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Atherosclerosis 195 (2007) e181–e190

Pioglitazone added to conventional lipid-lowering treatmentin familial combined hyperlipidaemia improves parameters

of metabolic control: Relation to liver, muscleand regional body fat content�

E. Louise Thomas a,∗, Elizabeth Potter b, Isabella Tosi b, Julie Fitzpatrick a,Gavin Hamilton a, Vian Amber c, Robert Hughes d, Christopher North e,

Paul Holvoet f, Mary Seed g, D. John Betteridge e,Jimmy D. Bell a, Rossi P. Naoumova b

a The Robert Steiner MR Unit, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UKb Lipid Clinic and Clinical Research Facility, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital,

Du Cane Road, London W12 0NN, UKc Division of Investigative Sciences, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK

d Department of Cardiology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UKe Department of Medicine, University College London, University College Hospitals, London, UK

f Department of Cardiovascular Diseases, Faculty of Medicine, Katholieke Universiteit Leuven, Belgiumg Lipid Clinic, Charing Cross Hospital, London, UK

Received 12 January 2007; received in revised form 9 March 2007; accepted 23 March 2007Available online 7 May 2007

bstract

Familial combined hyperlipidaemia (FCHL) is a complex genetic disorder conferring high risk of premature atherosclerosis, characterized byigh cholesterol and/or triglyceride, low high density lipoprotein (HDL) cholesterol and insulin resistance. We examined whether pioglitazone,dded to conventional lipid-lowering therapy, would favourably affect metabolic parameters and alter body fat content. We undertook aandomized, double blind, placebo-controlled study in 22 male patients with FCHL treated with pioglitazone or matching placebo 30 mg dailyor 4 weeks, increasing to 45 mg for 12 weeks. Magnetic resonance imaging and proton magnetic resonance spectroscopy were performed toeasure adipose tissue (AT) body content as well as intrahepatocellular lipids (IHCL) and intramyocellular lipids (IMCL) at baseline and after

reatment. Significantly improved in the pioglitazone group were: triglyceride/HDL (atherogenic index of plasma) −32.3% (p = 0.002), plasmalucose −4.4% (p = 0.03), alanine-aminotransferase (ALT) −7.7% (p = 0.005) and adiponectin 130.1% (p = 0.001). Pioglitazone treatment

esulted in a significant increase in total (5.3%, p = 0.02) and subcutaneous (7.1%, p = 0.003) adipose tissue as well as in soleus-IMCL levels47.4%, p = 0.02) without alteration in intra-abdominal AT or IHCL. Changes in ALT and AST and IHCL were strongly correlated (r = 0.72,< 0.01; r = .0.86, p < 0.01, respectively).In patients with FCHL on conventional lipid-lowering therapy, the addition of pioglitazone acts favourably on several metabolic parameters. 2007 Elsevier Ireland Ltd. All rights reserved.

eywords: Adipose tissue; Intrahepatic fat; Intramyocellular lipids; MRI

Abbreviations: AT, adipose tissue; IHCL, intrahepatocellular lipids; IMCL, inresonance imaging; 1H MRS, proton magnetic resonance spectroscopy; TE, echo t� This work was supported in part by the J. Coller Foundation.∗ Corresponding author at: Robert Steiner MR Unit, Hammersmith Hospital, Du

ax: +44 20 8383 3038.E-mail address: louise.thomas@csc.mrc.ac.uk (E.L. Thomas).

021-9150/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2007.03.043

tramyocellular lipids; IAAT, intra-abdominal adipose tissue; MRI, magneticime; TR, repetition time

Cane Road, London W12 0HS, UK. Tel.: +44 20 8383 3772;

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182 E.L. Thomas et al. / Athero

. Introduction

Familial combined hyperlipidaemia is a common, geneti-ally heterogeneous disorder that affects 1–3% of the Westernopulation and confers a substantially increased risk of pre-ature coronary artery disease (CAD) [1,2]. The FCHL

henotype is characterized by high serum levels of choles-erol and/or triglyceride, low high density lipoprotein (HDL)holesterol, high serum apolipoprotein B and small dense lowensity lipoproteins (LDL) as well as by insulin resistance3–6].

The complex nature of FCHL, involving genetic andnvironmental factors and the multiple lipid abnormali-ies that characterize FCHL make this condition difficulto treat, frequently necessitating combination drug therapyith different classes of lipid-lowering agents. Given the

ssociation of FCHL with insulin resistance and the find-ng that lipid abnormalities in this condition are similar tohose seen in patients with the metabolic syndrome andype 2 diabetes [6], there is a potential for improvement ofhe metabolic parameters in FCHL with thiazolidinediones.hese compounds activate the peroxisome proliferator-ctivated receptor (PPAR)-� on the nuclear membrane andmprove post-receptor insulin signalling and insulin sensi-ivity [7]. Thiazolidinediones have been shown to have aavourable effect on some serum lipid parameters in patientsith diabetes [8] and also in non-diabetic patients with hyper-

ension [9].The links between insulin resistance and components

f the metabolic syndrome, including combined hyperlip-daemia are complex. However, increased intra-abdominaldiposity and ectopic fat deposition have been implicated asredisposing factors in the pathogenesis of the biochemicalbnormalities and the development of metabolic and vascularomplications.

