning non-fasting blood glucose

(p ¼ 0.11 vs. t0; table 1). Over the course of 3 weeks,

non-fasting plasma insulin significantly increased in

placebo-treated mIGT ZR (p < 0.01 vs. t0) but remained

more or less unchanged in animals treated with P32/98

(p ¼ 0.37 vs. t0; table 1).

With exception of the fasting blood glucose value (at

17:00 hours), all the DNP values for the mIGT ZR in the

P32/98- and placebo groups followed the same circadian

rhythm before initiation of the study medication (fig-

ure 3c). During the 3 weeks of daily treatment, there

was a general trend towards declining blood glucose in

the rats in both groups, although it was more pro-

nounced in those treated with P32/98 (figure 3d). As

a consequence, the DNP parameters of placebo-treated

rats were not significantly altered during treatment

(p ¼ 0.06 vs. t0; table 1). However, mean blood glucose,

nBG and fasting blood glucose significantly decreased

following chronic P32/98 treatment (p < 0.01 vs. t0;

table 1), and the DNP profile was reasonably improved

compared with the placebo-treated rats (figure 3d).

There was a trend towards a decrease in gHb in mIGT

ZR during chronic placebo treatment; P32/98 produced

a more prominent reduction compared with placebo

(�0.86 � 0.15 vs. �0.35 � 0.15%; p < 0.05). Chronic

P32/98 treatment decreased NEFA in mIGT ZR (p <

0.05 vs. t0; table 1) and prevented the triglyceride eleva-

tion observed in placebo-treated rats (p ¼ 0.32 vs. t0;

table 1).

Acute and Chronic Effects in the oGTT

The chronic and acute effects of DPP-4 inhibition on IGT

were addressed by treatment with placebo or P32/98

bolus 30 min before oGTT in ZR chronically treated

(3 weeks) with placebo or P32/98.

oGTTs performed at study entry confirmed the mani-

festation of IGT (table 1 and figure 1). Chronic P32/98

administration did not affect the G-AUC or I-AUC com-

pared with placebo. However, acute administration of

a P32/98 bolus reduced the G-AUC in both the groups

both at study entry and after chronic treatment, an indi-

cation of improved glucose tolerance (table 1). P32/98

bolus increased the I-AUC in ZR from both groups when

administered at study entry. After chronic treatment,

Fig. 3 Blood glucose of day–night profiles in Zucker rat with incipient impaired glucose tolerance (a, b) and manifest

impaired glucose tolerance (c, d) before (a, c) and after (b, d) daily administration of placebo (s) or P32/98 (d). Data are the

mean � s.e.m. (n ¼ 10 for each group). **p < 0.01, *p < 0.05 vs. control.




this effect was observed in the placebo group only

(p ¼ 0.09; table 1).

Effects on Islet Morphology and Function

Compared with placebo group, P32/98 did not affect islet

size (p ¼ 0.32; data not shown), relative islet volume den-

sity (p ¼ 0.85; data not shown) or b-cell volume density

(p ¼ 0.97; figure 5a) in mIGT ZR. However, P32/98

decreased pancreatic insulin content (p < 0.05; figure 5

b). Islet insulin content was unaffected (p ¼ 0.44; data

not shown). The glucose responsiveness of isolated islets

was affected by chronic P32/98 treatment, as indicated

by an increase in the stimulation factor for islet insulin

secretion relative to placebo (p < 0.05; figure 5c).

Discussion

Enhancement of incretin through inhibition of DPP-4 has

beenproposed as apowerful newstrategy for treating type

2 diabetes [9]. Incretin enhancement can re-establish

glucose-stimulated insulin secretion, improve the insulin

Fig. 5 In situ and in vitro effects of chronic P32/98 treatment in Zucker rat (ZR) with incipient impaired glucose tolerance

(iIGT) and manifest impaired glucose tolerance (mIGT). b-cell volume density (a), pancreatic insulin content (b) and stimu-

lation factor of isolated pancreatic islets (c) from ZR with iIGT (after 8 weeks of treatment, 17 weeks old) and mIGT (after

8 weeks of treatment, 27 weeks old) treated once daily with P32/98 (dark) or placebo (white). The stimulation factor

describing the glucose responsiveness was defined as the quotient of islet insulin secretion at 20 and 2 mM glucose.

