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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.

Congenital disorders of glycosylation are a growing group of rare genetic disorders caused by deficient protein and lipid glycosylation. Here, we report the clinical, biochemical, and molecular features of seven patients from four families with GALNT2-congenital disorder of glycosylation (GALNT2-CDG), an O-linked glycosylation disorder. GALNT2 encodes the Golgi-localized polypeptide N-acetyl-d-galactosamine-transferase 2 isoenzyme. GALNT2 is widely expressed in most cell types and directs initiation of mucin-type protein O-glycosylation. All patients showed loss of O-glycosylation of apolipoprotein C-III, a non-redundant substrate for GALNT2. Patients with GALNT2-CDG generally exhibit a syndrome characterized by global developmental delay, intellectual disability with language deficit, autistic features, behavioural abnormalities, epilepsy, chronic insomnia, white matter changes on brain MRI, dysmorphic features, decreased stature, and decreased high density lipoprotein cholesterol levels. Rodent (mouse and rat) models of GALNT2-CDG recapitulated much of the human phenotype, including poor growth and neurodevelopmental abnormalities. In behavioural studies, GALNT2-CDG mice demonstrated cerebellar motor deficits, decreased sociability, and impaired sensory integration and processing. The multisystem nature of phenotypes in patients and rodent models of GALNT2-CDG suggest that there are multiple non-redundant protein substrates of GALNT2 in various tissues, including brain, which are critical to normal growth and development.

PM20D1 is a candidate thermogenic enzyme in mouse fat, with its expression cold-induced and enriched in brown versus white adipocytes. Thiazolidinedione (TZD) antidiabetic drugs, which activate the peroxisome proliferator-activated receptor-γ (PPARγ) nuclear receptor, are potent stimuli for adipocyte browning yet fail to induce Pm20d1 expression in mouse adipocytes. In contrast, PM20D1 is one of the most strongly TZD-induced transcripts in human adipocytes, although not in cells from all individuals. Two putative PPARγ binding sites exist near the gene’s transcription start site (TSS) in human but not mouse adipocytes. The −4 kb upstream site falls in a segmental duplication of a nearly identical intronic region +2.5 kb downstream of the TSS, and this duplication occurred in the primate lineage and not in other mammals, like mice. PPARγ binding and gene activation occur via this upstream duplicated site, thus explaining the species difference. Furthermore, this functional upstream PPARγ site exhibits genetic variation among people, with 1 SNP allele disrupting a PPAR response element and giving less activation by PPARγ and TZDs. In addition to this upstream variant that determines PPARγ regulation of PM20D1 in adipocytes, distinct variants downstream of the TSS have strong effects on PM20D1 expression in human fat as well as other tissues. A haplotype of 7 tightly linked downstream SNP alleles is associated with very low PMD201 expression and correspondingly high DNA methylation at the TSS. These PM20D1 low-expression variants may account for human genetic associations in this region with obesity as well as neurodegenerative diseases.

HDL cholesterol (HDL-C) remains a superior biochemical predictor of CVD risk, but its genetic basis is incompletely defined. In patients with extreme HDL-C concentrations, we concurrently evaluated the contributions of multiple large- and small-effect genetic variants. In a discovery cohort of 255 unrelated lipid clinic patients with extreme HDL-C levels, we used a targeted next-generation sequencing panel to evaluate rare variants in known HDL metabolism genes, simultaneously with common variants bundled into a polygenic trait score. Two additional cohorts were used for validation and included 1,746 individuals from the Montréal Heart Institute Biobank and 1,048 individuals from the University of Pennsylvania. Findings were consistent between cohorts: we found rare heterozygous large-effect variants in 18.7% and 10.9% of low- and high-HDL-C patients, respectively. We also found common variant accumulation, indicated by extreme polygenic trait scores, in an additional 12.8% and 19.3% of overall cases of low- and high-HDL-C extremes, respectively. Thus, the genetic basis of extreme HDL-C concentrations encountered clinically is frequently polygenic, with contributions from both rare large-effect and common small-effect variants. Multiple types of genetic variants should be considered as contributing factors in patients with extreme dyslipidemia.

Triglyceride-rich lipoproteins (TRLs) are circulating reservoirs of fatty acids used as vital energy sources for peripheral tissues. Lipoprotein lipase (LPL) is a predominant enzyme mediating triglyceride (TG) lipolysis and TRL clearance to provide fatty acids to tissues in animals. Physiological and human genetic evidence support a primary role for LPL in hydrolyzing TRL TGs. We hypothesized that endothelial lipase (EL), another extracellular lipase that primarily hydrolyzes lipoprotein phospholipids may also contribute to TRL metabolism. To explore this, we studied the impact of genetic EL loss-of-function on TRL metabolism in humans and mice. Humans carrying a loss-of-function missense variant in LIPG, p.Asn396Ser (rs77960347), demonstrated elevated plasma TGs and elevated phospholipids in TRLs, among other lipoprotein classes. Mice with germline EL deficiency challenged with excess dietary TG through refeeding or a high-fat diet exhibited elevated TGs, delayed dietary TRL clearance, and impaired TRL TG lipolysis in vivo that was rescued by EL reconstitution in the liver. Lipidomic analyses of postprandial plasma from high-fat fed Lipg-/- mice demonstrated accumulation of phospholipids and TGs harboring long-chain polyunsaturated fatty acids (PUFAs), known substrates for EL lipolysis. In vitro and in vivo, EL and LPL together promoted greater TG lipolysis than either extracellular lipase alone. Our data positions EL as a key collaborator of LPL to mediate efficient lipolysis of TRLs in humans and mice., Author summary Endothelial lipase (EL) plays a pivotal role in the breakdown of high-density lipoproteins (HDLs) by hydrolyzing phospholipids on HDL surfaces. Here we show through studies of humans and mice with genetic loss-of-function of EL activity that EL is also crucial in catabolizing triglyceride (TG)-rich lipoproteins, particularly in states of nutrient excess such as after refeeding or after feeding a high-fat diet. We demonstrate that EL collaborates with lipoprotein lipase (LPL), the predominant enzyme hydrolysing triglycerides, to promote the breakdown of triglycerides on lipoproteins. The mechanism for this may be due to an underappreciated ability of EL to hydrolyze TGs and cooperate with LPL to efficiently break down and clear these particles. Our data adds important insights into the mechanisms of circulating TG metabolism in mice and humans as informed by large-scale human genetics.

Background: Genome-wide association studies have uncovered common variants at many loci influencing human complex traits, such as high-density lipoprotein cholesterol (HDL-C). However, the contribu...

Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a novel regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, but the mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss-of-function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2 deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Additional glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs associated with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metabolism across mammals.

Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121*], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited.

Triglyceride-rich lipoproteins (TRLs) are causal contributors to the risk of developing coronary artery disease (CAD). Apolipoprotein C-III (apoC-III) is a component of TRLs that elevates plasma triglycerides (TGs) through delaying the lipolysis of TGs and the catabolism of TRL remnants. Recent human genetics approaches have shown that heterozygous loss-of-function mutations in APOC3, the gene encoding apoC-III, lower plasma TGs and protect from CAD. This observation has spawned new interest in therapeutic efforts to target apoC-III. Here, we briefly review both currently available as well as developing therapies for reducing apoC-III levels and function to lower TGs and cardiovascular risk. These therapies include existing options including statins, fibrates, thiazolidinediones, omega-3-fatty acids, and niacin, as well as an antisense oligonucleotide targeting APOC3 currently in clinical development. We review the mechanisms of action by which these drugs reduce apoC-III and the current understanding of how reduction in apoC-III may impact CAD risk.

3-Hydroxymethylglutaryl coenzyme A reductase inhibitors, commonly known as statins, are among the most widely prescribed drugs in the world. Indeed, In the United States at least one quarter of adults over the age of 40 years is prescribed a statin.1 Recent evidence suggests that wider prescription of statins to those with even moderate risk of cardiovascular disease may be greatly beneficial in primary risk prevention.2 Statins are highly efficacious in lowering low-density lipoprotein cholesterol (LDL-C) levels by inhibiting 3-hydroxymethylglutaryl coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway, resulting in increased transcription of hepatic LDL receptor levels.1 Yet despite their immense success, statin use is still limited, in part, by variable responsiveness with regard to LDL-C lowering and side effects, which include risk of developing insulin resistance, myopathy, cognitive impairment, and other morbidities.1 These concerns have prompted extensive studies focused on identifying the precise patients likely to benefit most from statins as well as those most at risk of developing adverse effects. Article, see p 223 Pharmacogenomics studies have been used to identify patients who would benefit the most from statins or are at risk of side effects. These studies relate natural genetic variants and associated genes and pathways to efficacy and tolerability of drugs at the individual level. At a basic level, these studies compare genotype frequencies for polymorphisms at individual genes or across multiple genomic loci among individuals with differing responsiveness for a given drug and identify those single-nucleotide polymorphisms (SNPs) whose allele frequencies differ across the drug-responsiveness groups. Pharmacogenomics approaches have been applied widely to identify genes that influence the LDL-C–lowering efficacy of many statins as well as their variable adverse effect profiles.3 Such efforts have been performed at times to compare the SNP–drug interactions across different statins and in …

Polyunsaturated fatty acids (PUFA) are essential constituents of the cell membrane. Brain PUFA content is critical for synaptic function and neuroinflammation and PUFA deficiency may underlie several neurological disorders. Prior work in murine models has showed that the predominant source of central nervous system (CNS) PUFAs is 2-acyl-lysophophatidylcholine (2-lysoPC), a class of phospholipids (PLs) circulating on blood lipoproteins that is formed from the enzymatic activity of phospholipases. Endothelial lipase (EL) is a phospholipase that is a critical determinant of HDL metabolism in humans and the predominant generator of 2-lysoPC on HDL particles. While the contribution of EL to circulating HDL metabolism is well-established, its contribution to CNS fatty acid availability and brain function has not been explored. In order to investigate the physiological contribution of EL to brain FA availability in vivo, we studied PL-derived PUFA uptake in mice with EL deficiency. We adopted a method for measuring EL activity using a fluorescent synthetic HDL particle containing sn2-TopFluor-PC, a fluorescent precursor of 2-lysoPC and demonstrated its utility in vitro. Next, we tested the clearance of this synthetic HDL-derived 2-lysoPC in C57BL/6 (WT) vs EL KO mice (n=12/group). Circulating plasma 2-lysoPC clearance was significantly impaired in in EL KO mice (-32%, p=0.024) and was accompanied by a slower remodeling of the HDL particles. Measurement of labeled PL content in tissues showed a significant reduction in hepatic uptake in KO mice (22% decrease, p=0.002). No significant differences were observed in total brain PL uptake, but the distribution of PL uptake differed between WT and KOs. Ongoing studies are evaluating the physiological consequences of reduced circulating 2-lysoPC clearance, reduced hepatic uptake, and altered distribution of brain PL uptake in EL KO mice. Collectively, our data demonstrate that EL is a key physiological regulator of systemic PL catabolism.

Humans with loss-of-function (LoF) variants in APOC3 , the gene encoding apolipoprotein C-III (apoC-III), have significantly reduced plasma triglycerides (TG) and protection from coronary disease. These findings suggest that apoC-III may be a viable therapeutic target for decreasing vascular risk through TG reduction, and that elucidation of the protective mechanism of APOC3 LoF variants would inform such strategies. We report here the protective mechanism of the APOC3 A43T missense variant, one of four recently identified CAD-protective variants. By genotyping >8,000 human participants with low TG, we identified 17 APOC3 A43T carriers and phenotyped 6 carriers and 54 matched controls. A43T heterozygotes demonstrate a significant reduction in apoC-III levels relative to non-carriers (50% reduction, P3-fold higher apoC-III clearance rate in vivo (Pin vivo . We are currently performing analogous studies of WT vs. A43T apoC-III turnover and VLDL clearance in human APOC3 A43T carriers. Collectively, our results support the rationale for therapeutic efforts to target circulating apoC-III through disruption of its binding to lipoproteins, mirroring the genetics-driven approaches for targeting PCSK9 that have recently yielded novel therapies.

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20–25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/−0.9 mg/dl vs. WT: 1.2+/−0.3 mg/dl, p

Elevated plasma concentrations of HDL cholesterol (HDL-C) are associated with protection from atherosclerotic cardiovascular disease. Animal models indicate that decreased expression of endothelial lipase (LIPG) is inversely associated with HDL-C levels, and genome-wide association studies have identified LIPG variants as being associated with HDL-C levels in humans. We hypothesized that loss-of-function mutations in LIPG may result in elevated HDL-C and therefore performed deep resequencing of LIPG exons in cases with elevated HDL-C levels and controls with decreased HDL-C levels. We identified a significant excess of nonsynonymous LIPG variants unique to cases with elevated HDL-C. In vitro lipase activity assays demonstrated that these variants significantly decreased endothelial lipase activity. In addition, a meta-analysis across 5 cohorts demonstrated that the low-frequency Asn396Ser variant is significantly associated with increased HDL-C, while the common Thr111Ile variant is not. Functional analysis confirmed that the Asn396Ser variant has significantly decreased lipase activity both in vitro and in vivo, while the Thr111Ile variant has normal lipase activity. Our results establish that loss-of-function mutations in LIPG lead to increased HDL-C levels and support the idea that inhibition of endothelial lipase may be an effective mechanism to raise HDL-C.

Triglyceride (TG) rich lipoproteins have emerged as causal risk factors for the development of coronary heart disease (CHD) independent of circulating low-density lipoprotein cholesterol levels. Apolipoprotein C-III (ApoC-III) is a modulator of TG metabolism through inhibition of lipoprotein lipase