The Relationship Between Plasma Angiopoietin-like Protein 4 Levels, Angiopoietin-like Protein 4 Genotype, and Coronary Heart Disease Risk
Objective—To investigate the relationship between angiopoietin-like protein 4 (Angptl4) levels, coronary heart disease (CHD) biomarkers, and ANGPTL4 variants.
Methods and Results—Plasma Angptl4 was quantified in 666 subjects of the Northwick Park Heart Study II using a validated ELISA. Seven ANGPTL4 single-nucleotide polymorphisms were genotyped, and CHD biomarkers were 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) and body fat mass (r=0.19, P=0.003) but negatively with plasma high-density lipoprotein cholesterol (r=−0.13, P=0.01). No correlation with triglycerides (TGs) was observed. T266M was independently associated with plasma Angptl4 levels (P<0.001) but was not associated with TGs or CHD risk in the meta-analysis of 5 studies (4061 cases/15 395 controls). E40K showed no independent association with plasma Angptl4 levels. In human embryonic kidney 293 and human hepatoma 7 cells compared with wild type, E40K and T266M showed significantly altered synthesis and secretion, respectively.
Conclusion—Circulating Angptl4 levels may not influence TG levels or CHD risk for the following reasons: (1) Angptl4 levels were not correlated with TGs; (2) T266M, although associated with Angptl4 levels, showed no association with plasma TGs; and (3) TG-lowering E40K did not influence Angptl4 levels. These results provide new insights into the role of Angptl4 in TG metabolism.
Lipoprotein lipase (LPL) plays a major role in the metabolism and transport of lipids. LPL hydrolyzes the core triglycerides (TGs) in chylomicrons and very-low-density lipoproteins1 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 changes.1,2 Data suggest that this regulation occurs at the transcriptional, translational, and posttranslational levels in a tissue-specific manner.3 In humans, angiopoietin-like protein 4 (Angptl4) is ubiquitously expressed, with the highest expression levels in the 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 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 Studies9,10 in more than 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 with E40 homozygotes. 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
Based on 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 coronary heart disease (CHD) biomarker levels in men participating in the prospective Northwick Park Heart Study II (NPHSII); and undertook a meta-analysis of 5 studies (4061 cases/15 395 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 the T266M variant (rs1044250)10 with plasma Angptl4 levels in the NPHSII. Plasma samples were grouped based on their T266M genotype; subsequently, 666 subjects were selected at random from each genotype group for this “bottom-up” approach. In addition to NPHSII, 3 prospective studies (Whitehall II [WHII], the British Women’s Heart and Health Study [BWHHS], and the British Heart Foundation Family Heart Study) and a nested case-control study (the European Prospective Investigation of Cancer–Norfolk) were included in a meta-analysis to study if T266M influences CHD risk. Details of the studies are presented in supplemental Table I (available online at http://atvb.ahajournals.org). Recruitment protocols and baseline characteristics of the 5 studies were previously published.11–15
Determination of Angptl4 Concentrations
Six tagging (t) single-nucleotide polymorphisms (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, the E40K (118G>A) genotype was determined. Genotyping in the NPHSII and the European Prospective Investigation of Cancer–Norfolk was performed following the methods previously reported.10 In silico data for T266M were obtained from WHII, BWHHS, and the British Heart Foundation Family Heart Study, in which genotyping was undertaken using a chip (50K Cardiovascular Human CVD BeadChip).18
Expression of Angptl4 Variants In Vitro
In brief, human hepatoma 7 and human embryonic kidney 293 cells were transiently transfected with Angptl4, wild type (WT), T266M, and E40K; and cloned into pcDNA3.1 vector. Medium was collected 24 hours after transfection, and cells were lysed. Aliquots from the medium and cell lysate were subjected to ELISA and Western blot analysis. For Western blotting, we used a rabbit polyclonal antibody specific for Angptl4 that recognizes full-length, C-terminal, and N-terminal Angptl4. Complete methods are provided in the supplemental data.
Analysis was performed using computer software (Intercooled Stata 10.2 for Windows). 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. More rare homozygotes and fewer heterozygotes were included in the sample compared with 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. Therefore, this weighting provides estimates relevant for the total population distribution rather than the sample distribution. CHD risk was estimated for a 1-SD decrease in 1/angptl4, adjusting for age and practice and then adjusting for additional covariates (eg, systolic blood pressure, body mass index, smoking, and cholesterol, TG, and HDL-C levels) in regression models. A backward stepwise logistic regression determined the independent association of selected variants with Angptl4 levels. A meta-analysis was conducted using the “metan” command in Stata. Pooled odds ratios were calculated using the inverse-variance method. Because there was significant heterogeneity between studies (I2=65.9, P=0.02), a random-effects estimate was also obtained using the method of DerSimonian and Laird.19 To account for multiple testing, the probability value cut off was P<0.01.
