Coenzyme A (CoA) is a vital cofactor involved in 8-10% of all metabolic reactions in human cells. Different inherited enzyme deficiencies in which the oxidation of acyl-CoAs is hampered have been hypothesised to share a phenotype characterised by toxic accumulation of acyl-CoA and a concomitant decline in free CoA (CoASH) levels, whereby CoASH becomes limiting for other metabolic reactions. This is referred to as CoASH sequestration. There is, however, limited experimental evidence for this hypothesis. Using a combination of approaches, we test this hypothesis in medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common deficiency of mitochondrial fatty acid oxidation (mFAO), under energetic stress. Both in vitro MCAD-knockout (KO) HepG2 cells and a kinetic model of mFAO showed decreased CoASH, elevated medium-chain acyl-CoA, and decreased long-chain acyl-CoA levels. MCAD-KO mice exposed to fasting and cold as energetic stressors had a significantly increased total CoA pool and increased expression of CoA biosynthetic enzymes in the liver, indicative of an upregulated CoA biosynthesis. Expression of carnitine acyltransferases and acyl-CoA thioesterases, enzymes that liberate CoASH from acyl-CoAs, was also upregulated, suggesting an adaptive response of CoA metabolism to decreased CoASH. Finally, computational model simulations showed that a combination of elevated total CoA and thioesterase activity led to normalisation of both CoASH and medium-chain acyl-CoA levels. Together, the results provide the first evidence for the CoA sequestration hypothesis in MCADD. The observed adaptation of CoA metabolism under energetic stress may act as a compensatory response that counteracts CoASH depletion and accumulation of toxic medium-chain acyl-CoAs.

Background and Aims: Metabolic-associated fatty liver disease is a worldwide health problem characterized by increased hepatic lipid accumulation, leading to conditions like steatohepatitis, cirrhosis, and liver cancer. Early growth response protein 1 (EGR1) gene encodes an immediate early transcription factor that regulates a wide variety of cellular processes in response to stress and injury. Whole-body Egr1 knockout studies suggest a role for EGR1 in metabolism, affecting insulin sensitivity, energy homeostasis, cholesterol biosynthesis, and circadian rhythm. However, its direct role in hepatic lipid metabolism remains unclear. This study aimed to investigate the function of EGR1 in the adult liver. Methods: Hepatocyte-specific EGR1-deficient male mice were generated using CRISPR/Cas9. Mice were maintained under chow-fed conditions or challenged with a high-fat diet (HFD). Hepatic lipid levels, zonation, and gene expression were analyzed. Oxygen consumption was measured in mouse and human hepatocytes. Results: After a HFD challenge, hepatic EGR1-deficient mice showed a significant increase in hepatic triglyceride levels. Transcriptome analysis revealed an upregulation of genes related to fatty acid oxidation and downregulation of mitochondrial respiration genes in livers of both chow and HFD hepatic EGR1-deficient mice. Functional analysis showed reduced maximum oxygen consumption in EGR1-deficient mouse and human hepatocytes. Fasting-induced hepatic lipid accumulation indeed indicated reduced fatty acid oxidation efficiency upon ablation of EGR1. Conclusion: Hepatic EGR1 deficiency significantly alters lipid metabolism and mitochondrial function, indicating a role of EGR1 in regulating the balance between mitochondrial fatty acid β-oxidation and respiration in the liver.

Farnesoid-X-receptor (FXR), a bile acid (BA)-activated nuclear receptor, is a therapeutic target for cholestatic and metabolic liver diseases. However, species differences in BA metabolism and FXR signaling hamper translation from mice to humans. The human FXR ligand-binding domain (LBD) structurally differs from the murine LBD, potentially impacting pharmacological responses. Therefore, we generated mice with "humanized" FXR by replacing the murine LBD by the human LBD (FXR-hLBD) and assessed its impact on BA and cholesterol metabolism. Male and female FXR-hLBD mice on wild-type (WT) or Cyp2c70-/- backgrounds were compared with FXR-mLBD controls under non-stimulated conditions. Additionally, WT mice expressing FXR-mLBD or FXR-hLBD received either vehicle or obeticholic acid (OCA; 40 mg/kg/day, p.o.) for 7 days. FXR humanization did not alter hepatic or intestinal FXR expression levels. Under basal conditions, physiological parameters, liver pathology markers, and hepatic transcriptomes were similar between FXR-hLBD and FXR-mLBD mice on a WT C57BL/6J background and in mice with a human-like BA composition (Cyp2c70-/-). OCA did, however, elicit markedly stronger transcriptional responses in FXR-hLBD mice, including more pronounced suppression of hepatic BA synthesis genes and differential regulation of BA transporters. Intriguingly, pathways involved in cell proliferation and fibrogenesis were induced in FXR-hLBD mice. Furthermore, OCA lowered plasma cholesterol to a greater extent in FXR-hLBD than FXR-mLBD mice, primarily due to a reduction in HDL-cholesterol. FXR-hLBD mice resemble FXR-mLBD controls under basal conditions but exhibit enhanced responses to FXR agonism by OCA. This model may improve preclinical evaluation of FXR-targeting drugs in a translation-relevant context.

