In regeneration mouse models and human livers, fat accumulation occurs transiently and is required for physiological hepatic regeneration after partial hepatectomy (PH). Lipid droplets are formed mainly by triglyceride (TG) levels peaking at 24–48 hours after PH and reducing to baseline at 72 hours after PH. Both overload and deficiency of lipid droplets are harmful to hepatocyte proliferation during liver regeneration.
7- Fernandez M.A.
- Albor C.
- Ingelmo-Torres M.
- Nixon S.J.
- Ferguson C.
- Kurzchalia T.
- Tebar F.
- Enrich C.
- Parton R.G.
- Pol A.
Caveolin-1 is essential for liver regeneration.
, 8- Fernandez-Rojo M.A.
- Restall C.
- Ferguson C.
- Martel N.
- Martin S.
- Bosch M.
- Kassan A.
- Leong G.M.
- Martin S.D.
- McGee S.L.
- Muscat G.E.
- Anderson R.L.
- Enrich C.
- Pol A.
- Parton R.G.
Caveolin-1 orchestrates the balance between glucose and lipid-dependent energy metabolism: implications for liver regeneration.
, 9- DeAngelis R.A.
- Markiewski M.M.
- Taub R.
- Lambris J.D.
A high-fat diet impairs liver regeneration in C57BL/6 mice through overexpression of the NF-kappaB inhibitor, IkappaBalpha.
, 10Trimming the fat from liver regeneration.
, 11- Kohjima M.
- Tsai T.H.
- Tackett B.C.
- Thevananther S.
- Li L.
- Chang B.H.
- Chan L.
Delayed liver regeneration after partial hepatectomy in adipose differentiation related protein-null mice.
, 12- Hamano M.
- Ezaki H.
- Kiso S.
- Furuta K.
- Egawa M.
- Kizu T.
- Chatani N.
- Kamada Y.
- Yoshida Y.
- Takehara T.
Lipid overloading during liver regeneration causes delayed hepatocyte DNA replication by increasing ER stress in mice with simple hepatic steatosis.
However, the mechanisms involved in lipid accumulation and lipid metabolism during liver regeneration remain obscure. Liver-specific knockout of the de novo lipid synthesis gene
Fasn showed no change in 5-bromo-2′-deoxyuridine (BrdU)-positive hepatocytes.
13- Newberry E.P.
- Kennedy S.M.
- Xie Y.
- Luo J.
- Stanley S.E.
- Semenkovich C.F.
- Crooke R.M.
- Graham M.J.
- Davidson N.O.
Altered hepatic triglyceride content after partial hepatectomy without impaired liver regeneration in multiple murine genetic models.
After PH, there was no time-dependent impairment in liver regeneration in
PPAR-α knockout mouse models.
13- Newberry E.P.
- Kennedy S.M.
- Xie Y.
- Luo J.
- Stanley S.E.
- Semenkovich C.F.
- Crooke R.M.
- Graham M.J.
- Davidson N.O.
Altered hepatic triglyceride content after partial hepatectomy without impaired liver regeneration in multiple murine genetic models.
Systemic knockdown of
PPAR-α resulted in a 12- to 24-hour lag to the G1/S checkpoint.
14- Anderson S.P.
- Yoon L.
- Richard E.B.
- Dunn C.S.
- Cattley R.C.
- Corton J.C.
Delayed liver regeneration in peroxisome proliferator-activated receptor-alpha-null mice.
Liver-specific deletion of
PPAR-α significantly reduced hepatocyte proliferation at 32 hours.
15- Xie G.
- Yin S.
- Zhang Z.
- Qi D.
- Wang X.
- Kim D.
- Yagai T.
- Brocker C.N.
- Wang Y.
- Gonzalez F.J.
- Wang H.
- Qu A.
Hepatocyte peroxisome proliferator-activated receptor alpha enhances liver regeneration after partial hepatectomy in mice.
The fatty acid (FA) transporter cluster of differentiation 36 (CD36) contributes to enhanced transient regeneration-associated steatosis.
16- Zhao Y.
- Tran M.
- Wang L.
- Shin D.J.
- Wu J.
PDK4-deficiency reprograms intrahepatic glucose and lipid metabolism to facilitate liver regeneration in mice.
However, the accumulation and utilization of lipid droplets during liver regeneration and the physiological significance of this process remain unclear.
Mammalian target of rapamycin 2 (mTORC2) is a master regulator of lipid synthesis and regulatory associated protein Of MTOR Complex 1 (RPTOR)-independent companion of mTOR complex 2 (Rictor) is a key component of the mTORC2 complex.
