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Diabetologia

, Volume 57, Issue 5, pp 878–890 | Cite as

The effects of ezetimibe on non-alcoholic fatty liver disease and glucose metabolism: a randomised controlled trial

  • Yumie Takeshita
  • Toshinari Takamura
  • Masao Honda
  • Yuki Kita
  • Yoh Zen
  • Ken-ichiro Kato
  • Hirofumi Misu
  • Tsuguhito Ota
  • Mikiko Nakamura
  • Kazutoshi Yamada
  • Hajime Sunagozaka
  • Kuniaki Arai
  • Tatsuya Yamashita
  • Eishiro Mizukoshi
  • Shuichi Kaneko
Article

Abstract

Aims/hypothesis

The cholesterol absorption inhibitor ezetimibe has been shown to ameliorate non-alcoholic fatty liver disease (NAFLD) pathology in a single-armed clinical study and in experimental animal models. In this study, we investigated the efficacy of ezetimibe on NAFLD pathology in an open-label randomised controlled clinical trial.

Methods

We had planned to enrol 80 patients in the trial, as we had estimated that, with this sample size, the study would have 90% power. The study intervention and enrolment were discontinued because of the higher proportion of adverse events (significant elevation in HbA1c) in the ezetimibe group than in the control group. Thirty-two patients with NAFLD were enrolled and randomised (allocation by computer program). Ezetimibe (10 mg/day) was given to 17 patients with NAFLD for 6 months. The primary endpoint was change in serum aminotransferase level. Secondary outcomes were change in liver histology (12 control and 16 ezetimibe patients), insulin sensitivity including a hyperinsulinaemic–euglycaemic clamp study (ten control and 13 ezetimibe patients) and hepatic fatty acid composition (six control and nine ezetimibe patients). Hepatic gene expression profiling was completed in 15 patients using an Affymetrix gene chip. Patients and the physician in charge knew to which group the patient had been allocated, but people carrying out measurements or examinations were blinded to group.

Results

Serum total cholesterol was significantly decreased in the ezetimibe group. The fibrosis stage and ballooning score were also significantly improved with ezetimibe treatment. However, ezetimibe treatment significantly increased HbA1c and was associated with a significant increase in hepatic long-chain fatty acids. Hepatic gene expression analysis showed coordinate downregulation of genes involved in skeletal muscle development and cell adhesion molecules in the ezetimibe treatment group, suggesting a suppression of stellate cell development into myofibroblasts. Genes involved in the l-carnitine pathway were coordinately downregulated by ezetimibe treatment and those in the steroid metabolism pathway upregulated, suggestive of impaired oxidation of long-chain fatty acids.

Conclusions/interpretation

Ezetimibe improved hepatic fibrosis but increased hepatic long-chain fatty acids and HbA1c in patients with NAFLD. These findings shed light on previously unrecognised actions of ezetimibe that should be examined further in future studies.

Trial registration University Hospital Medical Information Network (UMIN) Clinical Trials Registry UMIN000005250.

Funding The study was funded by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and research grants from MSD.

Keywords

Ezetimibe Fatty acid Gene expression Non-alcoholic fatty liver disease 

Abbreviations

ALT

Alanine aminotransferase

H-IR

Hepatic insulin resistance index

hsCRP

High-sensitivity C-reactive protein

ICG15

Indocyanine green retention rate at 15 min after venous administration

LXR

Liver-X-receptor

MCR

Glucose metabolic clearance rate

miR

MicroRNA

NAFLD

Non-alcoholic fatty liver disease

NAS

NAFLD activity score

NASH

Non-alcoholic steatohepatitis

NPC1L1

Niemann–Pick C1-like 1

PAI-1

Plasminogen activator inhibitor-1

RLP-C

Remnant-like particle cholesterol

sdLDL

Small dense LDL

SREBP

Sterol regulatory element binding protein

QUICKI

Quantitative insulin sensitivity check index

Introduction

Multiple metabolic disorders, such as diabetes [1], insulin resistance and dyslipidaemia [2], are associated with non-alcoholic fatty liver disease (NAFLD), ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH). Steatosis of the liver is closely associated with insulin resistance. However, the toxic lipids are not intrahepatic triacylglycerols but, rather, it is non-esterified cholesterol [3, 4] and some NEFA [5] that contribute to inflammation and insulin resistance in hepatocytes.

The level of cholesterol is tightly regulated by endogenous synthesis in the liver and dietary absorption/biliary reabsorption in the small intestine. Niemann–Pick C1-like 1 (NPC1L1) plays a pivotal role in cholesterol incorporation in enterocytes [6]. Ezetimibe, a potent inhibitor of cholesterol absorption, inhibits NPC1L1-dependent cholesterol transport at the brush border of the intestine and the liver [6]. This suggests that ezetimibe ameliorates toxic-lipid-induced inflammation and insulin resistance by inhibiting cholesterol absorption. Indeed, ezetimibe improves liver steatosis and insulin resistance in mice [7] and Zucker obese fatty rats [8], although the beneficial effects of ezetimibe are observed only when the animals are fed a high-fat diet. Ezetimibe can also ameliorate liver pathology in patients with NAFLD [9, 10]; however, these studies lack a control group, which precludes meaningful conclusions as liver pathology can improve over the natural course of the disease or with tight glycaemic control in some NAFLD patients [1]. In the present study, we investigated the efficacy of ezetimibe treatment in patients with NAFLD for 6 months in an open-label randomised control study by examining liver pathology, as well as hepatic enzymes, glucose metabolism, hepatic fatty acid composition and hepatic gene expression profiles.

Methods

Patient selection

Study staff recruited participants from outpatients at Kanazawa University Hospital, Ishikawa, Japan. Patients were recruited from April 2008 to August 2010, with follow-up visits during the 6 months thereafter. The study lasted from April 2008 to February 2011.

The inclusion criterion was a biopsy consistent with the diagnosis of NAFLD. Exclusion criteria included hepatic virus infections (hepatitis C virus [HCV] RNA–PCR-positive, hepatitis B and C, cytomegalovirus and Epstein–Barr virus), autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, haemochromatosis, α1-antitrypsin deficiency, Wilson’s disease, history of parenteral nutrition and use of drugs known to induce steatosis (e.g. valproate, amiodarone and prednisone) or hepatic injury caused by substance abuse and/or the current or past consumption of more than 20 g of alcohol daily. None of the patients had any clinical evidence of hepatic decompensation, such as hepatic encephalopathy, ascites, variceal bleeding or an elevated serum bilirubin level more than twofold the upper normal limit.

A random allocation sequence was computer-generated elsewhere and assigned participants in a 1:1 ratio to treatment with ezetimibe or to the control group. All patients and responsible guardians underwent an hour of nutritional counselling by an experienced dietitian before starting the 6 month treatment period. The experienced dietitians were unaware of the study assignments. In addition, all patients were given a standard energy diet (125.5 kJ/kg per day; carbohydrate 50–60%, fat 20–30%, protein 15–20%) and exercise (5–6 metabolic equivalent estimations for 30 min daily) counselling before the study. Patients remained on stable doses of medications for the duration of the study. The patients in the ezetimibe group received generic ezetimibe (10 mg/day; Zetia, [Merck, Whitehouse Station, NJ, USA]) for 6 months.

