original

article

original article

Diabetes, Obesity and Metabolism 15: 164174, 2013.

2012 Blackwell Publishing Ltd

Effects of the dual PPAR-/ agonist aleglitazar

on glycaemic control and organ protection in the Zucker
diabetic fatty rat
1

A. Benardeau
, P. Verry1 , E.-A. Atzpodien2 , J. M. Funk2 , M. Meyer1 , J. Mizrahi1 , M. Winter2 ,
1
M. B. Wright , S. Uhles1 & E. Sebokova1
1 pRED, Pharma Research & Early Development, DTA Cardiovascular & Metabolism, F. Hoffmann-La Roche AG, Basel, Switzerland
2 pRED, Non-Clinical Safety, F. Hoffmann-La Roche AG, Basel, Switzerland

Aims: To evaluate the effects of aleglitazar, a dual peroxisome proliferator-activated receptor-/ agonist, on the development of
diabetes-related organ dysfunction, in relation to glycaemic and lipid changes, in Zucker diabetic fatty (ZDF) rats.
Methods: Six-week-old, male ZDF rats received aleglitazar 0.3 mg/kg/day or vehicle as food admix for 13 weeks (n = 10 per group).
Age-matched male Zucker lean rats served as non-diabetic controls. Plasma and renal markers were measured at several time points.
Histopathology and quantitative immunohistochemistry were performed at 13 weeks.
Results: Glycated haemoglobin (5.4 vs. 9.2%) and blood glucose (8.3 0.3 vs. 26.1 1.0 mmol/l) were signicantly reduced at 12 weeks with
aleglitazar versus vehicle-treated ZDF rats (both p < 0.01), while aleglitazar preserved near-normal plasma insulin levels. Aleglitazar prevented
the development of hypertriglyceridaemia (1.4 0.1 vs. 8.5 0.9 mmol/l) and reduced plasma non-esteried fatty acids (0.09 0.02 vs.
0.26 0.04 mmol/l) relative to vehicle-treated animals (both p < 0.01). Urinary glucose and protein concentrations were signicantly reduced
at 13 weeks with aleglitazar versus vehicle-treated rats (both p < 0.01). Consistent with its effect on glycaemic control, aleglitazar protected
-cell morphology, as evidenced by preservation of islet integrity, and reduction of -cell apoptosis and islet brosis. Aleglitazar prevented
renal glomerular hypertrophy, podocyte degeneration, glomerulosclerosis, tubulo-interstitial lesions and development of cataracts.
Conclusions: Aleglitazar strongly improved glycaemic and lipid parameters while protecting key tissues, including the pancreas, kidneys and
eyes, against diabetes-associated structural and functional changes in the ZDF rat.
Keywords: cell, diabetes complications, dual PPAR- and - agonist, Zucker diabetic fatty rat
Date submitted 15 May 2012; date of first decision 12 June 2012; date of final acceptance 2 September 2012

Introduction
Landmark studies emphasize the need for comprehensive
management of multiple cardiovascular (CV) risk factors in
patients with type 2 diabetes mellitus (T2DM) to reduce CV
morbidity and mortality and microvascular complications [1].
While agonists of peroxisome proliferator-activated receptor
(PPAR)- (thiazolidinediones) provide effective glycaemic
control, and thus may reduce microvascular complications,
only pioglitazone in the PROactive trial showed a reduction
in CV events, as reflected by a reduction in the composite
(secondary) end point of all-cause mortality, non-fatal
myocardial infarction and stroke [2]. PPAR- agonists
(fibrates) lower triglycerides (TG) and increase high-density
lipoprotein cholesterol (HDL-C) [3], although their effects to
reduce risk for CV events have been modest [4].
Dual-selective agonists of PPAR- and - are of interest as
they may combine the glucose-lowering and lipid-modifying
Correspondence to: Elena Sebokova, PhD, pRED Pharma Research & Early Development,
DTA Cardiovascular & Metabolism, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, Basel
CH4070, Switzerland.
E-mail: elena.sebokova@roche.com

properties of both agents. Aleglitazar is a balanced dual

PPAR-/ agonist designed to optimize effects on glucose
and lipids. In diabetic rodent models, aleglitazar significantly
improved insulin sensitivity (Zucker fa/fa rats and db/db
mice) and improved plasma lipids (human apolipoprotein
A1 transgenic mice) [5]. In a Rhesus monkey model of T2DM,
aleglitazar more than doubled levels of HDL-C and significantly
reduced both low-density lipoprotein cholesterol (LDL-C) and
TG, while lowering glucose and glycated haemoglobin (HbA1c)
[6]. In a phase II dose-range-finding study (SYNCHRONY)
in T2DM patients, aleglitazar dose-dependently improved the
lipid profile (TG, LDL-C and HDL-C) and decreased glucose
(HbA1c and fasting plasma glucose), as well as reduced other
markers of CV risk, such as plasminogen activator inhibitor-1,
fibrinogen and C-reactive protein [7].
Zucker diabetic fatty (ZDF) rat is a useful animal model
of T2DM disease progression and end-organ dysfunction
[810]. Insulin resistance and slightly elevated plasma glucose
are typically evident by 7 weeks of age, with hyperinsulinaemia
occurring between weeks 7 and 10. Insulin levels eventually
decline, due to -cell loss [11], with overt diabetes developing
by 12 weeks. The disease course in the ZDF rat has similarities