Data are the mean � s.e.m. (n ¼ 10 for each group). *p < 0.05 vs. placebo.

Fig. 4 Acute and chronic effects of P32/98 treatment (d) vs. placebo (s) in oral glucose tolerance test (oGTT) in Zucker rat

(ZR) with incipient impaired glucose tolerance. Acute effects were investigated using a P32/98 bolus before oGTT. Chronic

effects were investigated using placebo bolus. Blood glucose (a, b) and plasma insulin (c, d) of oGTT with preceding

placebo (a, c) or P32/98 inhibitor (b, d) bolus. ZRs were treated once daily with P32/98 (d) or placebo (s). Data are the

mean � s.e.m. (n ¼ 10 for each group). *p < 0.05 vs. placebo. DPP-4, dipeptidyl peptidase IV.


# 2007 The Authors


secretory response to meals and suppress glucagon

secretion [9,12,25,26]. The present study investigated

the therapeutic efficacy of incretin enhancement in pre-

diabetes using the DPP-4 inhibitor P32/98 and glucose-

intolerant fatty ZR.

Effects of P32/98 on Body Weight and Food Intake

Recent clinical studies in type 2 diabetes demonstrated

stabilizing effects of incretin enhancement on food intake

and body weight [9,12]. A reduction of body weight was

even observed after administration of the DPP-4 inhibitor

vildagliptin and sitagliptin in patients continuing metfor-

min treatment [12,27,28]. In accordance with Pospisilik

et al. [29], we found that P32/98 tended to reduce food

intake and body weight gain in ZR with iIGT. In contrast,

we found that P32/98 did not affect body weight in ZR

with mIGT [22,30]. The stabilizing effects of incretin

enhancers on body weight might become important if

they are used on a large scale in prediabetes.

Effects of P32/98 on Hyperglycaemia and

Hyperinsulinaemia

Sitagliptin, vildagliptin (formerly LAF237) and FE999011

reduced hyperglycaemia in preclinical and clinical stud-

ies of type 2 diabetes [9,11,12,29,30]. Likewise, in this

study, chronic P32/98 treatment of glucose-intolerant

ZR opposed the increase in non-fasting blood glucose in

iIGT and mIGT. Consequently, rats demonstrated im-

proved daily blood glucose profiles and reduced gHb.

Confirming this finding, P32/98 also improved hyper-

glycaemia in 17- and 23-week-old Vancouver diabetic

fatty (VDF) rats [29]. Thus, DPP-4 inhibitors may have

therapeutic potential for prediabetes.

The effect of incretin enhancement on hyperinsulinae-

mia, a common feature of type 2 diabetes, is still under

investigation. In a 12-week core study, Ahren et al. [27]

observed no effects of LAF237 on plasma insulin; how-

ever, there was a significant reduction in the 52-week

extension. We showed that P32/98 prevented the pro-

gression of hyperinsulinaemia in ZR with iIGT. Like-

wise, VDF rats similar in age to our rats with iIGT

demonstrated reduced plasma insulin after 6 weeks of

P32/98 treatment [29]. In rats with mIGT, P32/98 had

a minor effect on hyperinsulinaemia relative to placebo.

This is consistent with previous studies reporting no

effect of the DPP-4 inhibitors P32/98 and FE999011 on

hyperinsulinaemia in old VDF and Zucker diabetic fatty

(ZDF) rats [22,29,30].

The American Diabetes Association defines prediabe-

tes as IFG and/or IGT [1]. Long-term treatment with vil-

dagliptin and sitagliptin reduced fasting blood glucose

[12,31], and as discussed above, P32/98 improves glu-

cose tolerance. In extension to earlier studies [15,22,29],

P32/98 also prevented the age-dependent rise in fasting

blood glucose in iIGT and mIGT, suggesting that incre-

tin enhancement may be useful for diabetes prevention.

