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The Journal of Nutrition Vol. 128 No. 8 August 1998,
pp. 1270-1275
Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48202 and * Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, TX 76107
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The regulation of plasma lecithin:cholesterol acyltransferase (LCAT) expression is not well understood. Although oleic acid increases both the secretion of triglycerides and LCAT by primary rat hepatocytes, the effect of other fatty acids (FA) on LCAT secretion is not known. This study was designed to examine the effect of FA on the hepatic secretion of LCAT, triglyceride and apolipoprotein A-1 (apoA-1). Primary rat hepatocytes were incubated with serum-free medium, supplemented with individual FA (0-1 mmol/L) for 22-24 h. Preliminary studies indicated a linear secretion of LCAT up to 24 h in both control and FA-treated cells. When hepatocytes were incubated with 1 mmol/L FA, the LCAT secretion increased 50-100% (P < 0.01) in the presence of the 18-carbon FA (stearic, oleic, elaidic and linoleic acids), whereas the presence of butyric, lauric and palmitic acids had no significant effect. LCAT secretion decreased (P < 0.01) in the presence of docosahexaenoic acid (DHA). All FA (except DHA) significantly enhanced triglyceride secretion; however, only the 18 carbon FA significantly stimulated the synthesis and secretion of apoA-1 and secretion of LCAT. The secretion of LCAT correlated with apoA-1 secretion (r = 0.88, P = 0.004) but not with triglyceride secretion (r = 0.55, P = 0.12). Treatment with oleic acid resulted in a 1.5-fold increase in hepatocyte LCAT mRNA accumulation, whereas butyrate and palmitate had no effect. These data indicate that FA that promote the apparent synthesis and secretion of apoA-1 also stimulate the secretion of LCAT in vitro, suggesting a coordinate regulatory mechanism for apoA-1 and LCAT expression.
KEY WORDS: lecithin:cholesterol acyltransferase · fatty acids · secretion · gene expression · rat hepatocytes
The molecular physiology of reverse cholesterol transport, including the role of lecithin:cholesterol acyltransferase (LCAT)5 in the metabolism of HDL has been recently reviewed (Fielding and Fielding 1995 To date, only limited information is available on the factors that influence LCAT expression. According to Warden et al. (1989) Golder-Novoselsky et al. (1995) Materials.
Collagen (rat-tail type I) was obtained from Collaborative Biomedical Products (Bedford, MA). Collagenase was from Worthington Biochemical (St. Louis, MO). William's E medium, Dulbecco's minimum essential medium (DMEM) and fetal bovine serum were obtained from Gibco (Grand Island, NY). Free FA, bovine serum albumin ( FA-free), insulin, antibiotics, Cab-o-sil and all other chemicals were obtained from Sigma Chemical (St. Louis, MO). [35S]Methionine and [32P]dCTP were obtained from Dupont NEN (Boston, MA). LCAT cDNA was obtained from Dr. R. Taramelli, University of Milan, Italy (Meroni et al. 1990 Cell culture and experimental design.
The protocol for the care and use of rats in this study received prior institutional approval and followed the NIH guidelines (NRC 1985). Livers were surgically isolated from male Sprague-Dawley rats (300-325 g) purchased from Harlan Sprague Dawley (Indianapolis, IN); rats had free access to a nonpurified Purina rodent diet (#5001,TMI Nutrition, St. Louis, MO). Hepatocytes were isolated and seeded by established methods (Berry et al. 1991 Measurement of LCAT activity in the medium.
Because the LCAT activity as measured by using a proteoliposome method is highly correlated with the LCAT mass determined by the RIA, the proteoliposome substrate method (Albers et al. 1981 Measurement of TG in medium.
For TG mass measurements in hepatocyte-conditioned media containing different FA, lipoproteins were isolated by density gradient ultracentrifugation (Terpstra et al. 1981 Determination of synthesis and secretion of apoA-1.
To investigate the effects of various FA on the synthesis of apoA-1, hepatocytes were preincubated overnight with FA. The incubation medium was removed and the cells were washed twice with methionine-free DMEM. The cells were then incubated with [35S]methionine (1.85 Bq/L) in methionine-free and FA-free medium for 30 min. During this period, the incorporation of the label into immunoprecipitable protein ([35S]apoA-1) was linear and was used to measure relative rates of synthesis (Dory 1989 Measurement of LCAT mRNA.
Total hepatocyte RNA was extracted by the method of Chomczynski and Sacchi (1987) Statistical methods.
