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Am J Physiol Endocrinol Metab 290: E463-E470, 2006. First published October 11, 2005; doi:10.1152/ajpendo.00593.2004
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Glycosylphosphatidylinositol-specific phospholipase D influences triglyceride-rich lipoprotein metabolism

Nandita S. Raikwar,1 Won Kyoo Cho,1,3,4 Rosario F. Bowen,1 and Mark A. Deeg1,2,4

1Departments of Medicine, 2Biochemistry and Molecular Biology, and 3Cellular and Integrative Physiology, Indiana University School of Medicine; and 4Department of Veterans Affairs, Indianapolis, Indiana

Submitted 15 December 2004 ; accepted in final form 4 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a minor HDL-associated protein. Because many minor HDL-associated proteins exchange between different lipoprotein classes during the postprandial state and are also involved in triglyceride (TG) metabolism, we hypothesized that GPI-PLD may play a role in the metabolism of TG-rich lipoproteins. To test this hypothesis, we examined the distribution of GPI-PLD among lipoprotein classes during a fat tolerance test in C57BL/6 and LDL receptor-deficient (LDLR–/–) mice fed either a chow or high-fructose diet. In the fasting state in wild-type mice fed a chow diet, GPI-PLD was only present in HDL, whereas in LDLR–/– mice GPI-PLD was present in HDL and intermediate-density lipoproteins (IDL)/LDL. During the fat tolerance test, there was no change in total serum GPI-PLD levels in either model; however, a significant amount of GPI-PLD appeared in both VLDL (0.5–1% of total GPI-PLD) and IDL/LDL (5–10% of total GPI-PLD) in both models. The high-fructose diet increased both fasting and postprandial TG and serum GPI-PLD levels in both strains as well as the amount of GPI-PLD in VLDL. To determine whether GPI-PLD plays a direct role in TG metabolism, we increased liver GPI-PLD expression in C57BL/6 mice by adenovirus-mediated gene transfer, which resulted in a sevenfold increase in serum GPI-PLD levels. This change was associated with an increase in fasting (30%) and postprandial TG (50%) and a twofold reduction in TG-rich lipoprotein catabolism compared with saline or control adenovirus-treated mice. These studies demonstrate that GPI-PLD affects serum TG levels by altering catabolism of TG-rich lipoproteins.

cholesterol; triglycerides; postprandial

ALTERED METABOLISM of triglyceride-rich lipoproteins [very-low-density lipoproteins (VLDL) and chylomicrons] leads to elevated fasting and/or postprandial triglycerides and is associated with an increased risk of coronary artery disease, particularly in women and patients with diabetes (6, 14). High levels of triglycerides in the postprandial state are not only associated with coronary artery disease but are also a more sensitive indicator of coronary artery disease risk compared with fasting triglycerides (16, 27, 36). Many minor high-density lipoprotein (HDL)-associated proteins are important for regulating the metabolism of triglyceride-rich lipoproteins, particularly during the postprandial state (31). These proteins typically reside on HDL in the fasting state and exchange onto triglyceride-rich lipoproteins during the postprandial state. These exchangeable HDL proteins include apolipoproteins A-IV, C-II, C-III, and E. We (9) and others (18) have described another minor HDL-associated protein, glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD).

GPI-PLD is an 814–816 amino acid protein present in minor quantities in various cell types and tissues and is most abundant in serum (8, 24). The highest level of GPI-PLD mRNA expression occurs in the liver (12, 22, 25, 26, 29, 33, 34). Because nearly all of the GPI-PLD in serum forms a small, 8-nm complex with apolipoproteins A-I and A-IV (9), GPI-PLD may be classified as an HDL protein. We have also observed that GPI-PLD associates with triglyceride-rich lipoproteins under various dietary, pathological (e.g., diabetes), or genetic conditions that alter lipoprotein metabolism (M. A. Deeg and R. F. Bowen, unpublished observations). However, its function is still unclear. Higher levels of serum GPI-PLD are associated with higher levels of serum triglycerides and insulin resistance in humans (21), but this observation does not provide direct evidence for a role of GPI-PLD in lipoprotein metabolism. To determine whether GPI-PLD is involved in triglyceride-rich lipoprotein metabolism, we studied the postprandial changes in serum GPI-PLD levels and its distribution among lipoprotein classes. In addition, we examined the effect of increasing hepatic expression and serum levels of GPI-PLD by use of adenovirus-mediated gene transfer on postprandial triglyceride levels. We found that during the postprandial state GPI-PLD was present in triglyceride-rich lipoproteins. In addition, increasing hepatic expression and serum levels of GPI-PLD resulted in an increase in fasting and postprandial triglycerides in association with a delay in catabolism of triglyceride-rich lipoproteins. These results suggest that GPI-PLD plays a role in triglyceride metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. C57BL/6 male mice (8 wk of age) were purchased from Harlan (Indianapolis, IN). Low-density lipoprotein receptor-deficient (LDLR–/–) male mice (8 wk of age), on a C57BL/6 genetic background, were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained in a temperature-controlled (25°C) atmosphere on a 12:12-h light-dark cycle with free access to food and water. Animals were fed specific diets for the times indicated in the table or figure legends. All diets used in this study were pellets and included a chow diet (protein 20%, fat 9%, and carbohydrates 71% of total kcal, Mouse Chow 20; Purina Mills, St. Louis, MO) and a high-fructose diet (protein 20%, fat 13%, fructose 60%, and complex carbohydrates 7% of total kcal, TD96130; Harlan-Teklad, Madison, WI). All animal studies were approved by the Institutional Animal Care and Use Committee of Indiana University.