The study was conducted with the approval of the Ethics Committee of Kanazawa University Hospital, Ishikawa, Japan, in accordance with the Declaration of Helsinki. Written informed consent was obtained from all individuals before enrolment. This trial is registered with the University Hospital Medical Information Network (UMIN) (Clinical Trials Registry, no. UMIN000005250).

Primary and secondary outcomes

The primary endpoint was change in serum alanine aminotransferase (ALT) level at month 6 from baseline. Secondary outcomes included changes in the histological findings for NAFLD, hepatic gene expression profiling, fatty acid compositions of plasma and liver biopsy samples, lipid profiles, insulin resistance and anthropometric measures, as well as assessment of ezetimibe safety. We had planned to enrol 80 patients in the trial, as we had estimated that with this sample size, the study would have 90% power at an α (two-tailed) value of 0.05 showing a 50% decrease of serum ALT values with 6 months of pioglitazone therapy on the basis of a previous study [11]. At the time of adverse event analyses, 32 of the targeted 80 patients had been randomly assigned and were included in the safety analyses.

Data collection

Clinical information, including age, sex and body measurements, was obtained for each patient. Venous blood samples were obtained after the patients had fasted overnight (12 h) and were used to evaluate blood chemistry. Insulin resistance was estimated by HOMA-IR, calculated as [fasting insulin (pmol/l) × fasting glucose (mmol/l)]/22.5 [12] and insulin sensitivity was estimated as the quantitative insulin sensitivity check index (QUICKI)[13]. The adipose tissue insulin resistance index (adipose IR) was calculated as fasting NEFA (mmol/l) × fasting insulin (pmol/l) [14, 15, 16]. The indocyanine green retention rate at 15 min after venous administration (ICG15) was assessed using standard laboratory techniques before and after treatment. Serum fatty acids were measured with a gas chromatograph (Shimizu GC 17A, Kypto, Japan) at SRL (Tokyo, Japan).

Evaluation of insulin sensitivity derived from an OGTT

After an overnight fast (10–12 h), a 75 g OGTT was performed at 08:30 hours. The OGTT-derived index of beta cell function, the insulinogenic index, computed as the suprabasal serum insulin increment divided by the corresponding plasma glucose increment in the first 30 min (ΔI30/ΔG30) [15, 17, 18] was calculated. From the OGTT data, the Matsuda index [19] was calculated. The hepatic insulin resistance index (H-IR) was calculated as the product of the total AUCs for glucose and insulin during the first 30 min of the OGTT (glucose 0–30 [AUC] [mmol/l] × insulin 0–30 [AUC] [pmol/l]). Skeletal muscle insulin sensitivity can be calculated as the rate of decline in plasma glucose concentration divided by plasma insulin concentration, as follows. Muscle insulin sensitivity index = dG/dt/mean plasma insulin concentration, where dG/dt is the rate of decline in plasma glucose concentration and is calculated as the slope of the least square fit to the decline in plasma glucose concentration from peak to nadir [20]. See the electronic supplementary material (ESM) for further details.

Evaluation of insulin sensitivity derived from the euglycaemic insulin clamp

Insulin sensitivity in 23 of the 31 patients (10 control and 13 ezetimibe patients) was also evaluated in a hyperinsulinaemic–euglycaemic clamp study [21]. Patients did not receive any medication on the morning of the examination. At ∼09:00 hours, after an overnight fast of at least 10 h, an intravenous catheter was placed in an antecubital vein in each individual for infusion, while a second catheter was placed in the contralateral hand for blood sampling. The euglycaemic–hyperinsulinaemic clamp technique was performed using an artificial pancreas (model STG-22; Nikkiso, Tokyo, Japan), as described previously [22]. See ESM for further details. The mean glucose metabolic clearance rate (MCR) in healthy individuals (n = 9; age, 26.6 0± 2.9 years; body mass index, 22.3 ± 2.1 kg/m2) was 13.5 ± 3.4 mg kg−1 min−1 [2].

Liver biopsy pathology

A single pathologist, who was blinded to the clinical information and the order in which the biopsies were obtained, analysed all biopsies twice and at separate times. The sections were cut from a paraffin block and stained with haematoxylin and eosin, Azan–Mallory and silver reticulin impregnation. The biopsied tissues were scored for steatosis (from 0 to 3), stage (from 1 to 4) and grade (from 1 to 3) as described [2], according to the standard criteria for grading and staging of NASH proposed by Brunt et al [23]. The NAFLD activity score (NAS) was calculated as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3) and ballooning (0–2), as reported by Kleiner et al [24].

Gene expression analysis of liver biopsied samples

Gene expression profiling was performed in samples from nine patients in the ezetimibe group and six in the control group. Liver tissue RNA was isolated using the RNeasy Mini kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s instructions. See ESM for further details. Data files (CEL) were obtained using the GeneChip Operating Software 1.4 (Affymetrix). Genechip data analysis was performed using BRB-Array Tools (http://linus.nci.nih.gov/BRB-ArrayTools.html). The data were log-transformed (log10), normalised and centred. To identify genetic variants, paired t tests were performed to define p values <0.05 and fold change > 1.5. Pathway analysis was performed using MetaCore (GeneGo, St Joseph, MI, USA). Functional ontology enrichment analysis was performed to compare the gene ontology (GO) process distribution of differentially expressed genes (p < 0.01).

Fatty acid composition of liver

Aliquots (0.2 mg) of liver samples snap-frozen by liquid nitrogen were homogenised in 1 ml normal NaCl solution (NaCl 154 mmol/l). Briefly, fatty acids were extracted by using pentadecanoic acid, and saponified with alkaline reagent (0.5 mmol/l KOH/ CH3OH). The fatty acid methyl esters were analysed in a gas chromatograph (Shimadzu GC-2014 AF/SPL; Shimadzu Corporation, Kyoto, Japan) equipped with a flame ionisation detector and an auto injector. See ESM for further details. Mass spectra were analysed using GC solution (v. 2.3) software (Shimadzu Corporation, Kyoto, Japan, www.shimadzu.com). The changes in hepatic fatty acid composition are expressed as 10−4 mg/mg liver.

Statistical analysis

Data are expressed as mean ± one standard error, unless indicated otherwise. The Statistical Package for the Social Sciences (SPSS; version 11.0; Chicago, IL, USA) was used for the statistical analyses. For univariate comparisons between the patient groups, Student’s t test or Mann–Whitney’s U test was used, as appropriate, followed by the Bonferroni multiple-comparison test. A value of p < 0.05 was considered to indicate statistical significance.

Results

Enrolment and discontinuation

The data and safety monitoring board recommended that the study intervention and enrolment be discontinued because of the higher proportion of adverse events (significant elevation in HbA1c) in the ezetimibe group than in the control group. At the time of adverse event analyses, 32 of the targeted 80 patients had been randomly assigned and were included in the safety analyses. In our open-label trial, 32 patients with NAFLD were enrolled. They were randomised to treatment with ezetimibe (n = 17) or a control (n = 15) with no significant clinical differences in variables between the groups. Of the 32 randomly assigned patients, 31 had completed the 6 month intervention period; one patient dropped out of the study. One case in the control group withdrew consent after randomisation and before intervention (ESM Fig. 1). The patient who withdrew was excluded from analysis because he did not start his course of treatment. Two analyses were conducted in the remaining patients. In the intention-to-treat analysis (ESM Tables 1 and 2), measures that were missing for participants who discontinued the study were replaced with baseline measures. In the second analysis, the only data included were from participants who completed the study to the end of the 6 month follow-up period. We performed a completed case analysis because there were few dropouts unrelated to baseline values or to their response.

Patient characteristics

The 31 study patients (mean age 52.7 ± 2.1 years; mean BMI 29.2 ± 1.0) included 14 randomised to the control group and 17 to the ezetimibe group (ESM Table 3).

At baseline, the characteristics of patients in the ezetimibe and control groups were comparable except for the waist circumference (p = 0.085) and the Matsuda index (p = 0.060). The histological features of the liver are summarised in Table 1. At baseline, neither the severity of the individual histological features nor the proportion of patients distributed in the three NAS categories was significantly different between the two groups. All 31 participants agreed to complete the follow-up venous blood samples including OGTT. The ICG15 was conducted in 24 patients (ten control and 14 ezetimibe patients).
Table 1

Histological characteristics of the livers of patients who completed the study at baseline and 6 months

Variable

Control

p a

Ezetimibe

p a

p b

Before

After

 

Before

After

  

Steatosis

1.42 ± 0.15

1.17 ± 0.17

0.082

1.56 ± 0.18

1.31 ± 0.15

0.300

0.989

Stage

1.71 ± 0.40

1.71 ± 0.39

1.000

1.75 ± 0.28

1.53 ± 0.26

0.048

0.163

Grade

0.88 ± 0.28

0.79 ± 0.26

0.339

0.84 ± 0.21

0.72 ± 0.15

0.362

0.628

Acinar inflammation

0.88 ± 0.20

0.83 ± 0.20

0.674

1.00 ± 0.13

0.97 ± 0.13

0.751

0.060

Portal inflammation

0.67 ± 0.19

0.71 ± 0.13

0.795

0.44 ± 0.16

0.56 ± 0.16

0.333

0.941

Ballooning

0.58 ± 0.23

0.58 ± 0.23

1.000

0.69 ± 0.20

0.41 ± 0.15

0.045

0.677

NAFLD activity score

3.25 ± 0.53

2.82 ± 0.59

0.139

3.71 ± 0.50

3.06 ± 0.45

0.185

0.705

Data are expressed as the means ± SE

a p value for the intergroup comparison (baseline vs 6 month)

b p value for the intergroup comparison (changes from baseline between groups)

Changes in laboratory variables

The primary study outcome, serum alanine aminotransferase levels, did not change after ezetimibe treatment (Table 2).
Table 2

Laboratory values, insulin sensitivity and insulin resistance derived from the euglycaemic insulin clamps and OGTTs of patients who completed the study at baseline and 6 months

Variable

Control

 

Ezetimibe

  

Before

After

p a

Before

After

p a

p b

Male/female

9/5

  

11/6

  

0.232

Age (years)

55.5 ± 3.0

50.4 ± 2.9

Body weight (kg)

74.4 ± 6.2

73.0 ± 5.6

0.144

81.5 ± 4.6

80.1 ± 4.2

0.367

0.983

BMI (kg/m2)

27.7 ± 1.7

27.3 ± 1.5

0.172

30.5 ± 1.2

30.0 ± 1.1

0.383

0.999

Waist circumference (cm)

93.1 ± 2.7

92.6 ± 3.4

0.709

99.9 ± 2.5

100.0 ± 2.6

0.956

0.713

Systolic blood pressure (mmHg)

125.2 ± 3.9

126.4 ± 4.9

0.771

124.0 ± 2.4

130.7 ± 2.8

0.048

0.269

Fasting plasma glucose (mmol/l)

7.15 ± 0.63

6.52 ± 0.40

0.240

6.62 ± 0.30

6.87 ± 0.34

0.411

0.131

HbA1c (%)

5.9 ± 0.2

6.0 ± 0.2

0.603

6.1 ± 0.2

6.5 ± 0.2

0.001

0.041

HbA1c (mmol/mol)

40.8 ± 2.2

41.6 ± 2.6

0.603

43.0 ± 2.6

48.0 ± 2.3

0.001

0.041

Hepaplastin test (%)

115.9 ± 5.8

117.1 ± 6.4

0.624

113.7 ± 4.6

111.8 ± 3.7

0.583

0.459

Glycated albumin (%)

15.9 ± 0.8

16.2 ± 1.0

0.397

15.7 ± 0.5

16.8 ± 0.5

0.014

0.196

Serum aspartate aminotransferase (μkat/l)

31.1 ± 4.4

30.3 ± 3.0

0.780

41.8 ± 6.7

33.7 ± 4.1

0.252

0.365

Serum ALT (μkat/l)

37.9 ± 6.8

38.0 ± 4.5

0.978

53.2 ± 8.6

49.3 ± 6.5

0.683

0.723

Plasma γ-glutamyltransferase (μkat/l)

74.9 ± 27.8

65.8 ± 19.5

0.345

71.4 ± 23.4

60.5 ± 16.1

0.220

0.892

Total cholesterol (mmol/l)

5.14 ± 0.21

5.20 ± 0.18

0.672

5.14 ± 0.20

4.65 ± 0.17

0.024

0.037

Triacylglycerols (mmol/l)

1.34 ± 0.12

1.17 ± 0.12

0.105

1.43 ± 0.11

1.46 ± 0.13

0.857

0.303

HDL-C (mmol/l)

1.40 ± 0.08

1.45 ± 0.06

0.914

1.36 ± 0.08

1.36 ± 0.06

0.942

0.903

sdLDL (mmol/l)

0.52 ± 0.07

0.54 ± 0.07

0.782

0.61 ± 0.10

0.50 ± 0.06

0.201

0.251

RLP-C (mmol/l)

0.13 ± 0.01

0.11 ± 0.01

0.163

0.12 ± 0.01

0.11 ± 0.01

0.601

0.365

Lathosterol × 10−3 (μmol/l)

2.27 ± 0.43

2.85 ± 0.52

0.001

3.52 ± 0.52

5.01 ± 0.67

<0.001

0.018

Campesterol × 10−3 (μmol/l)

4.32 ± 0.65

6.20 ± 0.68

0.004

3.78 ± 0.42

2.49 ± 0.30

0.007

<0.001

Sitosterol × 10−3 (μmol/l)

3.04 ± 0.47

3.89 ± 0.39

0.079

2.73 ± 0.28

1.81 ± 0.19

0.004

0.002

Ferritin (pmol/l)

412.1 ± 85.6

235.3 ± 47.0

0.009

395.7 ± 81.3

247.8 ± 56.8

0.005

0.689

Type IV collagen 7 s (μg/l)

4.52 ± 0.48

4.42 ± 0.45

0.622

4.23 ± 0.23

4.33 ± 0.20

0.592

0.465

NEFA (mmol/l)

0.50 ± 0.09

0.63 ± 0.06

0.160

0.51 ± 0.05

0.57 ± 0.03

0.835

0.447

Total bile acid (μmol/l)

12.5 ± 8.0

8.8 ± 5.2

0.214

5.0 ± 0.7

4.8 ± 1.3

0.893

0.267

hsCRP × 10−3 (μg/ml)

0.12 ± 0.02

0.09 ± 0.02

0.050

0.14 ± 0.04

0.13 ± 0.04

0.886

0.767

Adiponectin (μg/ml)

4.0 ± 0.5

4.6 ± 0.8

0.114

3.0 ± 0.6

3.3 ± 0.6

0.299

0.670

TNF-α ×10−5 (pmol/ml)