All analyses were performed using Stat View 4.5 (Abacus Concepts, Berkely, CA). Unless stated otherwise, data were analyzed by one-way ANOVA and means were compared by the least significant difference method. All experiments were repeated at least twice and a minimum of n = 3 concentrations of mRNA were compared with Student's t test. Differences were considered significant if P Dose dependence and time course of the effect of FA on LCAT.
During preliminary studies, the presence of the FA-albumin complex in the incubation mixture was shown not to have an effect on the activity of purified plasma or recombinant LCAT (data not shown). This finding was important because it excluded the possibility that FA in the medium had a nonspecific effect on LCAT activity measured with the use of the proteoliposome substrate.
Effect of FA on the secretion of LCAT into medium by primary rat hepatocytes.
For these experiments, hepatocytes were cultured with and without FA as summarized in the legend of Figure 3. Butyric, lauric and palmitic acids did not significantly affect LCAT secretion. Stearic and elaidic acid stimulated LCAT secretion by 50%, whereas oleic and linoleic acids stimulated the accumulation of LCAT by 150%. Inclusion of docosahexaenoic acid (DHA) in the medium decreased the secretion and accumulation of LCAT by 38% (P < 0.01).
Effect of FA on the secretion of TG by cultured rat hepatocytes.
All FA (except DHA) stimulated VLDL-TG secretion (Fig. 4), whereas only the addition of the 18-carbon FA resulted in enhancement of LCAT secretion (Fig. 3). The differential effect of the FA on TG secretion, relative to control conditions, was consistent with the results obtained after the incorporation of [3H]glycerol into hepatocyte- and conditioned medium-TG, incubated with the respective FA (data not shown). There was no significant correlation (r = 0.55; P = 0.12) between LCAT secretion and TG secretion (data not shown).
Synthesis and secretion of apoA-1 by cultured rat hepatocytes.
The relative rates of apoA-1 synthesis were determined by measuring the incorporation of [35S]methionine into immunoprecipitable apoA-1. The apparent synthesis of apoA-1 was not affected by butyric, lauric or palmitic acids. On the other hand, although the inclusion of DHA in the medium resulted in a 24% decrease in the apparent rates of apoA-1 synthesis, stearic, oleic and elaidic acids stimulated apoA-1 synthesis by 71, 44 and 41%, respectively (Fig. 5A). Similarly, the apparent rate of apoA-1 secretion was also increased during incubation with the same 18-carbon FA by 53, 56 and 72%, respectively (Fig. 5B). The 18-carbon FA stimulated both apoA-I secretion and LCAT secretion (Fig. 3), resulting in a correlation (r =0.88; P = 0.004) between the rate of LCAT and apoA-1 secretion (Fig. 6).
Effect of fatty acids on LCAT mRNA.
Total RNA was extracted from hepatocytes after incubation under control conditions or with selected FA that were found to either stimulate TG or LCAT secretion the most. The RNA were analyzed for LCAT mRNA concentration. The results, summarized in Figure 7, show a pattern consistent with the observed effects of these FA on LCAT secretion. Oleic acid treatment of hepatocytes resulted in a 1.5-fold increase in LCAT mRNA, whereas palmitic and butyric acids had no effect.
In humans, LCAT is synthesized in the liver and secreted into the blood where it is associated with HDL, specifically with particles containing only apoA-I (Cheung et al. 1986
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). The principal reaction catalyzed by LCAT is the transfer of an acyl group from the sn-2 position of phosphatidylcholine (PC) to cholesterol, producing lysoPC and cholesterol esters. This transesterification occurs preferentially on the surface of HDL, where the reaction is facilitated by apolipoprotein A-1 (apoA-1), the major apolipoprotein of HDL (Fielding and Fielding 1995, Glomset 1979). Although LCAT is expressed in a number of tissues, including the brain and testis, its mRNA is most abundant in the liver (Warden et al. 1989). The LCAT synthesized by parenchymal liver cells is secreted into the circulation in association with HDL (Fielding and Fielding 1995, Glomset 1979). Recent studies suggest that plasma HDL consists of discrete subpopulations of different densities and diameters. Most of the larger, lower density subfractions originate in the plasma compartment, whereas the liver is the primary source of the smaller, dense HDL particles (Blanche et al. 1981, Eisenberg 1984, Patsch et al. 1978). Data obtained by immunoaffinity chromatography of plasma suggest that LCAT is associated predominantly with small, apoA-1-rich HDL particles (Cheung et al. 1986).
, atherogenic diets or 
-hydroxy--methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have little or no effect on liver LCAT mRNA levels. Skretting et al. (1995) reported that LCAT is down-regulated in HepG2 cells by transforming growth factor- (TGF-) at a post-transcriptional level, involving increased RNA degradation. A similar observation was recently made when sodium butyrate was incubated with HepG2 cells by these investigators (Skretting et al. 1997). Fibrates and glucocorticoids, on the other hand, have also been shown to reduce plasma LCAT activity, as well as LCAT mRNA levels in rat livers (Jansen et al. 1992, Staels et al. 1992).
reported that overexpression of the human apoA-1 gene resulted in a parallel increase in apoA-I concentration and LCAT activity in mice. This observation (Golder-Novoselsky et al. 1995) suggests a relationship between the regulation of apoA-1 and LCAT expression. Previous studies suggested that the concentration and activity of LCAT in plasma increases with increased turnover of both endogenous and exogenous triglyceride (TG) (Akanuma et al. 1973, Quartfordt et al. 1993 and Wallentin and Vikrot 1975). Similarly, in vitro treatment of rat hepatocytes with oleic acid has been shown to increase the secretion of both TG and LCAT (Nordby et al. 1972). Although these data suggest a relationship between the secretion of TG and LCAT, it is not known how other FA influence LCAT expression and secretion. The aim of this investigation was to determine the respective roles of common dietary FA in the regulation of LCAT. The findings resulting from these studies suggest that there is no link between the hepatic secretions of TG and LCAT. However, additional correlative relationships between the effects of various FA on apoA-1 and LCAT secretion suggest a coordinate regulatory mechanism for apoA-1 and LCAT expression.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Antibody against rat apoA-1 was a gift from Dr. P. Roheim (Louisiana State University, New Orleans, LA ).
). Briefly, hepatocyte suspensions were prepared using collagenase and were separated from other cell types by four cycles of low speed centrifugation (50 × g for 3 min), followed by resuspension in the plating medium. Cells were resuspended at a density of 1.33 × 109 cells/L and seeded onto 60-mm dishes (3 mL per dish) precoated with rat-tail collagen. The plating medium consisted of an arginine-free, L-glutamine-containing DMEM, supplemented with 0.64 mmol/L L-ornithine, 38 mmol/L sodium bicarbonate, 10 mmol/L HEPES, 10 mmol/L dextrose and 20% (v/v) fetal bovine serum. Cell viability was assessed by trypan blue exclusion test (450-600 × 106 viable cells) with a viability >95%. After 4 h, the plating medium was replaced with serum-free William's E medium (control), or control medium supplemented with 1 mmol/L FA complexed to albumin (1:2) for up to 24 h. The FA used in this study included butyric (4:0), lauric (12:0), palmitic (16:0), stearic (18:0), oleic (18:1), elaidic (18:1), linoleic (18:2) and docosahexaenoic (22:6). In some experiments the medium was supplemented with a range of concentrations (0.2-2 mmol/L) of selected FA (4:0, 16:0, 18:1).
) was used to determine the apparent rate of LCAT secretion. Vesicles containing apoA-1, lecithin and cholesterol (including [3H]cholesterol) were prepared according to the method of Batzri and Korn (1973). To determine LCAT activity, 50 µL of the medium was added to 200 µL of the substrate and the mixture was incubated at 37°C for 5 h. The reaction was stopped by the addition of isopropanol. After centrifugation and evaporation of the extract, the radiolabeled unesterified (UC) and esterified cholesterol (EC) were separated by TLC (Kudchodkar and Sodhi 1976). The amount of EC formed was calculated as the product of UC (in picomoles) in the substrate mixture and the fractional rate of the formation of EC [1 LCAT unit (U) = 1 pmol of EC formed/(h·mg cell protein)].
). The VLDL fraction was adsorbed on Cab-o-sil (Sigma Chemical), washed with water and extracted with chloroform-methanol (Vance et al. 1984). The chloroform phase was evaporated to dryness and the lipid fractions were dissolved in 50% ethanol before the determination of TG by the glycerol phosphate oxidase method (DMA, Arlington, TX). In other experiments, medium was directly adsorbed on Cab-o-sil and total medium TG was measured as described above. The results are expressed as units of TG secreted per milligram of cell protein.
) and individual lipids were separated by TLC (Kudchodkar and Sodhi 1976). The relative rate of incorporation into TG was determined by scintillation counting.
). The extent of [35S]methionine incorporation was determined by counting the apoA-1-containing bands after resolution of the immunoprecipitate by SDS-PAGE and overnight solubilization in 0.4 mL hydrogen peroxide (30% wt/wt, Sigma Chemical) at 60°C.
).
and as previously described (Dory 1993). Total RNA (3-10 µg) was fractionated by formaldehyde-containing agarose (1.2%) gel electrophoresis; the separated RNA was transferred to nylon membranes (Zeta probe, Bio-Rad, Hercule, CA) overnight. For the detection of LCAT mRNA, a full-length rat LCAT probe was used (Meroni et al. 1990). The -actin probe was a DNA fragment of the chicken -actin gene (Dory 1993). DNA probes were labeled with [32P]dCTP (specific activity > 0.11 MBq/mmol) by nick translation for -actin and random prime labeling for LCAT to a specific activity >108 dpm/ng DNA (1.67 Mbq/ng DNA). The membranes were autoradiographed using XAR-5 film (Eastman Kodak, Rochester, NY). Autoradiograms were evaluated by densitometry.
0.05.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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Fig 1.
Time course of lecithin:cholesterol acyltransferase (LCAT) secretion from rat hepatocytes. Hepatocytes were isolated and cultured as described in Materials and Methods. At the indicated time points, the media from individual plates were assayed for LCAT activity. Data are presented as means ± SEM. Two independent experiments were performed, with four plates at each time point. The data obtained from both experiments were pooled (n =4). 1 LCAT unit (U) = 1 pmol of EC formed/h). *Different from control (P < 0.05).
0.05) on LCAT secretion at concentrations 0.8 mmol/L FA. In contrast to its inability to enhance LCAT secretion, palmitic acid was a potent stimulator of TG secretion at all concentrations tested (Fig. 2B). A significant increase (P 0.05) in TG secretion was also noted when oleic acid was used to supplement the medium at concentrations 0.6 mmol/L. The effects of butyric acid on TG secretion were significant only at concentrations 1 mmol/L.
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Fig 2.
Dose dependence of the effect of 1 mmol/L of fatty acids on the secretion of lecithin:cholesterol acyltransferase (LCAT) and triglycerides (TG) by primary rat hepatocytes. Hepatocytes were incubated overnight (20 h) with or without fatty acid (FA, 1 mmol/L). LCAT (panel A) and TG (panel B) secretion into the medium was assayed, as described in Materials and Methods. Data are presented as means ± SEM; (n = 6 plates at each concentration). Control = 100% [LCAT: 84.68 ± 2.67 pmol EC formed/(h·mg cell protein); TG: 81 ± 6 nmol/mg cell protein]. Although not visible, all data points have standard error bars. *Different from control, P < 0.05.
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Fig 3.
The effect of 1 mmol/L of fatty acids (FA) on lecithin:cholesterol acyltransferase (LCAT) secretion by rat hepatocytes. Hepatocytes were isolated and seeded as described in Materials and Methods. LCAT activity was assayed in the FA-containing medium as described in Materials and Methods. Data are presented as means ± SEM; (n = 3 plates per FA, pooled from three independent experiments). *Different from control (P < 0.05). Abbreviation: DHA, docosahexaenoic acid.
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Fig 4.
The effect of 1 mmol/L of fatty acids (FA) on the secretion of VLDL-triglyceride by primary rat hepatocytes. Hepatocytes were treated with FA as shown. VLDL were isolated from the hepatocyte-conditioned medium by density gradient ultracentrifugation, and VLDL-triglyceride was determined as described in Materials and Methods. Values are micrograms triglyceride secreted per milligram cell protein. To convert to µmol/mg protein, divide by 88.57. Data are presented as means ± SEM; (n = 3 plates per FA, pooled from three independent experiments). *Different from control (P < 0.05). Abbreviation: DHA, docosahexaenoic acid.
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Fig 5.
The effect of 1 mmol/L of fatty acids (FA) on the synthesis and secretion of apolipoprotein A-1(apoA-I) by primary hepatocytes. The apparent rates of synthesis (panel A) and secretion (panel B) of apoA-I were measured in hepatocytes previously incubated (24 h) with the respective FA as described in Materials and Methods. In panels A and B, data for linoeleic acid and docosahexaenoic acid (DHA), respectively, are not available due to spillage. The bars (Bq/mg protein) represent means ± SEM; (n = 4 plates per FA, pooled from two independent experiments). *Different from control (P < 0.05). Abbreviation: ND, not determined.
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Fig 6.
Relationship between lecithin:cholesterol acyltransferase (LCAT) and apolipoprotein A-I (apoA-1) secretion by primary rat hepatocytes. Data presented in Figures 3 and 6 were subjected to Spearman correlation analysis.
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Fig 7.
