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Journal of Lipid Research, Vol. 47, 2631-2639, December 2006 Human apolipoprotein A-II associates with triglyceride-rich lipoproteins in plasma and impairs their catabolism
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 505, Paris, F-75006 France; and Université Pierre et Marie Curie, Paris, F-75006 France Published, JLR Papers in Press, September 21, 2006.
1 To whom correspondence should be addressed. e-mail: athina.kalopissis-u505{at}bhdc.jussieu.fr
Postprandial hypertriglyceridemia and low plasma HDL levels, which are principal features of the metabolic syndrome, are displayed by transgenic mice expressing human apolipoprotein A-II (hapoA-II). In these mice, hypertriglyceridemia results from the inhibition of lipoprotein lipase and hepatic lipase activities by hapoA-II carried on VLDL. This study aimed to determine whether the association of hapoA-II with triglyceride-rich lipoproteins (TRLs) is sufficient to impair their catabolism. To measure plasma TRL residence time, intestinal TRL production was induced by a radioactive oral lipid bolus. Radioactive and total triglyceride (TG) were rapidly cleared in control mice but accumulated in plasma of transgenic mice, in relation to hapoA-II concentration. Similar plasma TG accumulations were measured in transgenic mice with or without endogenous apoA-II expression. HapoA-II (synthesized in liver) was detected in chylomicrons (produced by intestine). The association of hapoA-II with TRL in plasma was further confirmed by the absence of hapoA-II in chylomicrons and VLDL of transgenic mice injected with Triton WR 1339, which prevents apolipoprotein exchanges. We show that the association of hapoA-II with TRL occurs in the circulation and induces postprandial hypertriglyceridemia.
Supplementary key words postprandial hypertriglyceridemia transgenic mice apolipoprotein A-II knockout mice chylomicrons very low density lipoprotein plasma residence time Abbreviations: hapoA-II, human apolipoprotein A-II; KO, knockout; mapoA-II, murine apolipoprotein A-II; TG, triglyceride; TRL, triglyceride-rich lipoprotein
High plasma levels of triglyceride-rich lipoproteins (TRLs) are often associated with low HDL cholesterol concentrations, which are correlated with an increased risk of atherosclerosis (1). More recently, hypertriglyceridemia was also established as an independent risk factor for atherosclerosis, because decreasing plasma triglyceride (TG) without changing HDL and LDL cholesterol levels improved endothelial function (2). On the other hand, hypertriglyceridemia and low plasma HDL levels are features of the metabolic syndrome, a cluster of abnormalities comprising insulin resistance, hypertension, glucose intolerance, abdominal obesity, and the preponderance of small, dense LDLs (3, 4). The metabolic syndrome is diagnosed by the presence of any three of these abnormalities and confers an increased risk of cardiovascular disease.
Unexpectedly, we observed that several features of the metabolic syndrome were associated with moderate to high expression of human apolipoprotein A-II (hapoA-II) in transgenic mice generated in our laboratory. The transgenic lines
To establish whether the association of hapoA-II with TRL is sufficient to impair their catabolism and provoke hypertriglyceridemia, the present study aimed to i) determine the plasma residence time of TRL as a function of hapoA-II expression, and ii) establish whether hapoA-II is transferred to TRL in the plasma compartment. This point could not be assessed in our previous study (5), because both hapoA-II and VLDL originated from liver, the major source of VLDLs in the standard chow diet (carbohydrate-rich, with only 5% lipid) (9). Thus, hapoA-II may have associated with VLDLs either in the hepatocyte at some step of the secretory pathway or in the plasma compartment after secretion. Therefore, we dissociated in this study the sites of synthesis of hapoA-II and TRL by inducing chylomicron production after intragastric administration of sunflower oil mixed with [14C]triolein. To determine whether hapoA-II was carried by intestinal TRL in transgenic mice, we analyzed apolipoprotein compositions of chylomicrons and VLDLs of mice receiving the oral lipid load alone or together with Triton WR 1339, a nonionic detergent that coats the surface of lipoproteins and inhibits LPL activity and apolipoprotein exchanges (10, 11). In Triton-treated mice, the transfer of hapoA-II to TRL should be hindered. Finally, to ascertain that the metabolic effects studied were caused by hapoA-II only, we measured plasma TG concentrations after sunflower oil administration to
Animals The animals were housed in animal rooms with alternating 12 h periods of light (7 AM7 PM) and dark (7 PM7 AM). They were fed a chow diet (UAR) with free access to food and water. The generation of transgenic mice has been described previously (5). The transgenic lines and used in this study were backcrossed for at least eight generations to strain C57BL/6J, were hemizygous for the hapoA-II transgene, and were >8 weeks of age. Furthermore, and transgenic mice, backcrossed to C57BL/6J for at least 10 generations, were bred with mapoA-II-KO mice (12) to generate mice expressing hapoA-II only (subsequently called KOAII and KOAII, respectively). ApoA-II-KO mice had previously been backcrossed for 10 generations to strain C57BL/6J. Male and female transgenic mice were used in equal proportions in all studies. C57BL/6J male and female mice (Charles River) of the same age and maintained under the same nutritional conditions were used as controls. HapoA-II was measured in plasma from all animals by immunonephelometry using an antibody (Dade Behring) specific for hapoA-II and not recognizing mapoA-II. The procedures followed were in accordance with institutional guidelines.
Mice were identified by PCR analysis of tail DNA (Fig. 1
). The
In vivo clearance of plasma [14C]TG and total TG A kinetic study was performed with control and hapoA-II transgenic mice fed ad libitum. One control, one , and one transgenic mice were studied on any given day, between 9:30 AM and 6:00 PM, and six separate experiments were conducted. The production of chylomicrons was induced by intragastric administration of a bolus of 0.4 ml of sunflower oil mixed with [14C]triolein (New England Nuclear; specific activity 4.1440 GBq/mmol). Mice received 12 x 106 cpm of [14C]triolein. Six blood samples were taken from each mouse from the jugular vein into EDTA-containing tubes at 4°C: at 0 min (before lipid administration) and at 2, 3, 4, 5, and 8 h after the oral lipid load. In a separate experiment, four transgenic mice were studied for up to 24 h. Three blood samples were taken from each mouse at 0 min (before sunflower oil and triolein administration) and at 3 and 24 h after the lipid bolus. The mice were deprived of food after triolein administration. Plasma samples were extracted for lipids (14), and lipid classes were separated by TLC and counted. Total plasma TG was measured in duplicate with a commercial kit (Sigma Diagnostics 334-A).
TRL accumulation was also measured in
Production rate of chylomicrons and VLDLs after an oral lipid load
Effects of sunflower oil and Triton WR 1339 on apolipoprotein composition of TRL Three separate experiments were conducted with the six groups of mice (three genotypes + sunflower oil ± Triton). In each experiment, plasma was pooled from 12 control mice receiving sunflower oil only (having the lower chylomicron and VLDL concentrations in plasma) and from 8 mice from the other five groups. Plasma was supplemented with preservatives as described above and ultracentrifuged to isolate chylomicrons and VLDLs. After measuring protein content (15), chylomicrons and VLDLs were delipidated (16), and their apolipoprotein compositions were determined by SDS-PAGE using NuPAGE 412% Bis-Tris gels (Novex) (17). Electrophoresis was performed under nonreducing conditions to preserve the dimeric form of hapoA-II. The gels were stained with Coomassie Brilliant Blue (R250).
Statistical analysis
Effect of hapoA-II expression on plasma residence time of chylomicrons The transgenic and mice of this study expressed in the fed state hapoA-II at 50.1 ± 0.06 and 72.4 ± 0.11 mg/dl, respectively (usual concentration in humans, 2535 mg/dl). To minimize individual variability, TRL residence time was measured in the same mouse between 2 and 8 h after an oral lipid load consisting of sunflower oil mixed with [14C]triolein. Preliminary experiments established that [14C]TG appeared in plasma at 1.5 h after the oral lipid load. In all groups of mice, [14C]TG accounted for 87% of total radioactive plasma lipids. An important issue in this study was to distinguish between intestinal and hepatic TRL in plasma. Because intestinal and hepatic TRL mix immediately in the circulation upon secretion and both follow the same catabolic pathway initiated by hydrolysis of their core TGs by LPL, it is not possible to distinguish unlabeled intestinal from hepatic TRL in plasma. Besides, intestinal chylomicron production is induced by dietary lipid ingestion, whereas hepatic VLDL production is a constitutive pathway. Therefore, our strategy was to greatly enrich the proportion of intestinal TRL by administration of an oral 14C-labeled lipid bolus, the radioactivity allowing us to set the time of intestinal TRL secretion into the circulation, and to distinguish intestinal from hepatic TRL, at least during the initial time points.
Figure 2
shows the accumulation of [14C]TG and total TG in plasma between 0 and 8 h after triolein administration. In control mice, [14C]TG peaked at 3 h and decreased rapidly thereafter (Fig. 2A). In
Total plasma TG was substantially greater in transgenic compared with control mice at 0 time, before the lipid bolus (Fig. 2B). This is in accord with the hypertriglyceridemia displayed by hapoA-II transgenic mice in the fed state (5, 7). Similar to [14C]TG, total plasma TG increased more in transgenic compared with control mice after the oral lipid load. The pronounced and sustained TG accumulation in the circulation clearly indicated that the catabolism of chylomicrons was retarded in both transgenic mouse lines in relation to the expression level of hapoA-II.
To evaluate more accurately the plasma residence time of TRL in the high-expressing
To ascertain that the effects described above were attributable to hapoA-II without any confounding effects of endogenous mapoA-II, we measured TG accumulation in plasma of KOAII and KOAII mice gavaged with sunflower oil. Blood samples were taken from the same mouse at all time points studied, as above. Table 1
shows massive TG accumulation in KOAII and KOAII transgenic mice compared with controls that is related to the expression level of hapoA-II. Again, plasma TG of KOAII mice was maintained at peak levels even at 8 h after the oral lipid load. These results clearly show that hypertriglyceridemia after the oral lipid bolus was induced solely by hapoA-II.
TRL production is not altered in hapoA-II transgenic mice To validate our hypothesis that the hypertriglyceridemia of transgenic mice after the oral lipid load resulted from impaired TRL catabolism, we measured intestinal TRL production rate after injection of Triton WR 1339, which inhibits TRL-TG hydrolysis by LPL and allows in vivo estimation of TRL production (10, 11). Two hours after sunflower oil administration, total plasma TG was similar among Triton-treated and transgenic mice and controls (Table 2
). Chylomicron-TGs were two to three times greater than VLDL-TGs in all groups of mice, and both were comparable among transgenic and control mice. Thus, postprandial hypertriglyceridemia of transgenic mice resulted solely from impaired TRL catabolism.
Association of hapoA-II with TRL To assess whether hapoA-II is present in intestinal TRL, the apolipoprotein composition of chylomicrons and VLDLs was analyzed in control and transgenic mice 3 h after administration of either sunflower oil alone or both sunflower oil and Triton, which coats the surface of lipoproteins and impairs exchanges and catabolism by LPL (10, 11). The 3 h time point was chosen because it is the peak of TRL-[14C]TG in plasma and is reportedly the time point with the greatest enrichment of intestinal TRL (19). These experiments were performed with the original and transgenic lines as well as with the KOAII and KOAII lines, with identical results.
Three hours after sunflower oil administration, small amounts of chylomicrons and VLDLs were present in control mice, as expected from the rapid TG clearance shown in Fig. 2. Using a greater number of animals in this group (12 instead of 8), we analyzed VLDL apolipoproteins (Fig. 4B
), whereas chylomicron apolipoproteins were too low (data not shown). On the contrary, transgenic mice displayed appreciable amounts of chylomicrons and VLDLs, again in accord with the prolonged TG accumulation in plasma (Fig. 2). Both chylomicrons (Fig. 4A) and VLDLs (Fig. 4B) of
After sunflower oil and Triton administration, control mice displayed increased chylomicron and VLDL amounts, attributable to the inhibition of TRL catabolism by LPL. Thus, TRL of Triton-treated control and transgenic mice contained similar apolipoprotein amounts. Irrespective of genotype, chylomicrons and VLDLs essentially carried apoB-48 and little apoB-100, which is indicative of a great enrichment of intestinal TRL (Fig. 4A, B, respectively). The small apoB-100 amount of TRL corresponds to the presence of a small proportion of hepatic VLDL, because VLDL secretion is a constitutive pathway and cannot be stopped in vivo. Of note, the impairment of apolipoprotein exchanges by Triton resulted in the complete absence of hapoA-II in TRL. Interestingly, apoE and apoCs were also absent from TRL of animals treated with Triton for 3 h, implying that these apolipoproteins essentially associate with TRL in the plasma compartment. We verified that VLDL prepared from chow-fed or overnight-fasted
Together, these observations strongly suggest that TRLs of transgenic mice acquire hapoA-II after their secretion in the plasma compartment. The presence of hapoA-II in chylomicrons correlates with the hypertriglyceridemia measured after the oral lipid load and thus may account for the impaired hydrolysis of their TGs by LPL.
This study focused on the mechanisms of hapoA-II association with TRL and the effects on TRL catabolism. We show that TRLs produced after an oral lipid load accumulated in the plasma of hapoA-II transgenic mice but were rapidly cleared from the circulation of control mice. In transgenic mice, chylomicrons (produced by the intestine) carried hapoA-II (produced by the liver), strongly suggesting apoA-II transfer to TRL in the circulation. The absence of hapoA-II in TRL of transgenic mice injected with Triton, which coats the surface of lipoproteins, thereby hindering exchanges (11), confirmed that hapoA-II associates with TRL in plasma. Together, these results demonstrate that association of hapoA-II with TRLs in the circulation impairs their catabolism and induces postprandial hypertriglyceridemia, the extent and duration of which depend on the expression level of hapoA-II. Any confounding effects of endogenous mapoA-II were excluded by the use of hapoA-II transgenic mice deficient in mapoA-II.
The main objective of this study was to investigate whether the association of hapoA-II with TRLs is sufficient to impair their catabolism and induce hypertriglyceridemia. Because apoA-II is synthesized in liver, we sought to determine whether apoA-II would associate with intestinal TRLs and impair their catabolism. Thus, in this study, mice received a radioactive oral lipid bolus that served the dual purpose to set the time of food intake and to help differentiate intestinal from hepatic TRLs. The questions now arise of up to which time point do plasma [14C]TGs originate from intestine, and from which time point onward do they also originate from liver? Indeed, part of the fatty acids resulting from TRL-TG hydrolysis are taken up by the liver, esterified, and secreted as VLDL-TG. Secretion of exogenous fatty acids can account for 30% of VLDL-TG secreted by hepatocytes (18). A recent study in humans showed that [1-13C]palmitic acid included in a mixed meal started to appear in chylomicron remnants at 1.5 h after the meal and peaked after 3 h, whereas there was at least a 1.5 h delay before the label appeared in the VLDL-TG pool (19). Thus, it is reasonable to assume that in our study, plasma TG is essentially of intestinal origin at 3 h, at least in control mice. Plasma TG clearance is greatly decreased in transgenic
An important question is whether plasma TG accumulation resulted solely from the association of hapoA-II with TRL or whether this effect was also attributable to endogenous mapoA-II. Both hapoA-II and mapoA-II expression in transgenic mice induces hypertriglyceridemia, which is more pronounced in hapoA-II compared with mapoA-II transgenic mice (5, 7, 13, 20). However, hapoA-II expression decreases plasma HDLs that have smaller particle sizes and very low apoA-I contents (5, 7, 13), whereas high mapoA-II expression results in higher concentrations of large HDLs rich in apoA-I and mapoA-II (20). To answer this question, we used
What is the mechanism underlying the association of hapoA-II, a major HDL apolipoprotein, with TRL? The first possibility is that hapoA-II, synthesized mainly in the liver (5, 13), associates with nascent VLDL in the hepatocyte and is secreted in the circulation on VLDL. The second possibility is that hapoA-II is secreted by hepatocytes as a lipid-poor apolipoprotein and associates partly with TRL in the plasma compartment. In our first study, Because the intestinal TRL production rate was comparable among transgenic and control mice, it follows that hapoA-II expression only affected TRL catabolism. Coupled with the observation that TRL carried hapoA-II, this strongly suggests that hapoA-II on the surface of TRL inhibits LPL activity, probably by hindering LPL access to the lipoprotein surface. Indeed, addition of hapoA-II to postheparin plasma of control mice inhibited LPL and HL activities in a dose-dependent manner, and VLDL-TG from hapoA-II transgenic mice were hydrolyzed less efficiently by LPL compared with VLDL from control mice (5). Interestingly, in the course of a paradoxical hypertriglyceridemia obtained by fenofibrate treatment of independently established high-expressing hapoA-II transgenic mice, a similar defect in catabolism by LPL of hapoA-II-carrying VLDLs was reported (22). The apoA-II-KO mouse fits, in an inverse manner, our hypertriglyceridemic mice expressing hapoA-II. When apoA-II-KO mice were crossed with apoE-KO mice, which display dramatic accumulation of cholesterol-rich remnants, VLDL clearance was greatly increased (12). Thus, when apoA-II was absent, both LPL and HL apparently functioned at maximal activities. An important issue is whether apoA-II influences plasma TG levels in humans. The common 265T/C polymorphism in the promoter region of the hapoA-II gene was shown to be associated with a small decrease in plasma apoA-II in human carriers of the 265C/C allele compared with the 265T/T allele (23). The 265C allele reduced the basal rate of transcription in transfection studies compared with the 265T allele. Moreover, oral fat tolerance tests provided evidence that subjects homozygous for the 265C allele have faster clearance of postprandial large VLDLs compared with subjects homozygous for the 265T allele (23). Thus, small variations in plasma apoA-II levels are sufficient to influence postprandial triglyceridemia. Besides, variation of apoA-II plasma levels was associated with plasma TG in familial combined hyperlipidemia (24), whereas VLDL from patients with Tangier disease or type V dyslipidemia who carry apoA-II was less efficiently catabolized by LPL (8). What is the physiological relevance of the presence of apoA-II in TRL? A careful review of the literature reveals that apoA-II is partly/totally transported by TRL in metabolic situations with low/extremely low plasma HDL concentrations, such as i) Tangier disease and type V hyperlipoproteinemia patients, their VLDL carrying apoA-II (8), and ii) LPL-KO mice rescued at birth by transient adenovirus-mediated expression of LPL in liver. Adult LPL-KO mice practically lack HDL, and both apoA-II and apoA-I are present in TRL (25). We propose that a low number of HDL particles may not accommodate all newly secreted, lipid-poor apoA-II, so that part of apoA-II associates with TRL. The resulting impairment of TRL catabolism may participate in the increased cardiovascular risk conferred by low plasma HDL. Indeed, pronounced postprandial lipemia after a fatty meal impaired endothelium-dependent dilation of the brachial artery in men, which represents a surrogate functional marker for early atherosclerotic disease (26). Conversely, vascular smooth muscle function significantly improved after decreasing plasma TG by fenofibrate treatment, without changes in confounding lipoproteins or insulin resistance (2). Further studies are needed to establish the effects of hapoA-II-induced postprandial hypertriglyceridemia in vascular endothelial cell metabolism. In conclusion, a moderate overexpression of hapoA-II in mice led to postprandial accumulation of intestinal TRL for several hours, in a manner comparable to the accumulation of hepatic VLDL we reported previously (5, 7). The transfer of hapoA-II on the surface of TRL in the plasma compartment is probably the main mechanism underlying defective TRL catabolism by LPL and may be linked to low plasma HDL content (5, 7). The impaired LPL activity in turn results in decreased availability of TRL surface components for HDL formation, thus contributing to lower plasma HDL concentrations. Thus, we have identified in hapoA-II transgenic mice a novel mechanism of postprandial hypertriglyceridemia that is related to low plasma HDL levels.
The authors express their gratitude to Dr. J. L. Breslow for the generous gift of apoA-II-KO mice. The authors acknowledge the financial support of the Institut National de la Santé et de la Recherche Médicale. The authors thank Carole Lasne and Maryse Séau for excellent technical assistance. S.D-P. and X.R. are recipients of doctoral fellowships from the Ministère de la Recherche et Technologie (France). Manuscript received March 7, 2006 and in revised form September 12, 2006.
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