10.4 ± 2.3

15.6 ± 8.1

0.094

8.1 ± 0.6

30.0 ± 12.7

0.183

0.084

Leptin × 10−3 (μg/l)

8.1 ± 1.0

9.7 ± 1.3

0.044

10.8 ± 1.4

12.4 ± 1.5

0.085

0.982

PAI-1 (pmol/l)

400.0 ± 44.2

436.5 ± 44.2

0.401

550.0 ± 71.2

488.5 ± 67.3

0.217

0.136

8-Isoprostanes (pmol/mmol creatinine)

76.9 ± 14.3

57.0 ± 8.0

0.147

56.5 ± 6.6

68.0 ± 7.7

0.092

0.031

ICG15 (%)

8.7 ± 2.4

8.5 ± 2.0

0.662

7.7 ± 1.7

7.7 ± 1.5

0.984

0.796

HOMA-IR

10.1 ± 6.5

5.0 ± 2.1

0.471

9.5 ± 2.6

9.3 ± 2.2

0.839

0.479

QUICKI

0.32 ± 0.01

0.33 ± 0.01

0.443

0.30 ± 0.01

0.30 ± 0.01

0.984

0.019

Adipose IR

55.8 ± 15.5

78.8 ± 31.7

0.441

88.1 ± 25.5

107.5 ± 25.5

0.070

0.099

Insulinogenic index

0.43 ± 0.09

0.53 ± 0.11

0.307

0.41 ± 0.08

0.35 ± 0.09

0.501

0.765

H-IR × 106

1.82 ± 0.46

2.29 ± 0.44

0.568

2.29 ± 0.33

2.66 ± 0.41

0.221

0.796

Matsuda index

3.03±0.45

3.35±0.49

0.368

1.99±0.28

2.01±0.29

0.895

0.013

Muscle insulin sensitivity

0.039 ± 0.006

0.058 ± 0.016

0.210

0.036 ± 0.005

0.034 ± 0.004

0.560

0.067

MCR

4.86 ± 0.50

4.36 ± 0.45

0.174

4.70 ± 0.31

4.80 ± 0.35

0.827

0.352

Data are expressed as means ± SE

a p value for the intergroup comparison (baseline vs 6 month)

b p value for the intergroup comparison (changes from baseline between groups)

HDL-C, HDL-cholesterol

After 6 months of ezetimibe treatment, systolic blood pressure, HbA1c, glycated albumin, and lathosterol were significantly increased, while total cholesterol levels, campesterol, sitosterol and ferritin were significantly decreased. In contrast, body weight, BMI, fasting plasma glucose, plasma γ-glutamyltransferase, triacylglycerols, HDL-cholesterol, small dense LDL (sdLDL), remnant-like particle cholesterol (RLP-C), type IV collagen 7 s levels, NEFA, total bile acid, high-sensitivity C-reactive protein (hsCRP), adiponectin, TNF-α, plasminogen activator inhibitor-1 (PAI-1), 8-isoprostanes and ICG15 did not change after ezetimibe treatment (Table 2). Adipose IR tended to increase in the ezetimibe group (from 88.1 ± 25.5 to 107.5 ± 25.5, p = 0.070), but not in the control group.

When changes in the groups were compared, the ezetimibe group, but not the control group, had a significant decrease in total cholesterol (ezetimibe, −0.49 ± 0.19 vs control, 0.06 ± 0.14 mmol/l; p = 0.037), whereas the ezetimibe group, but not control group, showed a significant elevation in HbA1c (ezetimibe, 0.46 ± 0.12% [4.95 ± 1.28 mmol/mol] vs control, 0.08 ± 0.13% [0.78 ± 1.46 mmol/mol]; p = 0.041). Also, there were significant differences between the groups in cholesterol and HbA1c levels at 6 months. The multiple-comparison Bonferroni test revealed highly significant differences in the changes in total cholesterol (p = 0.037) and HbA1c (p = 0.040) between the ezetimibe and control groups.

Increased concentrations of the cholesterol synthesis markers lathosterol (ezetimibe, 1.49 ± 0.32 nmol/l vs control, 0.58 ± 0.14 nmol/l; p = 0.018) and decreased concentrations of the cholesterol absorption markers campesterol (ezetimibe, −1.28 ± 0.41 nmol/l vs control, 1.88 ± 0.54 nmol/l, p = 0.000) and sitosterol (ezetimibe, −0.91 ± 0.27 nmol/l vs control, 0.85 ± 0.45 nmol/l; p = 0.002) were observed on treatment. The ezetimibe group had an increase, whereas the control group had a decrease, in the level of 8-isoprostanes (ezetimibe, 11.6 ± 6.4 pmol/mmol creatinine vs control, −19.9 ± 12.9 pmol/mmol creatinine; p = 0.031).

When changes between groups were compared, the ezetimibe group had a greater decrease in the Matsuda index (ezetimibe = −0.78 ± 0.57 vs control = −1.35 ± 0.55, p = 0.013), QUICKI (ezetimibe = −0.02 ± 0.01 vs control = 0.03 ± 0.0, p = 0.019), and muscle insulin sensitivity (ezetimibe = −0.002 ± 0.004 vs control = 0.019 ± 0.014, p = 0.067) than the control group.

Changes in liver histology

Twenty-eight of 31 participants, 16 in the ezetimibe group and 12 in the control group, agreed to complete the follow-up and undergo a liver biopsy at 6 months, allowing for complete case analysis of the data (Table 1). After 6 months, the changes in staging score (from 1.75 ± 0.28 to 1.53 ± 0.26) and ballooning score (from 0.69 ± 0.20 to 0.41 ± 0.15) were significantly improved in the ezetimibe group compared with the control group, whereas the scores of steatosis, lobular inflammation and NAS were not significantly changed in either group. The degree of all of these histological features was not significantly different between the two groups (Table 1).

Serial changes in liver gene with ezetimibe treatment

Gene expression profiling was conducted in samples from nine patients in the ezetimibe group and six in the control group (ESM Table 4). In the ezetimibe group, 434 genes were upregulated and 410 genes downregulated, while in the control group, 643 genes were upregulated and 367 genes downregulated. Pathway analysis of the process network of differentially expressed genes showed coordinate downregulation of genes involved in skeletal muscle development and cell adhesion molecules in the ezetimibe group, suggesting a suppression of stellate cell development into myofibroblasts (Table 3). In addition, ezetimibe activated the immune response pathway. In contrast, genes involved in skeletal muscle development were upregulated and those in the immune response downregulated in the control group (Table 4). Pathway analysis of the metabolic network also revealed decreased l-carnitine pathway and increased steroid metabolism with ezetimibe treatment, but decreased CoA biosynthesis and increased glycerol 3-phosphate pathway in the control group (ESM Fig. 2).
Table 3

Signalling pathway gene expression changes in the ezetimibe group

Pathway

Gene symbol

Gene name

Affy ID

Up or down

Function

Development_skeletal muscle development

VEGFA

Vascular endothelial growth factor A

210512_s_at

Down

Angiogenesis

 

ACTA2

Actin, α2, smooth muscle, aorta1

200974_at

Down

Cytoskeleton and cell attachment

 

TCF3

Transcription factor 3

209153_s_at

Down

Differentiation

 

TTN

Titin

1557994_at

Down

Abundant protein of striated muscle

 

TPM2

Tropomyosin 2

204083_s_at

Down

Actin filament binding protein

 

