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Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USACincinnati VA Medical Center, Cincinnati, Ohio, USA
Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USACincinnati VA Medical Center, Cincinnati, Ohio, USA
Corresponding author. Address: Department of Pediatrics, Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, 3333 Burnett Avenue, Cincinnati, Ohio, 45229, USA, Tel.: +(513)-517-1090; fax: +(513)-558-8677.
Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USACincinnati VA Medical Center, Cincinnati, Ohio, USADepartment of Surgery, University of Cincinnati, Cincinnati, Ohio, USA
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.
A critical role of ALR in mitochondria is evident as its loss in vitro and in vivo causes lipid accumulation (steatosis), ATP depletion, oxidative stress, mitochondrial degeneration, and death of hepatocytes.
Non-alcoholic fatty liver disease (NAFLD) has become a major clinical challenge of recent times. NAFLD begins with simple steatosis that can progress to the more aggressive form, non-alcoholic steatohepatitis (NASH) in 10-30% of affected individuals.
it is apparent that ALR deficiency may play an important role in its pathogenesis and progression. In this review, we discuss the current understanding of the regulation of ALR expression and function, and we review the possible mechanisms by which ALR deficiency might contribute to aggressive NAFLD.
ALR is an evolutionally conserved protein that is abundantly expressed in hepatocytes.
Augmenter of liver regeneration gene and isoforms
Although known as augmenter of liver regeneration, hepatopoietin and hepatic stimulatory substance, these names are rather misnomers since ALR is present ubiquitously (in all major organs) and demonstrates functions other than liver cell proliferation.
indicate that the gene is evolutionally conserved. Homology between the rat, mouse and human ALR and the yeast scERV1 protein is shown in Fig. 1.
The mammalian ALR gene is named GFER (growth factor Erv1-like) because of its structural and functional similarities with the scERV1 (essential for respiration and vegetative growth-1) protein expressed by the yeast Saccharomyces cerevisiae.
However, the presence of multiple ATG codons in the ALR gene suggests the possibility of variants generated by alternative splicing. The entire mouse ALR gene is contained in a 6.7-kb HindIII fragment, comprising 3 exons and 2 introns.
Exon 1 contains a 5' untranslated sequence, ATG initiation codon and 6 amino acid-coding nucleotide sequence of s-ALR or 73 amino acid-coding sequence of l-ALR, which is followed by a 400 bp intron; exon 2 contains a 66 amino acid-coding sequence, followed by the second 480 bp intron; the third exon contains the remaining portion of the amino acid-coding sequence and the entire 3' untranslated sequence.
The N-terminal domain of l-ALR contains the mitochondrial leader sequence (absent in s-ALR), whereas the C-terminal domain is responsible for the flavin adenine dinucleotide (FAD)-linked functional activity.
ALR gene expression regulation
The ALR gene has a TATA-less promotor with features of housekeeping genes, oncogenes, growth factors and transcription factors.
Analysis of the ALR promoter identified positive regulatory elements between -416 and -608 nucleotide (nt), negative regulatory elements between -236 and -416 nt, and minimal core promoter activity between −22 and +27 nt. A “CTGGAGGC” sequence within the initiator (Inr)-like element and 2 other tandem flanking repeats comprise the core promoter that controls transcriptional initiation and the constitutive expression of the ALR gene. Activator protein 1/4 (AP1/AP4) is proposed to be responsible for basal ALR promoter activity.
ALR gene expression is positively regulated by nuclear factor erythroid 2-related factor 2 (NRF2), forkhead box A2 (FOXA2 also known as hepatocyte nuclear factor 3β [HNF3β]), HNF4α, early growth response protein-1 (EGR-1) and specificity protein 1 (SP1). An antioxidant response element (ARE) is located at −27/−19 nt from the initial ATG codon in the proximal promoter region. Upregulation of ALR expression by oxidative stress (due to an increase in nuclear NRF2 and its binding to ARE) suggests that ALR is an ARE-regulated gene.
The ALR promoter also contains 2 potential bile acid-binding response elements, and bile acids suppress ALR promoter activity induced by FOXA2, HNF4α (binding site at +421/+432 nt) and EGR-1c (binding site at +304/+314 nt) via activation of small heterodimer partner (SHP).
