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Nonalcoholic fatty liver disease (NAFLD) is form of chronic liver disease with high prevalence in the general population. As NAFLD progresses, the liver become inflamed, fibrotic, and eventually cirrhotic. The NLRC4 inflammasome produces inflammatory cytokines in response to bacterial component stimuli within the cell cytosol. We hypothesized that mild constitutive activation of the NLRC4 inflammasome would prevent hepatic fibrogenesis and induce hepatic regeneration after liver injury.Substitution of the A/J region of the genome into C57BL/6 (B6) mouse that encompasses the gene NLRC4 has shown to be hepatoprotective when mice were administered chronic and acute doses of hepatotoxin carbon tetrachloride (CCl4). Congenic mouse 17C-6 have two single nucleotide polymorphisms in NLRC4. Congenic 17C-6 have a portion of their 17th chromosome substituting the B6 form for the A/J form. The first polymorphism (rs74459439-T), previously unidentified, is situated in the promoter of NLRC4. Transcription factor binding analysis indicates this site is a potential binding site for transcription factor Cdx-1. This location appears to govern transcription of NLRC4 as the A/J allele displays increased transcriptional activity.The second polymorphism is located within the coding region of NLRC4 producing a nonsynonomous mutation leading to an amino acid substitution in the leucine rich repeat (LRR) domain of NLRC4. The LRR is known to regulate activation of NLRC4 and 17C-6 mice demonstrate increased production and maturation of the NLRC4 inflammasome downstream product, IL-1ß. Congenic 17C-6 are resistant to CCl4-induced hepatic fibrogenesis and have remarkably accelerated regeneration of hepatic mass after twothirds partial hepatectomy.Resistance to liver disease progression coupled with the acceleration of hepatic regeneration is attributable to the NLRC4 inflammasome response. Increased secretion of proinflammatory cytokines stimulates the release interleukin-6 (IL-6), an established initiator of hepatocyte cell survival and proliferation. Congenic 17C-6 have elevated plasma IL-6 after two-thirds partial hepatectomy. Through this increased activation of the NLRC4 inflammasome, hepatocytes display an increased rate of mitotic division to recover lost tissue after damage. This hepatoprotective and regenerative response through stimulation of the NLRC4 inflammasome complex may be a potential therapeutic mechanism for individuals with fibrotic liver disease potentially mitigating the progression of liver disease.

Alcoholic liver disease (ALD) ranks among the major causes of morbidity and mortality in the world and effects millions of patients each year. Progression of ALD is well characterized and is actually a spectrum of liver diseases, which progresses from steatosis, to inflammation and necrosis, to finally fibrosis and cirrhosis. However, the underlying mechanism(s) of ALD are not as well understood, and as a result there is no FDA-approved therapy to prevent or reverse the disease. With a better understanding of the mechanism(s) and risk factors that mediate the initiation and progression of ALD, a targeted therapy can be developed to treat or prevent it. The regenerative capacity of the liver is inhibited in fibrosis and cirrhosis. By enhancing the regenerative ability of the liver we may be able to reverse the effects of ALD. Indeed, recent in vivo studies by this group indicate that activating Aldehyde Dehydrogenase 2 (ALDH2) with Alda-1 (a novel ALDH2 activator) or Ethanol (EtOH) protects against oxidative stress and damage to the liver, as result enhancing regeneration. Because ALDH2 can protect mitochondria from oxidative stress that results from partial hepatectomy, oxidative stress from other sources should be protected against as well. To test this hypothesis, primary hepatocytes were isolated from mice and plated on 96-well plates for analysis via Cellomics High Content Screening. Some mice were administered EtOH for 3 days prior to hepatocyte isolation or exposed to Alda-1 to activate ALDH2. Cells were exposed to 4-Hydroxynonenal (4-HNE; 0-1000 µM) for 0-24 H. Activation of ALDH2, by pretreatment with EtOH or Alda-1 preexposure, clearly protected isolated hepatocytes from 4-HNE toxicity. Mitochondrial membrane potential was significantly increased in cells with activated ALDH2 and membrane permeability was decreased in these cells as well. These changes in hepatocyte viability indicate that activation of ALDH2 protects cells from oxidative stress.

Abstract: Phosphatidylcholine (PC) is the major component of mammalian membranes and the induction of PC biosynthesis has been shown to be an essential step in cell proliferation in various cell lines. In the liver, PC biosynthesis is regulated by two pathways: the CDP-choline and the phosphatidylethanolamine N-methyltransferase (PEMT) pathways. The major enzymes that regulate these two pathways are CTP:phosphocholine cytidylyltransferase (CT)α and PEMT, respectively. In our first study, we elucidated the role of PC biosynthesis during proliferation by monitoring liver regeneration after 70% partial hepatectomy (PH) in mice lacking hepatic CTα (LCTα-/- mice). In the liver, CDP-choline is the major pathway of PC biosynthesis. To our surprise, liver re-growth, DNA synthesis, and PC mass after surgery were not impaired in LCTα-/- mice despite reduced total PC synthesis. Furthermore, PC synthesis in control mice was not induced after PH. Thus, we concluded that CTα is not essential for hepatocyte proliferation in vivo and that basal hepatic PC biosynthesis is sufficient to sustain regeneration after PH. In the second study, we assessed hepatic PC to phosphatidylethanolamine ratio (PC/PE) as a predictor of non-alcoholic fatty liver disease (NAFLD) and post-operative complications after major hepatectomy. LCTα-/- mice, PEMT-deficient mice (Pemt-/- mice), and their respective controls were fed a high fat diet to induce various degrees of NAFLD, before PH was performed. We found significant correlation of decreased PC/PE (pre-surgery) with the progression of NAFLD and decreased survival rate after PH. Additionally, dietary choline supplementation increased hepatic PC/PE in Pemt-/- mice with NAFLD, decreased inflammation, and increased survival rate after partial hepatectomy. Thus, choline supplementation may serve as a potential therapy to prevent the progression of NAFLD and to improve post-operative outcome after liver surgery. Finally, we characterized some fundamental aspects of lipid and lipoprotein metabolism in primary human hepatocytes. In particular, we evaluated the synthesis of phospholipids in relation to lipoprotein metabolism. Overall, this work has contributed to the understanding of important processes required for liver regeneration as well as discovering the similarities and differences in lipid and lipoprotein metabolism in primary human hepatocytes compared to hepatoma cell lines and rodent primary hepatocytes.

This project focuses on elucidation of the role of microRNA 122 (miR-122) in liver regeneration and its implications in therapeutic intervention of liver diseases. MicroRNAs are small non-coding RNAs that downregulate gene expression of their specific targets mostly at the translational level. miR-122 is the most abundant liver specific microRNA that is involved in several important liver functions including in cholesterol and lipid metabolism as well as in growth and replication due to its function as a tumor suppressor. Liver regeneration (LR) is a highly controlled cell proliferation response that allows the liver weight to be restored to the original size after removal of 2/3 of the liver, termed partial hepatectomy (PH). Mice with a conditional knockout for miR-122 in hepatocytes (LKO) were previously developed in our laboratory. These mice were subjected to 2/3 PH along with control mice with the same genetic background. The main parameters studied were the histopathology in the regenerating livers, liver weight restoration kinetics, and a proliferation analysis.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor. Of the two genes that encode MAT, MAT1A is mainly expressed in adult liver and MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic SAMe content and spontaneously develop hepatocellular carcinoma. The current study examined the influence of chronic hepatic SAMe deficiency on liver regeneration. Despite having higher baseline hepatic staining for proliferating cell nuclear antigen, MAT1A knockout mice had impaired liver regeneration after partial hepatectomy (PH) as determined by bromodeoxyuridine incorporation. This can be explained by an inability to up-regulate cyclin D1 after PH in the knockout mice. Upstream signaling pathways involved in cyclin D1 activation include nuclear factor kappaB (NFkappaB), the c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and signal transducer and activator of transcription-3 (STAT-3). At baseline, JNK and ERK are more activated in the knockouts whereas NFkappaB and STAT-3 are similar to wild-type mice. Following PH, early activation of these pathways occurred, but although they remained increased in wild-type mice, c-jun and ERK phosphorylation fell progressively in the knockouts. Hepatic SAMe levels fell progressively following PH in wild-type mice but remained unchanged in the knockouts. In culture, MAT1A knockout hepatocytes have higher baseline DNA synthesis but failed to respond to the mitogenic effect of hepatocyte growth factor. Taken together, our findings define a critical role for SAMe in ERK signaling and cyclin D1 regulation during regeneration and suggest chronic hepatic SAMe depletion results in loss of responsiveness to mitogenic signals.