Retinol-binding protein 4: A new marker of virus-induced steatosis in patients infected with hepatitis c virus genotype 1

Abstract

Retinol-binding protein 4 (RBP4) is an adipocytokine associated with insulin resistance (IR). We tested serum levels of RBP4 to assess its link with steatosis in patients with genotype 1 chronic hepatitis C (CHC) or nonalcoholic fatty liver disease (NAFLD). Nondiabetic patients with CHC (n = 143) or NAFLD (n = 37) were evaluated by liver biopsy and anthropometric and metabolic measurements, including IR by the homeostasis model assessment. Biopsies were scored by Scheuer classification for CHC, and Kleiner for NAFLD. Steatosis was tested as a continuous variable and graded as absent-mild <30%, or moderate-severe ≥30%. Thirty nondiabetic, nonobese blood donors served as controls. RBP4 levels were measured by a human competitive enzyme-linked immunosorbent assay kit (AdipoGen). Mean values of RBP4 were similar in NAFLD and CHC (35.3 ± 9.3 μg/L versus 36.8 ± 17.6; P = 0.47, respectively), and both were significantly higher than in controls (28.9 ± 12.1; P = 0.02 and P = 0.01, respectively). RBP4 was higher in CHC patients with steatosis than in NAFLD (42.1 ± 19.7 versus 35.2 ± 9.3; P = 0.04). By linear regression, RBP4 was independently linked to steatosis only (P = 0.008) in CHC, and to elevated body mass index (P = 0.01) and low grading (P = 0.04) in NAFLD. By linear regression, steatosis was independently linked to homeostasis model assessment score (P = 0.03) and high RBP4 (P = 0.003) in CHC. By logistic regression, RBP4 was the only variable independently associated with moderate-severe steatosis in CHC (odds ratio, 1.045; 95% confidence interval, 1.020 to 1.070; P = 0.0004), whereas waist circumference was associated with moderate-severe steatosis in NAFLD (odds ratio, 1.095; 95% confidence interval, 1.007 to 1.192; P = 0.03). Conclusion: In nondiabetic, nonobese patients with genotype 1 CHC, serum RBP4 levels might be the expression of a virus-linked pathway to steatosis, largely unrelated to IR. (HEPATOLOGY 2008.)

Steatosis is the hallmark of nonalcoholic fatty liver disease (NAFLD),1 and is also a frequent histological finding in patients with chronic hepatitis C (CHC). Its prevalence in CHC ranges between 40% and 80% overall, and remains around 40% after exclusion of all known causes of fatty liver.2 The relevance of hepatic fat content comes from its effects on the course of viral and nonviral chronic liver damage. Steatosis is considered a major determinant of fibrosis in both NAFLD3 and CHC,4, 5 and negatively affects the rate of sustained virological response in patients with genotype 1 (G1) CHC treated with pegylated-interferon and ribavirin.5, 6

The biological mechanism underlying steatosis is not entirely understood and is probably multifactorial. In NAFLD patients, liver fat content is specifically related to insulin resistance (IR) and results from enhanced free fatty acid influx, together with de novo lipogenesis.1, 7 For CHC, in which the rate of steatosis is approximately two times higher than in chronic hepatitis B8 or autoimmune hepatitis,9 two distinct “viral” and “metabolic” pathways to steatosis probably exist (and possibly coexist in a large proportion of cases).

Among hepatitis C virus (HCV) genotypes, G1 HCV is generally considered devoid of any intrinsic steatogenic activity, as opposed to genotype 3 (G3) HCV.10 HCV may interfere with lipid metabolism through at least three distinct, nonmutually exclusive mechanisms, identified using G1-derived constructs (impaired secretion, impaired degradation, and increased synthesis).10 More recently, studies have focused on G3 CHC, in which steatosis is more prevalent and severe, correlates with the level of HCV replication,11 disappears in patients with sustained virological response to antivirals,12 and re-occurs after viral relapse.13 Conversely, in G1 CHC, hepatic fat content is associated with elevated body mass index (BMI), central adiposity, and other clinical risk factors for NAFLD,11 and it is not responsive to antiviral therapy.12 In line with these data, several studies of G1 CHC have identified IR as the main determinant in the pathogenesis of steatosis4 and fibrosis,14, 15 and have shown a higher prevalence of IR in HCV patients when compared to control populations.14 This evidence prompted a number of experimental studies showing that HCV per se is capable of inducing IR, possibly by interfering with insulin signaling.16, 17

