There is controversy over the extent to which fatty acids (FAs) derived from plasma free FAs (FFAs) or from hydrolysis of plasma triglycerides (TGFAs) form communal or separate pools and what the contribution of each FA source is to cellular FA metabolism. Chylomicrons and lipid emulsions were labeled with [3H]triolein, injected into mice, and appearance in plasma of [3H]oleic acid was estimated, either through a steady-state approach or by compartmental modeling. [14C]oleic acid was included to trace plasma FFA. Eighty to 90% of triglyceride (TG) label was recovered in plasma, irrespective of tracer method or TG source. The contribution of TG lipolysis to total plasma FA turnover was 10–20%. After infusion of [3H]TG and [14C]FA, the retention of these labels varied substantially among liver, adipose tissue, and skeletal and heart muscle. Retention of TG label changed during fasting in the same direction as lipoprotein lipase (LPL) activity is regulated. We propose a model that reconciles the paradoxical 80–90% loss of TG label into plasma with LPL-directed differential uptake of TGFA in tissues. In this model, TGFAs mix locally at the capillaries with plasma FFAs, where they would lead to an increase in the local FA concentration, and hence, FA uptake. Our data indicate that a distinction between TG-derived FA and plasma FFA cannot be made.

The transport of fatty acids (FAs) in plasma occurs in two ways: via (albumin-bound) free FAs (FFAs) and via triglycerides (TGs) that are transported in TG-rich lipoproteins. TGs in lipoproteins are hydrolyzed into FAs by the action of lipoprotein lipase (LPL), an enzyme expressed by tissues in need of the FA that is located at the capillary endothelium (1,2). The regulation of LPL is tissue-specific and dependent on the nutritional status, reflecting the FA requirements of the respective tissues at a specific time (1,35). For instance, postprandial LPL activity is high in white adipose tissue (WAT), whereas in the fasted state, LPL activity is high in muscle.

The mechanism(s) by which FA released by LPL activity enters the underlying tissues, and the interaction of this process with plasma FFA, is controversial. The literature appears to disagree regarding almost every aspect concerning the way FAs from both sources are transported into the underlying tissues. One issue is the role of proteins in the transport of FA over membranes (68). Another issue is the relative contribution of plasma FFA and TG to cellular FA metabolism (911). One particularly confusing issue is the extent to which FA derived from plasma TG (TGFA) is recovered in plasma (1113). Views vary from complete and direct uptake of TGFA via some sort of channeling, to some but relatively little escape of TGFA to the circulation (11), to complete mixing of labeled TGFA and FFA (13). It has also been suggested that the amount of FA escaping immediate uptake is a highly regulated process (14,15) and that impairment of this “extraction” or “entrapment” of TG-derived FA may be involved in the pathophysiology of insulin resistance and dyslipidemia.

The confusion may arise from methodological differences, different experimental models, nutritional status, and/or sources of TG-rich particles being employed. In this report, we have resolved some of the confusion by studying the fate of plasma FFA and TGFA in mice by employing different tracer methods, as described in the literature (11,13), and using different TG-rich substrates. Subsequently, we have studied the relative uptake of TGFA and FFA in liver, heart, and skeletal muscle, as well as in adipose tissue, because these organs have different LPL acitvities. In addition, we performed these experiments in the fed and fasted state because these conditions are known to specifically affect tissue LPL activity. We propose a model regarding the interactions between FFA and TGFA that incorporates and reconciles the data.

### Animals.

C57BL/6 mice, aged 3–6 months, were used for all experiments. All mice were housed under standard conditions (lights on 0700–1900 h) with free access to water and regular lab diet. For the isolation of lymph, a Wistar rat was obtained from Charles Rivers. All experiments were approved by the institutional animal care committee.

### Preparation of tracer-labeled materials

#### [3H]TG-labeled chylomicrons.

A male Wistar rat was orally given a mixture of 0.5 ml olive oil with 1 mCi of [9,10(n)-3H]oleic acid, after which the rat was anesthetized with 50 mg/kg nembutal i.p. A canula was placed in the thoracic lymph duct, and lymph was collected for the following 4 h. Lymph was mixed with a protease inhibitor cocktail (1 mmol/l benzamidine, 2 mg/l antipain, and 2 mg/l leupeptine; Sigma) and stored until the next day.

#### [3H]TG- and [3H]cholesteryl ether (CE)-labeled lipid emulsions.

Lipid emulsions were prepared as described by Rensen et al. (16) and separated into three different fractions (average size 45, 80, and 150 nm) by ultracentrifugation. After preparation of the particles, the small (45-nm) particles were enriched with 1 mg purified recombinant apoE (17) per 50 mg TG by incubation at 37°C for 30 min.

#### Preparation of infusion and bolus mixtures.

[14C]oleic acid was complexed to BSA as previously described (18). [14C]oleic acid was mixed with either lymph or lipid emulsions. Citrate was added to a final concentration of 3 μg/ml.

#### Infusion and bolus protocol.

Mice were fasted overnight; all experiments were performed in anesthetized mice (0.5 ml/kg Hypnorm; Janssen Pharmaceutica, Beerse, Belgium and 12.5 mg/kg midazolam; Roche, Mijdrecht, the Netherlands) between 0800 and 1100 h. An infusion needle was placed in the tail vein, attached to a minipump (Harvard microdialysis low flow 11; Harvard Apparatus, Holliston, MA), and chylomicrons or lipid emulsions were infused at a rate of 0.2 ml/h for 1.5–2 h, after which steady state was achieved. A 150-μl blood sample was taken by tail bleeding into paraoxon-coated capillaries to prevent ex vivo lipolysis (19).

For the bolus injection protocol, a single 0.2-ml injection into the tail vein was applied and small 30-μl blood samples were taken at regular time points after injection of the labeled mixture. At the last time point, a larger sample was taken for determination of TG and FA levels.

#### Plasma analysis.

TG, FA, and glucose were determined in plasma as previously described (20). Lipids were extracted from plasma according to Bligh and Dyer (21). The lipid fraction was dried under nitrogen, dissolved into chloroform/methanol (5:1 [vol/vol]) and subjected to TLC (LK5D gel 150; Whatman) using hexane:diethylether:acetic acid (83:16:1 [vol/vol/vol]) as mobile phase. Standards for FA, TG, and cholesteryl oleate were included during the TLC procedure to locate the spots of these lipids. Spots were scraped, lipids dissolved in hexane, and radioactivity measured.

#### Tissue uptake protocol.

One group of mice was fasted overnight and another was allowed to eat ad libitum. A mixture of 45-nm particles labeled with [3H]triolein and [14C]-labeled oleate was used as infusate. After 2 h of infusion, plasma was taken for specific activity measurements, mice were killed, and organs were quickly removed and frozen in liquid nitrogen. Tissues were dissolved in 5 mol/l potassium hydroxide in 50% (vol/vol) ethanol at 70°C for 1 h. After overnight saponification, an aliquot of the solution was neutralized with equimolar HCl, acidified with 1% trichloroacetic acid, and the FA extracted into methanol/chloroform according to Blight and Dyer. Radioactivity in this fraction was measured and corrected for the corresponding plasma specific activities of [3H]FA and [14C]FA.

### Calculations

In steady state, the rate of disappearance (Rd) (μmol/min) can be calculated from the steady-state specific activity of the infused tracer (22):

$R_{\mathrm{d}}{=}\mathit{V}_{\mathrm{in}}/(q/M)$

where Vin is the rate of label infusion (in Bq/min), q is the steady-state radioactivity of label in the total plasma pool (in Bq), and M is the poolsize (in μmol), i.e., q/M represent the specific activity of the compound being traced. The fractional catabolic rate (FCR) is defined as the Rd divided by the pool size:

$\mathrm{FCR}{=}\mathit{R}_{\mathrm{d}}/M$

Substituting Rd using equation 1 gives:

$\mathrm{FCR}{=}\mathit{V}_{\mathrm{in}}/q$

The extent to which [3H]FA from lipolysis of [3H]TG escapes immediate uptake was assessed by the assumption that once [3H]FA and [14C]FA have mixed, the rate of irreversible loss of label is equal for both FAs. The underlying assumption is that equilibration with albumin is much faster than the rate of FA release from LPL. Using the specific activity of [3H]FA and the FCR of [14C]FA, we can estimate the rate at which [3H]FA entered plasma (the “infusion rate of [3H]FA,” Vin,[3H]FA):

$\mathrm{plasma\ TGFA\ recovery}{=}V_{\mathrm{in},{[}3\mathrm{H}{]}\mathrm{FA}}/V_{\mathrm{in},{[}3\mathrm{H}{]}\mathrm{TG}}$
${=}(V_{\mathrm{in},{[}14\mathrm{C}{]}\mathrm{FA}}/q_{{[}14\mathrm{C}{]}\mathrm{FA})}/(V_{\mathrm{in},{[}14\mathrm{C}{]}\mathrm{TG}}/q_{{[}14\mathrm{C}{]}\mathrm{FA}})$

Dividing this rate with the infusion rate of [3H]TG gives the extent to which label that was infused into the circulation as TG ends up in the circulation as FA. This ratio is defined as plasma TGFA recovery:

$\mathrm{plasma\ TGFA\ recovery}{=}V_{\mathrm{in},{[}3\mathrm{H}{]}-\mathrm{FA}}/V_{\mathrm{in},{[}3\mathrm{H}{]}-\mathrm{TG}}$
${=}(V_{\mathrm{in},{[}14\mathrm{C}{]}-\mathrm{FA}}/q_{{[}14C{]}-FA})/(V_{\mathrm{in},{[}3\mathrm{H}{]}-\mathrm{TG}}/q_{{[}3\mathrm{H}{]}-\mathrm{FA}})$

The plasma TGFA recovery, as calculated in this manner, does not discriminate between FAs being hydrolyzed without mixing with albumin-bound FA and FAs that are taken up as TG, i.e., by endocytosis of the whole particle. Therefore, we included labeled CE as a marker for whole-particle uptake when we used the lipid emulsion and followed the bolus experimental protocol (see below).

### Compartmental modeling.

The changes in time of TG- and FA-associated labels were fitted with a compartmental model (Fig. 1), essentially as developed by Hultin et al. (13). Where Hultin used two compartments for the FA label and a variable fraction in blood (or volume of distribution), we found better fits for both [3H]FA and [14C]FA when we used three compartments for FA. Thus, we explicitly modeled the rapid loss of label by redistribution and set the fraction in blood to 1. A single compartment with a variable volume of distribution sufficed to describe the TG kinetics accurately. The metabolic simulation software package Gepasi (23) was employed to estimate initial values for the kinetic parameters of the model. Gepasi has powerful algorithms to obtain absolute regression minima without the need for good estimates of the initial parameters (24). WINSAAM (25) was subsequently used for fine-tuning by specifying errors in each time point.

The plasma TGFA recovery, as defined in equation 5, can be calculated from the compartmental modeling parameters as:

$\mathrm{plasma\ TGFA\ recovery}{=}k_{3,1}/(k_{0,1}{+}k_{3,1})$

The contribution of TG-derived FA to the total FA flux was estimated as:

$\mathrm{fraction\ FA\ flux\ from\ TG}{=}\mathrm{plasma\ TGFA\ recovery}{\times}(3R_{\mathrm{d,TG}}/R_{\mathrm{d,FA}})$

This equation assumes that 1 mol of TG releases 3 mol of FA. Theoretically, this should be considered as the upper boundary because formation of di- and monoacylglycerol are ignored. We cannot discriminate between label being taken up as acylglycerols or FA, but since the plasma TGFA recovery was high (see Table 1), assuming complete lipolysis of TGFA (with release into the plasma according to the plasma TGFA recovery) should quantitatively give a reasonable estimate of the contribution of TGFA lipolysis to the FA flux.

### Statistical analysis.

All data are presented as means ± SD of parameters obtained in individual mice. Nonparametric Mann-Whitney U tests were used for all statistical analyses. A P value of 0.05 was taken as boundary of significance.

[3H]TG-labeled rat chylomicrons were mixed with [14C]-labeled oleic acid, injected intravenously into anesthetized mice, and the behavior of the tracers were followed in plasma in time. [3H]TG activity decayed monoexponentially (Fig. 2). [14C]FA activity decreased very rapidly. [3H]FA activity initially increased in plasma and then started to decrease slightly. All data were fitted to a four-compartmental model (Fig. 1). Kinetic constants of [3H]FA and [14C]FA were forced to be identical. The solid lines in Fig. 2 show the fit of the model. The model (and all other models that we have tried) required (almost) all [3H]TG to be transferred to the FA compartments to get an optimal fit (Table 1). In three of five fits, k0,1 of the model even fell to zero during the fitting procedure (i.e., complete recovery of label in plasma was required). On average, the plasma TGFA recovery was 88%. From the model, FCR and Rd were computed for [14C]FA and [3H]TG (Table 1). From the Rd and plasma TGFA recovery, an estimate can be made to what extent TG-derived FAs contribute to the total FA flux (see equation 7). For the chylomicrons, it was estimated that 19% of the total FA flux was derived from lipolysis of TG.

Table 1 also shows the results of the steady-state infusion experiment. Steady state was reached within 1.5 h. In line with the result from the compartmental modeling, 81% of the infused [3H]TG activity was recovered as [3H]FA. The FCRs for TG and FA, calculated by the steady-state approach, also agreed well with those found with the compartmental modeling (Table 1).

After establishing that both tracer methods yielded the same results, we tested whether these results were specific for chylomicrons or whether they also apply to smaller TG-rich particles. Therefore, we used lipid emulsions and enriched these particles (average particle size 45 nm [16]) with apoE, which is a good ligand to the VLDL receptor (26). Because of the small size and presence of apoE, we expected that whole-particle uptake could lead to a quantitatively significant underestimation of the plasma recovery of TGFA (because TG-label not being lipolyzed but taken up as whole particle could not be distinguished from TG label being lipolyzed into FA and then being taken up without prior mixing with the plasma FA pool). Therefore, we included [3H]-labeled CE as a marker for particle uptake and used the bolus injection protocol to follow the CE kinetics.

Again, TG-associated label was lost from plasma in a monoexponential manner, whereas FA kinetics needed at least two compartments (Fig. 3). CE label decayed much slower than the TG label, indicating that in the first 15 min, lipolysis of TG dominated over whole-particle uptake. The initial volume of distribution was equal for both labels, which should be the case because they are part of the same particle. [3H]FA label rapidly appeared in plasma and subsequently disappeared. In accordance with the chylomicron data, 89% of the label had to enter the plasma FA compartment to obtain a good fit. It was estimated that 11% of the total FA flux was originating from TG-derived FA (Table 1).

To estimate the relative contribution of TG-lipolysis and plasma FFA to the total amount of FA taken up by tissues, we measured label retention after a 2-h infusion of [3H]TG-labeled lipid emulsion and [14C]FA in the liver, adipose tissue, and skeletal and heart muscle. [3H] counts originating from TG and [14C] counts from FFA were measured in tissue extracts and taken as representative of the uptake of the respective substrates. This experiment was carried out in fed animals and in mice that were fasted overnight. Large differences in retention of label existed between tissues, and significant differences between the fasted and fed state were observed for liver, skeletal muscle, and WAT but not for heart muscle (Fig. 4). Relative uptake of [3H]TG versus [14C]FA was highest in heart muscle (Fig. 4C) and lowest in liver (Fig. 4D), irrespective of nutritional status. In adipose tissue, absolute [3H] label in the TG store was twofold higher in the fed state (Fig. 4A), but because [14C]FA label retention was also increased, the relative [3H]TG retention did not significantly change. In skeletal muscle, [3H]TG retention was higher in the fasted state, both absolutely (Fig. 4B) and relative to [14C]FA retention (Fig. 4E).

In the present study, we have studied the fate of FA derived from lipolysis of plasma TG. We observed that 80–90% of infused TG tracer could be recovered as plasma FA, irrespective of tracer method or TG-rich particle used. It may appear puzzling that the small amount of [3H]FA label found in plasma in Figs. 2 and 3 indicates that most of the [3H]TG label entered plasma as [3H]FA. This is caused by the very rapid turnover of plasma FFA (see [14C]FA curves in Figs. 2 and 3). With the lower turnover of TG, the “infusion” rate of [3H]FA is relatively slow compared with the turnover of [3H]FA, and therefore much of the label is rapidly cleared. We have not included the individual rate constants of the FA kinetics, as they are very dependent on the model structure, whereas the overall kinetic constants, the FCR and Rd, are related to the area under the curve of the tracer and are therefore robust to details of the model. It is the FCR of FA that determines the plasma TGFA recovery, not the individual rate constants, an argument also made by Hultin et al. (13). Other studies have probed FA kinetics in mice in greater detail (27).

The high recovery of TGFA in plasma is in agreement with the compartmental modeling studies in rats (13). It does not agree, however, with the steady-state data presented by Wolfe et al. (11). Their conclusion was that only 5% of the total FA flux originated from VLDL-derived FA. This implies that the plasma TGFA recovery in their system was very small. However, the authors used the specific activity of the infusate (i.e., of FA in the VLDLTG infusate) to calculate the flux from VLDL to FFA rather than the specific activity of plasma VLDLTG itself. In our view, the latter is the proper precursor enrichment to calculate rate of production of FFA from TG.

When >80% of FA generated by LPL is lost in plasma, one may raise doubts as to the usefulness of tissue-specific regulation of LPL activity. Yet, we have measured enormous differences in the ratio of [3H]FA and [14C]FA retention between tissues, which were also dependent on the fed or fasting state (Fig. 4). Hultin et al. (13) found similar results, as well as observed that the ratio of [3H]FA and [14C]FA depended on the nutritional status. The observed [3H]/[14C] ratios and the changes therein upon fasting reflect well-documented differences in LPL activity in these tissues (3,5). Thus, liver does not have LPL activity, and its relative retention of [3H]TG is low (Fig. 4). The uptake of [3H] label in liver most probably reflects uptake of particle remnants that occurs primarily in liver. Skeletal muscle LPL, however, is upregulated during fasting and the relative amount of [3H]TG label measured was indeed higher in the fasted state (Fig. 4E). In WAT, the uptake of [3H]TG was expected to be higher in the fed state because LPL is insulin stimulated in WAT (5,28). This is also reflected in the total amount of [3H] counts (Fig. 4A) but not in the ratio of [3H] to [14C] label retention (Fig. 4E). In WAT, FA kinetics are complicated by the fact that in the fasted state, there is substantial intracellular FA hydrolysis of TG stores. It is clear from these data, however, that there is differential uptake of TGFA and FFA, which clearly follows the activity of LPL in these tissues. It is also important to note that in extrahepatic tissues, TGFA is (at least) equally important as plasma FFA, a conclusion that is in line with previous work (11,29).

The combination of near complete recovery of TGFA label in plasma and yet considerable differential uptake of these labels in the different tissues appears paradoxical. The paradox can be solved from the perspective that the labels actually enter the system at different sites. The simplest model that can solve the paradox is the capillary model (22). This model consists of three compartments: an arterial compartment (A), a venous compartment (V), and a compartment representing the capillary bed (C) (Fig. 5). In this model, we assume complete mixing of labels in compartment C, i.e., those (labeled) FA molecules that are released by LPL activity enter the same compartment, compartment C, as FFAs arriving from the circulation. The only difference for [14C] and [3H] label is the site of injection: for [14C]FA, label enters the general circulation via the vein; for [3H]TGFA, the site of “injection” is the capillary bed (where LPL is active).

