The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 477-484

Myristic Acid-Rich Fat Raises Plasma LDL by Stimulating LDL Production without Affecting Fractional Clearance in Gerbils Fed a Cholesterol-Free Diet^1,2

Tahar Hajri, Pramod Khosla, Andrzej Pronczuk, and K. C. Hayes³

Foster Biomedical Research Laboratory, Brandeis University, Waltham, MA 02254

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The imbalance that develops between low-density lipoprotein (LDL) production and clearance during saturated fat consumption is responsible for expanding the circulating LDL pool. To assess the imbalance attributable to fatty acids alone, i.e., without the interaction of dietary cholesterol, the most fat-sensitive species available (the gerbil) was challenged with either a 12:0+14:0 rich-fat (high coconut, low safflower) or high 18:2 (high safflower, low coconut) fat for 4-5 wks. The plasma lipoprotein cholesterol profile, including lipoprotein composition, particle size and ¹²⁵I-LDL turnover were measured. Although total plasma cholesterol (TC) was threefold higher with saturated fatty acid (SFA) feeding (230 vs. 70 mg/100 mL; 5.9 ± 0.1 vs. 1.8 ± 0.05 mmol/L, P < 0.0001) and LDL cholesterol (LDL-C) was fivefold greater (10 vs. 54 mg/100 mL; 0.26 ± 0.02 vs. 1.4 ± 0.02 mmol/L, P < 0.001), the high-density lipoprotein (HDL₂) fraction increased the most (27 vs. 79 mg/100 mL; 0.7 ± 0.02 vs. 2.0 ± 0.1 mmol/L, P < 0.05) with minimal HDL₃ (NS) difference (16 vs. 26 mg/100 mL; 0.43 ± 0.08 vs. 0. 7 ± 0.05 mmol/L). Particle composition and size did not differ between groups. LDL kinetic analyses revealed that the fractional catabolic rate did not differ between gerbils with these extreme fat intakes, implicating overproduction and not reduced clearance as the primary consideration in LDL expansion. Thus SFA-induced cholesterolemia can be severe in the absence of dietary cholesterol with a greater impact on high-density lipoprotein than LDL and without an appreciable role attributed to LDL clearance (receptors).

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The hypercholesterolemic effect of saturated relative to polyunsaturated fat is well documented, including its ability to raise plasma low-density lipoprotein (LDL)⁴ (Hegsted et al. 1965, Spady et al. 1993). Numerous studies (Connor et al. 1969, Cortese et al. 1983, Hayes and Khosla 1992, Mercer and Holub 1981, Nestel et al. 1975, Shepherd et al. 1980, Turner et al. 1981) have examined possible mechanisms that include a decrease in fecal excretion of cholesterol and bile acids (Connor et al. 1969, Nestel et al. 1975), stimulation of cholesterol synthesis (Mercer and Holub 1981) and altered production or clearance of plasma LDL (Cortese et al. 1983, Hayes et al. 1997, Shepherd et al. 1980, Turner et al. 1981). In the steady state, the plasma LDL concentration represents a balance between LDL production and removal from the plasma via specific (LDL-apolipoprotein B/E receptors) and nonspecific processes.

Clinical studies that specifically focused on LDL turnover during modulation of dietary fat saturation are of limited scope (Cortese et al. 1983, Shepherd et al. 1980, Turner et al. 1981) and provide conflicting results (Hayes et al. 1997). In all three human studies, the exchange between fats was extreme, representing simultaneous, opposing perturbations in saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA). Second, dietary cholesterol intake was typically a complicating factor, as was the differing lipoprotein profile of the subjects at the time of intervention. In fact, variability in dietary cholesterol intake and the host lipoprotein status make definitive interpretation of the fat saturation effect impossible. In general, saturated fat seemed to favor overproduction of LDL as opposed to impaired clearance (Hayes et al. 1997).