Previous studies using members of the thiazolidinedioneroup of drugs have shown improvements in insulin sen-itivity in type 2 diabetic patients in association with ae-distribution of adipose tissue away from metabolicallymportant sites such as intra-abdominal adipose depots10,11]. Other studies have shown that pioglitazone increaseshe insulin sensitivity of the liver and peripheral tissues,hrough a reduction in their triglyceride contents; termedntrahepatocellular lipids (IHCL) and intramyocellular lipidsIMCL), respectively [12,13]. Whilst those studies clarifyome of the mechanisms of action of thiazolidinediones inatients with type 2 diabetes, it is not known whether FCHLatients without diabetes, but with similar lipid abnormali-ies, would respond favourably to a thiazolidinedione, whendded to conventional lipid-lowering medication.

In this study we hypothesized that treatment with pioglita-one, added to standard lipid-lowering therapy, would further

mprove serum lipid and metabolic parameters in patientsith FCHL and that these changes would be associated with

lterations in skeletal muscle and liver triglyceride stores andhe re-distribution of body fat depots. We therefore under-

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is 195 (2007) e181–e190

ook a randomized, double blind, placebo-controlled study inCHL patients without diabetes, using magnetic resonancepectroscopy (1H MRS) to assess skeletal muscle and liverriglyceride content and magnetic resonance imaging (MRI)o assess body fat at baseline and following 16 weeks ofreatment with the thiazolidinedione pioglitazone.

. Materials and methods

.1. Patients

Index patients with FCHL from our well characterizedohort were invited to participate in the study. A descriptionf the FCHL cohort, recruitment and diagnostic criteriaave been published in detail previously [6,14]. In short,he index patients with FCHL had pre-treatment cholesterolnd triglyceride levels greater than age- and sex-specific5th and 90th percentile values, respectively, and a bloodelative with raised plasma cholesterol or triglyceride oroth, greater than age- and sex-specific 90th percentile.atients with other genetic hyperlipidaemias (e.g. familialypercholesterolaemia) or secondary hyperlipidaemiasue to obesity, hypothyroidism, liver and kidney diseases,lcohol abuse were excluded. Only male index patientsith FCHL were eligible to participate in this study ifespite lipid-lowering medication with a statin (with orithout other conventional lipid-lowering agents) oner more of their serum lipid parameters remained abovearget levels (total serum cholesterol > 5 mmol/l, triglyc-ride > 1.7 mmol/l, HDL-cholesterol < 1.0 mmol/l, totalholesterol/HDL-cholesterol ratio > 5.0). Patients werexcluded from the study on the following criteria: type 1r type 2 diabetes, obesity, hypothyroidism, abnormal liverunction tests (defined as ALT > 2.5 times the upper limitf the reference interval) and significant renal impairmentdefined as creatinine > 135 �mol/l).

In total 38 male Caucasian FCHL patients were invitednd agreed to participate in the study. Of these, 22 patientsmean age 53.5 ± 7.4 years; age range 42–73 years) com-leted the study and their data only were used in the statisticalnalysis. Due to a series of technical problems with theRI scanner, 13 patients only had a pre-treatment MRI scan

one and 3 were excluded from the final analysis becausef protocol violation. All patients had attended the sameipid Clinic for at least 2 years and had been on stableedical therapy and diet for at least 3 months prior to the

nclusion in the study. Therapy and dietary habits remainedhe same during the trial period. The clinical characteris-ics of the patients and their medication are presented inable 1.

All patients gave written informed consent before partici-

ation and the study protocol was approved by the Researchthics Committees of the two participating hospitals (Ham-ersmith Hospital, Imperial College and University Collegeospitals, University College London, UK). The study was

E.L. Thomas et al. / Atherosclerosis 195 (2007) e181–e190 e183

Table 1Characteristics of the study subjects (n = 22)

Pioglitazone group (n = 10) Placebo group (n = 12) p-Value

Age (years) 53.10 ± 6.37 53.83 ± 8.47 0.82Weight (kg) 81.79 ± 9.28 82.91 ± 9.02 0.78Height (m) 1.76 ± 0.07 1.75 ± 0.06 0.52BMI (kg/m2) 26.21 ± 1.89 27.14 ± 2.34 0.31Coronary artery disease (CAD) 1 4 NS

MedicationsACE inhibitors 5 6 NS�-blockers 0 3 NSCalcium channel blockers 3 4 NSAspirin 8 6 NSNitrates 0 0 NS

Lipid-lowering drugsStatins 10 12 NSFibrates 3 4 NS

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onducted according to the guidelines of the Declaration ofelsinki.The study was registered in a public access data base

or controlled clinical trials (register number ISRCTN4596899).

.2. Study design

The study protocol is shown in Fig. 1. The study wasesigned as a two-centre, double-blind, randomized, placebo-ontrolled trial; patients who met the inclusion and exclusionriteria at visit 1 were randomly allocated to receive treat-ent with pioglitazone or placebo from visit 2 (week 0) and

reated for a period of 16 weeks. For the first 4 weeks patientseceived 30 mg pioglitazone or placebo daily (both manu-actured by Takeda, Japan); for the remaining 12 weeks inoth the pioglitazone and placebo groups the dosage was

ncreased to 45 mg daily. If adverse events were observed,atients remained on 30 mg. Blood samples were taken at:aseline (week 0, visit 2), week 4 (visit 3), week 8 (visit 4),nd week 16 (visit 6). MRI and 1H MRS studies were per-

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ig. 1. Diagram depicting the study protocol with the relative timings. Small arrownd fasting blood sample obtained; big arrows, magnetic resonance imaging (MRItudy medication (placebo/pioglitazone) with randomization at visit 2; stethoscope

2 NS1 NS

ormed at baseline (week 0), before starting the medicationnd again at week 16 (visit 6).

