The rising rates of NASH make addressing the underlying causes of this serious condition more pressing. Hepatic steatosis is common in obese patients but only a subset of these patients develop NASH, emphasizing the contribution of genetic and potential environmental risk factors. Human NASH histopathology has been associated with steatosis, lobular and portal inflammation, hepatocyte ballooning, and fibrosis. Specifically, zone 3 predominant macrovesicular steatosis, ballooning, and perisinusoidal fibrosis is deemed consistent with adult or Type 1 NASH. Type 2 or pediatric NASH histopathology has been reported to have panacinar or periportal (zone 1) steatosis, rare ballooning and portal tract expansion by chronic inflammation or fibrosis. (37
). Individuals who have NASH with fibrosis have progressive disease and greater morbidity and mortality including the potential for cirrhosis, liver failure, and liver transplantation (3
). However, the specific biological determinants that lead to development of NASH with fibrosis are not well-defined.
Fructose consumption accounts for approximately 10.2% of all calories in our average diet in the United States (38
). In comparison to other simple sugars such as glucose, use of fructose for hepatic metabolism is not restricted by the rate-limiting step of phosphofructokinase, thus avoiding the regulating action of insulin (39
). Fructose intake is 2–3 fold higher in patients with NASH compared to BMI matched controls and recently daily fructose ingestion has been associated with increased hepatic fibrosis (40
). These epidemiologic data prompted us to investigate the potential mechanistic role that fructose and other simple sugars may play in the development of NASH.
The present manuscript was focused on the development of a dietary model of NASH. To this end we compared HF-fed mice to mice maintained on the same diet but also given ad libitum access to fructose in their drinking water (HFHC). While weight gain, body fat, insulin resistance and liver steatosis were similar between the two groups (and elevated relative to mice maintained on chow), mice fed the HFHC diet had increased hepatic oxidative stress, CD11b+F4/80+ Gr1+ macrophages in the liver, TGFβ1 driven fibrogenesis and collagen deposition compared to the weight matched controls in the HF fed group. Thus, while HF diets produce a range of the components of the metabolic syndrome, fructose consumption would appear necessary to move the process, from liver fat deposition alone to fibrogenesis.
ROS has been thought to be an important trigger for hepatic stellate cell activation and for promoting expression of fibrogenic molecules such as alpha-SMA, TGFβ1, and collagen 1 (15
). Recently, fructose-fed rats have been reported to develop hepatocyte damage with a decrease in the mitochondrial membrane potential similar to that induced by low non-cytotoxic doses of exogenous ROS (44
). In-vitro studies have also reported that the cytotoxic mechanism involving fructose driven ROS formation preceded the hepatocytotoxicity, and that this cell injury could be prevented by ROS scavengers (44
). We therefore investigated this as a potential process in our model and demonstrated that HFHC fed mice had significantly higher ROS levels compared to both HF diet and chow fed mice ().
Previous studies done with fructose diets have reported insulin resistance and severe necro-inflammatory NAFLD but not NASH with fibrosis (18
). In contrast to the ALIOS diet from Tetri et al which provided fructose water in gelatin form and long chain saturated trans-fats in their solid diet our high-fat-diet provided 58% calories from medium chain saturated trans-fats and fructose and sucrose in their regular drinking water. This diet resulted in 50% of the mice in our HFHC fed group having fibrosis with a minority having stage 2 fibrosis (). Karlmark et al highlighted the role of CD11+F4/80+Gr1+ monocytes in perpetuating hepatic stellate cell-driven TGFβ1 dependent fibrosis (7
). More recently Niedermeier et al reported that Gr1+ monocytes may be essential in production of murine fibrocytes (36
). In our experiment intrahepatic CD11+F4/80+Gr1+ monocyte-derived macrophages were 10 fold higher than either chow or HF diet fed mice, with 50% of the macrophages in HFHC livers being Gr1+ (). We propose that the conversion of CD11b+F4/80+Gr1+ monocytes into fibrocytes maybe responsible for the increased collagen 1 deposition through ROS driven TGFβ signaling and stellate cell activation.
In humans, studies have shown extensive mitochondrial damage including paracrystalline inclusion bodies, megamitochondria, damaged respiratory chain and low ATP production with NASH (24
). We have previously reported that increased ROS released from damaged mitochondrial respiratory chain is important in NAFLD development (45
). CoQ is an important element in the mitochondrial respiratory chain, contributing to the transport of electrons across complex III involving the Qo and Qi sites within the inner mitochondrial membrane to enable generation of ATP. Lower red
CoQ plasma levels are present in patients with cirrhosis and red
CoQ acts as a lipid soluble anti-oxidant in hepatocytes in culture (46
). Supplementation with CoQ has also been reported to inhibit liver fibrosis through suppression of TGFβ1 expression in mice (48
). We demonstrate that plasma levels of ox
CoQ9 correlate well with collagen 1 mRNA in liver tissue. We also present data that plasma levels of ox
CoQ9 can discriminate between NASH with fibrosis and NASH without fibrosis, with our HFHC (NASH with fibrosis) mice having higher levels compared to the HF (NASH without fibrosis) or chow (normal histology) mice ().
In summary, we believe therefore, that our ad libitum dietary model results in NASH with fibrosis in non-genetically modified obese mice. Our data suggest that the mechanism of fibrosis in this model may involve fructose producing an increased ROS signature in the liver associated with CD11b+F4/80+Gr1+ macrophage aggregation resulting in TGFβ1 signaled collagen deposition and histologically visible hepatic fibrosis.