Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH

Background/Aims

Non-alcoholic fatty liver disease (NAFLD) pathogenesis remains unknown. Due to the emerging role of free cholesterol (FC) in NAFLD, our aim was to examine the correlation between FC accumulation in patients with NAFLD and the expression of enzymes that regulate cholesterol homeostasis.

Methods

Filipin staining, indicative of FC accumulation, and real-time PCR analyses were performed in 31 NAFLD patients and in seven controls.

Results

All NASH patients (n = 14) and 4 out of 17 patients with steatosis exhibited filipin staining compared to controls (0 out of 7 subjects with normal liver histology and BMI). Sterol regulatory element-binding protein-2 (SREBP-2) mRNA levels were 7- and 3-fold higher in NASH and steatosis patients, respectively, compared to controls. Since hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the key enzyme in cholesterol synthesis and transcriptionally controlled by SREBP-2 we measured its mRNA levels, being 3- to 4-fold higher in NAFLD compared to controls, without any difference between NASH and steatosis patients. Fatty acid synthase (FAS) and SREBP-1c expression were not significantly induced in NAFLD, while ATP-binding cassette sub-family G member 1 (ABCG1), a transporter involved in cholesterol egress, and acyl-CoA-cholesterol acyltransferase mRNA levels were modestly increased (1.5- to 2.5-fold, p < 0.05), regardless of fibrosis. Interestingly, mRNA levels of steroidogenic acute regulatory protein (StAR), a mitochondrial-cholesterol transporting polypeptide, increased 7- and 15-fold in steatosis and NASH patients, respectively, compared to controls.

Conclusions

FC increases in NASH and correlates with SREBP-2 induction. Moreover, StAR overexpression in NASH suggests that mitochondrial FC may be a player in disease progression and a novel target for intervention.

Introduction

Non-alcoholic fatty liver disease (NAFLD), a common cause of chronic liver disease in Western countries, is associated with obesity and insulin resistance. NAFLD constitutes a spectrum of liver injury, ranging from fatty liver (steatosis) to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma [1], [2], [3]. NAFLD pathogenesis, especially the mechanisms contributing to the transition from steatosis to NASH are still unknown, although the “two-hit hypothesis” has been proposed to explain NASH progression from steatosis [4], [5]. The accumulation of lipids in the cytoplasm of hepatocytes, mostly free fatty acids (FFA) and triglycerides (TG) is considered as a first step in the development of NASH. However, NASH does not occur in the absence of a second hit that promotes oxidative stress, inflammation, cell death and fibrosis. In this regard, overexpression of proinflammatory cytokines, such as TNF, is considered to play a key role in disease progression as demonstrated both in experimental models and in humans [6], [7], [8], [9], [10].

The accumulation of lipids in the liver is secondary to the metabolic disturbances associated with obesity, involving mechanisms more complex than just the supply of FFA from the adipose tissue to the liver. In this regard, activation of endoplasmic reticulum (ER)-based transcription factors, sterol regulatory element-binding proteins-1c (SREBP-1c) and 2 (SREBP-2), in insulin-resistant and hyperinsulinemic states play a prominent role in the synthesis of FFA and cholesterol, respectively [11], [12], [13]. SREBPs reside bound to the ER membranes and their activation involves proteolytic activation by SP-1 and SP-2, two proteases located in the Golgi which generate the mature forms of either SREBP-1c and SREBP-2; these mature forms then translocate to the nuclei to induce the transactivation of specific target genes [11], [12]. One of the target genes induced by SREBP-2 is the hydroxymethylglutaryl-CoA (HMG-CoA) reductase, the key enzyme in cholesterol synthesis [14]. The free cholesterol (FC) synthetized de novo in the ER is then esterified by the action of the acyl-CoA-cholesterol acyltransferase (ACAT) and stored in different cell membranes [14]. In addition, cholesterol homeostasis is also regulated by the action of several enzymes involved in its transport (ABCG5 and ABCG8) and catabolism (CYP7A1, CYP27A1) via generation of oxysterols [15]. Unlike esterified cholesterol, FC is known to regulate membrane dynamics, which ultimately can modulate membrane’s protein functions [16], [17]. Cellular FC represents a fraction of total cholesterol content (free plus esterified) and total cholesterol in plasma has been shown to increase in human NAFLD [18], although the specific levels of plasma FC are not routinely determined in humans. In experimental models, however, it was shown that FC increases in both plasma and liver [19].

Consistent with the “two-hit hypothesis”, the extent of lipid accumulation within hepatocytes is considered as the first hit for NASH progression, and the current thinking is that the amount of fat determines the susceptibility to secondary hits. However, using nutritional and genetic models of hepatic steatosis we have recently observed that FC loading (particularly in mitochondria) but not FFA or TG sensitizes to TNF and Fas-induced steatohepatitis [17]. Mitochondrial FC accumulation resulted in mitochondrial GSH (mGSH) depletion, a critical antioxidant that determines the hepatocellular susceptibility to TNF/Fas by controlling mitochondrial membrane remodelling via cardiolipin peroxidation [17], [20]. Moreover, feeding an atherogenic diet enriched in cholesterol to mice for 12–24 weeks, Matsuzawa et al. reported the onset of hepatocellular signs that recapitulate those observed in human NASH, including fibrosis, oxidative stress, hepatocellular cell death and ballooning [19]. Finally, using lipidomic analyses, Puri et al. have observed a step-wise increment in FC and an increase in the FC to total phosphatidylcholine ratio in patients with NASH [18].

Thus, due to this emerging role of cholesterol in NASH and since cholesterol homeostasis reflects the balance between its synthesis, metabolism/transformation and transport, the current study was aimed to analyze the correlation of FC levels with the expression of proteins that regulate cholesterol homeostasis and its mitochondrial trafficking in liver biopsies from obese subjects with NAFLD.