Personalised modelling of clinical heterogeneity between medium-chain acyl-CoA dehydrogenase patients.

BACKGROUND: Monogenetic inborn errors of metabolism cause a wide phenotypic heterogeneity that may even differ between family members carrying the same genetic variant. Computational modelling of metabolic networks may identify putative sources of this inter-patient heterogeneity. Here, we mainly focus on medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common inborn error of the mitochondrial fatty acid oxidation (mFAO). It is an enigma why some MCADD patients-if untreated-are at risk to develop severe metabolic decompensations, whereas others remain asymptomatic throughout life. We hypothesised that an ability to maintain an increased free mitochondrial CoA (CoASH) and pathway flux might distinguish asymptomatic from symptomatic patients. RESULTS: We built and experimentally validated, for the first time, a kinetic model of the human liver mFAO. Metabolites were partitioned according to their water solubility between the bulk aqueous matrix and the inner membrane. Enzymes are also either membrane-bound or in the matrix. This metabolite partitioning is a novel model attribute and improved predictions. MCADD substantially reduced pathway flux and CoASH, the latter due to the sequestration of CoA as medium-chain acyl-CoA esters. Analysis of urine from MCADD patients obtained during a metabolic decompensation showed an accumulation of medium- and short-chain acylcarnitines, just like the acyl-CoA pool in the MCADD model. The model suggested some rescues that increased flux and CoASH, notably increasing short-chain acyl-CoA dehydrogenase (SCAD) levels. Proteome analysis of MCADD patient-derived fibroblasts indeed revealed elevated levels of SCAD in a patient with a clinically asymptomatic state. This is a rescue for MCADD that has not been explored before. Personalised models based on these proteomics data confirmed an increased pathway flux and CoASH in the model of an asymptomatic patient compared to those of symptomatic MCADD patients. CONCLUSIONS: We present a detailed, validated kinetic model of mFAO in human liver, with solubility-dependent metabolite partitioning. Personalised modelling of individual patients provides a novel explanation for phenotypic heterogeneity among MCADD patients. Further development of personalised metabolic models is a promising direction to improve individualised risk assessment, management and monitoring for inborn errors of metabolism.

Selective Hepatic Cbs Knockout Aggravates Liver Damage, Endothelial Dysfunction and ROS Stress in Mice Fed a Western Diet.

Cystathionine-ß-synthase (CBS) is highly expressed in the liver, and deficiencies in Cbs lead to hyperhomocysteinemia (HHCy) and disturbed production of antioxidants such as hydrogen sulfide. We therefore hypothesized that liver-specific Cbs deficient (LiCKO) mice would be particularly susceptible to the development of non-alcoholic fatty liver disease (NAFLD). NAFLD was induced by a high-fat high-cholesterol (HFC) diet; LiCKO and controls were split into eight groups based on genotype (con, LiCKO), diet (normal diet, HFC), and diet duration (12 weeks, 20 weeks). LiCKO mice displayed intermediate to severe HHCy. Plasma H2O2 was increased by HFC, and further aggravated in LiCKO. LiCKO mice fed an HFC diet had heavier livers, increased lipid peroxidation, elevated ALAT, aggravated hepatic steatosis, and inflammation. LiCKO mice showed decreased L-carnitine in the liver, but this did not result in impaired fatty acid oxidation. Moreover, HFC-fed LiCKO mice demonstrated vascular and renal endothelial dysfunction. Liver and endothelial damage correlated significantly with systemic ROS status. In conclusion, this study demonstrates an important role for CBS in the liver in the development of NAFLD, which is most probably mediated through impaired defense against oxidative stress.

Cargo-Specific Role for Retriever Subunit VPS26C in Hepatocyte Lipoprotein Receptor Recycling to Control Postprandial Triglyceride-Rich Lipoproteins.