BACKGROUND/OBJECTIVE:Glycogen storage disease type I (GSD I) is an autosomal recessive inborn error of carbohydrate metabolism. Patients with GSD type Ia and Ib exhibit overlapping and distinct symptoms and complications. Notably, GSD Ia patients show more severe hypertriglyceridemia and higher risk of hepatic tumors than GSD Ib patients. METHODS:Given the liver's pivotal role in these processes, this study utilized hepatocyte-specific CRISPR/Cas9-mediated somatic gene editing to explore the pathophysiological and biochemical adaptations in hepatic GSD Ia and Ib side-by-side. Additionally, hepatic histology, transcriptomics, and proteomics analysis was performed. RESULTS:Compared to controls, hepatic GSD Ia and Ib mice showed hepatomegaly, fasting hypoglycemia, hyperlactatemia, and increased uric acid in plasma, which was somewhat more pronounced in GSD Ia than Ib. Both GSD I subtypes showed similar reductions in hepatic acetyl-CoA precursor pool enrichment and increases in de novo biosynthesis of hepatic stearate and oleate. Interestingly, only GSD Ia mice showed mildly elevated plasma triglyceride and hepatic phosphate sugars. Metabolic changes were reflected at the transcriptomic and proteomic levels, with largely similar responses between GSD Ia and Ib livers. Moreover, altered mRNAs and protein levels related to nucleotide-binding oligomerization domain (NOD) signaling pathways, infection and inflammation, liver disease, and chemical carcinogenesis were somewhat more pronounced in hepatic GSD Ia than in GSD Ib mice. CONCLUSIONS:Overall, the metabolic disturbance was more severe in hepatocyte-specific GSD Ia than in GSD Ib mice, consistent with the clinical phenotype in patients. The metabolic disorders and specific metabolites, genes, and proteins identified in this study provided new insights into the pathophysiological and biochemical phenotypes of GSD I subtypes in the liver.

Background:Decreased hepatic removal of low density lipoproteins (LDL) and increased apolipoprotein B (apoB) production cause hypercholesterolemia, a major causal risk factor of atherosclerotic cardiovascular disease (ASCVD). By a genome-wide siRNA screen, we previously identified subunits of the Coat protein I (COPI) complex to limit LDL uptake into Huh-7 hepatocarcinoma cells. Methods:These findings were validated by targeted in vitro experiments as well as genetic association studies in humans and three mouse models with mutated or disrupted COPI genes. Results:Silencing of COPA, COPB1, COPB2, ARCN1, COPG1, and COPZ1 in Huh-7 cells resulted in decreased uptake of LDL and aberrant glycosylation and altered cell surface abundance of the LDL receptor (LDLR) as well as increased apoB secretion and cellular lipid storage. Single nucleotide polymorphisms of ARCN1 were associated with lower ARCN1 expression and higher levels of LDL-cholesterol (LDL-C). Rare variants of COPA and COPG1 were enriched among patients with LDL-C > 5 mmol/L. Patients and mice carrying other rare immunopathogenic missense variants of COPA and COPG1 did not present with elevated plasma levels of LDL-C, while hepatic knockdown of murine Copg1 increased the concentrations of non-HDL-cholesterol in plasma and triglycerides in the liver. Conclusions:The COPI coatomer regulates LDLR activity and apoB secretion as well as lipid content of liver cells. Loss of function of some variants of COPI genes are associated with higher LDL-C levels.

