Loss of nephric augmenter of liver regeneration facilitates acute kidney injury via ACSL4-mediated ferroptosis.

Ferroptosis, characterized by lipid accumulation in intracellular compartments, is related to acute kidney injury (AKI), but the mechanism remains obscure. In our previous study, the protective effect of augmenter of liver regeneration (ALR) on AKI was not fully clarified. In this study, we established an AKI mouse model by knocking out proximal tubule-specific ALR and an AKI cell model by inducing hypoxia, as well as enrolled AKI patients, to investigate the effects of ALR on ferroptosis and the progression of AKI. We found that ALR knockout aggravated ferroptosis and increased ROS accumulation and mitochondrial damage, whereas ALR overexpression attenuated ferroptosis through clearance of ROS and maintenance of mitochondrial morphology. Mechanistically, we demonstrated that ALR could directly bind to long-chain-fatty-acid-CoA ligase 4 (ACSL4) and further inhibit the expression of ACSL4 by interacting with certain regions. By resolution liquid chromatography coupled with triple quadruple mass spectrometry, we found that ALR could reduce the contents of polyunsaturated fatty acids, especially arachidonic acid. In addition, we showed that ALR binds to ACSL4 and attenuates oxylipin accumulation, exerting a protective effect against ferroptosis in AKI. Therefore, targeting renal ALR can attenuate ferroptosis and can offer a promising strategy for the treatment of AKI.

Augmenter of liver regeneration reduces mitochondria-derived ROS and NLRP3 inflammasome activation through PINK1/Parkin-mediated mitophagy in ischemia-reperfusion-induced renal tubular injury.

Ischemia-reperfusion (IR) injury is one of the main causes of acute kidney disease (AKI). Several studies have shown that mitochondrial damage, which leads to increased production of reactive oxygen species (ROS), plays a vital role in the pathogenesis of IR-induced AKI. Increased ROS production can cause oxidative damage and activate the inflammasome in renal tubular cells, ultimately resulting in apoptosis or necrosis. Mitophagy is a type of selective autophagy that plays a protective role in AKI by regulating the quality of mitochondria and reducing the production of ROS. We previously reported that the augmenter of liver regeneration (ALR) exhibits antiapoptotic and antioxidant functions, although the precise mechanisms of action need to be studied further. In the current study, ALR was overexpressed and an in vitro model of IR injury was constructed. The overexpression of ALR reduced the production of mitochondria-derived ROS (mtROS), the activation of the NLRP3 inflammasome, and the rate of apoptosis. Moreover, this suppression of mtROS production and inflammasome activation was mediated through the PTEN-induced kinase 1 (PINK1)/Parkin pathway of mitophagy. These results suggest that ALR can alleviate IR-induced apoptosis via the PINK1/Parkin mitophagy pathway to reduce the production of mtROS and limit the activation of the NLRP3 inflammasome.

Interleukin-1ß Attenuates Expression of Augmenter of Liver Regeneration (ALR) by Regulating HNF4α Independent of c-Jun.

Inflammasomes and innate immune cells have been shown to contribute to liver injury, thereby activating Kupffer cells, which release several cytokines, including IL-6, IL-1ß, and TNFα. Augmenter of liver regeneration (ALR) is a hepatotropic co-mitogen that was found to have anti-oxidative and anti-apoptotic properties and to attenuate experimental non-alcoholic fatty liver disease (NAFLD) and cholestasis. Additionally, hepatic ALR expression is diminished in patients with NAFLD or cholestasis, but less is known about the mechanisms of its regulation under these conditions. Therefore, we aimed to investigate the role of IL-1ß in ALR expression and to elucidate the molecular mechanism of this regulation in vitro. We found that ALR promoter activity and mRNA and protein expression were reduced upon treatment with IL-1ß. Early growth response protein-1 (Egr-1), an ALR inducer, was induced by IL-1ß but could not activate ALR expression, which may be attributed to reduced Egr-1 binding to the ALR promoter. The expression and nuclear localization of hepatocyte nuclear factor 4 α (HNF4α), another ALR-inducing transcription factor, was reduced by IL-1ß. Interestingly, c-Jun, a potential regulator of ALR and HNF4α, showed increased nuclear phosphorylation levels upon IL-1ß treatment but did not change the expression of ALR or HNF4α. In conclusion, this study offers evidence regarding the regulation of anti-apoptotic and anti-oxidative ALR by IL-1ß through reduced Egr-1 promoter binding and diminished HNF4α expression independent of c-Jun activation. Low ALR tissue levels in NAFLD and cholestatic liver injury may be caused by IL-1ß and contribute to disease progression.