BACKGROUND: The copper metabolism MURR1 domains/coiled-coil domain containing 22/coiled-coil domain containing 93 (CCC) complex is required for the transport of low-density lipoprotein receptor (LDLR) and LRP1 (LDLR-related protein 1) from endosomes to the cell surface of hepatocytes. Impaired functioning of hepatocytic CCC causes hypercholesterolemia in mice, dogs, and humans. Retriever, a protein complex consisting of subunits VPS26C, VPS35L, and VPS29, is associated with CCC, but its role in endosomal lipoprotein receptor transport is unclear. We here investigated the contribution of retriever to hepatocytic lipoprotein receptor recycling and plasma lipids regulation. METHODS: Using somatic CRISPR/Cas9 gene editing, we generated liver-specific VPS35L or VPS26C-deficient mice. We determined total and surface levels of LDLR and LRP1 and plasma lipids. In addition, we studied the protein levels and composition of CCC and retriever. RESULTS: Hepatocyte VPS35L deficiency reduced VPS26C levels but had minimal impact on CCC composition. VPS35L deletion decreased hepatocytic surface expression of LDLR and LRP1, accompanied by a 21% increase in plasma cholesterol levels. Hepatic VPS26C ablation affected neither levels of VPS35L and CCC subunits, nor plasma lipid concentrations. However, VPS26C deficiency increased hepatic LDLR protein levels by 2-fold, probably compensating for reduced LRP1 functioning, as we showed in VPS26C-deficient hepatoma cells. Upon PCSK9 (proprotein convertase subtilisin/kexin type 9)-mediated LDLR elimination, VPS26C ablation delayed postprandial triglyceride clearance and increased plasma triglyceride levels by 26%. CONCLUSIONS: Our study suggests that VPS35L is shared between retriever and CCC to facilitate LDLR and LRP1 transport from endosomes to the cell surface. Conversely, retriever subunit VPS26C selectively transports LRP1, but not LDLR, and thereby may control hepatic uptake of postprandial triglyceride-rich lipoprotein remnants.

Loss of hepatic SMLR1 causes hepatosteatosis and protects against atherosclerosis due to decreased hepatic VLDL secretion.

BACKGROUND AND AIMS: The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co-expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine-rich protein 1 ( SMLR1 ), encoding for small leucine-rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. APPROACH AND RESULTS: To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR-associated protein 9 gene editing to silence murine Smlr1 in hepatocytes ( Smlr1 -LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis-Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet-induced hyperlipidemia and atherosclerosis but causes NASH. On a high-fat, high-cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1 -LKO mice versus controls. CONCLUSIONS: We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis-Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.

Butyrate oxidation attenuates the butyrate-induced improvement of insulin sensitivity in myotubes.

Skeletal muscle insulin resistance is a key pathophysiological process that precedes the development of type 2 diabetes. Whereas an overload of long-chain fatty acids can induce muscle insulin resistance, butyrate, a short-chain fatty acid (SCFA) produced from dietary fibre fermentation, prevents it. This preventive role of butyrate has been attributed to histone deacetylase (HDAC)-mediated transcription regulation and activation of mitochondrial fatty-acid oxidation. Here we address the interplay between butyrate and the long-chain fatty acid palmitate and investigate how transcription, signalling and metabolism are integrated to result in the butyrate-induced skeletal muscle metabolism remodelling. Butyrate enhanced insulin sensitivity in palmitate-treated, insulin-resistant C2C12 cells, as shown by elevated insulin receptor 1 (IRS1) and pAKT protein levels and Slc2a4 (GLUT4) mRNA, which led to a higher glycolytic capacity. Long-chain fatty-acid oxidation capacity and other functional respiration parameters were not affected. Butyrate did upregulate mitochondrial proteins involved in its own oxidation, as well as concentrations of butyrylcarnitine and hydroyxybutyrylcarnitine. By knocking down the gene encoding medium-chain 3-ketoacyl-CoA thiolase (MCKAT, Acaa2), butyrate oxidation was inhibited, which amplified the effects of the SCFA on insulin sensitivity and glycolysis. This response was associated with enhanced HDAC inhibition, based on histone 3 acetylation levels. Butyrate enhances insulin sensitivity and induces glycolysis, without the requirement of upregulated long-chain fatty acid oxidation. Butyrate catabolism functions as an escape valve that attenuates HDAC inhibition. Thus, inhibition of butyrate oxidation indirectly prevents insulin resistance and stimulates glycolytic flux in myotubes treated with butyrate, most likely via an HDAC-dependent mechanism.