Acute and Chronic Effects of P32/98 on IGT

Long-term treatment with sitagliptin and vildagliptin

(LAF237) decreases glucose load after a test meal

[12,29,31,32]. As expected, placebo-treated rats demon-

strated improved IGT after acute DPP-4 inhibition [33].

Consistent with earlier studies with P32/98 in Wistar

and Vancouver ZR [34,35], P32/98 bolus before oGTT

reduced glucose load and increased insulin secretion,

thus confirming the insulinotropic effect of incretin

enhancers observed previously in mice treated with

valine-pyrrolidide [13]. Importantly, the efficacy of P32/

98 in reducing the glucose load was comparable in rats

with iIGT and mIGT, suggesting broad therapeutic effi-

cacy in IGT.

In parallel to the age-dependent rise in IGT, b-cell func-tion was weakened in rats with mIGT. Whereas P32/98

produced a fourfold increase in the insulin response dur-

ing oGTT of placebo-treated rats with iIGT, it produced

only a 1.5-fold increase in the insulin response in mIGT.

Apparently, the acute stimulation of b-cell function by

P32/98 is more effective in iIGT than inmIGT, suggesting

a dependence on the progression of disturbed b-cell func-tion. The acute effect of P32/98, based on indirect prolon-

gation of incretin action, is consistent with studies in

rodents and humans [13,33,34,36] and suggests that

incretin enhancement is a powerful strategy for treating

IGT, especially before diabetes onset.

To investigate the chronic effects of P32/98 on IGT,

oGTTs were performed after placebo bolus in rats chron-

ically treated with P32/98. Because P32/98 has a half-life

between 4 and 7 h [11], oGTTs were performed at least

15 h after the regular evening treatment with P32/98.

Surprisingly, following a placebo bolus, there was no

difference in the glucose tolerance curve between rats

chronically treated with P32/98 and placebo. However,

the requirement for insulin was halved in rats with iIGT

chronically treated with P32/98 vs. placebo, suggesting

increased insulin sensitivity with chronic P32/98 treat-

ment. P32/98-increased insulin sensitivity is suggested

by the earlier finding of increased glucose uptake

by soleus muscle after 12 weeks of treatment [29].

Potential mechanisms for this phenomenon are in-

creased insulin sensitivity because of reductions of tri-

glycerides and NEFA, improved b-cell function and




reduced hyperglucagonaemia [9,11,12]. The latter was

not measured in our study. In contrast, in mIGT rats

chronically treated with P32/98, there was no such

detectable effect on the insulin response during oGTT.

Combined acute and chronic P32/98 administration

produced additive effects in oGTT. In iIGT, the improve-

ment in IGT was further associated with a low insulin

response, which may also be interpreted as greater effi-

ciency of b-cell function. Apparently, acute and chronic

administration of P32/98 improved IGT through different

mechanisms. The acute effect is mediated by enhanced

glucose-stimulated insulin secretion and an insulino-

tropic action. Chronic administration has the potential

to increase insulin sensitivity and optimize b-cell func-tion, as observed following 12 weeks of treatment in VDF

rats [37]. Consistent with this idea, we observed a reduc-

tion of pancreatic insulin content in ZR with mIGT,

accompanied by improved islet function. Moreover,

long-term administration of vildagliptin in patients with

type 2 diabetes continuing metformin treatment im-

proved meal-related b-cell function and insulin sensi-

tivity [25]. Similar to our study, glucose tolerance was

improved without change in insulin secretion [25].

Nevertheless, the glucose tolerance and insulin

response observed after oGTT depend on the type and

dosage of DPP-4 inhibitor, the model, pharmacokinetic

factors and the administration schedule [13,30,36].

Moreover, different inhibitors may affect glucose toler-

ance through different mechanisms. For example, VDF

rats treated for 12 weeks with P32/98 responded with

higher plasma insulin after oGTT [11], whereas our

study found reduced insulin levels. However, the for-

mer study administered a lower P32/98 dose twice daily

[13,29,30,36].

