Date of Award
Doctor of Philosophy in Pharmaceutical Sciences
Nonalcoholic fatty liver disease (NAFLD) is an “umbrella” term for the broad spectrum of the disease that begins from the simple steatosis to more progressive stages of nonalcoholic steatohepatitis (NASH), that includes hepatocellular ballooning, inflammation, and fibrosis. NAFLD is a growing epidemic globally, with 25% of the population predicted to be diagnosed with this disease. Liver biopsy is the only definitive method of diagnosis, despite the widespread use of sonography and elastography to predict the disease state. There is no current FDA approved medication for NAFLD/NASH. This is partly due to the lack of translatable disease model to predict the whole spectrum of the disease in humans as well as lack of definitive biomarker to predict the disease state. The goal of this dissertation is (1) To build an in vitro model for NAFLD to study the influence of fat over-load on xenobiotic as well as lipid metabolizing proteins (2) To use the model to help identify novel biomarkers in liver tissue to characterize the early stages of disease.
Manuscript I: In recent times, there are numerous invitro models relevant to humans developed to predict the disease state. They involve monoculture, coculture and as well as multicellular culture in both 2D and 3D models to best represent the physiology and working of liver in the disease state. In this review, we have explored all the existing in vitro models of NAFLD relevant to humans as well as highlight the technological gaps in the current in vitro models for future development.
Manuscript II: Human hepatic carcinoma cell lines are commonly used in in vitro studies of lipid and xenobiotic metabolism, as well as glucose regulation in normal and disease state. However, their validity is still under debate due to the variable expression of proteins in the cell lines and human hepatocytes. In this present study, we used a data independent acquisition based total protein approach (DIA-TPA) to quantify the protein abundance in the different cell lines versus (vs.) cryopreserved human hepatocytes (cHH) and human liver tissue (HLT). For this purpose, the global proteome from the whole cell homogenates of HepaRG, HepG2, Huh7 cell lines were compared to cHH and HLT. The MS2 spectra for all detectable peptides were quantified using Spectronaut™. In summary, 2715, 2578, 2874, 2717 and 3083 proteins in, HepaRG, HepG2, Huh7, cHH and HLT, were identified at 1% FDR, respectively. The global proteome of the cHH significantly differed from the cancer hepatic cell lines. Within the cell lines, the global as well as ADME protein profile of HepaRG most closely correlated with cHH, with 84 out of 101 ADME proteins, identified in HepaRG cells. Within gluconeogenesis and glycolysis pathway, Huh7 cell line expressed proteins in high abundance in contrast to the other groups. Therefore, we show that the comparison demonstrates the capability of untargeted global proteomics to detect the differences in protein expression among the different groups. In addition, this study provides a comprehensive database of information to aid study design and model selection.
Manuscript III: To manage NAFLD and related co-morbidities, patients are administered with an array of pharmacological agents. Therefore, understanding of the effect of NAFLD on drug disposition is warranted. Using a HepaRG model, we aimed to mimic steatosis in vitro, and to examine its effects on drug-metabolizing enzymes (DME) and transporters. HepaRG cells, differentiated in-house, were exposed to a mixture of saturated and unsaturated fatty acids (1:2 ratio of 0.5 mM palmitate and oleate complex conjugated to BSA for 72h) and were subjected to RNA sequencing and proteomic analyses. Lipid accumulation was ascertained by Oil Red O (ORO) staining and triglyceride (TG) quantitation and cell viability by WST-1 determination. The treatment condition resulted in ~6-fold increase in TG concentration without reducing cell viability. RNA sequencing of lipid-loaded and control cells identified a total of 393 differentially expressed transcripts (89 up- and 304 down-regulated). Moreover, lipid loading resulted in significant downregulation of mRNA transcripts of transcription factors, NR1I2 (-1.18) and HNF4α (-0.55), phase 1 DMEs including CYP1A2 (-3.25), 2B6 (-2.02), 2C8 (-1.48), 2C9 (-2.00),2C19 (-1.32) and 3A4 (-1.87), phase 2 DMEs including UGT1A6 (-0.36) and 2B7 (-1.09), SULT2A1 (-0.75) and 1E1 (-1.41) as well as clinically relevant transporters such as ABCC11 (-1.36), ABCG5 (-1.66), SLC10A1(-1.63) and SLCO2B1(-1.49). However, the protein expression did not show a significant change. Furthermore, lipid loaded cells
significantly upregulated AKR1B10 mRNA (2.17) and protein (0.99) that may regulate lipid
as well as xenobiotic metabolism.
Manuscript IV: Non-alcoholic fatty liver disease (NAFLD) is a global epidemic, present in over 10% of the world population, despite the majority of the population being undiagnosed. Liver biopsies are the only gold standard available for the confirmation of disease state. Other non-invasive diagnostics such as ultrasound and MRI are either inaccurate or expensive for routine use. Many markers for disease state are available that detect the onset of inflammation and fibrosis from moderate-to-high accuracy. However, there is a huge gap in specific biomarkers that can distinguish the NAFLD liver tissue from normal in early stages of the disease. Using SWATH-MS based Data-independent acquisition (DIA) strategy the dysregulated proteins in the in vitro model of hepatic steatosis was compared with human liver tissue (n=116) showing progressive stages of NAFLD. More than 2,500 proteins were identified in HepaRG and human hepatocyte model as well as human liver tissue. Within the hepatocyte model, 40 proteins were dysregulated in steatosis. These proteins were screened in liver tissue and 6 common proteins were identified. The sensitivity and selectivity of the markers were analyzed using receiver-operative curve (ROC) for the following markers PLIN2 (0.77), ANXA1(0.70), H2AFY (0.80), SNX1 (0.67), GCHFR (0.69), APO (0.69) and all the above markers showed significance P <0.05.
Conclusion. This work demonstrates that in vitro human relevant disease model has the potential to explain the effect of NAFLD. Subsequently, the in vitro models when used in conjunction with human liver tissue aid in identification of novel biomarkers that may have therapeutic and diagnostic value.
Saravanakumar, Anitha, "AN OMICS BASED APPROACH FOR THE IDENTIFICATION OF BIOMARKERS IN NON-ALCOHOLIC FATTY LIVER DISEASE USING IN VITRO MODELS OF HEPATIC STEATOSIS" (2019). Open Access Dissertations. Paper 1103.