Mice with a deficiency in Peroxisomal Membrane Protein 4 (PXMP4) display mild changes in hepatic lipid metabolism.

Peroxisomes play an important role in the metabolism of a variety of biomolecules, including lipids and bile acids. Peroxisomal Membrane Protein 4 (PXMP4) is a ubiquitously expressed peroxisomal membrane protein that is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα), but its function is still unknown. To investigate the physiological function of PXMP4, we generated a Pxmp4 knockout (Pxmp4-/-) mouse model using CRISPR/Cas9-mediated gene editing. Peroxisome function was studied under standard chow-fed conditions and after stimulation of peroxisomal activity using the PPARα ligand fenofibrate or by using phytol, a metabolite of chlorophyll that undergoes peroxisomal oxidation. Pxmp4-/- mice were viable, fertile, and displayed no changes in peroxisome numbers or morphology under standard conditions. Also, no differences were observed in the plasma levels of products from major peroxisomal pathways, including very long-chain fatty acids (VLCFAs), bile acids (BAs), and BA intermediates di- and trihydroxycholestanoic acid. Although elevated levels of the phytol metabolites phytanic and pristanic acid in Pxmp4-/- mice pointed towards an impairment in peroxisomal α-oxidation capacity, treatment of Pxmp4-/- mice with a phytol-enriched diet did not further increase phytanic/pristanic acid levels. Finally, lipidomic analysis revealed that loss of Pxmp4 decreased hepatic levels of the alkyldiacylglycerol class of neutral ether lipids, particularly those containing polyunsaturated fatty acids. Together, our data show that while PXMP4 is not critical for overall peroxisome function under the conditions tested, it may have a role in the metabolism of (ether)lipids.

Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome.

BACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

Modeling Phenotypic Heterogeneity of Glycogen Storage Disease Type 1a Liver Disease in Mice by Somatic CRISPR/CRISPR-associated protein 9-Mediated Gene Editing.

BACKGROUND AND AIMS: Patients with glycogen storage disease type 1a (GSD-1a) primarily present with life-threatening hypoglycemia and display severe liver disease characterized by hepatomegaly. Despite strict dietary management, long-term complications still occur, such as liver tumor development. Variations in residual glucose-6-phosphatase (G6PC1) activity likely contribute to phenotypic heterogeneity in biochemical symptoms and complications between patients. However, lack of insight into the relationship between G6PC1 activity and symptoms/complications and poor understanding of the underlying disease mechanisms pose major challenges to provide optimal health care and quality of life for GSD-1a patients. Currently available GSD-1a animal models are not suitable to systematically investigate the relationship between hepatic G6PC activity and phenotypic heterogeneity or the contribution of gene-gene interactions (GGIs) in the liver. APPROACH AND RESULTS: To meet these needs, we generated and characterized a hepatocyte-specific GSD-1a mouse model using somatic CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing. Hepatic G6pc editing reduced hepatic G6PC activity up to 98% and resulted in failure to thrive, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly, hepatic steatosis (HS), and increased liver tumor incidence. This approach was furthermore successful in simultaneously modulating hepatic G6PC and carbohydrate response element-binding protein, a transcription factor that is activated in GSD-1a and protects against HS under these conditions. Importantly, it also allowed for the modeling of a spectrum of GSD-1a phenotypes in terms of hepatic G6PC activity, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly and HS. CONCLUSIONS: In conclusion, we show that somatic CRISPR/Cas9-mediated gene editing allows for the modeling of a spectrum of hepatocyte-borne GSD-1a disease symptoms in mice and to efficiently study GGIs in the liver. This approach opens perspectives for translational research and will likely contribute to personalized treatments for GSD-1a and other genetic liver diseases.

Taking One Step Back in Familial Hypercholesterolemia: STAP1 Does Not Alter Plasma LDL (Low-Density Lipoprotein) Cholesterol in Mice and Humans.

OBJECTIVE: STAP1, encoding for STAP1 (signal transducing adaptor family member 1), has been reported as a candidate gene associated with familial hypercholesterolemia. Unlike established familial hypercholesterolemia genes, expression of STAP1 is absent in liver but mainly observed in immune cells. In this study, we set out to validate STAP1 as a familial hypercholesterolemia gene. Approach and Results: A whole-body Stap1 knockout mouse model (Stap1-/-) was generated and characterized, without showing changes in plasma lipid levels compared with controls. In follow-up studies, bone marrow from Stap1-/- mice was transplanted to Ldlr-/- mice, which did not show significant changes in plasma lipid levels or atherosclerotic lesions. To functionally assess whether STAP1 expression in B cells can affect hepatic function, HepG2 cells were cocultured with peripheral blood mononuclear cells isolated from heterozygotes carriers of STAP1 variants and controls. The peripheral blood mononuclear cells from STAP1 variant carriers and controls showed similar LDLR mRNA and protein levels. Also, LDL (low-density lipoprotein) uptake by HepG2 cells did not differ upon coculturing with peripheral blood mononuclear cells isolated from either STAP1 variant carriers or controls. In addition, plasma lipid profiles of 39 carriers and 71 family controls showed no differences in plasma LDL cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, and lipoprotein(a) levels. Similarly, B-cell populations did not differ in a group of 10 STAP1 variant carriers and 10 age- and sex-matched controls. Furthermore, recent data from the UK Biobank do not show association between STAP1 rare gene variants and LDL cholesterol. CONCLUSIONS: Our combined studies in mouse models and carriers of STAP1 variants indicate that STAP1 is not a familial hypercholesterolemia gene.

A human-like bile acid pool induced by deletion of hepatic Cyp2c70 modulates effects of FXR activation in mice.

Bile acids (BAs) facilitate intestinal absorption of lipid-soluble nutrients and modulate various metabolic pathways through the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5. These receptors are targets for therapy in cholestatic and metabolic diseases. However, dissimilarities in BA metabolism between humans and mice complicate translation of preclinical data. Cytochrome P450 family 2 subfamily c polypeptide 70 (CYP2C70) was recently proposed to catalyze the formation of rodent-specific muricholic acids (MCAs). With CRISPR/Cas9-mediated somatic genome editing, we generated an acute hepatic Cyp2c70 knockout mouse model (Cyp2c70ako) to clarify the role of CYP2C70 in BA metabolism in vivo and evaluate whether its activity modulates effects of pharmacologic FXR activation on cholesterol homeostasis. In Cyp2c70ako mice, chenodeoxycholic acid (CDCA) increased at the expense of ßMCA, resulting in a more hydrophobic human-like BA pool. Tracer studies demonstrated that, in vivo, CYP2C70 catalyzes the formation of ßMCA primarily by sequential 6ß-hydroxylation and C7-epimerization of CDCA, generating αMCA as an intermediate metabolite. Physiologically, the humanized BA composition in Cyp2c70ako mice blunted the stimulation of fecal cholesterol disposal in response to FXR activation compared with WT mice, predominantly due to reduced stimulation of transintestinal cholesterol excretion. Thus, deletion of hepatic Cyp2c70 in adult mice translates into a human-like BA pool composition and impacts the response to pharmacologic FXR activation. This Cyp2c70ako mouse model may be a useful tool for future studies of BA signaling and metabolism that informs human disease development and treatment.

A common variant in CCDC93 protects against myocardial infarction and cardiovascular mortality by regulating endosomal trafficking of low-density lipoprotein receptor.

AIMS: Genome-wide association studies have previously identified INSIG2 as a candidate gene for plasma low-density lipoprotein cholesterol (LDL-c). However, we suspect a role for CCDC93 in the same locus because of its involvement in the recycling of the LDL-receptor (LDLR). METHODS AND RESULTS: Characterization of the INSIG2 locus was followed by studies in over 107 000 individuals from the general population, the Copenhagen General Population Study and the Copenhagen City Heart Study, for associations of genetic variants with plasma lipids levels, with risk of myocardial infarction (MI) and with cardiovascular mortality. CCDC93 was furthermore studied in cells and mice. The lead variant of the INSIG2 locus (rs10490626) is not associated with changes in the expression of nearby genes but is a part of a genetic block, which excludes INSIG2. This block includes a coding variant in CCDC93 p.Pro228Leu, which is in strong linkage disequilibrium with rs10490626 (r2 > 0.96). In the general population, separately and combined, CCDC93 p.Pro228Leu is dose-dependently associated with lower LDL-c (P-trend 2.5 × 10-6 to 8.0 × 10-9), with lower risk of MI (P-trend 0.04-0.002) and lower risk of cardiovascular mortality (P-trend 0.005-0.004). These results were validated for LDL-c, risk of both coronary artery disease and MI in meta-analyses including from 194 000 to >700 000 participants. The variant is shown to increase CCDC93 protein stability, while overexpression of human CCDC93 decreases plasma LDL-c in mice. Conversely, CCDC93 ablation reduces LDL uptake as a result of reduced LDLR levels at the cell membrane. CONCLUSION: This study provides evidence that a common variant in CCDC93, encoding a protein involved in recycling of the LDLR, is associated with lower LDL-c levels, lower risk of MI and cardiovascular mortality.

FXR overexpression alters adipose tissue architecture in mice and limits its storage capacity leading to metabolic derangements.

The bile acid-activated nuclear receptor, FXR (NR1H4), has been implicated in the control of lipid and energy metabolism, but its role in fat tissue, where it is moderately expressed, is not understood. In view of the recent development of FXR-targeting therapeutics for treatment of human metabolic diseases, understanding the tissue-specific actions of FXR is essential. Transgenic mice expressing human FXR in adipose tissue (aP2-hFXR mice) at three to five times higher levels than endogenous Fxr, i.e., much lower than its expression in liver and intestine, have markedly enlarged adipocytes and show extensive extracellular matrix remodeling. Ageing and exposure to obesogenic conditions revealed a strongly limited capacity for adipose expansion and development of fibrosis in adipose tissues of aP2-hFXR transgenic mice. This was associated with impaired lipid storage capacity, leading to elevated plasma free fatty acids and ectopic fat deposition in liver and muscle as well as whole-body insulin resistance. These studies establish that adipose FXR is a determinant of adipose tissue architecture and contributes to whole-body lipid homeostasis.

The hepatic WASH complex is required for efficient plasma LDL and HDL cholesterol clearance.

The evolutionary conserved Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex is one of the crucial multiprotein complexes that facilitates endosomal recycling of transmembrane proteins. Defects in WASH components have been associated with inherited developmental and neurological disorders in humans. Here, we show that hepatic ablation of the WASH component Washc1 in chow-fed mice increases plasma concentrations of cholesterol in both LDLs and HDLs, without affecting hepatic cholesterol content, hepatic cholesterol synthesis, biliary cholesterol excretion, or hepatic bile acid metabolism. Elevated plasma LDL cholesterol was related to reduced hepatocytic surface levels of the LDL receptor (LDLR) and the LDLR-related protein LRP1. Hepatic WASH ablation also reduced the surface levels of scavenger receptor class B type I and, concomitantly, selective uptake of HDL cholesterol into the liver. Furthermore, we found that WASHC1 deficiency increases LDLR proteolysis by the inducible degrader of LDLR, but does not affect proprotein convertase subtilisin/kexin type 9-mediated LDLR degradation. Remarkably, however, loss of hepatic WASHC1 may sensitize LDLR for proprotein convertase subtilisin/kexin type 9-induced degradation. Altogether, these findings identify the WASH complex as a regulator of LDL as well as HDL metabolism and provide in vivo evidence for endosomal trafficking of scavenger receptor class B type I in hepatocytes.

Partial Deletion of Tie2 Affects Microvascular Endothelial Responses to Critical Illness in A Vascular Bed and Organ-Specific Way.

