Graduate Thesis Or Dissertation
 

Western Diet-Induced Nonalcoholic Steatohepatitis (NASH): The Impact of Time on the Onset and Remission of Disease

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  • Nonalcoholic fatty liver disease (NAFLD) is characterized by an accumulation of neutral lipids in the form of triglycerides and cholesterol esters accounting for >5% of liver weight. Excess neutral lipids accumulate in large lipid droplets, i.e., macrosteatosis, displacing the nucleus of cells. Current theory suggests neutral lipid accumulation is the first hit of a multi-hit process promoting hepatic inflammation and oxidative stress causing liver injury. Nonalcoholic steatohepatitis (NASH) is the result of prolonged hepatic injury evidenced by the presence of fibrosis (scarring) that becomes irreversible. NAFLD parallels the obesity epidemic affecting up to 45% of the United States population and approximately 25% globally. Nonetheless, lean adults and children may also develop NAFLD. The dominant risk factors for NAFLD are obesity, dyslipidemia, metabolic syndrome, and type 2 diabetes mellitus. Nonenvironmental factors including genetics, sex, age, and ethnicity add to one’s risk of NAFLD and NASH. Up to 30% of those with NAFLD progress to NASH with 20-30% of NASH patients progressing to cirrhosis. Ludwig et al. identified NASH in 1980, yet there remains no Food and Drug Administration (FDA)-approved therapy effective at attenuating NASH pathology. The current approach includes lifestyle modifications targeting a reduction in dietary saturated-and trans- fats, cholesterol, and simple sugars, and increased ω3 polyunsaturated fatty acids (PUFA), low-glycemic foods, and monounsaturated fatty acids (MUFA) resulting in 3-10% reduction in body weight. Lifestyle is difficult to modify, thus the pharmacological approach aims to address specific NAFLD targets like pioglitazone, a PPARγ agonist, that targets insulin sensitivity involved in NASH pathology. However, off-target mechanisms are common in targeted therapies and can result in adverse effects thus inhibiting marketable FDA-approved products. Humans and mice with NAFLD and NASH have abnormal fat metabolism leading to increased neutral lipids combined with decreased concentration of blood and liver PUFA that can be attributed to a western diet (WD). The WD is low but sufficient in ω3 and ω6 essential fatty acids (EFA) that give rise to C20-22 PUFA. Less than 4% of α-linolenic acid (ALA) converts to C20-22 ω3 PUFA. Conversely, excess dietary docosahexaenoic acid (DHA) through peroxisomal retroconversion, increases blood and tissue levels of C20-22 ω3 PUFA. Importantly, C20-22 ω3 PUFA are key regulators of the mechanisms involved in lipid metabolism, fatty acid oxidation (FAO), de novo lipogenesis (DNL), fatty acid uptake and assimilation into lipids, and very low-density lipoprotein (VLDL) assembly and secretion and are precursors to regulatory oxylipins. Furthermore, there is a growing body of evidence that DHA attenuates NASH progression by reducing inflammation and the expression of genes involved in fibrosis. Therefore, a logical approach for treating NASH is to increase cellular C20-22 ω3 PUFA with dietary DHA supplementation. To examine the scope of recent evidence using ω3 PUFA in humans, I first conducted a review of clinical trials and meta-analyses published between 2016 and 2018 using various types, doses, and durations of ω3 PUFA therapy in adults and children. The broad range of ω3 PUFA therapies used made it difficult to ascertain the benefit of DHA on its own over the use of other ω3-PUFA products such as fish oils and nutraceutical products in improving NAFLD and NASH. Regardless, steatosis, plasma lipids, triglycerides, and plasma markers of hepatic injury (ALT) were reported to be significantly improved in meta-analyses. Blood and tissue levels of C20-22 ω3 PUFA, an indicator of therapy compliance, trended (meta-analyses) or significantly increased (clinical trials). Outcome limitations on the capacity of C20-22 ω3 PUFA centered on the mixed results in improving plasma markers of inflammation and fibrosis. Further, because biopsy is the gold standard for NASH diagnosis and fibrosis assessment, its inherent risks prohibit its widespread use in human studies. Overall, clinical evidence suggests C20-22 ω3 PUFA are well tolerated and effective in reducing hepatic fat, plasma triglycerides, and attenuating NAFLD progression in children and adults. Our preclinical studies in WD-induced Ldlr -/- male mice closely recapitulate human NASH. We use a WD containing 