MYH11

Myosin, heavy chain 11, smooth muscle

201496_x_at

Down

Smooth muscle myosin

Immune response_phagocytosis

FYB

FYN-binding protein

205285_s_at

Up

Platelet activation and IL2 expression

 

FCGR3A

Fc fragment of IgG, low affinity IIIA

204006_s_at

Up

ADCC and phagocytosis

 

LCP2

Lymphocyte cytosolic protein 2

244251_at

Up

T cell antigen receptor mediated signalling

 

CLEC7A

C-type lectin domain family 7, member A

221698_s_at

Up

T cell proliferation

 

MSR1

Macrophage scavenger receptor 1

214770_at

Up

Macrophage-associated processes

 

FCGR2A

Fc fragment of IgG, low affinity IIA

1565673_at

Up

Promotes phagocytosis

 

PRKCB

Protein kinase C, β

209685_s_at

Up

B cell activation, apoptosis induction

 

PLCB4

Phospholipase C, β4

240728_at

Up

Inflammation, cell growth, signalling and death

Cell adhesion_integrin priming

GNA12

G protein α12

221737_at

Down

Cytoskeletal rearrangement

 

ITGB3

Integrin, β3

204628_s_at

Down

Ubiquitously expressed adhesion molecules

 

PIK3R2

Phosphoinositide-3-kinase, regulatory subunit 2

229392_s_at

Down

Diverse range of cell functions

Cell adhesion_cadherins

PTPRF

Protein tyrosine phosphatase, receptor type, F

200636_s_at

Down

Cell adhesion receptor

 

BTRC

β-Transducin repeat containing E3 ubiquitin protein ligase

222374_at

Down

Substrate recognition component of a SCF E3 ubiquitin-protein ligase complex

 

CDHR2

Cadherin-related family member 2

220186_s_at

Down

Contact inhibition at the lateral surface of epithelial cells

 

SKI

V-ski sarcoma viral oncogene homologue

229265_at

Down

Repressor of TGF-β signalling

 

MLLT4

Myeloid/lymphoid or mixed-lineage leukaemia

214939_x_at

Down

Belongs to an adhesion system

 

VLDLR

Very low density lipoprotein receptor

209822_s_at

Down

Binds VLDL and transports it into cells by endocytosis

O-Hexadecanoyl-l-carnitine pathway

TUBB2B

Tubulin, β 2B class IIB

209372_x_at

Down

Major component of microtubules

 

TUBB2A

Tubulin, β2A class IIA

209372_x_at

Down

Major component of microtubules

 

PLCE1

Phospholipase C, epsilon 1

205112_at

Down

Hydrolyses phospholipids into fatty acids and other lipophilic molecules

 

CPT1B

Carnitine palmitoyltransferase 1B (muscle)

210070_s_at

Down

Rate-controlling enzyme of the long-chain fatty acid β-oxidation pathway

 

CPT1A

Carnitine palmitoyltransferase 1A (liver)

203634_s_at

Down

Carnitine-dependent transport across the mitochondrial inner membrane

 

NR1H4

Nuclear receptor subfamily 1, group H, member 4

243800_at

Down

Involved in bile acid synthesis and transport.

GalNAcbeta1-3Gal pathway

PLCB4

Phospholipase C, β4

240728_at

Up

Formation of inositol 1,4,5-trisphosphate and diacylglycerol

Steroid metabolism_cholesterol biosynthesis

CYP51A1

Cytochrome P450, family 51, subfamily A, polypeptide 1

216607_s_at

Up

Transforms lanosterol

 

SREBF2

Sterol regulatory element binding transcription factor 2

242748_at

Up

Transcriptional activator required for lipid homeostasis

 

SQLE

Squalene epoxidase

209218_at

Up

Catalyses the first oxygenation step in sterol biosynthesis

 

SC5DL

Sterol-C5-desaturase-like

215064_at

Up

Catalyses the conversion of lathosterol into 7-dehydrocholesterol

 

HMGCS1

3-Hydroxy-3-methylglutaryl-CoA synthase 1

205822_s_at

Up

Condenses acetyl-CoA with acetoacetyl-CoA to form HMG-CoA

Table 4

Signalling pathway gene expression changes in the control group

Pathway

Gene symbol

Gene name

Affy ID

Up or down

Function

Muscle contraction

MYH11

Myosin, heavy chain 11, smooth muscle

201497_x_at

Up

Smooth muscle myosin

 

CALM1

Calmodulin 1

241619_at

Up

Ion channels and other proteins by Ca2+

 

KCNJ15

Potassium inwardly-rectifying channel, subfamily J, member 15

211806_s_at

Up

Integral membrane protein, inward-rectifier type potassium channel

 

SRI

Sorcin

208920_at

Up

Modulates excitation–contraction coupling in the heart

 

ACTA2

Actin, α2, smooth muscle, aorta

215787_at

Up

Cell motility, structure and integrity

 

TTN

Titin

1557994_at

Up

Abundant protein of striated muscle

 

EDNRA

Endothelin receptor type A

204463_s_at

Up

Receptor for endothelin-1

 

TPM2

Tropomyosin 2

204083_s_at

Up

Actin filament binding protein

 

CRYAB

Crystallin, αB

209283_at

Up

Transparency and refractive index of the lens

Development_skeletal muscle development

GTF2IRD1

GTF2I repeat domain containing 1

218412_s_at

Up

Transcription regulator involved in cell-cycle progression, skeletal muscle differentiation

 

ADAM12

ADAM metallopeptidase domain 12

213790_at

Up

Skeletal muscle regeneration

 

MAP1B

Microtubule-associated protein 1B

226084_at

Up

Facilitates tyrosination of α-tubulin in neuronal microtubules

 

MYOM1

Myomesin 1

205610_at

Up

Major component of the vertebrate myofibrillar M band

Cell cycle_G1-S growth factor regulation

DACH1

Dachshund homologue 1

205472_s_at

Up

Transcription factor that is involved in regulation of organogenesis

 

FOXN3

Forkhead box N3

229652_s_at

Up

Transcriptional repressor, DNA damage-inducible cell cycle arrests

 

TGFB2

Transforming growth factor, β2

228121_at

Up

Suppressive effects on interleukin-2 dependent T cell growth

 

PIK3CD

Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta

203879_at

Up

Generate PIP3, recruiting PH domain-containing proteins to the membrane

 

EGFR

Epidermal growth factor receptor

1565484_x_at

Up

Antagonist of EGF action

 

CCNA2

Cyclin A2

203418_at

Up

Control of the cell cycle at the G1/S and the G2/M transitions

 

AKT3

v-Akt murine thymoma viral oncogene homologue 3

219393_s_at

Up

Metabolism, proliferation, cell survival, growth and angiogenesis

 

PRKD1

Protein kinase D1

205880_at

Up

Converts transient DAG signals into prolonged physiological effects

Regulation of metabolism_

INSR

Insulin receptor

226450_at

Down

Pleiotropic actions of insulin

Bile acid regulation of lipid metabolism and

SLC27A5

Solute carrier family 27, member 5

219733_s_at

Down

Bile acid metabolism

Negative FXR-dependent regulation of bile acids concentration

MBTPS2

Membrane-bound transcription factor peptidase

1554604_at

Down

Intramembrane proteolysis of SREBPs

 

PIK3R3

Phosphoinositide-3-kinase, regulatory subunit 3

202743_at

Down

During insulin stimulation, it also binds to IRS-1

 