Compliance with study medication was calculated basedn the dispensing and return of study medication tablets andhowed that the during the first 4 weeks the mean dose was9.0 ± 2.1 mg and 41.0 ± 6.3 mg daily for the rest of thetudy, which represents 97 and 93% compliance, respectively.

.3. Methods

.3.1. Laboratory analysesAll blood samples were obtained after 12 h fast at

aseline (week 0), week 4, week 8 and week 16. Totalerum cholesterol, triglyceride and HDL-cholesterol levels,lasma glucose and HbA1c were determined by automatedethods using large platform analysers and inter-assay

ontrols. Lipoprotein (a) [Lp(a)] was measured using an auto-

ated immunoturbidimetric assay (Beckman Instruments

nc., Galway, Ireland). Plasma insulin was measured usingn established radio-immuno assay (RIA) method [15]; allamples were frozen and assayed in duplicate and on a single

s, hospital visit when blood pressure, pulse and body weight were checked) scan and proton magnetic resonance spectroscopy (1H MRS); black bar,, physical examination.

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184 E.L. Thomas et al. / Athero

ccasion to avoid inter-assay variation. The detection limitf the insulin assay was 6 pmol/l and the intra-assay coef-cient of variation was 5.4%. Leptin was analyzed using

he Human Leptin RIA Kit (Catalogue Number HL-81K)rom Linco Research Inc., MO, USA. NEFA was measuredn serum using NEFA C, ACS-ACOD Method (Wako Chem-cals GmbH, Neuss, Germany). oxLDL was analysed usingmAB-4E6-based competition ELISA (oxLDL-M; Merco-ia, Uppsala, Sweden) [16]. Adiponectin was measured usingmmunoassay using the Quantikine kit (DRP 300) Humandiponectin/Acrp30 (R&D Systems Europe Ltd., Abingdon,K).Several parameters related to carbohydrate and lipid

etabolism were also calculated: Homeostatic Modelssessment (HOMA), steady state beta cell function (%B)

nd insulin sensitivity (%S) were assessed using the HOMA2alculator (http://www.dtu.ox.uk/homa/index.html). A quan-itative insulin sensitivity check index (QUICKI) wasetermined from fasting insulin and glucose values [17].he atherogenic index of plasma (AIP) was calculateds a triglyceride-to-HDL cholesterol molar ratio and wassed as an indicator of small dense LDL concentration18,19].

.3.2. Procedures to investigate total body lipid contentAll imaging and spectroscopy data were acquired with a

.5T Phillips Eclipse multinuclear system (Phillips, Cleve-and, OH, USA).

1H MRS of liver: Single voxel spectra were obtained fromhe liver using a PRESS sequence as previously described20]. Spectra were analyzed using AMARES. Peak areas forll resonances were obtained and lipid resonances quantifiedith reference to water after correcting for T1 and T2.

1H MRS of muscle: Spectra was obtained from the soleusS IMCL) and tibialis (T IMCL) muscles of the left calf, usingPRESS sequence as previously described [21]. Spectra werenalyzed using AMARES, lipid resonances quantified witheference to Crtot after correcting for T1 and T2.

Whole body MR imaging: Whole body MR images werecquired using a rapid T1-weighted spin echo sequence. Vol-nteers were scanned in a prone position with arms straightbove the head. Volunteers were scanned from their fingertipso their toes by acquiring 10 mm thick transverse images with0 mm gaps between slices in their arms and legs and 10 mmaps between slices in their abdomens [22]. Images werenalysed blind by an independent observer (VardisGroup,ondon, UK) using SliceOmatic (Tomovision, Montreal,anada).

.4. Statistical analyses

For each of the laboratory and demographic variables

xamined at four visits a repeated measure analysis of vari-nce was performed with factors treatment (pioglitazone orlacebo) and visit taken into account. The baseline value ofhe variable was included as a covariance. Log transforma-

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is 195 (2007) e181–e190

ions were performed on non-normal data, assessed fromhapiro and Francia’s test of normality. Bartlett’s test ofomogeneity of variance across the six combinations of treat-ent and visit was employed. Repeated analysis of varianceeasures using this transformation, including the baseline

alue as a covariate were performed, designed to controlor differences at baseline between study groups. The fac-or visit is a repeat measurement, so for the factor visitnd visit × treatment interaction the Huyn–Feldt corrected p-alues have been used, to take account of repeated measures.he MRI and MRS analyses were performed at baseline andnd of study. A value of p < 0.05 was considered to be sta-istically significant. Stepwise multiple regression analysisas performed using both forwards and backwards variable

election. Variables were retained in the model if p < 0.05.

. Results

.1. Baseline characteristics

The clinical characteristics of the patients in the placebond the pioglitazone groups and their medication are pre-ented in Table 1. There were no significant differences ataseline between the placebo and pioglitazone treated groupsor any of the parameters.

