Background
Chronic hepatitis C virus (HCV) infection is one of the main causes of chronic liver disease. Host factors such as obesity, insulin resistance (IR), and hepatic steatosis have been reported to contribute to the progression from chronic hepatitis C (CHC) to liver cirrhosis and hepatocellular carcinoma (HCC) [
1‐
3]. In addition, chronic HCV infection contributes to the development of hepatic steatosis and IR [
4]. Hepatic steatosis and IR have a significant impact on the acceleration of liver injury and hepatic fibrosis in patients with CHC. Furthermore, the pathogenesis of steatosis and IR depend on the viral genotype: increased steatosis has been linked to HCV genotypes 2 and 3 [
5,
6], and body mass index (BMI) and homeostasis model assessment of IR (HOMA-IR) directly contribute to steatosis in patients infected with HCV genotype 1 [
5]. IR is also independently associated with genotypes 1 and 4 [
6]. The mechanisms of these interactions, however, are not fully understood.
Apoptosis inhibitor of macrophage (AIM) was initially identified as an apoptosis inhibitor that supports the survival of macrophages against various apoptosis-inducing stimuli [
7]. Recently, it has been reported that an increase in the blood levels of AIM is a critical event in the initiation of macrophage recruitment into adipose tissue, which is followed by IR [
8]. Miyazaki et al. suggested that AIM is involved in the progression of metabolic syndrome, including obesity and IR, in both an advancing and inhibitory fashion [
9], but the impact of AIM on the pathogenesis of HCV-related chronic liver disease has not been investigated.
Adipose tissue, skeletal muscle, and the liver are the major insulin-sensitive organs in the human body. Adipogenesis is the process by which preadipocytes differentiate into adipocytes, which is induced by insulin. Adipose tissue–derived cytokines, so-called adipocytokines, theoretically play an important role in the development of IR [
10]. The more familiar adipocytokines include leptin, adiponectin, and resistin. Leptin is a proinflammatory cytokine that accelerates the progression of hepatic fibrosis and exacerbates the inflammatory process in the liver [
11]. In contrast, adiponectin reduces hepatic fibrosis and exerts a hepatoprotective effect [
12]. Further, resistin, a signaling molecule secreted from adipocytes and monocytes, is known to be involved in inflammatory processes [
13], and has recently been reported to be associated with hepatic fibrosis [
14,
15]. However, their role in HCV-related chronic liver disease is somewhat confusing and the results of various studies have been contradictory [
16‐
18].
In this study, we analyzed the association between serum levels of AIM, leptin, adiponectin, and resistin, and clarified the clinical significance of serum AIM levels in patients with CHC. We then determined whether serum levels of AIM are associated with histological features such as the degree of hepatic steatosis and hepatic inflammation and the stage of hepatic fibrosis. In addition, we determined whether serum levels of AIM and adipocytokines are associated with IR or sensitivity in patients with CHC.
Discussion
HCV infection is the most important cause of chronic hepatitis, liver cirrhosis, and HCC. The incidence of HCC increases with increasing severity of hepatic fibrosis in patients with HCV infection. Hepatic inflammation and steatosis are thought to affect the progression of hepatic fibrosis. In addition, IR and obesity are thought to be associated with histopathology of the liver. Several adipocytokines and macrophage-derived molecules are reported to be associated with IR and obesity; we examined the association between these molecules and histopathological features in the liver. In this study, we demonstrated that serum AIM levels in patients with CHC were positively associated with hepatic fibrosis, but leptin, adiponectin, or resistin levels did not show this association. In contrast, serum levels of adiponectin, leptin and resistin were associated with IR, but AIM was not. Although the pathophysiological mechanism of AIM in CHC patients remains unclear, our study is the first to illustrate the clinical significance of AIM in patients with CHC.
In this study, we showed that serum AIM levels as determined by ELISA were higher in CHC patients with severe hepatic fibrosis compared to those with no or mild hepatic fibrosis. In addition, we confirmed that six HCV-infected cirrhotic patients who underwent liver transplantation had serum AIM levels greater than 1.2 μg/ml (data not shown). Gangadharan et al. previously reported that serum levels of the AIM protein were elevated in hepatitis C patients with liver cirrhosis compared to healthy controls, using a proteomics method based on 2-dimensional gel electrophoresis (2-DE) [
22]. They speculated that the increased serum AIM levels observed in cirrhotic patients may be associated with HCV infection rather than cirrhosis. However, serum AIM levels determined by proteomics using 2-DE analysis were previously reported to reflect the severity of hepatic fibrosis in nonalcoholic fatty liver disease [
23]. ALT ≥43 IU/l was more strongly associated with advanced hepatic fibrosis compared to AIM levels ≥1.2 μg/ml in our study; this may suggest that any association is indeed weak, but it is well known that ALT levels in patients with liver cirrhosis are lower than levels in patients with chronic hepatitis. Further, ALT <43 IU/L was observed in half of the patients with HCV-associated liver cirrhosis who received liver transplantation (data not shown). Therefore, the serum concentration of AIM is potentially a marker of hepatic fibrosis in chronic liver disease. Further validation studies involving patients with chronic liver disease, including those with HCV-associated liver cirrhosis, chronic hepatitis B, autoimmune liver disease, and fatty liver disease, are needed.