Experiments in hamsters fed diets containing graded amounts of cholesterol (0.08-0.26%) revealed a progressive increase in plasma LDL when saturated fat (butter or coconut oil) was included with cholesterol, whereas LDL cholesterol (LDL-C) decreased when polyunsaturated fat (safflower, corn oil) was present (Horton et al. 1993, Spady and Dietschy 1985, 1988). The increase in LDL induced by the cholesterol-rich coconut oil diet was attributed to both stimulation of LDL production and inhibition of hepatic receptor-specific uptake of LDL (Horton et al. 1993, Spady and Dietschy 1985, 1988). By contrast, polyunsaturated fat countered the inhibitory effect of dietary cholesterol on the receptor-specific uptake of LDL. A major constraint of the model is that cholesterol must be added to the diet to down-regulate LDL receptors (LDLr) and induce the accumulation of plasma LDL particles (Ohtani et al. 1990, Spady and Dietschy 1988). On the other hand, in certain species such as the pig (Mustad et al. 1996) and guinea pig (Lin et al. 1994), the LDL pool may not increase with SFA feeding even though the degree of LDLr depression by cholesterol is substantial.

The data contained in the previous paragraph suggest that cholesterol intake complicates interpretation of the true fat effect. Accordingly, the present study measured the LDL pool size and LDL dynamics using cholesterol-free diets. The species (gerbil) most sensitive to dietary fat saturation (Hegsted and Gallagher 1967, Pronczuk et al. 1994) was studied to increase the likelihood of identifying the underlying cause of the fat effect. The assumption was that if LDL clearance was to be depressed by SFA and/or enhanced by PUFA in the absence of cholesterol, the extreme fatty acid polarity in the diets employed definitely should elicit the response in the ultrasensitive gerbil.

The hypothesis was that any increase in LDL induced by a high-SFA, low-PUFA diet largely would reflect impaired LDLr activity, similar to the situation with a high-cholesterol diet (Spady et al. 1993). In fact, the hypothesis was rejected because the fivefold greater pool of in LDL-C attributed to the 12:0+14:0-rich, low-18:2 diet was not associated with an altered fractional catabolic rate (FCR) but was attributed instead to a marked increase in LDL production.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals, diets and experimental design.  Adult male Mongolian gerbils (Meriones unguiculatus), weighing 75 ± 5 g, were obtained from Tumblebrook Farms (West Brookfield, MA) and housed in individual cages in an air-conditioned room with a 12-h reversed light-dark cycle (lights on at 18:00 h). They were assigned randomly to two groups (n = 24 per group) and were fed a cholesterol-free purified diet containing either a high proportion of linoleic acid (18:2 rich) or lauric and myristic acids (12:0+14:0 rich) for 4 wk. The basal composition of these diets (g/100 g of total dry weight) was as follows: 22.2 casein, 23 cornstarch, 13.3 glucose, 15.0 cellulose, 20 fat, 5.0 mineral mix (Ausman-Hayes), 1.2 vitamin mix (Hayes-Cathcart), and 0.3 choline chloride. Compositions of the mineral and vitamin mixes were detailed previously (Hayes et al. 1989). Fat provided ~40% of the total dietary energy and represented a blend of 99% coconut oil and 1% safflower oil (12:0+14:0 rich) or 1% coconut and 99% safflower oil (18:2 rich). The small increment of safflower oil added to the coconut oil diet was to guard against 18:2 insufficiency. Accordingly, the fatty acid composition of both diets, analyzed by gas liquid chromatography (GLC), differed in their proportions of linoleic acid (18:2) and myristic (14:0) + lauric (12:0) acids. The proportion of all other fatty acids was similar in the diets (Table 1) so that an exchange of ~27% energy occurred between 12:0+14:0 and 18:2. To document progressive changes in plasma cholesterol and triglyceride concentrations, blood samples were collected in gerbils under CO₂-O₂ anesthesia via cardiac puncture with a 30-gauge needle from both dietary groups after 1, 2, 3, and 4 wk of purified diet treatment.

In a first experiment, eight gerbils from each diet group were subjected to LDL turnover analysis after 4 wk with human LDL. Another eight gerbils from each group were food deprived overnight and exsanguinated by heart puncture under anesthesia. Collected plasma was used to measure lipid concentrations and to characterize lipoproteins. In a second experiment, LDL turnover again was investigated in gerbils fed both diets, this time with gerbil LDL. All experimental procedures were approved by the institutional animal care and use committee.