DIABETES, OBESITY AND METABOLISM

to T2DM in humans, including declining renal function

(glomerular and tubulo-interstitial alterations with proteinuria
and glucosuria) [10], presence of neuropathy (decreases in
peripheral nerve conduction and alterations in sensory
parameters) [12] and retinopathy (retinal vascular changes
and cataracts) [13].
The therapeutic effects of thiazolidinediones and fibrates
have been previously studied in the ZDF rat. Rosiglitazone
was shown to improve insulin sensitivity and glucose
control, leading to preservation of pancreatic -cell mass
and insulin content [14,15]. Rosiglitazone had protective
effects on kidney function, resulting in significantly reduced
proteinuria and improvements in glomerular and tubular
morphology effects that were superior to the angiotensinconverting enzyme inhibitor cilazapril [16]. Pioglitazone
has also been shown to reduce proteinuria in ZDF rats
because of glucose-dependent and -independent mechanisms.
Pioglitazone reduced glomerulosclerosis by reducing podocyte
activation and endothelial injury [17,18]. Effects were evident
despite minor reductions in glucose, suggesting they may occur
via anti-fibrotic PPAR-dependent mechanisms directly in the
kidney, where PPAR- is highly expressed [19].
Fibrate PPAR- agonists have also been evaluated in ZDF
rats. Fenofibrate reduced systolic blood pressure, and attenuated glomerular hypertrophy and fibrosis [20]. Fenofibrate
also reduced tubular injury, possibly by reducing interstitial
macrophages in the kidney, leading to suppression of
nuclear factor B and transforming growth factor-1/Smad3
signalling [21].
In this study, we investigated effects of the dual PPAR-/
agonist aleglitazar on glycaemic control and prevention of
diabetes-related organ dysfunction in ZDF rats, employing
quantitative immunohistochemistry with accepted and
emerging structural and functional markers.

Materials and Methods

Animals
Five-week-old male ZDF rats (ZDF fa/fa or ZDF) and lean,
aged-matched male Zucker rats (ZDF +/fa or lean [ZL])
from Charles River Laboratories (Wilmington, MA, USA)
were fed a diabetogenic diet (KLIBA 2437; Provimi KLIBA
AG, Kaiseraugst, Switzerland) immediately after weaning.
The animal study was performed under an animal research
protocol (ARP) authorization to F. Hoffmann-La Roche by the
Swiss Cantonal Veterinary Office Basel-Stadt. Animals were
housed individually under standard hygienic conditions in an
AAALACi-accredited facility with controlled temperature and
humidity, a 12 h/12 h light/dark cycle, and full access to food
and water.

Study Design and Blood/Urine Sampling

Rats were 6 weeks old when treatment commenced. Animals received aleglitazar (0.3 mg/kg/day; n = 10; Roche, Basel,
Switzerland) as food admix or vehicle (vehicle; n = 10) for
13 weeks. For each treatment group, 50 kg of food (KLIBA
2437) was homogenized in 10 l of vehicle buffer solution (0.89%