Tyrosine kinase receptor (Tie2) is mainly expressed by endothelial cells. In animal models mimicking critical illness, Tie2 levels in organs are temporarily reduced. Functional consequences of these reduced Tie2 levels on microvascular endothelial behavior are unknown. We investigated the effect of partial deletion of Tie2 on the inflammatory status of endothelial cells in different organs. Newly generated heterozygous Tie2 knockout mice (exon 9 deletion, ΔE9/Tie2) exhibiting 50% reduction in Tie2 mRNA and protein, and wild-type littermate controls (Tie2), were subjected to hemorrhagic shock and resuscitation (HS + R), or challenged with i.p. lipopolysaccharide (LPS). Kidney, liver, lung, heart, brain, and intestine were analyzed for mRNA levels of adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular cell adhesion molecule 1 (ICAM-1), and CD45. Exposure to HS + R did not result in different expression responses of these molecules between organs from Tie2 or Tie2 mice and sham-operated mice. In contrast, the LPS-induced mRNA expression levels of E-selectin, VCAM-1, and ICAM-1, and CD45 in organs were attenuated in Tie2 mice when compared with Tie2 mice in kidney and liver, but not in the other organs studied. Furthermore, reduced expression of E-selectin and VCAM-1 protein, and reduced influx of CD45 cells upon LPS exposure, was visible in a microvascular bed-specific pattern in kidney and liver of Tie2 mice compared with controls. In contrast to the hypothesis that a disbalance in the Ang/Tie2 system leads to increased microvascular inflammation, heterozygous deletion of Tie2 is associated with an organ-restricted, microvascular bed-specific attenuation of endothelial inflammatory response to LPS.

The COMMD Family Regulates Plasma LDL Levels and Attenuates Atherosclerosis Through Stabilizing the CCC Complex in Endosomal LDLR Trafficking.

RATIONALE: COMMD (copper metabolism MURR1 domain)-containing proteins are a part of the CCC (COMMD-CCDC22 [coiled-coil domain containing 22]-CCDC93 [coiled-coil domain containing 93]) complex facilitating endosomal trafficking of cell surface receptors. Hepatic COMMD1 inactivation decreases CCDC22 and CCDC93 protein levels, impairs the recycling of the LDLR (low-density lipoprotein receptor), and increases plasma low-density lipoprotein cholesterol levels in mice. However, whether any of the other COMMD members function similarly as COMMD1 and whether perturbation in the CCC complex promotes atherogenesis remain unclear. OBJECTIVE: The main aim of this study is to unravel the contribution of evolutionarily conserved COMMD proteins to plasma lipoprotein levels and atherogenesis. METHODS AND RESULTS: Using liver-specific Commd1, Commd6, or Commd9 knockout mice, we investigated the relation between the COMMD proteins in the regulation of plasma cholesterol levels. Combining biochemical and quantitative targeted proteomic approaches, we found that hepatic COMMD1, COMMD6, or COMMD9 deficiency resulted in massive reduction in the protein levels of all 10 COMMDs. This decrease in COMMD protein levels coincided with destabilizing of the core (CCDC22, CCDC93, and chromosome 16 open reading frame 62 [C16orf62]) of the CCC complex, reduced cell surface levels of LDLR and LRP1 (LDLR-related protein 1), followed by increased plasma low-density lipoprotein cholesterol levels. To assess the direct contribution of the CCC core in the regulation of plasma cholesterol levels, Ccdc22 was deleted in mouse livers via CRISPR/Cas9-mediated somatic gene editing. CCDC22 deficiency also destabilized the complete CCC complex and resulted in elevated plasma low-density lipoprotein cholesterol levels. Finally, we found that hepatic disruption of the CCC complex exacerbates dyslipidemia and atherosclerosis in ApoE3*Leiden mice. CONCLUSIONS: Collectively, these findings demonstrate a strong interrelationship between COMMD proteins and the core of the CCC complex in endosomal LDLR trafficking. Hepatic disruption of either of these CCC components causes hypercholesterolemia and exacerbates atherosclerosis. Our results indicate that not only COMMD1 but all other COMMDs and CCC components may be potential targets for modulating plasma lipid levels in humans.

NF-κB p65 serine 467 phosphorylation sensitizes mice to weight gain and TNFα-or diet-induced inflammation.

The NF-κB family of transcription factors is essential for an effective immune response, but also controls cell metabolism, proliferation and apoptosis. Its broad relevance and the high connectivity to diverse signaling pathways require a tight control of NF-κB activity. To investigate the control of NF-κB activity by phosphorylation of the NF-κB p65 subunit, we generated a knock-in mouse model in which serine 467 (the mouse homolog of human p65 serine 468) was replaced with a non-phosphorylatable alanine (S467A). This substitution caused reduced p65 protein synthesis and diminished TNFα-induced expression of a selected group of NF-κB-dependent genes. Intriguingly, high-fat fed S467A mice displayed increased locomotor activity and energy expenditure, which coincided with a reduced body weight gain. Although glucose metabolism or insulin sensitivity was not improved, diet-induced liver inflammation was diminished in S467A mice. Altogether, this study demonstrates that phosphorylation of p65 serine 467 augment NF-κB activity and exacerbates various deleterious effects of overnutrition in mice.

CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL.

The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking.

IL-1ß and TGFß2 synergistically induce endothelial to mesenchymal transition in an NFκB-dependent manner.

Endothelial to mesenchymal transition (EndMT) contributes to fibrotic diseases. The main inducer of EndMT is TGFß signaling. TGFß2 is the dominant isoform in the physiological embryonic EndMT, but its role in the pathological EndMT in the context of inflammatory co-stimulation is not known. The aim of this study was to investigate TGFß2-induced EndMT in the context of inflammatory IL-1ß signaling. Co-stimulation with IL-1ß and TGFß2, but not TGFß1, caused synergistic induction of EndMT. Also, TGFß2 was the only TGFß isoform that was progressively upregulated during EndMT. External IL-1ß stimulation was dispensable once EndMT was induced. The inflammatory transcription factor NFκB was upregulated in an additive manner by IL-1ß and TGFß2 co-stimulation. Co-stimulation also led to the nuclear translocation of NFκB which was sustained over long-term treatment. Activation of NFκB was indispensable for the co-induction of EndMT. Our data suggest that the microenvironment at the verge between inflammation (IL-1ß) and tissue remodeling (TGFß2) can strongly promote the process of EndMT. Therefore our findings provide new insights into the mechanisms of pathological EndMT.

Sex-dependent programming of glucose and fatty acid metabolism in mouse offspring by maternal protein restriction.

BACKGROUND: Nutritional conditions during fetal life influence the risk of the development of metabolic syndrome and cardiovascular diseases in adult life (metabolic programming). Impaired glucose tolerance and dysregulated fatty acid metabolism are hallmarks of metabolic syndrome. OBJECTIVE: We aimed to establish a mouse model of metabolic programming focusing on the sex-specific effects of a maternal low-protein diet during gestation on glucose and lipid metabolism in the adult offspring. METHODS: Pregnant C57BL/6 mice received a control or a low-protein diet (18% vs 9% casein) throughout gestation. Male and female offspring received a low-fat or a high-fat diet from 6 to 22 weeks of age. RESULTS: Maternal low-protein diet during gestation led to deteriorated insulin sensitivity on high-fat feeding in female offspring, as determined by biochemical and microarray analyses. Female offspring of control diet-fed dams were relatively resistant to high-fat diet-induced metabolic dysregulation. In contrast, maternal low-protein diet did not specifically affect the metabolic parameters addressed in male offspring. In males, the high-fat diet led to insulin insensitivity regardless of the diet of the dam. CONCLUSIONS: Our findings show that fetal malnutrition has a limited impact on male mouse offspring, yet it does influence the metabolic response to a high-fat diet in females. These findings may have implications for future early diagnostics in metabolic syndrome and for the development of sex-specific treatment regimens.