AIMS:In humans, reduced G-protein coupled receptor 146 (GPR146) expression is associated with reductions in both LDL and HDL cholesterol. While the effects on LDL cholesterol are mediated via the intracellular ERK/SREBP2 pathway, the mechanism explaining how GPR146 affects HDL cholesterol levels remains to be unravelled. METHODS AND RESULTS:Whole-body (Gpr146-/-) and liver-specific Gpr146 knockout (Gpr146 LKO) mice were used to explore changes in HDL metabolism. Wild-type mice were treated with a MEK1 inhibitor to block ERK signalling. HDL uptake and post-translational modification of scavenger receptor class B1 (SR-B1) were studied in murine primary hepatocytes. Genetic variants in GPR146 and SCARB1 served as instruments to examine HDL size and composition in human cohort studies. Investigation in both Gpr146-/- and Gpr146 LKO mice revealed a 20% reduction in HDL cholesterol and a concomitant 30% increase in hepatic SR-B1 protein (without changes in Scarb1 mRNA). This increase was driven by a 2.2-fold increase in cell surface SR-B1 via a mechanism that appears independent of ERK. In vitro studies show that loss of GPR146 increases SR-B1-mediated selective uptake of HDL lipid and HDL protein. Consistently, carriers of a GPR146 variant associated with loss-of-function and carriers of SCARB1 gain-of-function variant share reductions in apoA-I, HDL particle size, HDL cholesterol, and cholesteryl ester content compared to non-carriers. CONCLUSION:This study suggests that loss of GPR146 reduces HDL cholesterol via post-translational up-regulation of hepatic SR-B1 via an intracellular pathway that remains to be resolved. These findings imply that GPR146 inhibition to treat hypercholesterolaemia may not only lower plasma levels of LDL cholesterol but also HDL cholesterol.

BACKGROUND & AIMS:Hepatocellular carcinoma is the third leading cause of cancer-related mortality worldwide. Therapeutic options for hepatocellular carcinoma remain limited, and the mechanisms underlying hepatocellular carcinoma are not fully understood. Therefore, gaining a comprehensive understanding of the pathways that drive hepatocellular carcinoma is essential for improving treatments. Recent studies have identified vacuolar protein sorting 35, a component of the endosomal cargo sorting machinery called retromer, as a novel oncogene in various types of cancer, including hepatocellular carcinoma. However, its role in the initiation and progression of hepatocellular carcinoma is still unclear. METHODS:To study the role of vacuolar protein sorting 35 in hepatocellular proliferation and the development of hepatocellular carcinoma, we generated a liver-specific Vps35 knockout mouse model using the Cre-LoxP system (Vps35HepKO). Hepatocellular proliferation was studied in young and middle-aged mice, as well as during liver regeneration after two-thirds partial hepatectomy. Diethyl nitrosamine was used to induce hepatocellular carcinoma. Livers were analyzed at histological, transcriptional, and proteomic levels. RESULTS:Hepatic loss of vacuolar protein sorting 35 enhanced hepatocellular proliferation in post-natal livers via SRC and its downstream target signal transducer and activator of transcription 3. Pharmacologic inhibition of SRC with saracatinib normalized hepatocellular proliferation in Vps35HepKO mice. In contrast, hepatic vacuolar protein sorting 35 deficiency did not alter hepatocellular proliferation after partial hepatectomy in adult mice. Although vacuolar protein sorting 35-deficient postnatal livers exhibited an increased proliferative phenotype, hepatic loss of vacuolar protein sorting 35 reduced the number of diethyl nitrosamine-induced liver lesions without affecting tumor size. CONCLUSIONS:Our in vivo data identify murine vacuolar protein sorting 35 as a critical regulator of hepatocellular proliferation in postnatal livers, but not after partial hepatectomy. Although vacuolar protein sorting 35 deficiency mitigates diethyl nitrosamine-induced liver lesion formation, it does not affect tumor progression, arguing against a role for vacuolar protein sorting 35 as a canonical oncogene.