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di-sodium hydrogenphosphate-dihydrate; 0.4% polysorbate
80, 0.29% 1 N HCl in sterile water), with or without aleglitazar,
and the fluid phase was carefully evaporated. Final food was prepared in pellets by KLIBA. Average daily food consumption was
100 g/kg body weight during treatment (confirmed by recording of daily food intake), delivering aleglitazar at a daily dose of
0.3 mg/kg/day. The potential food aversion risk was explored
prior to starting chronic treatment by monitoring food and
water intake and body weight on diet over a 1-week period. No
food aversion behaviour was observed. Pharmacokinetic analysis confirmed that the expected plasma exposure of aleglitazar
was achieved (data not shown). Age-matched male ZL rats
(n = 10) received vehicle and served as non-diabetic controls.
Retro-orbital blood (200 l of blood with EDTA) was
taken from anaesthetized rats (isoflurane) into tubes coated
with EDTA, containing 1% aprotinin (to inhibit protein
degradation), and kept on ice. Animals were carefully
monitored both before and after blood sampling and no
signs of distress or complications were observed. Tubes were
then centrifuged (9279 g for 15 min at 4 C) and plasma was
aliquoted in different tubes or 96-well plates for analysis.
Blood samples were taken under postprandial conditions in
the morning 2 h following switch from dark to light cycle to
assess glucose, HbA1c, insulin, TG, non-esterified fatty acids
(NEFA), total cholesterol (TC) and adiponectin at several time
points under non-fasting conditions. Glucose and HbA1c were
measured in whole blood, while plasma was used for analysis
of insulin, TG, NEFA, TC and adiponectin. Urine samples
were taken after 5 weeks of treatment and at the end of the
treatment period (13 weeks); five representative animals per
group according to similar body weight and plasma parameters
were placed individually into metabolic cages, without access to
food but with free access to water, for 1617 h urine collection
using ice-refrigerated tubes.
An oral glucose tolerance test (OGTT) was carried out
at 12 weeks in overnight-fasted animals, with measurements
taken at 15, 30, 60 and 120 min after glucose challenge
(1 g/kg). Homeostasis model assessment of insulin resistance
(HOMA-IR) and -cell function (HOMA-B) were estimated
after overnight fasting and prior to performing OGTT
using the following calculations: HOMA-IR = fasting insulin
(ng/ml)/0.04 fasting blood glucose (mM)/22.5; HOMAB = 20 fasting insulin (ng/ml)/0.04/fasting blood glucose
(mM) 3.5 [22].
Blood glucose levels were measured using a glucometer
(GlucoTrend, Roche Diagnostics, Burgdorf, Switzerland).
Plasma concentrations of insulin (Rat Insulin, Mercodia,
Uppsala, Sweden) and adiponectin (Mouse/Rat ELISA kit,
K1002-1, B-Bridge, Cupertino, CA, USA) were quantified
by ELISA. Metabolic plasma profiles were established by the
Hitachi system using a fluorometric method: TC, TG, NEFA,
HbA1c/Hb (turbimetric assay). HbA1c (%) was calculated as
follows: 87.6 (HbA1c/Hb) + 2.27 [23].

Histopathology and Immunohistochemistry

Tissue samples were fixed in 10% neutral-buffered formalin
for at least 24 h. Eyes were fixed in modified Davidsons fixative
consisting of 13.3% ethyl alcohol, 6.3% glacial acetic acid and

doi:10.1111/dom.12006 165

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13.9% formalin solution in distilled water, for at least 48 h.
For histopathology examination, organs were embedded in
Paraplast. Tissue sections (23 m) were stained with haematoxylin eosin (pancreas, kidneys) or periodic acid-Schiff (PAS;
kidneys). Glycogen within distal tubular epithelial cells was
determined in adjacent kidney sections on two separate slides.
One section was pretreated with diastase, and both were stained
with PAS. Glycogen was determined in the non-diastase-treated
section. The following immunofluorescent stainings were carried out to assess islet morphology: anti-insulin (DAKO,
Glostrup, Denmark), anti-glucagon (R&D Systems, Minneapolis, MN, USA) and 4 , 6-diamidino-2-phenylindole (DAPI;
DAKO); to assess -cell apoptosis: anti-insulin (DAKO),
transferase-mediated dUTP nick-end labelling (TUNEL; Roche
Diagnostics) and DAPI (DAKO); and to assess islet fibrosis:
a cocktail of anti-insulin (DAKO), anti-glucagon (R&D Systems), anti-pancreatic polypeptide (Millipore, Billerica, MA,
USA), anti-somatostatin (Genetex, Irvine, CA, USA), antifibronectin (Sigma, St Louis, MO, USA) and DAPI (DAKO),
followed by respective secondary fluorescent antibodies (Invitrogen, Carlsbad, CA, USA). Digital imaging fluorescence
microscopy of the pancreas was performed using a scanning platform (MetaSystems, Altlussheim, Germany) with
a Zeiss Imager Z.2 microscope (Carl Zeiss MicroImaging,
Inc., Jena, Germany). Immunohistochemical staining of kidney sections was carried out using anti-desmin (Millipore),
anti-synaptopodin (Acris Antibodies, Herford, Germany),
anti-collagen IV (Millipore), anti-fibronectin (GeneTex, San
Antonio, TX, USA), anti-vimentin (Roche/Ventana) and
anti-T-cell immunoglobulin mucin/kidney injury molecule-1
(TIM/KIM1) (R&D Systems) antibodies. Anti-desmin was used
to detect glomerular podocyte damage; anti-synaptopodin,
anti-collagen IV and anti-fibronectin to detect further glomerular alterations; and anti-vimentin, anti-TIM/KIM1, anticollagen IV and anti-fibronectin to detect tubulo-interstitial
damage. Digital brightfield imaging of the kidneys was performed using the Aperio ScanscopeTM slidescanner (Aperio,
Vista, CA, USA). Quantitative image analysis of islet and
glomerular staining was performed using Definiens Architect
XD (Definiens AG, Munich, Germany).

Statistical methods
Statistical analysis was performed by analysis of variance
followed by Dunnetts post hoc test. Any between-group
differences versus vehicle were considered significant at
p 0.05. Data are reported as mean standard error of the
mean, unless otherwise stated.

Results
Glucose and Lipid Effects of Aleglitazar
ZDF rats at baseline had slightly elevated blood glucose levels
compared with their non-diabetic ZL counterparts (Table 1).
Blood glucose in untreated ZDF rats progressively increased
from baseline to 4 and 12 weeks, while blood glucose in
aleglitazar-treated ZDF rats remained normal. Aleglitazar
significantly lowered HbA1c compared with vehicle-treated

166 Benardeau
et al.

DIABETES, OBESITY AND METABOLISM

Table 1. Effect of aleglitazar on non-fasted metabolic parameters.

ZDF
(N = 10)
Baseline (age = 6 weeks)
Blood glucose, mmol/l
7.9 0.2
HbA1c, %
4.6 0.1
Plasma insulin, ng/ml
4.8 0.5
Plasma triglycerides, mmol/l
1.8 0.1
Plasma non-esterified fatty
0.07 0.01
acids, mmol/l
Plasma total cholesterol,
2.6 0.1
mmol/l
Body weight, g
204 3
At 4 weeks of treatment (age = 10 weeks)
Blood glucose, mmol/l
17.5 1.9
HbA1c, %
5.5 0.1
Plasma insulin, ng/ml
8.3 1.1
Plasma triglycerides, mmol/l
10.0 0.5
Plasma non-esterified fatty
0.32 0.03
acids, mmol/l
Plasma total cholesterol,
3.0 0.0
mmol/l
Body weight, g
354 6
At 12 weeks of treatment (age = 18 weeks)
Blood glucose, mmol/l
26.1 1.0
HbA1c, %
9.2 0.5
Plasma insulin, ng/ml
2.6 0.5
Plasma triglycerides, mmol/l
8.5 0.9
Plasma non-esterified fatty
0.26 0.04
acids, mmol/l
Plasma total cholesterol,
5.0 0.1
mmol/l
Plasma adiponectin, ng/ml
3.3
81.3 0.4
Glucosuria, mmol/l
4.4 1.2
Proteinuria, mg/ml
Body weight, g
428 16

ZDF +
aleglitazar
(N = 10)

ZL
(N = 10)

8.0 0.3
4.7 0.1
5.0 0.7
1.8 0.1
0.05 0.02

7.5 0.1
4.7 0.1
0.2 0.0
0.6 0.0
0.21 0.04

2.6 0.0

2.2 0.1

205 3

160 4

7.6 0.1
4.9 0.0
1.6 0.3
0.6 0.1
0.05 0.02

7.8 0.2
4.8 0.0
0.9 0.1
1.0 0.0
0.32 0.03

3.6 0.1

2.0 0.1

435 7

278 4

8.3 0.3
5.4 0.1
1.9 0.2
1.4 0.1
0.09 0.02

7.3 0.2
5.6 0.1
1.0 0.0
1.6 0.1
0.26 0.02

4.5 0.1

2.3 0.1

17.9
0.5 0.1
0.3 0.1
765 16

4.2
1.4 0.1
1.8 0.2
386 5

ZDF, Zucker diabetic fatty; ZL, Zucker lean.

Data are presented as means s.e.m.; ANOVA followed by Dunnetts post
hoc test.
N = 5 per group for glucosuria and proteinuria, evaluated at 13 weeks.
p < 0.01 ZDF + aleglitazar versus ZDF.

ZDF rats at both 4 and 12 weeks. At the end of the study period,
blood glucose and HbA1c were similar in both non-diabetic
ZL and aleglitazar-treated ZDF rats (Table 1). These data
indicate that aleglitazar effectively prevents the development
of hyperglycaemia.
At baseline, ZDF rats were already hyperinsulinaemic
(Table 1). In untreated ZDF rats, plasma insulin levels progressively increased up to 4 weeks and declined thereafter. Aleglitazar reversed the development of early hyperinsulinaemia,
maintaining near-normal plasma insulin levels at 4 weeks that
were significantly lower than in control ZDF rats and only
slightly elevated versus non-diabetic ZL rats. At 12 weeks, the
difference between plasma insulin levels in aleglitazar-treated
and vehicle-treated ZDF rats was not significant due to the
advanced state of disease in the untreated animals.
Aleglitazar strongly induced plasma adiponectin levels and
showed significant effects on non-fasted plasma lipids (Table 1).

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DIABETES, OBESITY AND METABOLISM

Table 2. Effects on fasted parameters of glucose homeostasis and oral

glucose tolerance test (OGTT) after 12 weeks of treatment.