17- Saxton R.A.
- Sabatini D.M.
mTOR signaling in growth, metabolism, and disease.
Liver-specific knockout Rictor (R-LKO) mouse models show liver-specific deficiency of mTORC2 and marked hyperglycemia, hyperinsulinemia, and hypolipidemia.
18- Hagiwara A.
- Cornu M.
- Cybulski N.
- Polak P.
- Betz C.
- Trapani F.
- Terracciano L.
- Heim M.H.
- Ruegg M.A.
- Hall M.N.
Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c.
Decreased lipid levels are reported in the mTORC2 knockout (KO) liver because of a remarkable deficiency in de novo lipid synthesis. The transcription of de novo lipid synthesis–related genes, such as
Fasn,
Acly, and
Srebp1c, is inhibited significantly under normal conditions in R-LKO mice. mTORC2 also promotes hepatic tumorigenesis through lipid synthesis, with sphingolipids and cardiolipin showing a causal effect during this process.
19- Guri Y.
- Colombi M.
- Dazert E.
- Hindupur S.K.
- Roszik J.
- Moes S.
- Jenoe P.
- Heim M.H.
- Riezman I.
- Riezman H.
- Hall M.N.
mTORC2 promotes tumorigenesis via lipid synthesis.
Prior studies have shown that mTORC2 is necessary for timely liver regeneration.
20- Xu M.
- Wang H.
- Wang J.
- Burhan D.
- Shang R.
- Wang P.
- Zhou Y.
- Li R.
- Liang B.
- Evert K.
- Utpatel K.
- Xu Z.
- Song X.
- Che L.
- Calvisi D.F.
- Wang B.
- Chen X.
- Zeng Y.
- Chen X.
mTORC2 signaling is necessary for timely liver regeneration after partial hepatectomy.
In this study, both male and female R-LKO mice showed accumulated lipid droplets in the regenerated liver. Further data suggest the FAs are mainly transported into hepatocytes. mTORC2 deficiency leads to significantly down-regulated peroxisome proliferator-activated receptor α (PPAR-α) levels and excess lipid droplet accumulation. The PPAR-α pathway down-regulation causes impaired hepatocyte proliferation and poor survival in R-LKO mice. Furthermore, we identified glucosylceramide (GluCer) as a novel activator of the PPAR-α pathway, which activated the pathway in vivo, promoted hepatocyte proliferation, and enhanced the survival rate in R-LKO mice after PH. Therefore, mTORC2 promoted liver regeneration via the GluCer–PPAR-α pathway, thereby establishing its newly discovered role in FA oxidation.
Discussion
The role and source of accumulated lipid droplets during liver regeneration are controversial.
7- Fernandez M.A.
- Albor C.
- Ingelmo-Torres M.
- Nixon S.J.
- Ferguson C.
- Kurzchalia T.
- Tebar F.
- Enrich C.
- Parton R.G.
- Pol A.
Caveolin-1 is essential for liver regeneration.
,13- Newberry E.P.
- Kennedy S.M.
- Xie Y.
- Luo J.
- Stanley S.E.
- Semenkovich C.F.
- Crooke R.M.
- Graham M.J.
- Davidson N.O.
Altered hepatic triglyceride content after partial hepatectomy without impaired liver regeneration in multiple murine genetic models.
,23- Abshagen K.
- Degenhardt B.
- Liebig M.
- Wendt A.
- Genz B.
- Schaeper U.
- Stumvoll M.
- Hofmann U.
- Frank M.
- Vollmar B.
- Kloting N.
Liver-specific Repin1 deficiency impairs transient hepatic steatosis in liver regeneration.
mTORC2 plays an important role in de novo lipid synthesis. The TG content in the livers of R-LKO mice was decreased significantly under normal conditions.
18- Hagiwara A.
- Cornu M.
- Cybulski N.
- Polak P.
- Betz C.
- Trapani F.
- Terracciano L.
- Heim M.H.
- Ruegg M.A.
- Hall M.N.
Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c.
However, we found that during liver regeneration, TG and FA contents in the R-LKO mouse livers were increased significantly after PH. Xu et al
20- Xu M.
- Wang H.
- Wang J.
- Burhan D.
- Shang R.
- Wang P.
- Zhou Y.
- Li R.
- Liang B.
- Evert K.
- Utpatel K.
- Xu Z.
- Song X.
- Che L.
- Calvisi D.F.
- Wang B.
- Chen X.
- Zeng Y.