Effects of P32/98 on Islet Morphology

In accordance with earlier P32/98 studies, b-cell mass

was unaffected in rats with iIGT and mIGT [22,29].

Apparently, DPP-4 inhibition produces a reasonable

increase in GLP-1 levels [11], although not sufficient to

affect the GLP-1-induced pathways of islet regeneration

[9]. However, whereas pancreatic insulin content was

unaffected by P32/98 in iIGT, it was reduced in mIGT.

Potentially, this could be because of restoration of b-cellfunction in association with increased insulin sensi-

tivity or reduced hyperglucagonaemia, consistent with

our findings of reduced plasma insulin and insulin

secretion after oGTT [9,25]. Moreover, P32/98 affected

glucose-stimulated insulin secretion in rats with mIGT.

There are two explanations for this finding. On one

side, P32/98 could have improved glucose-stimulated

insulin secretion, and on the other, prevented the

decline in glucose-stimulated insulin release with the

change from iIGT to mIGT. Similar findings were repor-

ted in mice following chronic DPP 728 treatment [36]. In

contrast, in ZDF rats, P32/98 had no effect on declining

pancreatic insulin content, probably because of more

aggressive diabetes progression in this model [22].

Effects of P32/98 on Dyslipidaemia

There are few and controversial data regarding effects of

incretin enhancement on dyslipidaemia, an important

atherogenic risk factor. Infusion of GLP-1 prevented the

postprandial rise of triglycerides and NEFA in humans;

the proposedmechanismswere delayed gastric emptying

and retarded triglyceride absorption [38]. Similarly, we

observed that chronic DPP-4 inhibition decreased NEFA

and triglycerides in rats with mIGT, and in another

study, the DPP-4 inhibitor FE999011 reduced plasma

levels of free fatty acids and triglycerides in ZDF rats

[30]. In contrast, P32/98 did not affect triglycerides in

ZDF rats [22], and 12 weeks of vildagliptin in combina-

tion with metformin failed to reduce any lipid parame-

ter in type 2 diabetes [27]. Future studies are necessary

to clarify whether DPP-4 inhibitors might affect a sub-

group of patients with severe dyslipidaemia. ZR with

a leptin receptor defect, hyperphagia and abdominal

adipositas might be a model to further investigate the

antidyslipidaemic effects of incretin enhancers [38,39].

Improvement of dyslipidaemia adds to the beneficial

effects of DPP-4 inhibition in type 2 diabetes [38].

Conclusion

Incretin enhancement has multiple beneficial effects for

the treatment of type 2 diabetes [38]. The incretin

enhancer P32/98 has the potential to improve both IFG

and IGT, a fact that supports its use for prevention of

diabetes. P32/98 had a strong effect on postprandial

blood glucose, a major contributor to abnormal haemo-

globin A1c levels in humans [40] and a factor that may

be independently associated with microvascular com-

plications in type 2 diabetes [41,42], as already demon-

strated for macrovascular disease [43]. Based on the

strength of current clinical evidence, there is reason to

target IFG and IGT, and this can be accomplished with

incretin enhancers. P32/98 also reduced glycaemic

excursion and significantly improved glycaemic control

in iIGT and mIGT. In addition, in mIGT, P32/98 had a

strong impact on dyslipidaemia, which contributes to

the excess morbidity and mortality in type 2 diabetes

[39]. Incretin enhancement could be an early intervention


# 2007 The Authors


strategy for prevention of diabetes-related complications,

which are often existent at diagnosis.

Acknowledgements

We thank Mrs Christiane Kauert and Mrs Kordula

Rudolph for excellent technical assistance. This work

was supported by grants fromProbiodrugAG, theGerman

Federal Ministry of Education and Research (BMBF; FKZ

03i2711) and the Ministerium fur Bildung, Wissenschaft

und Kultur Mecklenburg-Vorpommern (IDK 97 007 80/

SOM and IDK 97 007 80/HSP III).

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