Role of augmenter of liver regeneration on testicular ischemia and ischemia/reperfusion injury: An experimental study.

Background: Testicular torsion causes ischemic injury, and torsion causes reperfusion injury. Aim: Evaluating the role of augmenter of liver regeneration (ALR) in testicular ischemia and ischemia/reperfusion injury. Materials and Method(s): Seventy-eight (78) healthy Wistar albino male rats were randomly divided into four groups; control (C) (n = 6), sham (S) (n = 24), torsion (T) (n = 24), and torsion/detorsion (T/D) (n = 24). S, T, and T/D groups were divided into four subgroups (n = 6) as 1st, 2nd, 3rd, and 4th hours. Blood, tissue ALR, and histology analyses were performed between groups and subgroups. Results: The increase in plasma ALR values at the 3rd and 4th hours compared to the 1st hour in the T group were significant (P < 0.01, P < 0.001, respectively). In the T/D group, a significant increase was observed in plasma ALR values at the 3rd and 4th hours compared to the 1st hour (P < 0.05, P < 0.001, respectively). Plasma ALR values at the 1st, 2nd, 3rd, and 4th hours were higher in the T and T/D groups than in the C group (P < 0.001, P < 0.05, respectively). Plasma ALR values were higher in the T group at the 1st, 2nd, 3rd, and 4th hours than in the S group (P < 0.05). A significant increase was observed in tissue ALR at the 3rd and 4th hours than at the 1st hour in the T group (P < 0.05, P < 0.001, respectively). A significant increase was observed in tissue ALR at the 3rd and 4th hours than in the 1st hour in the T/D group (P < 0.05, P < 0.001, respectively). Discussion: ALR in plasma and testicular tissue has a potential role in the early diagnosis of testicular torsion and in predicting the prognosis of T and T/D.

Augmenter of liver regeneration: Mitochondrial function and steatohepatitis.

Augmenter of liver regeneration (ALR), a ubiquitous fundamental life protein, is expressed more abundantly in the liver than other organs. Expression of ALR is highest in hepatocytes, which also constitutively secrete it. ALR gene transcription is regulated by NRF2, FOXA2, SP1, HNF4α, EGR-1 and AP1/AP4. ALR's FAD-linked sulfhydryl oxidase activity is essential for protein folding in the mitochondrial intermembrane space. ALR's functions also include cytochrome c reductase and protein Fe/S maturation activities. ALR depletion from hepatocytes leads to increased oxidative stress, impaired ATP synthesis and apoptosis/necrosis. Loss of ALR's functions due to homozygous mutation causes severe mitochondrial defects and congenital progressive multiorgan failure, suggesting that individuals with one functional ALR allele might be susceptible to disorders involving compromised mitochondrial function. Genetic ablation of ALR from hepatocytes induces structural and functional mitochondrial abnormalities, dysregulation of lipid homeostasis and development of steatohepatitis. High-fat diet-fed ALR-deficient mice develop non-alcoholic steatohepatitis (NASH) and fibrosis, while hepatic and serum levels of ALR are lower than normal in human NASH and NASH-cirrhosis. Thus, ALR deficiency may be a critical predisposing factor in the pathogenesis and progression of NASH.

Downregulation of augmenter of liver regeneration impairs the therapeutic efficacy of liver epithelial progenitor cells against acute liver injury by enhancing mitochondrial fission.