Recently attention has been paid to the role of retinol-binding protein 4 (RBP4) in the pathogenesis of IR. This protein, a member of the lipocalin family,18 is secreted mainly by hepatocytes (80%), but also by adipose tissue (20%). It is the only specific transport protein for retinol19 and, by interacting with nuclear retinol X receptor (RXR), it takes part in the control of metabolic and proliferative cell functions,20 including steatogenesis.21 Mouse and human studies have highlighted a pathogenic link among IR, diabetes, and high serum and adipose levels of RBP4,21–24 identifying RBP4 as a novel adipocytokine. There is clinical evidence that circulating RBP4 levels are related to the severity of IR and to the various features of the metabolic syndrome.25–29 Specifically, raised serum levels of RBP4 have been linked to NAFLD, assessed by ultrasonography in a cohort of subjects with30 and without diabetes.31

A recent study found a direct relation between hepatic fat content, evaluated by magnetic resonance spectroscopy, and blood levels of RBP4 in healthy subjects,32 whereas other studies linked RBP4 levels to the inflammatory response in obese, insulin-resistant patients.33, 34

To our knowledge, no studies have specifically evaluated the serum levels of RBP4 in patients with histological proven NAFLD or with G1 CHC. The issue of HCV G1 inducing steatosis is of particular relevance, since a remarkable proportion of subjects chronically infected by this “nonsteatogenic” genotype do have steatosis, also in the absence of predisposing factors.35

The aim of the present study was to assess the possible role of serum levels of RBP4 in hepatic steatosis in both G1 CHC and NAFLD, and whether RBP4 levels were associated with the severity of steatosis in both conditions.

ALT, alanine aminotransferase; BMI, body mass index; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; γ-GT, γ-glutamyltransferase; HCV, hepatitis C virus; G1, genotype 1; G3, genotype 3; CHC, chronic hepatitis C; IR, insulin resistance; HOMA, homeostasis model assessment; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OR, odds ratio; RBP4, retinol-binding protein 4; RXR, retinol X receptor.

Patients and Methods

Patients.

The study was carried out on a convenient sample of 180 consecutive patients (143 with G1 CHC and 37 with NAFLD), recruited at the Gastrointestinal and Liver Unit of the University Hospital in Palermo, between January 2006 and March 2007 and fulfilling all inclusion and exclusion criteria detailed below. Patients were included if they had a histological diagnosis of G1 CHC or NAFLD on a liver biopsy performed less than 6 months before enrollment. G1 CHC patients were characterized by the presence of anti-HCV and HCV RNA, with persistently abnormal alanine aminotransferase (ALT) and liver histology of chronic hepatitis (any degree of fibrosis, including cirrhosis), and by alcohol consumption <20 g/day in the last year or more. The diagnosis of NAFLD was based on chronically elevated ALT for at least 6 months, alcohol consumption <20 g/day in the last year, steatosis (≥5% of hepatocytes) at histology with/without necroinflammation and/or fibrosis, and negative anti-HCV.

Exclusion criteria were as follows: (1) advanced cirrhosis (Child-Turcotte-Pugh B and C); (2) hepatocellular carcinoma; (3) other causes of liver disease or mixed etiologies (excessive alcohol consumption, hepatitis B, autoimmune liver disease, Wilson's disease, hemochromatosis, or α1-antitrypsin deficiency); (4) human immunodeficiency virus infection; (5) previous treatment with antiviral therapy, immunosuppressive drugs, and/or regular use of steatosis-inducing drugs, evaluated by a questionnaire (for example, corticosteroid, valproic acid, tamoxifen, amiodarone); (6) previous diagnosis of type 1 or type 2 diabetes mellitus, and/or fasting blood glucose ≥126 mg/dL; or (7) active intravenous drug addiction or use of cannabis.

Thirty nondiabetic, nonobese (BMI <30 kg/m²), apparently healthy blood donors were enrolled as controls. Alcohol consumption >20 g/day for at least the last year and/or concomitant use of any drugs were additional exclusion criteria. All had normal fasting glucose (<100 mg/dL), cholesterol (<200 mg/dL) and triglycerides (<150 mg/dL), normal ALT (<30 UI/mL), and no evidence of viral infection (HCV, human immunodeficiency virus, and hepatitis B surface antigen negativity) or steatosis at ultrasonography scan.

The study was performed in accordance with the principles of the Declaration of Helsinki, and its appendices, and with local and national laws. Approval was obtained from the hospital's Internal Review Board and the Ethical Committee, and written informed consent was obtained from all patients and controls.

Clinical and Laboratory Assessment.

Clinical and anthropometric data were collected at the time of liver biopsy (Table 1). BMI was calculated on the basis of weight (in kilograms) and height (in meters), and subjects were classified as normal weight (BMI 18.5 to 24.9 kg/m²), overweight (BMI 25 to 29.9 kg/m²), mildly obese (BMI 30 to 34.9 kg/m²), and moderately-severely obese (BMI ≥35 kg/m²). Hip circumference was measured at the widest point between hip and buttocks. Waist circumference was measured at the midpoint between the lower border of the rib cage and the iliac crest. Visceral obesity was diagnosed at a waist circumference >88 cm (women) and >102 cm (men).

Table 1. Demographic, Laboratory, Metabolic, and Histologic Features of 180 Consecutive Nondiabetic, Nondrinking Patients with Viral (HCV G1) and Nonviral (NAFLD) Chronic Liver Disease

Variable	HCV G1 (n = 143)	NAFLD (n = 37)	P
Data are given as mean ± SD or as number of cases (%). HCV-RNA indicates hepatitis C virus ribonucleic acid; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; NL, normal limit; and RBP4, retinol-binding protein 4.
Age (years)	50.2 ± 12.4	40.6 ± 11.4	<0.0001
Male gender	76 (53.1)	29 (78.4)	0.005
Body mass index (kg/m^2)	27.4 ± 4.3	28.9 ± 4.8	0.07
<25	35 (24.5)	9 (24.4)
25-29.9	79 (55.2)	15 (40.5)
≥30	29 (20.3)	13 (35.1)
Waist circumference (cm)	93.1 ± 11.4	96.0 ± 13.8	0.20
Visceral obesity (ATPIII criteria)	55 (38.5)	14 (37.9)	0.94
Hip circumference (cm)	104.7 ± 10.4	101.4 ± 13.2	0.11
Alanine aminotransferase (IU/L; NL < 30 IU/L)	91.2 ± 79.4	78.4 ± 53.9	0.25
Platelet count (×10^3/mm^3)	210.9 ± 52.9	217.6 ± 53.6	0.49
γ-Glutamyl transferase (IU/L)	53.6 ± 44.8	115.8 ± 142.1	0.01
Ferritin (ng/mL)	206.8 ± 189.4	275.0 ± 302.7	0.19
Cholesterol (mg/dL)	177.2 ± 34.9	209.5 ± 43.1	<0.0001
HDL cholesterol (mg/dL)	51.6 ± 15.8	51.2 ± 18.6	0.90
Triglycerides (mg/dL)	95.3 ± 37.8	136.6 ± 84.8	0.006
Blood glucose (mg/dL)	89.0 ± 15.7	95.0 ± 26.8	0.08
Insulin (μU/mL)	12.2 ± 6.7	16.5 ± 10.5	0.02
HOMA score	2.76 ± 1.73	4.19 ± 4.10	0.04
RBP4 (μg/L)	36.8 ± 17.5	35.2 ± 9.3	0.47
HCV-RNA (IU/mL)	765,680 ± 900,429	—	—
Histology at biopsy
Steatosis
Continuous variable	12.6 ± 17.8	39.2 ± 25.8	<0.0001
Categorical variable
<5%	68 (47.6)	0 (0)
≥5% to <30%	41 (28.6)	14 (37.8)
≥30%	34 (23.8)	23 (62.2)	<0.0001
Stage of fibrosis
0-1	68 (33.6)	31 (83.8)
2	48 (33.5)	4 (10.8)
3-4	27 (23.7)	2 (5.4)	<0.0001
Grade of activity
1	34 (23.8)	—	—
2	83 (58.0)	—	—
3	26 (18.2)	—	—

A 12-hour overnight fasting blood sample was drawn at the time of biopsy to determine serum levels of ALT, γ-glutamyltransferase (γ-GT), total cholesterol, high-density lipoprotein-cholesterol, triglycerides, ferritin, plasma glucose concentration, and platelet count. Serum insulin was determined by a two-site enzyme-linked immunosorbent assay (ELISA; Mercodia Insulin ELISA; Arnika). IR was determined by the homeostasis model assessment (HOMA) method, using the following equation36: IR (HOMA-IR) = fasting insulin (μU/mL) × fasting glucose (mmol/L)/22.5. HOMA-IR has been validated in comparison with the euglycemic/hyperinsulinemic clamp technique in both diabetic and nondiabetic subjects.37

Serum RBP4 levels were measured in duplicate by a human competitive ELISA kit (AdipoGen, Inc.). The coefficient of variation within each assay was less than 5%. The coefficient of variation between assays was less than 10%.

