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Nutrition Department, The Pennsylvania State University, University Park, PA 16802
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENT
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Desaturation of stearate and palmitate and its effect on cellular accumulation of oleate were determined in primary culture of rat hepatocytes. The rate of oleate synthesis as measured by the formation of monounsaturated fatty acids from stearate was significantly higher than that from palmitate. The rate of [1-14C]stearate incorporation into oleate [1208 ± 195 pmol/(mg protein·4 h)] was 80% higher than that of [1-14C]palmitate [(672 ± 82 pmol/(mg protein·4 h)]. Despite the different rates of desaturation, the cellular oleate concentrations did not differ in the cells treated with stearate and palmitate (i.e., 42.5 ± 4.5 vs. 40.8 ± 5.2 nmol/mg protein). On the other hand, oleate concentration in the cells incubated with exogenous oleate was 198.1 ± 9.5 nmol/mg protein. There was a dose-dependent increase in cellular stearate concentration by increasing stearate concentrations from 0.5 mmol/L to 4.0 mmol/L in culture medium. A linear increase in cellular stearate concentration was also achieved by increasing the duration of incubation with 1.0 mmol/L stearate from 2 to 24 h. Despite the marked increases in stearate concentrations under these conditions, oleate concentrations remained unchanged in the cells. These results do not support the contention that the hypocholesterolemic effect of stearate may be mediated by its conversion to oleate, although stearate is a more favorable substrate for desaturation than palmitate.
KEY WORDS: stearate · palmitate · oleate · desaturation · cultured rat hepatocytesINTRODUCTION
One of the major risk factors for developing coronary heart disease is a high level of plasma cholesterol, especially LDL cholesterol (American Council on Science and Health 1988, Levy and Feinleib 1984
). The elevated levels are largely determined by an increase in saturated fatty acid intake. However, unlike the hypercholesterolemic effects reported with shorter-chain saturated fatty acids, i.e., laurate (12:0), myristate (14:0) and palmitate (16:0), stearate (18:0) does not raise plasma cholesterol levels (Ahrens et al. 1957, Bonanome and Grundy 1988, Denke and Grundy 1991, Grundy and Vega 1988, Hegsted et al. 1965, Keys et al. 1965, Kris-Etherton et al. 1993, Mattson and Grundy 1985).
The mechanisms underlying the differential effects of saturated fatty acids on plasma lipids, particularly lipoproteins, have not been completely elucidated. Poor digestibility (Apgar et al. 1987, Imaizumi et al. 1993, Mitchell et al. 1989) and absorption through lymph (Bergstedt et al. 1990) of stearate compared with palmitate and oleate have been reported. However, these differences in absorption have not been consistently demonstrated by other investigators (Bonanome and Grundy 1989, Olubajo et al. 1986). Another important factor that has been extensively investigated is the production and catabolism of LDL (Woollett et al. 1992a and 1992b). The regulation of the plasma concentration of LDL cholesterol, which is mediated by LDL receptor activity, differs not only between unsaturated and saturated fatty acids (Woollett et al. 1992b) but also within saturated fatty acids of varying chain lengths (Woollett et al. 1992a). For example, in hamsters, a diet rich in stearate is hypocholesterolemic compared with diets high in laurate, myristate or palmitate (Woollett et al. 1992a). The hypocholesterolemic response is associated with the findings that in contrast to shorter-chain saturated fatty acids (12:0-16:0), stearate neither depresses hepatic LDL receptor activity nor increases the rate of LDL production (Woollett et al. 1992a). Most recently, studies have demonstrated that stearate is less efficiently esterified to triacylglycerol, phospholipids and cholester ester than other saturated and monounsaturated fatty acids in mouse liver (Kvilekval et al. 1994) and cultured rat hepatocytes (Pai and Yeh 1996). Together with poor utilization of stearate for 
-oxidation (Pai and Yeh 1996), this suggests that impaired metabolism of stearate may be involved in regulating plasma concentration of cholesterol (Kvilekval et al. 1994). Also, it is intriguing to note that a high stearate diet has been shown to be as effective as a high oleate diet in lowering the plasma levels of total and LDL cholesterol in humans (Bonanome and Grundy 1988). In view of potential desaturation of stearate by 
9-desaturase to oleate (Christiansen et al. 1991), Bonanome et al. (1992) postulated that this conversion may be a key mechanism underlying the hypocholesterolemic effect of stearate. If this is the case, one would expect a rapid formation and cellular accumulation of oleate from stearate.
The present study was undertaken to evaluate the contribution of saturated fatty acids via desaturation and elongation to the concentration of oleate in primary cultures of rat hepatocytes. Cultured hepatocytes permit a direct measurement of stearate conversion to oleate without potential interference by other fatty acids. The results showed that although the conversion of stearate to oleate was more rapid than the conversion of palmitate to oleate, the cellular oleate concentration was not increased by stearate treatment.
MATERIALS AND METHODS
as modified by Seglen (1973). As detailed elsewhere (Yeh and Yeh 1994), the procedure involved collagenase perfusion via hepatic portal vein, tissue mincing, filtration and centrifugation, and further purification by cell adherence. The isolated cells were resuspended in Dulbecco's modified Eagle medium (DMEM) supplemented with a 5.6 mmol/L glucose, 10% fetal bovine serum (FBS) and antibiotics (penicillin, 1 × 106 U/L and streptomycin, 100 mg/L). Cell viability was determined by trypan blue exclusion for each isolation. The cell preparations with 
95% viability were used throughout the experiments. Aliquots (2 mL) of cell suspension (0.7-0.8 × 109 cells/L) were transferred to culture plates, each containing six wells (3.5-cm diameter), and incubated at 37°C under an atmosphere of 95% air and 5% CO2. Six hours after incubation, the cells that adhered to the flask were refed with DMEM and incubated under the same conditions for 24 h.
Desaturation of fatty acids.
The cultured cells obtained after a 24-h incubation were washed three times with 2 mL of FBS-free DMEM followed by incubation in the same medium containing 0.5 mmol/L [1-14C]palmitate or [1-14C]stearate and nonlabeled fatty acid to yield a specific radioactivity of 37 MBq/mmol. Fatty acids were provided as an albumin complex in a molar ratio of 5.6 throughout the study. Stock solutions (100 mmol/L) were prepared from sodium salt of each fatty acid. An aliquot of the stock solution was added to DMEM containing 5.8 g/L bovine serum albumin followed by sonication before the incubation. After 4-h incubations, the cells and the medium were collected separately for lipid extraction according to the procedure of Folch et al. (1957). The lipid extracts were transmethylated in sealed ampules by using boron trifluoride in 12% methanol (Morrison and Smith 1964). 14C-Labeled methyl fatty acids were separated on 10% AgNO3-treated silica gel G 60 plates using a solvent system consisting of benzene/ethyl acetate/acetic acid (90:10:1) (Giron et al. 1989). The radioactivity recovered in the band corresponding to monounsaturated fatty acids was measured to determine the rate of fatty acid conversion to desaturation and/or elongation products (Elovson 1965). It has been previously determined that oleate was the major desaturation product from stearate, whereas palmitoleate and oleate were the predominant derivatives of palmitate (Elovson 1965). Thus, the synthesis of monounsaturated fatty acids from saturated fatty acids was determined by measuring the rate of [1-14C]stearate incorporation into oleate and [1-14C]palmitate incorporation into oleate and palmitoleate (Elovson 1965). The rates were expressed as nmol substrate incorporated/(mg cellular protein·4 h). Cellular protein was determined according to the procedure of Lowry et al. (1951).
Cellular accumulation of oleate.
To determine the effect of individual fatty acids on the cellular concentration of oleate, the cultured cells were incubated for 4 h with DMEM in the presence of 0.5 mmol/L myristate, palmitate, stearate or oleate. At the end of incubation, the cellular lipids were extracted and methylated as above for analysis of fatty acids by a gas chromatograph (Model 5980 Series II; Hewlett-Packard, Palo Alto, CA) equipped with a SP-2330 fused silica capillary column (30 m × 0.25 mm i.d., 20 µm film; Supelco, Bellefonte, PA) and a flame-ionization detector. The gas chromatographic conditions were as follows: temperature program starting at 150°C for 8 min and increased at a rate of 3°C/min to reach a final temperature of 190°C; injector temperature 220°C; detector temperature 250°C; flow rate 0.842 mL/min; and split ratio of 76:1. In a series of experiments, various stearate concentrations (0, 0.1, 0.5, 1.0, 2.0 and 4.0 mmol/L) and incubation times (2, 4, 8 and 24 h) were used to measure the cellular accumulation of oleate.
Materials.
Collagenase D was purchased from Boehringer Mannheim (Indianapolis, IN). [1-14C]Palmitate and [1-14C]stearate were obtained from Amersham (Arlington Heights, IL). Fatty acid-free bovine serum albumin and sodium salts of myristate, palmitate, stearate and oleate were from Sigma Chemical (St. Louis, MO). Dulbecco's modified Eagle medium, fetal bovine serum and antibiotics (penicillin and streptomycin) were provided by Life Technologies (Grand Island, NY). Silica gel G 60 plates were purchased from Alltech Associates (Deerfield, IL).
Statistical analysis.
Data are expressed as means ± SD. The significant differences in comparing four fatty acid treatments or different incubation times were analyzed by one-way ANOVA. Tukey's pairwise comparisons (Milton 1992) using Minitab Statistical software (Minitab, State College, PA) were applied to determine where the difference existed. Student's t test was used to compare the difference when only two treatments were included in the experimental design. P values <0.05 were judged to be significant.
RESULTS
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Table 1. The rate of [1-14C]palmitate and [1-14C]stearate conversion into monounsaturated fatty acids in cultured rat hepatocytes1,2 |
Table 2.
Fatty acid composition of cultured rat hepatocytes treated with exogenous fatty acids1,2
DISCUSSION 
Fig. 1.
Concentrations of stearate and oleate in rat hepatocytes treated with exogenous stearate. Cultured rat hepatocytes were incubated with medium containing exogenous stearate at 0, 0.1, 0.5, 1.0, 2.0 and 4.0 mmol/L. After 4-h incubations, cells were harvested and cellular lipids were extracted and methylated. Cellular concentrations of stearate and oleate were determined by the use of gas chromatography as described in Materials and Methods. The results are expressed as nanomoles of fatty acid per milligram cellular protein. Each data point represents a mean ± SD for 4 culture wells containing rat hepatocytes. Values with unnoticeable error bars indicate that the bars are smaller than data points. Values in a curve with different superscripts are significantly different, at P < 0.05.
[View Larger Version of this Image (16K GIF file)]
Fig. 2.
Changes in rat hepatocyte concentrations of stearate and oleate as a function of incubation time. Cultured rat hepatocytes were incubated with 1.0 mmol/L exogenous stearate for 0, 2, 4, 8 and 24 h. At the end of each incubation period, cells were harvested and cellular lipids were extracted and methylated. Cellular concentrations of stearate and oleate were determined by the use of gas chromatography as described in Materials and Methods. The results are expressed as nanomoles of fatty acid recovered per milligram cellular protein. Each data point represents a mean ± SD for 4 culture wells containing rat hepatocytes. Values with unnoticeable error bars indicate that the bars are smaller than data points. Values in a curve with different superscripts are significantly different at P < 0.05.
[View Larger Version of this Image (16K GIF file)]
), whereas saturated fatty acid and cholesterol reduce LDL receptor activity in the liver, leading to an increase in plasma cholesterol concentration (Grundy 1986, Spady and Dietschy 1988). Thus, a reduction of dietary saturated fat and cholesterol is desirable for the treatment and prevention of hypercholesterolemia. However, despite the hypocholesterolemic effect of stearate (Bonanome and Grundy 1988, Hegsted et al. 1965, Keys et al. 1965, Kris-Etherton et al. 1993), current diet recommendations do not differentiate stearate from other saturated fatty acids in their effect on plasma cholesterol level (American Heart Association 1988, NIH 1985, USDA/Department of Health and Human services 1985). This may be attributed in part to a lack of understanding of the mechanism(s) underlying the cholesterol-lowering effect of stearate.
