👤 Jan Albert Kuivenhoven

Chylomicron biosynthesis plays a vital role in supplying essential lipids and lipid soluble vitamins to peripheral tissues for various functions. Despite this, the intracellular synthesis, trafficking, and secretion of chylomicrons remains only partly understood. The purpose of this review is to summarize the role of established proteins in this process and bring attention to recently identified proteins to provide an up-to-date model of chylomicron biosynthesis. Recently, several proteins have been shown to play a role in the initial formation and lipidation of chylomicrons at the endoplasmic reticulum (ER), which include: TM6SF2, PLA2G12B, PRAP1, and SURF4. In addition, mitochondria have been implicated in chylomicron metabolism, but mechanistic insight is missing. The trafficking of chylomicrons from the ER to the Golgi, and the subsequent trafficking from the Golgi to the basolateral side of enterocytes, however, remains a mystery. Progress in the chylomicron biosynthesis field is largely associated with findings in VLDL biosynthesis. In addition, increased insight in events after prechylomicrons leave the ER is needed. Given the important role of chylomicron biosynthesis in whole-body lipid metabolism, further research into the molecular mechanisms is warranted. Show less

G-protein coupled receptor 146 (GPR146)-deficient mice exhibit a moderate 21 % reduction in plasma cholesterol. This is associated with decreased phosphorylation of ERK1/2 and reduced SREBP2 activity in the liver, which leads to lower VLDL secretion. Insight into the role of GPR146 in humans is however limited. We therefore set out to study rare genetic variants in GPR146 to improve our understanding of this new player in lipid metabolism. We used whole genome sequencing data from UK Biobank participants to search for rare coding variants in GPR146. We first carried out gene-based burden tests (using SAIGE-GENE-framework) and examined the association of individual variants with plasma cholesterol levels. One of the variants (P62L) was also studied using the Global Lipids Genetics Consortium (GLGC) data set and in a knock-in mouse model. We found that the combination of rare genetic variants identified in GPR146 is significantly associated with plasma cholesterol levels. Three rare variants, i.e. P62L, I129I, and A175T were individually associated with reduced plasma cholesterol. In the GLGC cohort, the P62L variant was associated with reductions in both HDL and LDL cholesterol. Follow-up experiments show lower plasma cholesterol levels in GPR146 This study shows that rare GPR146 gene variants are associated with lower plasma cholesterol levels in humans. One of these variants, P62L is associated with reductions of HDL cholesterol and LDL cholesterol in humans while the ortholog in mice confers a loss of GPR146 function leading to only reduced HDL cholesterol. How GPR146 affects HDL metabolism in humans and mice remains to be resolved. Show less

The small intestine plays a central role in lipid metabolism, most notably the uptake of dietary fats that are packaged into chylomicrons and secreted into the circulation for utilisation by peripheral tissues. While microsomal triglyceride transfer protein (MTP) is known to play a key role in this pathway, the intracellular assembly, trafficking, and secretion of chylomicrons is incompletely understood. Using human transcriptome datasets to find genes co-regulated with MTTP, we identified ERICH4 as a top hit. The gene encodes for glutamate-rich protein 4, a protein of unknown function. REACTOME gene-function prediction tools indicated that ERICH4 is involved in intestinal lipid metabolism. In addition, GWAS data point to a strong relationship between ERICH4 and plasma lipids. To validate ERICH4 as a lipid gene, we generated whole-body Erich4 knockout (Erich4 Despite prediction tools indicating ERICH4 as a strong candidate gene in intestinal lipid metabolism, we here show that ERICH4 does not play a role in intestinal lipid metabolism in mice. It remains to be established whether ERICH4 plays a role in human lipid metabolism. Show less

Within the human population, considerable variability exists between individuals in their susceptibility to develop obesity and dyslipidemia. In humans, this is thought to be caused by both genetic and environmental variation. APOE*3-Leiden.CETP mice, as part of an inbred mouse model in which mice develop the metabolic syndrome upon being fed a high-fat high-cholesterol diet, show large inter-individual variation in the parameters of the metabolic syndrome, despite a lack of genetic and environmental variation. In the present study, we set out to resolve what mechanisms could underlie this variation. We used measurements of glucose and lipid metabolism from a six-month longitudinal study on the development of the metabolic syndrome. Mice were classified as mice with either high plasma triglyceride (responders) or low plasma triglyceride (non-responders) at the baseline. Subsequently, we fitted the data to a dynamic computational model of whole-body glucose and lipid metabolism (MINGLeD) by making use of a hybrid modelling method called Adaptations in Parameter Trajectories (ADAPT). ADAPT integrates longitudinal data, and predicts how the parameters of the model must change through time in order to comply with the data and model constraints. To explain the phenotypic variation in plasma triglycerides, the ADAPT analysis suggested a decreased cholesterol absorption, higher energy expenditure and increased fecal fatty acid excretion in non-responders. While decreased cholesterol absorption and higher energy expenditure could not be confirmed, the experimental validation demonstrated that the non-responders were indeed characterized by increased fecal fatty acid excretion. Furthermore, the amount of fatty acids excreted strongly correlated with bile acid excretion, in particular deoxycholate. Since bile acids play an important role in the solubilization of lipids in the intestine, these results suggest that variation in bile acid homeostasis may in part drive the phenotypic variation in the APOE*3-Leiden.CETP mice. Show less

The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying the effects of defective glycosylation on plasma lipids in patients with B4GALT1-CDG, caused by a mutation in B4GALT1 with defective N-linked glycosylation. We studied plasma lipids, cholesteryl ester transfer protein (CETP) glyco-isoforms with isoelectric focusing followed by a western blot and CETP activity in three known B4GALT1-CDG patients and compared them with 11 age- and gender-matched, healthy controls. B4GALT1-CDG patients have significantly lowered non-high density lipoprotein cholesterol (HDL-c) and total cholesterol to HDL-c ratio compared with controls and larger HDL particles. Plasma CETP was hypoglycosylated and less active in B4GALT1-CDG patients compared to matched controls. Our study provides insight into the role of protein glycosylation in human lipoprotein homeostasis. The hypogalactosylated, hypo-active CETP found in patients with B4GALT1-CDG indicates a role of protein galactosylation in regulating plasma HDL and LDL. Patients with B4GALT1-CDG have large HDL particles probably due to hypogalactosylated, hypo-active CETP. Show less

It is well established that, besides facilitating lipid absorption, bile acids act as signaling molecules that modulate glucose and lipid metabolism. Bile acid metabolism, in turn, is controlled by several nutrient-sensitive transcription factors. Altered intrahepatic glucose signaling in type 2 diabetes associates with perturbed bile acid synthesis. We aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, glucose-6-phosphate (G6P), on bile acid metabolism. Hepatic gene expression patterns and bile acid composition were analyzed in mice that accumulate G6P in the liver, that is, liver-specific glucose-6-phosphatase knockout (L-G6pc Show less

The Metabolic Syndrome (MetS) is a complex, multifactorial disorder that develops slowly over time presenting itself with large differences among MetS patients. We applied a systems biology approach to describe and predict the onset and progressive development of MetS, in a study that combined in vivo and in silico models. A new data-driven, physiological model (MINGLeD: Model INtegrating Glucose and Lipid Dynamics) was developed, describing glucose, lipid and cholesterol metabolism. Since classic kinetic models cannot describe slowly progressing disorders, a simulation method (ADAPT) was used to describe longitudinal dynamics and to predict metabolic concentrations and fluxes. This approach yielded a novel model that can describe long-term MetS development and progression. This model was integrated with longitudinal in vivo data that was obtained from male APOE*3-Leiden.CETP mice fed a high-fat, high-cholesterol diet for three months and that developed MetS as reflected by classical symptoms including obesity and glucose intolerance. Two distinct subgroups were identified: those who developed dyslipidemia, and those who did not. The combination of MINGLeD with ADAPT could correctly predict both phenotypes, without making any prior assumptions about changes in kinetic rates or metabolic regulation. Modeling and flux trajectory analysis revealed that differences in liver fluxes and dietary cholesterol absorption could explain this occurrence of the two different phenotypes. In individual mice with dyslipidemia dietary cholesterol absorption and hepatic turnover of metabolites, including lipid fluxes, were higher compared to those without dyslipidemia. Predicted differences were also observed in gene expression data, and consistent with the emergence of insulin resistance and hepatic steatosis, two well-known MetS co-morbidities. Whereas MINGLeD specifically models the metabolic derangements underlying MetS, the simulation method ADAPT is generic and can be applied to other diseases where dynamic modeling and longitudinal data are available. Show less

Physiological adaptations resulting in the development of the metabolic syndrome in man occur over a time span of several decades. This combined with the prohibitive financial cost and ethical concerns to measure key metabolic parameters repeatedly in subjects for the major part of their life span makes that comprehensive longitudinal human data sets are virtually nonexistent. While experimental mice are often used, little is known whether this species is in fact an adequate model to better understand the mechanisms that drive the metabolic syndrome in man. We took up the challenge to study the response of male apoE*3-Leiden.CETP mice (with a humanized lipid profile) to a high-fat high-cholesterol diet for 6 months. Study parameters include body weight, food intake, plasma and liver lipids, hepatic transcriptome, VLDL - triglyceride production and importantly the use of stable isotopes to measure hepatic de novo lipogenesis, gluconeogenesis, and biliary/fecal sterol secretion to assess metabolic fluxes. The key observations include (1) high inter-individual variation; (2) a largely unaffected hepatic transcriptome at 2, 3, and 6 months; (3) a biphasic response curve of the main metabolic features over time; and (4) maximum insulin resistance preceding dyslipidemia. The biphasic response in plasma triglyceride and total cholesterol appears to mimic that of men in cross-sectional studies. Combined, these observations suggest that studies such as these can help to delineate the causes of metabolic derangements in patients suffering from metabolic syndrome. Show less

We have investigated how the natural LCAT[T147I] and LCAT[P274S] mutations affect the pathway of biogenesis of HDL. Gene transfer of WT LCAT in LCAT(-/-) mice increased 11.8-fold the plasma cholesterol, whereas the LCAT[T147I] and LCAT[P274S] mutants caused a 5.2- and 2.9-fold increase, respectively. The LCAT[P274S] and the WT LCAT caused a monophasic distribution of cholesterol in the HDL region, whereas the LCAT[T147I] caused a biphasic distribution of cholesterol in the LDL and HDL region. Fractionation of plasma showed that the expression of WT LCAT increased plasma apoE and apoA-IV levels and shifted the distribution of apoA-I to lower densities. The LCAT[T147I] and LCAT[P274S] mutants restored partially apoA-I in the HDL3 fraction and LCAT[T147I] increased apoE in the VLD/IDL/LDL fractions. The in vivo functionality of LCAT was further assessed based on is its ability to correct the aberrant HDL phenotype that was caused by the apoA-I[L159R]FIN mutation. Co-infection of apoA-I(-/-) mice with this apoA-I mutant and either of the two mutant LCAT forms restored only partially the HDL biogenesis defect that was caused by the apoA-I[L159R]FIN and generated a distinct aberrant HDL phenotype. Show less

Mining of the genome for lipid genes has since the early 1970s helped to shape our understanding of how triglycerides are packaged (in chylomicrons), repackaged (in very low density lipoproteins; VLDL), and hydrolyzed, and also how remnant and low-density lipoproteins (LDL) are cleared from the circulation. Gene discoveries have also provided insights into high-density lipoprotein (HDL) biogenesis and remodeling. Interestingly, at least half of these key molecular genetic studies were initiated with the benefit of prior knowledge of relevant proteins. In addition, multiple important findings originated from studies in mouse, and from other types of non-genetic approaches. Although it appears by now that the main lipid pathways have been uncovered, and that only modulators or adaptor proteins such as those encoded by LDLRAP1, APOA5, ANGPLT3/4, and PCSK9 are currently being discovered, genome wide association studies (GWAS) in particular have implicated many new loci based on statistical analyses; these may prove to have equally large impacts on lipoprotein traits as gene products that are already known. On the other hand, since 2004 - and particularly since 2010 when massively parallel sequencing has become de rigeur - no major new insights into genes governing lipid metabolism have been reported. This is probably because the etiologies of true Mendelian lipid disorders with overt clinical complications have been largely resolved. In the meantime, it has become clear that proving the importance of new candidate genes is challenging. This could be due to very low frequencies of large impact variants in the population. It must further be emphasized that functional genetic studies, while necessary, are often difficult to accomplish, making it hazardous to upgrade a variant that is simply associated to being definitively causative. Also, it is clear that applying a monogenic approach to dissect complex lipid traits that are mostly of polygenic origin is the wrong way to proceed. The hope is that large-scale data acquisition combined with sophisticated computerized analyses will help to prioritize and select the most promising candidate genes for future research. We suggest that at this point in time, investment in sequence technology driven candidate gene discovery could be recalibrated by refocusing efforts on direct functional analysis of the genes that have already been discovered. This article is part of a Special Issue entitled: From Genome to Function. Show less

The severe forms of hypertriglyceridaemia (HTG) are caused by mutations in genes that lead to the loss of function of lipoprotein lipase (LPL). In most patients with severe HTG (TG > 10 mmol L(-1) ), it is a challenge to define the underlying cause. We investigated the molecular basis of severe HTG in patients referred to the Lipid Clinic at the Academic Medical Center Amsterdam. The coding regions of LPL, APOC2, APOA5 and two novel genes, lipase maturation factor 1 (LMF1) and GPI-anchored high-density lipoprotein (HDL)-binding protein 1 (GPIHBP1), were sequenced in 86 patients with type 1 and type 5 HTG and 327 controls. In 46 patients (54%), rare DNA sequence variants were identified, comprising variants in LPL (n = 19), APOC2 (n = 1), APOA5 (n = 2), GPIHBP1 (n = 3) and LMF1 (n = 8). In 22 patients (26%), only common variants in LPL (p.Asp36Asn, p.Asn318Ser and p.Ser474Ter) and APOA5 (p.Ser19Trp) could be identified, whereas no mutations were found in 18 patients (21%). In vitro validation revealed that the mutations in LMF1 were not associated with compromised LPL function. Consistent with this, five of the eight LMF1 variants were also found in controls and therefore cannot account for the observed phenotype. The prevalence of mutations in LPL was 34% and mostly restricted to patients with type 1 HTG. Mutations in GPIHBP1 (n = 3), APOC2 (n = 1) and APOA5 (n = 2) were rare but the associated clinical phenotype was severe. Routine sequencing of candidate genes in severe HTG has improved our understanding of the molecular basis of this phenotype associated with acute pancreatitis and may help to guide future individualized therapeutic strategies. Show less

Hypertriglyceridemia and fatty liver are common in patients with type 2 diabetes, but the factors connecting alterations in glucose metabolism with plasma and liver lipid metabolism remain unclear. Apolipoprotein CIII (apoCIII), a regulator of hepatic and plasma triglyceride metabolism, is elevated in type 2 diabetes. In this study, we analyzed whether apoCIII is affected by altered glucose metabolism. Liver-specific insulin receptor-deficient mice display lower hepatic apoCIII mRNA levels than controls, suggesting that factors other than insulin regulate apoCIII in vivo. Glucose induces apoCIII transcription in primary rat hepatocytes and immortalized human hepatocytes via a mechanism involving the transcription factors carbohydrate response element-binding protein and hepatocyte nuclear factor-4α. ApoCIII induction by glucose is blunted by treatment with agonists of farnesoid X receptor and peroxisome proliferator-activated receptor-α but not liver X receptor, ie, nuclear receptors controlling triglyceride metabolism. Moreover, in obese humans, plasma apoCIII protein correlates more closely with plasma fasting glucose and glucose excursion after oral glucose load than with insulin. Glucose induces apoCIII transcription, which may represent a mechanism linking hyperglycemia, hypertriglyceridemia, and cardiovascular disease in type 2 diabetes. Show less

GPIHBP1 is an endothelial cell protein that binds lipoprotein lipase (LPL) and chylomicrons. Because GPIHBP1 deficiency causes chylomicronemia in mice, we sought to determine whether some cases of chylomicronemia in humans could be attributable to defective GPIHBP1 proteins. Patients with severe hypertriglyceridemia (n=60, with plasma triglycerides above the 95th percentile for age and gender) were screened for mutations in GPIHBP1. A homozygous GPIHBP1 mutation (c.344A>C) that changed a highly conserved glutamine at residue 115 to a proline (p.Q115P) was identified in a 33-year-old male with lifelong chylomicronemia. The patient had failure-to-thrive as a child but had no history of pancreatitis. He had no mutations in LPL, APOA5, or APOC2. The Q115P substitution did not affect the ability of GPIHBP1 to reach the cell surface. However, unlike wild-type GPIHBP1, GPIHBP1-Q115P lacked the ability to bind LPL or chylomicrons (d < 1.006 g/mL lipoproteins from Gpihbp1(-/-) mice). Mouse GPIHBP1 with the corresponding mutation (Q114P) also could not bind LPL. A homozygous missense mutation in GPIHBP1 (Q115P) was identified in a patient with chylomicronemia. The mutation eliminated the ability of GPIHBP1 to bind LPL and chylomicrons, strongly suggesting that it caused the patient's chylomicronemia. Show less

Apolipoprotein AV (apoAV) overexpression causes a decrease in plasma triglyceride (TG) levels, while deficiency of apoAV causes hypertriglyceridemia in both men and mice. However, contrary to what would be expected, plasma apoAV and TG levels in humans are positively correlated. To address this apparent paradox, we determined plasma apoAV levels in various mouse models with median TG levels ranging from 30 mg/dl in wild-type mice to 2089 mg/dl in glycosylphosphatidylinositol-anchored HDL binding protein 1-deficient mice. The data show that apoAV and TG levels are positively correlated in mice (r = +0.798, P < 0.001). In addition, we show that LPL gene transfer caused a simultaneous decrease in TG and apoAV in LPL-deficient mice. The combined data suggest that apoAV levels follow TG levels due to an intimate link between the apoAV molecule and TG-rich lipoproteins, comprising both secretion and removal of these lipoproteins. Taken together, the data suggest that higher plasma apoAV levels reflect an increased demand for plasma TG hydrolysis under normal physiological conditions. Show less

Variation in the apolipoprotein A5 (APOA5) gene has consistently been associated with increased plasma triglyceride (TG) levels in epidemiological studies. In vivo functionality of these variations, however, has thus far not been tested. Using adenoviral over-expression, we evaluated plasma expression levels and TG-lowering efficacies of wild-type human apoAV, two human apoAV variants associated with increased TG (S19W, G185C) and one variant (Q341H) that is predicted to have altered protein function. Injection of mice with adenovirus encoding wild-type or mutant apoAV resulted in an identical dose-dependent elevation of human apoAV levels in plasma. The increase in apoAV levels resulted in pronounced lowering of plasma TG levels at two viral dosages. Unexpectedly, the TG-lowering efficacy of all three apoAV variants was similar to wild-type apoAV. In addition, no effect on TG-hydrolysis-related plasma parameters (free fatty acids, glycerol and post-heparin lipoprotein lipase activity) was apparent upon expression of all apoAV variants. In conclusion, our data indicate that despite their association with hypertriglyceridemia and/or predicted protein dysfunction, the 19W, 185C and 341H apoAV variants are equally effective in reducing plasma TG levels in mice. Show less

The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible. We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant. The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression. Show less

The relevance of apolipoprotein A-V (apoA-V) for human lipid homeostasis is underscored by genetic association studies and the identification of truncation-causing mutations in the APOA5 gene as a cause of type V hyperlipidemia, compatible with an LPL-activating role of apoA-V. An inverse correlation between plasma apoA-V and triglyceride (TG) levels has been surmised from animal data. Recent studies in human subjects using (semi)quantitative immunoassays, however, do not provide unambiguous support for such a relationship. Here, we used a novel, validated ELISA to measure plasma apoA-V levels in patients (n = 28) with hypertriglyceridemia (HTG; 1.8-78.7 mmol TG/l) and normolipidemic controls (n = 42). Unexpectedly, plasma apoA-V levels were markedly increased in the HTG subjects compared with controls (1,987 vs. 258 ng/ml; P < 0.001). In the HTG group, apoA-V and TG were positively correlated (r = +0.44, P = 0.02). In addition, we noted an increased level of the LPL-inhibitory protein apoC-III in the HTG group (45.8 vs. 10.6 mg/dl in controls; P < 0.001). The correlation between apoA-V and TG levels in the HTG group disappeared (partial r = +0.09, P = 0.65) when controlling for apoC-III levels. In contrast, apoC-III and TG remained positively correlated in this group when controlling for apoA-V (partial r = +0.43, P = 0.025). Our findings suggest that in HTG patients, increased TG levels are accompanied by high plasma levels of apoA-V and apoC-III, apolipoproteins with opposite modes of action. This study provides evidence for a complex interaction between apoA-V and apoC-III in patients with severe HTG. Show less

In mouse models, apolipoprotein A-V (apoA-V) exhibits triglyceride (TG)-lowering effects. We investigated the apoA-V/TG relationship and the association of apoA-V with coronary artery disease (CAD) risk by determining serum apoA-V levels and genotypes in a nested case-control (n = 1,034/2,031) study. Both univariate and multivariate apoA-V levels showed no association with future CAD (P = 0.4 and 0.5, respectively). Unexpectedly, there was a significant positive correlation between serum apoA-V and TG in men and women (r = 0.36 and 0.28, respectively, P < 0.001 each) but a negative correlation between apoA-V and LPL mass (r = -0.14 and -0.12 for men and women respectively, P < 0.001 each). The frequency of the c.56C>G polymorphism did not differ between cases and controls despite significant positive association of c.56G with both apoA-V and TG levels. For -1131T>C, the minor allele was significantly associated with lower apoA-V yet higher TG levels and was overrepresented in cases (P = 0.047). The association of -1131T>C with CAD risk, however, was independent of apoA-V levels and likely acts through linkage disequilibrium with APOC3 variants. The positive correlation of apoA-V levels with TG levels, negative correlation with LPL levels, and lack of association with CAD risk highlight the need for further human studies to clarify the role of apoA-V. Show less