Research Article: Elevated Nrf-2 responses are insufficient to mitigate protein carbonylation in hepatospecific PTEN deletion mice

Date Published: May 25, 2018

Publisher: Public Library of Science

Author(s): Dennis R. Petersen, Laura M. Saba, Volkan I. Sayin, Thales Papagiannakopoulos, Edward E. Schmidt, Gary F. Merrill, David J. Orlicky, Colin T. Shearn, Manlio Vinciguerra.


In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model.

Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTENLKO) or control littermates were fed a standard chow diet for 45–55 weeks followed by analysis for liver injury, oxidative stress and inflammation.

Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTENLKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTμ, GSTπ and CBR1/3, but not HO-1, were elevated in PTENLKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTENLKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTENLKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-S-transferases (GST) M6, O1, and O2.

Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation.

Partial Text

In the United States, nonalcoholic steatohepatitis (NASH) resulting from unmitigated progression of non-alcoholic fatty liver disease (NAFLD) is rapidly becoming the major indicator for liver transplantation[1]. Increased lipid accumulation, inflammation and elevation of oxidative stress are hallmarks of NASH[2–4]. Pathologically, hepatic fat accumulation (steatosis) is frequently regarded as the initial insult during NAFLD and is hypothesized to be the prerequisite for progression to the inflammatory disease, steatohepatitis. Following steatosis, additional cellular processes including mitochondrial injury, oxidative stress and proinflammatory cytokines are all contributing factors in the progression of NAFLD to NASH[5].

Our data demonstrate increased lipid peroxidation in the periportal (zone 1) and centrilobular (zone 3) regions of PTENLKO livers, but not in the intervening zone 2 hepatocytes. This increase in lipid peroxidation correlated with both a co-localized increase in mitochondrial respiration and with increased infiltration of inflammatory cells; both likely contributing to a localized increase in ROS. Increased ROS, combined with the increase in lipid synthesis previously reported in PTENLKO, provide an environment that would favor formation of lipid peroxides. Interestingly, acrolein staining was significantly increased in proliferating cholangiocytes, yet these cells did not accumulate 4-HNE. Also, in zone 3, 4-HNE staining is adjacent to vesicular membranes where we detected no acrolein. This indicates that the source of short chain aldehydes (SCA) such as acrolein is different than that of longer aldehydes, such as 4-HNE, which has been hypothesized to originate from linoleic acid within the membranes[50]. Moreover, staining for both aldehydes contrasts with reported lipid peroxidation in early stages of NASH, which is primarily evident in the centrilobular (zone 3) region[51]. More work will be needed to resolve the causes of this apparent discrepancy.




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