Absence of Atg7 in the liver disturbed hepatic regeneration after liver injury

Autophagy is a critical process in cell survival and the maintenance of homeostasis. However, the implementation of therapeutic approaches based on autophagy mechanisms after liver damage is still challenging.


| INTRODUC TI ON
Macroautophagy (hereafter referred to as autophagy) is a cellular survival mechanism that targets cytosolic components to lysosomes.
Autophagy catabolizes and reutilizes energy generation and sustains cellular homeostasis. This delivery is performed by the autophagosome, a double-membraned spherical vesicle. The formation of the autophagosome is orchestrated by several evolutionarily wellconserved 'AuTophaGy' genes (ATGs). Autophagy is permanently active at low levels and is induced as a response to intra-or extracellular stimuli. Plenty of evidence indicates that autophagy participates in the pathogenesis of different human diseases, including neurodegenerative, cardiovascular and infectious diseases, metabolic disorders, and cancer. Nevertheless, the role of autophagy in several diseases is considered a 'double-edged sword.' Autophagy is considered of crucial importance in liver metabolism. Autophagy degrades and recycles protein and cell organelles supplying amino acids to cellular processes, delivers glycogen to vacuoles during starvation, and breaks down lipid droplets, providing free fatty acids and intracellular triglycerides. [1][2][3] Several studies have shown that hepatic autophagy is dysregulated in metabolic liver diseases such as nonalcoholic fatty liver disease and obesity. 4 Atg7 is involved in autophagosome formation and is thus an essential gene for autophagy. Therefore, there is no autophagy in Atg7deficient cells. 5 In this study, we used a hepatospecific Atg7-deficient murine model. We observed steady-state hepatocyte hyperproliferation. In contrast, after partial hepatectomy (PHx), hepatocyte proliferation was strongly decreased. Even more pronounced was the 50% mortality after this intervention that could be reversed by pharmacological mTOR inhibition. In accordance with hepatocyte hypoproliferation and impaired regeneration capacity after injury, Atg7-deficient hepatocytes failed to repopulate the liver in a Fah (fumarylacetoacetate hydrolase) hepatic injury model. Mechanistically, we showed that the triglyceride content varied with hepatic hypoproliferation and poor liver regeneration. Additionally, cell cycle-related proteins were specifically regulated after liver damage in the serum of Atg7-deficient mice.

| Mice
All animals were kept at the central animal facility of the Hannover Medical School (Hannover, Germany) under the previously described conditions. 6,7 Atg7 fl/fl mice were obtained from Masaaki Komatsu. 5 Atg7 fl/fl mice were bred with C57BL/6-Tg(Alb-Cre)21Mgn mice to generate Atg7 fl/fl Alb-cre + and Atg7 fl/fl Alb-cre − (controls) animals.

| Histology and immunostaining
Freshly dissected liver tissues were fixed, processed and paraffin embedded. Five-micron sections were stained with haematoxylin and eosin or processed further for immunohistochemistry.
Histological scoring for the modified hepatic activity index (mHAI) was performed by a pathologist in a blinded fashion as shown before. 8,9 Ki-67, BrdU, Sirius Red, Oil Red O, Fah and β-catenin staining were performed using standard immunohistochemistry protocols.
The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to identify apoptotic cells was performed as described previously. 6 mTOR inhibition overcame the impact of autophagy deficiency after liver damage and prevented mortality.

| Statistical analysis
The unpaired Student's two-tailed t-test, Kaplan-Meier survival analysis and two-way analysis of variance (ANOVA) with Tukey's post test were performed using Prism 8 software (GraphPad).
Data are presented as the mean ± standard deviation or as the median in violin plots. Alternatively, for multiple variables, the unpaired Student's two-tailed t-test with an implemented Benjamini-Hochberg multiplicity correction was performed using Qlucore Omics Explorer software 3.5 (Qlucore, Lund, Sweden). Heat maps represent the multiple protein expression profiles (P < .05; q < 0.05). Significant differences with P ≤ .05 are indicated by *, very significant differences (P ≤ .01) are indicated by **, extremely significant differences (P ≤ .001) by *** or by **** (P ≤ .0001). P > .05 was considered to be not significant (ns).

| Hepatocyte-specific autophagy loss leads to hepatic hypertrophy in Atg7 fl/fl Alb-cre + mice
To investigate the impact of autophagy on the liver as a whole and hepatocytes in detail, we crossed Atg7 fl/fl mice with mice harbouring the liver-specific albumin-cre (Alb-cre) transgene, thereby generating Atg7 fl/fl Alb-cre + animals expressing no Atg7 in hepatocytes (data not shown). The controls used were littermates that F I G U R E 1 Atg7 deficiency in hepatocytes produced hepatic hypertrophy in mice. Eight-and sixteen-week-old Atg7 fl/fl Alb-cre + males and their counterpart littermates were sacrificed. A, Atg7 fl/fl Alb-cre + males presented low body weight and extremely high liver weight; therefore, their liver/body weight ratio was elevated. B, Gross anatomical views of representative livers. C, Bilirubin levels in blood were unchanged between groups. D, Representative pictures of haematoxylin & eosin staining. The magnification bar represents 100 μm. n = 6-14 express no Alb-cre (Atg7 fl/fl Alb-cre -) and therefore have normal Atg7 levels (data not shown). Autophagy deficiency was validated by an impaired LC3-II formation ( Figure S1). This newly generated model is slightly different from published Atg7 fl/fl Mx-cre or Atg5 fl/fl Mx-cre models with a less specific promotor and/or different targeted gene. 5,10 Nonetheless, and in line with these models, examination of the Atg7 fl/fl Alb-cre + mice revealed that they were significantly lighter than the controls at 8 weeks of age ( Figure 1A). At 16 weeks of age, this difference persisted; the Atg7 fl/fl Alb-cre + mice displayed a 40% body weight gain in 8 weeks, while the control mice displayed a 32% gain. Upon closer examination of the liver, we observed that the liver weight of Atg7 fl/fl Alb-cre + mice was significantly increased compared to that of controls at 8 weeks of age, and the difference was even greater at the sixteenth week of life ( Figure 1A). The Atg7 fl/fl Alb-cre + mice gained a mean liver weight of 92% in 8 weeks, whereas the control mice gained only 19%. Overall, the liver weight gain in Atg7 fl/fl Alb-cre + mice was ten times more rapid than that in control mice. Due to the reciprocal relationship between body weight and liver weight, the proportion of liver to total body weight increased from 4.7% in controls to 14.2% in Atg7 fl/fl Alb-cre + mice and increased even more with advancing age ( Figure 1A). This extraordinary liver enlargement was also macroscopically visible ( Figure 1B), but the bilirubin values were rather unremarkable ( Figure 1C). Interestingly, a significant increase in hepatocyte size was evident at 8 weeks of age with Atg7 loss, but this difference was not observed 8 weeks later ( Figure 1D). These observations led to the question of whether the hepatic hypertrophy in   Figure 2D). However, this proliferation was lower at 16 weeks of age than at 8 weeks but was still exceptionally elevated. These data suggest that Atg7 deficiency promotes hepatocyte hyperproliferation and cellular hypertrophy but only transient.

| The regenerative capacity of the hepatocytes of Atg7 fl/fl Alb-cre + mice is severely impaired despite greater steady-state proliferation of hepatocytes
The strong proliferation of hepatocytes and the extreme liver weight in Atg7 fl/fl Alb-cre + mice suggest a very high regenerative capacity.
Therefore, we were interested in determining whether hepatocyte proliferation could be further increased by additional regenerative stimuli, such as PHx. To address this question, we partially hepatectomized Atg7 fl/fl Alb-cre + and control mice at the time of peak baseline proliferation. In contrast to the controls, approximately 50% of Atg7 fl/fl Alb-cre + mice died within 48 hours after surgery ( Figure 3A).
Interestingly, an analysis of the liver weight of the survivors showed that the liver mass was always higher in these mice than in the controls after PHx ( Figure 3B). As the initial size and mass of the Atg7 fl/fl Alb-cre + livers were well above those of the controls, data normalization was performed. Therefore, the 'basal' time point in all animals corresponds to a value of 1 in Figure 3C. Immediately after PHx, this value dropped to 0.3 at time point 0 hours as a result of the two-thirds F I G U R E 3 Atg7 loss compromised survival and impaired liver mass recovery after PHx. Eight-week-old Atg7 fl/fl Alb-cre + males and their counterpart littermates underwent PHx. A, Survival was dramatically decreased in Atg7 fl/fl Alb-cre + males. B, Liver weight of males after PHx at the determined time points. C, Atg7 fl/fl Alb-cre + livers did not completely reach the original mass after intervention. D, Bilirubin levels in blood were unaffected. h = hours, 1 w = 1 week. The 0-h data correspond to 8-wk-old samples. n = 6-14 PHx. We noticed that the Atg7 fl/fl Alb-cre + livers, only regained 47% of the starting weight one week after PHx, whereas the controls regained 70% and 3%-4% of the total body weight ( Figure 3C; data not shown). This observation implies that liver regeneration was reduced to less than 50% of that in the controls. Importantly, at time points after 48 hours, the Atg7 fl/fl Alb-cre + mice included in the analysis represent a positive selection, since half of the animals have already died ( Figure 3A). This also explains the inconspicuous bilirubin levels in Atg7 fl/fl Alb-cre + animals, as only surviving animals were tested, resulting in a form of positive selection ( Figure 3D).
To exclude environmental influences and to examine only the effect on hepatocytes, we have established a hepatocyte transplantation model using immunosuppressed Fah -/-Rag2 -/-Il2rg -/-(FRG) mice. 6,11 These mice lack B, T and NK cells and have a genetic defect that leads to an accumulation of a toxic metabolite during tyrosine catabolism, which subsequently causes acute liver failure. The drug NTBC blocks this accumulation and therefore prevents liver damage in mice. 12 One million hepatocytes from 8-week-old Atg7 fl/fl Alb-cre + or Atg7 fl/fl Alb-cremice were intrasplenically transplanted into FRG mice, which allows nearly complete liver repopulation with wild-type F I G U R E 4 Atg7-deficient hepatocytes did not repopulate Fah immunosuppressive livers. One million hepatocytes isolated from 8-wk-old Atg7 fl/fl Alb-cre + males and their counterpart littermates were intrasplenically injected into FRG mice. A, Illustration of the hepatocyte transplantation approach. B, Survival of FRG mice after hepatocyte transplantation. C, Representative pictures of Fah immunohistochemistry and quantification of repopulated livers. Red-stained hepatocytes were the transplanted hepatocytes. n = 3-7 hepatocytes. Following transplantation, NTBC treatment was withdrawn, creating a strong proliferative environment for transplanted Fah-expressing cells ( Figure 4A). After 8 weeks, 62% of the FRG mice transplanted with control hepatocytes survived; however, the survival after transplantation with Atg7 fl/fl Alb-cre + hepatocytes was significantly lower (37%; Figure 4B). Fah immunostaining revealed multiple proliferating hepatocytes in liver sections of both groups 4 weeks after transplantation. Interestingly, 8 weeks after transplantation, 50% of the livers were repopulated with control hepatocytes; in contrast, Atg7 fl/fl Alb-cre + hepatocytes were unable to repopulate the FRG livers ( Figure 4C). Together, these data imply that Atg7 deficiency in hepatocytes significantly impairs their proliferation and regenerative capacity.

| Hepatocyte proliferation after liver damage in Atg7-deficient mice is severely compromised, and cellular hypertrophy is reversible
The discrepancy between hepatocellular hyperproliferation during homeostasis and less liver regeneration after PHx and transplantation is conspicuous and requires further investigation.
Therefore, we additionally examined the regeneration and proliferation capacity of hepatocytes after PHx. The Ki-67 and BrdU immunostaining results revealed that the proliferation of hepatocytes in the Atg7-deficient mice was still greatly increased at the time of PHx (8 weeks of age). Surprisingly, this picture was reversed after 48 hours, and furthermore, hepatocyte proliferation returned to a level comparable to that in control mice after one week ( Figure 5A).
Notably, at 48 hours and later, only up to half of the mice survived and served as a benchmark. Immunostaining quantification revealed a strong increase in hepatocyte proliferation in the control livers that was not observed in Atg7 fl/fl Alb-cre + mice. As expected, control mice presented a hepatocyte proliferation peak at 48 hours, reaching levels of proliferation after one week similar to those before PHx. In contrast, Atg7 fl/fl Alb-cre + livers exhibited reduced hepatocyte proliferation after intervention compared to the basal time point (Figure 5B and 5D). Normalization of the data to fold change revealed the timeline of hepatocyte proliferation in control and Atg7-deficient livers ( Figure 5C and 5E). Here, the data show that PHx did not induce the proliferation of Atg7 fl/fl Alb-cre + hepatocytes.
In addition, the hepatocyte enlargement seen in Atg7 fl/fl Alb-cre + livers was highly reversible, since the hypertrophic hepatocytes halved in size 48 hours after PHx, shifting back to normal size ( Figure 5F).

| Atg7 deficiency promotes apoptosis and thereby enhances the transaminases and modified hepatic activity index in Atg7 fl/fl Alb-cre + mice
Homeostasis in the liver requires balanced hepatocyte apoptosis and mitosis in healthy individuals. Therefore, we wondered how Atg7 deficiency modulates apoptosis sensitivity in the liver. The TUNEL assay and its quantification revealed that approximately 10% of hepatocytes in Atg7 fl/fl Alb-cre + mice at 8 weeks of age were apoptotic, level which remained high with increasing age ( Figure 6A).
The increased apoptosis rate of hepatocytes was correlated with a strong increase in aspartate transaminase and alanine transaminase (ALT) in Atg7 fl/fl Alb-cre + mice ( Figure 6B). Additionally, pathological evaluation of liver sections showed a high mHAI ( Figure 6C), almost exclusively due to apoptosis without any inflammation (data not shown).
After PHx, the number of apoptotic hepatocytes remained high in Atg7 fl/fl Alb-cre + livers ( Figure 6D). At 48 hours, the transaminase levels were higher than the levels before PHx. Unexpectedly, after one week, the transaminase levels were only moderately increased ( Figure 6E) and significantly lower than the levels before PHx ( Figure 6A and 6E). In summary, the high pathological mHAI is due to a significantly increased hepatocyte apoptosis that is in line with the elevated transaminases in Atg7-deficient mice.

| Hyperproliferation and the high apoptosis rate are regulated at the molecular level
To elucidate the mechanism by which high hepatocyte proliferation and apoptosis occurred in Atg7 fl/fl Alb-cre + mice, we analysed the expression of 92 selected proteins in the serum. They are related to different biological processes, including cellular proliferation, apoptosis and metabolism. The steady-state hepatocyte hyperproliferation, cellular hypertrophy and increased hepatocyte apoptosis rate of Atg7 fl/fl Alb-cre + mice were accompanied by 57 differentially regulated proteins in the serum (Table S1A). The most interesting severely upregulated proteins are shown in Figure 7A and 7C.
While HGF, PAK4 and NOTCH3 regulate p53 and therefore cell proliferation as well as HCC and metastasis, YES1 is part of the protooncogene tyrosine-protein (Src) kinase family. Nevertheless, increased apoptosis was associated with a 5-fold increase in FAS expression. Interestingly, the short-lived hepatocyte-stimulating factor IL-6 was also boosted in Atg7 fl/fl Alb-cre + mice. The second comparison shows the changes occurred between 0 and 48 hours after PHx in Atg7 fl/fl Alb-cre + serum ( Figure 7B and 7D). A pool of 37 proteins was significantly regulated (Table S1B). Mechanistically, most of these proteins shown in Figure 7C were reversely regulated 48 hours after PHx, where hepatocyte proliferation was diminished ( Figure 5A-5E). Contrary to expectations, FAS was also expressed at lower levels after 48 hours, while the apoptosis rate was unchanged ( Figure 6A and 6D).

| mTOR inhibition improves survival in mice with Atg7 deficiency
Mechanistic target of rapamycin (mTOR) is a protein kinase identified as a central regulator of cell growth, proliferation and Taken together, these observations imply a mechanism for autophagy modulation since mTOR is a key factor and clinical target due to its inhibition improving the survival of Atg7 fl/fl Alb-cre + mice.

| Fewer protective triglycerides in Atg7 fl/fl Alb-cre + mice
Triglycerides in the liver protect against apoptosis and other damage by preventing the formation of free radicals. 14,15 Therefore, we were interested in how Atg7 deficiency affects triglyceride levels in the blood and liver. At 8 weeks of age, the triglyceride content was inconspicuous in the blood of Atg7 fl/fl Alb-cre + mice. Noticeably, the triglyceride content was significantly reduced at 16 weeks of age when compared to younger Atg7 fl/fl Alb-cre + mice and controls ( Figure 8E). Transient hepatic steatosis is known to occur within one day after PHx. 16 This process is essential for the induction of hepatocyte proliferation and thus liver regeneration. 17 Therefore, we analysed mice one day after PHx. In contrast to controls and our hypothesis, triglyceride levels were not increased in Atg7-deficient mice. As shown, Oil Red O staining revealed a significantly diminished amount of lipid droplets in hepatocytes in Atg7 fl/fl Alb-cre + mice compared to the amount in controls ( Figure 8F). Mitochondria are known to regulate F I G U R E 6 Apoptosis levels and liver damage were higher in Atg7 fl/fl Alb-cre + mice. (A-C) Eight-and sixteen-weekold Atg7 fl/fl Alb-cre + males and their counterpart littermates were sacrificed. A, The TUNEL assay displayed higher steadystate hepatic apoptosis in Atg7-deficient mice. B, Aspartate and ALTs in the blood of those mice were increased. C, The mHAI was elevated in Atg7 fl/fl Alb-cre + livers. D and E, Eight-week-old Atg7 fl/fl Alb-cre + males and their counterpart littermates underwent PHx. After 48 h, liver and blood were collected. (D) Apoptosis was higher in Atg7-deficient livers. (E) Transaminase levels in Atg7 fl/fl Alb-cre + mice were lower than the levels before PHx. The magnification bar represents 100 μm. h = hours, 1 w = 1 week. n = 5-11 hepatic triglyceride metabolism, and mitochondrial stress and damage contribute to the development of fibrosis and inflammation in the liver following cell death. Consequently, we evaluated the mitochondrial respiration status in Atg7 fl/fl Mx-cre + hepatocytes. No significant differences in the oxygen consumption rate were observed ( Figure S2).

| D ISCUSS I ON
In this study, we report the importance of autophagy for hepatocellular regeneration in a new model with the liver-specific albumin promotor and Atg7 deficiency. We demonstrated for the first time an increased proliferation of hepatocytes in Atg7-deficient mice ( Figure 2). Furthermore, we show that the extent of some liver injury abnormalities intensified with age (e.g. hepatocyte size, proliferation and fibrosis; Figure 1 and 2), while other pathological and biochemical parameters did not change. However, the most striking effects were shown for liver regeneration and repopulation after PHx (Figures 3,5 and 8) and cellular Tx (Figure 4). Impaired liver regeneration was most prominent in the poor survival rate of Atg7 fl/fl Alb-cre + mice accompanied by impaired lipid droplet formation that might be causative. Nonetheless, the survivors had better transaminases, decreased proliferation, and reversal of transient cellular hypertrophy with a low hepatic triglyceride content. Surprisingly, poor survival could be fully recovered by pharmacological mTOR inhibition ( Figure 8). Mechanistically, we observed the modulation of cell cycle-related proteins before and after PHx in serum ( Figure 7).
The disproportional hyperproliferation of hepatocytes was also described by other authors for autophagy-deficient models. 18,19 Notably, others have used different animal models. 19 The models utilized were not based on Alb-cre but on MX1-cre system. The Alb-cre system is hepatospecific, while MX1 is also highly expressed in the lung, 20 spleen, lymph nodes, heart, nervous system and other organs. 21 This effect might cause interference within the organism, resulting in a discrepancy in the results.
According to Biogps.org, albumin mRNA levels in the liver are approximately 90,000-fold higher than in the reference, while MX1 is approximately 5-fold higher. This observation is in line with our own findings; even though Atg7 fl/fl Mx-cre + mice had hepatocyte hyperproliferation, this increase was less pronounced than that in Atg7 fl/fl Alb-cre + mice ( Figure S3). Although the observed F I G U R E 7 Several cell cycle-related proteins were modulated in Atg7 fl/fl Alb-cre + serum after PHx. Ninety-two proteins were quantified in the serum of Atg7-deficient and control mice before and after PHx. A, Heat map showing selected highly regulated proteins in Atg7 fl/fl Alb-cre + serum compared to controls at 0 h. B, Regulated proteins in Atg7 fl/fl Alb-cre + serum at 0 h compared to 48 h after PHx. C-D, Bar charts displaying the modulation of several proteins C, at 0 h for Atg7 fl/fl Alb-cre + and control mice and D, at 0 and 48 h after PHx for Atg7 fl/fl Alb-cre + mice. n = 4-19 hyperhepatoproliferation was slightly reduced in Atg7 fl/fl Alb-cre + mice over time, the level of hepatocyte proliferation remained high (>10%), as also seen by others. 22 In contrast, other studies/models could not reproduce this finding 5,10,2 3 or have shown the opposite. 3 However, cellular hypertrophy and fibrosis have already been shown in other autophagy-deficient models. 5,22 Corroborating our previous observations (Figure 1) and other studies, 10,19 we were able to attest that there was strong hepatocyte hypertrophy at the eighth week of life, which was associated with a disarray of liver cell plates (Figure 2A). However, we were able to demonstrate for the first time that while this disarray remained, the difference in hepatocyte size could no longer be noticed at 16 weeks of age and therefore appeared to be transient.
The recovery rate after liver damage by PHx is a crucial parameter to study. A benign effect of PHx in our model was likely because the transaminases were strikingly reduced. However, since approximately 50% of Atg7 fl/fl Alb-cre + mice died after this intervention ( Figure 3A), it cannot be considered; although, pharmacological mTOR inhibition resulted in complete survival after PHx ( Figure 8A). Here, we described a disturbed tissue mass restoration ( Figure 3C). Another autophagy study reported opposing results, but these mice were also Atg5-deficient as in the MX1-cre line, making a comparison difficult. 10 Nevertheless, in our Atg7 fl/fl Mx-cre + mice, hepatocyte proliferation after PHx was also disturbed ( Figure S4), but this disturbance was less pronounced than that observed with hepatocyte-specific Atg7 deficiency. The reduced liver regeneration in mice with disordered F I G U R E 8 Pharmacological inhibition of mTOR improved the survival of Atg7-deficient mice, and triglyceride levels varied in those mice. Eight-week-old Atg7 fl/fl Alb-cre + males and their counterpart littermates were treated with everolimus and underwent PHx. A, Everolimus treatment sustained Atg7-deficient mouse survival after PHx. B, Hepatocyte proliferation assessed by Ki-67 with or without everolimus treatment. C and D, Liver function parameters were not improved after mTOR inhibition and PHx in Atg7 fl/fl Alb-cre + mice. The Atg7 fl/fl Albcre + data for survival correspond to Figure 3. E, Blood triglyceride levels were measured in 8-and 16-wk-old Atg7 fl/fl Alb-cre + males and their counterpart littermates. F, Eight-week-old Atg7 fl/fl Alb-cre + males and their counterpart littermates underwent PHx. Representative pictures showing Oil Red O immunohistochemistry before and after PHx. The magnification bar represents 100 μm. h = hours. n = 3-10 autophagy might be due to various reasons: decreased hepatocyte proliferation or increased hepatocyte apoptosis. In contrast to wildtype mice 13 and other compromised models, [6][7]11,24 we observed a fulminant drop in hepatocyte proliferation after PHx in our Atg7 fl/fl Alb-cre + mice ( Figure 5). Coincidentally, the apoptosis rate was slightly increased after PHx (Figure 6A and 6D). Others have shown that impaired hepatocyte regeneration in Atg5-deficient MX1-cre mice is due to premature senescence but not due to apoptosis activation. 10 Our data after PHx displayed a decreased hepatocyte proliferation rate. This suggests that the remaining liver tissue might be sufficient to fulfil hepatic function and allow survival. Liver mass may limit regeneration through a negative feedback loop.
However, Toshima et al showed that a massive hepatectomy (90% PHx) in Atg5-deficient mice increases mortality to 100% within one day. 10 We and others also observed cellular hypertrophy in autophagy-deficient mice. 5,18,19 Here, we could show that this cellular hypertrophy is only a transient effect. Contrary to our results, in Atg5-deficient mice, hepatocyte hypertrophy was observed one to two days after PHx 10 ; however, no later time points were analysed. Therefore, this is not the cause of the disturbed hepatocyte function and regeneration.
Whether the doubled liver mass in Atg7-deficient mice after PHx hampers regeneration is a difficult question to address. Therefore, we transplanted Atg7 fl/fl Alb-cre + hepatocytes into FRG mice to specifically analyse how the loss of Atg7 affects cell cycle progression. 6,13 We hypothesized that the transplanted Atg7-deficient hepatocytes should repopulate the damaged livers more efficiently than control hepatocytes because of their steady-stage hyperproliferation.
Surprisingly, Atg7-deficient hepatocytes failed to repopulate the liver. Therefore, we indirectly showed that disturbed liver regeneration is very unlikely to be due to a pure mass effect but is more likely due to a metabolic disorder. 6,13,24,25 Together, these findings explain the reduction in regenerative capacity. To support this, we have shown a differential regulation of proteins involved in cell cycle control (Figure 7). Although little has been studied in the liver, PAK4 has been shown to play a role in cell proliferation and cytoskeletal architecture modulation. Similar to NOTCH3, 26 upregulation of PAK4 has been associated with uncontrolled cell proliferation and cancer in different organs. 27 Interestingly, cell proliferation activation by PAK4 and NOTCH3 is performed through p53. 28,29 Furthermore, YES1, a member of the SRC kinase family, interacts with YAP, facilitating its translocation to the nucleus and therefore its activation. YAP overexpression in the liver has been correlated with hepatocyte proliferation and hepatomegaly. 30 Notably, these proteins were inversely regulated after PHx, where hepatocyte proliferation was largely decreased. This effect explains the impaired liver regeneration after PHx. HGF is associated with sustained hepatocyte proliferation in cancer. 31 However, a study revealed that HGF stimulates PAK4, possibly contributing to HGF-induced changes in cytoskeletal architecture in epithelial cells. 32 The modulation of PAK4 and HGF before and after PHx could elucidate transient cellular hypertrophy.
Although most of these findings are not clinically useful, they have important implications for the underlying mechanism of liver regeneration and autophagy; therefore, further detailed molecular characterization is needed. Therefore, we evaluated the therapeutic options involving the inhibition of mTOR. One effect of the inhibition of mTOR is the activation of autophagy. 33,34 We used everolimus to overcome the effect of Atg7 deficiency in hepatocytes. Although this therapy did not improve the mHAI, it led to complete survival of Atg7-deficient mice after PHx. This is the opposite effect described in Atg5/mTOR double knockout mice. 35 These observations suggest that the pharmacological inhibition of mTOR might activate an Atg5/Atg7independent autophagy process, leading to better mouse survival.
An alternative Atg5/Atg7-independent process of autophagy without microtubule-associated protein 1A/1B light chain 3 (LC3) lipidation has been detected in several embryonic tissues. 36 This revealed an important pharmacological mechanism to overcome the high mortality after liver injury.
There is controversy over the causality between autophagy and lipid storage. While some studies have shown an increase in lipid storage and triglyceride content in lipid droplets due to a lack of autophagy, 19,37,38 others have shown the opposite. 22,39,40 Impaired lipid droplet formation is considered to be a causative mechanism responsible for poor hepatic regeneration and hepatocyte hypoproliferation after liver injury. We have shown an almost complete absence of these hepatic lipid droplets after PHx in Atg7-deficient hepatocytes/mice (Figure 8). This observation is very remarkable in connection with the disturbed regeneration after PHx. This transient hepatic steatosis by lipid droplet formation is a crucial mechanism for successful hepatic regeneration after PHx 16,41 and starvation. 42 LC3 and ATG7 contribute to the fusion and growth of lipid droplets. 43 LC3 is a central protein in autophagosome biogenesis. It has been shown that LC3-II is localized not only on the membrane of autophagosomes but also on the surface of lipid droplets in hepatocytes. 39 These observations suggest that a nonfunctional ATG7 protein during autophagy leads to a nonfunctional LC3 conjugation system and lipid droplet formation and therefore to an impaired regeneration capacity after PHx.
Furthermore, triglycerides are radical scavengers and fulfil a protective function against toxicity. 15 Different studies suggest that triglyceride deposition may be a guiltless witness or an initial cellular defence during lipotoxicity. 44,45 Losing this protection mechanism results in the observed apoptosis of hepatocytes. This increase in hepatocyte cell death was confirmed by an increase in the mHAI and a disproportional increase in transaminase levels. Interestingly, specific autophagy deficiency in stellate cells attenuates fibrogenesis in the liver during CCl 4 treatment, 46 indicating that autophagy appears to be involved in hepatofibrogenesis. In this context, one might speculate whether the decrease in triglyceride content with age in Atg7 fl/fl Albcre + mice (Figure 8) is also responsible for the decrease in hepatocyte proliferation and perisinusoidal/pericellular fibrosis (Figure 2).
In conclusion, we demonstrated the important role of autophagy in the regeneration capacity of hepatocytes. We showed the causative relationship between autophagy and triglyceride content, which is essential for preventing hepatocyte cell death and