Augmenter Of Liver Regeneration Gene Research Articles

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.

Augmenter of liver regeneration-mediated mitophagy protects against hepatic ischemia/reperfusion injury

Augmenter of liver regeneration (ALR) is an anti-apoptotic protein found mainly in mitochondria. It protects hepatocytes from ischemia-reperfusion (I/R) injury, but the underlying mechanism is not clear. We found that in rats, delivery of the ALR gene alleviated hepatic I/R injury during orthotopic liver transplantation as evidenced by reduced serum aminotransferase, oxidative stress and apoptosis, and increased expression of autophagy markers. In an in vitro hypoxia/reoxygenation (H/R) model, overexpression of the ALR gene activated autophagy and relieved defective mitophagy via the PINK1/Parkin pathway. Mechanistically, ALR transfection induced the expression of mitofusin 2 (Mfn2) in the H/R model, which led to PINK1 accumulation and mitochondrial translocation of Parkin. Deletion of Mfn2 abolished mitophagy activation induced by ALR transfection, promoted mitochondrial dysfunction, and eventually increased cell apoptosis. Mfn2 administration prevented the inhibition of mitophagy in ALR-knockout (KO) cells, thus attenuated mitochondrial dysfunction and cell apoptosis. In heterozygous ALR-knockout mice treated with a warm I/R injury, marked aggravation of liver injury was associated with mitophagy inhibition and reduction in Mfn2 expression. Taken together, our results confirm that ALR accelerated Parkin translocation and mitophagy via Mfn2, and protected hepatocytes from I/R-induced injury. Our findings provide a novel rationale for the treatment of hepatic I/R injury.

Alleviation of Ischemia-Reperfusion Injury in Liver Steatosis by Augmenter of Liver Regeneration Is Attributed to Antioxidation and Preservation of Mitochondria.

Fatty liver is one of the major impediments to liver surgery and liver transplantation because steatotic hepatocytes are more susceptible to ischemia-reperfusion injury (IRI). In this study, the effects of augmenter of liver regeneration (ALR) on hepatic IRI in steatotic mice were investigated. In vivo, liver steatosis of mice was induced by feeding a methionine-choline-deficient diet for 2 weeks. Three days before hepatic partial warm IRI, mice were transfected with the ALR-containing adenovirus. In an in vitro study, the protective effect of ALR on steatotic HepG2 cells was analyzed after hypoxia/reoxygenation (HR) treatment. The transfection of the ALR gene into steatotic mice attenuated liver injury, inhibiting hepatic oxidative stress, increasing antioxidation capacities, promoting liver regeneration, and consequently suppressing cell apoptosis/death. Furthermore, resistance to HR injury was notably increased in ALR-transfected cells compared with the vector-transfected cells. The HR-induced rise in the mitochondrial reactive oxygen species was reduced, and cellular antioxidant activities were enhanced. The ALR transfection prevented cells from apoptosis, which can be attributed to the preservation of the mitochondrial membrane potential, enhancement of oxygen consumption rate and production of adenosine triphosphate. ALR protects steatotic hepatocytes from IRI by attenuating oxidative stress and mitochondrial dysfunction, as well as improving antioxidant effect. ALR may be used as a potential therapeutic agent when performing surgery and transplantation of steatotic liver.

Deceleration of liver regeneration by knockdown of augmenter of liver regeneration gene is associated with impairment of mitochondrial DNA synthesis in mice.

