Susceptibility of Hepatic ProgenitoInhibition by acetazolamide of reactivated carbonic anhydrase activity in a receptor protein tyrosine phosphatase γ (RPTPγ) mutantr Cells to Lipotoxicity as Contributor for Impaired Liver Regeneration and Disease Advancement In Pediatric Nonalcoholic Fatty Liver Disease (PNAFLD)

Abstract

BackgroundIn PNAFLD mechanisms underlying early onset and rapid disease progression have been unclear. Active liver growth in preadolescent children imparted by facultative progenitor cells would increase genome damage following oxidative stress and lipotoxicity, leading to DDR and further hepatic injury events. To reveal this we considered hTERT‐FH‐B hepatocytes expressing progenitor properties will be ideal for modeling growth‐related lipotoxicity and DDR of PNAFLD.Methods/ResultsThe FH‐B cells were derived from late‐gestation liver with doubling time of 24h, including 20‐30% in S phase, which was similar to pH3 histone +ve proliferating liver cell fractions in 5‐8‐yr old healthy children. Moreover, FH‐B cells express hepatobiliary markers (G6P, Glycogen, GGT, DPPIV) along with progenitor markers (SOX9, FOXA2, Cx43, EpCAM) and P450 profiles indicating hepatic immaturity. To induce lipotoxicity in cultured cells 50 μM palmitic acid (PA) was added with and without oxidized collagen matrix or 2,5‐10 ng/ml TNFa. This dose‐dependently caused oxidative stress (dihydrorhodamine dye for ROS) and lower mitochondrial membrane potential (MMP, JC‐1 dye) in 1‐4h and onset of steatosis (Oil red O or Bodipy staining) and decreased cell viability (MTT assay) in 18‐24h. The contribution of ATM signaling during lipotoxicity was apparent from pNBS1 accumulation, which marks DNA strand break sites. Moreover, lipotoxicity and DNA damage greatly increased in presence of ATM‐specific kinase inhibitor, KU‐60019. Additionally, ATM insufficiency exacerbated cell growth‐arrest through downstream p53‐dependent p21 activation, which increased polyploidy indicated by Hoechst dye‐stained nuclear DNA content analysis, and inhibited cell number increases in growth assays over few days. To test therapeutic feasibility, we studied obeticholic acid (OCA) and found this drug decreased ATM insufficiency‐related exacerbation of lipotoxicity. The improvements included steatosis, ROS, MMP. DDR, cell viability, as well as less growth‐arrest or polyploid accumulations and continued cycling.ConclusionsThe successful modeling of PNAFLD in hTERT‐FH‐B cells establishes the critical susceptibility of actively growing facultative progenitor hepatocytes to mitochondrial dysfunction and impaired DDR related to ATM insufficiency. The potential of OBC in improving lipotoxic outcomes will be relevant for PNAFLD. These pathophysiology‐based models offer opportunities to advance therapy in PNAFLD.