- Chen X.
mTORC2 signaling is necessary for timely liver regeneration after partial hepatectomy.
also showed an accumulation of lipid droplets after 3 days. Furthermore, the delayed lipid accumulation was estimated by Oil Red O staining, similar to our study, in which we have used the same technique to evaluate the accumulation tendency and reconfirmed it by 2 other methods, namely, biochemical analyses and lipidomics using liquid chromatography. Liver-specific KO of AKT1/2 results in an antisteatotic effect leading to increased lethality,
24- Pauta M.
- Rotllan N.
- Fernandez-Hernando A.
- Langhi C.
- Ribera J.
- Lu M.
- Boix L.
- Bruix J.
- Jimenez W.
- Suarez Y.
- Ford D.A.
- Baldan A.
- Birnbaum M.J.
- Morales-Ruiz M.
- Fernandez-Hernando C.
Akt-mediated foxo1 inhibition is required for liver regeneration.
resembling the downstream effects of mTORC2 KO. However, AKT1/AKT2 KO
24- Pauta M.
- Rotllan N.
- Fernandez-Hernando A.
- Langhi C.
- Ribera J.
- Lu M.
- Boix L.
- Bruix J.
- Jimenez W.
- Suarez Y.
- Ford D.A.
- Baldan A.
- Birnbaum M.J.
- Morales-Ruiz M.
- Fernandez-Hernando C.
Akt-mediated foxo1 inhibition is required for liver regeneration.
might differ from mTORC2 KO in terms of AKT activity. For example, in AKT1/AKT2 KO mice, the activity of both AKT phosphorylated site at serine 473 (pSer473) and AKT phosphorylated site at threonine 308 (pThr308) is lost, whereas in mTORC2 KO mice, only AKT pSer473 activity is almost lost. In line with this notion, only AKT (Ser473) was reported to rescue fatty acid synthesis in R-LKO mice. AKT Thr308 activated by pyruvate dehydrogenase kinase 1 (PDK1). PDK1 could phosphorylate ribosomal protein S6 kinase (S6K) and mTORC1,
25- Gonzalez A.
- Hall M.N.
- Lin S.C.
- Hardie D.G.
AMPK and TOR: the yin and yang of cellular nutrient sensing and growth control.
inhibit which could strongly provoke PPAR-α ketogenesis pathway,
26- Sengupta S.
- Peterson T.R.
- Laplante M.
- Oh S.
- Sabatini D.M.
mTORC1 controls fasting-induced ketogenesis and its modulation by ageing.
,27S6 kinase 2 deficiency enhances ketone body production and increases peroxisome proliferator-activated receptor alpha activity in the liver.
which might be a main cause of antisteatotic effect.
28- Mora A.
- Lipina C.
- Tronche F.
- Sutherland C.
- Alessi D.R.
Deficiency of PDK1 in liver results in glucose intolerance, impairment of insulin-regulated gene expression and liver failure.
Our data also showed that mRNA expression levels of de novo FA synthesis genes, such as
Fasn,
Acly, and
Srebp1c, were decreased significantly in regenerated R-LKO livers 36 hours after PH, which is the same time point at which lipid accumulation also occurred. We concluded that de novo FA synthesis did not correlate with TG accumulation. These results are consistent with normal liver regeneration in
Fasn KO mice.
13- Newberry E.P.
- Kennedy S.M.
- Xie Y.
- Luo J.
- Stanley S.E.
- Semenkovich C.F.
- Crooke R.M.
- Graham M.J.
- Davidson N.O.
Altered hepatic triglyceride content after partial hepatectomy without impaired liver regeneration in multiple murine genetic models.
Furthermore, the expression of the lipid transport-related genes
Cd36 and
Fabp4 peaked at 24 hours after PH. To ensure the effective use of lipid droplets, the PPAR-α–FA oxidation pathway also peaked at 24 hours. However, lipid overloading delays DNA replication in hepatocytes.
9- DeAngelis R.A.
- Markiewski M.M.
- Taub R.
- Lambris J.D.
A high-fat diet impairs liver regeneration in C57BL/6 mice through overexpression of the NF-kappaB inhibitor, IkappaBalpha.
