- Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of excess neutral lipids within the parenchymal cells of the liver. The primary etiology is diet-induced and most often exists as a comorbidity with obesity, metabolic syndrome, and/or Type 2 Diabetes. The development of excess liver lipid can be self-limiting but may also progress to nonalcoholic steatohepatitis (NASH). NASH features include hepatic inflammation, oxidative stress, hepatocyte ballooning and cell death. NASH promotes deposition of aberrant extra cellular matrix resulting in fibrosis and is a risk factor for cirrhosis and hepatocellular carcinoma. Currently there is no FDA approved drug treatment for NASH. General standard of care is to treat the underlying comorbidities in these patients and encourage lifestyle alterations (diet, exercise and weight loss). However, there remains a need to empirically define what treatment strategies are effective at either reversing NASH or simply halting any further disease progression. The most intuitive avenue for reversing NASH is simply weight loss through dietary intervention (i.e. removing the western diet and providing a "healthy diet"). Another promising avenue of therapy is the use of supplemental Docosahexaenoic acid (DHA), a long chain omega-3 polyunsaturated fatty acid (ω3 PUFA). This molecule has broad effects on hepatic function including repressing inflammation, oxidative stress, and reducing fatty acid synthesis and inducing fatty acid oxidation. Previous work from our lab demonstrated that DHA alone at a physiologically relevant dose was able to reduce the severity of NASH when provided as a preventative measure. Collectively, this makes DHA an attractive candidate for addressing the multiple levels of hepatic and metabolic perturbations seen in NASH.
In order to better understand the impact of supplemental DHA or dietary interventions on NASH pathogenesis, we utilize a model of Ldlr-/- mice fed western diet (WD) (calories are 30% sucrose, 41% fat, and .21% cholesterol by weight). WD-fed Ldlr-/- mice develop a whole body metabolic phenotype consistent with metabolic syndrome and a fatty, inflamed and fibrotic liver consistent with NASH in humans. We started by extending analysis of a previous study in which DHA was provided concurrently with WD feeding in Ldlr-/- mice to test prevention of WD-induced NASH. We also assessed the ability of DHA to target fibrotic pathways in LX2 cells, an immortalized cell line of the primary collagen producing hepatic cell type. The key findings from this paper were that while DHA had little to no effect on circulating endocrine and inflammatory factors, it repressed most hepatic fibrosis associated genes induced by WD- feeding. DHA inclusion also repressed total hepatic hydroxyproline content, a marker of fibrosis formation, as well as expression of signaling molecules known to drive fibrosis progression (i.e., Tgf-β subtypes and associated receptors). Increases in Tgf-β following WD feeding increased phosphorylation and subsequent nuclear localization of Smad3, resulting in increases in Col1A1 expression, the main collagen subtype in NASH-induced fibrosis formation. DHA repressed the increases in phospho-Smad3. Mechanistic studies further demonstrated that DHA was able to directly suppress Tgf-β driven expression of Col1A1 in LX2 cells.
Moving from prevention studies, I then assessed several treatment options. First, I determined the capacity of two separate low-fat low-cholesterol diets to reverse pre-established NASH in long term WD- fed Ldlr-/- mice. Results from these studies demonstrated that while using low-fat low-cholesterol diets to induce weight loss may reverse many of the features associated with WD- induced NASH, this intervention was unable to fully reverse hepatic fibrosis or alterations in hepatic fatty acid content. These studies also established a temporal link between resolution of plasma parameters and hepatic parameters, demonstrating that plasma resolution occurs quicker than resolution of hepatic parameters. Furthermore, we established that in NASH mice, hepatic PUFA content is negatively correlated to a plasma inflammatory marker (TLR2 activation) and hepatic fibrosis formation (Loxl2 transcript abundance).
Finally I assessed the capacity of DHA as a treatment of WD-induced NASH in continued presence of WD feeding or coupled with a low-fat low cholesterol diet to promote improved remission over diet change alone. Addition of DHA to WD in mice with pre-established NASH was able to blockade further progression of hepatic and metabolic markers of NASH as well as reduce existing hepatosteatosis. As expected, providing the low-fat low-cholesterol diet alone normalized most of the hepatic and metabolic perturbations induced by WD-feeding but failed to reverse fibrosis and alterations in hepatic fatty acids. Combining DHA with a low-fat low-cholesterol diet and weight loss increased hepatic content of long-chain ω-3 PUFA, repressed hepatic content of ω6 PUFA and altered expression of genes related to inflammation, collagen cross-linking, and liver repair. Furthermore, these studies provided preliminary evidence for a dual role of Notch and Hedgehog signaling pathways in both the NASH disease process and the remission phase. Overall, inclusion of DHA in low-fat low- cholesterol diets for remission provided little discernable benefit over diet change alone.
In conclusion, I established that low-fat low-cholesterol diets leading to weight loss are an efficacious treatment for reversing many of the metabolic and hepatic abnormalities that occur in WD-induced NASH. However, this approach was unable to reverse existing fibrosis and normalize other hepatic perturbations. The addition of DHA showed little to no definitive benefit over simply returning mice to a low-fat low-cholesterol diet. I also demonstrated that DHA is a viable therapeutic approach for blocking further NASH progression in Ldlr-/- mice with pre-established NASH. DHA has wide repressive effects both in vivo and in vitro on pathways involved in the pathophysiology of NASH development and progression. Taken together this data suggests that dietary alterations and weight loss provide the most efficacious outcome in the context of NASH prevention and therapy, DHA has beneficial effects even in the absence of dietary changes but should be a second line approach. However, following onset of NASH, neither DHA supplementation nor dietary improvements can fully reverse pathologies apparent in the liver.