Characteristics of the subset of 666 NPHSII men in whom 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 had lower HDL-C levels.
Distribution of Angptl4 in Study Subjects
Plasma Angptl4 levels displayed high variability, with a skewed distribution; levels ranged from 3.2 to 232.4 ng/mL (Figure 1). The median Angptl4 level was 7.7 (interquartile range, 5.9 to 11.0) ng/mL; the weighted mean±SD level was 10.0±11.0 ng/mL.
Correlation of Angptl4 With Clinical and Biochemical Parameters
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 body mass index (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).
Relationship Between ANGPTL4 Genotype, Angptl4 Levels, and TG Levels
The genotype distributions of all SNPs were in Hardy-Weinberg equilibrium in the whole of the NPHSII.10 A map of ANGPTL4 is shown in supplemental Figure I, demonstrating strong linkage disequilibrium (LD) across the gene. The minor allele frequencies for the 7 Angptl4 SNPs in the subpopulation and the whole NPHSII are provided in supplemental Table II. There are differences in the minor allele frequency in the subpopulation compared with the study as a whole because subjects for Angptl4 measures were chosen based on 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 few E40K carriers (n=41) showed a borderline association with Angptl4 (P=0.01).
To identify those SNPs that independently associated with Angptl4 levels, a stepwise logistic regression was used. T266M showed a consistent independent association with higher Angptl4 levels (MM homozygotes compared with TT homozygotes; standardized for a 1-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 a borderline significant association with Angptl4. This effect probably reflects the strong LD with T266M because all E40K carriers were heterozygous or homozygous for T226 mol/L.10 Because of the bias in T266M genotype frequency, haplotype analysis was not appropriate.
E40K and T266M Interfere With Protein Secretion
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 human embryonic kidney 293 and human hepatoma 7 cells. As shown in Figure 2A, E40K displayed reduced Angptl4 levels in the cells (by approximately 50%) and the media (Figure 2B) compared with WT protein. In contrast, cellular levels of T266M Angptl4 were similar to those of WT Angptl4 (Figure 2A). However, T266M Angptl4 levels were significantly higher (15% on average) in media (P<0.05) (Figure 2B), consistent with the plasma Angptl4-increasing 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 3 forms of Angptl4 under reducing conditions: full-length, C-terminal, and N-terminal fragments. No significant differences in proteolytic processing between the 3 Angptl4 variants tested (Figure 2C) were observed. Densitometry of the Western blot patterns displayed similar differences between the mutant variants and WT Angptl4 as obtained with ELISAs (Figure 2D). Gel electrophoresis performed under nonreducing conditions revealed an absence of oligomers for the E40K variant, an observation that agrees well 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 the media of all 3 genetic variants (supplemental Figure II).
Angplt4 Levels and CHD Risk
Considering those men with Angptl4 measures, a 1-SD increase in weighted Angptl4 levels was associated with reduced risk for CHD (hazard ratio, 0.79; 95% CI, 0.68 to 0.92) (P=0.003) when adjusting for age and recruitment center. However, after further adjustment for the classic risk factors of age, systolic blood pressure, body mass index, smoking, and cholesterol, TGs, and HDL-C levels, the association no longer remained statistically significant (hazard ratio, 0.80; 95% CI, 0.65 to 0.99) (P=0.04) (supplemental Table IV).
Relationship Between ANGPTL4 Genotypes and CHD Risk
A meta-analysis of 5 studies, with more than 4000 cases and 15 000 controls, demonstrated no association of the T266M variant with CHD risk: combined odds ratio, 1.02 (95% CI, 0.91 to 1.14) (P=0.84) (Figure 3). Adjustment for age and sex (where appropriate), TGs, and HDL-C did not affect this meta-analysis (data not shown). Previously, E40K, which has been consistently associated with lower TG levels, was associated with increased CHD risk in a meta-analysis.10
The major findings of this study were as follows: (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 human embryonic kidney 293 and human hepatoma 7 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.
Angptl4 Levels and CHD Traits
Baseline plasma Angptl4 levels varied greatly, with 92% of the study subjects displaying levels <20 ng/mL, an observation that is in line with other published data.16 Based on in vitro studies that have clearly established an inhibitory role of Angptl4 on LPL activity,7 we anticipated that plasma Angptl4 levels would show a positive correlation with TG levels. However, plasma Angplt4, although 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 those of the study by Robciuc et al16 and a small study of healthy controls (n=108), in which no correlation was seen between Angptl4 and TG levels.21 By using the same commercial ELISA,21 Stejskal et al22 reported a positive correlation between TG levels and Angptl4 in a study of patients with metabolic syndrome (n=115). However, they reported a negative correlation between HDL and Angptl4,22 which is in agreement with the result of the current study.
LPL-mediated hydrolysis of TG-rich lipoproteins primarily occurs on the endothelial cell surface where LPL is anchored. Heparin sulfate proteoglycans were thought to play a key role in this process; however, recently, the novel glycosylphosphatidylinositol-anchored HDL-binding protein was identified as an alternate anchor of LPL and chylomicrons.23 Glycosylphosphatidylinositol-anchored HDL-binding protein, required for the lipolytic processing of TG-rich lipoproteins, stabilizes LPL and prevents its inhibition by Angptl4.24 In a set of experiments on rat adipose tissue after feeding/fasting experiments, Sukonina et al7 observed an inverse relationship between Angptl4 expression and LPL activity, leading them to conclude that the change in extracellular LPL activity occurred in the subendothelial space or at the endothelium, where Angptl4 destabilized the active LPL dimer. Furthermore, Nilsson et al25 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 of an effect on LPL activity than tissue-bound Angptl4. Combined with our observations, these data strongly support the view that circulating Angptl4 does not inhibit endothelial-bound LPL; 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 ratio.16
ANGPTL4 Genotype and Angptl4 Levels
Our results show that T266M rare homozygotes have significantly higher Angptl4 levels than T266 carriers. This is in agreement with the in vitro studies that show that T266M protein is more readily secreted. However, T266M shows no association with TG levels under fasting conditions in healthy subjects10; as confirmed in our meta-analysis, T266M 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 which T266M resides is still not clear. Our in vitro data showed that E40K was expressed at lower levels compared with WT and T266M; consequently, less protein was secreted into the cell media. Yin et al8 reported less E40K in the media but comparable levels expressed in the cell. Researchers for both the current study and the study by Yin et al observed a reduction or nonexistence 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; we did not have sufficient statistical power to be confident of the association of Angptl4 levels with CHD risk in this subcohort. However, the meta-analysis in nearly 20 000 individuals showed no association of T226M with CHD risk, despite its strong association with Angptl4 levels. Although no correlation of TG 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 TGs.
In conclusion, ANGPTL4–T266M, but not E40K, was independently associated with Angptl4 levels, yet circulating Angptl4 levels showed no correlation with TG levels. The in vitro data provide a biological basis for the association of T266M with circulating Angplt4 levels, therefore giving new insights into the role of Angptl4 in TG metabolism.
We thank Helen Hobbs for kindly providing the pcDNA 3.1/human WT ANGPTL4 Angptl4 construct; the participants, general practitioners, and staff in NPHSII, European Prospective Investigation of Cancer–Norfolk, WHII, BWHHS, and British Heart Foundation (BHF) Family Heart Study. The BWHHS is coordinated by Shah Ebrahim (principal investigator), Debbie Lawlor, and Juan-Pablo Casas, with BHF cardiochip work performed by TRG (principal investigator), IND, DL, SE, George Davey Smith, Yoav Ben-Shlomo, and Santiago Rodriguez.
Sources of Funding
Smart-Halajko is supported by a Unilever/BBSRC Case studentship. This study was supported by grant RG2008/014 from the British Heart Foundation (BHF) (Drs Cooper, Humphries, and Talmud); Finska Lakaresallskapet; Magnus Ehnrooth; the National Institute for Health and Welfare of Finland (M Robciuc, Dr Jauhiainen and Dr Ehnholm); and grant R01HL036310 from the National Heart, Lung, and Blood Institute, US National Institutes of Health (NIH) (Dr Kumari). NPHSII was supported by the Medical Research Council (MRC) UK, grant NHLBI 33014 from the US NIH, and Du Pont Pharma. The European Prospective Investigation of Cancer–Norfolk was supported by MRC UK and Cancer Research UK, with additional support from the European Union, Stroke Association, grant PG2000/015 from the BHF, the Department of Health, the Food Standards Agency, and Wellcome Trust. WHII was supported by grants PG/07/133/24260, RG/08/008, SP/07/007/23671 from the BHF and was previously supported by grants from MRC; Health and Safety Executive, Department of Health; grant AG13196 from the National Institute on Aging, US NIH; grant HS06516 from the Agency for Health Care Policy Research; and the John D. and Catherine T. MacArthur Foundation Research Networks on Successful Midlife Development and Socio-economic Status and Health. The BWHHS was supported by funding from the BHF and the UK Department of Health Policy Research Programme, with cardiochip work funded by grant PG/07/131/24254 from the BHF.
M.C. Smart-Halajko and M. Robciuc contributed equally to this article.
Received on: April 8, 2010; final version accepted on: August 16, 2010.
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