Hypoxia signaling and cholesterol/steroidogenic acute regulatory protein 1 axis: interplay and role in alcohol and non-alcohol-related liver diseases

Metabolic zonation in the liver carries out the maintenance of organ and body homeostasis. Hypoxia is an inherent physiological feature of the liver and contributes to the zonal properties of the hepatic parenchyma. As a master regulator of hypoxia, the transcription factor hypoxia-inducing factor (HIF) is stabilized primarily by oxygen availability, and it is thought to contribute to steatohepatitis due to alcohol-related (ASH) and non-alcohol-related liver disease (NASH). Cholesterol has emerged as an important player in both diseases, and hypoxia increases hepatic cholesterol levels. Steroidogenic acute regulatory protein 1 (STARD1) is a mitochondrial outer membrane protein that transfers cholesterol to mitochondrial inner membrane for metabolic processing and acts as the rate-limiting step in the alternative pathway of bile acid synthesis in hepatocytes. STARD1 expression increases in ASH and NASH and determines the accumulation of cholesterol in mitochondria, which impacts the physico-chemical mitochondrial membranes properties and as a consequence impairs the activity of specific mitochondrial solute carriers, such as the 2-oxoglutarate carrier (2-OGC), limiting the exchange between cytosolic glutathione and mitochondrial 2-oxoglutarate (2-OG). Although HIF-1 is stabilized in hypoxia largely due to the requirement of prolylhydroxylases (PHDs) for oxygen to signal HIF degradation, PHDs are also dependent on 2-OG, and therefore it is conceivable that impairment of 2-OGC by STARD1-mediated cholesterol accumulation may contribute to HIF-1 stabilization due in part to decreased availability of cytosolic 2-OG. In this perspective, this review explores the interplay between HIF-1 stabilization and STARD1 induction and the potential contribution of this functional relationship to ASH and NASH

Beneficial effect of ursodeoxycholic acid in patients with acyl-CoA oxidase 2 (ACOX2) deficiency-associated hypertransaminasemia

Background and aims: A variant (p.Arg225Trp) of peroxisomal acyl-CoA oxidase 2 (ACOX2), involved in bile acid (BA) side-chain shortening, has been associated with unexplained persistent hypertransaminasemia and accumulation of C27-BAs, mainly 3?,7?,12?-trihydroxy-5?-cholestanoic acid (THCA). We aimed to investigate the prevalence of ACOX2 deficiency-associated hypertransaminasemia (ADAH), its response to ursodeoxycholic acid (UDCA), elucidate its pathophysiological mechanism and identify other inborn errors that could cause this alteration. Methods and results: Among 33 patients with unexplained hypertransaminasemia from 11 hospitals and 13 of their relatives, seven individuals with abnormally high C27-BA levels (>50% of total BAs) were identified by high-performance liquid chromatography-mass spectrometry. The p.Arg225Trp variant was found in homozygosity (exon amplification/sequencing) in two patients and three family members. Two additional nonrelated patients were heterozygous carriers of different alleles: c.673C>T (p.Arg225Trp) and c.456_459del (p.Thr154fs). In patients with ADAH, impaired liver expression of ACOX2, but not ACOX3, was found (immunohistochemistry). Treatment with UDCA normalized aminotransferase levels. Incubation of HuH-7 hepatoma cells with THCA, which was efficiently taken up, but not through BA transporters, increased reactive oxygen species production (flow cytometry), endoplasmic reticulum stress biomarkers (GRP78, CHOP, and XBP1-S/XBP1-U ratio), and BAX? expression (reverse transcription followed by quantitative polymerase chain reaction and immunoblot), whereas cell viability was decreased (tetrazolium salt-based cell viability test). THCA-induced cell toxicity was higher than that of major C24-BAs and was not prevented by UDCA. Fourteen predicted ACOX2 variants were generated (site-directed mutagenesis) and expressed in HuH-7 cells. Functional tests to determine their ability to metabolize THCA identified six with the potential to cause ADAH. Conclusions: Dysfunctional ACOX2 has been found in several patients with unexplained hypertransaminasemia. This condition can be accurately identified by a noninvasive diagnostic strategy based on plasma BA profiling and ACOX2 sequencing. Moreover, UDCA treatment can efficiently attenuate liver damage in these patients.Funding information: This study was supported by the following grants: CIBERehd (EHD15PI05/2016); Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain (PI19/00819 and PI20/00189), co-funded by European Regional Development Fund/European Social Fund, “Investing in your future”; “Junta de Castilla y León” (SA074P20); Fundació Marato TV3 (201916–31); AECC Scientific Foundation (2017/2020), Spain; and “Centro Internacional sobre el Envejecimiento” (OLD-HEPAMARKER, 0348_CIE_6_E), Spain. We also acknowledge support from grants PID2019-111669RBI-100, PID2020-115055RB-I00 from Plan Nacional de I+D funded by the “Agencia Estatal de Investigación” (AEI) and the center grant P50AA011999 Southern California Research Center for ALPD and Cirrhosis funded by NIAAA/NIH, as well as support from AGAUR of the “Generalitat de Catalunya” SGR-2017-1112, European Cooperation in Science & Technology (COST) ACTION CA17112 Prospective European Drug-Induced Liver Injury Network. Marta Alonso-Peña was the recipient of a predoctoral fellowship from “Ministerio de Educación, Cultura y Deporte” (BOE-A-2015-9456; FPU-14/00214) and a Mobility Grant for Short Stays from “Ministerio de Ciencia, Innovación y Universidades” (EST17/00186). Ricardo Espinosa-Escudero is the recipient of a predoctoral fellowship from “Junta de Castilla y León” and “Fondo Social Europeo” (EDU/574/2018). The funding sources were not involved in the research design or preparation of the article

Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD), however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities

Lysosomal and Mitochondrial Liaisons in Niemann-Pick Disease

Lysosomal storage disorders (LSD) are characterized by the accumulation of diverse lipid species in lysosomes. Niemann-Pick type A/B (NPA/B) and type C diseases Niemann-Pick type C (NPC) are progressive LSD caused by loss of function of distinct lysosomal-residing proteins, acid sphingomyelinase and NPC1, respectively. While the primary cause of these diseases differs, both share common biochemical features, including the accumulation of sphingolipids and cholesterol, predominantly in endolysosomes. Besides these alterations in lysosomal homeostasis and function due to accumulation of specific lipid species, the lysosomal functional defects can have far-reaching consequences, disrupting intracellular trafficking of sterols, lipids and calcium through membrane contact sites (MCS) of apposed compartments. Although MCS between endoplasmic reticulum and mitochondria have been well studied and characterized in different contexts, emerging evidence indicates that lysosomes also exhibit close proximity with mitochondria, which translates in their mutual functional regulation. Indeed, as best illustrated in NPC disease, alterations in the lysosomal-mitochondrial liaisons underlie the secondary accumulation of specific lipids, such as cholesterol in mitochondria, resulting in mitochondrial dysfunction and defective antioxidant defense, which contribute to disease progression. Thus, a better understanding of the lysosomal and mitochondrial interactions and trafficking may identify novel targets for the treatment of Niemann-Pick disease

Mitochondrial–Lysosomal Axis in Acetaminophen Hepatotoxicity

Acetaminophen (APAP) toxicity is the most common cause of acute liver failure and a major indication for liver transplantion in the United States and Europe. Although significant progress has been made in understanding the molecular mechanisms underlying APAP hepatotoxicity, there is still an urgent need to find novel and effective therapies against APAP-induced acute liver failure. Hepatic APAP metabolism results in the production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which under physiological conditions is cleared by its conjugation with glutathione (GSH) to prevent its targeting to mitochondria. APAP overdose or GSH limitation leads to mitochondrial NAPQI-protein adducts formation, resulting in oxidative stress, mitochondrial dysfunction, and necrotic cell death. As mitochondria are a major target of APAP hepatotoxicity, mitochondrial quality control and clearance of dysfunctional mitochondria through mitophagy, emerges as an important strategy to limit oxidative stress and the engagement of molecular events leading to cell death. Recent evidence has indicated a lysosomal–mitochondrial cross-talk that regulates APAP hepatotoxicity. Moreover, as lysosomal function is essential for mitophagy, impairment in the fusion of lysosomes with autophagosomes-containing mitochondria may compromise the clearance of dysfunctional mitochondria, resulting in exacerbated APAP hepatotoxicity. This review centers on the role of mitochondria in APAP hepatotoxicity and how the mitochondrial/lysosomal axis can influence APAP-induced liver failure