All patients were tested at the time of biopsy for HCV-RNA by qualitative polymerase chain reaction (Cobas Amplicor HCV Test, version 2.0; limit of detection 50 IU/mL). HCV-RNA–positive samples were quantified by Versant HCV RNA 3.0 bDNA (Bayer, Tarrytown, NY), expressed in IU/mL. Genotyping was performed by INNO-LiPA HCV II (Bayer).

Assessment of Histology.

Slides were coded and read by a single pathologist (D.C.) unaware of the patient's identity and history. A minimum length of 15 mm of biopsy specimen or the presence of at least 10 complete portal tracts was required.38 The percentage of hepatocytes containing fat was determined for each 10× field. An average percentage of steatosis was then determined for the entire specimen.

Steatosis was assessed as the percentage of hepatocytes containing fat droplets (minimum 5%), and evaluated as continuous variable. Steatosis was also classified as: absent-mild <30%; moderate-severe ≥30%.

In the NAFLD group, the Kleiner classification39 was used to compute the NAFLD activity score (from 0 to 8, on a scale including separate scores for steatosis, lobular inflammation, and hepatocellular ballooning) and to stage fibrosis from 0 to 4. Nonalcoholic steatohepatitis (NASH) was defined and graded according to the Brunt global grade of activity.40

Biopsies of CHC patients were classified for grade and stage according to the Scheuer system.41

Statistics.

Continuous variables were summarized as mean ± standard deviation and categorical variables as frequency and percentage. The t test and analysis of variance test were used as appropriate. Multiple linear regression analysis was performed to identify independent predictors of serum RBP4 levels as continuous dependent variables in both CHC and NAFLD patients. In these models, we selected as explanatory variables age, gender, BMI, waist and hip circumference, baseline ALT, platelet count, γ-GT, ferritin, total and high-density lipoprotein-cholesterol, triglycerides, blood glucose, insulin, HOMA score, HCV viral load, fibrosis, steatosis, activity score (in CHC patients), and lobular inflammation, hepatocellular ballooning, and NAFLD activity score in NAFLD patients.

Multiple logistic regression models were used to assess the relationship of steatosis to demographic, virological (in CHC patients), metabolic and histological characteristics of both CHC and NAFLD patients. In these models, the dependent variable was steatosis coded as 0 = absent-mild (<30%); or 1 = moderate-severe (≥30%). As candidate risk factors for moderate-severe steatosis we selected the same independent variables included in the RBP4 model, with RBP4 added as an additional independent variable. Multiple linear regression analysis was also performed to identify independent predictors of steatosis as continuous dependent variables. In CHC patients, multiple logistic models were also used to identify independent risk factors of steatosis, coded as: 0 = absent (<5%); 1 = mild (≥5% to <30%); 2 = moderate-severe (≥ 30%).

Variables found to be associated with the dependent variable at univariate analyses (probability threshold, P ≤ 0.10) were included in multivariate regression models. To avoid the effect of the colinearity, HOMA score, blood glucose levels, and insulin levels, as well as waist circumference, hip circumference, and BMI, were not included in the same multivariate model. Regression analyses were performed using PROC LOGISTIC, PROC REG, and subroutines in SAS (SAS Institute, Inc., Cary, NC).42

Results

Patient Features and Histology.

CHC patients were characterized by older age (by 10 years) and different gender and weight class distribution, compared with NAFLD (Table 1). Visceral obesity was identified in about 40% of cases in both groups. Metabolic abnormalities were more common in NAFLD, with higher cholesterol and triglycerides and a higher HOMA score.

At liver biopsy (Table 1), steatosis was present in half of the CHC patients, but a moderate-severe grade was identified only in approximately a quarter of the cases, less frequently than in NAFLD patients (62%). Twenty-seven subjects with NAFLD (72.9%; 95% CI, 59 to 87) were histologically diagnosed as NASH, graded as mild in 12 cases (44.4%), moderate in 12 (44.4%), and severe in three (11.2%). According to Kleiner et al.,39 43% of NAFLD were classified as NASH, 12% as non-NASH, and 43% were indeterminate (Table 2).