Hepatic stimulator substance, also known as augmenter of liver regeneration (ALR), is a novel hepatic mitogen that stimulates liver regeneration after partial hepatectomy (PH). Recent work has indicated that a lack of ALR expression inhibited liver regeneration in rats, and the mechanism seems to be related to increased cell apoptosis. The mitochondria play an important role during liver regeneration. Adequate ATP supply, which is largely dependent on effective mitochondrial biogenesis, is essential for progress of liver regeneration. However, ALR gene expression during liver regeneration, particularly its function with mitochondrial DNA synthesis, remains poorly understood. In this study, ALR expression in hepatocytes of mice was suppressed with ALR short-hairpin RNA interference or ALR deletion (knockout, KO). The ALR-defective mice underwent PH, and the liver was allowed to regenerate for 1 wk. Analysis of liver growth and its correlation with mitochondrial biogenesis showed that both ALR mRNA and protein levels increased robustly in control mice with a maximum at days 3 and 4 post-PH. However, ALR knockdown inhibited hepatic DNA synthesis and decelerated liver regeneration after PH. Furthermore, both in the ALR-knockdown and ALR-KO mice, expression of mitochondrial transcription factor A and peroxisome proliferator-activated receptor-γ coactivator-1α were reduced, resulting in impaired mitochondrial biogenesis. In conclusion, ALR is apparently required to ensure appropriate liver regeneration following PH in mice, and deletion of the ALR gene may delay liver regeneration in part due to impaired mitochondrial biogenesis.

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl4-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl4 in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl4-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.

304 HUMAN AUGMENTER OF LIVER REGENERATION TRANSGENIC OVINE EMBRYO DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

The aim of this research was to develop an efficient screening technique to detect transgenic ovine embryos using neomycin resistance (NeoR) and enhanced green fluorescence protein (EGFP) genes as genetic markers. A 0.8-kb fragment of the ovine β-lactoglobulin promoter sequence (BLG) and 1.8 kb of the human augmenter of liver regeneration (ALR) genomic sequence were derived by PCR amplification. These 2 fragments were inserted into the MCS of pGEM-7zf(+) plasmid; this vector was named p7Z-BA. The coding sequence of NeoR was derived by PCR amplification from the plasmid pIRES2-EGFP and was assembled into the MCS of the pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a NeoR gene coding sequence and an EGFP coding sequence linked by an internal ribosome entry site (IRES) sequence downstream of the CMV promoter. The vector pNIE was excised as an NsiI-SspI fragment and inserted into the vector p7Z-BA. In the end, we had a vector named pNEA, which contained the NeoR gene and the EGFP gene regulated by a CMV promoter for expression in a non-tissue specific mode, and the human ALR gene regulated by the BLG promoter for expression specifically in mammary gland. Sheep fetal fibroblast (SFFB) cells were isolated by attachment of tissue pieces from the ear skin of a 1- to 2-month ovine fetus. Karyotypes of the cells at the third passage and after 15 passages were analyzed. The cells proliferated well and more than 72% of the cells maintained a diploid karyotype after 15 passages. Therefore, the SFFB cells are amenable for transgenic cloning manipulations. For transfection, third-passage SFFB cells at 70% confluency were transfected in a 100-mm dish with pNEA (0.5, 1.0, 2.0, 3.0, 5.0, and 7.0 µg) using Lipofectamine 2000 (2, 4, 6, 8, 10, and 12 µL; Invitrogen, Carlsbad, CA). Cells were checked 24 to 48 h after transfection under fluorescence microscopy for GFP expression, and G418 selection (800 µg mL–1) was applied at that time. After 2 weeks, selected colonies were counted and propagated in culture medium containing 300 µg mL–1 G418 for 2 to 3 passages and cryopreserved. A small portion of the cells was analyzed by PCR for gene integration. Bright green fluorescence could be detected 24 to 48 h after transfection. More colonies were selected when transfection parameters were 2 µg of DNA and 10 µL of Lipofectamine. The results of PCR detection showed that the foreign gene was integrated into the genome. A total of 612 oocytes were aspirated from 2- to 5-mm follicles of ovine ovaries collected from an abattoir; 78% of them were matured after 18 h in culture. Four hundred forty-three oocytes were enucleated, and 332 enucleated oocytes were treated for electrofusion with green fluorescence cells. Of these, 180 (54.2%) couplets were fused. A total of 172 reconstructed embryos were stimulated and cultured in vitro, 31 (18%) of which developed to the blastocyst stage, and 19 blastocysts expressed GFP. In conclusion, we established an effective method to select transgenic embryos formed by nuclear transfer using transfected donor cells.

Insights on augmenter of liver regeneration cloning and function.