.2. Effect of pioglitazone on blood parameters of lipidnd carbohydrate metabolism and related calculatedarameters

The effect of 16 weeks treatment on demographic, labo-atory and related calculated parameters is summarised inable 2 for the pioglitazone and for the placebo groups,espectively (mean ± S.D.). Patients in the active treat-ent group demonstrated significant changes in plasma

riglycerides (−26.5%, p = 0.006), HDL cholesterol (10.7%,= 0.02), triglyceride to HDL-cholesterol molar ratio (AIP)

−32.3%, p = 0.002), plasma glucose (−4.4%, p = 0.03) andLT (−7.7%, p = 0.005). Changes were also observed in CRP

−12.7%, p = 0.04) and adiponectin (130.1%, p = 0.001). Ithould be stressed that although baseline levels of adiponectiniffer between the two groups, the statistical analysismployed included the baseline as covariate and controlledor such baseline differences between study groups. No sig-ificant changes were observed in the following parameters:xLDL, expressed as either absolute values or as a per-ent of LDL, plasma insulin, leptin levels or any of thendirect measurements used to assess insulin resistance andensitivity.

.3. Effect of pioglitazone treatment on body

omposition, IHCL and IMCL

Significant increases in both total (5.3%, p = 0.02) andubcutaneous (7.1%, p = 0.003) AT depots were observed

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therosclerosis195

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e185

Table 2Clinical data at baseline (V2), follow-ups (V3, V4) and final visit (V6)

Pioglitazone group Placebo group p-Values

Baseline Visit 3 Visit 4 Final visit Baseline Visit 3 Visit 4 Final visit Treatment Visit* Treatment× visit*

Weight (kg) 81.79 ± 9.28 82.68 ± 9.18 82.57 ± 8.59 83.43 ± 9.58 82.91 ± 9.02 83.38 ± 9.68 83.15 ± 9.85 82.62 ± 9.00 0.08 0.8 0.11BMI† (kg/m2) 26.21 ± 1.89 26.45 ± 1.88 26.43 ± 1.68 26.71 ± 2.16 27.15 ± 2.34 27.33 ± 2.46 27.24 ± 2.58 27.07 ± 2.35 0.10 0.8 0.11Waist (cm) 95.20 ± 5.56 95.73 ± 5.60 95.38 ± 5.25 96.38 ± 7.40 96.85 ± 6.33 97.38 ± 6.95 97.25 ± 7.18 95.27 ± 7.05 0.3 0.5 0.08Waist:hip 0.92 ± 0.03 0.93 ± 0.03 0.92 ± 0.04 0.93 ± 0.06 0.93 ± 0.04 0.94 ± 0.04 0.93 ± 0.05 0.93 ± 0.05 0.7 0.4 0.5Total cholesterol†

(mmol/l)5.05 ± 0.47 5.69 ± 1.11 5.09 ± 0.54 5.01 ± 0.96 4.80 ± 0.80 4.80 ± 0.63 4.74 ± 1.10 4.62 ± 0.65 0.09 0.08 0.5

Triglycerides† (mmol/l) 2.44 ± 0.72 1.90 ± 0.56 1.77 ± 0.49 1.69 ± 0.50 2.52 ± 1.50 2.34 ± 0.98 2.53 ± 1.09 2.23 ± 0.85 0.006 0.5 0.7HDL (mmol/l) 1.31 ± 0.23 1.47 ± 0.22 1.42 ± 0.41 1.46 ± 0.39 1.20 ± 0.32 1.23 ± 0.28 1.13 ± 0.23 1.20 ± 0.27 0.02 0.3 0.9Triglyceride/HDL

cholesterol ratio (AIP)1.97 ± 0.75 1.34 ± 0.51 1.39 ± 0.92 1.25 ± 0.60 2.28 ± 1.44 2.04 ± 1.04 2.31 ± 1.12 1.98 ± 0.88 0.002 0.4 0.7