Table 2. Histological Features by Kleiner et al.'s40 Classification of 37 Consecutive Nondiabetic Patients with Nonalcoholic Fatty Liver Disease

Histology	NAFLD (n = 37)
Data are given as number of cases (%).
NAFLD activity score
0-2	5 (13.6)
3-4	16 (43.2)
5-8	16 (43.2)
Lobular inflammation
0	10 (27.0)
1	20 (54.0)
2	7 (19.0)
3	0 (0)
Steatosis grade
0 (<5%)	0 (0)
1 (5%-33%)	22 (59.5)
2 (>33%-66%)	6 (16.2)
3 (>66%)	9 (24.3)
Hepatocellular ballooning
0	8 (21.6)
1	9 (24.3)
2	20 (54.1)

The control subjects had a mean age of 40.0 ± 12.5 years, and 63.4% were males. Their mean BMI was 24.6 ± 2.8 kg/m². All had normal ALT (24.6 ± 2.8 IU), fasting glucose (76.6 ± 10.4 mg/dL), cholesterol (163.8 ± 22.4 mg/dL), and triglycerides (78.2 ± 30.8 mg/dL). The mean HOMA score was 1.98 ± 0.60 μU/mL (range, 0.56 to 2.55). None had arterial hypertension. Visceral obesity was not assessed.

Serum RBP4 Levels.

Mean serum values of RBP4 were similar in NAFLD and CHC (36.8 ± 17.5 versus 35.2 ± 9.3; P = 0.4; Fig. 1), but both were significantly higher than controls (28.9 ± 12.1; P = 0.01 and P = 0.02, respectively; Fig. 1). CHC patients with steatosis had higher RBP4 levels than in NAFLD (42.1 ± 19.7 versus 35.2 ± 9.3; P = 0.04; Fig. 2). In the CHC cohort only, a progressive increase of RBP4 levels was observed from patients without steatosis to patients with mild (<30%) and with moderate-severe steatosis (≥30%; P = 0.0002; Fig. 3).

Figure 1. Serum RBP4 levels in viral (G1 CHC) and nonviral (NAFLD) chronic liver disease, and in healthy control subjects.

Figure 2. Serum RBP4 levels in viral (G1 CHC) and nonviral (NAFLD) steatosis.

Figure 3. Serum RBP4 levels according to the degree of steatosis in G1 CHC and NAFLD.

In the subset of 34 CHC patients with moderate-severe steatosis, 21 (61.7%) were males, 24 (70.6%) had a BMI of <30 kg/m², and 50% had visceral obesity. CHC subjects with moderate-severe steatosis without visceral obesity had lower HOMA score (2.61 ± 1.27 versus 3.90 ± 2.36; P = 0.05) but higher RBP4 levels (57.5 ± 24.0 versus 35.0 ± 10.6 P = 0.003), compared to viscerally obese patients.

High γ-GT levels (P = 0.06) and hepatic steatosis (P = 0.008), evaluated as continuous variables, were associated with higher RBP4 levels in CHC (Table 3), but only steatosis was an independent factor by multiple linear regression (P = 0.008). In NAFLD, elevated BMI, low ALT and γ-GT, and high cholesterol, and low degree of fibrosis and necroinflammation were associated with high RBP4, but only elevated BMI (P = 0.01) and low necroinflammatory activity (P = 0.04) were maintained in multiple linear regression analysis.

Table 3. Univariate and Multivariate Analysis of Host Factors Associated with Elevated RBP4 Levels in Patients with Genotype 1 Chronic Hepatitis C and with Nonalcoholic Fatty Liver Disease

Variable	Univariate Analysis			Multivariate Analysis
	β	SE	P Value	β	SE	P Value
SE indicates standard error.
Genotype 1 chronic hepatitis C
γ-Glutamyl transferase (IU/L)	0.060	0.032	0.06	0.042	0.032	0.19
Steatosis	0.214	0.80	0.008	0.215	0.079	0.008
Nonalcoholic fatty liver disease
Body mass index (kg/m^2)	0.584	0.337	0.09	0.750	0.297	0.01
Alanine aminotransferase (IU/L)	−0.066	0.029	0.03	−0.055	0.032	0.10
γ-Glutamyl transferase (IU/L)	−0.026	0.010	0.02	−0.014	0.014	0.32
Cholesterol (mg/dL)	−0.077	0.036	0.03	0.000	0.094	0.98
Histology at biopsy
Stage of fibrosis	−3.127	1.830	0.08	−1.468	2.079	0.48
Lobular inflammation	−5.733	2.368	0.02	−5.297	2.472	0.04

Finally, in the NAFLD cohort, RBP4 levels were not significantly different between patients with NASH and all the others (37.1 ± 7.4 versus 34.5 ± 5.8; P = 0.31).