BACKGROUND: Reverse cholesterol transport by HDLs (high-density lipoproteins) is considered an antiatherogenic metabolic pathway. Hepatocytes are the main contributors to the efficacy of this pathway by the production of apoA-I (apolipoprotein A1) and its lipidation by ABCA1 (ATP-binding cassette transporter A1), selective uptake of cholesterol via SR-BI (scavenger receptor class B type 1), and uptake of entire HDL particles. The molecular determinants of the latter step are not well understood. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of HDL fluorescently labeled at its protein moiety into Huh-7 hepatocarcinoma cells. Top hit genes were validated by targeted in vitro experiments and the analysis of associations between their variants and HDL-C (HDL-cholesterol) levels in the databases of the Global Lipids Genetics Consortium and the UK Biobank, as well as inborn errors of metabolism and their respective mouse models. RESULTS: The knockdown of 128 genes significantly inhibited HDL uptake. Six of them encode components of the COPI (coat protein I) coatomer, namely, COPA , COPB1 , COPB2 , COPG1 , ARCN1 , and COPZ1 . Knocking down any of them decreased the uptake of both fluorescently labeled proteins and lipids of HDL, the cell surface abundance of SR-BI, and APOA1 expression and apoA-I secretion but increased the cell surface abundance of ABCA1. Common single-nucleotide polymorphisms of ARCN1 and COPB1 were associated with significantly higher HDL-C levels in the population, while rare COPA and COPG1 variants causing immunopathies were associated with rather lower levels of HDL-C in both affected patients and the corresponding genetically modified mice. CONCLUSIONS: In hepatocytes, the COPI coatomer regulates HDL holoparticle uptake, selective lipid uptake, apoA-I secretion, and cholesterol efflux, and thereby, it influences plasma levels of HDL-C.

BACKGROUND AND AIMS:Liver sinusoidal endothelial cells (LSECs) acquire a proinflammatory phenotype in metabolic dysfunction-associated steatohepatitis (MASH), characterized by enhanced expression of adhesion molecules and inflammatory mediators, a process termed lipotoxic endotheliopathy. However, the molecular drivers of this transformation remain incompletely defined. APPROACH AND RESULTS:We employed complementary models of lipotoxicity and MASH, including palmitate-treated primary human LSECs (in vitro), cultured precision-cut liver slices (PCLS) from MASH mice and normal human liver treated with MASH-inducing media (ex vivo), and diet-induced MASH models (in vivo). GeoMx digital spatial profiling of human MASH livers revealed enriched proinflammatory and fibrogenic signaling between LSECs and myeloid cells. Among the top upregulated genes and ligand-receptor pairs was ICAM1, whose expression correlated with disease severity and was increased in human MASH by immunostaining. ATAC-seq on primary mouse LSECs showed enhanced chromatin accessibility at the Icam1 promoter in MASH. Chromatin immunoprecipitation confirmed enrichment of the active epigenetic mark H3K27ac and BRD4 binding at the ICAM1 promoter in human LSECs under lipotoxic stress. Mechanistically, we identified the GSK3β/c-Jun/BRD4 axis as a key regulator of ICAM1 induction in LSECs. In vivo, ICAM1-neutralizing antibody or endothelial-specific epigenetic suppression reduced hepatic inflammation, injury, and fibrosis in MASH mice. CONCLUSIONS:ICAM1 is epigenetically upregulated in LSECs during lipotoxic stress and promotes myeloid cell recruitment in MASH. Targeting the epigenetic regulation of ICAM1 may offer a novel therapeutic strategy for treating human MASH.

BACKGROUND AND AIMS:Decreased removal and increased production of apolipoprotein B (apoB) containing lipoproteins cause hypercholesterolemia, a major causal risk factor of atherosclerotic cardiovascular disease. By a genome-wide siRNA screen, we previously identified subunits of the Coat protein I (COPI) complex to limit low density lipoprotein (LDL) uptake into Huh-7 hepatocarcinoma cells. This study investigated the underlying mechanism and the role of impaired COPI function for hypercholesterolemia. METHODS:Targeted loss of function experiments in vitro as well as genetic association studies in humans and three mouse models with mutated or disrupted COPI genes were performed. RESULTS:Silencing of COPA, COPB1, COPB2, ARCN1, COPG1, and COPZ1 in Huh-7 cells resulted in decreased uptake of LDL and aberrant glycosylation and reduced cell surface abundance of the LDL receptor (LDLR) as well as increased apoB secretion and cellular lipid storage. Single nucleotide polymorphisms of ARCN1 were associated with lower ARCN1 expression and higher levels of LDL-cholesterol. While patients and mice carrying rare immunopathogenic missense variants of the WD40- or appendage domains of COPA and COPG1, respectively, had normal LDL-cholesterol levels, rare variants altering other domains of these proteins were enriched among patients with hypercholesterolemia. The hepatic knockdown of Copg1 increased the concentrations of nonHDL-cholesterol in plasma and triglycerides in the liver of mice. CONCLUSIONS:The COPI coatomer regulates LDLR activity and apoB secretion as well as hepatic lipid content. Deficiency of Copg1 in mice and some but not all rare damaging COPI gene variants in humans are associated with higher LDL-cholesterol levels.