Zonal expression of StARD1 and oxidative stress in alcoholic-related liver disease

Alcoholic-related liver disease (ALD) is one of the leading causes of chronic liver disease and morbidity. Unfortunately, the pathogenesis of ALD is still incompletely understood. StARD1 has emerged as a key player in other etiologies of chronic liver disease, and alcohol-induced liver injury exhibits zonal distribution. Here, we report that StARD1 is predominantly expressed in perivenous (PV) zone of liver sections from mice-fed chronic and acute-on-chronic ALD models compared to periportal (PP) area and is observed as early as 10 days of alcohol feeding. Ethanol and chemical hypoxia induced the expression of StARD1 in isolated primary mouse hepatocytes. The zonal-dependent expression of StARD1 resulted in the accumulation of cholesterol in mitochondria and increased lipid peroxidation in PV hepatocytes compared to PP hepatocytes, effects that were abrogated in PV hepatocytes upon hepatocyte-specific Stard1 KO mice. Transmission electron microscopy indicated differential glycogen and lipid droplets content between PP and PV areas, and alcohol feeding decreased glycogen content in both areas while increased lipid droplets content preferentially in PV zone. Moreover, transmission electron microscopy revealed that mitochondria from PV zone exhibited reduced length with respect to PP area, and alcohol feeding increased mitochondrial number, particularly, in PV zone. Extracellular flux analysis indicated lower maximal respiration and spared respiratory capacity in control PV hepatocytes that were reversed upon alcohol feeding. These findings reveal a differential morphology and functional activity of mitochondria between PP and PV hepatocytes following alcohol feeding and that StARD1 may play a key role in the zonal-dependent liver injury characteristic of ALD

Impact of Liver Inflammation on Bile Acid Side Chain Shortening and Amidation

Bile acid (BA) synthesis from cholesterol by hepatocytes is inhibited by inflammatory cytokines. Whether liver inflammation also affects BA side chain shortening and conjugation was investigated. In human liver cell lines (IHH, HepG2, and HepaRG), agonists of nuclear receptors including the farnesoid X receptor (FXR), liver X receptor (LXR), and peroxisome proliferator-activated receptors (PPARs) did not affect the expression of BA-related peroxisomal enzymes. In contrast, hepatocyte nuclear factor 4α (HNF4α) inhibition down-regulated acyl-CoA oxidase 2 (ACOX2). ACOX2 was repressed by fibroblast growth factor 19 (FGF19), which was prevented by extracellular signal-regulated kinase (ERK) pathway inhibition. These changes were paralleled by altered BA synthesis (HPLC-MS/MS). Cytokines able to down-regulate cholesterol-7α-hydroxylase (CYP7A1) had little effect on peroxisomal enzymes involved in BA synthesis except for ACOX2 and bile acid-CoA:amino acid N-acyltransferase (BAAT), which were down-regulated, mainly by oncostatin M (OSM). This effect was prevented by Janus kinase (JAK) inhibition, which restored BA side chain shortening and conjugation. The binding of OSM to the extracellular matrix accounted for a persistent effect after culture medium replacement. In silico analysis of four databases (n = 201) and a validation cohort (n = 90) revealed an inverse relationship between liver inflammation and ACOX2/BAAT expression which was associated with changes in HNF4α levels. In conclusion, BA side chain shortening and conjugation are inhibited by inflammatory effectors. However, other mechanisms involved in BA homeostasis counterbalance any significant impact on the serum BA profile