Hepatic stimulator substance (HSS) has been referred to as a liver-specific but species non-specific growth factor. Gradient purification and sequence analysis of HSS protein indicated that it contained the augmenter of liver regeneration (ALR), also known as hepatopoietin (HPO). ALR, acting as a hepatotrophic growth factor, specifically stimulated proliferation of cultured hepatocytes as well as hepatoma cells in vitro, promoted liver regeneration and recovery of damaged hepatocytes and rescued acute hepatic failure in vivo. ALR belongs to the new Erv1/Alr protein family, members of which are found in lower and higher eukaryotes from yeast to man and even in some double-stranded DNA viruses. The present review article focuses on the molecular biology of ALR, examining the ALR gene and its expression from yeast to man and the biological function of ALR protein. ALR protein seems to be non-liver-specific as was previously believed, increasing the necessity to extend research on mammalian ALR protein in different tissues, organs and developmental stages in conditions of normal and abnormal cellular growth.

Effect of naked eukaryotic expression plasmid encoding rat augmenter of liver regeneration on acute hepatic injury and hepatic failure in rats

To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl(4)) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 microg/kg) was injected into the rats with acute hepatic injury intravenously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl(4) administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 microg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl(4) after 4 h of CCl(4) administration, respectively. Rats living over 96 h were considered as survivals. The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the most effective group after four times of injection of recombinant pcDNA3-ALR plasmid. The indexes of PCNA significantly increased in the recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The level of serum AST and ALT remarkably reduced in recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The results showed that the effect of 200 microg/kg recombinant pcDNA3-ALR plasmid in the rats with acute liver injury was stronger than that of 50 microg/kg pcDNA3-ALR DNA. The effect of intravenous injection of recombinant pcDNA3-ALR plasmid was better. After the rats with acute hepatic failure were treated with recombinant pcDNA3-ALR plasmid, the survival rate (40%) significantly increased in treatment groups compared to control group (15%, P<0.01). The ALR gene may play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing level of AST and ALT in CCl(4)-intoxicated rats.

The augmenter of liver regeneration induces mitochondrial gene expression in rat liver and enhances oxidative phosphorylation capacity of liver mitochondria.

The mammalian augmenter of liver regeneration gene encodes a protein involved in the unique process of liver regeneration. The augmenter of liver regeneration respective protein stimulates hepatocyte proliferation in hepatectomized rats and inhibits cytotoxic activity of liver-derived Natural Killer cells from intact rats. Augmenter of liver regeneration protein shares homology with a Saccharomyces Cerevisiae protein essential for the viability, oxidative phosphorylation and cell-division cycle. To demonstrate if augmenter of liver regeneration protein, like the homologous in the yeast, plays a role in the regulation of biogenesis of mitochondria. Augmenter of liver regeneration protein was injected in intact rats and, in the hepatic tissue, the expression of two genes located in two different regions of the mitochondrial genome, mitochondrial ATPase 6/8, and ND1 subunit, and of a nuclear gene, mitochondrial Transcription Factor A, were considered. In addition, cytochrome content and oxidative phosphorylation capacity of liver-derived mitochondria were evaluated. The augmenter of liver regeneration protein administration induces an increase in the mitochondrial gene expression and enhances cytochrome content and oxidative phosphorylation capacity of liver-derived mitochondria. The present data demonstrate a comparable role in the regulation of mitochondria biogenesis in the eukaryotic cell like the yeast protein. This phenomenon could be part of the complex mechanism through which augmenter of liver regeneration regulates hepatocyte proliferation.

A fresh look at augmenter of liver regeneration in rats.