To overcome this side effect, lipid export-related genes such as
Mttp21The regulation of hepatic fatty acid synthesis and partitioning: the effect of nutritional state.
also showed a peak in expression after lipid oxidation to remove extra fat after supplying energy. This removal might be downstream of the PPAR-α pathway and not the signal derived from lipid overload. As in R-LKO mice, despite lipid overloading, the
Mttp genes remained silent, similar to PPAR-α pathway genes. Moreover, in the control group, the genes responsible for hepatocyte transport were up-regulated after activation of the PPAR-α pathway. Consequently, it can be concluded that lipid metabolism is a fine-tuned process during liver regeneration. Overall, TGs and FAs are transported into hepatocytes, and the PPAR-α pathway metabolizes FAs to provide energy for hepatocyte proliferation. These results are consistent with previously obtained findings that cluster of differentiation 36 contributes to the enhancement of transient regeneration-associated steatosis.
16- Zhao Y.
- Tran M.
- Wang L.
- Shin D.J.
- Wu J.
PDK4-deficiency reprograms intrahepatic glucose and lipid metabolism to facilitate liver regeneration in mice.
Hepatocytes are selected to increase lipid transport rather than synthesis, which requires more energy and raw materials, as a survival mode in emergencies such as PH.
Liver regeneration is a highly precise and efficient process.
29- Fausto N.
- Campbell J.S.
- Riehle K.J.
Liver regeneration.
Hepatocyte size starts increasing after PH, reaches a peak value at 36 hours, and then returns to normal 96 hours after PH.
30- Caldez M.J.
- Van Hul N.
- Koh H.W.L.
- Teo X.Q.
- Fan J.J.
- Tan P.Y.
- Dewhurst M.R.
- Too P.G.
- Talib S.Z.A.
- Chiang B.E.
- Stunkel W.
- Yu H.
- Lee P.
- Fuhrer T.
- Choi H.
- Bjorklund M.
- Kaldis P.
Metabolic remodeling during liver regeneration.
The hepatocyte population also increases from 36 hours onward after PH and is sustained until the end of liver regeneration.
30- Caldez M.J.
- Van Hul N.
- Koh H.W.L.
- Teo X.Q.
- Fan J.J.
- Tan P.Y.
- Dewhurst M.R.
- Too P.G.
- Talib S.Z.A.
- Chiang B.E.
- Stunkel W.
- Yu H.
- Lee P.
- Fuhrer T.
- Choi H.
- Bjorklund M.
- Kaldis P.
Metabolic remodeling during liver regeneration.
Reconstruction requires high bioenergy to ensure liver cell repopulation. However, the mechanism of metabolic pattern transformation during different stages of liver regeneration remains unclear. Glucose levels decreased significantly during liver regeneration after PH, similar to the results of previous studies.
30- Caldez M.J.
- Van Hul N.
- Koh H.W.L.
- Teo X.Q.
- Fan J.J.
- Tan P.Y.
- Dewhurst M.R.
- Too P.G.
- Talib S.Z.A.
- Chiang B.E.
- Stunkel W.
- Yu H.
- Lee P.
- Fuhrer T.
- Choi H.
- Bjorklund M.
- Kaldis P.
Metabolic remodeling during liver regeneration.
Oxidative phosphorylation levels have been reported to decrease after PH
31- Vendemiale G.
- Guerrieri F.
- Grattagliano I.
- Didonna D.
- Muolo L.
- Altomare E.
Mitochondrial oxidative phosphorylation and intracellular glutathione compartmentation during rat liver regeneration.
; accordingly, during liver regeneration, increased cell size is accompanied by down-regulation of mitochondrial and lipid biosynthesis-related genes. Furthermore, the glycolysis rate increases with cell size.
30- Caldez M.J.
- Van Hul N.
- Koh H.W.L.
- Teo X.Q.
- Fan J.J.
- Tan P.Y.
- Dewhurst M.R.
- Too P.G.
- Talib S.Z.A.
- Chiang B.E.
- Stunkel W.
- Yu H.
- Lee P.
- Fuhrer T.
- Choi H.
- Bjorklund M.
- Kaldis P.
Metabolic remodeling during liver regeneration.
,32Crabtree effect and the anaerobic glycolysis of the regenerating rat liver.
,33[Study of some aspects of liver regeneration after partial hepatectomy. I. Oxygen consumption and glycolysis of regenerating tissue].
For lipid metabolism, lipid droplets started to accumulate 12 hours after PH, reaching a peak value at 36 hours, and were reduced to the basal level during the advanced stage of liver regeneration. Systemic knockdown of PPAR-α causes a 12- to 24-hour lag at the G1/S checkpoint,
14- Anderson S.P.
- Yoon L.
- Richard E.B.
- Dunn C.S.
- Cattley R.C.
- Corton J.C.