CCAAT/enhancer binding protein-β (C/EBPβ) is another negative regulator of ALR gene transcription. In HepG2 cells, electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis revealed a C/EBPβ-binding site at −292/−279 nt, and the epidermal growth factor (EGF) was found to downregulate ALR expression via C/EBPβ.
This suggested that ALR released after 70% hepatectomy stimulates synthesis of growth mediators. Thus, the augmenting effect of exogenous ALR is proposed to be due to ALR-induced synthesis of tumour necrosis factor (TNF)α and IL-6 by Kupffer cells,
Furthermore, ALR reduced oxidative stress, autophagy and apoptosis, and at the same time increased oxidative phosphorylation and mitochondrial expression of ATPase 6/8, ND1 subunit and mitochondrial transcription factor A (TFAM) in partially hepatectomised rats.
However, increased hepatic synthesis of ALR and powerful mitogens, such as hepatocyte growth factor (HGF) and TGFα (transforming growth factor α), after portacaval shunt in rats indicated that first pass of gastrointestinal-derived growth factors is critical for liver cell size maintenance and function.
Accumulation of toxic bile acids during cholestasis causes oxidative stress and death of hepatocytes. Reduced hepatic ALR levels in human cholestatic liver disease (presumably via bile acid-induced SHP activation and suppression of ALR promoter activity),
ALR is essential for proper folding of mitochondrial imported proteins, electron transport chain activity and iron/sulphur maturation of cytosolic proteins.
Importance of ALR in mitochondrial integrity and function
Mutations in the scERV1 gene or depletion of scERV1 protein caused loss of the inner mitochondrial membrane and eventually the entire organelle, indicating the critical importance of ERV1 in mitochondrial integrity, survival and function.
Most of the mitochondrial proteins are synthesised in the cytosol and transported into the mitochondria as precursors, aided by the TOM (translocase of the outer membrane) complex. Appropriate oxidative folding of several of these proteins is essential for their functions and is catalysed by the Mia40 (mitochondrial intermembrane space import and assembly protein 40 kDA)/ALR-sulfhydryl relay system
(Fig. 2). Mia40, with its redox-active cysteine-proline-cysteine disulphide bond, oxidises cysteine residues of the imported polypeptides; these stably folded proteins are prevented from transport through the outer membrane.
Two essential redox-active cysteine-x-x-cysteine pairs in ALR shuttle electrons from Mia40 to FAD. ALR can be directly re-oxidised by oxygen in vitro in a reaction yielding H2O2. In vivo, ALR is re-oxidised by passing its electrons through FAD to cytochrome c of the respiratory chain; these electrons are then accepted by molecular oxygen to produce water, thus preventing generation of H2O2 in the intermembrane space.
Mechanistically, the Mia40/ALR pathway has been shown to facilitate import of ABCB8 (ATP-binding cassette-B8), an inner mitochondrial membrane protein necessary for cytoplasmic Fe/S cluster maturation.
The pathophysiological implication of ALR as a regulator of iron homeostasis is evidenced by excessive iron accumulation in the liver of hepatocyte-specific ALR-deficient mice upon alcohol consumption.
Clinically, the importance of ALR in the mitochondrial sulfhydryl relay system is exemplified by mitochondriopathy, decreased activity of respiratory complexes I, II and IV, congenital cataract, muscular hypotonia and developmental delay observed in patients with a rare (R194H) mutation in the ALR gene.
The mutation reduces stability of the ALR protein, as well as the expression and activity of cytochrome oxidase. The R194H mutation also causes defective accumulation of Mia40 in mitochondria suggesting that ALR regulates Mia40 localisation.
In line with this observation, ALR-knockdown in mice reduces expression of TFAM as well as PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α), impairs mitochondrial biogenesis, and delays liver regeneration after partial hepatectomy.
In a rat model of obstructive jaundice, administration of human ALR led to increased expression of TFAM and nuclear respiratory factor 1, mtDNA damage repair and improvement of mitochondrial functions.
Increased ALR expression also imparts protection against irradiation-induced mitochondrial and cellular damage by increasing mitochondrial membrane potential, inhibiting cytochrome c release and preventing ATP loss.