Cell-based therapeutic approaches have been proven to be effective strategies for the treatment of acute liver injury (ALI). However, widespread application of these procedures is limited by several key issues, including rapid loss of stemness in vitro, aberrant differentiation into undesirable cell types, and low engraftment in vivo. In this study, liver epithelial progenitor cells (LEPCs) were characterized and transfected with augmenter of liver regeneration (ALR). The results revealed that in ALI mice with CCl4 , the transplantation of ALR-bearing LEPCs into the liver markedly protected mice against ALI by decreasing the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), thus relieving hepatic tissue injury and attenuating inflammatory infiltration. Mechanistically, the knockdown of ALR in LEPCs activated the phosphorylation of dynamin-related protein 1 (Drp1) at the S616 site and thereby enhanced mitochondrial fission. In contrast, the transfection of ALR into LEPCs significantly inhibited Drp1 phosphorylation, thereby favoring the maintenance of mitochondrial integrity and the preservation of adenosine triphosphate contents in LEPCs. Consequently, the ALR-bearing LEPCs transplanted into ALI mice exhibited substantially greater homing ability to the injured liver via the SDF-1/CXCR4 axis than that of LEPCs-lacking ALR. In conclusion, we demonstrated that the transplantation of ALR-transfected LEPCs protected mice against CCl4 -induced ALI, thus offering immense curative potential in the clinic.

Alleviation of CCCP-induced mitochondrial injury by augmenter of liver regeneration via the PINK1/Parkin pathway-dependent mitophagy.

The occurrence of liver diseases is attributed to mitochondrial damage. Mitophagy selectively removes dysfunctional mitochondria, thereby preserving mitochondrial function. Augmenter of liver regeneration (ALR) protects the mitochondria from injury. However, whether ALR protection is associated with mitophagy remains unclear. In this study, mitochondrial damage was induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and long-form ALR (lfRNA)-mediated protection against this damage was investigated. Treatment of HepG2 cells with CCCP elevated the level of intracellular ROS, inhibited ATP production, and increased the mitochondrial membrane potential and cell apoptotic rate. However, in lfALR-transfected cells, CCCP-induced cell injury was clearly alleviated, the apoptosis and ROS levels clearly declined, and the ATP production was significantly enhanced as compared with that in vector-Tx cells. Furthermore, lfALR overexpression promoted autophagy and mitophagy via a PINK1/Parkin-dependent pathway, whereas knockdown of ALR suppressed mitophagy. In lfALR-transfected cells, the phosphorylation of AKT was decreased, thus, downregulating the phosphorylation of the transcription factor FOXO3a at Ser315. In contrast, the phosphorylation of AMPK was enhanced, thereby upregulating the phosphorylation of FOXO3a at Ser413. Consequently, FOXO3a's nuclear translocation and binding to the promoter region of PINK1 was enhanced, and the accumulation of PINK1/Parkin in mitochondria increased. Meanwhile, short-form ALR (sfALR) also increased PINK1 expression through FOXO3a with the similar pathway to lfALR. In conclusion, our data suggest a novel mechanism through which both lfALR and sfALR protect mitochondria by promoting PINK1/Parkin-dependent mitophagy through FOXO3a activation.

Augmenter of Liver Regeneration (ALR) regulates bile acid synthesis and attenuates bile acid-induced apoptosis via glycogen synthase kinase-3ß (GSK-3ß) inhibition.

Bile acid synthesis is restricted to hepatocytes and is rate-limited by CYP7A1 (cholesterol 7α hydroxylase). CYP7A1 expression undergoes tight regulation and is repressed after partial hepatectomy to prevent the accumulation of toxic bile acids. Augmenter of Liver Regeneration (ALR) is a hepatotrophic factor shown to support liver regeneration by augmenting cell proliferation and reducing apoptosis. Nevertheless, less is known about ALR's role in protecting hepatocytes from bile acid accumulation and bile acid-induced apoptosis. Therefore, HepG2 and Huh-7 cells were incubated with recombinant human ALR (rALR) and the expression of CYP7A1, bile acid-induced apoptosis as well as potential molecular mechanisms were analyzed. We found that rALR reduces CYP7A1 expression by increasing nuclear NFκB levels. Moreover, rALR reduced glycochenodeoxycholate (GCDC)-induced-apoptosis by decreased expression of pro-apoptotic Bax and enhanced expression of anti-apoptotic Mcl-1, which is regulated by phosphatidylinositol-3-kinase (PI3K)/Akt activation and glycogen synthase kinase-3ß (GSK3ß) phosphorylation. Inhibitors for PI3K/Akt (GSK690693) and GSK3ß (SB415286) confirmed the specificity of rALR treatment for this pathway. In addition, rALR reduces pro-death signaling by decreasing GCDC-induced JNK phosphorylation. Taken all together, rALR might contribute to protecting hepatocytes from toxic concentrations of bile acids by down-regulating their denovo synthesis, attenuating apoptosis by activation of PI3K/Akt - GSK3ß pathway and inhibition of JNK signaling. Thereby this suggests a new role of ALR in augmenting the process of liver regeneration.