MTTP

Microsomal triacylglycerol transfer protein

205675_at

Down

Catalyses the transport of triglyceride, cholesteryl ester, and phospholipid

 

PPARA

Peroxisome proliferator-activated receptor α

226978_at

Down

Ligand-activated transcription factor

 

CYP7A1

Cytochrome P450, family 7, subfamily A

207406_at

Down

Catalyses cholesterol catabolism and bile acid biosynthesis

 

FOXA3

Forkhead box A3

228463_at

Down

Transcription factor

Immune response_phagosome in antigen presentation

HLA-B

Major histocompatibility complex, class I, B

211911_x_at

Down

Foreign antigens to the immune system

 

CD14

CD14 molecule

201743_at

Down

Mediates the innate immune response to bacterial lipopolysaccharide

 

LBP

Lipopolysaccharide binding protein

211652_s_at

Down

Binds to the lipid A moiety of bacterial lipopolysaccharides

 

CTSS

Cathepsin S

202901_x_at

Down

Thiol protease

 

DERL1

Derlin 1

222543_at

Down

Functional component of endoplasmic reticulum-associated degradation

 

CFL2

Cofilin 2

224352_s_at

Down

Reversibly controls actin polymerisation and depolymerisation

 

PAK1

p21 protein (Cdc42/Rac)-activated kinase 1

230100_x_at

Down

Activated kinase acts on a variety of targets

Vitamin, mediator and cofactor

SLC1A2

Solute carrier family 1, member 2

1558009_at

Down

Transports l-glutamate and also l- and d-aspartate

Metabolism_CoA biosynthesis and transport

PANK3

Pantothenate kinase 3

218433_at

Down

Physiological regulation of the intracellular CoA concentration

 

PANK1

Pantothenate kinase 1

226649_at

Down

Physiological regulation of the intracellular CoA concentration

 

VNN1

Vanin 1

205844_at

Down

Membrane-associated proteins

 

ACSL5

Acyl-CoA synthetase long-chain family member 5

222592_s_at

Down

Synthesis of cellular lipids and degradation via β-oxidation

 

ACOT1

Acyl-CoA thioesterase 1

202982_s_at

Down

Catalyses the hydrolysis of acyl-CoAs to the NEFA and coenzyme A

 

ACOT2

Acyl-CoA thioesterase 2

202982_s_at

Down

Catalyses the hydrolysis of acyl-CoAs to the NEFA and coenzyme A

 

ENPP1

Ectonucleotide pyrophosphatase/phosphodiesterase 1

229088_at

Down

Involved primarily in ATP hydrolysis at the plasma membrane

Phatidic acid pathway

GPR63

G protein-coupled receptor 63

220993_s_at

Up

Orphan receptor. May play a role in brain function

2-Oleoyl-glycerol_3-phosphate pathway

LPAR1

Lysophosphatidic acid receptor 1

204037_at

Up

Receptor for LPA, a mediator of diverse cellular activities

Changes in plasma fatty acid composition and fatty acid composition extracted from liver tissue

The changes in plasma fatty acid composition are shown in Table 5. Compared with baseline levels, only eicosatrienoic acid was significantly increased in the ezetimibe group.
Table 5

Changes in plasma fatty acid composition

Fatty acid

Control

p a

Ezetimibe

p a

p b

 

Before

After

 

Before

After

  

C12:0 (lauric acid)

1.9 ± 0.5

1.2 ± 0.2

0.177

2.3 ± 0.6

2.1 ± 0.5

0.753

0.301

C14:0 (myristic acid)

24.9 ± 2.5

23.6 ± 2.9

0.575

27.1 ± 2.8

29.5 ± 3.7

0.441

0.352

C16:0 (palmitic acid)

698.0 ± 24.7

690.0 ± 38.2

0.827

714.3 ± 32.5

717.0 ± 36.2

0.991

0.893

C16:1n-7 (palmitoleic acid)

68.6 ± 6.5

72.5 ± 9.6

0.643

62.4 ± 5.0

69.9 ± 6.2

0.219

0.721

C17:0 (margaric acid)

NE

NE

 

NE

NE

  

C18:0 (stearic acid)

203.3 ± 9.4

196.7 ± 6.9

0.488

207.2 ± 7.7

211.0 ± 9.9

0.854

0.571

C18:1n-9 (oleic acid)

560.2 ± 31.3

556.4 ± 30.3

0.914

547.3 ± 23.9

578.8 ± 32.1

0.475

0.550

C18:2n-6 (linoleic acid)

745.8 ± 26.3

750.6 ± 34.4

0.910

735.8 ± 34.2

713.5 ± 31.4

0.558

0.629

C18:3n-6 (γ‐linolenic acid)

9.8 ± 1.3

9.2 ± 1.0

0.506

9.8 ± 0.9

11.1 ± 1.5

0.402

0.300

C18:3n-3 (α-linolenic acid)

21.7 ± 1.6

20.1 ± 1.4

0.285

23.0 ± 2.2

21.6 ± 1.5

0.507

0.924

C20:0n-6 (arachidic acid)

7.0 ± 0.4

6.9 ± 0.3

0.671

7.2 ± 0.2

7.0 ± 0.3

0.410

0.642

C20:1n-9 (eicosenoic acid)

4.8 ± 0.3

4.8 ± 0.4

0.323

4.3 ± 0.2

4.2 ± 0.3

0.831

0.343

C20:2n-6 (eicosadienoic acid )

6.1 ± 0.4

6.1 ± 0.3

0.899

5.6 ± 0.2

5.7 ± 0.3

0.774

0.770

C20:3n-6 (dihomo-γ-linolenic acid)

36.6 ± 3.0

37.3 ± 2.8

0.784

36.5 ± 2.4

40.6 ± 3.7

0.247

0.438

C20:3n-9 (eicosatrienoic acid)

2.5 ± 0.4

2.4 ± 0.4

0.941

1.9 ± 0.2

2.7 ± 0.5

0.034

0.079

C20:4n-6 (arachidonic acid)

135.7 ± 8.4

138.8 ± 6.0

0.689

143.8 ± 11.1

151.1 ± 11.0

0.538

0.787

C20:5n-3 (eicosapentaenoic acid)

67.0 ± 9.0

71.3 ± 9.3

0.640

64.4 ± 7.2

59.1 ± 5.7

0.385

0.369

C22:0 (behenic acid)

16.6 ± 0.8

18.3 ± 1.0

0.035

17.1 ± 0.8

17.9 ± 1.3

0.623

0.468

C22:1n-9 (erucic acid)

1.6 ± 0.1

1.3 ± 0.1

0.066

1.3 ± 0.1

1.3 ± 0.1

0.914

0.170

C22:2n-6 (docosadienoic acid)

NE

NE

 

NE

NE

  

C22:4n-6 (docosatetraenoic acid)

3.9 ± 0.2

4.2 ± 0.2

0.252

4.4 ± 0.3

4.9 ± 0.6

0.262

0.689

C22:5n-3 (docosapentaenoic acid)

20.0 ± 1.4

20.7 ± 1.7

0.657

20.7 ± 1.7

21.5 ± 1.7

0.839

0.887

C22:6n-3 (docosahexaenoic acid)

128.7 ± 9.8

138.6 ± 9.3

0.231

126.5 ± 10.0

128.3 ± 10.8

0.936

0.456

C24:1 (nervonic acid)