Considering the importance of ALR in mitochondrial biogenesis, survival and function, a deficiency of, or an abnormality in, the ALR protein might be a predisposing condition in the development of NASH, the aggressive form of NAFLD. NASH is characterised by steatosis, hepatocyte ballooning and injury, inflammation, and pericellular fibrosis.
Obesity, sedentary lifestyle, type 2 diabetes mellitus/insulin resistance, altered gut microbiota, and genetic and environmental factors are all considered as contributors to NAFLD/NASH and NASH-induced cirrhosis.
Despite extensive clinical and experimental research, there is still a significant knowledge gap regarding the pathogenesis and progression of NAFLD, for which no pharmacological treatment has been approved.
Genetic ablation of Alr in mice leads to robust steatosis and cell death followed by “lean” NASH-like progression to hepatocellular carcinoma.
Ultrastructural mitochondrial lesions, increased production of ROS and lipid peroxidation (due to decreased activity of respiratory chain complexes), decreased fatty acid β-oxidation, and reduced ability to resynthesise ATP have all been observed in patients with NASH.
However, mechanisms underlying deterioration of mitochondrial structure and function are not completely understood. It should be noted that hepatic mitochondria of obese individuals with or without NAFL exhibit an increased respiration rate, but this adaptation is lost in those with NASH who exhibit a significantly reduced rate of respiration in association with increased oxidative stress and DNA damage.
It will be important to elucidate whether this transition occurs as patients progress from NAFL to NASH, or if patients who progress to NASH are distinct from those with NAFL who do not develop NASH.
Hepatocyte-specific Alr-knockout (Alr-H-KO) mice were generated to investigate ALR’s role in liver physiology. Alr-H-KO mice showed severe mitochondriopathy (degenerating or enlarged mitochondria with loss of cristae, and defect at complex II of the electron transport chain), and ATP depletion due to significant loss of ALR between 1 and 2 weeks postpartum.
These pathologies led to strong ductular reaction, regeneration of hepatocytes from the surviving cells, as well as from cells of the biliary compartment at 4 weeks, and regression of steatosis. It was observed that the surviving/regenerating cells expressed ALR, albeit at significantly lower magnitude than cells in the wild-type (WT) mouse liver, but there was continued inflammation and modest fibrosis. Eventually, nearly 70% of Alr-H-KO mice developed liver tumours (60% being HCC) by 1 year when their ALR expression was similar to the WT mice.
Since cells from the biliary compartment seem to transform into ALR-expressing hepatocytes following pronounced apoptosis at 2 weeks postpartum, they are the likely source of malignancy. Although NAFLD is generally associated with central obesity, patients with a normal body mass index developing “lean” NASH have been identified.
Our analysis showed physiological serum ALR concentrations in the 32-380 pg/ml range (n = 27), indicating interindividual variability; ALR concentrations were significantly lower in NASH (0-336 pg/ml; p <0.05 vs. Control; n = 25) and NASH-cirrhosis (24-380 pg/ml; p <0.05 vs. Control; n = 22). In contrast, serum ALR increases in acute, chronic and fulminant hepatitis due to hepatitis virus A, B or C infection.
Discrepancies in these values may be due to differential specificity of the ALR antibodies used and differences in assay procedures. Thus, a more comprehensive investigation is required to ascertain the serum ALR range in NAFL, NASH and NASH-cirrhosis. Such research might lead to determination of serum ALR concentrations predictive or diagnostic of NASH. Similar or greater hepatic ALR levels in some patients with NASH-cirrhosis compared to healthy individuals
how it regulates the expression of proteins involved in lipid homeostasis is unclear. The loss of ALR in vivo and in vitro reduces the expression of carnitine palmitoyl transferase a (CPT1a), sterol regulatory element-binding protein (SREBP)1c, peroxisome proliferator-activated receptor-α (PPARα), peroxisomal membrane protein 70 (PMP70) and acyl-CoA oxidase 1 (ACOX1).
The mechanism of ALR depletion-induced dysregulated lipid homeostasis appears to involve altered expression of several microRNAs (miRNAs) with binding sequences on mRNAs encoding CPT1a, SREBP1c, PPARα, PMP70 and ACOX1.