Bile acid-induced apoptosis and bile acid synthesis are reduced by over-expression of Augmenter of Liver Regeneration (ALR) in a STAT3-dependent mechanism.

Cholestasis represents pathophysiologic syndromes defined as an impaired bile flow from the liver. As an outcome, bile acids accumulate and promote hepatocytes injury followed by liver cirrhosis and liver failure. Bile acids induce apoptosis, ER stress and mitochondrial membrane instability. In this study we aimed to investigate the role of cytosolic short form of ALR (Augmenter of Liver Regeneration) in the synthesis of bile acids and bile acid-induced apoptosis. Human hepatoma cells over-expressing the short form of ALR (sfALR, 15 kDa) were incubated with glycochenodeoxycholic acid (GCDCA), and then primary bile acids' production and apoptosis were analyzed. High levels of cytosolic sfALR reduced CYP7A1 mRNA expression and bile acids levels, the rate-limiting enzyme in the classic pathway of bile acid synthesis. This reduction was attributed to STAT3 (signal transducer and activator of transcription 3) activation and reduction of HNF4α (Hepatocyte nuclear factor 4α). Furthermore, apoptosis induction by GCDCA and TRAIL was reduced in cells over-expressing sfALR which was attributed to reduced expression of death receptor 5 (DR5). We found decreased hepatic mRNA levels of ALR and FOXA2 (Forkhead Box A2), an inducer of ALR expression, in human cholestatic liver samples which might explain the increased accumulation of bile acids and bile acid-induced apoptosis in cholestasis patients.

Augmenter of liver regeneration protects the kidney from ischaemia-reperfusion injury in ferroptosis.

Acute kidney injury (AKI) is a common and severe clinical condition with high morbidity and mortality. Ischaemia-reperfusion (I/R) injury remains the major cause of AKI in the clinic. Ferroptosis is a recently discovered form of programmed cell death (PCD) that is characterized by iron-dependent accumulation of reactive oxygen species (ROS). Compelling evidence has shown that renal tubular cell death involves ferroptosis, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR) is a widely distributed multifunctional protein that is expressed in many tissues. Our previous study demonstrated that ALR possesses an anti-oxidant function. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. Here, to elucidate the role of ALR in ferroptosis, ALR expression was inhibited using short hairpin RNA lentivirals (shRNA) in vitro model of I/R-induced AKI. The results suggest that the level of ferroptosis is increased, particularly in the shRNA/ALR group, accompanied by increased ROS and mitochondrial damage. Furthermore, inhibition of system xc- with erastin aggravates ferroptosis, particularly silencing of the expression of ALR. Unexpectedly, we demonstrate a novel signalling pathway of ferroptosis. In summary, we show for the first time that silencing ALR aggravates ferroptosis in an in vitro model of I/R. Notably, we show that I/R induced kidney ferroptosis is mediated by ALR, which is linked to the glutathione-glutathione peroxidase (GSH-GPx) system.

Overexpression of augmenter of liver regeneration (ALR) mitigates the effect of H2O2-induced endoplasmic reticulum stress in renal tubule epithelial cells.