Plasma lipoprotein separation and characterization.  Plasma was separated from red cells by centrifugation at 1000 × g for 20 min at 4°C. Lipoproteins were separated from pooled plasma of three gerbils (3 mL) per diet group by ultracentrifugation in a preformed saline (KBr) density gradient as described by Goulinet and Chapman (1993). Ultracentrifugation occurred at 36,000 rpm (54 × 10⁷ g/min) and 15°C for 48 h in a SW 41 Beckman rotor. Lipoprotein fractions were collected according to their density (kg/L): very low-density lipoprotein (VLDL, d < 1. 018), LDL (1.018 < d <1 .063), high-density lipoprotein (HDL₂; 1.063 < d < 1.103) and HDL₃ (1.103 < d < 1.21). Particle diameter of each plasma lipoprotein class was calculated from the core-to-surface volume ratio as described by Van Heek and Zilversmit (1991) according to the following formulas: In the above formulas, R, radius of particle; D, diameter; TG, triglyceride mass; CE, cholesteryl ester mass; FC, free cholesterol mass; PL, phospholipid mass; PR, protein mass; 2.15 is the assumed thickness (nm) of the lipoprotein surface layer; and 1.093, 1.044, 0.968, 0.97 and 0.705 are the corresponding specific volumes as reported by Van Heek and Zilversmit (1991).

Preparation of LDL tracers and kinetic analyses.  In preparation for two separate LDL turnover experiments, LDL (d: 1.019-1.055 kg/L) were isolated from plasma of humans (experiment 1) or gerbils fed commercial pellets (experiment 2) by sequential ultracentrifugation in a 70 Ti rotor (Beckman Instruments, Palo Alto, CA). LDL was washed and concentrated by ultracentrifugation at the proper density before dialyzing at 4°C against 0.15 mol/L NaCl per 1 mmol/L EDTA (pH 7.4) for 24 h. LDL protein concentration was determined using the method of Markwell et al. (1978) adapted from the procedure of Lowry et al. (1951). Half of the concentrated human LDL was labeled with iodine ¹³¹I, and the other half with iodine ¹²⁵I (Na¹³¹I and Na ¹²⁵I, Amersham, Arlington Heights, IL) according to the McFarlane method (1958) as previously described (Khosla and Hayes 1991). Free iodine was removed by passing the labeled lipoprotein through a small disposable column (0.5 × 10 cm of sephadex G-50 and eluting with 0.15 mol/L NaCl per 1 mmol/L EDTA (pH 7.4) followed by exhaustive dialysis (3 changes during 24 h) against saline. Human LDL labeled with ¹²⁵I was methylated according to the procedure of Weisgraber et al. (1978) to modify the lysine residues of apolipoprotein B. Such modification blocks specific recognition of LDL by the apB/E receptor and allows assessment of LDL particles by nonreceptor uptake. The final specific radioactivity of ¹³¹I-LDL and ¹²⁵I-LDL were 0.2 and 0.3 kBq/µg of protein, respectively. For the second turnover study, gerbil LDL were labeled with ¹²⁵I without methylation as described earlier to determine kinetic parameters for native gerbil LDL. The proportion of radioactivity associated with LDL apolipoprotein, measured as previously described (Khosla and Hayes 1991), was >95%, whereas the lipid-bound radioactivity was <3%. The drinking water of gerbils in the kinetic studies was supplemented with potassium iodide (KI) at the final concentration of 1 g/L 24 h before starting the kinetic experiments.

Turnover of human native (¹³¹I) and methylated (¹²⁵I) LDL was studied in the first experiment. Gerbils were anesthetized with pentobarbital and given an intrajugular injection of 100 µL of labeled LDL . Each gerbil received ~30 µg of LDL protein corresponding to 5.5 and 9.2 kBq of ¹³¹I-LDL and ¹²⁵I-LDL. Blood samples (~50 µL) were withdrawn by heart puncture with 30-gage needle at 2 min and 1, 3, 5, 8, 12 and 24 h after injection with the gerbil briefly anesthetized with CO₂-O₂ for 1 min. The plasma radioactivity (¹³¹I and ¹²⁵I) was counted in a gamma counter and expressed as percent of 2-min radioactivity. In the second turnover, native gerbil LDL (¹²⁵I) was assessed after the injection of 100 µL of labeled LDL as in the first turnover. Plasma from the last bleeding (24 h) was pooled according to diet group, and lipoproteins were separated in a discontinuous density gradient as described earlier. Distribution of radioactivity was determined in each lipoprotein fraction.

The decay of plasma ¹³¹I and ¹²⁵I (gerbil and human LDL) was biexponential and was analyzed by the two-pool model (Matthews 1957). The fractional catabolic rate (FCR) was calculated from the slopes after decomposition of each curve as described previously (Khosla and Hayes 1991). The fractional catabolic rate (FCR_t) calculated from the ¹³¹I curve of native human LDL and from the ¹²⁵I curve of gerbil native LDL represents the total LDL uptake (specific uptake by the LDL receptor plus nonspecific uptake). The fractional catabolic rate (FCR_ns) calculated from the ¹²⁵I curve of methylated human LDL represents the nonspecific LDL uptake. Apo LDL pool size was calculated as the plasma volume (4 × 5% of body weight) × plasma apo LDL concentration (Fernandez et al. 1992). The transport rate (TR) was calculated as the FCR of native gerbil LDL × apo LDL pool.

Fatty acid analysis.  Fatty acid composition of formulated diets and lipoprotein samples (LDL total lipids and VLDL triglycerides) were determined by GLC according to the one-step transesterification procedure (Lepage and Roy 1986). VLDL and LDL were separated by gradient ultracentrifugation as described earlier. VLDL triglycerides were isolated by thin layer chromatography after a Folch extraction (Yao and Vance 1988), whereas LDL lipids were extracted and analyzed in toto. Fatty acid analysis was performed in a Shimadzu GC9-AM GLC using a 100-m fused capillary column (SP-2560, Supelco, Bellefonte, PA).

Plasma lipid and lipoprotein analysis.  Total cholesterol and triglycerides were determined by enzymatic assay (kit Nos. 352 and 336, respectively) from Sigma (St. Louis, MO). Phospholipids and free cholesterol concentrations were determined by enzymatic assay (free cholesterol C and phospholipids B kits from Wako Chemicals, Richmond, VA). Cholesteryl ester was calculated as the difference between the free and total cholesterol concentration. Protein concentration in lipoprotein fractions was determined using the Markwell assay (1978) adapted from the Lowry et al. procedure (1951) using bovine serum albumin as standard.

Statistics.  Results are expressed as means ± SEM, and significant differences between groups were assessed by the Student's t test, except for time course changes of plasma cholesterol and triglyceride concentrations (Fig. 1) for which repeated measure analysis of variance and Scheffé's F-test were used. Significance was set at P < 0.05. 

View larger version (15K): [in this window] [in a new window]   Fig 1. Time course changes in gerbil plasma cholesterol and triglyceride concentrations during 1-mo consumption of purified diets rich in 12:0+14:0 or 18:2. Values are means ± SEM, n = 8-10 gerbils. Values with different superscripts are significantly different (P < 0.05). The conversion facror of cholesterol concentrations from mg/100 mL to mmol/L is 0.02586.

	RESULTS

Abstract Introduction Methods Results Discussion References

Plasma lipids and lipoproteins.  Because the gerbils initially were fed commercial pellets, entry plasma values for total cholesterol (79 mg/100 mL; 2.0 mmol/L) and triglycerides (45 mg/100 mL; 0.5 mmol/L) concentrations were low. The 18:2-rich purified diet induced only a minor (nonsignificant, NS) change in these initial lipid values, whereas the 12:0+14:0-rich diet caused a dramatic increase (P < 0.05, paired t test) in both plasma cholesterol and triglycerides. After 3 wk, total cholesterol and triglycerides were elevated threefold in 12:0+14:0-fed gerbils compared with those fed 18:2-rich diet and remained at that level (Fig. 1).

Both plasma-free and esterified cholesterol were elevated approximately threefold in gerbils fed the 12:0+14:0-rich diet, whereas plasma triglycerides were more than doubled when compared with gerbils fed the 18:2-rich diet (Table 2). Dietary fatty acid saturation affected both LDL (d: 1.018-1.063 kg/L) and HDL₂ (d: 1.063-1.103 kg/L) (Table 3 and Fig. 2). All the constituents of these lipoprotein classes were significantly greater in gerbils fed the 12:0+14:0-rich diet, total cholesterol increasing fivefold in LDL and about threefold in HDL₂, respectively, whereas the mass of HDL₃ cholesterol increased only 50% (NS). Despite the large relative percent increase in LDL, the absolute increase in plasma cholesterol mass was 40% greater for HDL than LDL due to the fact that HDL represents the major transport system for cholesterol in gerbils (Table 3). In contrast to the mass difference in lipid fractions, the percent composition was not modified by the dietary fatty acids. Also, the mean size (diameter) of all lipoprotein classes in 12:0+14:0-fed gerbils (VLDL+IDL: 47.9 ± 2.6 nm, LDL: 16.5 ± 1.3 nm, HDL₂: 9.8 ± 0.5 nm and HDL₃: 7.7 ± 0.2 nm) did not differ from those isolated from gerbils fed 18:2-rich diets (VLDL + IDL: 43.2 ± 3.3 nm, LDL: 17.0 ± 3.1 nm, HDL₂: 10.5 ± 0.5 nm and HDL₃: 7.2 ± 0.3 nm).