Augmenter of liver regeneration (ALR) is a hepatotrophic protein originally identified by bioassay in regenerating rat and canine livers following partial hepatectomy and in the hyperplastic livers of weanling rats, but not in resting adult livers. The ALR gene and gene product were subsequently described, but little is known about the cellular/subcellular sites of ALR synthesis in the liver, or about the release and dissemination of the peptide. To obtain this information in rats, we raised antibodies in rabbits against rat ALR for development of an enzyme-linked immunosorbent assay (ELISA). ALR concentrations were then determined in intact livers of unaltered weanling and adult rats; in regenerating residual liver after partial hepatectomy; in cultured hepatocytes and nonparenchymal cells (NPCs); and in culture medium and serum. ALR in the various liver cells was localized with immunohistochemistry. In addition, hepatic ALR and ALR mRNA were assayed with Western blotting and reverse-transcriptase polymerase chain reaction (RT-PCR), respectively. The hepatocyte was the predominant liver cell in which ALR was synthesized and stored; the cultured hepatocytes secreted ALR into the medium in a time-dependent fashion. Contrary to previous belief, the ALR peptide and ALR mRNA were present in comparable concentrations in the hepatocytes of both weanling and resting adult livers, as well as in cultured hepatocytes. A further unexpected finding was that hepatic ALR levels decreased for 12 hours after 70% hepatectomy in adult rats and then rose with no corresponding change in mRNA transcripts. In the meantime, circulating (serum) ALR levels increased up to 12 hours and declined thereafter. Thus, ALR appears to be constitutively expressed in hepatocytes in an inactive form, and released from the cells in an active form by unknown means in response to partial hepatectomy and under other circumstances of liver maturation (as in weanling rats) or regeneration.

Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization

The yeast scERV1 gene product is involved in the biogenesis of mitochondria and is indispensable for viability and regulation of the cell cycle. Recently the general importance of this gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (Augmenter of Liver Regeneration) genes from man, mouse and rat are involved in the phenomenon of liver regeneration. A low expression rate of the genes is found in all investigated cells and mammalian tissues but it is specifically induced after damage of liver organs and is especially high during spermatogenesis. The alignment of the different proteins identifies a highly conserved carboxy terminus with more than 40% identical amino acids between yeast and mammals. The conserved carboxy terminus is functionally interchangeable between distantly related species like yeast and man. In contrast, the amino terminal parts of the proteins display a high degree of variability and significant differences even among closely related species. This finding leads to the problem whether the amino termini have comparable or divergent functions in different species. In this study we demonstrate by heterologous complementation experiments in yeast that the complete human ALR protein with its own amino terminus is not able to substitute for the yeast scERV1 protein. Fusion proteins of Alrp and scErv1p with the green fluorescence protein were created to investigate the respective subcellular localizations of these homologous proteins in yeast and human cells. In yeast cells human Alrp accumulates in the cytoplasm in contrast to yeast scErv1p that is preferentially associated with yeast mitochondria. Comparable studies with human cells clearly show that the homologous human Alrp is located in the cytosol of these cells. Fractionation experiments and antibody tests with yeast and human mitochondria and cellular extracts verify these findings.

Analysis of the Structure and Expression of the Augmenter of Liver Regeneration (ALR) Gene

The gene encoding the hepatotrophic factor Augmenter of Liver Regeneration (ALR) has recently been cloned in the rat. The availability of the mouse form of ALR would allow the analysis of the role of this factor in the physiology of liver and other organs, while the identification of the human homolog would allow the transfer of the great wealth of information that has been generated in animal models to clinically oriented pilot trials, and eventually the therapeutic application of this information. Standard molecular biology approaches have been used to determine the genomic structure of the ALR gene in the mouse, and to characterize the ALR transcript and its protein product. The human ALR cDNA was also isolated and the amino acid sequence of the human gene product deduced. The mapping of mouse and human ALR genes on mouse and human chromosomes was then completed. The protein coding portion of the mouse ALR gene is comprised of three exons, the first containing the 5' untranslated sequence and the initial 18 bases after the ATG translation initiation codon, the second exon encompasses 198 bases, and the third exon contains the remaining portion of the protein coding sequence. Rat, mouse, and human ALR genes (and protein products) were found to be highly conserved and preferentially expressed in the testis and in the liver. The ALR gene maps to the mouse chromosome 17, in a region syntenic with human chromosome 16, where the T/t region has also been mapped. ALR appears to be a protein with important physiologic properties, not exclusively limited to liver regeneration, with roles that are involved in the synthesis or stability of the nuclear and mitochondrial transcripts that are present in actively regenerating cells, particularly the germ cells of the testes.