Liver regeneration is a well-orchestrated process, in which complex signaling pathways coordinate the progression of distinct stages, including withdrawal of hepatocytes from quiescence (“priming phase”), cell-cycle entry and progression, cessation of cell division, and return of hepatocytes to quiescence. Although hepatocytes rarely divide under normal circumstances, the liver has a remarkable ability to regenerate after surgical removal or after viral or chemical injury.[1-3] For example, after partial hepatectomy (PHx) of two thirds of the liver, a rodent model of liver regeneration, the remaining third grows rapidly to restore the liver's original mass, structure, and function within a few days. Indeed, >95% of mature hepatocytes synchronously exit the G0 phase and re-enter the cell cycle.[2, 3] Hepatocytes are the first cells to replicate, followed sequentially by biliary epithelial cells, Kupffer cells, stellate cells, and sinusoidal endothelial cells.[1-3] Hepatocyte proliferation in response to cytokines and growth factors plays a central role in liver regeneration.[1-3]
Early stress signals occurring after PHx may be the result of an increase in energy demand per unit liver mass. Remnant tissue retains liver-specific functions, such as gluconeogenesis and ureagenesis, and continues to produce adenosine 5'-triphosphate (ATP) for synthesis of proteins, nucleic acids, and other cell constituents.[4, 5] There was a marked decline in ATP within 6 hours after PHx, which was maintained throughout the prereplicative period. Moreover, the change in hepatocytes from the quiescent to replicative mode during the early phase of recovery was accompanied by reorganization of intracellular proteins and organelles.[1-3] However, the mechanisms underlying restoration of normal liver function, including the role of systemic metabolism, and reorganization of intracellular content throughout the replicative period after PHx remain unknown.
Autophagy is a homeostatic mechanism that regulates turnover of long-lived or damaged proteins and organelles, buffers intracellular constituents, and supplies amino acids taken from degradation products of the autolysosome. The first step of autophagy involves the formation of a lipid bilayer structure, which sequesters cytoplasmic materials to form autophagosomes. These autophagosomes engulf organelles and then fuse with lysosomes to form mature autolysosomes, in which the sequestered proteins are digested into amino acids by lysosomal enzymes. Based on its ability to reorganize intracellular proteins and organelles, and to modulate intracellular energy,[6-8] autophagy may be essential for liver regeneration, and its activities may be critical for mitotic or hypertrophic hepatocytes.
In a mouse model with liver-specific knockout (KO) of autophagy-related gene 7, livers exposed to long-term surveillance exhibited significant hepatomegaly with aggregates of unfolded proteins, which is apparently inconsistent with the role of autophagy in promoting cell proliferation. To investigate the mechanism by which autophagy regulates hepatocyte proliferation and liver growth, we developed a mouse model of liver regeneration with liver-specific KO of autophagy-related gene 5 (L-Atg5 KO mice). Unexpectedly, liver regeneration after PHx was significantly delayed and was accompanied by delays in DNA synthesis and cell-cycle arrest during liver regeneration. Furthermore, we found that impaired hepatocyte proliferation was biologically distinct from cell-cycle arrest because it represented cellular senescence accompanied by increased cell size (hypertrophy) and accumulation of senescence-associated enzyme β-galactosidase (SA-β-gal). These results suggest that autophagy plays critical roles in liver regeneration after PHx by maintaining intracellular energy production, as well as constitutive proteins and organelles, to prevent hepatocyte senescence.
We have used L-Atg5 KO mice to investigate the roles of autophagy-associated pathways in liver regeneration after PHx. We found that liver regeneration was severely impaired by 70% PHx in these mice. Their livers showed an impaired postoperative mitotic response, with quiescent hepatocytes becoming senescent and hypertrophic. Moreover, PHx was followed by considerable damage to mitochondria, reduced β-oxidation, and reduced intrahepatic ATP generation. Thus, autophagy during the early phase of liver regeneration is critical for maintaining healthy mitochondria capable of producing ATP and prevents hepatocytes from becoming senescent.
Growth factors, such as HGF and IL-6, activate phosphatidylinositol 3-phosphate, which, in turn, phosphorylates Akt at Thr308 and Ser473 in hepatocytes.[17, 18] Akt also plays a critical role in proliferation by phosphorylating mTOR1, which suppresses autophagy by inhibiting TOR-dependent phosphorylation of Atg13 in a rapamycin-sensitive complex containing raptor.[17, 18] During proliferation, Atg13 is phosphorylated by phosphorylated AMPK-α, which is activated by an increase in the AMP/ATP ratio caused by cellular/environmental stressors, such as energy deficiency, hypoxia, and ischemia, during proliferation.[6-8] To date, a few reports have shown a relationship between HGF and autophagic activity. A recent report showed that HGF has a pivotal role in directly promoting autophagic activity for clearance of advanced glycation endproducts, which are involved in the pathogenesis of diabetic vascular complications, in primary mouse nonparenchymal cells (NPCs). This previous report showed that LC3-II levels in NPCs are increased by HGF, similar to our results, and that this effect is inhibited by cotreatment with an anti-HGF neutralizing antibody. In some ongoing clinical trials, pharmacological inhibitors of autophagy, such as CQ, which is an autophagolysosomal inhibitor, have been used for treating solid cancers (e.g., pancreatic adenocarcinoma and breast cancer), based on the knowledge that growth-factor–induced autophagic activity promotes proliferation of cancer cells through important components of cellular metabolism. In proliferating hepatocytes, the level of phosphorylated AMPK is increased, activating autophagy to overcome the burden of various stressors, especially those that cause endoplasmic reticulum stress or oxidative stress (OS). Therefore, HGF indirectly increases LC3-II levels by enhancing phosphorylated AMPK levels.
Mitophagy, the autophagy-dependent degradation of mitochondria, is a defensive mechanism that involves selective sequestration and subsequent degradation of dysfunctional mitochondria. Occurrence of mitophagy in livers of patients with Reye's syndrome suggests its importance for normal mitochondrial turnover and function. Accumulation of mitochondria caused by disruption of autophagy is thought to lead to increased levels of reactive oxygen species and DNA damage, resulting in mitochondrial depolarization and permeability transition.[23-25] In this study, we showed that selective degradation of dysfunctional mitochondria in proliferating hepatocytes was impaired after PHx in L-Atg5 KO mice, resulting in a disruption of mitochondrial functions, such as maintenance of intracellular ATP levels, by mitochondrial β-oxidation. However, with the exception of hepatocyte senescence, which was accompanied by increased p21 expression, we were unable to detect any phenotypes of mitophagy in proliferating hepatocytes. Further detailed research is required to clarify the pivotal role of mitophagy in proliferating hepatocytes.
Recent studies have elucidated the mechanisms involved in cellular senescence in hepatocytes adapted to stress stimuli.[10, 26-30] As a stress response, senescence is a dynamic process involving multiple effector mechanisms, whose combination ultimately determines cellular phenotype. This process is characterized by a number of biological, biochemical, and molecular changes, including cell hypertrophy, up-regulation of SA-β-gal, irreversible growth arrest, and expression of a specific SASP.[31, 32] SASP components, such as IL-6, IL-8, and matrix metalloproteinases, can promote tissue repair by preventing the generation of a persistent acute inflammatory response and by attracting immune cells that kill and clear senescent cells by adhesion molecules expressed on the latter. Stimuli, such as OS, can induce autophagy and senescence and may be part of the same physiological process, known as the autophagy-senescence transition.[33, 34] However, the pivotal relationship between these two cellular responses has not been elucidated. In particular, it is unknown whether induction of autophagy is involved in the induction of senescence or vice versa.[26, 27] The results of our study suggest that autophagy regulates cellular senescence during liver regeneration. A subset of autophagy-related genes was up-regulated during liver regeneration in association with negative feedback in p21-activated senescence. Furthermore, inhibition of autophagy augmented the senescence phenotype, including senescence-associated secretion of IL-6 and IL-8. We reasoned that rapid protein turnover, which involves autophagy coupled with active protein synthesis, may facilitate this process by allowing the remodeling of proteins needed for liver regeneration. Indeed, senescent cells are usually hypertrophic, and we confirmed that the fall into senescence was dependent on cellular quality maintained by autophagy. Thus, autophagy may be complementary to epigenetic regulation in preventing the specific biochemical alterations observed during acute induction of senescence in the regenerating liver. Our findings suggest that autophagy, and its maintenance of cellular quality by rapid protein turnover during hepatocyte regeneration, negatively regulates the acquisition of the senescence phenotype.
Several genetic and pharmacological interventions can suppress transient hepatic steatosis, a characteristic of the early regenerative response accompanied by hepatic energy deficiency, resulting in impaired liver regeneration. Subsequent to liver injury, liver mass is maintained or recovers in proportion to body mass.[1, 21] Moreover, hypoglycemia after PHx induces systemic lipolysis, which is followed by accumulation of fat derived from peripheral stores, during early regeneration. These observations, and the central role of the liver as the principal intermediary between fatty acid metabolism and intrahepatic energy maintenance,[35, 36] prompted us to investigate the regulation and functional role of autophagy, a systemic metabolic phenomenon, during liver regeneration after PHx. In our study, we found that hepatocytes with activated autophagy, without senescence, stimulated mitochondrial metabolism during liver regeneration by maintaining healthy mitochondria and the production of high-energy mitochondrial fuels to support hepatocyte proliferation. The remnant liver relies on mitochondrial oxidative phosphorylation to satisfy its energy demands, and defects in mitochondrial permeability transition have been reported in mitochondria isolated from remnant livers after PHx. In addition, autophagy is critical for regulating hepatocellular lipid stores. Future studies should address whether systemic adipose stores during normal liver regeneration are needed as fuel sources to support regeneration, as lipid precursors for membrane synthesis, and/or act as specific signals that initiate the regenerative response itself.[37, 38] Mitochondrial oxidative damage is a fundamental component of liver regeneration, and multiple studies have shown that it is attenuated by autophagic activity to regulate hepatocellular lipid stores. A recent study showed that sirtuin-1 (SIRT1), a nicotinamide adenine dinucleotide–dependent deacetylase, was involved in the protective effects of calorie restriction against hypoxia in the aged kidney, which is linked to calorie restriction-related longevity by mitochondrial autophagy. Adult-onset and long-term calorie restriction in mice promoted increased SIRT1 expression in the aged kidney and attenuated hypoxia-associated mitochondrial and renal damage by enhancing mitophagy. Here, we have shown that autophagic activity in proliferating hepatocytes markedly diminished mitochondrial dysfunction in terms of increased mitochondria permeability transition and decreased β-oxidation. These data highlight the role of autophagy-dependent systemic lipolysis, including β-oxidation, in hepatocellular adaptation to mitochondrial oxidative damage, delineate a molecular mechanism of the autophagy-mediated antiaging effect, and could potentially direct the design of new therapies to promote liver regeneration after damage associated with OS and aging.
In our study, the peak percentage of Ki67-positive cells, as determined by immunohistochemistry (IHC), was 7.4% at 12 hours after PHx, 11.2% at 24 hours, and 31.2% at 36 hours (Supporting Fig. 10). These values are higher than those reported in previous studies.[5, 15] All of the mice subjected to 70% PHx were 6 weeks old in our study, whereas other studies induced PHx at 10-12 weeks. Hepatocyte senescence strongly down-regulates cellular proliferation by decreasing farnesoid X receptor levels or SIRT1 levels. Therefore, the difference in the percentage of BrdU-positive cells at 12 hours after PHx between our study and previous studies may be the result of a difference in the effect of senescence on proliferating hepatocytes labeled with BrdU.[41, 42]
Autophagy during the early phase of liver regeneration is critical for maintaining healthy mitochondria, which can produce ATP through β-oxidation after hypoglycemia-induced hepatic steatosis. Therefore, autophagy may be essential for preserving cellular quality by preventing hepatocytes from becoming senescent and hypertrophic.
The authors are grateful to T. Yoshimori (Osaka University, Osaka, Japan) for kindly providing the inactive mutant of Atg4B (Atg4BC74A) and N. Mizushima (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) for the Atg5flox/flox mice. The authors also thank N. Yamashita (Kyushu University, Fukuoka, Japan) for her expert advice related to statistical analysis.