![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
The Journal of Nutrition Vol. 129 No. 1 January 1999,
pp. 126-131
,,, and
* Department of Animal Science, Faculty of Agriculture, Tohoku University, Sendai-shi 981, Japan and National Institute of Animal Health, Tsukuba-shi 305, Japan
 |
ABSTRACT |
|---|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
To identify the substrate specificity of lipoprotein lipase (LPL) for triacylglycerol-rich lipoproteins with monoacid-rich triacylglycerols, monoacid-rich lipoproteins were prepared and kinetic parameters of LPL were characterized. Male broiler chickens were fed 8 g/100 g fat diets differing only in the fat source: palm oil (tripalmitin-rich), olive oil (triolein-rich), safflower oil (trilinolein-rich) and linseed oil (trilinolenin-rich). After diets were fed for 3 d, chickens were starved for 2 d and then force-fed emulsions containing one of the monoacid-triacylglycerols: tripalmitin, triolein, trilinolein or trilinolenin. The triacylglycerols in chylomicrons and very low density lipoprotein (VLDL) of chickens force-fed tripalmitin, triolein or trilinolein contained the corresponding acid at more than 70% of total acids. Linolenic acid was incorporated into chylomicrons and VLDL to a lower extent (51.2 and 57.2%, respectively) in chickens force-fed trilinolein. Major apolipoproteins and lipid compositions were not significantly different among all lipoproteins isolated from chickens fed the different fats. Vmax of LPL was significantly higher (P < 0.05) for palmitic acid-rich chylomicrons and VLDL and decreased with increasing chain length and unsaturation of monoacid: 16:0>18:1>18:2>18:3. The electron spin resonance analysis, order parameter (S), decreased with monoacid chain length and unsaturation. In addition, the Vmax of LPL increased linearly (P < 0.01, r = 0.912) with an increase in the palmitic acid content of the lipoprotein triacylglycerols. These findings suggest that lipoprotein catalysis by LPL is modulated by the palmitic acid content of the lipoprotein triacylglycerol, which affects the fluidity of lipoproteins.
KEY WORDS: lipoprotein lipase · triacylglycerol-rich lipoprotein · fatty acid · chickens · electron spin resonance analysis
The obligatory step in the transport of triacylglycerol fatty acids from circulating chylomicrons and very low density lipoprotein (VLDL)3 into tissues is the hydrolysis of the triacylglycerol core in lipoprotein particles by lipoprotein lipase (LPL, EC 3.1.1.3) (Nilsson-Ehle et al. 1980 Chylomicrons and VLDL form micelle structures with an inner core consisting of triacylglycerol and cholesteryl esters and an outer monolayer of phospholipids, unesterified cholesterols and proteins (Bradley and Gotto 1978 Bouziane et al. (1994) In the present work with growing chickens, we show the preparation and structure of chylomicrons and VLDL rich in various mono fatty acids. In addition, the substrate specificity of LPL for triacylglycerol-rich lipoproteins was found by determining the kinetic parameters of LPL for lipoproteins composed of monoacid-rich triacylglycerols.
The 1 mL of each lipoprotein (10 g/L) was combined with 5 µL (1 g/L) of 5-DSA solution in ethanol. The mixtures were incubated at 37°C for 15 min and then the residual labels were removed using a PD-10 column (Pharmacia Fine Chemicals, Uppsala, Sweden). The ESR spectra were recorded on a JES-RE1X (JEOL, Tokyo, Japan) operated at a microwave frequency of 9.4 GHz. The temperature of the microwave cavity was 20°C.
Animals.
Male broiler chickens (Ross Strain) were housed in a battery brooder with an electric heater and fed on a commercial broiler starter diet (22 g crude protein/kg diet, 13.0 MJ metabolisable energy/kg diet) until 5 wk of age (body weight ranging from 1,000 to 1,200 g). The chickens were randomly divided into four experimental groups of 5 chickens each and were housed individually in wire cages in a room with a controlled temperature (25 ± 3°C).
Preparation of chylomicrons and VLDL.
Plasma lipoproteins, chylomicrons (d < 0.96 kg/L) and VLDL (d = 0.96-1.006 kg/L), were prepared by the method of Lindgren (1975) Preparation of LPL.
Adipose LPL from chickens was purified by a procedure that included the three chromatography steps of heparin-sepharose 4B, hydroxyapatite and con A-sepharose 4B described previously by Sato et al. (1997) Fatty acid analysis of triacylglycerol fraction in lipoproteins.
Lipoprotein lipids were extracted according to the method of Folch et al. (1957) Determination of different classes of lipids and phospholipids in lipoproteins with monoacid-rich triacylglycerol.
The different classes of lipids in lipoproteins were quantified according to Spios and Ackman (1978) Electrophoretic evaluation of chylomicrons and VLDL apolipoproteins.
After partial delipidation, chylomicrons and VLDL apolipoproteins were estimated using SDS-PAGE (12.5 and 17.5%) by the method of Bouziane et al. (1994) Electron spin resonance (ESR) analysis of lipoproteins with mono fatty acid-rich triacylglycerol.
The measurements of the order parameter (S) as the index of lipoprotein surface fluidity were performed using the 5-doxyl-stearic acid (5-DSA; Sigma Chemical Co., St. Louis, MO) (Foucher et al., 1996 Enzyme kinetics.
Kinetic constants (Apparent Km and Vmax) of LPL for the hydrolysis of chylomicrons and VLDL were determined using double reciprocal plots (Dixon and Webb, 1979 Other assays.
The protein contents of enzymes were determined by the method of Lowry et al. (1951) Statistical analysis.
A computer generated SAS applications package was used for statistical calculations (Statistical Analysis System Version 6.03, SAS Institute Inc., Cary, NC). Group data for multiple comparisons were analyzed by ANOVA using a general linear model procedure and were followed by Duncan's multiple range test. To demonstrate the difference among groups force-fed the different monoacids, the comparisons of chemical properties of lipoproteins were made within chylomicron and VLDL. The data for LPL kinetics were compared among all eight groups (chylomicron + VLDL) for the investigation of factors regulating lipoprotein catalyzed hydrolysis. The level of significance used in all comparisons was P < 0.05.
Fatty acid composition of triacylglycerols in chylomicrons and VLDL.
Force-feeding chickens triacylglycerols containing 16:0, 18:1, 18:2 or 18:3 resulted in a higher proportion of the corresponding fatty acid in both chylomicrons and VLDL (Table 2). However, incorporation of 18:3 into lipoproteins in chicken force-fed trilinolenin was proportionately less than that of 16:0, 18:1 or 18:2 in chickens force-fed triacylglycerols composed of the respective monoacid.
Different classes of lipids and phospholipids in chylomicrons and VLDL with monoacid-rich triacylglycerol.
Triacylglycerols comprised approximately 85% and 65% of chylomicrons and VLDL, respectively, followed by phospholipids at less than 25% (Table 3). However, the lipid composition of plasma chylomicrons and VLDL were not significantly affected by the force-feeding of triacylglycerols containing different monoacids. Of the phospholipid species, phosphatidylcholine was proportionally high (>65%) in chylomicrons and VLDL with different monoacid-rich triacylglycerols. The proportion of phosphatidylcholine in 18:3-rich chylomicrons was lower than lipoproteins from chickens force-fed other monoacids, whereas the phospatidylserine and sphingomyelin contents were high.
Apolipoproteins.
Four major apolipoprotein bands, apo B100, apo 52 kDa (unknown protein), apo A-I and apo C were detected on SDS-PAGE analysis of lipoproteins (Fig. 1). No significant differences due to the different monoacid-rich triacylglycerols were detected except apo 52 kDa (unknown protein) was highest in 16:0-rich lipoproteins and generally decreased with the increase in unsaturation.
ESR analysis.
The order parameter (S) of lipoproteins isolated from chickens force-fed the different fats was highest in 16:0-rich lipoproteins and gradually decreased with increasing monoacid chain length and unsaturation (Table 4).
Kinetic parameters in LPL for monoacid-rich lipoproteins.
The Vmax of LPL for 16:0-rich chylomicrons and VLDL were significantly higher than those of 18:1, 18:2 and 18:3-rich lipoproteins, whereas the apparent Km values were lower (Table 5). Chylomicrons rich in 18:3 were characterized by the highest apparent Km and the lowest Vmax for LPL among all lipoproteins studied.
Refeeding starved chickens with 16:0, 18:1, 18:2 or 18:3 triacylglycerols resulted in higher levels of the corresponding fatty acid in the triacylglycerol of lipoproteins, suggesting that the dietary fatty acid composition influenced not only fatty acid composition in plasma chylomicrons but also that in VLDL, and confirmed an earlier observation by Bouziane et al. (1994)
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). LPL is located both at capillary endothelial surfaces and in parenchymal cells of heart and adipose tissue (Pedersen and Schotz 1980).
). Dolphin and Rubinstein (1974) reported that the hydrolysis of VLDL by rat LPL was higher than that of serum chylomicrons, partly because of the high apolipoprotein content of VLDL. To determine the substrate specificity of LPL for chylomicrons and VLDL, the fatty acid profiles in lipoprotein triacylglycerol must be considered. Using artificial substrates of monoacid triacylglycerol emulsions, Wang et al. (1993) found that the preferential order of bovine milk LPL catalysis was 8:0>10:0>4:0>12:0>18:1>6:0. There is, however, less information available on the relative catalytic activity of LPL for triacylglycerol-rich lipoproteins, chylomicrons and VLDL with various fatty acyl-chain lengths and saturation of triacylglycerol because of the difficulty changing the fatty acid composition of triacylglycerols in lipoproteins.
observed that feeding growing rats a diet of salmon oil raised the (n-3) fatty acid level in serum and VLDL triacylglycerol fractions. Suarez et al. (1996) reported that, in weaning rats fed a diet with high oleic acid, ~30% of the total fatty acids in VLDL was comprised of oleic acid. These findings suggested that the fatty acid composition of triacylglycerols in chylomicrons and VLDL could be changed by dietary fat. However, to determine the substrate specificity of LPL on monoacid-rich lipoproteins, it might be necessary to provide lipoproteins with the monoacid constituting more than 50% of total acids as the substrate. Nir et al. (1973) reported that the refeeding of a diet following a period of food deprivation increased plasma triacylglycerols in geese. We anticipated, therefore, that in chickens force-fed the monoacid triacylglycerol following starvation, chylomicrons would be synthesized in the intestine from the exogenous fat and subsequently transported into liver for VLDL synthesis, thereby allowing the preparation of lipoproteins rich in a particular monoacid.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Composition of experimental diets
using ultracentrifugation (Kontron instrument K. K., Zurich, Switzerland) with a TFT65.13 rotor.
. The specific activity of purified LPL was 64.87 U/mg protein. The LPL activity was determined by measuring released free fatty acid (FFA) following in vitro incubation at 37°C with substrates and LPL as described previously (Sato et al. 1995). One unit of enzyme activity was defined as 1 mmol of FFA released per hour.
. Triacylglycerol fractions were isolated by 100 mesh silicic acid (Mallinckrodt, Paris, KY) according to the method of Fleischer et al. (1967). Fatty acids were derivatized using phenacyl bromide at 50°C for 2h and analyzed by high pressure liquid chromatography (Borch 1975), using a Shimadzu 6A system, Zorbax ODS (4.6 × 250 mm) (Shimadzu Co., Kyoto, Japan), with increasing acetonitrile concentrations from 85 to 90% and measuring absorbance at 254 nm. Flow rate was set at 1.0 mL/min. Identification of fatty acids was performed by comparison with commercial standards of known relative retention times. Areas were calculated with a Shimadzu C-R6A integrator. All lipids samples were stored under N2 gas in the dark at -20°C to prevent peroxidation of unsaturated fatty acids.
using TLC-FID equipment (IATROSCAN TH-10, Iatron Laboratories, Tokyo, Japan) connected to an electronic integrator. Standards of tripalmitin, dipalmitin, cholesterol, cholesterol palmitate, palmitic acid and phospatidyl choline were used to standardize the FID detector. The phospholipid species were determined by the method of Nakamura and Hanada (1984) using thin-layer chromatography (TLC). The phospholipids were applied to Silica gel 60 (Merck, Darmstadt, Germany) and developed with chloroform/methanol/NH4OH (60:35:8, by volume). Developed TLC plates were air-dried and immersed in a staining solution consisting of 0.03% Brilliant blue R (Sigma) in 20% methanol. After 2h, the plates were removed out of the staining solution, immersed in 20% methanol and scanned with a densitometer (CS-9300PC, Shimadzu Co., Kyoto, Japan) under the reflectance mode at 580 nm. Standards of phosphatidylcholine, phosphatidylethanolamine, phospatidylserine, phosphatidylinositol and shingomyelin were used for sample identification.
. Electrophoresis was performed in a Mini-PROTEAN II electrophoresis cell (Bio-Rad Laboratories, Hercules, CA) at 4°C for 3h with 25 mA/gel slab. Gels were then stained with Coomassie brilliant blue G250. Destained gels were scanned at 595 nm with a densitometer (CS-9300PC, Shimadzu Co., Kyoto, Japan). Apolioproteins were determined semiquantitatively with the densitometer tracing. To estimate the concentration of each apolipoprotein, the percentage of the area of each apolipoprotein was multiplied by the total apolipoprotein content of each sample. Results were expressed as arbitrary units.
; Fretten et al., 1980).
T
; outer hyperfine splitting, T 
; inner hyperfine splitting
). Incubations were performed at 37°C with various amounts of substrate (0.5, 0.75, 1.5, 2.5 and 4 mmol triacylglycerols/L) in a total volume of 0.6 mL of 10 mmol sodium barbital-HCl buffer/L, pH 8.6, containing 20 mg defatted bovine serum albumin (BSA) (fraction V, Sigma, St. Louis, MO). All experiments were performed in triplicate.
using BSA as the standard. The triacylglycerol concentrations of plasma lipoprotein were quantified by the method of Fletcher (1968).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 2.
Fatty acid composition of triacylglycerol in mono fatty acid- rich lipoproteins prepared from plasma of chickens force-fed tripalmitin, triolein, trilinolein or trilinolenin1,2
View this table:
Table 3.
Lipid composition and phospholipid species in mono fatty acid-rich lipoproteins prepared from plasma of chickens force-fed tripalmitin, triolein, trilinolein or trilinolenin1,2
View larger version (28K):
[in a new window]
Fig 1.
Apolipoprotein distribution in chylomicrons (A) and VLDL (B) prepared from plasma of chickens force-fed tripalmitin, triolein, trilinolein or trilinolenin. Results are expressed as means ± SD, n = 5. Bars with different superscript letters are significantly different, P < 0.05.
View this table:
Table 4.
Electron spin resonance analysis of mono fatty acid-rich lipoproteins prepared from plasma of chickens force-fed tripalmitin, triolein, trilinolein or trilinolenin1
View this table:
Table 5.
Kinetic parameters of chicken LPL for monoacid-rich lipoproteins prepared from plasma of chickens force-fed tripalmitin, triolein, trilinolein or trilinolenin1,2
View larger version (11K):
[in a new window]
Fig 2.
The relationship between the Vmax of chicken lipoprotein lipase (LPL) for lipoproteins prepared from chickens force-fed tripalmitin, triolein, trilinolein or trilinolenin and the palmitic acid level in lipoprotein triacylglycerol (A) or the order parameter of lipoprotein particles (B) (P < 0.01). One unit of enzyme activity was defined as 1 mmol of FFA released per hour.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
that the fatty acids composition in diet reflected that in VLDL triacylglycerols. The present findings that the VLDL fatty acid composition was similar to that in chylomicrons may indicate that chylomicrons synthesized in intestinal cells are transported to be incorporated in part by lipoprotein receptor-mediated endocytosis. Suarez et al. (1996) demonstrated that monoacid-rich VLDL were prepared by dietary modification in rats, whereas only 30% of total acids in VLDL was comprised of oleic acid in rats fed a diet with high oleic acid. The present study may be the first to demonstrate that chickens force-fed triacylglycerols containing 16:0, 18:1 or 18:2 incorporate more than 70% of the corresponding fatty acid into VLDL triacylglycerols, which are relevant for the kinetic study of LPL specificity on lipoproteins differing in the fatty acid composition. Incorporation of 18:3 into chylomicrons and VLDL in chickens force-fed trilinolenin was lower than in those force-fed triacylglycerols, the values being 51.2 and 57.2%, respectively. The explanation for these differences is currently lacking, but one worth considering is that highly unsaturated fatty acids, like 18:3, are poor at keeping the micelle structure of lipoproteins. Wang et al. (1993) showed that LPL-catalyzed hydrolysis of the artificial substrate with monoacid triacylglycerol emulsion increased in the order 8:0>10:0>4:0>12:0>18:1>6:0. McLean et al. (1986) demonstrated that the fatty acyl-chain specificity of phospholipase A1 activity of bovine LPL followed the sn-1 acyl-chain length of the artificial substrates with an order of 14:0>16:0>18:0. In the present experiment with chylomicrons and VLDL, we first demonstrated that the Vmax of LPL for 16:0-rich lipoproteins exceeded those of 18:1, 18:2 and 18:3-rich lipoproteins. Our data reveal the impact of the degree of unsaturation on substrate specificity of LPL. In addition, the Vmax of LPL for chylomicrons and VLDL increased linearly with an increase of palmitic acid content. It is possible that the palmitic acid content in chylomicrons and VLDL triacylglycerols determines the Vmax value of LPL for lipoproteins.
. We also determined that force-feeding chickens 16:0, 18:1 or 18:2 triacylglycerols did not modify the lipid composition and phospholipid species. Hence, it may indicate that the substrate specificity of LPL in lipoprotein hydrolysis observed in the present study is not associated with lipid classes and phospholipid specificity of the substrate, except for 18:3-rich lipoproteins.
reported that an amino-terminal fragment of Apo B binds to LPL. Baum et al. (1990) observed that refeeding rats a high carbohydrate diet for 48 h following 48 h of starvation increased the expression of apolipoprotein-48 mRNA in the liver. These results suggested that the change of apolipoprotein composition affects LPL catalyzed hydrolysis. In the present study, Apo B100, Apo A-I and Apo C in plasma chylomicrons and VLDL were not significantly affected by force-feeding chickens triacylglycerols of 16:0, 18:1, 18:2 or 18:3. Our data showed that the substrate specificity of LPL for monoacid-rich lipoproteins was due to the difference in fatty acid compositions, but not to phospholipid and apolipoprotein species in lipoproteins. On the other hand, the unidentified apolipoprotein, Apo 52 kDa, was quantitatively changed by monoacid species of the lipoprotein. It should be noted that Apo 52 kDa increased in lipoproteins rich in 16:0 but decreased in those rich in 18:3, thereby correlating with the Vmax of LPL. The explanations for properties of Apo 52 kDa in lipoprotein metabolism and for the inclusion into LPL kinetics await further study.
). The electron spin resonance analysis order parameter (S) of monoacid-rich lipoproteins in the present study decreased in the order 16:0>18:1>18:2>18:3. These results may imply that the fluidity of lipoproteins is partly dependent upon the fatty acid composition of the triacylglycerol, which decreases with carbon number and the unsaturation of mono fatty acids. To be emphasized in the present study are the findings that the Vmax of LPL increased with the increase of the order parameter (S) for lipoproteins. It is, therefore, likely that the 16:0 content in lipoprotein triacylglycerols is one of the major factors that determine the fluidity of lipoprotein, and the low fluidity of lipoprotein enhances the affinity between lipoproteins and LPL.
observed that feeding rats (n-3) fatty acids decreases the epididymal fat pad weight through a decrease in plasma triacylglycerol concentration. Dietary polyunsaturated fatty acids regulates fat accumulation via a decrease in plasma triacylglycerol through the reduction in hepatic fatty acid synthesis (Fukuda et al. 1997). We suggest that the decrease of fat accumulation by polyunsaturated fatty acids stimulates not only the reduction in hepatic fatty acid synthesis but also in part the decrease in the Vmax of adipose tissue LPL.
| |
FOOTNOTES |
|---|
Manuscript received 17 December 1997. Initial reviews completed 26 January 1998. Revision accepted 4 September 1998.
| |
ACKNOWLEDGMENTS |
|---|
We thank KAO Corporation, Wakayama Research Laboratories, Wakayama 640, Japan, for providing monoacid triacylglycerols.
| |
LITERATURE CITED |
|---|
|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  M. S. Kim, F. Wang, P. Puthanveetil, G. Kewalramani, E. Hosseini-Beheshti, N. Ng, Y. Wang, U. Kumar, S. Innis, C. G. Proud, et al. Protein Kinase D Is a Key Regulator of Cardiomyocyte Lipoprotein Lipase Secretion After Diabetes Circ. Res., August 1, 2008; 103(3): 252 - 260. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Qi, D. An, G. Kewalramani, Y. Qi, T. Pulinilkunnil, A. Abrahani, U. Al-Atar, S. Ghosh, R. B. Wambolt, M. F. Allard, et al. Altered cardiac fatty acid composition and utilization following dexamethasone-induced insulin resistance Am J Physiol Endocrinol Metab, August 1, 2006; 291(2): E420 - E427. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