LDL†-C (mmol/l) 2.63 ± 0.59 3.37 ± 1.10 2.87 ± 0.52 2.78 ± 0.78 2.59 ± 0.78 2.61 ± 0.63 2.59 ± 0.82 2.41 ± 0.74 0.05 0.11 0.4Total cholesterol/HDL 3.95 ± 0.73 3.94 ± 0.79 3.82 ± 0.95 3.58 ± 0.70 4.17 ± 0.96 4.06 ± 0.95 4.27 ± 0.94 4.02 ± 1.15 0.4 0.15 0.4AST† (IU/l) 25.00 ± 2.87 23.44 ± 6.06 23.10 ± 3.78 26.50 ± 9.19 24.17 ± 4.86 24.92 ± 4.54 25.92 ± 4.21 26.17 ± 9.30 0.12 0.3 0.4ALT† (IU/l) 30.70 ± 6.38 24.30 ± 4.95 22.60 ± 3.27 27.70 ± 12.60 29.00 ± 11.08 31.92 ± 11.42 30.92 ± 10.42 31.17 ± 13.83 0.005 0.5 0.4Glucose (mmol/l) 5.55 ± 0.46 5.35 ± 0.51 5.38 ± 0.46 5.28 ± 0.34 5.60 ± 0.86 5.64 ± 0.85 5.77 ± 0.69 5.62 ± 0.84 0.03 0.4 0.9HbA1c (%) 5.41 ± 0.36 5.45 ± 0.29 5.37 ± 0.48 5.46 ± 0.19 5.54 ± 0.51 5.70 ± 0.45 5.81 ± 0.47 5.83 ± 0.42 0.04 0.6 0.5ApoB† (mg/dl) 97.70 ± 21.80 109.70 ± 36.22 103.70 ± 29.29 96.20 ± 28.62 99.00 ± 25.96 98.17 ± 18.83 99.92 ± 34.46 96.08 ± 24.92 0.4 0.3 0.7Lp(a)† (mg/dl) 47.30 ± 51.07 49.00 ± 49.84 47.70 ± 49.65 48.20 ± 45.31 24.67 ± 29.29 24.08 ± 30.31 26.00 ± 30.41 25.33 ± 27.52 0.2 0.6 0.9ApoA1 (mg/dl) 125.80 ± 17.29 118.60 ± 15.88 124.60 ± 29.67 119.40 ± 27.95 117.25 ± 27.80 119.08 ± 17.27 117.75 ± 17.88 114.25 ± 26.27 0.9 0.7 0.8Insulin† (pmol/l) 70.62 ± 23.80 61.97 ± 19.25 59.48 ± 26.05 58.04 ± 27.41 78.59 ± 33.01 69.47 ± 21.23 74.27 ± 21.44 64.93 ± 28.88 0.4 0.2 0.4Leptin (ng/ml) 6.14 ± 1.95 5.64 ± 1.70 6.20 ± 2.20 5.66 ± 2.63 5.47 ± 1.70 6.08 ± 1.81 5.36 ± 1.46 5.49 ± 1.57 0.6 0.7 0.14NEFA† (mmol/l) 0.73 ± 0.41 0.45 ± 0.09 0.55 ± 0.24 0.53 ± 0.27 0.47 ± 0.19 0.47 ± 0.15 0.48 ± 0.15 0.54 ± 0.18 0.5 0.5 0.7

oxLDL-M (U/l) 68.00 ± 21.50 70.00 ± 22.82 63.80 ± 19.66 63.10 ± 21.86 57.67 ± 11.06 59.50 ± 12.42 56.75 ± 14.99 51.91 ± 10.97 0.4 0.2 0.9Adiponectin† (ng/ml) 13260.60 ± 17851.50 18404.30 ± 11899.91 19446.20 ± 13388.13 16670.90 ± 13150.67 8562.25 ± 8372.06 9062.25 ± 9002.40 8564.50 ± 8812.47 5242.33 ± 4151.00 0.001 0.14 0.6CRP† (mg/dl) 0.96 ± 1.00 0.89 ± 1.03 1.08 ± 1.15 0.85 ± 1.02 1.21 ± 0.61 1.49 ± 1.36 1.63 ± 1.83 2.78 ± 4.56 0.04 0.6 0.3HOMA2%B 91.64 ± 14.07 90.15 ± 15.38 86.80 ± 21.68 88.57 ± 25.42 106.48 ± 50.58 94.17 ± 30.36 91.22 ± 21.08 89.71 ± 33.76 0.6 0.7 0.9HOMA2%S 83.58 ± 32.97 92.89 ± 29.10 102.35 ± 37.10 106.98 ± 41.78 77.10 ± 29.55 87.14 ± 45.08 76.18 ± 24.37 97.08 ± 45.03 0.4 0.16 0.3HOMA2 IR 1.35 ± 0.46 1.18 ± 0.37 1.12 ± 0.49 1.08 ± 0.51 1.48 ± 0.60 1.33 ± 0.40 1.44 ± 0.41 1.24 ± 0.53 0.4 0.4 0.6ApoB:ApoA1† 0.80 ± 0.26 0.94 ± 0.32 0.88 ± 0.32 0.87 ± 0.39 0.89 ± 0.35 0.85 ± 0.25 0.86 ± 0.32 0.90 ± 0.39 0.3 0.7 0.5QUICKI 0.39 ± 0.03 0.40 ± 0.03 0.41 ± 0.03 0.41 ± 0.03 0.39 ± 0.02 0.39 ± 0.03 0.38 ± 0.02 0.40 ± 0.03 0.2 0.16 0.5

Results presented as mean ± S.D. Abbreviations: AIP, atherogenic index of plasma; BMI, body mass index; CRP, C-reactive protein; AST, aspartate amino transferase; ALT, alanine amino transferase; HbA1c, glycated haemoglobin; HOMA, Homeostatic

Model Assessment.* Huyn–Feldt corrected p-values.† Log-transformed data.

e186 E.L. Thomas et al. / Atherosclerosis 195 (2007) e181–e190

Table 3MRI data at baseline (visit 2) vs. final visit (visit 6) for pioglitazone and placebo groups

Pioglitazonegroup (n = 10)

Placebo group(n = 12)

Adjusted difference(placebo − pioglitazone)

