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RESEARCH PAPER
Pioglitazone improves lipid and insulin levels inoverweight rats on a high cholesterol and fructosediet by decreasing hepatic inflammation bph_671 1892..1902
Massimo Collino1, Manuela Aragno2, Sara Castiglia1, Gianluca Miglio1, Chiara Tomasinelli2,
Giuseppe Boccuzzi3, Christoph Thiemermann4* and Roberto Fantozzi1*
1Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Turin, Italy, 2Department of ExperimentalMedicine and Oncology, University of Turin, Turin, Italy, 3Department of Clinical Pathophysiology, University of Turin, Turin,Italy, and 4Centre for Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the LondonSchool of Medicine and Dentistry, Queen Mary University of London, London, UK
Background and purpose: Nutrient overload leads to obesity and insulin resistance. Pioglitazone, a selective peroxisomeproliferator-activated receptor (PPAR)g agonist, is currently used to manage insulin resistance, but the specific molecularmechanisms activated by PPARgare not yet fully understood. Recent studies suggest the involvement of suppressor of cytokinesignalling (SOCS)-3 in the pathogenesis of insulin resistance. This study aimed to investigate the hepatic signalling pathwayactivated by PPARg activation in a non-genetic insulin-resistant animal model.Experimental approach: Male Wistar rats were maintained on a high-cholesterol fructose (HCF) diet for 15 weeks. Pioglita-zone (3 mgkg-1) was administered orally for the last 4 weeks of this diet. At the end of the treatment, serum was collected forbiochemical analysis. Levels of PPARg, SOCS-3, pro-inflammatory markers, insulin receptor substrate-2 and Akt/glycogensynthase kinase-3b phosphorylation were assesed in rat liver.Key results: Rats fed the HCF diet exhibited hyperlipidemia, hyperinsulinemia, impaired glucose tolerance and insulin
resistance. Pioglitazone administration evoked a significant improvement in lipid metabolism and insulin responsiveness. Thiswas accompanied by reduced hepatic expression of SOCS-3, interleukin-6, tumour necrosis factor-aand markers of neutrophilinfiltration. Diet-induced PPARgexpression was unaffected by the pioglitazone treatment.Conclusion and implications: Chronic pioglitazone administration reduced hepatic inflammatory responses in rats fed a HCFdiet. These effects were associated with changes in hepatic expression of SOCS-3, which may be a crucial link between thereduced local inflammation and the improved insulin signalling.
British Journal of Pharmacology(2010) 160,18921902; doi:10.1111/j.1476-5381.2010.00671.x; published online 8March 2010This article is commented on by Chatterjee, pp. 18891891 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00739.x
Keywords:pioglitazone; high cholesterol and fructose diet; insulin resistance; inflammation; PPARg; SOCS-3
Abbreviations: COX-2, cyclooxygenase-2; ELISA, enzyme-linked immunosorbent assay; FAS, fatty acid synthase; GSK-3 b,
glycogen synthase kinase-3b; HCF, high-cholesterol fructose; HDL, high-density-lipoprotein; ICAM-1, inter-cellular adhesion molecule-1; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; IRS, insulin receptorsubstrate protein; LDL, low-density-lipoprotein; LGK, liver glucokinase; MPO, myeloperoxidase; OGTT, oralglucose tolerance test; PGC-1a, peroxisome proliferator-activated receptor-gcoactivator-1a; PPAR, peroxi-some proliferator-activated receptor; PPRE, PPAR response element; SOCS-3, suppressor of cytokinesignalling-3; TC, total cholesterol; TG, triglyceride; TNF-a, tumour necrosis factor-a; TZD, thiazolidinedione
Introduction
Dietary energy intake has increased steadily in Western soci-
eties resulting in increased obesity, diabetes and cardiovascu-
lar disease (Grosset al., 2004). Simple sugars and saturated fats
are believed to be the major components of the Western diet
that promote obesity and insulin resistance (Bessesen, 2001;Axen et al., 2003). Sugar-sweetened beverages on the market
Correspondence: Massimo Collino, Department of Anatomy, Pharmacology
and Forensic Medicine, University of Turin, via P. Giuria 9, 10125 Torino, Italy.
E-mail: [email protected]
*Both authors contributed equally to this work.