Increased miR-540 (miR-6801 in humans) was found to affect the expression of CPT1a, SREBP1c, PPARα, PMP70 and ACOX1, and treatment with anti-miR-540 or recombinant ALR between 1 and 2 weeks mitigated steatosis and pericellular fibrosis in Alr-H-KO mice.
Although mitochondrial injury-related oxidative stress might be a causal factor for the altered expression of miRNAs and mRNAs, whether ALR influences the binding and activity of nuclear transcription factors responsible for the expression of miRNAs remains to be determined.
Several single nucleotide polymorphisms found in the human ALR gene cause developmental delay, progressive multiorgan pathologies and death.
The GFER (ALR) gene contains several single nucleotide polymorphisms, some of which are pathogenic (https://www.ncbi.nlm.nih.gov/snp/) (Table 1). It is noteworthy that children receiving the hypofunctional GFER allele (R194H mutation; rs121908192) from both healthy heterozygous parents develop severe mitochondriopathy, progressive myopathy and partial combined respiratory chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay.
Other investigations found that homozygous or heterozygous mutations in GFER (rs121908192; rs1555486560; rs1597063051; rs1597063303; rs771809901) in the same patient induced similar congenital progressive multiorgan pathologies.
However, ultrasound evaluation for steatosis is only accurate when >25% of the liver is affected, and biopsy is essential to diagnose steatohepatitis. Importantly, liver biopsy of a patient with compound heterozygous mutation (frameshift variants c.219delC [p.(Cys74Alafs∗76)] and [c.259-25_259-24delCA]) showed mitochondrial damage, and centrolobular and portal fibrosclerosis, and electron microscopy revealed a number of pleiomorphic mitochondria containing paracrystalline inclusions.
it is likely that individuals with inherent ALR deficiency or dysfunction might be predisposed to develop aggressive NASH. Alternatively, steatosis-induced downregulation of ALR might be an important contributing factor to the severity of disease progression.
A high-fat high-carbohydrate (HF/HC) diet induced greater obesity and hepatic steatosis (increased de novo lipogenesis and depressed lipolysis) in Alr-H-HET mice than in WT mice, with relevant changes in the expression of related enzymes (SREBP1c, ACACA, and FASN).
The authors went on to show that inactivation of AMPK (AMP-activated protein kinase) leads to increased cholesterol in high-fat diet-fed Alr-HET mice. Accumulation of free cholesterol induces endoplasmic reticulum stress and mitochondrial injury by inhibiting glutathione transport. Furthermore, mitochondrial accumulation of cholesterol causes JNK activation and subsequent apoptosis/necrosis. Thus, based on the cholesterol accumulation observed in liver biopsies of patients with NASH, which correlates with severity of NASH and NASH-fibrosis, it is proposed that increased accumulation of cholesterol is a major contributor to ongoing lipotoxicity in experimental and human NASH.
These findings provide further support for the impact of ALR deficiency on NASH development, and are supported by the greater magnitude of reduction in hepatic ALR in HF/HC diet-fed Alr-H-HET mice than WT mice.
HF/HC-fed Alr-H-HET mice had increased inflammation in the liver (greater incidence of TNFα-, IL-6- and IL17-producing cells and lower incidence of FoxP3+ immunosuppressive regulatory T cells) and in white adipose tissue. This model is relevant to human NASH since both female and male mice developed hepatocyte injury, inflammation, stellate cell activation, and fibrosis. Alr-H-KO mice with underlying modest inflammation and fibrosis are resistant to HF/HC-induced obesity and hepatic steatosis but progressed to cirrhosis.
The importance of ALR deficiency in steatohepatitis is exemplified by lower hepatic ALR in human alcohol-related cirrhosis and the occurrence of alcohol-induced cirrhosis within 4 weeks in mice fed the Lieber de Carli diet (while WT mice fed the same diet showed only modest steatosis at the same timepoint).
Like humans, most animal models are resistant to more aggressive ALD, and steatosis is readily reversed upon termination of alcohol ingestion. The Lieber Di Carli liquid alcohol diet, which has been used extensively to study ALD in mice, caused steatosis in control mice, but promoted aggressive liver disease leading to cirrhosis, accompanied by reduced expression of alcohol dehydrogenases-1 and aldehyde dehydrogenases-1, in ALR-deficient mice.