Fasting blood glucose, mmol/l

Fasting plasma insulin, ng/ml
Glucose AUC0-120 , mmol/
l.min
HOMA-IR
HOMA-B

ZDF
(N = 10)

ZDF +
aleglitazar
(N = 10)

ZL
(N = 10)

9.3 1.1
2.84 0.86
2054 265

5.8 0.1
1.70 0.19
971 68

4.4 0.0
0.95 0.03
883 38

26.8 5.9
322 143

11.0 1.3
379 36

4.6 0.2
564 22

HOMA-B, homeostasis model assessment of -cell function; HOMA-IR,

homeostasis model assessment of insulin resistance; ZDF, Zucker diabetic
fatty; ZL, Zucker lean.
Data are presented as means s.e.m. ANOVA followed by Dunnetts post
hoc test.
p < 0.01 ZDF + aleglitazar versus ZDF.

At baseline, ZDF rats had elevated plasma TG and lower plasma

NEFA levels compared with non-diabetic ZL controls. In ZDF
rats, plasma TG progressively increased over time an effect
that was largely prevented by aleglitazar. At the end of the
treatment period, aleglitazar-treated ZDF rats had plasma TG
comparable to the ZL controls. Plasma cholesterol was elevated
in controlled ZDF rats compared with ZL rats but was not
significantly changed by aleglitazar.
Fasting Parameters and OGTT. Aleglitazar significantly
reduced fasting blood glucose compared with vehicle-treated
ZDF rats at 12 weeks (Table 2). Following an OGTT, aleglitazar
significantly reduced glucose area under the curve compared
with vehicle-treated ZDF rats at 12 weeks (Table 2) and normalized glucose excursions to a level comparable to vehicle-treated
ZL rats. Fasting plasma insulin levels were slightly lower
with aleglitazar compared with vehicle-treated ZDF rats after
12 weeks of treatment, but were higher than in non-diabetic
ZL rats (Table 2; not significant vs. vehicle-treated ZDF rats).
Reductions in HOMA-IR indicated a trend to improved insulin
sensitivity with aleglitazar compared with vehicle-treated ZDF
rats (Table 2; not significant vs. vehicle-treated ZDF rats).
Body Weight. At baseline, body weight was increased in ZDF
rats compared with ZL controls (Table 1). Aleglitazar was
associated with significantly increased body weight compared
with vehicle-treated ZDF rats at both 4 weeks and 12 weeks.

Effects of Aleglitazar on Islet Morphology, -Cell

Markers and -Cell Function
Severe -cell destruction was notable in vehicle-treated ZDF
rats. As shown in figure 1 and Table 3, loss of islet structural
integrity and insulin content occurred in vehicle-treated ZDF
rats after 13 weeks of treatment. Islets were enlarged, containing irregular projections into the exocrine pancreas, with
-cells scattered throughout individual islets and vacuolated
endocrine cells dispersed within the exocrine tissue. In contrast,
aleglitazar preserved islet integrity, maintaining normal -cell
localization at the islet periphery while preserving -cell area

Volume 15 No. 2 February 2013

and number (figure 1 and Table 3). In aleglitazar-treated ZDF

rats, -cell area, number and insulin content were higher than
in non-diabetic ZL rats (figure 1).
Aleglitazar significantly prevented islet fibrosis (indicated
by percentage fibrotic islet area and fibronectin staining
intensity) to a level comparable to that of ZL rats (figure 2).
Aleglitazar treatment significantly protected -cells from
apoptosis (indicated by number of apoptotic -cells and
percentage apoptotic -cell number per islet cross-section)
compared with vehicle-treated ZDF rats (figure 2).

Effect of Aleglitazar on Cataract Formation

Aleglitazar protected against cataract formation, which
occurred in vehicle-treated ZDF rats (see figure 3 and Table 3).
Clinical examination revealed vehicle-treated ZDF rats had lens
opacity indicative of diabetic cataracts as compared with ZL
rats. Histologically, cataracts were characterized by degeneration and swelling of lens fibres, with formation of Morgagnian
globules, abnormally retained nuclei (bladder cells) and
hyperplasia of the lens epithelium underlying the anterior
capsule. Lens opacity or histopathologic changes indicative of
cataract formation were absent in aleglitazar-treated rats.