95% CI fordifference

p-Value

Baseline Final visit Baseline Final visit

Adipose tissue (l)Total adipose tissue 27.03 ± 4.27 28.35 ± 4.04 28.46 ± 4.39 27.90 ± 4.61 −1.8 −3.2 to −0.4 0.02Subcutaneous AT 17.53 ± 3.28 18.68 ± 2.95 18.22 ± 3.32 18.04 ± 3.28 −1.3 −2.1 to −0.5 0.003Subcut abdominal AT 4.62 ± 1.33 5.11 ± 1.31 4.57 ± 0.85 4.73 ± 0.95 −0.3 −0.6 to −0.03 0.04Peripheral subcut AT 12.92 ± 1.98 13.56 ± 1.75 13.65 ± 2.57 13.31 ± 2.45 −0.9 −1.5 to −0.3 0.008Internal AT 9.50 ± 1.81 9.68 ± 1.96 10.24 ± 1.52 9.86 ± 1.71 −0.5 −1.4 to 0.3 0.2Intra-abdominal AT 5.05 ± 1.09 4.99 ± 1.14 5.80 ± 1.09 5.45 ± 1.14 −0.2 −0.8 to 0.3 0.4Peripheral internal AT 4.45 ± 0.96 4.69 ± 1.07 4.44 ± 0.79 4.42 ± 1.06 −0.3 −0.9 to 0.3 0.4

Adipose tissue as percentage of body weightTotal adipose tissue (%) 29.69 ± 2.74 30.58 ± 2.75 30.88 ± 2.99 30.33 ± 3.04 −1.3 −2.7 to 0.2 0.08Subcutaneous (%) 19.19 ± 2.07 20.10 ± 1.89 19.72 ± 2.16 19.59 ± 2.16 −1.0 −1.8 to −0.2 0.02Subcut abdominal AT (%) 5.02 ± 1.06 5.46 ± 0.97 4.95 ± 0.56 5.13 ± 0.68 −0.3 −0.5 to 0.02 0.07Peripheral subcut AT (%) 14.18 ± 1.10 14.93 ± 1.21 14.78 ± 1.78 14.40 ± 1.61 −1.0 −1.7 to −0.3 0.007

Ectopic fatS IMCL 19.80 ± 9.42 26.88 ± 18.36 15.83 ± 5.77 11.88 ± 5.76 −12.6 −22.8 to −2.5 0.02T IMCL 9.26 ± 3.51 9.45 ± 4.92 8.56 ± 3.83 9.39 ± 7.74 0.2 −4.5 to 4.8 0.9

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IHCL 6.34 ± 6.17 4.34 ± 3.56 7.97 ±esults presented as mean ± SD.

ollowing pioglitazone treatment (Table 3). SubcutaneousT increased in both the abdominal (12.0%, p = 0.04) anderipheral (5.5%, p = 0.008) regions. There were no signif-cant changes in internal AT either peripherally or in thentra-abdominal depot.

IMCL levels increased significantly in the soleus mus-le (47.4%, p = 0.02) following pioglitazone treatment. Noignificant changes were observed in the tibialis muscle norn IHCL levels following pioglitazone treatment. However,here were highly significant correlations between changes in

HCL content and alterations in both ALT (r = 0.72, p < 0.01)Fig. 2) and AST (p = 0.86, p < 0.01) levels.

ig. 2. Associations between changes in the intrahepatocellular lipidsIHCL) and alanine-aminotransferase (ALT) in the pioglitazone and placeboroups.

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Multiple regression analysis of the data revealed the onlyarameters to influence changes in IMCL in the soleus mus-le were changes in triglyceride-to-HDL cholesterol molaratio (AIP) and CRP. Whereas changes in several adipose tis-ue compartments, including total adipose tissue, peripheralubcutaneous adipose tissue, intra-abdominal and peripheralnternal adipose tissue, and body weight, influenced changesn tibialis IMCL levels. Changes in IHCL were significantlyssociated with changes in plasma triglycerides, and totaldipose tissue. There was no relationship between changesn IHCL and regional adiposity.

. Discussion

The major novel findings in this study are that pioglitazonedded to conventional lipid-lowering therapy in non-diabeticCHL patients leads to: (1) additional beneficial effects onerum lipid and metabolic parameters over and above thoseeen with standard lipid-lowering therapy; (2) the changesn the blood parameters are observed despite an increase inotal and subcutaneous adipose depots, although there waso effect on internal adipose tissue depots; (3) highly sig-ificant correlations were documented between changes inHCL content and alterations in both ALT and AST levels.

.1. Effect of pioglitazone on blood parameters

The insulin-sensitizing thiazolidinediones have beenested as experimental therapies with variable success [7] inther groups of patients with non-diabetic insulin-resistanceuch as hypertension [9], non-alcoholic fatty liver disease

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23] and lipodystrophy [24]. Patients with FCHL have numer-us metabolic abnormalities, similar to those in the metabolicyndrome, and require complex management. The additionf pioglitazone to the therapeutic regimen of our FCHLatients, all of whom were on statins and nearly 30% of themn a statin/fibrate combination, resulted in further reductionn triglyceride and increase of HDL cholesterol, leading to

significant decrease by more than a third in the triglyc-ride/HDL cholesterol molar ratio. This ratio, also knowns AIP, is a good indicator of LDL particle size in that aatio >1.33 has been shown in 90% of patients with smallDL particles [18]. It is of interest that despite the increase

n HDL cholesterol following treatment with pioglitazone,he serum apolipoprotein A-I levels remained unchanged.his observation is in agreement with apolipoprotein A-I

urnover studies performed in the New Zealand White Rabbithich showed that pioglitazone lead to enhanced productionf small HDL3c particles, despite unchanged apolipoprotein-I concentration [25]. It has also been suggested that piogli-

azone acts as a partial PPAR alpha agonist which could alsoontribute to the increase in serum HDL cholesterol levels26].