40% fat, 43% carbohydrate 17% protein, and 1.5 mg/g of diet to induce NASH. Mice develop a metabolic syndrome phenotype, including obesity and a fatty, inflamed, and fibrotic liver. We recently demonstrated in a proof-of-concept study that DHA supplementation at 2% of total calories attenuates WD-induced NASH progression by decreasing hepatic fat, inflammation, and gene expression involved with fibrosis in male mice. These studies were limited as they revealed changes at a single timepoint and examined only male mice. Thus, my studies built upon our previous reports by employing the use of two separate time-course studies to: 1) examine the events occurring in the onset and progression of WD-induced NASH in female and male mice; and 2) to use a time-course approach to identify the sequences of event associated with the capacity of DHA to attenuate pre-existing NASH in female and male mice. The onset and progression of WD-induced NASH was assessed using anthropometric and plasma markers of disease, hepatic lipidomic and oxylipidomic, and RNAseq transcriptomic approaches. Chronic WD consumption promoted rapid decreases in ω3 and ω6 EFA, C20-22 PUFA, and several pro- and anti-inflammatory oxylipins derived from these PUFA and increased the expression of transcripts involved in inflammation and oxidative stress. These events preceded histological and transcriptional evidence of steatosis and fibrosis. Further, many of the aforementioned areas revealed sex-based differences suggesting a personalized and sex-based approach may be necessary in NASH assessment and diagnosis. Similarly, I used a time-course approach to assess how quickly WD-induced NASH mice respond to DHA therapy and the longevity of DHA efficacy. Lipidomic and oxylipidomic assessment revealed DHA supplemented female and male mice with WD-induced NASH rapidly increased hepatic DHA, DPA, EPA, and anti-inflammatory oxylipin levels while repressing pro-inflammatory oxylipin production. Only female mice had rapid changes in the expression of transcripts involved in inflammation and fibrosis. Male mice were less responsive to DHA supplementation overall and its effect tended to wane in histological evidence compared to females. Nevertheless, fibrosis was not fully resolved in either female or male mice with 12 weeks of DHA supplementation. These outcomes suggests that the timing of DHA therapy relative to NASH progression may determine its efficacy, and male mice may need a higher dose based on differential sex-dependent body weight. In conclusion, clinical and preclinical studies reveal the role diet plays in the manifestation of chronic disease like NAFLD and NASH. Lifestyle modification, including dietary changes, are challenging to adopt and achieve permanent improvement of NAFLD and NASH. Likewise, targeted pharmacologic approaches are known for potential harmful off-target effects. Nutraceutical DHA therapy on its own or in combination with other ω3 PUFA, is well tolerated and effective at attenuating many pathological aspects of NASH but does not fully resolve NASH. I identified the rapid dietary effects that a WD and WD with DHA supplementation have on NASH onset and treatment, respectively. Hepatic fatty acid content likely plays a major role in the development of NASH, and likewise its prevention. I further observed the early involvement of inflammation that preceded the accumulation of hepatosteatosis. This suggests molecular and cellular level events prime the liver for the accumulation of neutral lipids and hepatic injury that are progressive. The paradigm shift revealed in this model of WD-induced NASH in female and male mice highlights the importance of a healthy diet that limits saturated- and trans-fat and simple sugars. Since NAFLD and NASH take decades in most cases to develop, it may also be prudent to incorporate ω3 PUFA-rich foods like salmon or marine-derived DHA supplements to protect against the early inflammation and oxidative stress that may not be detected prior to a diagnosis of NAFLD or NASH. Finally, diagnostic, and therapeutic approaches that account for sex-based differences in NAFLD and NASH may improve outcomes that could reduce the incidence of advancing disease leading to transplantation.
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  • This work was supported by the National Institutes of Health grant DK112360.
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  • Pending Publication
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  • 2022-05-24 to 2022-12-25

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