Generation of adenoviral vectors expressing GPI-PLD or beta-galactosidase. The murine full-length cDNA of GPI-PLD isolated from a pancreatic {alpha}-cell library (22) was modified by removing the 5'- and 3'-untranslated regions and adding the Kozak consensus sequence upstream of the ATG initiation codon to enhance translation. The expression cassettes, comprising murine pancreatic GPI-PLD [CMV-GPI-PLD-poly(A)] or beta-galactosidase [CMV-LacZ-poly(A)], under the transcriptional control of a constitutively active CMV promoter and an SV40 polyadenylation signal (excised from pCMVbeta; Clontech, Palo Alto, CA), were subcloned into an adenoviral transfer vector p{Delta}E1sp1A (Microbix Biosystems, Toronto, ON, Canada). Recombinant replication-deficient adenoviral vectors AdGPI-PLD and AdLacZ were generated by cotransfecting the respective adenoviral transfer vectors along with the adenoviral vector pJM17 (Microbix Biosystems) in low-passage human embryonic kidney 293 cells (kindly provided by Dr. Chinghai Kao, Indiana University, Indianapolis, IN). The viral plaques were rescued on day 14 following homologous recombination between the two plasmids. The plaques were subsequently subjected to three rounds of plaque purification and were amplified in 293 cells. The virus was purified by two rounds of cesium chloride gradient ultracentrifugation followed by extensive dialysis. The viral titers were determined by plaque assay. Expression of GPI-PLD by AdGPI-PLD was confirmed by transducing Hep G2 cells in vitro and observing an increase in media GPI-PLD activity (data not shown).

The time course of adenoviral-mediated overexpression of GPI-PLD on serum GPI-PLD activity was examined in mice. C57BL/6 mice were administered saline, AdLacZ (109 pfu) or AdGPI-PLD (108 or 109 pfu; n = 2 for each condition) via tail vein. A nonfasting serum GPI-PLD activity was determined every morning for 7 days and then every week or two for an additional 7 wk. AdGPI-PLD (109 pfu) increased serum GPI-PLD activity within 24 h and the peak serum GPI-PLD activity occurred between days 7 and 14 after infection (Fig. 1). Serum GPI-PLD levels were still elevated at 8 wk. The tissue distribution of the adenovirus infections was determined by injecting C57BL/6 mice with saline, AdLacZ, or AdGPI-PLD (n = 2 for each condition) as described above. After 7 days, total DNA was isolated from liver, kidney, lung, pancreas, spleen, small intestine, aorta, and heart by use of the DNeasy Tissue Kit (Qiagen, Valencia, CA). The following primers, designed using Primer Express software (Applied Biosystems, Foster City, CA), were used to detect the adenoviral genome: forward primer 5'-CACCACCTCCCGTACCATA-3', reverse primer 5'-CCGCACCTGGTTTTGCTT-3'. The PCR reaction was done in 25 µl containing 10 mM Tris·HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 nM each of dCTP, dGTP, dATP, and dTTP, 200 nM primers and 1 U of Taq DNA polymerase. Cycling conditions were as follows: 1 cycle of 5 min at 94°C for initial denaturation followed by 30 cycles of 1 min denaturation at 94°C, 1 min annealing, and 1 min extension at 72°C. The expected product was 500 bp and was verified by agarose gel electrophoresis (not shown). Adenoviral genome was identified in all tissues with ~50-fold higher levels in liver compared with the other tissues (data not shown). Distribution of virus within the liver was determined by removing the liver and staining sections with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. Staining was observed in all hepatocytes for AdLacZ-infected mice (data not shown).