Risk Factors for Steatosis.

The characteristics of CHC patients according to steatosis grade are listed in Table 4. Elevated BMI and waist circumference, as well as high RBP4 and HOMA score were associated with moderate-severe steatosis, but only higher RBP4 levels [odds ratio (OR), 1.045; 95% confidence interval (CI), 1.020 to 1.070; P = 0.0004] were maintained in multivariate analysis.

Table 4. Univariate and Multivariate Analysis of Host and Viral Factors Associated with Moderate-Severe Steatosis (≥30% Hepatocytes Involved) in 143 Consecutive Nondrinking, Nondiabetic Patients with Chronic Hepatitis C Genotype 1

Variable	Absent-Mild Steatosis (<30% Hepatocytes; n = 109)	Moderate-Severe Steatosis (≥30% Hepatocytes; n = 34)	Univariate Analysis (P Value)	Multivariate Analysis
				OR (95% CI)	P Value
Data are given as mean ± standard deviation or as number of cases (%). HCV-RNA indicates hepatitis C virus ribonucleic acid; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; NL, normal limit; and RBP4, retinol-binding protein 4.
Age (years)	50.0 ± 12.1	51.1 ± 13.5	0.64		—
Male gender	55 (50.4)	21 (61.7)	0.25		—
Body mass index (kg/m^2)	27.1 ± 4.4	28.5 ± 3.8	0.09		—
Waist circumference (cm)	92.1 ± 11.5	96.5 ± 10.3	0.05	1.034 (0.992-1.079)	0.11
Hip circumference (cm)	104.3 ± 10.4	106.3 ± 10.1	0.37		—
Alanine aminotransferase (IU/L; NL < 30 IU/L)	87.6 ± 71.4	103.3 ± 101.8	0.32		—
Platelet count (×10^3/mm^3)	214.6 ± 53.7	198.9 ± 49.5	0.13		—
γ-Glutamyl transferase (IU/L)	52.2 ± 47.0	58.2 ± 36.8	0.49		—
Ferritin (ng/mL)	193.2 ± 169.9	254.5 ± 243.6	0.12		—
Cholesterol (mg/dL)	175.0 ± 32.9	184.0 ± 40.4	0.18		—
HDL cholesterol (mg/dL)	51.8 ± 16.6	50.8 ± 13.1	0.74		—
Triglycerides (mg/dL)	93.1 ± 34.3	102.9 ± 47.3	0.19		—
HOMA score	2.61 ± 1.63	3.25 ± 1.98	0.07	1.148 (0.886-1.488)	0.29
RBP4 (μg/L)	33.8 ± 15.0	46.3 ± 21.5	0.0009	1.045 (1.020-1.070)	0.0004
HCV-RNA (IU/mL)	752,361 ± 924,554	803,687 ± 843,391	0.83		—
Histology at biopsy
Stage of fibrosis
1-2	88 (80.7)	28 (82.3)
3-4	21 (19.3)	6 (17.7)	0.84		—
Grade of activity
Mild	27 (24.8)	7 (14.9)
Moderate-severe	82 (75.2)	27 (85.1)	0.97		—

By multiple logistic regression analysis, carried out according to steatosis grade and the presence and the degree of steatosis (absent versus mild versus moderate-severe) were independently linked to HOMA score (OR, 1.379; 95% CI, 1.094 to 1.738; P = 0.006) and high RBP4 levels (OR, 1.046; 95% CI, 1.024 to 1.068; P < 0.0001). These data were confirmed by multiple linear regression analyses in CHC patients, in whom the degree of steatosis was predicted quantitatively by HOMA score (P = 0.03) and high RBP4 (P = 0.003).

In NAFLD (Table 5), elevated BMI, waist and hip circumference, and HOMA score were all related to moderate-severe steatosis. By multivariate analysis, only a large waist circumference (OR, 1.095; 95% CI, 1.007 to 1.192; P = 0.03) was independently associated with moderate-severe steatosis. By multiple linear regression analysis, the degree of steatosis was quantitatively predicted by solely the waist circumference (P = 0.009).