). Moreover, oleate also appears to be as effective as linoleic acid in lowering LDL cholesterol levels in humans (Bonanome and Grundy 1988, Mattson and Grundy 1985, Mensink and Katan 1989). Additionally, oleate may be more beneficial than polyunsaturated fatty acids in reducing the risk for coronary heart disease because it does not decrease the plasma concentration of HDL cholesterol (Mancini and Parillo 1991). Further, elevated oleate concentrations have been found in plasma of subjects fed a liquid diet high in stearate (Bonanome and Grundy 1988).
, Katz 1986). However, despite the higher rate of oleate synthesis from [1-14C]stearate than [1-14C]palmitate, the concentration of oleate in the stearate-treated cells was not different than that in palmitate-treated cells (Tables 1 and 2). Although the cause is not completely clear, the difference in the synthesis of oleate from stearate compared with oleate from palmitate was not associated with a lack of substrate transport because the rate of palmitate uptake was estimated to be 1.5-fold that of stearate (Pai and Yeh 1996).
). The present results, however, are consistent with a feeding study that demonstrated that the ratios of hepatic stearoyl-CoA and oleoyl-CoA, the substrate and product of 9-desaturase, respectively, were the same in two groups of rats fed diets containing corn oil or cocoa butter rich in stearate (Monsma and Ney 1993). Therefore, it was suggested that the hepatic conversion of stearate to oleate is not the main cause of the hypocholesterolemic effect of stearate (Monsma and Ney 1993). Aside from the liver, the intestine, adipose tissue, muscle and brain are capable of desaturation of stearate and hence could contribute to tissue levels of oleate (Klingenberg et al. 1995). Interestingly, Emken et al. (1993) estimated that only 9.2% of deuterated stearate administered to human subjects as converted to oleate. They concluded that stearate conversion to oleate could not fully account for the hypocholesterolemic effect of stearate.
-oxidation and/or slower rate of uptake of stearate compared with other fatty acids. However, this possibility is unlikely because when the cellular stearate concentrations were approximately tripled by increasing the fatty acid concentration in the culture medium (Fig. 1) or increasing duration of incubation (Fig. 2), the concentration of oleate in the cells remained unchanged. Furthermore, a previous study demonstrated that the rate of stearate oxidation was markedly lower than that of other fatty acids and was only one third that for palmitate (Pai and Yeh 1996). Similarly, stearate was incorporated into glycerolipids (the sum of phospholipids, tri-, di- and monoacylglycerols) at a rate which was <10% of that for palmitate incorporation (Pai and Yeh 1996). On the contrary, a greater proportion of stearate than palmitate and other fatty acids taken up by the hepatocytes remained free and was not metabolized in the cells, suggesting that stearate was not efficiently activated to stearoyl CoA by fatty acylCoA synthetase (Pai and Yeh 1996). These findings, together with the centrally important role of fatty acylCoA synthesis in fatty acid metabolism (Schoonjans et al. 1993), have led us to speculate that the unaltered oleate concentration in the hepatocytes treated with exogenous stearate is due in part to its slow rate of activation to stearoyl CoA. Alternatively, the newly synthesized oleate from stearate may be preferentially incorporated into VLDL lipids and secreted into the medium. However, this possibility is not supported by the present data, which show a comparable rate of monounsaturated fatty acids released into the medium by the cells treated with different fatty acids (Table 1).
ACKNOWLEDGMENT
FOOTNOTES
Manuscript received 25 September 1996. Initial reviews completed 15 November 1996. Revision accepted 21 January 1997.
LITERATURE CITED
-oxidation in cultured rat hepatocytes.
Lipids
1996;
31:159-164
[Medline]
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