View larger version (21K): [in this window] [in a new window]   Fig 2. Total cholesterol concentration in lipoprotein fractions isolated from discontinuous density gradient of plasma in gerbils fed 12:0+14:0- or 18:2-rich diet for 4 wk. Values are means ± SEM, n = 5 pooled plasma samples as described in Materials and Methods. The conversion factor of cholesterol concentrations from mg/100 mL to mmol/L is 0.02586.

Fatty acid composition.  As expected, triglycerides from VLDL of gerbils fed 12:0+14:0-rich diet were enriched in saturated (12:0, 16:0, 14:0, 18:0) and monounsaturated fatty acid (18:1) to the detriment of 18:2 (Table 4). Fatty acid composition of LDL lipids (Table 4) revealed that the 12:0+14:0-rich diet increased the proportions of 12:0, 14:0, 18:0, 16:1 and 18:1 while decreasing polyunsaturated fatty acids (18:2 and 20:4).

Receptor-dependent and receptor-independent LDL metabolism.  The distribution of radioactivity in the lipoprotein fractions isolated from plasma of gerbils 24 h after injection of native gerbil ¹²⁵I-LDL (Fig. 3) revealed that ~93% of the radioactivity injected was isolated between the densities 1.018 and 1.063 kg/L corresponding to LDL. This suggests that transfer of radioactivity from LDL to other lipoproteins was minimal and did not confound the measurement of LDL radioactivity.

View larger version (17K): [in this window] [in a new window]   Fig 3. Distribution of radioactivity (125I-LDL) in lipoprotein fractions from gerbils fed diets rich in 12:0+14:0 or 18:2 demonstrates concentration in low-density lipoprotein (LDL). Plot represents the mean of 4 samples collected 24 h after injection of native gerbil LDL into gerbils fed 12:0+14:0.

Plasma radioactivity declined in both diet groups after the simultaneous injection of human native ¹³¹I-LDL and methylated ¹²⁵I-LDL (experiment 1) or after the injection of native gerbil ¹²⁵I-LDL (experiment 2) (Fig. 4). For each category of LDL, the plasma radioactivity decay was similar in the two dietary groups. As expected, native gerbil LDL cleared at a faster rate than human native LDL, suggesting that gerbil LDL receptors had a lower affinity for human LDL apoB. Human LDL altered by methylation to prevent binding to the LDL receptor, was cleared most slowly. Kinetic parameters (Table 5) indicated that fractional catabolic rates for native gerbil LDL and human LDL as well as for methylated human LDL did not differ between dietary groups. However, pool size of LDL protein was four times greater in the 12:0+14:0-fed gerbils than those fed 18:2. Consequently, the absolute mass of LDL cleared, and thus the transport rate of LDL protein, was increased fourfold by 12:0+14:0.

View larger version (26K): [in this window] [in a new window]   Fig 4. Decline in plasma LDL radioactivity after the injection of native gerbil LDL, native human LDL, and methylated human LDL in gerbils fed 12:0+14:0- or 18:2-rich diets. Values are means ± SEM of 4-6 gerbils.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The results of this study confirm the previous observation (Mercer and Holub 1981, Nicolosi et al. 1981, Pronczuk et al. 1994) that even in the absence of dietary cholesterol, 12:0+14:0-rich fat dramatically increased total plasma cholesterol, including VLDL, LDL and HDL₂ levels in gerbils. Furthermore, neither the size nor the composition of LDL particles were affected by fat saturation, despite the extreme alteration in fatty acid composition. Most notably, neither specific nor nonspecific LDL clearance (FCR) was affected by fat saturation, indicating that the expansion of the plasma LDL pool induced by 12:0+14:0-rich diet resulted from increased production rather than impaired clearance linked to depressed LDL receptor activity.