Delayed liver regeneration in peroxisome proliferator-activated receptor-alpha-null mice.
whereas liver-specific deletion of PPAR-α significantly reduces hepatocyte proliferation at 32 hours.
15- Xie G.
- Yin S.
- Zhang Z.
- Qi D.
- Wang X.
- Kim D.
- Yagai T.
- Brocker C.N.
- Wang Y.
- Gonzalez F.J.
- Wang H.
- Qu A.
Hepatocyte peroxisome proliferator-activated receptor alpha enhances liver regeneration after partial hepatectomy in mice.
Acceleration of lipid metabolism by a PPAR-α agonist before PH accelerates liver regeneration.
34- Aoyama T.
- Ikejima K.
- Kon K.
- Okumura K.
- Arai K.
- Watanabe S.
Pioglitazone promotes survival and prevents hepatic regeneration failure after partial hepatectomy in obese and diabetic KK-A(y) mice.
In accordance with previous findings, the PPAR-α activator WY14643 significantly increased liver size and proliferation during liver regeneration.
35- Fan S.
- Gao Y.
- Qu A.
- Jiang Y.
- Li H.
- Xie G.
- Yao X.
- Yang X.
- Zhu S.
- Yagai T.
- Tian J.
- Wang R.
- Gonzalez F.J.
- Huang M.
- Bi H.
YAP-TEAD mediates peroxisome proliferator-activated receptor alpha-induced hepatomegaly and liver regeneration in mice.
In summary, we hypothesized that glycolysis is a pivotal energy source for the early cell size growth phase, and FA oxidation is a major energy source for the increase in both hepatocyte size and proliferation.
Fatty acid oxidation is an essential factor that promotes cell proliferation during liver regeneration. PFT-α treatment every 48 hours after PH only inhibited p53 expression in the nucleus and had no effect on p21 expression.
36- Eipel C.
- Schuett H.
- Glawe C.
- Bordel R.
- Menger M.D.
- Vollmar B.
Pifithrin-alpha induced p53 inhibition does not affect liver regeneration after partial hepatectomy in mice.
Conversely, daily pretreatment of mice with the p53 inhibitor PFT-α 1 week before PH significantly reduced the levels of p21 protein in hepatocytes.
37- Inoue Y.
- Tomiya T.
- Yanase M.
- Arai M.
- Ikeda H.
- Tejima K.
- Ogata I.
- Kimura S.
- Omata M.
- Fujiwara K.
p53 May positively regulate hepatocyte proliferation in rats.
However, acceleration of the cell cycle by p21 inhibition did not promote FA oxidation and could not drive hepatocyte proliferation and liver regeneration. Consistently, it did not rescue the survival rate of the R-LKO mice. In turn, the PPAR-α agonist increased the oxidation of accumulated lipids in the mTORC2-deficient liver during liver regeneration. Finally, improvement in lipid oxidation promoted hepatocyte proliferation and rescued survival rate.
Previous studies have shown that FAs and their metabolites are activators of the PPAR-α signaling pathway. Considering the accumulated FA and TG contents and depressed PPAR-α activation in R-LKO livers after PH, a lipidomics analysis was performed to screen for potential activators of PPAR-α. The most down-regulated SPs were considered. SPT is a key enzyme in SP synthesis, and liver-specific SPT KO mice show disrupted liver regeneration after PH.
38- Li Z.
- Kabir I.
- Jiang H.
- Zhou H.
- Libien J.
- Zeng J.
- Stanek A.
- Ou P.
- Li K.R.
- Zhang S.
- Bui H.H.
- Kuo M.S.
- Park T.S.
- Kim B.
- Worgall T.S.
- Huan C.
- Jiang X.C.
Liver serine palmitoyltransferase activity deficiency in early life impairs adherens junctions and promotes tumorigenesis.
However, the role of GluCer (one of the terminal products of SP synthesis) during liver regeneration remains unclear because the GluCer content showed no change in SPT-deficient livers. GluCer is a key factor for mTORC2 that induces tumorigenesis.
19- Guri Y.
- Colombi M.
- Dazert E.
- Hindupur S.K.
- Roszik J.
- Moes S.
- Jenoe P.
- Heim M.H.
- Riezman I.
- Riezman H.
- Hall M.N.
mTORC2 promotes tumorigenesis via lipid synthesis.
Furthermore, the SPT inhibitor myriocin significantly decreased GluCer levels in hepatic tumors
19- Guri Y.
- Colombi M.
- Dazert E.
- Hindupur S.K.
- Roszik J.
- Moes S.
- Jenoe P.
- Heim M.H.
- Riezman I.
- Riezman H.
- Hall M.N.
mTORC2 promotes tumorigenesis via lipid synthesis.
and inhibited hepatocyte cell proliferation. In addition, our study showed that GluCer could activate the PPAR-α pathway and significantly rescue the controlled proliferation in the mTORC2-deficient livers after PH.
mTORC2 regulates glucose and lipid metabolism through AKT.
18- Hagiwara A.
- Cornu M.
- Cybulski N.
- Polak P.
- Betz C.
- Trapani F.
- Terracciano L.
- Heim M.H.
- Ruegg M.A.
- Hall M.N.
Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c.
In turn, AKT has been reported to facilitate hepatocyte proliferation by inhibiting Forkhead box O1 expression
24- Pauta M.
- Rotllan N.
- Fernandez-Hernando A.
- Langhi C.
- Ribera J.
- Lu M.
- Boix L.
- Bruix J.
- Jimenez W.
- Suarez Y.
- Ford D.A.
- Baldan A.
- Birnbaum M.J.
- Morales-Ruiz M.
- Fernandez-Hernando C.
Akt-mediated foxo1 inhibition is required for liver regeneration.
; however, its relationship with PPAR-α currently is unknown. We showed that after SC79 (an AKT activator) treatment in R-LKO mice, the PPAR-α pathway also was activated, possibly because of the promotion of GluCer synthesis by SC79, which is supported by evidence from human liver biopsy specimens showing that AKT (Ser473) activity is correlated positively with GluCer level.
19- Guri Y.
- Colombi M.
- Dazert E.
- Hindupur S.K.
- Roszik J.
- Moes S.
- Jenoe P.
- Heim M.H.
- Riezman I.
- Riezman H.
- Hall M.N.
mTORC2 promotes tumorigenesis via lipid synthesis.
Therefore, we speculated that the down-regulation of the PPAR-α pathway in the R-LKO liver is dependent on AKT activation.
Therefore, using R-LKO mice in the present study, we elucidated the mechanisms involved in lipid metabolism and function during liver regeneration. Furthermore, we showed that the generalized lipid accumulation observed in the mTORC2-deficient liver during regeneration after PH was associated with down-regulation of the PPAR-α pathway. Conversely, we showed that enhanced lipid metabolism by GluCer through the activation of the PPAR-α pathway can improve liver regeneration, and that GluCer may be a key factor associated with the activation of mTORC2 and PPAR-α during liver regeneration.
Materials and Methods
Animals
Generation of Rictor conditional KO (Rictor
flox/flox) mice has been described previously.
18- Hagiwara A.
- Cornu M.
- Cybulski N.
- Polak P.
- Betz C.
- Trapani F.
- Terracciano L.
- Heim M.H.
- Ruegg M.A.
- Hall M.N.
Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c.
Rictor
flox/flox mice were crossed with albumin-Cre (provided by Lu Zhongjie) mice to generate LKO mice with the genotype Rictor
flox/flox–albumin-Cre mice (R-LKO). Rictor
flox/flox mice were used as the littermate control. All animals received humane care according to the Animal Research: Reporting of In Vivo Experiments guidelines, and all animal experiments were approved by the Animal Care and Ethics Committee of Jinan University. Animals used for the experiments were 8- to 12-week-old males or females. All mice had a C57 background. Both male and female mice were used indistinctively. Two thirds of the PH procedures were performed according to a previously described method, the left and median lobes were resected for detail.
39- Mitchell C.
- Willenbring H.
A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice.
,40- Zhang L.
- Liu L.
- He Z.
- Li G.
- Liu J.
- Song Z.
- Jin H.
- Rudolph K.L.
- Yang H.
- Mao Y.
- Zhang L.
- Zhang H.
- Xiao Z.
- Ju Z.
Inhibition of wild-type p53-induced phosphatase 1 promotes liver regeneration in mice by direct activation of mammalian target of rapamycin.
The PPAR-α activator WY-14643 (S8029; Selleck) was administered at 50 mg/kg/d 2 weeks before PH. For GluCer (131304P; Sigma-Aldrich) administration, animals received daily intraperitoneal injections of 1.5 μg/dose for 3 days before PH.
41- Zigmond E.
- Preston S.
- Pappo O.
- Lalazar G.
- Margalit M.
- Shalev Z.
- Zolotarov L.
- Friedman D.
- Alper R.
- Ilan Y.
Beta-glucosylceramide: a novel method for enhancement of natural killer T lymphocyte plasticity in murine models of immune-mediated disorders.
Animals were injected with the p53 inhibitor, pifithrin-α (S2929, 2.2 mg/kg; Selleck), or control vehicle, dimethyl sulfoxide, dissolved in physiological saline 3 times per week for 2 weeks before PH. SC79 (S7863; Selleck) was applied via intraperitoneal injection at a concentration of 0.04 mg/g of body weight, and dimethyl sulfoxide was used as a control vehicle. Myriocin (HY-N6798; Med Chem Express) was administered intraperitoneally to 8-week-old R-LKO and control mice at a dosage of 0.3 mg/kg, every alternate day for 2 weeks. All animals were injected intraperitoneally with 100 mg/kg body weight BrdU (B9285; Sigma-Aldrich) every day, along with 0.8 mg/mL BrdU water before PH until 2 hours before they were killed. Liver remnants were removed, weighed, snap-frozen in liquid nitrogen, or immediately processed for histologic analysis.
Real-Time Quantitative Polymerase Chain Reaction Analysis
Total RNA was isolated from liver samples using TRIzol (15596-026; Invitrogen), reverse-transcribed, and analyzed using real-time quantitative polymerase chain reaction with the SYBR Green master mix (1725272; Bio-Rad) and target-specific primers (
Table 1) on a Bio-Rad CFX96 Touch. Liver complementary DNA samples were pooled from 3 mice per group at 24 and 36 hours after PH.
Table 1Primer Table
F, forward; mRNA, messenger RNA; R, reverse.
Liver Histology and Immunohistochemical Analyses
Liver tissues were fixed in 0.01 mol/L phosphate-buffered saline (pH 7.4) containing 10% formalin, embedded in paraffin, sectioned, and stained with H&E for histologic examination. Immunohistochemistry was performed with mouse anti-BrdU (B2531; Sigma-Aldrich) and Ki-67 (9129S; CST) antibodies at 1:1500 dilution. For Oil Red O staining, the freshly isolated tissues were fixed in optimal cutting temperature compound and sliced at −20°C. Frozen sections were fixed in cold methanol, rinsed with 60% isopropanol, and stained for 20 minutes with Oil Red O solution (E607319; Sangon Biotech) at 37°C. After 2 rinses with distilled water, the slides were stained with hematoxylin for 1 minute, rinsed with distilled water, and mounted. Ten fields per section from 3 sections per mouse were randomly chosen and examined.
Western Blot Analysis
Liver tissues were homogenized and lysed in radio immunoprecipitation assay buffer (P0013B; Beyotime, Co) containing protease and phosphatase inhibitors. The liver tissue homogenate was centrifuged at 15,000 ×
g at 4°C, and the supernatant was collected. Protein concentration was measured using the bicinchoninic acid method. Proteins were separated on 8% and 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to 0.2-μm nitrocellulose membranes for 100 minutes at 4°C. The antibodies used are listed in
Table 2 and were incubated overnight at 4°C. This was followed by incubation with the secondary goat anti-rabbit- (Ultra Sensitive Chemiluminescent Immunoassay Substrate) horseradish-peroxidase antibody at a 1:3000 dilution in 5% bovine serum albumin in Tris buffered saline Tween (TBST) and developed with enhanced chemiluminescence Western chemiluminescent horseradish-peroxidase substrate. The Bio-Rad VersaDoc imaging system and software were used to visualize the proteins. Signal intensities were quantified and normalized to tubulin levels using the ImageJ Pro Plus software (National Institutes of Health, Bethesda, MD).
Table 2Antibodies Used in Western Blot
Measurement of Triglycerides and Free Fatty Acid in the Serum and Liver Tissue
For the serum samples, reagents for biochemical analyses were purchased from DiaSys Diagnostic Systems GmbH (Holzheim, Germany). Serum samples were tested on a HITACHI Clinical Analyzer 7180 according to the manufacturer’s protocol. For liver tissue, the extraction and tests were performed using Biovision-free fatty acid (K612-100) and triglyceride (K622-100) quantification kits. Liver tissues (100 mg) were homogenized in 1 mL solution containing 5% Nonidet P-40 in water, slowly heated in a water bath at 80°C–100°C until the liquid became cloudy, followed by cooling. This process was repeated 2 or 3 times. The samples then were centrifuged at 15,000 × g for 2 minutes, and the supernatant was collected.