Several prospective studies have demonstrated that smallense LDL particles are the best predictors of future car-iovascular events in non-diabetic subjects. The associationemains significant even after adjustment for confoundingactors such as serum levels of triglyceride, HDL- and LDL-holesterol [27]. The triglyceride/HDL cholesterol molaratio is a strong predictor of myocardial infarction with a riskactor-adjusted relative risk of 16 for the highest as comparedith lowest quartile [28]. The mean baseline level of triglyc-

ride/HDL cholesterol molar ratio in our FCHL patients wasaised, suggesting that despite conventional lipid-loweringreatment the residual CAD risk from the presence of smallDL particles was still high and underestimated; the ratioas reduced to 1.25 ± 0.60 following the addition of piogli-

azone. In prior studies, treatment with pioglitazone has beenhown to have a substantial benefit on diabetic dyslipidaemia,ncluding increasing HDL-C levels and reducing triglyc-rides levels [29]. Both of these effects were also observedn a recent study and could have contributed to improvementn carotid intima-media thickness in patients with type 2 dia-etes [30]. The beneficial effect of pioglitazone on carotidntima-media thickness in the latter study is consistent withesults of the PROactive Study [31]. While treatment withioglitazone did not significantly reduce the risk of the com-osite primary end point it did significantly reduce by 16%he risk of the main secondary end point (a composite ofll-cause mortality, MI or stroke).

The slight, but significant increase in LDL cholesterolbserved following treatment with pioglitazone may be offsety the favourable changes in the triglyceride/HDL choles-

erol molar ratio, indicating an increase in LDL particle sizes well as the reduction in total cholesterol/HDL cholesterolatio. The small increase observed in LDL cholesterol witho change in apolipoprotein B would also suggest increased

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is 195 (2007) e181–e190 e187

DL particle size. In addition, it is well recognized thatsually, but not always, when triglyceride decrease on treat-ent, for example with fibrates, LDL cholesterol increases

ue to increased conversion of very-low density lipoproteinsVLDL) to LDL [32].

Furthermore, addition of pioglitazone led to a markedncrease of plasma adiponectin and a significant decrease inerum CRP levels (Table 2), suggesting an improvement inardiovascular risk. Previous studies have shown that levelsf adiponectin can be significantly increased by weight loss,aloric restriction and treatment with TZD such pioglitazone.ndeed it is believed that much of the benefit derived fromZD treatment may arise from their effects on adiponectinene expression and the association between adiponectinnd HDL cholesterol levels [33]. Interestingly, the increasen adiponectin took place in the absence of altered plasmansulin and IAAT levels. TZDs have been previously shown toave a significant effect on adiponectin levels, which correlatetrongly with some of the reported changes in body adiposity,nd insulin resistance [34,35]. However, levels of adiponectinre known to increase in the absence of any other appar-nt metabolic and anatomical (fat content) modification byZD. Indeed, the study by Yu et al. has shown that in diabet-

cs TZD “non-responders”, an increase in adiponectin levelsas still observed [36]. These and other studies suggest that

he increase in adiponectin following administration of TZDrises independently of changes in total and regional adipoc-ty. It has been suggested that the degree of insulin resistancend hyperinsulinemia rather than the degree of adiposity mayave greater influence on adiponectin levels. However, in theurrent study insulin levels were unaltered. It is possible thatZD may influence adiponectin levels through adipose tis-ue remodeling (reduced adipocyte hypertrophy) [37]. Thisould happen independently of changes of insulin and IAAT

evels. Clearly further work is needed in this area, but ourtudy appears to support the possibility of TZD modulationf adiponectin independently of insulin and IAAT levels.

There was no change in oxLDL level in our study possiblys a result of the already long-term treatment with statin,hich is associated with a reduction in oxLDL in addition to

ts cholesterol lowering [38,39].None of the parameters assessing insulin sensitivity or beta

ell function indirectly (HOMA2, HOMA%B, HOMA%S,UICKI) were significantly altered. This is possibly becauseur FCHL patients were non-diabetic or could also be dueo a lack of sensitivity in detection of minor changes withinhe normal range. The lack of association between changesn serum lipid parameters and those of insulin resistance sug-ests that in a lean, non-diabetic population the effect ofioglitazone may be independent of effect on insulin resis-ance.

Abbink et al. [40] observed that treatment of FCHL

atients with pioglitazone alone led to advantageous changesf the LDL subfraction profile, but no other changes in theipid-profile or in the flow-mediated dilatation in the brachialrtery were documented. The authors documented increased

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nsulin sensitivity, using the hyperinsulinaemic euglycaemiclamp procedure, but found no significant effect on BMI,asting glucose, HbA1c, serum lipid levels or body fat distri-ution and concluded that the changes in insulin sensitivityight have been too small to induce changes in serum param-

ters of lipid and carbohydrate metabolism as well as in bodyat distribution. The differences with our current study may beue to the smaller dose of pioglitazone used by Abbink et al.nd the lack of concomitant conventional lipid-lowering med-cation; the latter might have facilitated and/or augmented theffects of pioglitazone we observed.

.2. Effect of pioglitazone treatment on bodyomposition, IMCL and IHCL

Following pioglitazone treatment of patients with FCHL, aignificant increase in both total and subcutaneous AT depotsere observed. This was accompanied by increased IMCL

evels in the soleus muscle. There were no changes in internalT either peripherally or in the intra-abdominal depot.