Ischemia/reperfusion is a major cause of acute kidney injury and can induce apoptosis in renal epithelial tubule (HK-2) cells. Accumulating evidence indicates that endoplasmic reticulum (ER) stress is a major contributor to apoptosis in acute kidney injury. We previously reported that augmenter of liver regeneration (ALR) functions as an anti-apoptotic factor in H2O2-treated HK-2 cells although the precise mechanism underlying this action remains unclear. In the present study, we investigate the role of ALR in H2O2-induced ER stress-mediated apoptosis. We overexpressed ALR and established a H2O2-induced ER stress model in HK-2 cells. Overexpression of ALR reduced the level of reactive oxygen species and the rate of apoptosis in H2O2-treated HK-2 cells. Using confocal microscopy and western blot, we observed that ALR colocalized with the ER and mitochondria compartment. Moreover, ALR suppressed ER stress by maintaining the morphology of the ER and reducing the levels of the ER-related proteins, glucose-regulated protein 78 (GRP78), phospho-protein kinase-like ER kinase (p-PERK), phospho-eukaryotic initiation factor 2α (p-eIF2α) and C/EBP-homologous protein (CHOP) significantly (p < 0.05). Mechanistically, ALR promoted Bcl-2 expression and suppressed Bax and cleaved-caspase-3 expression significantly during ER-stress induced apoptosis (p < 0.05). Furthermore, ALR attenuated calcium release from the ER, and transfer to mitochondria, under ER stress. To conclude, ALR alleviates H2O2-induced ER stress-mediated apoptosis in HK-2 cells by suppressing ER stress response and by maintaining calcium homeostasis. Consequently, ALR may protect renal tubule epithelial cells from ischemia/reperfusion induced acute kidney injury.

Augmenter of liver regeneration enhances cell proliferation through the microRNA-26a/Akt/cyclin D1 pathway in hepatic cells.

AIM: Hepatocytes can proliferate and regenerate when injured by toxins, viral infections, and so on. Augmenter of liver regeneration (ALR) is a key regulator of liver regeneration, but the mechanism is unknown. The role of ALR in other cell types is not known. In the present study, we investigated the relationship between microRNA (miRNA)-26a and ALR in the Huh7 cell line and adipose tissue-derived mesenchymal cells from chronic liver disease patients and healthy individuals. METHODS: Huh7 cells were transfected independently with ALR and miRNA-26a expression vectors, and their effects on cell proliferation, the expression of miRNA-26a, and activation of the phosphatase and tensin homolog and Akt signaling pathways were determined. The experiments were repeated on mesenchymal stem cells derived from healthy individuals and chronic liver disease patients to see whether the observations can be replicated in primary cells. RESULTS: Overexpression of ALR or miRNA-26a resulted in an increase of the phosphorylation of Akt and cyclin D1 expression, whereas it resulted in decreased levels of p-GSK-3ß and phosphatase and tensin homolog in Huh7 cells. The inhibition of ALR expression by ALR siRNA or anti-miR-26a decreased the Akt/cyclin D1 signaling pathway, leading to decreased proliferation. Mesenchymal stem cells isolated from the chronic liver disease patients had a higher ALR expression, while the mesenchymal stem cells isolated from healthy volunteers responded to the growth factor treatments for increased ALR expression. It was found that there was a significant increase in miRNA-26a expression and proliferation. CONCLUSIONS: These data clearly showed that ALR induced the expression of miRNA-26a, which downregulated phosphatase and tensin homolog, resulting in an increased p-Akt/cyclin D1 pathway and enhanced proliferation in hepatic cells.

Evaluation of 3D re-cellularized tissue engineering: a drug-induced hepatotoxicity model for hepatoprotectant research.

