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To investigate the relationship between Angiopoietin-like protein 4 (Angptl4) levels, CHD biomarkers and ANGPTL4 variants.
Plasma Angptl4 was quantified in 666 subjects of the Northwick Park Heart Study II using a validated ELISA. Seven ANGPTL4 SNPs were genotyped and CHD biomarkers assessed in the whole cohort (n=2775). Weighted mean (±SD) plasma Angptl4 levels were 10.0(±11.0) ng/ml. Plasma Angptl4 concentration correlated positively with age (r=0.15, P<0.001), body fat mass (r=0.19, P=0.003) but negatively with plasma HDL-cholesterol (r=−0.13, P=0.01). No correlation with triglycerides was observed. T266M was independently associated with plasma Angptl4 levels (P<0.001), but not associated with triglycerides or with CHD risk in the meta-analysis of five studies (4,061 cases/15,395 controls). E40K showed no independent association with plasma Angptl4 levels. In HEK293 and Huh7 cells compared to wild-type, E40K and T266M showed significantly altered synthesis and secretion, respectively.
These data suggest that circulating Angptl4 levels do not influence triglyceride levels or CHD risk since (1) Angptl4 levels were not correlated with triglycerides, (2) T266M, although associated with Angptl4 levels, showed no association with plasma triglycerides (3) Triglyceride-lowering E40K did not influence Angptl4 levels. These results provide new insights into the role of Angptl4 in triglyceride metabolism.
Lipoprotein lipase (LPL) plays a major role in the metabolism and transport of lipids. LPL hydrolyses the core triglycerides (TG) in chylomicrons and very low-density lipoproteins (VLDLs) 1 and regulates the supply of fatty acids to various tissues for either storage or oxidation. LPL enzymatic activity is regulated in a complex manner in response to energy requirements and hormonal changes1, 2 Data suggest that this regulation occurs at the transcriptional, translational, and post-translational levels in a tissue-specific manner.3 In humans, Angptl4 is ubiquitously expressed with the highest expression levels in liver.4 Full length Angptl4 is cleaved into N- and C-terminal fragments. Both the N- and C-terminal fragments together with full length protein can be detected in plasma.5, 6 Current evidence, from in vitro studies, suggests that the coiled-coil domain is a potent inhibitor of LPL and converts the catalytically active dimeric form of the enzyme into inactive monomers,7 thus, reducing the hydrolysis of TG-rich lipoproteins.
A role for Angptl4 as an inhibitor of LPL-mediated TG catabolism in humans is derived from genetic studies. In particular the E40K variant prevents Angptl4 oligomer formation which is essential in Angptl4-mediated inhibition of LPL.8 Studies in over 30,000 individuals revealed that carriers of the K40 allele have significantly lower TG levels and, in some studies, higher high density lipoprotein cholesterol (HDL-C) levels compared to E40 homozygotes.9, 10 Another coding variant, T266M, which is more prevalent than E40K, affected TG metabolism only during the postprandial state and showed no significant effect on TG or HDL-C levels in the fasting state in healthy individuals.10
On the basis of biochemical and epidemiological evidence for a role of Angptl4 in lipid metabolism, we examined the relationship between plasma Angptl4 levels, ANGPTL4 gene variants and common CHD biomarker levels in men participating in the prospective Northwick Park Heart Study II (NPHSII), and undertook a meta-analysis in five studies (4061 cases/ 15395 controls) examining the association of T266M with CHD risk. We also present in vitro studies to validate our findings.
Our primary aim was to examine the association of T266M variant (rs1044250)10 with plasma Angptl4 levels in Northwick Park Heart Study II (NPHSII). Plasma samples were grouped on the basis of their T266M genotype and subsequently 666 subjects selected at random from each genotype group for this ‘bottom-up’ approach. In addition to NPHSII, three prospective studies; Whitehall II (WHII), the British Women's Heart and Health Study (BWHHS), the British Heart Foundation Family Heart Study (BHF-FHS) and a nested case: control study; European Prospective Investigation of Cancer (EPIC)-Norfolk were included in meta-analysis to study if T266M influences CHD risk. Details of the studies are presented in Supplemental Table I. Recruitment protocols and baseline characteristics of the 5 studies have been published before.11–15
Plasma Angptl4 concentration was measured using a non-competitive direct enzyme-linked immunosorbent assay (ELISA)16 using a goat polyclonal antibody specific for Angptl4 (RnD Systems, Minneapolis, USA) that recognizes the full length Angptl4 in human plasma17.
Six tagging (t)SNPs were used to genotype NPHSII; rs4076317 (−207C>G), rs7255436 (3991A>C), rs1044250 (6959C>T, T266M), rs11672433 (9511A>G), rs7252574 (12574C>T), and rs1808536 (12651G>A).10 These tSNPs captured >92% of the genetic variation in ANGPTL4. In addition, E40K (118G>A) genotype was determined. Genotyping in NPHSII and EPIC-Norfolk was performed following the methods previously reported10 In silico data for T266M was obtained from WHII, BWHHS and BHF-FHS where genotyping had been undertaken using the 50K cardiovascular Human CVD BeadChip (Illumina).18
In brief, human hepatoma cells (Huh7) and human embryonic kidney (HEK293) cells were transiently transfected with Angptl4, wild-type (WT), T266M and E40K, cloned into pcDNA3.1 vector (pcDNA3.1/human WT ANGPTL4 was kindly provided by Professor Helen Hobbs, Dallas, USA). Medium was collected 24 hours after transfection and cells lysed. Aliquots from the medium and cell lysate were subjected to ELISA and western blot analysis. For western blot we used a rabbit polyclonal antibody specific for Angptl4 (BioVendor, Modrice, Czech Republic) that recognizes full length, C-terminal and N-terminal Angptl4. Full methods are provided in the Supplemental Methods (available online at http://atvb.ahajournal.org).
Analysis was performed using Intercooled Stata 10.2 for Windows (StataCorp LP, Texas, USA). Angptl4 levels were normalized using a reciprocal transformation (1/angptl4). To account for the sampling design, weighted estimates were obtained from the regression models. Each observation was weighted according to the inverse of its probability of being included the sample. A greater proportion of rare homozygotes and fewer heterozygotes were included in the sample as compared to the total NPHSII population. For this reason a weighted analysis was performed with each observation weighted according to the inverse of its probability of being included in the sample. This weighting therefore gives us estimates relevant for the total population distribution rather than the sample distribution. CHD risk was estimated for a 1 standard deviation decrease in 1/angptl4 adjusting for age and practice and then adjusting for additional covariates; such as systolic blood pressure, body mass index (BMI), smoking, cholesterol, TG and HDL-C in regression models. A backward stepwise logistic regression determined independent association of selected variants with Angptl4 levels. Meta-analysis was conducted using the ‘metan’ command in Stata. Pooled odds ratios were calculated using the inverse-variance method. As there was significant heterogeneity between studies (I-squared=65.9, P=0.02), a random effects estimate was also obtained using the DerSimonian and Laird method.19 To account for multiple testing p-value cut off was P<0.01.
Characteristics of the subset of 666 NPHSII men in which plasma Angptl4 levels were determined and those of the whole cohort are presented in Table 1. The clinical and biochemical characteristics of the study subjects did not differ significantly from the whole NPHSII cohort (Table 1). The baseline characteristics for the whole cohort, stratified by CHD status, have been published elsewhere;20 men who developed CHD during follow-up (n=275) were older, had higher plasma total cholesterol, TG and blood pressure levels, and lower HDL-C levels.
Plasma Angptl4 levels displayed high variability, with a skewed distribution, and with levels ranging from 3.2 to 232.4 ng/ml (Figure 1). Median Angptl4 levels were 7.7 (IQR 5.9–11.0) ng/ml and weighted mean levels were 10.0 (±11.0) ng/ml.
Plasma Angptl4 levels were positively correlated with measures of obesity such as body fat percentage (r=0.17, P=0.02), fat mass (r=0.19, P=0.003) and BMI (r=0.09, P=0.001). Angptl4 levels were also positively correlated with age (r=0.15, P<0.001) and negatively correlated with HDL-C (r=−0.13, P=0.01). However, there was no correlation between Angptl4 and plasma TG levels (Table 2).
The genotype distributions of all SNPs were in Hardy-Weinberg equilibrium in the whole of NPHSII.10 A map of ANGPTL4 is shown in Supplemental Figure I, demonstrating strong LD across the gene. The minor allele frequency (MAF) for the 7 Angptl4 SNPs in the sub-population and whole of NPHSII are provided in Supplemental Table II. There are differences in the MAF in the subpopulation compared to the study as a whole, since subjects for Angptl4 measures were chosen on the basis of their T266M genotype. The association of these 7 SNPs with plasma Angptl4 levels in the 666 men is presented in Table 3. The association of these 7 SNPs with plasma TG levels in the whole cohort is presented in Supplemental Table III. T266M and rs11672433 were significantly associated with weighted mean Angptl4 levels. The small number of E40K carriers (n=41) showed borderline association with Angptl4 (P=0.01).
To identify those SNPs which independently associated with Angptl4 levels, a stepwise logistic regression was used. T266M, showed consistent independent association with higher Angptl4 levels (MM homozygotes compared to TT homozygotes; standardized for a one SD increase in 1/Angptl4; β (SE) 0.455 (0.063), P<0.001). In addition rs1808536 (β (SE) 0.638 (0.229), P<0.005) and rs11672433 (β (SE) 0.712 (0.191), P<0.001) showed independent recessive effects. E40K did not enter the model (P=0.75) despite showing borderline significant association with Angptl4. This effect probably reflects the strong LD with T266M, since all E40K carriers were heterozygous or homozygous for T226M.10 Because of the bias in T266M genotype frequency, haplotype analysis was not appropriate.
To determine whether E40K and T266M variants affect protein processing, we compared the expression and secretion of the variant proteins E40K and T266M with WT Angptl4 in cultured HEK293 and Huh7 cells. As shown in Figure 2 (Panel A) E40K displayed reduced Angptl4 levels in the cells (by about 50%) as well as in the media (Figure 2, Panel B), in comparison to WT protein. In contrast, cellular levels of T266M Angptl4 were similar to that of WT Angptl4 (Figure 2, Panel A). However, T266M Angptl4 levels were significantly higher (with 15% on average) in media (P<0.05) (Figure 2, Panel B), consistent with the plasma Angptl4-raising effect observed in the association studies. To study differences in the proteolytic cleavage and oligomerization of the mutants and WT Angptl4, we performed Western blot analysis using a polyclonal antibody that recognized all three forms of Angptl4 under reducing conditions: full length, C-terminal and N-terminal fragments. No significant differences in proteolytic processing between the three Angptl4 variants tested (Figure 2, Panel C) were observed. Densitometry of the Western blot patterns displayed similar differences between the mutant variants and WT Angptl4 as that obtained with ELISA analyses (Figure 2, Panel D). Gel electrophoresis performed under non-reducing conditions revealed an absence of oligomers for E40K variant, an observation that well agrees with the data in a recent study.8 In addition, we verified the effect of heparin on Angptl4 mutants and WT protein. Heparin, when present in the cell media for 24 hours, increased the Angptl4 levels in media of all three genetic variants (Supplemental Figure II).
Considering those men with Angptl4 measures, a one standard deviation increase in weighted Angptl4 levels was associated with reduced risk for CHD (HR 0.79 (95% CI 0.68, 0.92) (P=0.003) when adjusting for age and recruitment centre. However, after further adjustment for classical risk factors of age, systolic blood pressure, BMI, smoking, cholesterol, TGs and HDL-C the association no longer remained statistically significant, HR 0.80 (95% CI 0.65, 0.99) (P=0.04) (Supplemental Table IV).
A meta-analysis of 5 studies, with over 4,000 cases and 15,000 controls demonstrated no association of the T266M variant with CHD risk; combined OR 1.02 (95% CI 0.91, 114) (P=0.843) (Figure 3). Adjustment for age and gender (where appropriate), TGs and HDL-C did not affect this meta-analysis (data not shown). We previously reported that E40K, which has been consistently associated with lower triglyceride levels, was associated with increased CHD risk in meta-analysis.10
The major findings of this study were 1) circulating Angptl4 levels did not correlate with plasma TG levels; 2) E40K, a variant affecting plasma TG levels, was not associated with plasma Angptl4 levels 3) the T266M variant was associated with plasma Angptl4 levels 4) expression of E40K and T266M in HEK 293 and Huh7 cells revealed altered synthesis and secretion, respectively and 5) T266M, although associated with plasma Angptl4 levels, was not associated with CHD risk in meta-analysis.
Baseline plasma Angptl4 levels varied greatly, with 92% of the study subjects displaying levels below 20 ng/ml, an observation which is in line with other published data.16 On the basis of in vitro studies that have clearly established an inhibitory role of Angptl4 on LPL activity7 we anticipated that plasma Angptl4 levels would show a positive correlation with TG levels. However, plasma Angplt4, while significantly correlated with age and measures of obesity and negatively correlated with HDL-C, showed no correlation with plasma TG levels. Our data are in agreement with the study by Robciuc et al.16 and a small study of healthy controls (n=108), where no correlation was seen between Angptl4 and triglyceride levels.21 Using the same commercial ELISA,21 Stejskal et al.22 reported a positive correlation between triglyceride levels and Angptl4 in a study of patients with the metabolic syndrome (n=115). However, they reported a negative correlation between HDL and Angptl422, which is in agreement with our current study.
LPL-mediated hydrolysis of TG-rich lipoproteins primarily takes place on the endothelial cell surface where LPL is anchored. Heparin sulphate proteoglycans (HSPG) were thought to play the key role in this, but recently the novel glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) was identified as an alternate anchor of LPL and chylomicrons.23 GPIHBP1, required for the lipolytic processing of TG-rich lipoproteins, stabilises LPL and prevents its inhibition by Angptl4.24 In a set of experiments on rat adipose tissue after feeding/fasting experiments Sukonina et al.7 observed an inverse relationship between Angptl4 expression and LPL activity which led them to conclude that the change in extracellular LPL activity occurred in the subendothelial space or at the endothelium, where Angptl4 destabilised the active LPL dimer. Furthermore, Nilsson et al. recently reported that the inactivation of LPL by Angptl4 was significantly weakened in the presence of TG-rich lipoproteins, and concluded that circulating Angptl4 may have less effect on LPL activity than tissue bound Angptl4.25 Combined with our observations, these data strongly support the view that circulating Angptl4 does not inhibit endothelial bound LPL, and this in turn may preserve the tissue specific regulation of TG hydrolysis by Angptl4. It remains to be determined whether circulating Angptl4 plays a role in the inactivation of circulating LPL.
We report a modest positive correlation of Angptl4 levels with body weight and body fat mass in our middle aged men. This supports the report that Angptl4 levels were positively correlated with waist: hip ratio16.
Our results show that T266M rare homozygotes have significantly higher Angptl4 levels than T266 carriers. This is in agreement with the in vitro studies which show that T266M protein is more readily secreted. However T266M shows no association with TG levels under fasting condition in healthy subjects10 and as confirmed in our meta-analysis, is not associated with CHD risk. These results taken together suggest that circulating Angptl4 levels do not support the possible action of Angptl4 at the cell surface. The function of the soluble carboxy-fragment in whichT266M resides, is still not clear. Our in vitro data showed that E40K was expressed at lower levels compared to WT and T266M and as a consequence less protein was secreted into the cell media. Yin et al.8 reported less E40K in the media but comparable levels expressed in the cell. Both the current study and Yin et al.8 observed a reduction or non existence of monomers or oligomers of the N-terminal fragments, only the C-terminal fragment and a slight presence of full-length E40K was evident in the medium. Our results show a borderline association of E40K with Angptl4 levels, but this result hinges on just 41 K40 carriers. In addition all of these K40 carriers are M266 carriers. The E40K carriers may be merely reflecting the complete LD with M266 which shows significant association with Angptl4 levels.
There are certain limitations in the present study. First, Angplt4 levels were only measured in a subset of men from NPHSII and we did not have sufficient statistical power to be confident of the association of Angptl4 levels with CHD risk in this sub-cohort. However, the meta-analysis in nearly 20,000 individuals showed no association of CHD risk with the T226M with CHD risk, despite its strong association with Angptl4 levels. Although no correlation of triglyceride levels with full length Angptl4 was observed it is possible, using an antibody specific for the N-terminal portion of the protein, that the N-terminal fragment of Angptl4 may be associated with plasma triglycerides.
In conclusion, ANGPTL4-T266M, but not E40K, was independently associated with Angptl4 levels, yet circulating Angptl4 levels showed no correlation with triglyceride levels. The in vitro data provides a biological basis for the association of T266M with circulating Angplt4 levels and therefore providing new insights into the role of Angptl4 in triglyceride metabolism.
The authors would like to thank Professor Helen Hobbs for kindly providing the WT Angptl4 construct. The authors would also like to thank the participants, general practitioners, and staff in NPHSII, EPIC-Norfolk, WH-II, BWHHS and BHF-FHS. The BWHHS is coordinated by Shah Ebrahim (PI), Debbie Lawlor and Juan-Pablo Casas, with BHF cardiochip work by TRG (PI), INMD, DL, SE, George Davey Smith, Yoav Ben-Shlomo and Santiago Rodriguez.
Sources of Funding
MCSH is supported by a Unilever/BBSRC Case studentship. SEH, JAC, and PJT are supported by the British Heart Foundation (RG2008/014); MRR, MJ and CE are supported by the Finska Läkaresällskapet, Magnus Ehnrooth and the National Institute for Health and Welfare, Finland. MK is supported by the National Heart, Lung, and Blood Institute (R01HL036310), US NIH. NPHSII was supported by MRC UK, US NIH (grant NHLBI 33014), and Du Pont Pharma, Wilmington, USA. EPIC-Norfolk was supported by MRC UK and Cancer Research UK, with additional support from the EU, Stroke Association, British Heart Foundation (Grant PG2000/015), Department of Health, Food Standards Agency, and Wellcome Trust. WH-II was supported by the British Heart Foundation (BHF) PG/07/133/24260, RG/08/008, SP/07/007/23671. The WH-II study has also been supported by grants from the Medical Research Council; Health and Safety Executive; Department of Health; National Institute on Aging (AG13196), US, NIH; Agency for Health Care Policy Research (HS06516); and the John D and Catherine T MacArthur Foundation Research Networks on Successful Midlife Development and Socio-economic Status and Health. The British Women's Heart and Health Study (BWHHS) has been supported by funding from the BHF and the UK Department of Health Policy Research Programme, with cardiochip work funded by the BHF (PG/07/131/24254).
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