The effect of 1 mmol/L of fatty acids (FA) on the induction of lecithin:cholesterol acyltransferase (LCAT) mRNA in cultured primary rat hepatocytes. Rat hepatocytes were cultured for 24 h in the absence or presence of the indicated FA. Total RNA was extracted, purified and analyzed for LCAT mRNA by Northern analysis, as described in Materials and Methods. Values for LCAT were corrected for differences in loading by normalizing the data to -actin. The bars represents normalized mRNA levels ± SEM; (n = 3 blots from three independent experiments, expressed as a percentage of control). Control, 100 ± 5%. *Different from control (P < 0.05).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Fielding and Fielding 1995, Glomset 1979). It is now recognized that LCAT is an important component of the reverse cholesterol transport pathway (Fielding and Fielding 1995), and increase in the activity of LCAT has been shown to be anti-atherogenic (Hoeg et al. 1996). During this process, the nascent HDL particle takes up unesterified cholesterol from cell membranes, which is esterified by LCAT, leading to the mature HDL. At present, the molecular mechanism(s) that regulate the circulating concentrations of LCAT are not well understood. In this study, evidence is presented that 18-carbon FA stimulate the secretion of both LCAT and apoA-I, independently of increased TG secretion.
). According to Gaziano and colleagues (1997), high TG alone increases the risk of heart attack nearly threefold. These investigators report that in a study of 340 heart attack patients and 340 healthy age-matched controls, individuals with the highest ratio of TG to HDL have 16 times the risk of heart attack as those with the lowest ratio of TG to HDL. Previous studies suggest that plasma LCAT does not correlate with the levels of plasma HDL, but may correlate with plasma TG concentrations (Akanuma et al. 1973, Wallentin and Vikrot 1975 ). Furthermore, it has been shown that plasma LCAT activity is elevated under those conditions that lead to increased turnover of both endogenous (Akanuma et al. 1973, Quartfordt et al. 1993, Wallentin and Vikrot 1975) and exogenous TG (Wallentin and Vikrot 1976, Quartfordt et al. 1993). The link between secretion of TG and LCAT was first suggested by Nordby and co-workers (1972) who found increased secretion of both LCAT and TG by rat hepatocytes in the presence of oleic acid. The secretion of LCAT correlated with the secretion of TG but not with that of cholesterol (Nordby et al. 1972). The findings reported here are consistent with those of the last-mentioned study regarding the effects of oleic acid on LCAT and TG secretion. However, when these studies were extended to include other FA, such as butyric, lauric, palmitic, elaidic, linoleic and docosahexaenoic acids, the pattern of secretion of LCAT was found to be different from that of TG. Accordingly, although all FA (except DHA) stimulate TG secretion by the hepatocytes, only the 18-carbon FA effectively stimulate LCAT secretion. Our data thus indicate that the regulation of LCAT synthesis and secretion is not linked to synthesis and secretion of TG.
). Taken together, these findings suggest that the expression of LCAT and apoA-I may be coordinated, especially in the presence of several common dietary FA.
), more recent studies point to a relationship with apoA-I. For example, incubation of hepatocytes with cytokines simultaneously reduced the concentration of apoA-I and LCAT activity in the medium after 24 h (Ettinger et al. 1994). Similarly, HepG2 cells treated with TGF-, showed a simultaneous decrease in LCAT and apoA-I mRNA levels (Skretting et al. 1995). The implication is that in apoA-I deficiency, there may be reduced LCAT activity in plasma. Similarly, patients with familial LCAT deficiency have less apoA-I in plasma (Assmann et al. 1991, Ettinger et al. 1994, Franceschini et al. 1990, Funke et al. 1991).
demonstrated that diets high in fat and cholesterol raise HDL concentrations in transgenic mice. Although the diets do not increase apoA-I mRNA abundance per se, an increase is seen in the fraction of apoA-I mRNA in the polysomal pool that leads to increased synthesis and secretion of apoA-I. Similarly, Britton et al. (1990 and 1994) reported that in humans, high fat/high cholesterol diets increase HDL-cholesterol in part by stimulating apoA-I transport in plasma. However, it is not clear if specific FA are responsible for the stimulatory effects of these high fat diets.
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FOOTNOTES |
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-hydroxy--methylglutaryl coenzyme A; LCAT, lecithin:cholesterol acyltransferase; PC, phosphatidylcholine; TG, triglyceride; TGF-, transforming growth factor-; UC, unesterified cholesterol.
Manuscript received 17 October 1997. Initial reviews completed 8 December 1997. Revision accepted 3 April 1998.
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ACKNOWLEDGMENT |
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The authors acknowledge the expert technical assistance of Darla Rudledge.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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and interleukin-6.
Biochim. Biophys. Acta
1995;
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