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Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.

ABSTRACT: Levels of augmenter of liver regeneration (ALR), a multifunctional protein, are reduced in steatohepatitis. ALR depletion from ALR ^flox/flox/Alb-Cre [ALR-L-knockout (KO)] mouse causes robust steatosis and apoptosis of hepatocytes, and pericellular fibrosis between 1 and 2 wk postbirth. Steatosis regresses by 4 wk upon reappearance of ALR-expressing hepatocytes. We investigated mechanisms of ALR depletion-induced steatosis. ALR-L-KO mice (1-, 2-, and 4 wk old) and Adeno-Cre-transfected ALR ^flox/flox hepatocytes were used for in vivo and in vitro studies. ALR depletion from hepatocytes in vivo downregulated peroxisome proliferator-activated receptor (PPAR)-α, carnitine palmitoyl transferase I (CPT1)a, peroxisomal membrane protein 70 (PMP70) (modest down-regulation), and acyl-CoA oxidase 1 (ACOX1). The markedly up-regulated (20X) novel microRNA-540 (miR-540) was identified to target PPARα, PMP70, ACOX1, and CPT1a. ALR depletion from primary hepatocytes increased oxidative stress, miR-540 expression, and steatosis and down-regulated PPARα, ACOX1, PMP70, and CPT1a expression. Anti-miR-540 mitigated ALR depletion-induced steatosis and prevented loss of PPARα, ACOX1, PMP70, and CPT1a expression. Antioxidant N-acetylcysteine and recombinant ALR (rALR) both inhibited ALR depletion-induced miR-540 expression and lipid accumulation in hepatocytes. Finally, treatment of ALR-L-KO mice with rALR between 1 and 2 wk prevented miR-540 expression, and arrested steatosis and fibrosis. We conclude that ALR deficiency-mediated oxidative stress induces generation of miR-540, which promotes steatosis by dysregulating peroxisomal and mitochondrial lipid homeostasis.-Kumar, S., Rani, R., Karns, R., Gandhi, C. R. Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.

SUBMITTER: Kumar S

PROVIDER: S-EPMC6404588 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and aimsThe augmenter of liver regeneration (ALR) protein is critical for lipid homeostasis and mitochondrial function. We investigated high-fat/high-carbohydrate (HF/HC) diet-induced nonalcoholic fatty liver disease (NAFLD) in wild-type (WT), hepatocyte-specific ALR-knockout (ALR-H-KO), and ALR-heterozygous (ALR-H-HET) mice. ALR was measured in serum of human nonalcoholic steatohepatitis (NASH) and NASH-induced cirrhosis (serum and liver).Approach and resultsHF/HC feeding decreased ALR expression in all groups of mice. The otherwise normal ALR-H-HET mice gained more weight and steatosis than WT mice when challenged metabolically with the HF/HC diet; ALR-H-KO mice gained the least weight and had the least steatosis. These findings were consistent with correspondingly increased triglycerides and cholesterol and altered expression of carnitine palmitoyltransferase 1a, sterol regulatory element-binding protein, acetyl coenzyme A carboxylase, and fatty acid synthase. All HF/HC-fed mice developed insulin resistance, the magnitude being lower in ALR-H-KO mice. HF/HC-fed ALR-H-HET mice were more resistant to glucose challenge than WT or ALR-H-KO mice. The frequency of tumor necrosis factor alpha-producing, interleukin 6 (IL6)-producing, and IL17-producing cells was greater in ALR-H-KO than ALR-H-HET and lowest in WT mice. HF/HC feeding did not increase their number in ALR-H-KO mice, and the increase in ALR-H-HET was greater than that in WT mice except for IL17 cells. Cluster of differentiation 25-positive (CD25+ ) forkhead box P3-positive CD4+ regulatory T-cell frequency was lower in ALR-H-HET than WT mice and further reduced in ALR-H-KO mice; HF/HC reduced regulatory T-cell frequency only in WT mice. HF/HC-fed ALR-H-HET, but not WT, mice developed fibrosis; and ALR-H-KO mice progressed to cirrhosis. White adipose tissue of HF/HC-fed ALR-deficient mice developed strong inflammation, indicating bidirectional interactions with the liver. Hepatic and serum ALR levels were significantly reduced in patients with NASH-cirrhosis. Serum ALR was also significantly lower in patients with NASH.ConclusionsHepatic ALR deficiency may be a critical predisposing factor for aggressive NAFLD progression.

Project description:Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.

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Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.

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