Investigating the intracellular biology of very low-density lipoproteins (VLDL) remains a challenge. We therefore generated apoB100-FLAG mice, which display normal lipid levels and concentration of FLAG-tagged apoB100 in plasma. However, we were unable to detect either apoB or FLAG in primary hepatocytes using immunofluorescence. Blocking proteasomal degradation alone or combined with inhibition of the secretory pathway only marginally improved apo ss detection. Our study suggests that the physiological concentration of apoB100 in isolated hepatocytes is too low and heterogeneous to allow for studies into the subcellular localisation of apoB.

BACKGROUND & AIMS:Bile Salt Export Pump (BSEP) deficiency is a rare genetic cholestatic liver disease, often necessitating liver transplantation. The p.E297G missense mutation is associated with residual BSEP function in vitro and delayed need for transplantation in patients. We aimed to generate a p.E297G BSEP knock-in (BSEPE297G) mouse model to evaluate interventions to improve residual BSEP function. METHODS:We generated BSEPE297G mice by CRISPR-Cas9 technology. BSEPE297G mice and wild type (WT) littermates were characterized for BSEP expression and liver pathology at 14 weeks of age. Maximal BSEP transport capacity without and after 4-phenylbutyrate (4-PB) treatment were determined in vivo by quantification of biliary bile acid secretion during intravenous infusion of increasing dosages of tauroursodeoxycholic acid (TUDCA) in WT, BSEPE297G and BSEP-/- mice. RESULTS:Western blot analysis showed immature BSEP protein in BSEPE297G livers. Median plasma AST was three-fold higher in BSEPE297G mice (Males: 198 vs. 60 U/L; Females: 188 vs. 50 U/L; each p < 0.001) while plasma bile acid levels were higher in female BSEPE297G mice compared to WT (Females: 36 vs. 6 μM, p < 0.001; Males: 11 vs. 3 μM, p = 0.07). Histological analysis revealed features of cholestatic liver pathology in BSEPE297G mice. TUDCA infusion strongly increased biliary bile acid secretion in WT but not in BSEPE297G and BSEP-/- mice. 4-PB treatment did not enhance bile acid transport capacity in BSEPE297G mice. CONCLUSIONS:BSEPE297G mice display a BSEP deficiency phenotype with a strongly reduced hepatobiliary bile acid transport capacity. The expression of immature BSEP protein suggests the potential to assess correctors of the BSEP functionality in vivo.

Background and aims: G-protein coupled receptor 146 (GPR146)-deficient mice exhibit a moderate 21% reduction in plasma cholesterol. This is associated with decreased phosphorylation of ERK1/2 and reduced SREBP2 activity in the liver, which leads to lower VLDL secretion. Insight into the role of GPR146 in humans is however limited. We therefore set out to study rare genetic variants in GPR146 to improve our understanding of this new player in lipid metabolism. Methods: We used whole genome sequencing data from UK Biobank participants to search for rare coding variants in GPR146. We first carried out gene-based burden tests (using SAIGE-GENE-framework) and examined the association of individual variants with plasma cholesterol levels. One of the variants (P62L) was also studied using the Global Lipids Genetics Consortium (GLGC) data set and in a knock-in mouse model. Results: We found that the combination of rare genetic variants identified in GPR146 is significantly associated with plasma cholesterol levels. Three rare variants, i.e. P62L, I129I, and A175T were individually associated with reduced plasma cholesterol. In the GLGC cohort, the P62L variant was associated with reductions in both HDL and LDL cholesterol. Follow-up experiments show lower plasma cholesterol levels in Gpr146(P61L) male and female mice (-13%, p<0.05 and -15%, p<0.005, respectively) when compared to controls due to a reduction in HDL cholesterol. The Gpr146(P61L) mice did not exhibit a change in VLDL secretion. In line, the ERK1/2 signalling pathway and Srebp2 mRNA expression in liver homogenates, and the secretion of apoB by primary hepatocytes of Gpr146(P61L) and wild-type mice were unchanged. Conclusions: This study shows that rare GPR146 gene variants are associated with lower plasma cholesterol levels in humans. One of these variants, P62L is associated with reductions of HDL cholesterol and LDL cholesterol in humans while the ortholog in mice confers a loss of GPR146 function leading to only reduced HDL cholesterol. How GPR146 affects HDL metabolism in humans and mice remains to be resolved.