35.4 ± 2.2

36.1 ± 2.1

0.656

31.6 ± 1.8

30.3 ± 1.9

0.275

0.263

Data are expressed as means ± SE

a p value for the intergroup comparison (baseline vs 6 month)

b p value for the intergroup comparison (changes from baseline between groups)

NE, not estimated

Fatty acid composition in extracted liver tissue was available for 16 NAFLD patients treated with ezetimibe and 12 controls (Table 6). Ezetimibe treatment for 6 months significantly and markedly increased hepatic lauric, myristic, palmitic, palmitoleic, margaric and stearic acids compared with the control group. The changes in hepatic fatty acid composition did not correlate with the changes in serum fatty acid composition before and after ezetimibe treatment (ESM Table 5).
Table 6

Changes in hepatic fatty acid composition

Fatty acid

Control

p a

Ezetimibe

p a

p b

Before

After

 

Before

After

  

C12:0 (lauric acid)

7.7 ± 1.2

15.2 ± 5.6

0.219

6.3 ± 1.8

18.8 ± 4.7

0.019

0.494

C14:0 (myristic acid)

19.9 ± 2.5

33.0 ± 10.1

0.228

17.6 ± 2.2

56.6 ± 13.0

0.014

0.148

C16:0 (palmitic acid)

185.9 ± 23.8

303.9 ± 118.2

0.334

169.7 ± 22.9

583.9 ± 176.8

0.042

0.202

C16:1n-7 (palmitoleic acid)

24.2 ± 4.5

37.3 ± 13.4

0.362

22.3 ± 4.3

51.9 ± 13.2

0.031

0.368

C17:0 (margaric acid)

4.6 ± 0.7

3.5 ± 0.1

0.400

5.3 ± 0.8

16.0 ± 4.1

0.024

0.025

C18:0 (stearic acid)

45.9 ± 4.4

54.4 ± 8.9

0.283

56.0 ± 7.1

125.1 ± 30.2

0.017

0.042

C18:1n-9 (oleic acid)

166.4 ± 25.1

250.2 ± 91.6

0.367

173.9 ± 30.6

381.9 ± 84.3

0.017

0.288

C18:2n-6 (linoleic acid)

80.4 ± 12.3

87.9 ± 22.5

0.556

73.9 ± 8.5

147.3 ± 36.1

0.035

0.066

C18:3n-6 (γ‐linolenic acid)

ND

ND

 

ND

ND

  

C18:3n-3 (α-linolenic acid)

0.6 ± 0.4

0.0 ± 0.0

0.171

0.6 ± 0.4

0.0 ± 0.0

0.178

0.981

C20:0n-6 (arachidic acid)

ND

ND

 

ND

ND

  

C20:1n-9 (eicosenoic acid)

5.5 ± 1.1

4.7 ± 1.9

0.639

5.7 ± 1.0

13.1 ± 4.8

0.170

0.168

C20:2n-6 (eicosadienoic acid )

ND

ND

 

ND

ND

  

C20:3n-6 (dihomo-γ-linolenic acid)

ND

ND

 

ND

ND

  

C20:3n-9 (eicosatrienoic acid)

ND

ND

 

ND

ND

  

C20:4n-6 (arachidonic acid)

ND

ND

 

ND

ND

  

C20:5n-3 (eicosapentaenoic acid)

ND

ND

 

ND

ND

  

C22:0 (behenic acid)

ND

ND

 

ND

ND

  

C22:1n-9 (erucic acid)

14.2 ± 2.5

11.7 ± 2.7

0.474

16.2 ± 2.4

19.2 ± 1.0

0.664

0.468

C22:2n-6 (docosadienoic acid)

2.8 ± 1.0

1.8 ± 1.0

0.433

22.3 ± 0.7

62.3 ± 2.9

0.176

0.152

C22:4n-6 (docosatetraenoic acid)

ND

ND

 

ND

ND

  

C22:5n-3 (docosapentaenoic acid)

ND

ND

 

ND

ND

  

C22:6n-3 (docosahexaenoic acid)

13.6 ± 3.5

7.8 ± 3.3

0.232

14.2 ± 3.7

48.7 ± 19.9

0.109

0.097

C24:1 (nervonic acid)

ND

ND

 

ND

ND

  

The data are expressed as 10−4 mg/mg liver, means ± SE

a p value for the intragroup comparison (baseline vs 6 month)

b p value for the intergroup comparison (changes from baseline between groups)

ND, not determined

Discussion

This is the first report of the efficacy of ezetimibe treatment on liver pathology in patients with NAFLD in an open-label randomised controlled trial. Treatment with 10 mg/day ezetimibe for 6 months did not alter the primary study outcome, serum aminotransferase levels. Ezetimibe significantly decreased serum cholesterol levels and cholesterol absorption markers as expected, whereas, in contrast to previous reports, ezetimibe treatment did not decrease serum levels of triacylglycerol. Our initial hypothesis was that ezetimibe treatment ameliorates liver pathology by inhibiting the absorption of toxic lipids such as oxidised cholesterol and palmitate. In our animal model, cholesterol feeding to mice increased not only cholesterol but also triacylglycerols in the liver, and upregulated the gene for sterol regulatory element binding protein (SREBP)-1c that governs fatty acid synthesis [3], probably via activation of liver-X-receptor (LXR) in the liver [25]. Therefore, in experimental models of high-cholesterol-diet-induced steatohepatitis, ezetimibe ameliorated liver steatosis by reducing cholesterol-induced activation of LXR and SREBP-1c [26, 27]. In the present study, however, treatment with ezetimibe unexpectedly ameliorated liver fibrosis staging and ballooning scores without significantly changing hepatic steatosis and insulin resistance.

One possible explanation for the improvement of hepatic fibrosis by ezetimibe treatment may be related to the direct effect of cholesterol on hepatic fibrogenesis. The cholesterol molecule affects membrane organisation and structure, which are critical determinants of membrane bilayer permeability and fluidity [28]. Altered cholesterol metabolism has several toxic effects on hepatocytes, resident macrophages, Kupffer cells and hepatic stellate cells, which promote NASH through diverse mechanisms. Hepatic stellate cells, in particular, are responsible for liver fibrosis in NASH. It has recently been reported that intracellular cholesterol accumulation directly activates hepatic stellated cells through a toll-like receptor-4-dependent pathway and triggers hepatic fibrosis [29]. These effects might be more evident in humans because, unlike rodents, where NPC1L1 is primarily expressed in the intestine, in humans NPC1L1 mRNA is highly expressed both in the small intestine and liver. Therefore, ezetimibe is estimated to inhibit not only dietary and biliary cholesterol absorption through the small intestine, but also reabsorption of biliary cholesterol in the liver [30, 31]. Thus, ezetimibe may inhibit liver fibrosis by ameliorating cholesterol-induced activation of hepatic stellate cells in patients with NAFLD. This hypothesis was well supported by the hepatic gene expression profile induced by ezetimibe administration. Ezetimibe treatment coordinately downregulated genes involved in skeletal muscle development and cell adhesion molecules, suggesting that ezetimibe suppressed stellate cell development into myofibroblasts and thereby inhibited fibrogenesis.

