Pioglitazone Decreases Hepatic Inflammation

8/9/2019 Pioglitazone Decreases Hepatic Inflammation

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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);


fication at 95C for 60 s, 51C for 60 s, 72C for 90 s (IL-6);


fication at 95C for 60 s, 53C for 60 s, 72C for 90 s (TNF-a);


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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Pioglitazone Decreases Hepatic Inflammation

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