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Background: Common genetic variation in close proximity to the ILRUN gene are significantly associated with coronary artery disease as well as with plasma lipid traits. We recently demonstrated that hepatic inflammation and lipid regulator with ubiquitin-associated domain-like and NBR1-like domains (ILRUN) regulates lipoprotein metabolism in vivo in mice. However, whether ILRUN, which is expressed in vascular cells, directly impacts atherogenesis remains unclear. We sought to determine the role of ILRUN in atherosclerosis development in mice. Methods: For our study, we generated global Ilrun -deficient ( Ilrun KO) male and female mice on 2 hyperlipidemic backgrounds: low density lipoprotein receptor knockout ( Ldlr KO) and apolipoprotein E knockout ( Apoe KO; double knockout [DKO]). Results: Compared with littermate control mice (single Ldlr KO or Apoe KO), deletion of Ilrun in DKO mice resulted in significantly attenuated both early and advanced atherosclerotic lesion development, as well as reduced necrotic area. DKO mice also had significantly decreased plasma cholesterol levels, primarily attributable to non-HDL (high-density lipoprotein) cholesterol. Hepatic-specific reconstitution of ILRUN in DKO mice on the Apoe KO background normalized plasma lipids, but atherosclerotic lesion area and necrotic area remained reduced in DKO mice. Further analysis showed that loss of Ilrun increased efferocytosis receptor MerTK expression in macrophages, enhanced in vitro efferocytosis, and significantly improved in situ efferocytosis in advanced lesions. Conclusions: Our results support ILRUN as an important novel regulator of atherogenesis that promotes lesion progression and necrosis. It influences atherosclerosis through both plasma lipid-dependent and lipid-independent mechanisms. These findings support ILRUN as the likely causal gene responsible for genetic association of variants with coronary artery disease at this locus and suggest that suppression of ILRUN activity might be expected to reduce atherosclerosis.

SummaryObesity is a growing worldwide epidemic that carries numerous metabolic complications including increased risk of type 2 diabetes (T2D), cardiovascular disease (CVD), and non-alcoholic fatty liver disease (NAFLD). Multiple genome-wide association studies (GWAS) have associated thePPP1R3Blocus with cardiometabolic traits including fasting glucose and insulin levels (T2D traits), plasma lipids (CVD traits), and indications of hepatic steatosis and liver damage (NAFLD traits)1–5. ThePPP1R3Bgene encodes the glycogen regulatory protein PPP1R3B (also known as GL) which has an established role in liver glycogen metabolism and plasma glucose homeostasis6,7. The metabolic and NAFLD GWAS single nucleotide polymorphisms (SNPs) in this region, which are all in high linkage disequilibrium, result in increased liverPPP1R3Bexpression and hepatic glycogen accumulation, but have provided conflicting results on the impacts on hepatic steatosis and liver damage. Here we investigate the consequences of bothPpp1r3boverexpression and deletion in mouse and cell models and find that dysregulatedPpp1r3bexpression in either direction promotes metabolic dysfunction and liver injury. Hepatocyte overexpression ofPpp1r3bincreases hepatic glycogen storage, prolongs fasting blood glucose levels, and confers protection from hepatic steatosis, but increases plasma ALT in aged animals. Conversely, deletion of hepatocytePpp1r3beliminates hepatic glycogen, causes impaired glucose disposal, and results in hepatic steatosis with age or high sucrose diet. We investigated the metabolic pathways contributing to steatosis and found thatPpp1r3bdeletion and diminished glycogenesis diverts the storage of exogenous glucose to hepatic triglycerides (TG), and stored liver lipids are preferentially used for energy during fasting through lipid oxidation and ketogenesis. Further, we interrogated two large human biobank cohorts and found carriers of SNPs associated with increasedPPP1R3Bexpression have increased plasma glucose, decreased hepatic fat, and lower plasma lipids, while putative loss-of-function (pLoF) variant carriers have increased hepatic fat and elevated plasma ketones and lipids, consistent with the results seen in our mouse models. These findings suggest hepatic PPP1R3B serves as a metabolic switch favoring hepatic energy storage as glycogen instead of TG.

© 2021 American Society of Human Genetics, EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG., This work was supported by the EU FP7 large-scale integrating project Genetic and Epigenetic Networks in Cognitive Dysfunction (241995) (to H.v.B.); National Institutes of Health (NIH) grants 5R01GM115730-03 (to M.H.), U54 NS115198 (to A.C.E. and M.H.), and T32GM008638 (to A.C.E.); and the Transatlantic Network of Excellence grant (10CVD03) from the Fondation Leducq and NIH NHGRI U01HG006398 (to D.J.R.). Family 4 was enrolled in the CAUSES Study; investigators include Shelin Adam, Christele Du Souich, Alison Elliott, Anna Lehman, Jill Mwenifumbo, Tanya Nelson, Clara Van Karnebeek, and Jan Friedman; it is funded by Mining for Miracles, British Columbia Children’s Hospital Foundation (grant number F15-01355) and Genome British Columbia (grant number F16-02276). D.L.P. is recipient of a CAPES Fellowship (99999.013311/2013-01). X.B. is supported by an AHA career development award (19CDA34630032).