Background: Application of hepatoprotectants, such as drugs or cytokines, can reduce drug-induced hepatotoxicity (DIH). Due to species-specific differences and abnormal cell polarity and drug-metabolizing enzymes (DMEs), in vivo animal models and in vitro 2D plastic dishes are not good DIH models. The aim of this study was to evaluate whether 3D re-cellularized liver is a sensitive, accurate and efficient DIH model for evaluation of hepatoprotectants. Methods: 2D plastic dishes and 3D decellular liver scaffolds were perfused with HepG2 cells or augmenter of liver regeneration (ALR)-HepG2 cells. These two cell lines were exposed to 4 µM troglitazone (TRO) or 20 µM diclofenac sodium (DIC) on day 8. DME-related genes were analyzed by quantitative reverse transcription polymerase chain reaction; morphological images were revealed by immunohistochemistry, scanning electron microscopy, transmission electron microscopy, and hematoxylin and eosin staining. Results: DME activity and cell polarity were retained and lower doses of TRO and DIC led to DIH in 3D re-cellularized liver. This DIH model reflected the protective effects and mechanism of ALR, which is one of the hepatoprotectants. ALR reduced mitochondrial damage, decreased transaminase level, and alleviated inflammation in TRO-DIH and DIC-DIH. Our re-cellularized liver lobe also showed the effect of ALR in suppressing expression of DMEs. Conclusions: Drug-induced 3D re-cellularized tissue engineering is a sensitive, accurate, and efficient DIH model for evaluation of hepatoprotectants.

Alleviation of palmitic acid-induced endoplasmic reticulum stress by augmenter of liver regeneration through IP3R-controlled Ca2+ release.

The aberrant release of Ca2+ from the endoplasmic reticulum (ER) contributes to the onset of ER stress, which is closely related to the pathogenesis of non-alcoholic fatty liver disease. We previously reported that augmenter of liver regeneration (ALR) alleviates ER stress and protects hepatocytes from lipotoxicity. However, the link between ALR protection and the suppression of ER stress remains unclear. In this study, we investigated whether the protection against liver steatosis afforded by ALR is related to its inhibition of calcium overflow from the ER to the mitochondria. The treatment of HepG2 cells with palmitic acid (PA) upregulated IP3R expression, triggering ER-luminal Ca2+ release and inducing ER stress. However, in ALR-transfected (ALR-Tx) HepG2 cells, PA-induced cell injury was clearly alleviated compared with that in vector-Tx cells. After exposure to PA, IP3R expression was downregulated and ER stress was effectively inhibited in the ALR-Tx cells, and ER-Ca2+ release and simultaneous mitochondrial Ca2+ uptake were lower than those in vector-Tx cells. The knockdown of ALR expression with shRNA abolished the protective effects afforded by ALR transfection. PA treatment also suppressed the interaction between BCL-2 and IP3R in HepG2 cells, whereas this interaction was massively enhanced in the ALR-Tx cells, effectively reducing the IP3R-mediated ER-Ca2+ release and thus mitochondrial Ca2+ influx. Our results suggest that the inhibition of ER stress by ALR is related to the interruption of the interaction between BCL2 and IP3R, demonstrating a novel mechanism of ER stress resistance in ALR-Tx cells.

Augmenter of liver regeneration promotes mitochondrial biogenesis in renal ischemia-reperfusion injury.

Mitochondria are the center of energy metabolism in the cell and the preferential target of various toxicants and ischemic injury. Renal ischemia-reperfusion (I/R) injury triggers proximal tubule injury and the mitochondria are believed to be the primary subcellular target of I/R injury. The promotion of mitochondrial biogenesis (MB) is critical for the prevention I/R injury. The results of our previous study showed that augmenter of liver regeneration (ALR) has anti-apoptotic and anti-oxidant functions. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. To gain further insight into the role of ALR in MB, human kidney (HK)-2 cells were treated with lentiviruses carrying ALR short interfering RNA (siRNA) and a model of hypoxia reoxygenation (H/R) injury in vitro was created. We observed that knockdown of ALR promoted apoptosis of renal tubular cells and aggravated mitochondrial injury, as evidenced by the decrease in the mitochondrial respiratory proteins adenosine triphosphate (ATP) synthase subunit ß, cytochrome c oxidase subunit 1, and nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) beta subcomplex 8. Meanwhile, the production of reactive oxygen species was increased and ATP levels were decreased significantly in HK-2 cells, as compared with the siRNA/control group (p < 0.05). In addition, the mitochondrial DNA copy number and membrane potential were markedly decreased. Furthermore, critical transcriptional regulators of MB (i.e., peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, mitochondrial transcription factor A, sirtuin-1, and nuclear respiratory factor-1) were depleted in the siRNA/ALR group. Taken together, these findings unveil essential roles of ALR in the inhibition of renal tubular cell apoptosis and attenuation of mitochondrial dysfunction by promoting MB in AKI.