Figure 1
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  		Fig. 1. Time course of overexpressing glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) on serum GPI-PLD activity. C57BL/6 mice were treated with saline (bullet), or infected with AdLacZ [109 pfu ({triangleup})] or AdGPI-PLD [108 ({blacktriangledown})or 109 pfu ({circ})], and nonfasting morning serum was collected for determining GPI-PLD activity as described in MATERIALS AND METHODS. Results are the average of 2 mice.

 
Adenoviral infection of the liver was confirmed by measuring serum alanine transferase activity 7 days after infection. Serum alanine transferase activity was 72 ± 30 (n = 8) in control, 285 ± 190 (n = 13) in AdLacZ-treated, and 468 ± 320 (n = 17) in AdGPI-PLD-treated mice (P < 0.05 for AdLacZ or AdGPI-PLD vs. control, 1-way ANOVA).

The effect of overexpressing GPI-PLD on liver cholesterol and triglyceride content was determined by injecting mice with saline, AdLacZ, or AdGPI-PLD as described above. After 8 days, the mice were fasted for 4 h, and hepatic cholesterol and triglycerides were determined as previously described (13).

Fat tolerance test. Mice were fed either a normal chow diet or a high-fructose diet for 2 wk. On the morning of the fat tolerance test, mice were fasted for 4 h and then given 0.25 ml of corn oil (Mazola) via gavage. Blood and liver samples were obtained at 0, 1, 2, 3, and 4 h after gavage, and serum triglycerides and GPI-PLD mass were determined. The distribution of GPI-PLD among lipoproteins was determined by separating lipoproteins by gel filtration (Superose 6; Amersham Bioscience, Piscataway, NJ) as previously described (11). Fractions (0.5 ml) were collected and assayed for cholesterol and GPI-PLD mass by Western blotting, as described below. The amount of GPI-PLD in each fraction was estimated as (%total GPI-PLD mass in respective fraction) x (total serum GPI-PLD mass). Total RNA from isolated liver was extracted with TriPure (Roche Diagnostics, Indianapolis, IN) followed by further purification with an Absolutely RNA RT-PCR Miniprep kit (Stratagene, La Jolla, CA). GPI-PLD mRNA was quantitated by real-time PCR as described below.

The catabolism of triglyceride-rich lipoproteins was followed by incorporating 0.5 MBq of [11,12-3H]retinol (American Radiolabeled Chemicals, St. Louis, MO) in the corn oil, and [3H]retinol in serum was measured by liquid scintillation for each sample.

Liver perfusion studies. C57BL/6 and LDLR–/– mice were fed a high-fructose diet as described above, fasted for 4 h, and then administered 0.25 ml of corn oil as described above. After 1 h, mice were anesthetized with pentobarbital sodium (50 mg/kg body wt), and the liver was perfused in situ as previously described (3, 7). The liver was perfused in a nonrecirculating, single-pass mode, and the core body temperature of the mice was maintained with a heater blanket and heat from a light bulb. The liver was perfused for 90 min with RPMI 1640 (BioWhitaker, Rockland, ME) containing 1 mM oleic acid, 0.43 mM fatty acid-free BSA, 1 mM vitamin C, and 5 mM glutathione at 0.9 ml/min at 37°C. The perfusate during the initial 10-min stabilization period was discarded. The perfusate from 10 to 90 min was collected and concentrated to ~0.5 ml with a Centriprep 50 ultrafiltration device (Amicon, Beverly, MA). Lipoproteins were fractionated by gel filtration (Superose 6) and fractions (0.5 ml) assayed for cholesterol and GPI-PLD activity and mass.

VLDL synthesis. The rate of VLDL synthesis was estimated by measuring the accumulation of plasma triglyceride following injection of Triton WR-1339 (Sigma Chemical, St. Louis, MO), a detergent that blocks the hepatic and peripheral clearance of triglyceride-rich lipoproteins. Mice were injected with AdGPI-PLD, AdLacZ, or saline, as described above, and maintained on a chow diet. On day 7 after the virus injection, mice were fasted for 4 h followed by Triton WR-1339 (500 mg/kg as 15 g/dl in 0.9% saline) infusion via tail vein. Blood samples were collected from tail veins for measurement of triglycerides at 0, 30, 60, and 90 min. Triglyceride accumulation rates were calculated using the following formula: triglyceride synthetic rate (mg/min) = 1/3[(TG30 – TG0)/30 + (TG60 – TG0)/60 + (TG90 – TG0)/90] x plasma volume, where TG0, TG30, TG60, and TG90 are triglyceride concentrations at 0, 30, 60, and 90 min, respectively. The plasma volume was estimated as 5.77% of body weight (23).

Real-time quantitative PCR. Taqman quantitative real-time (qRT)-PCR was used for the quantitation of GPI-PLD mRNA. Primers and probes were designed using the Primer Express software to function according to the Taqman technology. GPI-PLD specific primers and probes used for qRT-PCR were as follows: forward 5'-TGGAGAACGGGACCAGTGA-3' (corresponding to +793 to +812), reverse 5'-ACTGAGGGTGTGGTTCCTGC-3' (corresponding to +872 to +853), and probe 5'-FAM-CAACCTGCCTGAGAACCCCCTGTTC-TAMRA-3' and were synthesized by Integrated DNA Technologies (Coralville, IA). The expected amplicon was 80 bp, and the PCR product was verified by agarose gel electrophoresis (data not shown). The qRT-PCR reactions were performed in Stratagene’s Mx4000 Multiplex Quantitative PCR System using a Brilliant Plus Single-Step Quantitative RT-PCR Core Reagent Kit in a total reaction volume of 25 µl. The reaction contained 5 U of StrataScript reverse transcriptase, 1.25 U of SureStart Taq DNA polymerase, 0.2 mM GAUC mix, 2 mM MgCl2, 300 nM forward primer, 500 nM reverse primer, and 400 nM of probe for GPI-PLD. Cycling conditions were as follows: 1 cycle for 90 min at 45°C, 1 cycle for 10 min at 95°C followed by 40 cycles of 15 s denaturation at 95°C, and a 1-min annealing/extension at 55°C. Standard curves were generated for GPI-PLD from serial dilutions of mouse liver total RNA (Stratagene). Unknown sample values were determined from the standard curve, and data were expressed as arbitrary units and normalized to total RNA as determined by RiboGreen assay (Molecular Probes, Eugene, OR).

Analytic assays. GPI-PLD activity in serum and Superose 6 column fractions was determined using the membrane form of the variant surface glycoprotein radiolabeled with [3H]myristate as the substrate (10). One unit of GPI-PLD activity was defined as the amount of enzyme that cleaves 1% of the substrate in 1 min. GPI-PLD mass was quantitated by Western blotting with anti-GPI-PLD771, using purified mouse serum GPI-PLD as the standard (10). Cholesterol, triglycerides, and alanine transferase assays were performed by the Indiana University Endocrinology Analyte Core using commercially available kits (Sigma). Apolipoprotein A-I mass was determined by Western blotting and normalized to serum obtained from a fasting C57BL/6 mouse maintained on a chow diet and expressed as fold of control (11).

Statistical analysis. All values are presented as means ± SE (unless indicated otherwise). Multiple-group comparisons were done by one-way or two-way ANOVA, as indicated in the figure legends, followed by multiple pairwise comparisons (Tukey’s test). Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Postprandial changes in GPI-PLD serum levels and hepatic mRNA. To determine whether GPI-PLD is an exchangeable apolipoprotein, we examined the changes in serum GPI-PLD levels and distribution of GPI-PLD among lipoprotein classes during the postprandial state when numerous proteins and lipids exchange between HDL and triglyceride-rich lipoproteins. Because VLDL levels are low in mice, we used two strategies to increase VLDL. First, we compared chow-fed C57BL/6 and LDLR–/– mice during the postprandial state. VLDL catabolism is delayed in LDLR–/– mice. Second, we compared the postprandial changes in both strains of mice fed either a chow or a high-fructose diet. High-fructose diets increase hepatic production and decrease clearance of VLDL (17, 30, 35).

On a chow diet, LDLR–/– mice had higher levels of fasting cholesterol and triglycerides compared with wild-type mice (Table 1). The fructose diet increased cholesterol and triglycerides in both strains approximately twofold (Table 1), which was reflected in both strains by an increase in VLDL and LDL (data not shown). Serum levels of GPI-PLD in C57BL/6 mice on a chow diet were 468 ± 99 µg/ml or 4.2 µM, which is similar to the concentration of apolipoprotein C-III of 5.6 µM in mice (15). The fructose diet increased serum levels of GPI-PLD in C57BL/6 mice (Table 1). Apolipoprotein A-I and GPI-PLD levels did not differ between strains (Table 1).


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  		Table 1. Effect of diet on lipid parameters in wild-type and LDLR–/– mice

 
During the fat tolerance test in wild-type mice on a chow diet, serum triglycerides increased approximately twofold with a peak at 2 h and returned to baseline by 4 h (Fig. 2A). LDLR–/– on a chow diet showed a similar change in serum triglycerides with an approximately twofold increase at 1 h but did not return to baseline at 4 h (Fig. 2B). This is consistent with delayed VLDL catabolism in these mice (20). There was no statistically significant change in total serum GPI-PLD during the fat tolerance test in wild-type mice and LDLR–/– mice fed a chow diet (Fig. 2, C and D).


Figure 2
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  		Fig. 2. Effect of diet on postprandial triglycerides, GPI-PLD serum levels, and hepatic mRNA in C57BL/6 and LDL receptor-deficient (LDLR–/–) mice. C57BL/6 (A, C, and E) and LDLR–/– (B, D, and F) were fed a chow or high-fructose diet for 2 wk. After a 4-h fast, mice were fed 0.25 ml of corn oil via gavage and serum triglycerides (A and B), and GPI-PLD mass (C and D) were determined from 0 to 4 h. A 2nd experiment was performed to quantitate liver GPI-PLD mRNA (E and F) as described in MATERIALS AND METHODS. Results are expressed as means ± SE; n = 3- 5 at each time point. *P < 0.05 vs. 0 h, {dagger}P < 0.05 vs. 4 h, §P < 0.05 vs. chow by 2-way ANOVA.

 
Fructose feeding significantly altered the postprandial response for serum triglycerides and GPI-PLD. In wild-type mice, fructose feeding was associated with higher fasting (Table 1) and peak serum triglycerides (Fig. 2A) during the fat tolerance test. Overall, serum GPI-PLD was significantly higher in the fructose group compared with chow-fed wild-type mice (P < 0.005) but changed little during the fat tolerance test (Fig. 2C). In LDLR–/– mice, fructose feeding was associated with a continuous increase in serum triglycerides during the fat tolerance test (Fig. 2B). In contrast, serum GPI-PLD increased and peaked at 1 h followed by a return to baseline at 4 h (Fig. 2D). The liver GPI-PLD mRNA did not change during the fat tolerance test (Fig. 2, E and F) in either strain on either diet.

Postprandial distribution of GPI-PLD among lipoprotein classes. To determine the distribution of GPI-PLD during the fat tolerance test, lipoproteins were separated by gel filtration, and the GPI-PLD content was determined in each lipoprotein class by Western blotting. Greater than 98% of GPI-PLD associates with lipoproteins, and its distribution among lipoproteins does not differ between plasma and serum (data not shown). Under fasting conditions, >95% of serum GPI-PLD elutes slightly earlier (fractions 24–32; Figs. 3A and 4B) than the bulk of HDL cholesterol (fractions 26–32; Fig. 4A). In both strains and diets, the GPI-PLD content was greatest in HDL at all time points during the fat tolerance test (Fig. 3). The GPI-PLD content of HDL paralleled the changes in total serum GPI-PLD (compare Fig. 2, C and D, with 3B). Although the majority of GPI-PLD remained with HDL, GPI-PLD appeared in triglyceride-rich lipoproteins (fractions 14–17) and IDL/LDL (fractions 18–22) during the fat tolerance test (Fig. 3A).


Figure 3
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  		Fig. 3. Distribution of serum GPI-PLD among lipoprotein classes during a fat tolerance test. Serum from all animals at each time point in Fig. 2 was pooled, and lipoproteins were fractionated by gel filtration as described in MATERIALS AND METHODS. Fractions were assayed for cholesterol and GPI-PLD mass. VLDL/chylomicrons are present in fractions 14–17, intermediate-density lipoproteins (IDL) and LDL in fractions 18–25, and HDL in fractions 26–32, as indicated. A: GPI-PLD mass by Western blotting in gel filtration fractions from LDLR–/– mice at time 0 and 1 h after the corn oil gavage. B: distribution of GPI-PLD among lipoprotein fractions for each strain consuming a chow or high-fructose diet during the fat tolerance test.

 

Figure 4
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  		Fig. 4. Comparison of GPI-PLD complexes by gel filtration from serum and liver perfusate. Lipoproteins from serum from a fasting C57BL/6 mouse and liver perfusate from an LDLR–/– and C57BL/6 (not shown) mouse 1 h after gavage were separated by gel filtration. Fractions were assayed for cholesterol (A) and GPI-PLD activity (B) as described in MATERIALS AND METHODS. GPI-PLD mass in the FPLC fractions was below the level of detection by Western blotting (not shown). Results were similar for liver perfusate from C57BL/6 and LDLR–/– mice. The positions VLDL, LDL, HDL, and pre-beta-HDL are indicated in A.

 
In wild-type mice on a chow diet, there was no significant change in the GPI-PLD content of HDL during the fat tolerance test (Fig. 3B). There was no detectable GPI-PLD comigrating with triglyceride-rich lipoproteins or IDL/LDL under fasting conditions. However, GPI-PLD was detected in triglyceride-rich lipoproteins at 1 h and was absent by 2 h. GPI-PLD was also detected with IDL/LDL at 2 h and continued to increase at 4 h. The fructose diet exacerbated the increase of GPI-PLD in the triglyceride-rich lipoproteins fractions (Fig. 3B). In the IDL/LDL fraction, GPI-PLD was detectable even under fasting conditions and increased over twofold after 4 h.

In LDLR–/– mice on a chow diet, there was an approximately 30% increase in the HDL content of GPI-PLD at 1 h that returned to baseline by 4 h (Fig. 3B). Although GPI-PLD was not detected in triglyceride-rich lipoproteins during fasting, a detectable amount of GPI-PLD in triglyceride-rich lipoproteins was present at 1 and 2 h and returned to baseline. Interestingly, GPI-PLD was detected in LDL during fasting (Fig. 3, A and B) and increased threefold by 2 h and returned to baseline at 4 h. Compared with a chow diet, the fructose diet in LDLR–/– mice increased the GPI-PLD content of HDL and triglyceride-rich lipoproteins during the fat tolerance test. In contrast, there was no change in the GPI-PLD content of IDL/LDL (Fig. 3B).

Triglyceride-rich lipoprotein-associated GPI-PLD could be derived from the small intestine and/or be secreted with VLDL from the liver. The former likely makes only a minor contribution to serum GPI-PLD, as the GPI-PLD mRNA in small intestine, as measured by qRT-PCR, is ~30-fold lower than in the liver (data not shown). To determine whether GPI-PLD is secreted from liver in association with VLDL, we identified GPI-PLD complexes secreted from perfused liver. LDLR–/– mice were fed a fructose diet, and a fat tolerance test was performed as described above. After 1 h, the liver was perfused in situ. The liver perfusate contained VLDL and a cholesterol particle smaller than plasma HDL (Fig. 4A). This complex likely represents a nascent or pre-beta-HDL. GPI-PLD complexes were identified in the perfusate but were smaller than serum GPI-PLD and the bulk of HDL cholesterol (Fig. 4B). No GPI-PLD was detected in the VLDL region.

Overexpressing hepatic GPI-PLD alters serum triglycerides. To determine whether GPI-PLD plays a direct role in triglyceride-rich lipoprotein metabolism, we utilized an adenovirus to overexpress hepatic GPI-PLD and increase serum GPI-PLD levels. C57BL/6 mice were treated with either saline control adenovirus expressing beta-galactosidase (AdLacZ) or GPI-PLD (AdGPI-PLD). AdLacZ infection had no effect on fasting triglycerides or serum GPI-PLD levels compared with saline-treated animals. Infecting animals with AdGPI-PLD increased fasting serum GPI-PLD levels sevenfold, which is similar in magnitude to the increased serum GPI-PLD observed in diabetic vs. nondiabetic NOD mice (5-fold) (11). This increase in serum GPI-PLD was associated with a significant increase (30%) in fasting triglycerides [75 ± 11 mg/dl (n = 10) for control, 85 ± 14 (n = 17) for AdLacZ-treated, and 110 ± 23 (n = 19) for AdGPI-PLD-treated mice, means ± SD, P < 0.001 for AdGPI-PLD vs. control or AdLacZ by 1-way ANOVA]. In contrast, the fasting cholesterol did not differ between treatments [112 ± 11 mg/dl (n = 7) for control, 110 ± 22 (n = 8) for AdLacZ-treated, and 134 ± 35 (n = 9) for AdGPI-PLD-treated mice, means ± SD, P = 0.09 by 1-way ANOVA]. There was no difference in liver content of triglycerides [10.5 ± 4.4 (n = 4), 10.2 ± 2.4 (n = 10), and 9.2 ± 3.3 mg/g wet wt (n = 12), means ± SD] or cholesterol [1.3 ± 0.4 (n = 4), 1.3 ± 0.4 (n = 10), and 1.3 ± 0.7 mg/g wet wt (n = 12)] between control, AdLacZ, or AdGPI-PLD, respectively, suggesting that overexpressing GPI-PLD does not alter intrahepatic triglyceride levels. These results demonstrate that overexpressing hepatic GPI-PLD and increasing serum levels of GPI-PLD is associated with an increase in fasting triglycerides.

To determine the effect of increased hepatic expression and serum levels of GPI-PLD on postprandial triglycerides, mice were treated with saline, AdLacZ, or AdGPI-PLD, and a fat tolerance test was performed 7 days after infection. The postprandial triglycerides were similar between control and AdLacZ-infected animals (Fig. 5A). In control animals, serum triglycerides peaked at 1 h and returned to baseline by 3 h. In the AdGPI-PLD-infected animals, fasting triglycerides were significantly elevated, peaked at 2 h, and returned toward baseline by 3–4 h. The triglyceride area under the curve for AdGPI-PLD-infected mice was significantly higher than control or AdLacZ-infected animals [561 ± 170 (n = 11) vs. 345 ± 80 (n = 6) and 411 ± 70 (n = 9), respectively, P < 0.05 vs. control or AdLacZ by one-way ANOVA].


Figure 5
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  		Fig. 5. Effect of overexpression of hepatic GPI-PLD on postprandial triglycerides and remnant accumulation. C57BL/6 mice were treated with saline (n = 6) or infected with AdLacZ (n = 9) or AdGPI-PLD (n = 11) as described in MATERIALS AND METHODS. Seven days after infection, mice were fasted for 4 h and then given 0.25 ml of corn containing [3H]retinol via gavage, and serum triglycerides (A) and [3H]retinol (B) were determined. Results are means ± SE. {dagger}P < 0.05 vs. control or AdLacZ; *P < 0.01 vs. 0 h, ¶P < 0.001 vs. 1 h by 2-way ANOVA. Baseline serum GPI-PLD activity was 14.4 ± 2.1 U/µl for control, 14.1 ± 1.6 U/µl for AdLacZ-, and 105 ± 1.7 U/µl for AdGPI-PLD-treated mice (means ± SD).

 
The increases in serum triglycerides during the postprandial state could be derived from delayed catabolism of triglyceride-rich lipoproteins and/or an increase in VLDL synthesis. To determine whether catabolism of triglyceride-rich lipoproteins was affected, [3H]retinol was included in the corn oil. [3H]retinol accumulated in serum in a linear fashion in all groups. There was no difference between control and AdLacZ-treated animals (Fig. 5B), but [3H]retinol accumulated twice as fast in the AdGPI-PLD-treated animals compared with the controls (Fig. 5B). In contrast, the rate of VLDL synthesis, as determined by Triton WR-1339, was similar between control (13.5 ± 3.50 mg/min), AdLacZ-treated (15.3 ± 1.40 mg/min), and AdGPI-PLD-treated mice (13.9 ± 4.6 mg/min) (means ± SD).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Many minor HDL-associated proteins play an important role in the metabolism of triglyceride-rich lipoproteins. In this study, we demonstrated the dynamics of GPI-PLD movement among lipoprotein subclasses in the postprandial state and provided evidence that GPI-PLD plays a role in triglyceride metabolism.

Total serum GPI-PLD appears to be influenced by numerous mechanisms. Diet appears to be one mechanism, as diets high in fructose or fat (M. A. Deeg and R. F. Bowen, unpublished observation) are associated with higher levels of fasting serum GPI-PLD. Levels of hepatic GPI-PLD mRNA have generally correlated to fasting serum GPI-PLD levels in mouse models of types 1 and 2 diabetes (2, 11), suggesting that transcriptional control of GPI-PLD expression in the liver may play a role in regulating serum GPI-PLD levels. Although liver is likely the major source of serum GPI-PLD, hepatic GPI-PLD mRNA levels are likely not the only factor regulating serum levels, since the fructose diet increased serum GPI-PLD levels without effects on liver GPI-PLD mRNA levels. The discord between liver mRNA and serum levels may result from differences in mRNA translation, other cells contributing to serum GPI-PLD in LDLR–/– mice, and/or altered catabolism of GPI-PLD in the plasma compartment. This is illustrated in our observation that, compared with wild-type mice, LDLR–/– mice have higher serum levels of GPI-PLD during the postprandial state without changes in liver mRNA levels. Because the bulk of GPI-PLD remains associated with HDL while fasting and in the postprandial state, the differences between the wild-type and LDLR–/– mice with respect to total and HDL-associated GPI-PLD may reflect the altered metabolism of certain subspecies of HDL that occurs in LDL receptor-deficient animals (4, 19). The influence of GPI-PLD on HDL metabolism per se will require additional experiments.

During the postprandial state, a small amount of GPI-PLD appears in triglyceride-rich lipoproteins. This GPI-PLD is not derived from the liver or small intestine; hence, it likely results from the exchange of GPI-PLD between HDL and triglyceride-rich lipoproteins within the plasma compartment. Triglyceride-rich lipoproteins containing GPI-PLD may represent chylomicrons, VLDL, and/or their remnants. It is possible that GPI-PLD associates with a small subpopulation of triglyceride-rich lipoproteins, as the maximum amount of GPI-PLD found in chylomicrons/VLDL is <1% of the total serum GPI-PLD.

The fate of the GPI-PLD associated with triglyceride-rich lipoproteins is unknown. Some GPI-PLD may exchange back to HDL as the triglycerides are hydrolyzed to form VLDL and chylomicron remnants. In the postprandial state, a population of GPI-PLD was also present in IDL/LDL. This was particularly evident in the LDLR–/– mice. The amount of GPI-PLD in the IDL/LDL region did increase and peak after the GPI-PLD changes in the triglyceride-rich lipoprotein region. One possible explanation is that the GPI-PLD in the VLDL stays with VLDL while it is being converted to IDL and LDL. Another possibility is that GPI-PLD exchanges between HDL and IDL/LDL independently of the conversion of VLDL to IDL/LDL. It is possible that the GPI-PLD associated with IDL or LDL is cleared via the LDL receptor, as the GPI-PLD in IDL/LDL is higher in the LDLR–/– mice compared with wild-type mice. Additional kinetic experiments are needed to determine the details of GPI-PLD exchange between lipoproteins and metabolism.

The maximum amount of GPI-PLD associated with triglyceride-rich lipoproteins and LDL ranges from 6 to 8% of the total amount of GPI-PLD in serum. This represents the minimum amount of GPI-PLD associated with HDL that can exchange between HDL and other lipoproteins. This is considerably lower than the 80–85% of exchangeable apolipoprotein C-II and C-III found in human HDL (5, 32). This may reflect the relatively low abundance of VLDL in mice compared with humans. Consistent with this hypothesis is our observation that elevations in triglyceride-rich lipoproteins by dietary fructose or deletion of the LDL receptor are associated with higher levels of GPI-PLD in chylomicrons/VLDL, particularly in the postprandial state. Our observations suggest that GPI-PLD is an exchangeable, HDL-associated protein and demonstrate that increasing hepatic expression and serum levels of GPI-PLD are associated with an increase in fasting and postprandial triglycerides. Unfortunately, our experimental design does not allow us to differentiate an effect of changing GPI-PLD within the hepatocyte per se vs. an effect to increase serum GPI-PLD. Although overexpressing GPI-PLD does delay catabolism of triglyceride-rich lipoprotein, the mechanism for this effect is not clear. One possibility is that increasing serum GPI-PLD alters the lipoproteins within the plasma compartment. Binding of GPI-PLD to triglyceride-rich lipoproteins could influence the composition of chylomicrons/VLDL. Increased levels of apolipoprotein C-III displace apolipoprotein E from VLDL, which is required for VLDL binding to hepatocytes (1, 3). However, given the small amount of GPI-PLD present in triglyceride-rich lipoproteins, this mechanism seems unlikely. It is clear that the GPI-PLD effect on serum triglycerides is more complicated than simply changing total serum GPI-PLD or the GPI-PLD content of particular lipoproteins, since the time course of change in serum triglycerides does not parallel the changes in total or lipoprotein content of GPI-PLD in LDLR–/– mice.

A second possibility is that overexpressing hepatic GPI-PLD changes the ability of triglyceride-rich lipoprotein remnants to bind to the hepatocyte. Consistent with this possibility is our observation that the magnitude of the increase in fasting triglycerides and postprandial triglycerides and the twofold decrease in catabolism of triglyceride-rich lipoproteins by overexpressing hepatic GPI-PLD are comparable to the changes seen in mice lacking the LDLR (20) or hepatic LDL receptor-related protein (28).

In summary, these studies demonstrate the dynamics of GPI-PLD exchange among lipoprotein classes during the postprandial state and indicate that overexpression of hepatic GPI-PLD induces an increase in fasting and postprandial triglycerides secondary to a decrease in catabolism of triglyceride-rich lipoproteins. Given our previous observations with respect to GPI-PLD and insulin resistance, GPI-PLD may play a role in the dyslipidemia of the metabolic syndrome. Further studies are needed to elucidate the functional significance of GPI-PLD as well as to gain further insight into the molecular mechanism by which GPI-PLD influences lipoprotein metabolism.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by funds from Indiana University (M. A. Deeg).


   ACKNOWLEDGMENTS
 
We thank Brad Poteat for excellent technical assistance with the liver perfusion experiments, and Drs. Robert Considine and Dona Gray for critical review of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Deeg, Endocrinology 111E, Indiana University, 1481 W. 10th St., Indianapolis, IN 46202 (e-mail: mdeeg{at}iupui.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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