There was also significant mitochondrial damage and iron accumulation (lower glutaredoxin-5 and hepcidin expression) in alcohol-fed ALR-deficient mice. The clinical significance of these findings is indicated by significantly lower hepatic ALR expression in patients with alcohol-related cirrhosis.
However, Alr-H-KO mice already have underlying modest oxidative stress, inflammation and fibrosis that are accelerated/augmented by alcohol, as demonstrated by further increases in oxidative stress, robust lipid peroxidation and mtDNA damage. Such underlying conditions in humans are likely a prerequisite for aggressive ALD. ALR was also shown to protect mice from alcohol-induced acute liver injury by promoting autophagy through repression of mTOR (mammalian target of rapamycin).
ALR deficiency or dysfunction may be an important risk factor for NASH.
Conclusions and future prospects
Despite being an evolutionally conserved fundamental life protein with varied functions, understanding of the role of ALR in physiology and pathophysiology has been inadequate. ALR is critically important for mitochondrial biogenesis, protein folding (Mia40-sulfhydryl relay system), and respiratory chain activity, disruption of which is implicated in the pathogenesis and progression of both NAFLD and ALD. In vivo and in vitro studies of ALR-knockdown and hepatocyte-specific ALR deficiency have provided crucial evidence of ALR’s role in lipid homeostasis and in promoting the expression of several genes, including those involved in alcohol and iron metabolism. The clinical significance of ALR in NASH and ASH is inferred from its lower hepatic concentration in human NASH- and ASH-cirrhosis. Because of its lack of a DNA-binding sequence, ALR may not be directly involved in gene transcription but may act as a promoter or suppressor of certain transcription factors. In this regard, ALR immunoprecipitates with TFAM (unpublished observation), and deficiency of ALR downregulates TFAM expression. Several pathogenic single nucleotide polymorphisms are found to cause severe mitochondrial damage and progressive multiorgan disease in humans. Liver biopsy of 1 patient with mutations in both ALR alleles showed hepatic mitochondrial damage and fibrosis. Thus, humans with heterozygous mutations in the ALR gene could be predisposed to chronic liver diseases such as NASH and ASH. Future investigations to further delineate mechanisms by which ALR deficiency or dysfunction promotes NAFLD or ALD progression will be important.
ACACA, acetyl-CoA carboxylase alpha; ACOX1, acyl-CoA oxidase 1; ALD, alcohol-related liver disease; ALR, augmenter of liver regeneration; CCl4, carbon tetrachloride; C/EBPβ, CCAAT/enhancer binding proteins; CPT1a, carnitine palmitoyl transferase I a; Egr-1, Early growth response protein-1; ERV1, essential for respiration and vegetative growth-1; ESLD, end-stage liver disease; FAD, flavin adenine dinucleotide; FASN, fatty acid synthase; FoxA2, forkhead box A2; HCC, hepatocellular carcinoma; HF/HC, high-fat high carbohydrate; HNF4α, hepatocyte nuclear factor 4 alpha; l-ALR, long form ALR; IMS, intermembrane space; JAB1, Jun activation domain-binding protein 1; Mia40, mitochondrial IMS import and assembly protein 40 kDa; miRNA, microRNA; mtDNA, mitochondrial DNA; NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NRF2, nuclear factor erythroid 2-related factor 2; nt, nucleotide; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1-alpha; PMP70, peroxisomal membrane protein 70; PPARα, peroxisome proliferator-activated receptor alpha; rALR, recombinant ALR; ROS, reactive oxygen species; s-ALR, short form ALR, scERV1, Saccharomyces cerevisiae essential for respiration and vegetative growth-1; SHP, small heterodimer partner; SREBP, sterol regulatory element-binding protein; TFAM, mitochondrial transcription factor A.
Department of Defense grant #W81XWH2010477 .
All authors contributed to the writing of this manuscript.
Conflict of interest
The authors declare no conflicts of interest that pertain to this work.
Please refer to the accompanying ICMJE disclosure forms for further details.
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