Bile acids down-regulate the expression of Augmenter of Liver Regeneration (ALR) via SHP/HNF4α1 and independent of Egr-1.

Bile acids (BA) are signaling molecules that activate nuclear factors and g-protein coupled receptors signaling to maintain metabolic homeostasis. However, accumulation of toxic BA promotes liver injury by initiating inflammation, inducing apoptosis and causing oxidative stress leading to cirrhosis and liver failure. Augmenter of Liver Regeneration (ALR) is a hepatotrophic growth factor with anti-apoptotic and anti-oxidative properties that has been shown to improve mitochondrial and hepatic functions in rats after bile duct ligation. In the current study we aimed to analyze the regulation of the pro-survival protein, ALR, under conditions of cytotoxic concentrations of BA. Promoter studies of ALR (-733/+527 bp) revealed potential binding sites for various transcription factors like Egr-1, HNF4α and two bile acid response elements (BARE). Using a full-length and several truncated promoter constructs for ALR we analyzed promoter activity and showed that BA reduce ALR promoter activity whereas Egr-1 transfection induces it. EMSA and supershift analysis confirmed the specific binding of Egr-1 to its response element within ALR promoter and this binding was reduced upon simultaneous stimulation with BA. We also showed that ALR promoter activity and protein expression are induced by HNF4α1 and repressed by SHP. In conclusion, these results indicate that BA negatively regulate ALR expression by SHP activation.

Augmenter of liver regeneration regulates autophagy in renal ischemia-reperfusion injury via the AMPK/mTOR pathway.

Autophagy may have protective effects in renal ischemia-reperfusion (I/R) injury, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR), a widely distributed multifunctional protein that is originally identified as a hepatic growth factor, may participate in the process of autophagy. To investigate the role of ALR in autophagy, ALR expression is knocked-down in human kidney 2 (HK-2) cells with short hairpin RNA lentivirals. Then, the level of autophagy is measured in the shRNA/ALR group and the shRNA/control group in an in vitro model of ischemia-reperfusion (I/R). The results indicate that the level of autophagy in two groups increase, accompanied by increased reactive oxygen species production, especially in the shRNA/ALR group. The AMPK/mTOR signaling pathway is hyperactive in the shRNA/ALR group. Inhibition of autophagy with the AMPK inhibitor compound C induce apoptosis, especially in the shRNA/ALR group. These findings collectively indicate that ALR negatively regulates the autophagy process through an association with the AMPK/mTOR signaling pathway. Autophagy inhibit apoptosis and play a protective role under conditions of oxidative stress.

Augmenter of liver regeneration (ALR) exhibits a dual signaling impact on hepatic acute-phase response.

The acute-phase response (APR) is an inflammatory process triggered mainly by IL-6 in response to neoplasm, tissue injury, infection or inflammation. Signaling of IL-6 is transduced by activating STAT3 which rapidly results in production of acute-phase proteins (APPs) such as fibrinogen ß (FGB) and haptoglobin (HP). Augmenter of liver regeneration (ALR), a hepatotrophic factor supporting liver regeneration, was reported to be upregulated after liver damage. In this study we analyzed the role of ALR for IL-6 signaling and APR. Thus, we investigated the expression and release of APPs in human liver cells under conditions of increased exogenous or endogenous ALR. HepG2 cells and ALR-reexpressing HepG2 cells were treated with IL-6 in the presence or absence of exogenous ALR for different time points. The mRNA expression and release of both FGB and HP were measured by RT-PCR and ELISA. We found that exogenously applied ALR attenuated the IL-6-induced mRNA expression and protein secretion of both FGB and HP. In contrast, IL-6 stimulation in HepG2 cells which re-express ALR, revealed elevated APR shown by increased mRNA expression and secretion of FGB and HP. Furthermore, we found that ALR-mediated regulation of IL-6-induced APP production is accompanied by altered STAT3 activity. While exogenous ALR reduced the IL-6-induced phosphorylation of STAT3, endogenous ALR enhanced STAT3 activity in liver cells. In conclusion, ALR, dependent on its localization, changes APR at least in part, by modifying STAT3 activation. This study shows a dual signaling of ALR and suggests that ALR is pivotal for the regulation of APR, a crucial event in liver injury and regeneration.

Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Augmenter of liver regeneration (ALR, encoded by GFER) is a widely distributed pleiotropic protein originally identified as a hepatic growth factor. However, little is known about its roles in hepatic physiology and pathology. We created mice with liver-specific deletion of ALR to study its function. METHODS: We developed mice with liver-specific deletion of ALR (ALR-L-KO) using the albumin-Cre/LoxP system. Liver tissues were collected from ALR-L-KO mice and ALR(floxed/floxed) mice (controls) and analyzed by histology, reverse-transcription polymerase chain reaction, immunohistochemistry, electron microscopy, and techniques to measure fibrosis and lipids. Liver tissues from patients with and without advanced liver disease were determined by immunoblot analysis. RESULTS: Two weeks after birth, livers of ALR-L-KO mice contained low levels of ALR and adenosine triphosphate (ATP); they had reduced mitochondrial respiratory function and increased oxidative stress, compared with livers from control mice, and had excessive steatosis, and hepatocyte apoptosis. Levels of carbamyl-palmitoyl transferase 1a and ATP synthase subunit ATP5G1 were reduced in livers of ALR-L-KO mice, indicating defects in mitochondrial fatty acid transport and ATP synthesis. Electron microscopy showed mitochondrial swelling with abnormalities in shapes and numbers of cristae. From weeks 2-4 after birth, levels of steatosis and apoptosis decreased in ALR-L-KO mice, and numbers of ALR-expressing cells increased, along with ATP levels. However, at weeks 4-8 after birth, livers became inflamed, with hepatocellular necrosis, ductular proliferation, and fibrosis; hepatocellular carcinoma developed by 1 year after birth in nearly 60% of the mice. Hepatic levels of ALR were also low in ob/ob mice and alcohol-fed mice with liver steatosis, compared with controls. Levels of ALR were lower in liver tissues from patients with advanced alcoholic liver disease and nonalcoholic steatohepatitis than in control liver tissues. CONCLUSIONS: We developed mice with liver-specific deletion of ALR, and showed that it is required for mitochondrial function and lipid homeostasis in the liver. ALR-L-KO mice provide a useful model for investigating the pathogenesis of steatohepatitis and its complications.

Amelioration of nonalcoholic fatty liver disease by hepatic stimulator substance via preservation of carnitine palmitoyl transferase-1 activity.

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease and so far is supposed to be related with mitochondrial impairment. Hepatic stimulator substance (HSS) has been defined as a liver-protective factor promoting hepatocyte DNA synthesis and hepatic proliferation after liver intoxication. We previously reported that HSS ameliorated hepatocyte death, probably because of its preservation of mitochondria. This study aims to explore whether HSS could protect carnitine palmitoyl transferase-1 (CPT-1), an essential enzyme responsible for ß-oxidation of free fatty acids in mitochondria, from lipotoxicity, thus alleviating hepatic lipid deposition. To test this, the HSS gene was delivered into C57BL/6J mice and efficiently expressed in the liver. NASH mice were prepared with high-fat diet or methionine-choline-deficient diet. The results showed that hepatic inflammation and liver functions were alleviated in the HSS-transfected mice; meanwhile, the activity of CPT-1 was obviously protected. Moreover, oleic acid (OA) treatment resulted in remarkable lipid accumulation in HepG2 cells; this deposition was improved by HSS transfection. Simultaneously, the CPT-1 activity, which was impaired by OA treatment, was profoundly rescued in the HSS-expressing cells. CPT-1 activity was more severely impaired if the OA treatment was combined with S15176, a CPT-1 inhibitor. However, this impairment was effectively reduced by the HSS transfection, and the effect was enhanced by C75, a CPT-1 activator. Interestingly, if the cells were transfected with HSS-siRNA, the preservation of CPT-1 provided by HSS was again diminished. In conclusion, HSS reduces lipotoxicity to mitochondria most likely via preservation of CPT-1.