Lipoprotein composition and fat saturation.  Changes in the composition of plasma lipoproteins, such as the ratio of core-to-surface components or the cholesteryl ester-to-triglyceride ratio within the core, affect the physical properties of the particles (Deckelbaum et al. 1977). This can alter intravascular processing of the particles (Swinkels et al. 1988) and affect their in vivo turnover (Fernandez et al. 1992, Swinkels et al. 1988). Because the extremes in fat saturation fed to these gerbils had no effect on the composition or size of LDL or other plasma lipoproteins, the observed elevation in plasma LDL cholesterol induced by the 12:0+14:0-rich diet is assumed to reflect an increase in the number of otherwise typical LDL particles. Our compositional data agree with previous studies in gerbils (Nicolosi et al. 1981) and cebus monkeys (Nicolosi et al. 1992) fed similar cholesterol-free diets. By contrast, changes in LDL size and composition have been induced by differences in fat saturation when accompanied by dietary cholesterol in guinea pigs (Fernandez et al. 1992), cynomolgus monkeys (Johnson et al. 1985) and humans (Baudet et al. 1984). In guinea pigs (Abdel-Fattah et al. 1995, Fernandez et al. 1992) and hamsters (Ohtani et al. 1990), LDL composition and size were affected by dietary cholesterol and fat in an interactive manner. In fact, adding an excessive amount of cholesterol (0.5%) to the diet of gerbils along with a saturated fat (beef tallow) increased the ratio of cholesterol-to-phospholipids in LDL when compared with safflower oil (Leach and Holub 1984), suggesting that even in gerbils, dietary cholesterol can interact with fats to modify LDL composition. In the absence of cholesterol, it may be a different story.

LDL kinetics and fat saturation.  The expansion in LDL cholesterol associated with the 12:0+14:0-rich, casein-based purified diet is in keeping with a report in cebus monkeys, which were fed a similar cholesterol-free diet based on coconut oil, in which LDLr activity was found to be depressed (Nicolosi et al. 1992). However, in gerbils, the increased LDL mass was not associated with reduced FCR (clearance), as reported in cebus monkeys (Nicolosi et al. 1992), but rather reflected exaggerated LDL production. This was true for both heterologous (human) and homologous (gerbil) LDL particles, suggesting that tracer particle composition was not the critical issue. Using the same tracer for both dietary groups allowed direct comparison of hepatic clearance involved in LDL catabolism, including receptor-mediated catabolism. In addition, comparable clearance of methylated human LDL indicated that non-receptor-mediated catabolism was unaffected by fat. Heterologous LDL (e.g., human into gerbils) clear more slowly than homologous LDL (gerbil into gerbils) because of differences in particle composition (especially apolipoprotein B) between species. Using a standard LDL preparation for both test diet groups was appropriate because liver functions related to LDL (production and clearance) greatly outweigh any particle effect (Fernandez et al. 1992). Using two different sources of LDL (human and gerbil) resulted in slightly different kinetics but showed that the responses of the host to both preparations were comparable, in spite of the difference in LDL composition (human vs. gerbil). When dietary fat or cholesterol depress hepatic LDLr activity, the effect clearly is demonstrated with a common LDL tracer (Fernandez et al. 1992).

These gerbil data indicate that a major SFA (12:0+14:0)-induced expansion of the LDL pool in the absence of dietary cholesterol can occur by means other than impaired LDL removal. The difference between these gerbil results and the previous cebus monkey data (Nicolosi et al. 1992) may be a matter of degree. On the one hand, the cebus were fed pure coconut oil (only 0.6% energy from 18:2) for 5 y and may have experienced a shortage of 18:2 that depressed LDLr activity (clearance) independent of any saturated fatty acid effect (Hayes et al. 1997). We previously reported that either the lack of 18:2 or excess 12:0+14:0 can each raise TC in gerbils (Pronczuk et al. 1994). It is possible that the relative lack of 18:2 (referred to elsewhere in physiological terms as "below the 18:2 threshold") (Hayes 1995, Hayes and Khosla 1992, Hayes et al. 1995) is the only fatty acid perturbation that actually modulates LDLr activity in the absence (or possibly even in the presence) of dietary cholesterol. In this model, 18:2 (or its metabolites) would be the only fatty acid capable of mobilizing the regulatory pool of hepatic free cholesterol sufficiently to enhance LDL uptake. This could explain why no significant difference in plasma LDL or hepatic LDLr activity was found in hamsters fed commercial pellets and hydrogenated coconut oil or safflower oil when cholesterol was not added to the diets (Spady and Dietschy 1988), i.e., cholesterol must be added to down-regulate LDL receptors to allow for an 18:2 effect. By the same token, an exaggerated intake of 18:2 in a cholesterol-rich diet often precludes any rise in apoB-rich lipoproteins, including LDL (Horton et al. 1993, Lin et al. 1994, Spady and Dietschy 1985, 1988), indicating that "extra" 18:2 can overcome the cholesterol-induced depression of LDLr activity.

