Previous work has supported roles for oxidative stress and ER stress in the etiology of ALD [4
]. These pathological processes are intrinsically linked, with each capable of activating the other [16
]. In order to elucidate the mechanisms involved in the initiation of ALD hepatopathy, our aim was to focus on the earliest initiating stages of the disease where therapeutic intervention is typically most effective. This 6-week Lieber-DeCarli model for early-state ALD represents the earliest initiating stages of disease progression as shown through the documented sustained ingestion of substantial quantities of alcohol resulting in significant elevations in plasma ALT, liver triglycerides and increased liver
body weight ratio. Histological analyses revealed a marked increase in pan-lobular hepatic lipid accumulation consistent with the early signs of steatosis. Collectively, these data demonstrate a reliable model for early-stage ALD, which allowed for further investigation into the parameters associated with the initiating stages of disease progression.
Oxidative stress is a well-recognized outcome of chronic ethanol ingestion and is likely to play a major role in ethanol-mediated liver damage [4
]. Previous works utilizing N-acetylcysteine have demonstrated a protective role for antioxidant therapies in ethanol-mediated liver injury [47
]. These studies revealed a significant decrease in ALT measurements, steatosis, and TBARS content. While these data do not definitively prove that oxidative stress is responsible for the early-initiating stages of disease development, a strong correlation can be established. Data presented here reveals a significant decrease in GSH following 6 weeks of ethanol consumption, indicating a decrease in the overall antioxidant capacity of the liver at this time point. Further, a significant decrease in the activity of CBS was observed at the week 6 time point, demonstrating a potential decrease in the GSH precursor cysteine. Previous work conducted on CBS has shown a decrease in enzymatic activity following other hepatic insults such as a methionine-choline-deficient diet and other cellular stresses [49
]. We postulate that the effects on CBS activity in the control animals may be due to the high fat content in the control diet. More importantly, the effects of this diet on CBS activity appear to be exacerbated following ethanol consumption. The availability of cysteine is often the rate-limiting determinant in GSH biosynthesis, and these effects on CBS activity offer a potential novel mechanism for the observed decrease in GSH following sustained ethanol ingestion.
The decrease in GSH was consistent with an increase in lipid peroxidation, as demonstrated by increased staining of 4-HNE modified proteins in the livers of ethanol-fed mice. Previous reports by Backos et al. have highlighted the effects of 4-HNE on the activity of GCL utilizing cultured cells; GCL activity was found to be significantly increased following treatment with 4-HNE, despite a significant depletion of cellular GSH [30
]. Our findings revealed a similar result, where GCL activity was found to be significantly increased despite no change in the overall expression of the enzyme. Although the effects of 4-HNE on GCL activity are noted, the precise role of aldehyde adduction in the alcoholic liver remains to be fully characterized.
Following 6 weeks of ethanol ingestion, hepatic pan-lobular steatosis is observed, with larger lipid droplets present throughout zones 1 and 2. These macrosteatotic vesicles are likely contributing to the increased lipid peroxidation observed, as demonstrated by staining with 4-HNE throughout these zones. It should be noted as well that CYP2E1 is found almost exclusively throughout zone 3 and is completely absent in zones 1 and 2. This may, in part, provide a likely explanation for the observed increase in zone 3 staining of protein-SSG, whereby these modifications act to protect critical thiol residues. Oxidative stress occurring in zone 3 has been documented by numerous other groups; however, the staining of ubiquitinated proteins in this region remains a novel finding. It is conceivable that the increased oxidative stress in zone 3 is resulting in other damaging oxidative modifications stemming from CYP2E1-mediated free radical generation. At the current time, the precise mechanisms and rationale behind the increased ubiquitin staining in zone 3 are unknown. Reports from our laboratory utilizing the Lieber-DeCarli model in rats, however, have identified the ER-resident protein disulfide isomerase (PDI) to be a target for modification by 4-HNE in vivo
; this leads to a decrease in enzymatic activity and impaired protein folding [51
]. Collectively, these data suggest a potential mechanism for oxidative-stress-induced UPR signaling in rodent models for ALD [51
In the past decade, ER stress has been associated with an increasing number of hepatic disease states, most notably ALD [10
]. Previous research in mice by Ji et al. has suggested a role for ER stress in an intragastric model of ALD [13
]. However, the intragastric model represents a model for more severe pathologies of ALD, as shown by a roughly 8-fold increase in serum ALT (versus an approximate doubling here) and a significant increase in inflammation (inflammation was not seen in our model). The transition from steatosis to steatohepatitis is considered to be the critical pathogenic determinant in ALD furthering a need for therapeutic intervention during the initiating stages of disease progression [20
]. Recent reports have suggested an association between the inflammatory response and UPR signaling, outlining a mechanism similar to that with oxidative stress [54
]. Consistent with early-stage ALD, our data demonstrate clearly that the inflammatory response does not precede disease pathogenesis and UPR induction in this model.
To further understand the mechanisms behind ALD progression, our research focused on the relative pathogenic contributions of ER stress and oxidative stress during the initiating stages of the disease. Previous work in rodent models of ALD has hypothesized a role for the ER stress response in the activation of lipogenic pathways. This was shown to occur through SREBP1 activation and this mechanism was thought to be a major contributor to the observed hepatic steatosis [13
]. To investigate these reports, our studies utilized the Lieber-DeCarli model for ALD. Immunoblotting for both hallmark and lipogenic UPR signaling cascades revealed no significant activation during our time course model for ALD despite the generation of significant pathologies, such as increased lipid accumulation and serum ALT levels. Taken together, these data validate a minimal role for the UPR during the development of ethanol-induced steatosis in a murine model for early-stage ALD.
Oxidative stress has gained considerable attention as a possible mechanism for the induction of various cellular responses, including the ER stress response [8
]. Additionally, UPR signaling has been shown to lead to induction of the Ero1 proteins and Nox4, furthering the oxidative stress burden in the cell. This oxidative protein folding relay has been estimated by Tu and Weissman to account for up to 25% of total cellular ROS [59
]. This creates a vicious cycle of cellular derangements, where each response propagates the other. Regarding the alcoholic liver, UPR signaling and oxidative stress have been intimately associated with the progression of ALD. To date, these responses have not been investigated in relation to the initiation of ALD and therefore the relative role each has during early-stage ALD is not understood.
Our data demonstrate a clear delineation between the ER stress response and oxidative stress in early-stage ALD, indicating that oxidative stress is a primary initiating factor responsible for the progression of ALD. The observed increase in protein glutathionylation demonstrates a potential involvement for this posttranslational modification following increased oxidative stress and provides interesting avenues for research in this field. The effects of oxidative stress in our model were determined to be independent of UPR induction, indicating that the ER stress response may play a pivotal role during more advanced stages of disease progression.