Effects of Aleglitazar on Renal Function, and

Glomerular and Tubulo-Interstitial Markers
ZDF rats developed renal dysfunction, as indicated by high
levels of urinary protein. At 13 weeks, aleglitazar significantly
reduced urinary protein and glucose compared with vehicletreated ZDF rats to levels characteristic of non-diabetic ZL rats
(Table 1).
Vehicle-treated ZDF rats exhibited evidence of glomerular hypertrophy, PAS + glomerular absorption droplets within
degenerating podocytes, mesangial expansion and glomerulosclerosis (Table 3). PAS staining revealed minimal focal
segmental glomerulosclerosis in vehicle-treated ZDF rats,
which was prevented by treatment with aleglitazar (figure 4).
Glomerular desmin staining was increased in vehicle-treated
ZDF rats, suggesting podocyte damage, and was largely prevented by aleglitazar treatment (figure 4). Quantitative image
analysis of desmin demonstrated that both desmin marker
area and percentage desmin marker area per glomerulum
was reduced in aleglitazar-treated ZDF rats to levels comparable to those in non-diabetic ZL rats (figure 5). Other
markers, most notably of fibrosis and glomerular dysfunction [collagen IV and fibronectin (figure 4) and synaptopodin
(data not shown)] were not significantly different in any of
the groups.
Aleglitazar prevented tubular basophilia, dilation and
lumenal proteinaceous casts accompanied by interstitial
fibrosis, and vacuolation associated with PAS + droplets
indicative of glycogen storage within renal distal tubular
epithelial cells, all of which were present in vehicle-treated
ZDF rats (Table 3). Immunohistochemistry for vimentin,
TIM/KIM1, collagen IV and fibronectin revealed increased
tubular expression of these markers in vehicle-treated
ZDF rats (figure 6), correlating with tubular damage, also
observed with haematoxylin eosin and PAS staining. These

doi:10.1111/dom.12006 167

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DIABETES, OBESITY AND METABOLISM

Figure 1. Preservation of islet structure in aleglitazar-treated rats. Assessment of islet morphology, -cell area, -cell number and insulin staining
intensity by digital imaging quantitative fluorescence microscopy using anti-insulin or anti-glucagon antibodies, and 4 , 6-diamidino-2-phenylindole
(DAPI) staining. Scale bar, 100 m. N = 179291 islets/group, ***p < 0.001 versus vehicle; data are shown as mean s.e.m.; ANOVA followed by Dunnetts
test.

168 Benardeau
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DIABETES, OBESITY AND METABOLISM

Table 3. Summary of histopathologic findings after 13 weeks of treatment.

Pancreas
Islets enlarged with irregular projections into exocrine pancreas, endocrine cells scattered within
exocrine tissue (HE)
Vacuolated endocrine cells (HE) corresponding to reduced insulin content (insulin IHC)
-cells at periphery of islet (glucagon IHC)
-cells scattered throughout islet (glucagon IHC)
Eyes (HE)
Cataract, cortical
Kidneys (HE & PAS)
Kidney weight relative to brain weight
Glomerular hypertrophy
PAS + absorption droplets within degenerating glomerular podocytes
Glomerular mesangial expansion
Glomerulosclerosis, focal, segmental
Vacuolation and PAS + droplets within distal tubular epithelial cells (corresponding to glycogen
storage)
Tubular dilation and lumenal proteinaceous casts
Tubular basophilia

ZDF (N = 10)

ZDF + aleglitazar
(N = 10)

ZL (N = 10)

+++

++++

Present

Present

Present

++

2.2
++
++
+
+
++

2.0

1.5

+++
+++

(+)

(+)

HE, haematoxylin eosin; IHC, immunohistochemistry; PAS, periodic acid-Schiff; ZDF, Zucker diabetic fatty; ZL, Zucker lean.
= absent; (+) = minimal, individual animals only; + = minimal; ++ = slight; +++ = moderate; ++++ = marked.

markers were decreased in aleglitazar-treated animals to levels

comparable to non-diabetic ZL rats, indicating prevention of
tubulo-interstitial fibrosis and pathologic changes.

Discussion
Several landmark studies have demonstrated a relationship
between improvements in metabolic abnormalities and
prevention of diabetic complications in patients with T2DM.
The direct relationship between glycaemia and diabetes-related
complications was shown in an epidemiologic analysis from
the UKPDS [24], while, in Steno-2, patients with T2DM
and microalbuminuria who were receiving intensive therapy
for control of glucose, lipids and blood pressure had a
significantly lower risk of CV disease, nephropathy, retinopathy
and autonomic neuropathy [25]. Results from these studies
suggest that a significant portion of the beneficial effect of the
multifactorial intervention on CV disease was attributable to
lipid lowering on top of effective blood pressure and glycaemic
control [26].
Dual PPAR-/ agonists that combine glycaemic control
with improvements in lipid profile may provide a multifactorial
approach to reduce CV morbidity and mortality, as well as other
diabetes-related complications. This study demonstrated the
protective effect of aleglitazar, a novel dual PPAR-/ agonist,
on end-organ damage and the development of T2DM-related
complications in ZDF rats. In concordance with previous
studies of PPAR ligands in the ZDF model [1318,20,21],
aleglitazar significantly improved both glycaemic and lipid
parameters, with concurrent preservation of pancreatic islet
structure and renal structure and function. Aleglitazar was also
associated with significantly increased body weight compared
with vehicle-treated ZDF rats, a typical effect of PPAR-

Volume 15 No. 2 February 2013

activation in rodents and humans [27,28]. PPAR- agonists

increase fat mass, associated with a reduction in circulating
free fatty acids (FFAs) due to FFA repartitioning towards fat
rather than muscle and pancreas [28]. This may contribute to
increased insulin sensitivity and reduction of glucose, lipids
and inflammatory markers, which may in turn affect organ
protection.
The strengths of this study are its evaluation of multiple
immunohistochemical markers of pancreatic -cell health,
and renal glomerular and interstitial changes, in a wellestablished rodent disease model of T2DM. A limitation of
this study is inability to dissect the relative contributions of
improved glycaemic control versus improved lipids, or of
the contributions of direct mechanisms on -cell or renal
protection conferred by aleglitazar.
Aleglitazar prevented -cell apoptosis in this study by up to
87%. Histologic and immunofluorescent examination of the
pancreas showed that aleglitazar was able to maintain -cell
area and number to levels comparable to prediabetic ZDF
rats. After 13 weeks of treatment, aleglitazar preserved islet
integrity, maintained insulin content and significantly reduced
islet fibrosis. These effects are likely the result of multiple factors
leading to reduced gluco- and lipotoxicity, related to improved
insulin sensitivity, as well as significantly reduced glucose, TG
and NEFA. These beneficial effects on -cell morphology and
function are in agreement with previous studies of PPAR-
agonists, which showed that rosiglitazone preserves -cell mass
by preventing the rise in net -cell death [14,15]. In one study,
rosiglitazone, but not metformin, preserved -cell mass while
both agents reduced glucose levels, suggesting a protective effect
of PPAR- that is independent of the effects on glucose [14].
Podocyte injury plays an early and important role in
progressive renal dysfunction in the ZDF model [10,29].

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DIABETES, OBESITY AND METABOLISM

Figure 2. Aleglitazar inhibits islet fibrosis and -cell apoptosis in the pancreas. Assessment of islet fibrosis and -cell apoptosis by digital imaging
quantitative fluorescence microscopy using anti-insulin, anti-glucagon, anti-pancreatic polypeptide, anti-somatostatin antibodies and anti-fibronectin
antibody (for fibrosis) and anti-insulin antibody, transferase-mediated dUTP nick-end labelling (TUNEL) and 4 , 6-diamidino-2-phenylindole (DAPI)
staining (for apoptosis). Scale bar, 100 m. Fibrosis: N = 227562 islets/group, Apoptosis: N = 190344 islets/group, * p < 0.05, **p < 0.01 versus vehicle;
data are shown as mean s.e.m.; ANOVA followed by Dunnetts test.

170 Benardeau
et al.

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DIABETES, OBESITY AND METABOLISM

original article

Figure 3. Aleglitazar prevents development of cataracts. Histologic characterization of the lens following haematoxylin eosin (HE) staining, showing
development of cortical cataracts in untreated Zucker diabetic fatty (ZDF) rats but prevention of cataracts in aleglitazar treated ZDF rats. Scale bar, 50 m.

Figure 4. Aleglitazar prevents glomerulosclerosis. Assessment of glomerular pathology by periodic acid-Schiff (PAS) staining and immunohistochemical
staining. Evidence of glomerulosclerosis indicated by black arrow. Evidence of podocyte damage indicated by PAS+ absorption droplets (open arrow)
and by immunohistochemical staining with anti-desmin antibody. Extent of fibrosis was assessed by immunohistochemical staining with anti-collagen IV
or anti-fibronectin antibodies. Scale bar, 50 m.

Although early podocyte damage may be influenced by high

glucose and dyslipidaemia [10], agonism of PPAR- [20,21]
and PPAR- [1618] may also have direct renoprotective
actions. PPAR- and PPAR- are both expressed in the kidney,
with PPAR- predominantly expressed in proximal tubules and
medullary thick ascending limbs and PPAR- in medullary
collecting ducts, pelvic urothelium and glomerular mesangial
cells [19]. In diabetic PPAR--knockout mice, diabetic
nephropathy is more severe than in wild-type mice [30]. There
is substantial evidence that PPAR- and PPAR- agonists have
direct effects on several critical pathways contributing to the
development and progression of diabetic kidney disease, as
reviewed by Thomas et al. [30]. For example, both gemfibrozil
and rosiglitazone have been shown to reduce albuminuria,
glomerulosclerosis, tubulointerstitial expansion and collagen
IV deposition in streptozotocin-induced diabetes, where the
effects on glycaemic control and lipids are minimal, suggesting
direct protective actions [30]. Other glucose-independent

Volume 15 No. 2 February 2013

renoprotective mechanisms that have been attributed to PPAR agonism include reductions in blood pressure, oxidative
stress, inflammation, fibrosis and positive effects on podocyte
function, including increased expression of nephrin [30].
In this study, aleglitazar prevented renal glomerular hypertrophy, podocyte degeneration and glomerulosclerosis, as well
as tubular basophilia, dilation and lumenal proteinaceous
casts accompanied by interstitial fibrosis. Aleglitazar also prevented vacuolation associated with PAS + droplets, indicative
of hyperglycaemia-related glycogen storage within renal distal
tubular epithelial cells that may, in part, be consistent with the
ArmanniEbstein phenomenon in diabetic states [31]. Quantitative immunohistochemistry using an extensive panel of
markers provided detailed insights regarding both glomerular
and tubular protection. In the glomerulus, aleglitazar normalized desmin levels, suggesting prevention of podocyte dysfunction. Our data also indicated a tubulo-interstitial-protective
effect of aleglitazar, evidenced by decreases in a range of markers

doi:10.1111/dom.12006 171

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DIABETES, OBESITY AND METABOLISM

Figure 5. Aleglitazar decreases glomerular desmin expression. Quantitative image analysis of glomerular desmin staining measured as average desmin
marker area (m2 /glomerulum) and percentage marker area per glomerulum. N = 240 glomeruli/group; ***p < 0.001 versus Zucker diabetic fatty (ZDF);
data are shown as mean s.e.m.; ANOVA followed by Dunnetts test.

Figure 6. Prevention of kidney fibrosis with aleglitazar. Assessment of tubular pathology by immunohistochemical staining of kidney sections using
anti-vimentin, anti-KIM-1, anti-collagen IV and anti-fibronectin antibodies. Scale bar, 200 m.

for tubular damage, including vimentin, TIM/KIM1, collagen

IV and fibronectin. These results concur with previous findings
in studies with both PPAR- and - agonists [1618,21,32],
although, to our knowledge, previous investigations have not
explored the effect of PPAR agonists on such a comprehensive
panel of markers. In the case of fenofibrate, one mechanism
that has been hypothesized to explain its effects is the inhibition
of transforming growth factor 1/Smad3 and nuclear factor B
signalling pathways, resulting in suppression of inflammation
and fibrosis [21].
The reduction in renal fibrosis may, in part, be mediated by
decreased fibrinogen, which has been observed in preclinical
and clinical studies of aleglitazar [7,33]. This concurs with

172 Benardeau
et al.

recent data suggesting that fibrinogen promotes renal fibrosis

by triggering resident fibroblast proliferation [34]. These effects
are similar to observations from studies [35] of fenofibrate
such as the Fenofibrate Intervention and Event Lowering
in Diabetes (FIELD) trial, in which fenofibrate reduced
albuminuria and slowed estimated glomerular filtration rate
loss over 5 years, indicating a potential to retard loss of kidney
function despite initial reversible increases in serum creatinine
[36]. Serum creatinine levels were not monitored in the
present study; however, aleglitazar has been shown to increase
serum creatinine in clinical studies [7,37]. Notwithstanding,
these effects have been shown to be fully reversible, even
at the dose of 600 g (four times the dose currently being

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DIABETES, OBESITY AND METABOLISM

evaluated in phase III trials), with creatinine levels returning

to pretreatment levels following cessation of treatment within
a 4- to 8-week follow-up period [37].
In conclusion, aleglitazar strongly improves glycaemic and
lipid parameters in ZDF rats while protecting key tissues,
including the pancreas, eyes and kidneys, against diabetesassociated structural and functional changes. Aleglitazar protected -cell morphology, as evidenced by effects on islet
integrity, -cell apoptosis and islet fibrosis. Aleglitazar prevented the development of cataracts and prevented renal
glomerular hypertrophy, podocyte degeneration, glomerulosclerosis and tubulo-interstitial lesions. Together, these data
suggest that aleglitazar may be effective in reducing the progression of T2DM, and macrovascular and microvascular
complications of the disease.

Acknowledgements
This study was funded by F. Hoffmann-La Roche AG. We would
like to thank Mathieu Brecheisen and Claudia Richardson, F.
Hoffmann-La Roche, Basel, for their assistance in preparation
of immunofluorescence data, as well as Emmanuelle Hainaut
and Andree Roeckel for conducting the pharmacology study
and analysis. Editorial support was provided by Cindy
Macpherson, MediTech Media, UK, funded by F. Hoffmann-La
Roche AG.

Conict of Interest
A. B., M. M., J. M., E. S., P. V. and M. B. W. designed the
study. E.-A. A., J. M. F., A. B., S. U., P. V. and M. W. conducted
experiments. E.-A. A., A. B., J. M. F., E. S., S. U., P. V., M. W.
and M. B. W. analysed the study. E.-A. A., A. B., J. M. F., M. M.,
J. M., E. S., S. U., M. W. and M. B. W. wrote the manuscript.
All the authors are employees of F. Hoffmann-La Roche AG.

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