Another important finding of the present study was that treatment with ezetimibe significantly deteriorated glycaemic control. Ezetimibe therapy also altered the hepatic profile of fatty acid components by significantly increasing hepatic levels of lauric, myristic, palmitic, palmitoleic, margaric, stearic, oleic and linoleic acids. Experimentally, palmitate induces interleukin-8 [32], endoplasmic reticulum stress, and c-Jun amino-terminal kinase activation and promotes apoptosis in the liver [5, 33, 34]. Lipid-induced oxidative stress and inflammation are closely related to insulin resistance [3, 5], which could be relevant to the ezetimibe-induced deterioration of glucose homeostasis. Indeed, urinary excretion of 8-isoprostanes was significantly increased in the ezetimibe group compared with the control, and showed significant negative correlation with insulin sensitivity indices such as the Matsuda index and QUICKI in the present study (ESM Table 6). Moreover, hepatic gene expression in the ezetimibe group showed coordinated upregulation of genes involved in the immune response compared with those in the control group, suggestive of oxidative stress caused by ezetimibe treatment.

Pathway analysis of the metabolic network showed unique metabolic changes in the ezetimibe group compared with the control group. In the control group, genes involved in the CoA-biosynthesis pathway were coordinately downregulated, and those in the glycerol-3 phosphate pathway coordinately upregulated, suggesting activated triacylglycerols biosynthesis. In the ezetimibe group, genes involved in the l-carnitine pathway, including CPT1A, were coordinately downregulated. A decreased l-carnitine pathway could be associated with reduced β-oxidation of palmitic acids in mitochondria, resulting in an increase in long-chain fatty acids (lauric, myristic, palmitic, palmitoleic, margaric, stearic, oleic and linoleic acids). Unbalanced fatty acid composition could induce oxidative stress and lead to insulin resistance in the ezetimibe group. In addition, genes involved in the cholesterol and NEFA biosynthesis, including SREBF2, were coordinately upregulated in the ezetimibe group (Table 3), probably as a result of deceased absorption of exogenous cholesterol. Upregulation of SREBF2 potentially represses the expression of hepatocyte nuclear factor 4, which is required for CPT1 transcription [35]. Moreover, recent reports have demonstrated that microRNA (miR)-33, encoded by an intron of Srebp2 [36], inhibits translation of transcripts involved in fatty acid β-oxidation, including CPT1 [37]. miR-33 is also implicated in decreased insulin signalling by reducing insulin receptor substrate-2 [38, 39]. Hepatic gene expression profiles may, to some extent, explain hepatic fatty acid composition and impaired glycaemic control in the ezetimibe group. These novel SREBP-2-mediated pathways in the gene expression network may be relevant to a recent report that a polymorphism in the SREBF2 predicts incidence and the severity NAFLD and the associated glucose and lipid dysmetabolism [15]. These unique hypotheses should be confirmed in future in vitro and in vivo studies.

Our study has some limitations. First, the number of patients is relatively small because the data and safety monitoring board recommended that the study intervention and enrolment be discontinued in light of the higher proportion of adverse events in the ezetimibe group than in the control group. Second, our trial was a 6 month open-label study that resulted in subtle changes in liver pathology compared with previous reports [40]. Indeed, a 6 month duration may be too short a period to expect improvement of fibrosis, which is a slowly progressive process [40]. Third, the average serum aminotransferase levels were lower than those in previous studies [9, 10], and most of the patients had mild steatosis, fibrosis and lower NAS at baseline before ezetimibe treatment. Serum ALT levels did not decrease with ezetimibe treatment in the present study, in contrast to the significant improvement reported previously [9, 10]. And finally, secondary outcomes are always at risk of false-positive associations. Therefore, we not only presented the changes in HbA1c (p = 0.001 for ezetimibe treatment and p = 0.041 for the intergroup difference at the end of the study), but also showed the signature of hepatic fatty acid composition and hepatic gene expression profiles that support the hypothesis that ezetimibe increases HbA1c and hepatic fatty acids contents possibly through the SREBP-2–miR33 pathway. No previous studies have raised this issue, which is worth investigating. The same mechanism may underlie a statin-induced deterioration of glucose tolerance, which remains a serious concern. Furthermore, the SREBP-2–miR33 pathway may raise a concern for a safety issue of combination therapy with ezetimibe and statins because these agents may additively upregulate SREBF2 expression [41]. Future large-scale, long-duration studies involving more severely affected patients are required to determine the definite efficacy and risks of ezetimibe in the treatment of NAFLD.

In conclusion, the present study represents the first randomised controlled clinical trial of the efficacy of ezetimibe on liver pathology, energy homeostasis, hepatic fatty acid composition and hepatic gene expression profiles in patients with NAFLD. The lipid profile and liver histology of cell ballooning and fibrosis were significantly improved by ezetimibe treatment. However, our findings suggest an increase in oxidative stress, insulin resistance and HbA1c on treatment with ezetimibe, which should be taken into consideration in NAFLD patients.

Notes

Acknowledgements

We thank M. Kawamura (Kanazawa University Graduate School of Medical Sciences) for technical assistance.

Funding

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and research grants from MSD (to TT and SK).

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Contribution statement

YT designed the study, recruited the patients, analysed the data and wrote the manuscript. TT designed the study, recruited the patients, interpreted the data and edited the manuscript. MH analysed the hepatic gene expression profiles. YK performed the statistical analyses. YZ analysed all the biopsies. KK, HM and TO recruited the patients and collected the clinical information. HS, KA and TY performed the liver biopsies and histological examinations. MN performed the DNA chip experiments. KY and EM analysed the hepatic fatty acid compositions. SK initiated and organised the study. All authors contributed to the acquisition, analysis and interpretation of data and the drafting and editing of the manuscript. All of the authors approved the final version of the manuscript.

Supplementary material

125_2013_3149_MOESM1_ESM.pdf (55 kb)
ESM Methods (PDF 55 kb)
125_2013_3149_MOESM2_ESM.pdf (32 kb)
ESM Fig. 1 (PDF 31 kb)
125_2013_3149_MOESM3_ESM.pdf (98 kb)
ESM Fig. 2 (PDF 98 kb)
125_2013_3149_MOESM4_ESM.pdf (93 kb)
ESM Table 1 (PDF 92 kb)
125_2013_3149_MOESM5_ESM.pdf (118 kb)
ESM Table 2 (PDF 118 kb)
125_2013_3149_MOESM6_ESM.pdf (86 kb)
ESM Table 3 (PDF 86 kb)
125_2013_3149_MOESM7_ESM.pdf (87 kb)
ESM Table 4 (PDF 86 kb)
125_2013_3149_MOESM8_ESM.pdf (143 kb)
ESM Table 5 (PDF 142 kb)
125_2013_3149_MOESM9_ESM.pdf (59 kb)
ESM Table 6 (PDF 59 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yumie Takeshita
    • 1
  • Toshinari Takamura
    • 1
  • Masao Honda
    • 1
  • Yuki Kita
    • 1
  • Yoh Zen
    • 2
  • Ken-ichiro Kato
    • 1
  • Hirofumi Misu
    • 1
  • Tsuguhito Ota
    • 1
  • Mikiko Nakamura
    • 1
  • Kazutoshi Yamada
    • 1
  • Hajime Sunagozaka
    • 1
  • Kuniaki Arai
    • 1
  • Tatsuya Yamashita
    • 1
  • Eishiro Mizukoshi
    • 1
  • Shuichi Kaneko
    • 1
  1. 1.Department of Disease Control and HomeostasisKanazawa University Graduate School of Medical SciencesKanazawaJapan
  2. 2.Histopathology Section, Institute of Liver StudiesKing’s College HospitalLondonUK

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