Table 5. Univariate and Multivariate Analysis of Host Factors Associated with Moderate-Severe Steatosis in 37 Consecutive Patients with Nonalcoholic Fatty Liver Disease

Variable	Mild Steatosis (<30% Hepatocytes; n = 14)	Moderate-Severe Steatosis (≥30% Hepatocytes; n = 23)	Univariate Analysis (P Value)	Multivariate Analysis
				OR (95% CI)	P Value
Data are given as mean ± standard deviation or as number of cases (%). HDL indicates high-density lipoprotein; HOMA, homeostasis model assessment; NL, normal limit; and RBP4, retinol-binding protein 4.
Age (years)	43.4 ± 13.8	39.0 ± 9.6	0.25		—
Male gender	10 (71.4)	19 (82.6)	0.42		—
Body mass index (kg/m^2)	26.1 ± 4.4	30.5 ± 4.3	0.01		—
Waist circumference (cm)	87.0 ± 15.0	101.2 ± 10.0	0.01	1.095 (1.007-1.192)	0.03
Hip circumference (cm)	92.8 ± 17.6	105.5 ± 8.1	0.02		—
Alanine aminotransferase (IU/L; NL < 30 IU/L)	62.5 ± 37.9	88.1 ± 60.4	0.18		—
Platelet count (×10^3/mm^3)	220.3 ± 45.7	215.9 ± 58.8	0.80		—
γ-Glutamyl transferase (IU/L)	124.6 ± 139.0	110.4 ± 146.7	0.76		—
Ferritin (ng/mL)	191.7 ± 95.7	325.6 ± 370.7	0.25		—
Cholesterol (mg/dL)	208.0 ± 30.0	210.5 ± 50.1	0.86		—
HDL cholesterol (mg/dL)	55.6 ± 17.2	48.6 ± 11.9	0.17		—
Triglycerides (mg/dL)	118.0 ± 46.4	147.9 ± 100.7	0.30		—
HOMA score	2.60 ± 1.64	5.16 ± 4.83	0.06	1.527 (0.826-2.825)	0.17
RBP4 (μg/L)	33.5 ± 7.7	36.3 ± 10.1	0.38		—
Histology at biopsy
Stage of fibrosis
0-2	13 (92.8)	22 (95.6)
3-4	1 (7.2)	1 (4.4)	0.81
Lobular inflammation
0-1	12 (85.7)	18 (78.2)
2-3	2 (14.3)	5 (21.8)	0.28		—

Discussion

This is the first study that provides evidence of an association between elevated RBP4 levels and steatosis in G1 CHC patients, largely independent of obesity and the degree of IR, pointing to a possible direct involvement of the virus in RBP4 abnormalities. In contrast, in NAFLD the elevated RBP4 levels are more closely associated with IR and obesity, in keeping with a general metabolic disorder.

These conclusions are based on several findings: (1) both HCV-infected and NAFLD patients have higher serum levels of RBP4 than nonobese, nondiabetic healthy blood donors without metabolic disturbances; (2) HCV-infected patients with steatosis are at low metabolic risk, but their RBP4 levels are higher compared to NAFLD subjects; (3) RBP4 levels increase progressively in direct relation to the histological degree of steatosis in G1 CHC, but not in NAFLD; and (4) in patients with CHC and moderate-severe steatosis, subjects without metabolic disturbances have higher RBP4 levels than their counterparts with metabolic risk factors, and an independent link exists between the degree of steatosis and RBP4 levels.

In patients with G1 HCV infection the mechanism(s) underlying the occurrence of liver steatosis are not entirely understood. A high hepatic fat content is associated with IR4 due to metabolic and viral factors, and with its clinical expressions, such as elevated BMI and central adiposity.11 However, G1 HCV seems also able to induce steatosis by direct interference with lipid metabolism,10 and the present study is fairly in keeping with this hypothesis. In G1 CHC patients (all nondrinkers, naive to antiviral therapy and with a low rate of obesity, and independently of the classic measures of visceral adiposity), 52% had histological steatosis, which was in the moderate-severe range in 24%.