Rationale: Single-nucleotide polymorphisms near the ILRUN (inflammation and lipid regulator with ubiquitin-associated–like and NBR1 [next to BRCA1 gene 1 protein]-like domains) gene are genome-wide significantly associated with plasma lipid traits and coronary artery disease (CAD), but the biological basis of this association is unknown. Objective: To investigate the role of ILRUN in plasma lipid and lipoprotein metabolism. Methods and Results: ILRUN encodes a protein that contains a ubiquitin-associated–like domain, suggesting that it may interact with ubiquitinylated proteins. We generated mice globally deficient for Ilrun and found they had significantly lower plasma cholesterol levels resulting from reduced liver lipoprotein production. Liver transcriptome analysis uncovered altered transcription of genes downstream of lipid-related transcription factors, particularly PPARα (peroxisome proliferator-activated receptor alpha), and livers from Ilrun -deficient mice had increased PPARα protein. Human ILRUN was shown to bind to ubiquitinylated proteins including PPARα, and the ubiquitin-associated–like domain of ILRUN was found to be required for its interaction with PPARα. Conclusions: These findings establish ILRUN as a novel regulator of lipid metabolism that promotes hepatic lipoprotein production. Our results also provide functional evidence that ILRUN may be the casual gene underlying the observed genetic associations with plasma lipids at 6p21 in human.

Background & Aims Recently, novel inborn errors of metabolism were identified because of mutations in V-ATPase assembly factors TMEM199 and CCDC115. Patients are characterized by generalized protein glycosylation defects, hypercholesterolemia, and fatty liver disease. Here, we set out to characterize the lipid and fatty liver phenotype in human plasma, cell models, and a mouse model. Methods and Results Patients with TMEM199 and CCDC115 mutations displayed hyperlipidemia, characterized by increased levels of lipoproteins in the very low density lipoprotein range. HepG2 hepatoma cells, in which the expression of TMEM199 and CCDC115 was silenced, and induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells from patients with TMEM199 mutations showed markedly increased secretion of apolipoprotein B (apoB) compared with controls. A mouse model for TMEM199 deficiency with a CRISPR/Cas9-mediated knock-in of the human A7E mutation had marked hepatic steatosis on chow diet. Plasma N-glycans were hypogalactosylated, consistent with the patient phenotype, but no clear plasma lipid abnormalities were observed in the mouse model. In the siTMEM199 and siCCDC115 HepG2 hepatocyte models, increased numbers and size of lipid droplets were observed, including abnormally large lipid droplets, which colocalized with lysosomes. Excessive de novo lipogenesis, failing oxidative capacity, and elevated lipid uptake were not observed. Further investigation of lysosomal function revealed impaired acidification combined with impaired autophagic capacity. Conclusions Our data suggest that the hypercholesterolemia in TMEM199 and CCDC115 deficiency is due to increased secretion of apoB-containing particles. This may in turn be secondary to the hepatic steatosis observed in these patients as well as in the mouse model. Mechanistically, we observed impaired lysosomal function characterized by reduced acidification, autophagy, and increased lysosomal lipid accumulation. These findings could explain the hepatic steatosis seen in patients and highlight the importance of lipophagy in fatty liver disease. Because this pathway remains understudied and its regulation is largely untargeted, further exploration of this pathway may offer novel strategies for therapeutic interventions to reduce lipotoxicity in fatty liver disease., Graphical abstract

Plasma triglycerides (TGs) are an independent predictor of the risk for CAD, the leading cause of death worldwide. TGs are also positively associated with risk and severity of hyperTG-induced acute pancreatitis (HTG-AP). Current therapies are often insufficient in reducing extremely elevated TGs. We believe that apolipoprotein A-V (apoA-V, encoded by APOA5) can fill this unmet medical need. ApoA-V is a potent modulator of TG metabolism; it enhances lipoprotein lipase TG hydrolysis. We hypothesize that naturally occurring human APOA5 variants can inform ApoA-V function and identify novel ApoA-V based therapeutic axes. We used the Penn Medicine Biobank (PMBB) to identify and measure plasma TGs of carriers of APOA5 variants predicted to change ApoA-V structure-function. Then, we used hydrogen-deuterium exchange mass spectroscopy to determine the secondary structure of ApoA-V, thereby identifying putative functional domains onto which we mapped our variants of interest. Finally, we characterized the plasma lipid effects of these mutants using adeno-associated viral (AAV) vectors in apoa5 knockout (KO) mice. We identified APOA5 variants associated with changes in plasma TGs. These variants primarily fall near the central heparin binding domain or C-terminal lipid binding domain. We selected APOA5 Q275X, which removes the entire lipid binding domain, for further interrogation. Apoa5 KO mice that received APOA5 Q275X AAV had higher plasma TGs than mice treated with WT APOA5 AAV. While WT ApoA-V protein associated with VLDL and HDL particles, Q275X ApoA-V protein appeared in lipoprotein free fractions. We have identified APOA5 variants associated with plasma TG phenotypes in humans, and mapped them to an experimentally determined ApoA-V secondary structure to identify the functional domains likely impacted. We have identified APOA5 Q275X as a loss of function variant that fails to bind lipoprotein particles and is associated with elevated plasma TGs. Continued study of this and other interesting naturally occurring variants will provide insight into the function of ApoA-V in TG metabolism. These insights can help us to therapeutically enhance ApoA-V to rapidly reduce TG levels during acute HTG-AP and to help prevent recurrent HTG-AP.

Nonalcoholic fatty liver disease (NAFLD) is a growing cause of chronic liver disease. Using a proxy NAFLD definition of chronic alanine aminotransferase elevation (cALT) without other liver diseases, we performed a trans-ancestry genome-wide association study in the Million Veteran Program including 90,408 cALT cases and 128,187 controls. In the Discovery stage, seventy-seven loci exceeded genome-wide significance – including 25 without prior NAFLD or ALT associations – with one additional locus identified in European-American-only and two in African-American-only analyses (P-8). External replication in cohorts with NAFLD defined by histology (7,397 cases, 56,785 controls) or liver fat extracted from radiologic imaging (n=44,289) validated 17 SNPs (P-4) of which 9 were novel (TRIB1, PPARG, MTTP, SERPINA1, FTO, IL1RN, COBLL1, APOH, and IFI30). Pleiotropy analysis showed that 61 of 77 trans-ancestry and all 17 validated SNPs were jointly associated with metabolic and/or inflammatory traits, revealing a complex model of genetic architecture. Our approach integrating cALT, histology and imaging reveals new insights into genetic liability to NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a growing cause of chronic liver disease. Using a proxy NAFLD definition of chronic elevation of alanine aminotransferase (cALT) levels without other liver diseases, we performed a multiancestry genome-wide association study (GWAS) in the Million Veteran Program (MVP) including 90,408 cALT cases and 128,187 controls. Seventy-seven loci exceeded genome-wide significance, including 25 without prior NAFLD or alanine aminotransferase associations, with one additional locus identified in European American-only and two in African American-only analyses (P < 5 × 10(−8)). External replication in histology-defined NAFLD cohorts (7,397 cases and 56,785 controls) or radiologic imaging cohorts (n = 44,289) replicated 17 single-nucleotide polymorphisms (SNPs) (P < 6.5 × 10(−4)), of which 9 were new (TRIB1, PPARG, MTTP, SERPINA1, FTO, IL1RN, COBLL1, APOH and IFI30). Pleiotropy analysis showed that 61 of 77 multiancestry and all 17 replicated SNPs were jointly associated with metabolic and/or inflammatory traits, revealing a complex model of genetic architecture. Our approach integrating cALT, histology and imaging reveals new insights into genetic liability to NAFLD.

Genetic variants near the TRIB1 gene are highly significantly associated with plasma lipid traits and coronary artery disease. While TRIB1 is likely causal of these associations, the molecular mechanisms are not well understood. Here we sought to investigate how TRIB1 influences low density lipoprotein cholesterol (LDL-C) levels in mice. Hepatocyte-specific deletion of Trib1 (Trib1(Δ)hep) in mice increased plasma cholesterol and apoB and slowed the catabolism of LDL-apoB due to decreased levels of LDL receptor (LDLR) mRNA and protein. Simultaneous deletion of the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα) with TRIB1 eliminated the effects of TRIB1 on hepatic LDLR regulation and LDL catabolism. Using RNA-seq, we found that activating transcription factor 3 (Atf3) was highly upregulated in the livers of Trib1(Δ)hep but not Trib1(Δ)hep Cebpa(Δ)hep mice. ATF3 has been shown to directly bind to the CEBPα protein, and to repress the expression of LDLR by binding its promoter. Blunting the increase of ATF3 in Trib1(Δ)hep mice reduced the levels of plasma cholesterol and partially attenuated the effects on LDLR. Based on these data, we conclude that deletion of Trib1 leads to a posttranslational increase in CEBPα, which increases ATF3 levels, thereby contributing to the downregulation of LDLR and increased plasma LDL-C.

In humans, apolipoprotein C-III (apoC-III) plasma levels have been associated with increased risk of cardiovascular disease. This association is in part explained by the effects of apoC-III on triglyceride (TG) metabolism; apoC-III raises plasma TG by increasing very low density lipoprotein (VLDL) secretion, inhibiting lipoprotein lipase (LPL)-mediated TG lipolysis, and impairing the removal of triglyceride-rich lipoprotein (TRL) remnants from the circulation. In this study, we explored the structure-function relationship the interaction of apoC-III with plasma lipoproteins and its ultimate impact on LPL activity. The structural and functional properties of wild-type (WT) apoC-III were compared with two missense variants previously associated with lower (A23T) and higher (Q38K) plasma TG. ApoC-III in the lipid-free state is unstructured but its helix content and stability increases when bound to lipid. Lipid-bound apoC-III contains two alpha helices spanning residues amino acids 11 - 38 (helix 1) and 44 – 64 (helix 2). Investigation of the structural and functional consequences of the A23T and Q38K variants showed that these amino acid substitutions within helix 1 do not significantly alter the stability of the helical structure but affect its hydrophilic-lipophilic properties. The A23T substitution impairs lipoprotein binding capacity, reduces LPL inhibition, and ultimately leads to lower plasma TG levels. Conversely, the Q to K substitution at position 38 enhances the lipid affinity of helix 1, increases TRL binding capacity and LPL inhibition, and is associated with hypertriglyceridemia. This study indicates that structural modifications that perturb the hydrophilic/lipophilic properties of the alpha helices can modulate the hypertriglyceridemic effects of apoC-III.

Isolated left ventricular noncompaction (LVNC) results from excessive trabeculation and impaired myocardial compaction during heart development. The extracellular matrix (ECM) that separates endocardium from myocardium plays a critical but poorly understood role in ventricular trabeculation and compaction. In an attempt to characterize solute carrier family 39 member 8-null (Slc39a8-null) mice, we discovered that homozygous null embryos do not survive embryogenesis and exhibit a cardiac phenotype similar to human LVNC. Slc39a8 encodes a divalent metal cation importer that has been implicated in ECM degradation through the zinc/metal regulatory transcription factor 1 (Zn/MTF1) axis, which promotes the expression of ECM-degrading enzymes, including Adamts metalloproteinases. Here, we have shown that Slc39a8 is expressed by endothelial cells in the developing mouse heart, where it serves to maintain cellular Zn levels. Furthermore, Slc39a8-null hearts exhibited marked ECM accumulation and reduction of several Adamts metalloproteinases. Consistent with the in vivo observations, knockdown of SLC39A8 in HUVECs decreased ADAMTS1 transcription by decreasing cellular Zn uptake and, as a result, MTF1 transcriptional activity. Our study thus identifies a gene underlying ventricular trabeculation and compaction development, and a pathway regulating ECM during myocardial morphogenesis.

Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (GL) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion (Ppp1r3bΔhep) or liver-specific overexpression of Ppp1r3b. The Ppp1r3b deletion significantly reduced glycogen synthase protein abundance, and the remaining protein was predominantly phosphorylated and inactive. As a consequence, glucose incorporation into hepatic glycogen was significantly impaired, total hepatic glycogen content was substantially decreased, and mice lacking hepatic Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3bΔhep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12–36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3bΔhep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis.

Widespread technological advances have propelled human genetics into a "big data" era, in which genome-wide data from extremely large cohorts can be integrated with other "-omics" datasets from humans and model systems. Li et al. (2020) demonstrate the power of applying multiple computational analyses to publicly available data to prioritize the study of genes with novel trait associations.

Dyslipidemias are strongly linked to the development of atherosclerotic cardiovascular disease. Most dyslipidemias find their origin in the liver. In recent years, the differentiation of induced pluripotent stem cells (iPSCs) into hepatocyte-like cells has provided a versatile platform for the functional study of various dyslipidemias, both rare genetic dyslipidemia as well as common lipid disorders associated with insulin resistance or non-alcoholic fatty liver disease. In addition, iPSC-derived hepatocytes can serve as a cell model for developing novel lipid lowering therapies and have the potential of regenerative medicine. This review provides an overview of these developments.

Background: The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. Methods: We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)–deficiency CDG and PMM2 (phosphomannomutase 2)–deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models—gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells—by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. Results: We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. Conclusions: Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor–mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

Deciphering the impact of genetic variation on gene regulation is fundamental to understanding common, complex human diseases. Although histone modifications are important markers of gene regulatory regions of the genome, any specific histone modification has not been assayed in more than a few individuals in the human liver. As a result, the impacts of genetic variation that direct histone modification states in the liver are poorly understood. Here, we generate the most comprehensive genome-wide dataset of two epigenetic marks, H3K4me3 and H3K27ac, and annotate thousands of putative regulatory elements in the human liver. We integrate these findings with genome-wide gene expression data collected from the same human liver tissues and high-resolution promoter-focused chromatin interaction maps collected from human liver-derived HepG2 cells. We demonstrate widespread functional consequences of natural genetic variation on putative regulatory element activity and gene expression levels. Leveraging these extensive datasets, we fine-map a total of 77 GWAS loci that have been associated with at least one complex phenotype. Our results contribute to the repertoire of genes and regulatory mechanisms governing complex disease development and further the basic understanding of genetic and epigenetic regulation of gene expression in the human liver tissue.

SORT1 is strongly associated with plasma lipid traits and coronary artery disease. The protein it encodes, sortilin is a multi-ligand receptor involved in trafficking of proteins from the Golgi to the lysosome. Hepatic sortilin overexpression reduces VLDL secretion, but the effects of loss of sortilin function on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing. Conflicting studies have variously reported increased and decreased apoB/VLDL secretion in response to loss of sortilin, but used a variety of different models and methods of ablating sortilin function. To resolve this, we first measured VLDL secretion in Sort1 knockout (KO) mice on a chow diet and observed no differences in either apoB or TG secretion. ApoB secretion was similarly unaffected in Sort1 KO primary hepatocytes or in McA-RH7777 cells treated with siRNA to knockdown sortilin. We concluded that in a basal secretory state, loss of sortilin alone does not impact apoB secretion, leading us to test if differences in secretory state might explain the discrepancies in published results. We found that when hAPOB was overexpressed, Sort1 knockdown further increased its secretion. The reports of increased VLDL secretion with Sort1 knockout occurred with apoB overexpression, or on high fat diet (HFD); consistent with our hypothesis that secretory overload or metabolic stress are required to uncover the deficiency phenotype. We placed Sort1 KO mice on a 45% HFD for 12 weeks and observed a significant increase in VLDL secretion compared to controls. In addition, when we lipid-loaded hepatocytes with oleic acid or palmitic acid, apoB secretion was increased, and Sort1 knockdown further increased this compared to control. We treated cells with Tunicamycin to induce ER stress, and found that apoB secretion was decreased in the control group was observed, but not in the absence of Sort1, suggesting that sortilin regulates apoB secretion the cell is stressed. Based on these data, we propose that hepatic sortilin regulates the post-ER fate of apoB for either degradation or export; thereby coordinating intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi, and the level of post-ER pre-secretory proteolysis activity.

Single nucleotide polymorphisms (SNPs) in the human 8q24 locus have repeatedly been associated by genome-wide association studies (GWAS) with multiple human metabolic traits such as plasma lipids and coronary artery disease. These SNPs lie in a non-coding region ~30kb downstream of the gene Tribbles-1 ( TRIB1 ). While a large body of in vivo evidence from Trib1 gain- and loss-of-function mouse models strongly supports TRIB1 as the gene of interest at this locus, there has been no demonstrated association between SNPs in the GWAS region and the expression of the neighboring TRIB1 gene to date. To address this, we performed RNA-seq and genome-wide genotyping on 42 human cadaveric liver samples, and next performed allele-specific expression (ASE) analysis of the TRIB1 locus in 23 samples that harbored heterozygous coding SNPs in the TRIB1 gene, allowing for allelic discrimination. While subjects homozygous for the major allele of the lead GWAS SNP (rs2954029) had even allelic expression ( p =0.29 by non-parametric t-test), samples heterozygous for the minor allele at rs2954029 exhibited imbalanced allelic expression ( p TRIB1 locus do affect TRIB1 gene expression; however, it remains unclear which SNP(s) is causal. To that end, we used ENCODE data from primary hepatocytes and HepG2 cells to identify 10 genomic regions of interest that contained SNPs with significant GWAS p-values and epigenetic markers consistent with enhancer activity. Two regions (R2, R9) exhibited strong enhancer activity when cloned in front of a minimal promoter driving a luciferase reporter (pGL4.23), increasing luciferase activity 2-5-fold as compared to empty vector ( p TRIB1 gene expression, as measured by ASE in human livers. We also identified multiple enhancer elements that exhibit reduced activity when the minor alleles of GWAS SNPs are introduced into them. We are currently pursuing modification of the endogenous locus in HepG2 cells via CRISPR/Cas genome editing to further elucidate the roles of both these enhancer elements and the SNPs contained in them.

Genome-wide association studies have identified a signal at the SLC22A1 locus for serum acylcarnitines, intermediate metabolites of mitochondrial oxidation whose plasma levels associate with metabolic diseases. Here, we refined the association signal, performed conditional analyses, and examined the linkage structure to find coding variants of SLC22A1 that mediate independent association signals at the locus. We also employed allele-specific expression analysis to find potential regulatory variants of SLC22A1 and demonstrated the effect of one variant on the splicing of SLC22A1. SLC22A1 encodes a hepatic plasma membrane transporter whose role in acylcarnitine physiology has not been described. By targeted metabolomics and isotope tracing experiments in loss- and gain-of-function cell and mouse models of Slc22a1, we uncovered a role of SLC22A1 in the efflux of acylcarnitines from the liver to the circulation. We further validated the impacts of human variants on SLC22A1-mediated acylcarnitine efflux invitro, explaining theirassociation with serum acylcarnitine levels. Our findings provide the detailed molecular mechanisms of the GWAS association for serum acylcarnitines at the SLC22A1 locus by functionally validating the impact of SLC22A1 and its variants on acylcarnitine transport.

Genetic variants at the solute carrier family 39 member 8 (SLC39A8) gene locus are associated with the regulation of whole-blood manganese (Mn) and multiple physiological traits. SLC39A8 encodes ZIP8, a divalent metal ion transporter best known for zinc transport. Here, we hypothesized that ZIP8 regulates Mn homeostasis and Mn-dependent enzymes to influence metabolism. We generated Slc39a8-inducible global-knockout (ZIP8-iKO) and liver-specific-knockout (ZIP8-LSKO) mice and observed markedly decreased Mn levels in multiple organs and whole blood of both mouse models. By contrast, liver-specific overexpression of human ZIP8 (adeno-associated virus-ZIP8 [AAV-ZIP8]) resulted in increased tissue and whole blood Mn levels. ZIP8 expression was localized to the hepatocyte canalicular membrane, and bile Mn levels were increased in ZIP8-LSKO and decreased in AAV-ZIP8 mice. ZIP8-LSKO mice also displayed decreased liver and kidney activity of the Mn-dependent enzyme arginase. Both ZIP8-iKO and ZIP8-LSKO mice had defective protein N-glycosylation, and humans homozygous for the minor allele at the lead SLC39A8 variant showed hypogalactosylation, consistent with decreased activity of another Mn-dependent enzyme, β-1,4-galactosyltransferase. In summary, hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes. This work provides a mechanistic basis for the association of SLC39A8 with whole-blood Mn, potentially linking SLC39A8 variants with other physiological traits.

UNLABELLED A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ∼90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy, we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Second, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, lifelong depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated overexpression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l, and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. CONCLUSION Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified; our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization, and these studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis, and disease. (Hepatology 2016;64:599-615).

Sortilin is a multi-ligand sorting receptor involved in trafficking of proteins from the Golgi apparatus to the lysosome and has been widely shown to be associated with plasma lipid traits and coronary artery disease. While over expression of sortilin in the liver reduces VLDL production, the reported effects of the genetic loss of sortilin on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing; loss of sortilin has been shown in different studies to result in both increased and decreased apoB/VLDL secretion. These conflicting studies were carried out in a variety of different models and used different methods of knocking down sortilin expression. To attempt to further clarify the role of sortilin, we explored the role of sortilin deficiency on apoB secretion in the hepatocyte by utilizing 2 in vitro models of sortilin knockdown; primary hepatocytes from Sort1-/- mice and siRNA -treated McA-RH7777 (McA) cells. In both primary hepatocytes and McA cells, loss of sortilin alone was not associated with any change in apoB secretion. The previously reported increases in VLDL secretion occurred on either the background of apoB over expression or in livers of mice on a high fat diet, suggesting the requirement for a metabolic stress. We found that apoB secretion was increased with Sort1 knockdown as compared to control in isolated primary hepatocytes from Apobec1-/-; hAPOB Tg mice and McA cells stably over expressing apoB. We then sought to increase apoB secretion by lipid loading with oleic acid (OA). While OA increased apoB secretion in all cells, there was no effect of Sort1 knockdown in this context. However, when the cells were further treated with either palmitic acid, proteasomal inhibitors, or tunicamycin (an ER stress inducer), there was an observed increase in apoB secretion with Sort1 knockdown, suggesting that sortilin regulates apoB secretion only when both apoB secretion is increased and the cell is stressed. Based on this data, we propose that hepatic sortilin regulates the post-ER fate of apoB for degradation and export and acts to coordinate intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi and the level of post-ER pre-secretory proteolysis activity.

The lack of reliable noninvasive diagnostic biomarkers of biliary atresia (BA) results in delayed diagnosis and worsened patient outcome. Circulating microRNAs (miRNAs) are a new class of noninvasive biomarkers with encouraging diagnostic utility.We examined the ability of serum miRNAs to distinguish BA from other forms of neonatal hyperbilirubinemia. BA-specific serum miRNAs were identified using a microfluidic array platform and validated in a larger, independent sample set.The miR-200b/429 cluster was significantly increased in the sera of patients with BA relative to infants with non-BA cholestatic disorders.Circulating levels of the miR-200b/429 cluster are elevated in infants with BA and have promising diagnostic clinical performance.

Biliary atresia is a pediatric liver disease of unknown underlying etiology, in which fibro-inflammatory destruction of the extrahepatic biliary system leads to obstructive cholestasis. MicroRNAs are a class of short (18–23 nucleotide), non-coding RNA molecules which act as negative regulators of target mRNA stability and translation. The importance of these molecules in normal and diseased liver has been demonstrated, but their potential role in the pathogenesis of biliary atresia has not been addressed. We have profiled changes in liver microRNA levels in an established mouse model of the disease, identified significantly altered transcripts, and defined the spatial expression patterns of selected microRNAs. Two of these, miR-29a/29b1 are up-regulated in experimental biliary atresia. Using antisense oligonucleotide-mediated inhibition in mice, we have delineated the full set of hepatic genes regulated by miR-29 and identified two mRNA targets of potential pathological relevance in experimental biliary atresia, Igf1 and Il1RAP. We have used reporter assays to confirm that Igf1 and Il1RAP are direct targets of miR-29.

The gold standard for the diagnosis and evaluation of Crohn disease (CD) is endoscopy/colonoscopy, although this is invasive, costly, and associated with risks to the patient. Recently, circulating microRNAs (miRNAs) have emerged as promising noninvasive biomarkers. Here, we examined the utility of serum miRNAs as biomarkers of CD in children.Studies were conducted using sera samples from patients with pediatric CD, healthy controls, and a comparison group of patients with pediatric celiac disease. Serum miRNA levels were explored initially using a microfluidic quantitative reverse transcription-polymerase chain reaction array platform. Findings were subsequently validated using quantitative reverse transcription-polymerase chain reaction in larger validation sample sets. The diagnostic utility of CD-associated serum miRNA was examined using receiver operating characteristic analysis.A survey of miRNA levels in the sera of control and patients with CD detected significant elevation of 24 miRNAs, 11 of which were chosen for further validation. All of the candidate biomarker miRNAs were confirmed in an independent CD sample set (n = 46). To explore the specificity of the CD-associated miRNAs, they were measured in the sera of patients with celiac disease (n = 12); none were changed compared with healthy controls. Receiver operating characteristic analyses revealed that serum miRNAs have promising diagnostic utility, with sensitivities for CD above 80%. Significant decreases in serum miRNAs were observed in 24 incident patients with pediatric CD after 6 months of treatment.The present study identifies 11 CD-associated serum miRNA with encouraging diagnostic potential. Our findings suggest serum miRNAs may prove useful as noninvasive biomarkers in CD.

Genome-wide association studies have provided a rich collection of ≈58 coronary artery disease (CAD) loci that suggest the existence of previously unsuspected new biology relevant to atherosclerosis. However, these studies only identify genomic loci associated with CAD, and many questions remain even after a genomic locus is definitively implicated, including the nature of the causal variant(s) and the causal gene(s), as well as the directionality of effect. There are several tools that can be used for investigation of the functional genomics of these loci, and progress has been made on a limited number of novel CAD loci. New biology regarding atherosclerosis and CAD will be learned through the functional genomics of these loci, and the hope is that at least some of these new pathways relevant to CAD pathogenesis will yield new therapeutic targets for the prevention and treatment of CAD.

Human genetics has contributed to the development of multiple drugs to treat hyperlipidemia and coronary artery disease (CAD), most recently including antibodies targeting PCSK9 to reduce LDL cholesterol. Despite these successes, a large burden of CAD remains. Genetic and epidemiological studies have suggested that circulating triglyceride (TG)-rich lipoproteins (TRLs) are a causal risk factor for CAD, presenting an opportunity for novel therapeutic strategies. We discuss recent unbiased human genetics testing, including genome-wide association studies (GWAS) and whole-genome or -exome sequencing, that have identified the lipoprotein lipase (LPL) and hepatic lipogenesis pathways as important mechanisms in the regulation of circulating TRLs. Further strengthening the causal relationship between TRLs and CAD, findings such as these may provide novel targets for much-needed potential therapeutic interventions.

The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli , an H. volcanii Δ secFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.

Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect. The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1. Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1 -like phenotypes. Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1 : cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation. Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon , suggesting a fundamental link between the phenotypes. We speculate that the common properties of the suppressors relate to secretion defects. The ygdP gene (also known as nudH ) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase. Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion. Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner.

Background and aims Changes in intestinal microRNAs have been reported in adult patients with ulcerative colitis or Crohn's disease. The goal of this study was to identify changes in microRNA expression associated with colitis in children with inflammatory bowel disease. Methods Rectal mucosal biopsies (n = 50) and blood samples (n = 47) were collected from patients with known or suspected inflammatory bowel disease undergoing endoscopy. Rectal and serum microRNA levels were profiled using the nCounter® platform and the TaqMan® low-density array platform, respectively. Significantly altered microRNAs were validated in independent sample sets via quantitative RT-PCR. In vitro luciferase reporter assays were performed in the human colorectal Caco-2 cell line to determine the effect of miR-192 on NOD2 expression. Results Profiling of rectal RNA identified 21 microRNAs significantly altered between control, UC, and colonic CD sample groups. Nine of the ten microRNAs selected for validation were confirmed as significantly changed. Rectal miR-24 was increased 1.47-fold in UC compared to CD samples (p = 0.0052) and was the only microRNA altered between IBD subtypes. Three colitis-associated microRNAs were significantly altered in sera of disease patients and displayed diagnostic utility. However, no serum microRNAs were found to distinguish ulcerative colitis from Crohn's colitis. Finally, miR-192 inhibition did not affect luciferase reporter activity, suggesting that miR-192 does not regulate human NOD2 . Conclusion This study has demonstrated that rectal and serum microRNAs are perturbed in pediatric inflammatory bowel disease. Future studies identifying targets of inflammatory bowel disease-associated microRNAs may lead to novel therapies. * Abbreviations : IBD : inflammatory bowel disease UC : ulcerative colitis CD : Crohn's disease L2 CD : Crohn's colitis miRNA : microRNA UTR : untranslated region SAM : Significance Analysis of Microarrays TuD : tough decoy NOD2 : nucleotide-binding oligomerization domain containing 2 ROC : receiver operating characteristic AUC : area under the ROC curve

MicroRNAs have been found to play a profound role in embryonic and post-natal development through their regulation of processes such as cell proliferation, differentiation, and morphogenesis. The microRNA-30 (miR-30) family is necessary for vertebrate hepatobiliary development; however, the mechanism through which miR-30 regulates these processes is not fully understood. Here, we identify genes directly regulated by miR-30 that have been characterized as key developmental factors. The targets were confirmed via a luciferase reporter assay, following exogenous over-expression of miR-30a and miR-30c2 in cultured cells. Five novel miR-30ac2 targets were identified using this approach, all of which play crucial roles in hepatobiliary development or are involved in hepatocellular carcinoma and cholangiocarcinoma.

We report a 29,445 bp sequence from the right arm of yeast chromosome XV. It contains the genes MYO2, SNC2, PDR10, SCD5 (also called FTB1), MIP1, VMA4, MRS2, ALA1, KRE5, TEA1, and a homologue of YAL034c. Several discrepancies with previously published sequences were found. PDR10 encodes a protein highly similar to the pleiotropic drug resistance protein Pdr5p. This sequence contig forms part of a region of extended similarity to part of the left arm of chromosome I, which is a relic of an ancient duplicated chromosomal region.

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression through partial or complete complementarity with target messenger RNAs. The function of miRNAs in normal liver physiology is largely unknown. We address the role of Dicer1 in the differentiated liver. We derived mice lacking Dicer1 function in hepatocytes and assessed the loss of mature miRNA via quantitative polymerase chain reaction. Gene expression microarray analysis was performed on liver RNA from mutant and control mice. Liver sections from mutant and control mice were examined and liver function tests were performed. Mice lacking Dicer1 function in hepatocytes appeared and behaved normally. Despite the loss of mature miRNAs, hepatic function was maintained, as reflected by normal blood glucose, albumin, cholesterol, and bilirubin. However, mutant mice between 2 and 4 months of age exhibited progressive hepatocyte damage with elevated serum alanine aminotransferase and aspartate aminotransferase. Liver mass was increased in mutant mice, as were cellular markers of both proliferation and apoptosis. Microarray analysis indicated large-scale changes in gene expression, with increased expression of many miRNA targets, particularly imprinted genes. Conclusions: Loss of miRNA processing in the liver at late gestation has a remarkably mild phenotype, suggesting that miRNAs do not play an essential role in hepatic function. However, miRNA deficiency results in hepatocyte apoptosis, hepatocyte regeneration, and portal inflammation. Finally, microarray analysis of gene expression in the mutant liver supports a previously hypothesized role for Dicer1 in the repression of imprinted genes. (HEPATOLOGY 2008.)

Background & Aims: The function of microRNA (miRNA) in liver development is unknown. To address this issue, we characterized miRNA expression in the embryonic mouse liver, performed functional miRNA analysis in zebrafish larvae, and identified novel hepatic miRNA targets. Methods: Hepatic RNA isolated from mice at embryonic days 15.5, 18.5, and postnatal day 2 was hybridized to a mouse miRNA microarray. The microarray results were confirmed by Northern blot hybridization and quantitative reverse-transcription polymerase chain reaction. The spatial distribution of selected miRNAs was determined by in situ hybridization. Functional analysis of miR-30a was performed in zebrafish using antisense-mediated miRNA knockdown. Targets of miR-30a were identified by microarray analysis of gene expression following knockdown in cultured cells. Results: A set of 38 differentially expressed fetal hepatic miRNAs was identified. Several of these miRNAs were found to exhibit distinct temporal and spatial patterns of expression in hepatocytes, cholangiocytes, and nonepithelial cells within the liver. Two (miR-30a and miR-30c) are the first examples of ductal plate and bile duct-specific hepatic miRNAs. Knockdown of miR-30a in the zebrafish larva results in defective biliary morphogenesis. Several newly identified targets of miR-30a are known regulators of liver development and function. Conclusions: We have identified miRNAs whose spatial and temporal patterns of expression are suggestive of functional roles in hepatic development and/or function. One of these, the biliary miRNA miR-30a, is required for biliary development in zebrafish. This is the first demonstration of a functional role for miRNA in hepatic organogenesis.

Toxin–antitoxin systems consist of a stable toxin, frequently with endonuclease activity, and a small, labile antitoxin, which sequesters the toxin into an inactive complex. Under unfavorable conditions, the antitoxin is degraded, leading to activation of the toxin and resulting in growth arrest, possibly also in bacterial programmed cell death. Correspondingly, these systems are generally viewed as agents of the stress response in prokaryotes. Here we show that prlF and yhaV encode a novel toxin–antitoxin system in Escherichia coli. YhaV, a ribonuclease of the RelE superfamily, causes reversible bacteriostasis that is counteracted by PrlF, a swapped-hairpin transcription factor homologous to MazE. The two proteins form a tight, hexameric complex, which binds with high specificity to a conserved sequence in the promoter region of the prlF-yhaV operon. As homologs of MazE and RelE, respectively, PrlF and YhaV provide an evolutionary connection between the two best-characterized toxin–antitoxin systems in E. coli, mazEF and relEB.

All cells need to transport proteins across hydrophobic membranes. Several mechanisms have evolved to facilitate this transport, including: (i) the universally-conserved Sec system, which transports proteins in an unfolded conformation and is thought to be the major translocation pathway in most organisms and (ii) the Tat system, which transports proteins that have already obtained some degree of tertiary structure. Here, we present the current understanding of these processes in the domain Archaea, and how they compare to the corresponding pathways in bacteria and eukaryotes.

Publisher Summary This chapter discusses the construction and use of lac fusions in Escherichia coli. It discusses the applications of β-galactosidase. β-galactosidase is known as the best transcriptional reporter. Using appropriate media, mutations that increase or decrease the expression of an operon fusion of interest can be isolated easily. Conversely, screening pools of random LacZ chromosomal insertions can identify targets of a regulator (either transcriptional or translational). β-galactosidase is also useful in the study of translational regulation. The chapter presents the protocols for making specific or random lac fusions (both transcriptional and translational). Transcriptional fusions are also known as promoter or operon fusions, whereas translational fusions are alternatively referred to as gene or protein fusions. Two classes of LacZ fusions cover most uses: fusions created to specific cis-acting regulatory regions (including inflame protein fusions) and fusions created by random chromosomal insertion of the lac operon. The chapter also presents a protocol for creating a fusion of a specific DNA fragment to the lac operon and the subsequent isolation of the fusion in single copy on the bacterial chromosome for analysis.

SummaryType 2 diabetes mellitus (T2DM) is a complex disease characterized by the inability of the insulin-producing β cells in the endocrine pancreas to overcome insulin resistance in peripheral tissues. To determine if microRNAs are involved in the pathogenesis of human T2DM, we sequenced the small RNAs of human islets from diabetic and nondiabetic organ donors. We identified a cluster of microRNAs in an imprinted locus on human chromosome 14q32 that is highly and specifically expressed in human β cells and dramatically downregulated in islets from T2DM organ donors. The downregulation of this locus strongly correlates with hypermethylation of its promoter. Using HITS-CLIP for the essential RISC-component Argonaute, we identified disease-relevant targets of the chromosome 14q32 microRNAs, such as IAPP and TP53INP1, that cause increased β cell apoptosis upon overexpression in human islets. Our results support a role for microRNAs and their epigenetic control by DNA methylation in the pathogenesis of T2DM.

Loss of two hepatic nuclear receptors muddles the Golgi and the sugarcoat on proteins., Nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP) are key regulators of metabolism. Here, we report a previously unknown function for the hepatic FXR-SHP axis in controlling protein N-linked glycosylation. Transcriptome analysis in liver-specific Fxr-Shp double knockout (LDKO) livers revealed induction of genes encoding enzymes in the N-glycosylation pathway, including Mgat5, Fut8, St3gal6, and St6gal1. FXR activation suppressed Mgat5, while Shp deletion induced St3gal6 and St6gal1. Increased percentages of core-fucosylated and triantennary glycan moieties were seen in LDKO livers, and proteins with the “hyperglycoforms” preferentially localized to exosomes and lysosomes. This up-regulation of N-glycosylation machinery was specific to the Golgi apparatus and not the endoplasmic reticulum. The increased glycan complexity in the LDKO correlated well with dilated unstacked Golgi ribbons and alterations in the secretion of albumin, cholesterol, and triglycerides. Our findings demonstrate a role for the FXR-SHP axis in maintaining glycoprotein diversity in the liver.