Increased LDL production can result indirectly from increased secretion and conversion of VLDL to LDL and/or the direct production and secretion of LDL by the liver (Goldberg et al. 1983, Huff and Telford 1985, Janus et al. 1980). The latter has been described in humans and animal models, occasionally contributing significantly to LDL entry into the plasma (Goldberg et al. 1983, Huff and Telford 1985). No data are available concerning this process in gerbils, let alone its modulation by fat saturation in this species. However, previous studies in pigs (Huff and Telford 1989) and monkeys (Khosla and Hayes 1991) revealed that direct secretion of LDL can be modulated by dietary fat composition. In miniature pigs, fish oil decreased direct production of LDL and simultaneously increased indirect LDL formation through VLDL lipolysis (Huff and Telford 1989). Dual isotope kinetics with ¹²⁵I -VLDL and ¹³¹I-LDL performed in rhesus monkeys revealed that a 12:0+14:0-rich diet increased the LDL pool by direct production of LDL, whereas 16:0+18:1 used the indirect route (Khosla and Hayes 1991). These data suggest that fat saturation may modulate both routes of plasma LDL formation (direct production as well as the secretion and conversion of VLDL to LDL) independently.

The rate of LDL formation from VLDL depends on the rate of VLDL secretion as well as lipolysis (catabolism). Slowed hepatic lipase mediated-lipolysis prolongs VLDL catabolism and favors conversion to LDL (Tato et al. 1997). In agreement with previous studies (Nicolosi et al. 1976, 1991), VLDL concentration was greater in plasma of food-deprived gerbils that had been fed a diet with 12:0+14:0 as compared with 18:2 (Table 3). However, VLDL-TG secretion was reduced by 12:0+14:0 in this comparison (Nicolosi et al. 1976), suggesting delayed lipolysis as the underlying cause of the triglyceridemia. In fact, studies in humans and animals indicate that VLDL triglycerides rich in saturated fatty acids are hydrolyzed less rapidly than those rich in polyunsaturated fatty acids and remain longer in circulation (Bergeron and Havel 1995, Weintraub et al. 1988), enhancing their chances for conversion to LDL (Tato et al. 1997). Because production rate must equal the catabolic rate of LDL in the steady state, the absolute mass of LDL uptake by the gerbil liver was increased by 12:0+14:0, apparently without compromising LDL receptor activity as measured by the FCR. It would appear that only when a strategic pool of 18:2 (or its metabolites) becomes limited by excessive SFA intake (approximating essential fatty acid depletion at <1% of energy from 18:2) or when cholesterol is present in the diet, do LDL receptors become inhibited sufficiently to depress FCR (Horton et al. 1993, Nicolosi et al. 1992, Spady and Dietschy 1986, 1988). A synergism between saturated fat and cholesterol may encumber the available pool of 18:2, thereby decreasing LDL clearance while adding to the primary problem of LDL overproduction.

A final point made by these data is that the SFA-induced cholesterolemia was not restricted to (in fact, least affected) the LDL pool in this model. This suggests that the nature of the cholesterolemia is species dependent, typically raising HDL more than LDL in an animal species in which HDL is the predominant lipoprotein (Hayes et al. 1995). Thus the resulting expansion in the plasma CE pool in different animal species is more complicated than a reduced number of receptors for a specific clearance site (such as LDL receptors). Accordingly, the definitive resolution of the saturated fatty acid effect must encompass a broader based explanation than LDL receptor abundance. Furthermore, it is obvious that much of the variability in the human cholesterol response to diet derives from the fact that in some cases, the major effect is from fat (e.g., South Sea islanders), whereas in most Western cultures, it is a fat × cholesterol interaction. Assuming the fat effect is primarily on LDL production should augment interpretation of past and future human studies. In most world populations (developing countries), the consumption of cholesterol is still low (true vegetarians, none). So the question how saturated fat behaves in the absence of cholesterol is an important public health issue and more than esoteric, especially to individuals who suffer from hypercholesterolemia and coronary heart disease.

	FOOTNOTES

Manuscript received 3 July 1997. Initial reviews completed 15 August 1997. Revision accepted 15 October 1997.

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