The steady-state solution of this system for [14C]FA tracer is:

$q_{\mathrm{A}}/M_{\mathrm{A}}{=}q_{\mathrm{c}}/M_{\mathrm{c}}{=}V_{\mathrm{in}}/R_{\mathrm{d}}$

where q/M is the specific activity of tracer (see research design and methods), Vin the tracer infusion rate, and Rd is the rate of disappearance into tissues, in this system k0CMC. This is the classical solution to estimate Rd from specific activities of infusion experiments (i.e., equation 1).

For [3H]TGFA tracer, however, “injection” is at the capillary bed, where LPL is located, and the steady-state solution of such a system is different:

$q_{\mathrm{A}}/M_{\mathrm{A}}{=}V_{\mathrm{LPL}}/R_{\mathrm{d}}{\times}k_{\mathrm{vc}}/(k_{\mathrm{vc}}{+}k_{0\mathrm{c}})$
$q_{\mathrm{c}}/M_{\mathrm{c}}{=}V_{\mathrm{LPL}}/R_{\mathrm{d}}$

Here, VLPL rather than Vin is used to stress the fact that the “infusion rate” is determined by the local LPL activity. It follows that the capillary specific activity (qC/MC) differs from the one at the sampling site (qA/MA) with a factor that depends on the relative amount of label escaping immediate uptake. It should be noted that this ratio corresponds to the plasma TGFA recovery defined in equation 5. It is also related to the FA entrapment, as defined by Evans et al. (15).

In a system with multiple organs, the differences between qc/Mc and qA/MA become even larger because the local “infusion rates” VLPL are determined by local LPL activity and may therefore differ greatly from the overall systemic “infusion rate” that determines the arterial specific activity. This model can therefore explain how a steady-state infusion model with complete local mixing of [14C] and [3H] label can give rise to differential uptake of labels according to LPL activity.

Figure 6 illustrates a proposal of the physiology underneath the behavior of the tracers. The activity of LPL is proposed to increase the local pool of FA close to the endothelium (Fig. 6). The relative sensitivity of the uptake rate toward this increase in local FA therefore decides how much of the newly formed FA will be extracted and metabolized and how much will end up in venous plasma. Thus, tissue-specific regulation of LPL would allow for the production of a local increase in FA that then drives the (facilitated) diffusion of FA into the underlying cells.

This mechanism has implications for the current methods in experimental animals to measure tissue-specific uptake of FA, i.e., by FA analogs (BMIPP, bromopalmitate, or palmitate only [30,31]). These methods do not take into account the tissue-specific contribution of LPL to the local FA pool that may affect the local rate of label transfer. The proposed mechanism may have further implications for the interpretation of traditional tracer studies of plasma FFA turnover and oxidation. The release of FAs from adipose tissue and plasma fatty oxidation has been studied intensively by primed continuous infusions of labeled palmitate. Our data indicate that the release of FAs by adipose tissue in these studies may have been overestimated by the extent to which TGFA have contributed to the plasma FFA pool. Accordingly, the data on plasma FA oxidation include the contribution of unlabeled plasma TG-derived FAs to the plasma FA pool. However, the quantitative impact of the present data on these parameters in humans is uncertain.

In conclusion, our data indicate that the distinction between TGFA and plasma FFA is artificial. Traditionally, plasma TG metabolism and FFA metabolism are being studied in separate research areas; TG metabolism is mainly being studied in the context of cardiovascular disease and FFA metabolism in the context of diabetes. We propose a model in which FAs released from LPL are locally in rapid exchange with plasma FFAs and serve the important purpose of maintaining a driving force for (facilitated) diffusion. This model therefore embraces the importance of LPL and combines it with that of other factors involved in FA metabolism, such as acylation stimulating protein (32) and FAT/CD36 (6,30). Understanding these interactions between lipoprotein metabolism and FFA metabolism is crucial for understanding complex metabolic diseases such as the insulin resistance syndrome.

FIG. 1.

Compartmental model used for the fitting of the TG and FA tracer kinetics. One compartment was used for TG and three for FA. Ki,j, rate constant for label transfer; dashed line with open circle, site of sampling (i.e., plasma compartment). Sites of injection of [3H] (white) and [14C] (gray) are indicated by the syringe.

FIG. 1.

Compartmental model used for the fitting of the TG and FA tracer kinetics. One compartment was used for TG and three for FA. Ki,j, rate constant for label transfer; dashed line with open circle, site of sampling (i.e., plasma compartment). Sites of injection of [3H] (white) and [14C] (gray) are indicated by the syringe.

FIG. 2.

Kinetic behavior of TG and FA tracers after a bolus injection of a mixture of rat chylomicrons labeled with [3H]triolein and albumin-bound FA labeled with [14C]oleate. Data are presented as percentage of injected label found back as TG or FA in plasma. Solid lines indicate the best fit of the model of Fig. 1. □, [3H]TG; ○, [3H]FA; •, [14C]FA.

FIG. 2.

Kinetic behavior of TG and FA tracers after a bolus injection of a mixture of rat chylomicrons labeled with [3H]triolein and albumin-bound FA labeled with [14C]oleate. Data are presented as percentage of injected label found back as TG or FA in plasma. Solid lines indicate the best fit of the model of Fig. 1. □, [3H]TG; ○, [3H]FA; •, [14C]FA.

FIG. 3.

Kinetic behavior of TG, CE, and FA tracers after a bolus injection of a mixture of lipid emulsion labeled with [3H]CE (▴), [3H]TG (□), [3H]FA (○), and [14C]FA (•). Solid lines indicate the best fit of the model of Fig. 1.

FIG. 3.

Kinetic behavior of TG, CE, and FA tracers after a bolus injection of a mixture of lipid emulsion labeled with [3H]CE (▴), [3H]TG (□), [3H]FA (○), and [14C]FA (•). Solid lines indicate the best fit of the model of Fig. 1.

FIG. 4.

Retention of [3H]TG and [14C]FA label in different tissues in the fed (□) and fasted (▪) state. After primed continuous infusion for 2 h, label content in lipids was measured and corrected for the specific activities in plasma of [3H]FA and [14C]FA, respectively. A: WAT; B: skeletal muscle; C: heart muscle; D: liver; E: ratio of label retention. *P < 0.05.

FIG. 4.

Retention of [3H]TG and [14C]FA label in different tissues in the fed (□) and fasted (▪) state. After primed continuous infusion for 2 h, label content in lipids was measured and corrected for the specific activities in plasma of [3H]FA and [14C]FA, respectively. A: WAT; B: skeletal muscle; C: heart muscle; D: liver; E: ratio of label retention. *P < 0.05.

FIG. 5.

Capillary model explaining the difference between [3H]FA derived from TG and plasma [14C]FFA. A, artery; C, capillary bed; V, vein. Other symbols are explained in Fig. 1.

FIG. 5.

Capillary model explaining the difference between [3H]FA derived from TG and plasma [14C]FFA. A, artery; C, capillary bed; V, vein. Other symbols are explained in Fig. 1.

FIG. 6.

Model of the interactions between plasma FFA and LPL-derived TGFA. A: FA released by LPL rapidly mix with plasma FFA (bound to albumin, not drawn). B: Putative profile of FA concentration in space: LPL activity increases the local concentration of FAs close to the endothelium, driving the uptake of FAs into the underlying tissues (indicated by large arrows). C: Absence of LPL activity results in lower uptake of FAs (indicated by dashed arrows).

FIG. 6.

Model of the interactions between plasma FFA and LPL-derived TGFA. A: FA released by LPL rapidly mix with plasma FFA (bound to albumin, not drawn). B: Putative profile of FA concentration in space: LPL activity increases the local concentration of FAs close to the endothelium, driving the uptake of FAs into the underlying tissues (indicated by large arrows). C: Absence of LPL activity results in lower uptake of FAs (indicated by dashed arrows).

