Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplantation, with no approved therapeutics available. Despite years of research, the exact molecular mechanism of NASH progression is not well understood, but fat accumulation is believed to be the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long term gene silencing with single subcutaneous administration. Here we identified a hyper-functional, fully chemically stabilized GalNAc conjugated siRNA targeting DGAT2 (Dgat2-1473) that upon injection elicits up to three months of DGAT2 silencing (>80-90%, p<0.0001) in wild-type and NSG-PiZ “humanized” mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (> 50%, p:0.0008), resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat didn’t translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

Liver mitochondria play a crucial role in intermediary metabolism and main metabolic pathways. We evaluated the pharmacological effect on liver mitochondria in vitro using two novel anticancer drugs: 3-bromopyruvate and α-tocopheryl succinate. Metabolic influence on liver mitochondria was performed in vivo by high fat and high cholesterol diet. Toxicity of both drugs was evaluated in cell cultures of hepatocytes isolated from rat and mouse liver. The effect of anticancer drugs on liver mitochondrial functions in vitro was studied on suspensions of isolated liver mitochondria, tissue homogenate and permeabilized hepatocytes. Mitochondrial respiration was measured using high-resolution respirometry. 3-bromopyruvate caused morphological and functional damage of primary rat and mouse hepatocytes in cell cultures; this toxic effect was accompanied by an increase of reactive oxygen species production and mitochondrial dysfunction. 3-bromopyruvate decreased the oxygen consumption of mitochondria energized by substrates for complex I and complex II. α-Tocopheryl succinate caused a decrease of succinate-dependent respiration in all experimental models both in coupled and in uncoupled states. The most pronounced effect of α-tocopheryl succinate was apparent in isolated mitochondria and the least pronounced...

Dietary supplementation of nicotinamide adenine dinucleotide (NAD+) precursors has been suggested as a treatment for non-alcoholic fatty liver disease and obesity. In the liver, NAD+ is primarily generated by nicotinamide phosphoribosyltransferase (NAMPT), and hepatic levels of NAMPT and NAD+ have been reported to be dependent on age and body composition. The aim of the present study was to identify time course-dependent changes in hepatic NAD content and NAD+ salvage capacity in mice challenged with a high-fat diet (HFD). We fed 7-week-old C57BL/6JBomTac male mice either regular chow or a 60% HFD for 6, 12, 24, and 48 weeks, and we evaluated time course-dependent changes in whole body metabolism, liver steatosis, and abundance of hepatic NAD-associated metabolites and enzymes. Mice fed a 60% HFD rapidly accumulated fat and hepatic triglycerides with associated changes in respiratory exchange ratio (RER) and a disruption of the circadian feeding pattern. The HFD did not alter hepatic NAD+ levels, but caused a decrease in NADP+ and NADPH levels. Decreased NADP+ content was not accompanied by alterations in NAD kinase (NADK) abundance in HFD-fed mice, but NADK levels increased with age regardless of diet. NAMPT protein abundance did not change with age or diet. HFD consumption caused a severe decrease in protein lysine malonylation after six weeks, which persisted throughout the experiment. This decrease was not associated with changes in SIRT5 abundance. In conclusion, hepatic NAD+ salvage capacity is resistant to long-term HFD feeding, and hepatic lipid accumulation does not compromise the hepatic NAD+ pool in HFD-challenged C57BL/6JBomTac male mice.

C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects. Our initial work identified CTRP3’s promise as an effective treatment for Nonalcoholic fatty liver disease (NAFLD). Specifically, we demonstrated that mice fed a high fat diet failed to develop NAFLD when treated with CTRP3. The purpose of this current project is to identify putative receptors which mediate the hepatic actions of CTRP3. Methods We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line. Results Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells Conclusion The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. Relevance The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

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Thesis / Doctor of Philosophy (PhD)

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Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced.

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels.

Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced.