The survival pathways phosphatidylinositol-3 kinase (PI3-K)/phosphoinositide-dependent protein kinase 1 (PDK1)/Akt modulate liver regeneration through hepatocyte size rather than proliferation

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Liver regeneration comprises a series of complicated processes. The current study was designed to investigate the roles of phosphoinositide-dependent protein kinase 1 (PDK1)-associated pathways in liver regeneration after partial hepatectomy (PH) using liver-specific Pdk1-knockout (L-Pdk1KO) and Pdk1/STAT3 double KO (L-DKO) mice. There was no liver regeneration, and 70% PH was lethal in L-Pdk1KO mice. Liver regeneration was severely impaired equally in L-Pdk1KO and L-DKO mice, even after nonlethal 30% PH. There was no cell growth (measured as increase of cell size) after hepatectomy in L-Pdk1KO mice, although the post-PH mitotic response was the same as in controls. As expected, hepatectomy did not induce hepatic Akt-phosphorylation (Thr308) in L-Pdk1KO mice, and post-PH phosphorylation of Akt, mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase (p70S6K), and S6 were also reduced. To examine the specific role of PDK1-associated signals, a “pif-pocket” mutant of PDK1, which allows PDK1 only to phosphorylate Akt, was used. Liver regeneration was recovered in L-Pdk1KO mice with a “pif-pocket” mutant of PDK1. This re-activated Akt in L-Pdk1KO mice liver and induced post-PH cell growth, without affecting cell proliferation. Further deletion of STAT3 (L-DKO mice) did not further deteriorate liver regeneration, although this certainly reduced post-PH mitotic response. These findings indicate that PDK1/Akt contribute to liver regeneration by regulating cell size. Regarding phosphatidylinositol-3 kinase (PI3-K), immediate upstream signal of PDK1, activation of PI3-K induced cell proliferation via STAT3 activation in the liver of L-Pdk1KO mice but did not improve impaired liver regeneration. This confirmed the pivotal role of PDK1 in liver regeneration and cell growth. Conclusion: PDK1/Akt-mediated responsive cell growth is essential for normal liver regeneration after PH, especially when cell proliferation is impaired. (HEPATOLOGY 2009;49:204-214.)

Liver regeneration is a physiopathological phenomenon of quantitative recovery from loss of liver mass to compensate for decreased hepatic volume and impaired function. The liver has a unique ability to restore lost volume, rarely seen in other organs.1, 2 It is well established that normal adult hepatocytes are usually quiescent but have the potential ability to replicate. After surgical procedures that reduce liver mass, such as partial hepatectomy (PH) or live donor liver transplantation, rapid enlargement of the residual or grafted liver commonly takes place to restore liver mass and function. Clinically, liver regeneration has important implications because many therapeutic strategies for surgical treatment of liver diseases such as removal of liver tumors and liver transplantation depend on the ability of liver to regenerate physically and functionally. Poor or insufficient liver regeneration may be potentially fatal for these patients.3–5 Therefore, better understanding of the physiopathological features of liver regeneration could lead to clinical benefits.

Numerous studies so far have sought to elucidate the mechanisms responsible for liver regeneration, investigating the regulation of cell proliferation in simple rodent hepatectomy models.6–11 The importance of interleukin-6/signal transducer and activator of transcription-3 (STAT3) pathway in liver regeneration has been established, as reported by a number of researchers12, 13 using liver-specific Interleukin-6 or Stat3 knockout mice. Regarding STAT3, it was found that the defective mitotic response to hepatectomy in liver-specific Stat3-KO (L-Stat3KO) mice did not affect physical and functional liver regeneration at all.6 Although the hepatocyte mitotic response was suppressed or greatly delayed in L-Stat3KO mice, Akt and its associated signaling molecules such as p70 ribosomal S6 kinase (p70S6K), mammalian target of rapamycin (mTOR), and glycogen synthase kinase-3 were immediately phosphorylated after PH. These findings suggest that when cell proliferation is impaired, these molecules mediate a possible compensatory mechanism of liver regeneration.

The phosphatidylinositol-3 kinase (PI3-K)/phosphoinositide-dependent protein kinase 1 (PDK1)/Akt pathway, known as a survival pathway, targets various molecules involved in anti-apoptosis, anti-oxidation, and protein synthesis.14–18 Recent reports have shown that PI3-K/PDK1/Akt pathway and its associated molecules are responsible for determining cell size and functions.6, 19–24 Many of these studies used animals with targeted gene disruption to demonstrate the crucial roles of these molecules (mTOR, p70S6K, and glycogen synthase kinase-3) in determining the “inherent size of cells” in the specific organ. In contrast, liver-specific knockout of phosphatase tensin homolog deleted on chromosome 10, a negative regulator of PI3-K/Akt pathway, induced enlargement of the organ. Persistent stimulation of PI3-K/Akt pathway in hepatocytes resulted in an enlarged liver mass mainly by stimulating glycogen/fatty acid synthesis.25 Because the PI3-K/Akt pathway is certainly involved in glucose/fat metabolism as well as protein metabolism in the liver,26, 27 these findings suggest a crucial role for these molecules in cell growth and liver regeneration during an acute response, such as occurs post-PH. However, the role of this pathway in acute responses to such stimuli has not yet been clearly determined.

We hypothesized that PI3-K/PDK1 plays a pivotal role in liver regeneration by positively regulating cell growth, especially in cases in which cell proliferation is severely impaired. The current study was therefore designed to further examine the roles of PI3-K/PDK1 and associated molecules in liver regeneration and to determine the molecule(s) critically responsible for post-PH liver regeneration in mice.

Abbreviations

BrdU, bromodeoxyuridine; L-DKO, liver-specific Pdk1/Stat3 double knockout; L-Pdk1KO, liver-specific Pdk1 knockout; L-Stat3KO, liver-specific Stat3 knockout; mTOR, mammalian target of rapamycin; myr-p110, myristoylated form of p110 (catalytic subunit) of PI3-K; PCNA, proliferating cell nuclear antigen; PDK1, phosphoinositide-dependent protein kinase 1; PH, partial hepatectomy; pif, PDK1-interacting fragment; PI3-K, phosphatidylinositol-3 kinase; p70S6K, p70 ribosomal S6 kinase; SEM, standard error of the mean; STAT3, signal transducer and activator of transcription protein 3; S6, ribosomal S6; WB, western blot

Materials and Methods

Generation of Liver-Specific Knockout Mice.

We generated liver-specific Pdk1-knockout (L-Pdk1KO) mice and liver-specific Pdk1/Stat3 double knockout (L-DKO) mice,28 which harbor a transgene for Cre-recombinase under the control of albumin gene promoter and are homozygous for a floxed allele of Pdk1 or both Pdk1 and Stat3, respectively, by crossing Alb-Cre mice29 with Pdk1-flox mice and Stat3-flox; Pdk1-flox mice, respectively. We used Stat3-flox/flox and Pdk1-flox/flox littermates as controls for L-Pdk1KO and L-DKO mice, respectively. Although mice with a liver-specific deficiency of PDK1 were previously shown to develop a severe condition characterized by prominent edema and premature death,27 L-Pdk1KO mice generated in the current study appeared normal until at least 6 months of age. The metabolic phenotypes of L-Pdk1KO and L-DKO mice have been described elsewhere.28

Animal Experiments.

L-Pdk1KO, L-DKO, and C57BL/6 mice (male, 8–10 weeks) were used for simple 70%/30% PH experiments. Anesthesia was induced with an intraperitoneal injection of Nembutal (pentobarbital sodium, 60 mg/100 g body weight). Mice were fasted overnight before the experiments. After laparotomy, the left and median liver lobes were surgically resected for 70% PH, and the left lobe for 30% PH. The mice were sacrificed for collection of liver specimens at the indicated times before or after hepatectomy, and the liver/body weight ratios were calculated to estimate the recovery of liver mass. The animals were maintained under standard conditions and treated according to the Guidelines for the Care and Use of Laboratory Animals of Hokkaido University School of Medicine.

Cell Proliferation Assay.

To evaluate proliferation of hepatocytes after PH, proliferating cell nuclear antigen (PCNA)-positive, bromodeoxyuridine (BrdU)-labeled hepatocytes and mitotic hepatocytes were counted. Liver tissues were removed before and 48 hours or 72 hours after hepatectomy (for PCNA and BrdU or mitosis assessment, respectively), fixed in 10% buffered formalin, and paraffin embedded. Hematoxylin-eosin staining and immunohistochemical staining with anti-PCNA were performed. For BrdU labeling assay, BrdU labeling reagent was injected intravenously into mice at 1 mL/100 g body weight 1 hour before sacrifice. BrdU was immunostained with anti-BrdU antibody according the manufacturer's recommendations (Roche, Basel, Switzerland). At least 500 hepatocytes were counted for mitotic or PCNA/BrdU positivity at least three times in different sections in each group.

Electrophoretic Mobility Shift Assay.

STAT3 DNA-binding activity was assayed using mutant 67 of serum inducible element of c-fos gene promoter (SIE-m67) oligonucleotide as a probe (5′-actgGGATTTTTCCCGTAAATGGTC-3′). The reaction mixture contained nuclear protein extract (5 μg), dithiothreitol (2 mM), poly(deoxyinosinic-deoxycytidylic) acid sodium salt (dI-dC) (2 μg), single-stranded DNA (10 μg/mL), and 32P-labeled SIE-m67 probe (5 × 105 cpm). The specimens were electrophoresed on 5% polyacrylamide native gels at 4°C in 0.25× Tris-Borate/ethylenediaminetetra-acetic acid buffer.

Western Blot Analysis.

Thirty micrograms whole liver protein extract was separated by 10% sodiumk dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The following antibodies were used as primary antibodies: PDK1, STAT3/phospho-STAT3 (Santa Cruz, CA), Akt/phospho-Akt (Thr and Ser), p70S6K/phospho-p70S6K, mTOR/phospho-mTOR (Cell Signaling, MA), and PCNA (Santa Cruz, CA).

Measurement of Cell Size.

The method to measure the size of hepatocytes in liver sections was described previously.6 Briefly, individual hepatocytes were outlined and cross-sectional area was determined with a computer-assisted image analysing system (LSM Image Browser, Carl Zeiss GmBH, Jena, Germany). Cell areas of at least 500 hepatocytes were randomly selected in zone 2 and calculated in triplicate using different sections in each group.

Adenoviral Vectors (LacZ, L155E, and myr-p110).

The replication-deficient adenovirus encoding β-galactosidase was used as a control vector (LacZ). An adenovirus vector encoding PDK1-interacting fragment (pif)-pocket mutant of Pdk1(L155E) was generated, where Leu155 was replaced by Glu, allowing PDK1 signaling exclusively to Akt, but not to p70S6K or any others.30 An adenovirus vector encoding a hemagglutinin-tagged myristoylated form of p110 (catalytic subunit) of PI3-K (myr-p110) was generated as described previously.31 All viruses were produced in human embryonic kidney 293 cells, purified on double cesium chloride gradients, and plaque-titered. All adenoviruses were injected intravenously via tail vein 72 hours before the experiments.

Statistical Analysis.

Results are expressed as means ± standard error of the mean (SEM). Statistical analyses were performed with Fishers' test, and P values less than 0.05 were considered significant.

Results

L-Pdk1KO Mice and L-DKO Mice.

Western blot (WB) analyses showed the expression of PDK1 and PDK1/STAT3 in the livers of L-Pdk1KO and L-DKO mice, respectively (Fig. 1A). Only a trace amount of PDK1 was detected in L-Pdk1 KO liver, but no PDK1 and STAT3 were detected in L-DKO liver. These mice, however, showed normal liver structure and morphology (Fig. 1B), as reported previously.27 Liver and body weights of these knockout mice were no different from control mice at 8 to 10 weeks of age (data not shown).

Figure 1.

Expression of PDK1 and STAT3 in liver tissues (A) and liver histology (B) in L-Pdk1KO and L-DKO mice. (A) WB showing liver-specific deletion of PDK1 and PDK1/STAT3 in L-Pdk1KO and L-DKO mice, respectively. (B) Hematoxylin-eosin staining showing normal liver structure of both L-Pdk1KO and L-DKO mice (original magnification ×200)

Survival and Liver Regeneration After Hepatectomy in L-Pdk1KO Mice and L-DKO Mice.

Six of 10 L-Pdk1KO mice with conventional 70% PH died within 12 hours. Liver regeneration of the remaining four L-Pdk1KO mice was severely impaired (Fig. 2A). However, 30% PH allowed all mice to survive until at least 14 days post-PH (Fig. 2B). Liver/body weight ratios showed that estimated liver regeneration was significantly and persistently suppressed at this time in L-Pdk1KO mice. Because albumin is produced exclusively by hepatocytes, serum levels of albumin were measured to evaluate functional liver regeneration after PH in L-Pdk1KO mice (Fig. 2C). It was found that serum albumin levels were maintained in the normal range even after PH in control mice, but were still reduced 2 weeks post-PH in L-Pdk1KO mice. These data thus suggest that PDK1 is essential for the recovery of liver mass and function after hepatectomy. Regarding the other markers of liver function/injury, serum levels of glutamic oxaloacetic transaminase/glutamate pyruvate transaminase/lactate dehydrogenase and bilirubin did not differ between control and L-Pdk1KO mice before PH, and were moderately increased after PH, showing somewhat higher levels in the latter mice (Fig. 2D). Blood glucose levels were decreased after PH but did not show any difference between the two groups.

Figure 2.

Impaired liver regeneration after PH in L-Pdk1KO mice. (A) Seventy percent PH was lethal within 12 hours with no liver regeneration. (B) Thirty percent PH allowed the L-Pdk1KO mice to survive at least until 14 days post-PH with no liver regeneration. (C) Serum albumin levels were reduced in L-Pdk1KO mice transiently after PH. (D) Serum levels of glutamic oxaloacetic transaminase/glutamate pyruvate transaminase/lactate dehydrogenase and bilirubin and blood glucose level were measured pre-PH and post-PH. No significant differences were observed between control and L-Pdk1KO mice. Data are from at least three independent experiments, expressed as mean ± SEM.

Apoptotic cell death was very mildly induced by PH equally in control and L-Pdk1 KO liver, but did not differ between the groups (Fig. 2E). This fact indicates that apoptotic cell death did not contribute to the reduced liver mass recovery in L-Pdk1 KO mice.

Mitotic Responses in L-Pdk1KO Mice After Hepatectomy.

Mitotic responses began immediately after PH in L-Pdk1KO mice even though liver regeneration was severely impaired (Fig. 3). Mitotic hepatocytes 3 days post-PH (Fig. 3A) and BrdU/PCNA-positive hepatocytes 2 days post-PH (Fig. 3B and C) were equally apparent in control and L-Pdk1KO mice. The numbers of mitotic cells, BrdU-positive cells, and PCNA-positive cells observed at each time (48 hours, 72 hours) in the post-PH liver tissues were not statistically different between the groups. BrdU incorporation into nuclei, as well as PCNA expression, also remained identical. These data suggest that deletion of PDK1 in liver did not affect the initial mitotic response of hepatocytes after PH.

Figure 3.

Normal mitotic response after PH in L-Pdk1KO mice. (A) Hematoxylin-eosin staining showing mitotic hepatocytes 48 and 72 hours post-PH in control and L-Pdk1KO liver. (Arrowheads indicate mitotic hepatocytes. Original magnification ×200) Counts of mitotic, BrdU-positive, and PCNA-positive hepatocytes (immunohistochemistry and WB) in the regenerating liver document equivalent mitotic responses in control and L-Pdk1KO liver (A, B, C). Each photo is representative of at least three independent experiments. Data are expressed as mean ± SEM. (D) WB and electrophoretic mobility shift assays showing STAT3 phosphorylated (Tyr705 and Ser727) and activated to the same extent in control and L-Pdk1KO liver. Each blot is representative of at least three independent experiments.

In support of these observations, STAT3, which is a pivotal transcription factor in the post-PH mitotic response, showed similar activity in both control and L-Pdk1KO mice (Fig. 3D). Phosphorylation of STAT3 at Tyr705 and Ser727 and its binding to DNA occurred immediately after PH, and recovered to basal levels by 72 hours in both knockout and control mice. STAT3 activation after PH also did not differ in its intensity and timing in control and L-Pdk1KO mice.

Cell Size (Cell Growth) After Hepatectomy and Activation of PDK1-Associated Signals.

Hepatocyte cell size in L-Pdk1KO mice without PH was slightly smaller than in controls, but the difference was not statistically significant (Fig. 4A). Cell size in normal livers increased significantly after PH, but not in L-Pdk1KO mice; rather, there was a tendency to decrease in the latter.

Figure 4.

Post-PH cell growth and activation of Akt, mTOR, p70S6K, and S6 proteins in control and L-Pdk1KO liver (A, B). Responsive cell growth did not occur after PH in L-Pdk1KO mice. Akt, mTOR, p70S6K, and S6 were all activated by PH in control mice, but hardly at all in L-Pdk1KO mice. The data at each time point consist of two representative blots from at least three independent experiments.

PDK1 specifically phosphorylates Akt at Thr308, and its activity can be assessed in this way (Fig. 4B). We found that Akt was phosphorylated at Thr308 immediately but transiently after PH in controls, but not in L-Pdk1KO livers up to 14 days post-PH. Akt phosphorylation at Thr308 was completely abolished in L-Pdk1 KO liver. This means that the liver of L-Pdk1 KO mice is enough to study the function of PDK1, although PDK1 expression is slightly detected in L-Pdk1 KO liver (Fig. 1A). Interestingly, Akt was strongly and persistently phosphorylated at Ser473 in L-Pdk1KO liver even in the quiescent state, as well as after PH, although this is normally carried out by PI3-K–regulated kinases other than PDK1. Also, the increased amounts of total Akt were observed in L-Pdk1 KO liver, which may be the result of increased production of Akt in response to the reduced signal from PDK1.

Among the molecules downstream of PDK1, Akt, mTOR, p70S6K, and S6 may potentially contribute to the maintenance of normal liver regeneration when cell proliferation is suppressed.6 To confirm the involvement of these molecules in the impairment of liver regeneration in L-Pdk1KO mice, phosphorylation (activation) of mTOR, p70S6K, and S6, downstream targets of p70S6K, as well as Akt, was also examined (Fig. 4B). Mtor and p70S6K were weakly phosphorylated even in quiescent liver. Although mTOR, p70S6K, and S6 were all phosphorylated markedly from 4 hours after PH up until 72 hours in controls, they were not or were only very weakly phosphorylated in L-Pdk1KO liver after PH. S6 was markedly phosphorylated even at quiescence, which was slightly and transiently increased after PH in control livers. Deletion of PDK1 suppressed post-PH phosphorylation of S6, which quickly returned to the pre-PH level up to 72 hours post-PH in L-Pdk1KO liver regardless of liver mass recovery. p70S6K responded immediately to PH and possibly phosphorylated S6, but the latter is presumably also phosphorylated by kinases other than p70S6K. Taken together, these findings may suggest the possibility that PDK1-Akt(/mTOR) pathway plays a more important role in post-PH liver regeneration by cell growth, even though p70S6K/S6 pathway is certainly involved in cell growth in other types of cells.32, 33

PDK1/Akt-Mediated Liver Regeneration by Cell Growth Rather than Cell Proliferation.

To confirm the role of PDK1-Akt/mTOR pathway in liver regeneration, we employed the “pif-pocket” mutant of PDK1, which allows PDK1 to signal exclusively to Akt but not to p70S6K or others.30, 34–36 Adenovirus-mediated introduction of the pif-pocket mutant (L155E) did re-phosphorylate Akt (Thr308), but not p70S6K(Thr389), 4 hours after PH in L-Pdk1KO mice (Fig. 5A). Re-activation of Akt, but not p70S6K, in L-Pdk1KO mice allowed responsive cell growth again in the post-PH liver without affecting cell proliferation, and eventually improved liver regeneration in L-Pdk1KO mice (Fig. 5A, B).

Figure 5.

Effects of Akt and p70S6K on cell growth or proliferation in liver regeneration. (A) Introduction of pif-pocket mutant of PDK1 (L155E) to L-Pdk1KO liver allowed re-phosphorylation of Akt (Thr308), but not p70S6K(Thr389), and normal regeneration of L-Pdk1KO liver 72 hours post-PH. (B) By introducing pif-pocket of PDK1 (L155E) into PDK1-deficient livers, hepatocytes re-grow in size, but post-PH mitotic cell counts are not affected. (C) Introduction of pif-pocket mutant of PDK1 (L155E) to L-Pdk1KO improved liver regeneration even in the 70% PH model. (D) Phosphorylation of p70S6K (Thr389) suppressed by sodium salicylate (NaSal), an inhibitor of p70S6K phosphorylation, in phorbol myristate acetate (PMA)-stimulated AML12 cells. NaSal was added to the culture media 16 hours before the experiment, and then AML12 cells were stimulated by PMA for 1 hour. Inhibition did not affect cell size, but slightly suppressed cell proliferation in phorbol myristate acetate–stimulated AML12 cells. Data are means ± SEM. Each blot is representative of at least three independent experiments.

We also performed the experiment of 70% PH with hepatic introduction of the pif-pocket mutant (L155E), to confirm the effect of Akt on liver cell growth or proliferation after PH (Fig. 5C). Hepatic introduction of pif-pocket mutant let the 70% PH mice survive at least until 72 hours post-PH and recovered liver sufficiently. Post-PH mitosis and cell growth in L-Pdk1 KO liver were observed to the same degree as the post-PH liver of control mice.

Furthermore, we performed an additional experiment to ask whether p70S6K indeed has no influence on hepatocyte cell size. Sodium salicylate, a known inhibitor of p70S6K,37, 38 clearly suppressed phorbol myristate acetate–induced phosphorylation of p70S6K in hepatocytes (Fig. 5D) but had no effect on Akt (data not shown). Inhibition of p70S6K did not affect hepatocyte cell size but slightly suppressed cell proliferation (Fig. 5D). This may suggest that p70S6K is involved mainly in transmitting mitotic signals, not cell growth signals. These data are consistent with our hypothesis that the PDK1–Akt pathway, but not PDK1–p70S6K, plays a pivotal role in liver regeneration mainly by regulating cell size.

Activation of PI3-K Does Not Improve Post-PH Liver Regeneration in L-Pdk1KO Mice.

To confirm the pivotal role of PDK1 in liver regeneration, we performed additional experiments to investigate the effects of PI3-K, a signaling molecule immediately upstream of PDK1, on liver regeneration in L-Pdk1KO mice.

In controls, activation of PI3-K by transducing myr-p110 leads to phosphorylation of Akt (Thr308 and Ser473) and STAT3 (Tyr705) in a dose-dependent manner and results in enlarged livers without PH (Fig. 6A). This may indicate that PI3-K signals to STAT3 for cell proliferation as well as Akt for cell growth, and contributes cooperatively to liver regeneration. An adenovirus vector of myr-p110 was injected to L-Pdk1KO mice at the time of PH (1 × 108 pfu/body). It was found that myr-p110 phosphorylated Akt at Thr308 and Ser473 in control mice, but only at Ser473 in L-Pdk1KO mice, because Akt Thr308 is phosphorylated only by PDK1 whereas Ser473 can be phosphorylated by PI3-K–regulated kinases other than PDK1 (Fig. 6B). Hepatic activation of PI3-K clearly induced more PCNA-positive and mitotic hepatocytes in the liver of L-Pdk1KO mice after PH than in controls (Fig. 6B). However, this was not sufficient to improve impaired liver regeneration and albumin synthesis in L-Pdk1KO mice (Fig. 6C). In contrast, additional deletion of STAT3 in the liver of L-Pdk1KO mice (L-DKO mice) did not affect its regeneration (Fig. 6D). These data indicate that PI3-K is important for the increase of liver mass by signaling STAT3 for cell proliferation and PDK1/Akt for cell growth, but cannot mediate liver regeneration if PDK1/Akt pathway is disturbed. PDK1-mediated signals for cell growth are thus crucial for liver regeneration.

Figure 6.

Effects of PI3-K on liver regeneration in L-Pdk1KO mice. (A) Expression of constitutively active PI3-K (myr-p110) induced phosphorylation of Akt (Thr308 and Ser473) and STAT3 (Tyr705) in the liver and enlarged the liver mass. The amount of adenovirus was adjusted to 1 × 109 plaque-forming units in total by adding LacZ in each mouse. (B) Expression of constitutively active PI3-K (myr-p110) did not phosphorylate Akt at Thr308 and more strongly at Ser473 72 hours post-PH in L-Pdk1KO liver, and induced more hepatocyte proliferation after PH in L-Pdk1KO mice (Hematoxylin-eosin staining and immunostaining for PCNA. Arrowheads indicate mitotic hepatocytes. Original magnification ×200). (C) Activation of PI3-K failed to regenerate L-Pdk1KO liver after PH, despite efficacy in control liver. It also increased serum albumin levels in control mice, but not in L-Pdk1KO mice. Adenovirus vector encoding myr-p110 was intravenously injected at the time of surgery in this experiment (1 × 108 plaque-forming units/body). (D) Additional deletion of STAT3 in PDK1-deficient liver (L-DKO mice) did not further exacerbate liver regeneration in L-Pdk1KO mice. (E) Rescue experiment using myr-p110 showed that activation of PI3-K did not affect post-PH cell size, mitosis, or liver regeneration in L-DKO mice.

To confirm the contribution of PI3-K/STAT3 to post-PH cell growth, we performed 30% PH in DKO mice with hepatic introduction of myr-p110. Constitutive activation of PI3-K failed to increase post-PH cell proliferation and growth, or to improve the impaired liver regeneration in DKO mice (Fig. 6E).

Discussion

Important roles for interleukin-6/STAT3 in liver regeneration have been reported previously.7, 8, 10, 12, 13, 39 However, deletion of STAT3 in liver fails to affect liver regeneration despite almost completely suppressing the mitotic response, where Akt, p70S6K, and mTOR signals are markedly activated.6 It has therefore been suggested that “survival signals” such as Akt, p70S6K, and mTOR may be responsible for the acute response in post-PH liver regeneration, possibly by regulating cell growth and thus may contribute to the initiation and maintenance of liver regeneration when the mitotic response is impaired.6 Conversely, p70S6K/S6, for example, is suggested to be involved in cell proliferation40–42 and post-PH liver regeneration.42 So far, the involvement and the roles of these proteins in cell proliferation and cell growth in liver regeneration have not been well delineated. In the current study, we investigated the roles of PI3-K/PDK1-mediated signaling pathways in liver regeneration after PH from the perspective of cell growth and proliferation. PI3-K signaled STAT3 for cell proliferation and PDK1/Akt for cell growth. We propose that PDK1/Akt-mediated signals (but not p70S6K/S6) are essential contributors to physical and functional liver regeneration mainly by regulating cell size, but not cell proliferation.

PDK1-deficiency in liver had a critical effect on both physical and functional post-PH liver regeneration. Seventy percent PH was lethal to L-Pdk1KO mice; remaining liver tissue did not regenerate in either 70% or 30% PH models in L-Pdk1KO mice despite normal cell proliferation. The increase of cell size (cell growth) that occurred after PH in control mice was completely absent in L-Pdk1KO mice and in fact was even reduced (Fig. 4A). This finding may account for the impaired liver regeneration in L-Pdk1KO mice.

Regarding the molecules downstream of PDK1, Akt and mTOR were very weakly phosphorylated at quiescence, whereas they were markedly phosphorylated 4 hours after PH. In L-Pdk1 KO mice, phosphorylation of these molecules was significantly reduced. In contrast, S6 (and p70S6K) were phosphorylated even at quiescence in control mice, were phosphorylated 4 hours after PH, and had recovered to pre-PH levels by 72 hours post-PH. In L-Pdk1 KO mice, phosphorylation of S6 was suppressed 4 hours after PH but recovered to pre-PH levels despite lack of recovery of liver mass. Considering these findings, it seems that PDK1 signals weakly to Akt/mTOR under quiescent conditions but signals strongly immediately after PH in a transient manner for the liver regeneration response. In contrast, S6 (and p70S6K) may be phosphorylated even when PDK1/p70S6K signals are not fully activated. S6 was phosphorylated after PH but returned to pre-PH levels regardless of liver mass recovery. These dynamics of protein phosphorylation may indicate that PDK1/Akt is more responsive than p70S6K to PH and plays more roles in liver regeneration. Canceling PDK1-mediated signals other than Akt in L-Pdk1KO liver led to full recovery of liver mass and responsive cell growth after PH. These findings suggest that PDK1/p70S6K signals may not be directly involved in cell growth–associated liver regeneration.

Stimulation of PI3-K induced activation of STAT3 and Akt, which enlarged liver and increased levels of serum albumin. This, however, failed to regenerate post-PH liver physically and functionally in L-Pdk1KO mice, despite marked proliferation of hepatocytes (Fig. 6B, C). Furthermore, liver regeneration of Pdk1KO and DKO mice was equally suppressed after PH (Fig. 6D). These findings clearly indicate the potential importance of PDK1 and PDK1-mediated cell growth in liver regeneration but suggest that STAT3-mediated cell proliferation is not involved. It was reported previously that Akt and associated signals are strongly activated immediately after PH in L-Stat3KO mice, in which the post-PH mitotic response is greatly suppressed,6 and it was suggested that activation of these signals may be responsible for compensation of the impaired liver regeneration. Recent evidence indicates an essential role of certain molecules in determining cell size in genetically manipulated animals. Akt, mTOR, p70S6K, and glycogen synthase kinase-3β are all good candidates for determining the original (inborn) cell size in various cells or organs.19–21, 23 This is consistent with our proposal that PDK1/Akt and associated signals contribute to liver regeneration mainly by inducing cell growth (cell size), but not by inducing cell proliferation.

Inhibition of p70S6K by salicylic acid mildly suppressed hepatocyte proliferation, although it was not seen to be involved in cell growth in vitro (Fig. 5C). It has been reported that p70S6K/S6 deletion suppresses cell proliferation in different types of cells,40–42 consistent with our results in vitro. However, hepatic suppression of p70S6K by deletion of PDK1 did not suppress hepatocyte proliferation after PH. Because PI3-K/STAT3 mitotic signals possibly function well enough to initiate/maintain cell proliferation in the post-PH liver, inhibition of p70S6K may not affect post-PH hepatocyte proliferation. Interestingly, mitotic responses and STAT3 activation after PH occurred to the same degree in L-Pdk1KO and control mice (Fig. 3A-D), despite the marked difference in liver regeneration (Fig. 2A, B). This indicates that, at least in the impaired liver regeneration induced by deficient PDK1, the interleukin-6/STAT3 pathway is activated but not sufficient to maintain normal liver regeneration.

Seventy percent PH was lethal in L-Pdk1KO mice (Fig. 2A) but not in L-Stat3KO mice.6 Most L-Pdk1KO mice died within 12 hours of PH with no signs of liver regeneration. Serum markers of liver damage (glutamic oxaloacetic transaminase/glutamate pyruvate transaminase/lactate dehydrogenase and bilirubin) were moderately elevated 72 hours after PH even in 30% PH L-Pdk1KO mice (Fig. 2D), suggesting the possibility of post-PH extended liver failure in 70% PH L-Pdk1KO mice. The exact cause of death is not yet clear; however, remaining liver function certainly seems responsible for any survival. Liver function assessed by serum albumin level showed persistent reduction 14 days after PH. Reduced liver function of L-Pdk1KO mice may therefore be one of the major causes of death.

In conclusion, we have demonstrated a pivotal role for PDK1/Akt-mediated acute responsive cell growth in liver regeneration, and for PI3-K/STAT3-mediated mitotic pathway in a rodent PH model (Fig. 7). PDK1/Akt signals are especially crucial for avoiding post-PH liver failure as well as for maintaining normal liver regeneration capacity. Although further studies are necessary, the current report has begun to elucidate the molecular mechanisms underlying liver regeneration.

Figure 7.

Schematic illustration of the pivotal signaling pathways in liver regeneration. Janus kinase and PI3-K signaling to STAT3 for hepatocyte proliferation. PDK1/Akt plays a central role in cell growth–mediated liver regeneration and anti-apoptosis. p70S6K mediates protein synthesis and cell proliferation but may not be essential for liver regeneration.

Ancillary