Although this study was not designed to clarify the pathogenesis of elevated RBP4 levels in G1 CHC, a few hypotheses may be put forward on the basis of the present and literature data. G1 HCV might induce the hepatic overexpression of RBP4, and this protein may in turn have an important role in the control of hepatic fat infiltration. Tsutsumi et al.43 showed that HCV core protein enhances the transcriptional activity regulated by RXRα homodimers and heterodimers, also inducing steatosis. Retinol is the precursor for the synthesis of ligands of these nuclear hormone receptors; RBP4 is the only transport protein of retinol in the blood,19 and its expression in animal models is associated with elevation of messenger RNA of several retinoic acid-responsive genes. HCV, by interacting with the same metabolic pathway, might induce the hepatic expression of RBP4, favoring the RXR-retinol interaction and therefore determining hepatic fat infiltration.

However, other mechanism(s) linking RBP4 to steatosis may be operative. Ost et al.44 found that RBP4 interferes with insulin signaling in adipocytes. Accordingly, RBP4 might also interact with a specific, so far unidentified, hepatocyte membrane receptor, acting as a modulator of steatogenesis. Finally, the evidence that both HCV-infected and NAFLD patients have RBP4 levels higher than healthy controls could stem from totally independent mechanism(s), for example, adipose overexpression of RBP4 in NAFLD subjects with metabolic disturbances, and virus-induced hepatic overproduction of RBP4 in G1 CHC.

We did not find a direct relation between HCV-RNA levels and serum RBP4, which, given the fluctuating levels of viremia in HCV infection,45 does not preclude a direct association of RBP4 with infection with G1 HCV. Similarly, in keeping with literature data,4 we did not observe a direct relation between the degree of steatosis and serum HCV-RNA levels.10

The elevated RBP4 levels of the NAFLD cohort are more easily explained. Our study confirms the link between steatosis and IR and identifies a pivotal role of visceral fat. Recent studies in experimental animals and in humans22–24 have suggested that RBP4, when overexpressed by visceral fat, is implicated in the pathogenesis of IR and is directly associated with many features of the metabolic syndrome,25–29 including NAFLD.30 According to these data, we found a direct relationship between RBP4 levels and BMI in NAFLD, but could not confirm the recently proven association of RBP4 levels with AST and γ-GT levels.31 Differences in age, anthropometric indices, and ALT and γ-GT levels among studies may explain this discrepancy.

In our setting, RBP4 was inversely associated with hepatic necroinflammatory activity. Conflicting data are available on this issue. Clinical studies have reported a direct association between adipose or serum expression of RBP4 and adipose or systemic inflammation,33, 34 whereas in an experimental mouse model RXRα-deficiency was reported to promote inflammatory-related liver injury.46 The effects of RBP4 on liver cells might differ from those on adipose tissue33 and on systemic inflammation,34 thus the results observed in our small NAFLD group need to be confirmed in a larger study. The main limitation of this study lies in its cross-sectional nature and its inability to dissect the temporal relation between RBP4 and steatosis. A further methodological issue is the potentially limited external validity of the results to different populations and settings, particularly for NAFLD. Our study included a cohort of Italian subjects enrolled at a tertiary care center, who may be different from the majority of prevalent cases of NAFLD in the general population.

Lack of data on the liver expression of RBP4, and on other potential confounders, such as proinflammatory cytokines and adipocytokines, could also affect the interpretation of the results. In addition, we cannot exclude the possibility that hidden abuse of alcohol may be responsible for steatosis in a few subjects, although this bias, if present, might be equally shared by the two groups, as well as by controls, given the similar methodology of ascertainment.

In conclusion, this study has shown a remarkable association between the degree of hepatic steatosis and RBP4 levels, restricted to G1 CHC patients and unrelated to abnormal metabolic features. This would suggest a viral pathway to steatosis, involving direct overexpression of RBP4 by the hepatocytes. Experimental studies are needed to determine the virus-induced mechanism(s) responsible for the association between steatosis and RBP4 in G1 HCV.

Acknowledgements

We thank Warren Blumberg for his help in editing this article. S. Petta designed the study, contributed to data acquisition, was responsible for writing the manuscript, and participated in statistical analysis. C. Cammà participated in the writing of the manuscript and was responsible for statistical analysis. V. Di Marco, A. Craxì (Director of the Gastrointestinal and Liver Unit), and G. Marchesini were responsible for writing the manuscript and have seen and approved the final version. N. Alessi, F. Barbaria, D. Cabibi, R. Caldarella, S. Ciminnisi, A. Licata, F. Massenti, A. Mazzola, and G. Tarantino participated in patient management and data collection. All authors have seen and approved the final version of the manuscript.