AIM is a member of the scavenger receptor cysteine-rich superfamily that was initially identified as an inhibitor of apoptosis that supports the survival of macrophages against different types of pro-apoptotic stimuli [
5]. It is reportedly solely produced by tissue macrophages, including Kupffer cells [
24,
25]. In addition, we observed that mRNA expression of AIM in the human hepatic stellate cell line LI90 was weak, but it was strong in the human macrophage cell line THP-1 (data not shown). Which cell types express AIM in chronic liver disease was not determined in this study; Kupffer cells should be the main source of AIM in liver with hepatic fibrosis. Kupffer cells are resident macrophages of the liver. Activated Kupffer cells play a pivotal role in triggering and maintaining inflammation in chronic liver disease including CHC and nonalcoholic steatohepatitis (NASH) [
26,
27]. Persistent hepatic inflammation and hepatic stellate cell resistance to apoptosis are some of the mechanisms involved in progressive hepatic fibrogenesis [
28]. AIM induced by Kupffer cells may contribute to these mechanisms, and we speculate that elevated serum AIM levels may be due to increased production in vivo. However, our study was cross-sectional, and a longitudinal study involving a larger number of patients with an even distribution among the different stages of fibrosis is needed to elucidate the causal relationship between AIM and hepatic fibrosis. In addition, the association between serum AIM levels and hepatic reserve or renal function should be closely examined.
IR is independently associated with the severity of fibrosis in chronic liver disease, including CHC and NASH [
29,
30]. In addition, patients may have advanced hepatic fibrosis complicated by IR, abnormal glucose metabolism, or diabetes mellitus [
31]. IR is thought to directly activate profibrogenic signaling pathways [
32,
33]. However, the molecular mechanisms through which IR influences hepatic fibrosis are not fully elucidated. Recently, it was reported that the whole-body glucose intolerance and IR observed in obese AIM wild-type mice were less severe in obese AIM knockout mice, as shown by intraperitoneal glucose and insulin tolerance tests [
8,
9]. In our study, serum levels of AIM were tended to be correlated with HOMA-IR, although this correlation was not significant and AIM levels were not associated with the Matsuda index of whole-body insulin sensitivity [
19]. Simple indices based on fasting levels of glucose and insulin (e.g. HOMA-IR) assesses hepatic IR more than peripheral insulin sensitivity [
34]. Although it is unclear whether serum levels of AIM affect hepatic IR, high serum levels of AIM associated with hepatic fibrosis potentially connect hepatic fibrosis to IR. Further examination in patients with the same stage of fibrosis is needed.
In this study, serum levels of leptin were associated with HOMA-IR and hepatic steatosis, but not hepatic fibrosis and inflammation. Previous studies similarly showed that serum leptin levels are associated with IR [
18] and hepatic steatosis [
35]. In contrast, Cua et al. also reported that serum leptin levels are not associated with histological findings [
18], but Piche et al. showed an association between serum leptin levels and the severity of hepatic fibrosis [
36]. Resistin levels were reported to be inversely associated with hepatic fibrosis and resistin may stimulate fibrogenesis directly or indirectly through hepatic inflammation [
14,
16], but our study indicates that serum levels of resistin are not associated with hepatic fibrosis. In addition, Jonsson et al. reported that serum adiponectin levels were correlated with HOMA-IR and inversely correlated with steatosis in HCV-infected male subjects [
37]. However, Liu et al. reported that adiponectin levels do not correlate with histological features such as hepatic steatosis, although low adiponectin levels are associated with HOMA-IR in patients with CHC [
38]. Thus, prior studies on the role of the adipocytokines including adiponectin, leptin, and resistin, in the pathogenesis of histological changes in the liver and IR in patients with CHC have yielded conflicting results [
16]. It is noteworthy that an association between BMI and steatosis has been reported [
5,
6]. Further, more severe steatosis is associated with more rapid progression of fibrosis [
39]. In our study population, BMI is lower and hepatic steatosis is less severe compared to a previous study [
21]. In addition to BMI and histological findings, many other factors such as study population and sample size should be carefully considered when interpreting the data.
There are several limitations to this study. First, there were no F4 patients among the 77 HCV-infected patients analyzed in whom ultrasound-guided liver biopsy was performed. This selection bias was likely due to the fact that patients with suspected liver cirrhosis usually do not undergo liver biopsy for histological examination. In addition, the number of patients with OGTT data was small (only 39 patients). Further examination using a large number of patients with OGTT data available, including patients with compensated cirrhosis, is needed. Second, we employed a 5% cut-off for the amount of fat within the liver; some reproducible histological scales use the categories of mild (5–10%), moderate (11–30%), and severe steatosis (>30%) [
39]. Itoh et al. also divided patients into groups with 0–10% and >10% hepatic steatosis [
40]. However, the proportion of patients with ≥5% or ≥11% hepatic steatosis in our study population was small (18/77 [23.4%] or 3/77 [3.9%], respectively) compared to earlier studies. The reason for this difference in the distribution of hepatic steatosis severity is unclear, but there might be regional differences. In addition, the cut-off of 5% steatosis may not be clinically significant, and we should keep in mind that factors associated with steatosis might depend on the cut-off value used to define steatosis. Thus, the association between serum AIM levels and hepatic steatosis should be re-evaluated in studies with more subjects that include patients with severe hepatic steatosis. Third, several confounders such as alcohol consumption, previous interferon treatment, HCV genotype, and genetic factors including interleukin 28B polymorphism [
41], were not considered. The association between examined cytokines and hepatic fibrosis or steatosis in patients with CHC should be further examined in large-scale nationwide studies in the future.
Competing interests
HT holds endowed faculty positions in research for HGF tissue repair and regenerative medicine and has received funds from Eisai Co., Ltd.
Authors’ contributions
KM and HU: principal investigator, data collection, subject evaluation and manuscript preparation. SM, AI, YY and TN: subject evaluation and statistical analysis. KO and KT: subject recruitment and subject evaluation. KK, TT, AM, MO, YS, MH and SE: subject recruitment and data collection. HU and HT: guarantor of the article. All authors read and approved the final manuscript.