Liquid Chromatography Analysis for Lipidomics
Samples were injected onto a C18 Charged Surface Hybrid column (100 mm × 2.1 mm, 1.7 μm; Waters, Manchester, UK) using a 20-minute linear gradient at a flow rate of 0.4 mL/min. The column temperature was set to 60°C. The mobile phase buffer A was constitute by 60% water and 40% acetonitrile, whereas buffer B was constitute by 10% acetonitrile isopropanol (1/9), with 10 mmol/L ammonium formate and 0.1% formic acid in each solution. The solvent gradient was set as follows: 40% B, initial; 43% B, 2 minutes; 50% B, 2.1 minutes; 54% B, 12 minutes; 70% B, 12.1 minutes; 99% B, 18 minutes; and re-equilibration for 2 minutes at 40% B. After separation using ultra performance liquid chromatography, mass spectrometry (MS) was performed using a Xevo G2-S Q-TOF instrument with an electrospray ionization source (Waters). Dynamic range enhancement was implemented in the MS method using Xevo G2-S Q-TOF to improve isotopic distribution and mass accuracy and reduce high ion intensities. In the positive-ion mode, MS parameters were as follows: capillary voltage, 2.5 kV; cone voltage, 24 V; source temperature, 100°C; desolvation temperature, 400°C; desolvation gas flow, 800 L/h; and cone gas flow, 50 L/h. Acquisition was performed from 100 to 1500 daltons. In the negative-ion mode, MS parameters were as follows: capillary voltage, 2.5 kV; cone voltage, 25 V; source temperature, 100°C; desolvation temperature, 500°C; desolvation gas flow, 600 L/h; and cone gas flow, 10 L/h. Acquisition was performed from 100 to 1500 daltons (Novogene Co., Ltd). The raw data were imported to Progenesis QI (Waters) for peak alignment to obtain a peak list containing the retention time, mass-to-charge ratio (m/z), and the peak area of each sample.
Transactivation Assays
We co-transfected the PPRE X3-TK-luc (1015; Addgene) and pSG5 PPAR-α (22751; Addgene) plasmid using Lipo8000 (C0533; Beyotime) into HEK293T cells according to the Lipo8000 protocol; then, we added appropriate concentrations of GluCer (131304P; Sigma-Aldrich). We used a dual-luciferase reporter assay system (E1910; Promega) and a MIX Microtiter plate luminometer (Thermo Scientific) to determine luciferase activity in cell lysates.
Statistical Analyses
Statistical significance analyses were performed using the Student t test, the Wilcoxon test, and the log-rang test. Differences with calculated P values less than .05 were considered statistically significant. For Western blot, we used the Image Pro-Plus software to analyze the density of the bands, which were obtained from the Western blot (WB) experiments.
All authors had access to the study data and have reviewed and approved the final manuscript.
CRediT Authorship Contributions
Lingling Zhang (Conceptualization: Lead; Investigation: Supporting; Supervision: Lead; Writing – original draft: Lead; Writing – review & editing: Lead)
Yanqiu Li (Investigation: Lead)
Ying Wang (Investigation: Lead)
Yugang Qiu (Methodology: Equal; Writing – original draft: Equal)
Hanchuan Mou (Investigation: Equal)
Yuanyao Deng (Investigation: Equal)
Jiyuan Yao (Investigation: Equal)
Zhiqing Xia (Investigation: Equal)
Wenzhe Zhang (Investigation: Equal)
Di Zhu (Investigation: Equal)
Zeyu Qiu (Data curation: Equal)
Zhongjie Lu (Methodology: Equal)
Jirong Wang (Methodology: Equal)
Zhouxin Yang (Methodology: Equal)
GenXiang Mao (Methodology: Supporting)
Dan Chen (Validation: Equal)
Leimin Sun (Project administration: Supporting)
Leiming Liu (Conceptualization: Equal; Investigation: Equal; Writing – original draft: Equal; Writing – review & editing: Equal)
Zhenyu Ju (Conceptualization: Lead; Funding acquisition: Lead)
Data Availability Statement
All data, analytic methods, and study materials have been made available in the results, figures, and materials and methods of this article.
Article info
Publication history
Published online: August 02, 2022
Accepted:
July 15,
2022
Received:
September 2,
2021
Footnotes
Conflicts of interest The authors disclose no conflicts.
Funding This study was supported by National Natural Science Foundation of China grants 92049304, 81801372, 91949125, 82171545, and 82030039; National Key R&D Program of China grant 2021YFA0804903; Program for Guangdong Introducing Innovative and Entrepreneurial Teams grant 2017ZT07S347; and Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory grant 2018GZR110103002. Writing Assistance was provided by the National Natural Science Foundation of China grant 91949125.
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© 2022 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute.