Integrating Single Cell RNAseq and Computational Modeling to Infer Hepatocyte Metabolic Functionalities during Liver Regeneration.

Hepatocytes make up over 80% of liver mass and carry out all essential liver functions such as protein synthesis and detoxification, among others. Recent studies have shown distinct metabolic functions of proliferating and non-proliferating hepatocytes as a compensatory mechanism during liver regeneration. However, this mechanism has not been studied in the case of alcoholic liver disease (ALD), which is known to disrupt the regenerative capacity and alter the metabolic processes of the liver. We seek to determine if alcohol consumption alters the balance in metabolic functionality between proliferating and non-proliferating hepatocyte populations during liver regeneration. In the present study, rats were pair-fed either a chronic ethanol diet or a carbohydrate control diet per Lieber DeCarli calorie-matched liquid diet protocol. We performed 70% partial hepatectomy (PHx) and liver samples were collected at the baseline and at 24 hours post PHx. Single hepatocytes were isolated by laser capture microdissection (LCM) where the subset of proliferating hepatocytes were identified by Ki67 staining. Single-cell RNA-sequencing (scRNA-seq) was employed for the purpose of identifying transcriptional differences across hepatocyte subtypes (i.e. PHx, diet, proliferation). Principal component analysis (PCA) of all samples showed distinct separation of cells into clusters based on liver regeneration (baseline and PHx samples), but not on diet (control and ethanol) and cellular proliferation (Ki67-positive vs. negative staining). PCA of baseline samples showed separation of proliferating and non-proliferating hepatocytes in the ethanol group. We characterized metabolic differences between hepatocyte subtypes using a computational model of genome-scale metabolic reaction activities. We found that the metabolic compartmentalization separating proliferating from non-proliferating hepatocytes in the carbohydrate group is absent in the ethanol group. Several functional differentiating factors arose favoring reactive oxygen species detoxification and steroid metabolism in proliferating hepatocytes from ethanol-adapted animals at baseline, which were absent post PHx. Furthermore, proliferating cells from the carbohydrate group showed higher reaction activity in CoA synthesis, NAD metabolism and inositol phosphate metabolic pathways as compared to their non-proliferating counterparts, both at baseline and following PHx. Our results also indicate that these pathways are also active in the non-proliferating hepatocytes following PHx in the ethanol group. Taken together, our combinatorial transcriptomic analysis and computational modeling showed distinct metabolic pathways differentiating proliferating from non-proliferating hepatocytes at baseline, which is absent in hepatocytes following PHx in the ethanol group. These results point towards an alternative mechanism of metabolic compensation in ethanol-fed animals such that similar metabolic pathways are active in both proliferating and non-proliferating hepatocytes. This lack of metabolic compartmentalization may hinder the regenerative response to PHx in the ethanol-fed animals. Targeting the metabolic division of labor may be a potential intervention to promote liver regenerative capacity in alcoholic liver disease.