Received 11 September 2009; revised 29 October 2009; accepted 29 Novem-ber 2009
British Journal of Pharmacology (2010), 160, 18921902 2010 The AuthorsJournal compilation 2010 The British Pharmacological Society All rights reserved 0007-1188/10
www.brjpharmacol.org
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today contain 1015% sucrose, which might contribute to the
development of insulin resistance. The rat model described in
this article mimics a typical unhealthy Western diet as it
featured both high cholesterol and fructose (HCF) intake, an
insult that induced dyslipidemia and insulin resistance with
hyperinsulinemia but not yet frank hyperglycaemia.Many lines of evidence have convincingly shown that
chronic inflammation is an important pathogenic factor in
the development of insulin resistance in key metabolic
tissues, including the liver (Schenket al., 2008). Both systemic
and locally produced cytokines are known to activate intrac-
ellular kinases capable of inhibiting key elements of the
insulin signalling pathway, including the insulin receptor
substrate proteins (IRS) and the phosphatidylinositol 3-kinase
(PI3K)-Akt-glycogen synthase kinase (GSK)-3b cascade (Hota-
misligil, 2006). Recently, the family of suppressors of cytokine
signalling (SOCS) proteins has been suggested to be involved
in both inflammation and insulin resistance. This family of
proteins is characterized by their ability to cause feedbackinhibition of interleukin-6 (IL-6) and tumour necrosis factor-a
(TNF-a) activity and the isoform SOCS-3 has been reported to
be highly expressed in the livers of obese animals (Ueki et al.,
2004). Furthermore, it has been demonstrated that SOCS-3
binds to the insulin receptor in the liver and prevents the
coupling of IRS-2 with the insulin receptor, thus reducing
insulin sensitivity (Farrell, 2005).
Peroxisome proliferator-activated receptor (PPAR)g is a
member of the nuclear receptor superfamily of ligand-
activated transcription factors. The most clinically used PPARg
agonists, rosiglitazone and pioglitazone, which belong to the
thiazolidinedione (TZD) class of anti-diabetic drugs, are able
to manage obesity-linked insulin resistance and type 2 diabe-
tes (Miyazaki et al., 2002; Gerstein et al., 2006). A large
number of studies have revealed a broad spectrum of action
for PPARg agonists beyond the control of glucose and lipid
metabolism, including anti-inflammatory properties (Moller
and Berger, 2003). However, to date, very few studies have
been carried out on the effects of PPARgagonism in a non-
genetic, pre-diabetic animal model of insulin resistance and
the molecular mechanism(s) of PPARgagonists effects in the
liver has not been investigated. Hence, the present study was
undertaken to determine the effects of chronic treatment with
the selective PPARgagonist pioglitazone in HCF-fed rats. We
focused on the SOCS-3 signalling pathway in order to ascer-
tain its potential involvement in PPARgeffects in the liver of
rats fed with a HCF diet for 15 weeks.
Methods
Animals and diets
Animal care and experimental procedures complied with
Italian regulations on the protection of animals used for
experimental and other scientific purposes (D.M. 116/92).
Four-week-old male Wistar rats (Harlan-Italy; Udine, Italy)
(170200 g body weights) were housed in a controlled envi-
ronment at 25 2C with alternating 12-h light and dark
cycles. They were provided with a Piccioni pellet diet (n.48,
Gessate Milanese, Italy) and water ad libitum. All the animalswere fed with a normal pellet diet for 1 week prior to the
experiment. The animals were then allocated to one of two
dietary regimens, either normal (control) or a high-
cholesterol diet with 10% fructose solution (HCF) for 15
weeks. The high-cholesterol diet, recently described (Aragno
et al., 2009), contained 10.1% fat (5% coconut oil and 5.1%
linoleic acid), 17% protein, 51.6% carbohydrate and 4% cho-lesterol. All diets contained a standard mineral and vitamin
mixture. Body weight, water and food intake were recorded
weekly. Feed efficiency was expressed as body change in g
100/cumulated food intake.
Drug administration
After the initial period of 11 weeks of dietary manipulation,
each diet group was further subdivided into different treat-
ment groups: normal diet (control), normal diet with piogli-
tazone (control+Pio), HCF diet alone and HCF diet with
pioglitazone (HCF+Pio). Pioglitazone was suspended in 0.5%
methylcellulose and administered daily for 4 weeks by oral
gavage at a dose of 3 mgkg-1 of body weight. This low dose of
pioglitazone closely resembles that used in patients treated
with TZDs and it was previously found to improve insulin
sensitivity and plasma lipid profile in rats fed a high fat diet
(Yoshimoto et al., 1997; Yang et al., 2005). The rats were
allowed to continue to feed on their respective diets until the
end of the study.
Oral glucose tolerance test (OGTT)
One day before the rats were due to be killed, the OGTT was
performed after overnight fasting by administering glucose
(2 gkg-1) by oral gavage. Blood was obtained from the tail
vein before administration and 30, 60, 90, 120 and 150 minafterwards, and glucose concentration was measured on a
conventional Glucometer (Accu-Check Compact kit, Roche
Diagnostics Gmbh, Mannheim, Germany). The food was
removed from the cages on the day before measurement to
ensure accurate fasting glucose values.
Biochemical analysis
At 4 weeks after the start of the drug treatment (15 weeks of
dietary manipulation), the rats were anaesthetized with i.p.
injection (30 mgkg-1) of Zoletil 100 (Laboratoires Virbac,
France), and killed by aortic exanguination. Blood samples
were collected and plasma was isolated. Glycaemia was mea-
sured using the Accu-Check Compact kit. The liver was iso-
lated, weighed and rapidly freeze-clamped with liquid
nitrogen and stored at -80C. The plasma lipid profile was
determined by measuring the content of triglyceride (TG),
total cholesterol (TC), high-density lipoprotein (HDL) and
low-density lipoprotein (LDL) by standard enzymatic proce-
dures using reagent kits (Hospitex Diagnostics, Florence,
Italy). Plasma insulin, levels were measured using enzyme-
linked immunosorbent assay (ELISA) kits (Rat Insulin ELISA,
Sylveniusgaten, Sweden).
Tissue extracts
Liver extracts were prepared using the Meldrum method(Meldrum et al., 1997) with modification. Briefly, rat livers
High-fat/fructose-fed rats and PPARgCollino et al 1893
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were homogenized at 10% (wv-1) in a Potter Elvehjem
homogenizer (Wheaton, NJ, USA) using a homogenization
buffer containing 20 mM HEPES, pH 7.9, 1 mM MgCl2,
0.5 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT),
0.5 mM phenylmethyl sulphonyl fluoride (PMSF), 5 mgmL-1
aprotinin and 2.5mgmL-
1 leupeptin. Homogenates were cen-trifuged at 1000 g for 5 min at 4C. Supernatants were
removed and the protein content was determined using a
BCA protein assay following the manufacturers instructions.
Samples were stored at -80C until use.
Liver triglyceride level
Hepatic TG was extracted from total tissue homogenate and
assayed using reagent kits (Hospitex Diagnostics, Florence,
Italy).
Western blot analysisAbout 15mg total proteins were loaded for Western blot
experiments. Proteins were separated by 8% sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to a polyvinyldenedifluoride (PVDF) membrane,
which was then incubated with a primary antibody (rabbit
anti-PPARg, dilution 1:1000; rabbit anti-cyclooxygenase-2
(COX-2), dilution 1:200; rabbit anti-inducible nitric oxide
synthase (iNOS), dilution 1:200; rabbit anti-total GSK-3b,
dilution 1:500; goat anti-pGSK-3bSer9, dilution 1:500; rabbit
anti-total Akt, dilution 1:500; mouse anti-pAkt Ser 473, dilution
1:1000; goat anti-intercellular adhesion molecule (ICAM)-1,
dilution 1:200). Blots were then incubated with a secondary
antibody conjugated with horseradish peroxidase (dilution
1:10 000) and developed using the ECL detection system. The
immunoreactive bands were visualized by autoradiography
and the density of the bands was evaluated densitometrically
using Gel Pro Analyzer 4.5, 2000 software (Media Cybernet-
ics, Silver Spring, MD, USA). The membranes were stripped
and incubated with b-actin monoclonal antibody (dilution
1:5000) and subsequently with an anti-mouse antibody (dilu-
tion 1:10 000) to assess gel-loading homogeneity.
Myeloperoxidase (MPO) activity
Samples were homogenized in a solution containing 0.5%
(wv-1) hexadecyltrimethyl-ammonium bromide dissolved in
10 mM potassium phosphate buffer (pH 7) and centrifugedfor 30 min at 20 000 g at 4C. An aliquot of the supernatant
was then allowed to react with a solution of 1.6 mM tetram-
ethylbenzidine and 0.1 mM H2O2. The rate of change in
absorbance was measured spectrophotometrically at 650 nm.
MPO activity was defined as the quantity of enzyme degrad-
ing 1mmol of peroxide per min at 37C and was expressed in
milliunits (gwet tissue-1).
Reverse transcriptase-polymerase chain reaction
(RT-PCR) analysis
Total RNA was extracted from the rat liver by using Omnizol
(Euroclone, Milan, Italy) and precipitated with isopropanol.mRNA was reverse-transcribed by using the RevertAid
HMinus Synthesis kit and oligo(dT)18primers (Fermentas Life
Science, Milan, Italy). cDNA were PCR-amplified using
EuroTaq (EuroClone) and sequence-specific oligonucleotide
primers (Sigma-Genosys, Milan Italy): SOCS-3 (amplicon size
310 bp) forward 5-CAGCTCCAAGAGCGAGTACCAG-3,
reverse 5-CATGTAGTGGTGCACCAACTTGA-3;18S (amplicon size 489 bp) forward 5-TCAAGAACGAAA
GTCGAAGGT-3, reverse 5-GGACATCTAAGGGCATCACAG-
3; IL-6 (amplicon size 479 bp) forward 5-CCGGAGAGGAGA
CTTCACAG-3, reverse 5-TGGTCTTGGTCCTAAGCCAC-3;
TNF-a (amplicon size 254 bp) forward 5-CACGCTCTTCT
GTCTACTGA-3, reverse 5-GTACCACCAGTTGGTTGTCT-3;
PGC-1a (amplicon size 668 bp) forward 5-GGAGCTGGAT
GGCTTGGGAC-3, reverse 5-GTGAGGAGGGTCATCGTTTG
TG-3; LGK (amplicon size 310 bp) forward 5 -GTGGTGCTTT
TGAGACCCGTT-3, reverse 5-TTCGATGAAGGTGATTTCG
CA-3; FAS (amplicon size 459 bp) forward 5 -GCCTCACTC
CGAGGAACAAACA-3, reverse 5-CCCGGCATTCAGAAGG
TGATTTCGCA-3. Thermal cycling conditions were as follows:activation of 94C for 5 min, followed by 39 cycles of ampli-
fication at 94C for 15 s, 60C for 30 s, 72C for 30 s (18S);
activation of 95C for 5 min, followed by 35 cycles of ampli-
fication at 95C for 30 s, 60C for 30 s, 72C for 30 s (SOCS-3);
activation of 95C for 2 min, followed by 34 cycles of ampli-
fication at 95C for 60 s, 51C for 60 s, 72C for 90 s (IL-6);
activation of 95C for 2 min, followed by 34 cycles of ampli-
fication at 95C for 60 s, 53C for 60 s, 72C for 90 s (TNF-a);
activation of 95C for 10 min, followed by 35 cycles of ampli-
fication at 62C for 45 s, 53C for 30 s, 72C for 120 s (PGC-
1a); activation of 94C for 5 min, followed by 35 cycles of
amplification at 94C for 30 s, 53C for 30 s, 72C for 60 s
(LGK); activation of 94C for 5 min, followed by 35 cycles of
amplification at 94C for 30 s, 57C for 30 s, 72C for 60 s
(FAS). RT-PCR amplicons were resolved in an ethidium
bromide-stained agarose gel [1.8% (wtvol-1)] by electrophore-
sis, and signals were quantified using analysis software (NIH
Image J 1.41).
Statistical analysis
All values in both the text and figures are expressed as mean
SD for n observations. One-way analysis of variance with
Dunnetts post test was performed using the GraphPad Prism
version 4.02 for Windows (GraphPad Software, San Diego,
CA, USA) and P-values
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antibodies and Luminol ECL were from Amersham (Bucking-
hamshire, UK). Nomenclature for the receptors and molecular
targets studied here follows Alexander et al. (2009).
Results
Characteristics of the experimental groups
After 15 weeks of feeding, the body weights of the HCF group
were significantly increased (>20%) compared with those of
the control group (Table 1) and daily administration of piogli-
tazone did not induce any significant changes in body weight.
Feed efficiencies in the HCF and HCF+Pio groups were signifi-
cantly increased in comparison with those in the control
group (P < 0.05). Rats fed with the experimental diet had a
greater liver and adipose tissue (epididymal fat) weight than
control diet-fed rats (Table 1) and pioglitazone treatment sig-nificantly reduced liver weight, but not epididymal fat. The
triglyceride content was doubled in the liver of HCF rats in
comparison with control rats, whereas hepatic triglyceride
accumulation was reduced by pioglitazone administration
(Table 1). Basal glucose levels in the plasma, measured on
completion of the protocol, did not show any difference
among the groups (Table 2). A threefold increase in insulin
levels was detected in HCF rats, relative to controls, and
chronic pioglitazone administration significantly decreased
plasma insulin levels in HCF rats (P
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Figure 3 Effects of pioglitazone on insulin signal transduction in the liver of rats fed a HCF diet. IRS-2 (A), total Akt protein expression andSer473 phosphorylation (B), and total GSK-3b protein expression and Ser9 phosphorylation (C) were analysed by Western blot on the liverobtained from Wistar rats fed with a standard (Control) or HCF diet for 15 weeks and treated with pioglitazone (Pio; 3 mgkg-1, p.o.) duringthe last 4 weeks (Control+Pio; HCF+Pio). Densitometric analysis of the bands is expressed as relative optical density (O.D.), corrected for the
correspondingb-actin contents and normalized using the related Control band. The data are means SD of three separate experiments.P
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