Studies in subjects with type 2 diabetes have shown var-ed responses to pioglitazone therapy on body composition.mith et al. found similar changes in adiposity as in our study,ith increases in peripheral and abdominal subcutaneous AT,

nd no changes in intra-abdominal AT (IAAT) [41]. Someave reported significant reductions in IAAT or in the ratio ofAAT/abdominal-subcutaneous AT [11], whilst others havehown non-significant changes in adiposity during treatmentith pioglitazone [42,43]. We did find a significant reduc-

ion in the ratio of IAAT/abdominal-subcutaneous AT, buthis is most likely to be related to the increase observed inbdominal-subcutaneous AT.

Pioglitazone has previously been reported to significantlyeduce IMCL in the vastus lateralis muscle of subjects withype 2 diabetes [13], with no changes in IMCL reported inhe soleus, tibialis or gastrocnemius muscles in patients withCHL [40]. In the current study we also found no change

n tibialis IMCL but observed a significant increase in soleusMCL. The reasons for this are not immediately apparent, butay be related to changes in fat oxidation and redistribution.egression analysis suggested that the changes in the soleus

MCL might be predicted by changes in the triglyceride/HDLolar ratio and CRP. This may imply an increase in fat

tilization and therefore supply to the muscle as it is seques-rated from the circulation to the muscle prior to oxidation.ndeed Pruchnic et al. showed that increasing capacity forat oxidation through exercise training increases IMCL con-ent [44]. The higher oxidative capacity of the soleus relativeo the tibialis muscle may explain the differences observedetween the two muscle groups. However, although levelsf intramuscular lipid (IMCL) are associated with insulinesistance, there is a growing body of evidence consistent

ith the hypothesis that IMCL stores alone are not sufficient

o account for impaired insulin action [45]. Indeed, insulinensitivity can be modified independently of IMCL levels46] and this and other studies suggest that IMCL level are a

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is 195 (2007) e181–e190

eflection of underlying alteration in lipid metabolism asso-iated with insulin resistance rather than the cause. Hence,he changes in IMCL observed in the present study are likelyo reflect a shift in fat distribution rather than alterations inipid metabolism associated with insulin resistance.

In this study we observed a decrease in IHCL lev-ls following pioglitazone treatment (although it did noteach formal significance) and highly significant correlationsetween changes in IHCL content and alterations in both ALTr = 0.72, p < 0.01) and AST (p = 0.86, p < 0.01). Regressionnalyses showed that only changes in plasma triglyceridesnd total adipose tissue content predicted change in IHCL.

A number of studies have reported reductions in IHCL inubjects with type 2 diabetes and NASH following TZD inter-entions [12,23,47]; in some of these studies the changes inHCL are observed together with changes in intra-abdominalT, and IMCL. This may well occur since intra-abdominalT, IMCL and IHCL levels tend to be significantly increased

n these subjects before starting treatment with pioglitazone.owever, in the current study, subjects with FCHL had beenn treatment with conventional lipid-lowering medication foreveral years preceding the current intervention with piogli-azone. Probably as a result of this long-standing therapyone of these parameters (IAAT, IMCL and IHCL as wells liver function tests) were significantly outside the rangeound in our control populations (data not shown), possiblyaking a reduction in any of these parameters with piogli-

azone more difficult to achieve and/or to detect. Moreover,e observed no correlation between IHCL and IAAT in these

ubjects, as previously reported in control volunteers, indicat-ng that conventional lipid-lowering drugs may affect IHCLndependently of other fat depots. Interestingly, significantorrelations between the change in measures of liver func-ion (ALT and AST) following treatment and IHCL werebserved, suggesting a beneficial effect of pioglitazone treat-ent on the liver. The lack of significant reduction in IHCL

n our study may also be a reflection of the short durationf treatment for a period of 4 months; 6 months interventionith pioglitazone has been shown to improve non-alcoholic

teatohepatitis [47].In summary, the results of the present study suggest that

he addition of pioglitazone at a dose of 45 mg daily to thetandard lipid-lowering medication of patients with FCHLmproves serum triglyceride and HDL cholesterol, plasmalucose and liver enzyme levels with favourable changes inRP and adiponectin. These changes in blood parameters aressociated with slight increase in body weight, due primarilyo increases in the subcutaneous depots. Although this smalltudy, in which only male FCHL patients were involved, can-ot be used as a basis for changing routine clinical practice,t suggests that in selected patients of high risk, especiallyn secondary prevention, in whom a combination of conven-

ional lipid-lowering drugs fails to fully control abnormalipid profile, the addition of pioglitazone may result in achiev-ng target lipid levels. Long-term studies are needed to assesshether pioglitazone would lead not only to reduction of

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arameters of cardiovascular risk but also to improvementf patients’ survival. Such studies are being undertaken andheir results are eagerly waited for.

cknowledgements

We would like to thank Ashwin Algudkar, William-Parrymith and Mark Punyanitya from the Image Reading Centrend research nurses Clare Neuwirth and Yvonne Tan fromhe Clinical Research Facility at the MRC Clinical Sciencesentre for their assistance with this work. We would also

ike to acknowledge the invaluable contribution of Carolineore for her statistical expertise, Ms. Rocio Lale-Montes for

xcellent assistance with the manuscript and Sonia Isauraorambuena Cruz por inspiratio.

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