TABLE 1

Comparison of kinetic parameters and plasma TGFA recovery for chylomicrons and lipid emulsions

Kinetic parameterChylomicrons
Lipid emulsions (bolus)
FCR TG (min−10.15 ± 0.06 0.29 ± 0.10 0.09 ± 0.03
Rd TG (μmol · min−1 · kg−14.9 ± 0.9 ND 2.1 ± 0.7
FCR FA 3.02 ± 0.45 3.52 ± 0.90 3.26 ± 0.91
Rd FA (μmol · min−1 · kg−166 ± 3 ND 57 ± 13
Plasma TGFA recovery 0.88 ± 0.15 0.81 ± 0.22 0.89 ± 0.14
Fraction FA flux from TG 0.19 ± 0.03 ND 0.11 ± 0.03
Kinetic parameterChylomicrons
Lipid emulsions (bolus)
FCR TG (min−10.15 ± 0.06 0.29 ± 0.10 0.09 ± 0.03
Rd TG (μmol · min−1 · kg−14.9 ± 0.9 ND 2.1 ± 0.7
FCR FA 3.02 ± 0.45 3.52 ± 0.90 3.26 ± 0.91
Rd FA (μmol · min−1 · kg−166 ± 3 ND 57 ± 13
Plasma TGFA recovery 0.88 ± 0.15 0.81 ± 0.22 0.89 ± 0.14
Fraction FA flux from TG 0.19 ± 0.03 ND 0.11 ± 0.03

Data are means ± SD. Parameters were calculated for each mouse. ND, not determined.

This work was conducted in the framework of the Leiden Center for Cardiovascular Research LUMC-TNO and supported by the Dutch Heart Foundation, (grant NHS 96011) and the Netherlands Organization for Research NWO (grants 903-39-194 and 903-39-291).

The authors thank anonymous referees for useful comments and discussion.

1.
Zechner R: The tissue-specific expression of lipoprotein lipase: implications for energy and lipoprotein metabolism.
Curr Opin Lipidol
8
:
77
–88,
1997
2.
Olivecrona T, Hultin M, Bergo M, Olivecrona G: Lipoprotein lipase: regulation and role in lipoprotein metabolism.
Proc Nutr Soc
56
:
723
–729,
1997
3.
Goldberg IJ, Merkel M: Lipoprotein lipase: physiology, biochemistry, and molecular biology.
Front Biosci
6
:
D388
–405,
2001
4.
Zechner R, Strauss J, Frank S, Wagner E, Hofmann W, Kratky D, Hiden M, Levak-Frank S: The role of lipoprotein lipase in adipose tissue development and metabolism.
Int J Obes Relat Metab Disord
24 (Suppl. 4)
:
S53
–S56,
2000
5.
Olivecrona G, Olivecrona T: Triglyceride lipases and atherosclerosis.
Curr Opin Lipidol
6
:
291
–305,
1995
6.
Febbraio M, Hajjar DP, Silverstein RL: CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism.
J Clin Invest
108
:
785
–791,
2001
7.
Glatz JF, Storch J: Unravelling the significance of cellular fatty acid-binding proteins.
Curr Opin Lipidol
12
:
267
–274,
2001
8.
Hamilton JA, Kamp F: How are free fatty acids transported in membranes? Is it by proteins or by free diffusion through the lipids?
Diabetes
48
:
2255
–2269,
1999
9.
Linder C, Chernick SS, Fleck TR, Scow RO: Lipoprotein lipase and uptake of chylomicron triglyceride by skeletal muscle of rats.
Am J Physiol
231
:
860
–864,
1976
10.
Ferraro RT, Eckel RH, Larson DE, Fontvieille AM, Rising R, Jensen DR, Ravussin E: Relationship between skeletal muscle lipoprotein lipase activity and 24-hour macronutrient oxidation.
J Clin Invest
92
:
441
–445,
1993
11.
Wolfe RR, Durkot MJ: Role of very low density lipoproteins in the energy metabolism of the rat.
J Lipid Res
26
:
210
–217,
1985
12.
Scow RO: Metabolism of cyhlomicrons in perfused adipose and mammary tissue of the rat.
Fed Proc
36
:
182
–185,
1977
13.
Hultin M, Savonen R, Olivecrona T: Chylomicron metabolism in rats: lipolysis, recirculation of triglyceride-derived fatty acids in plasma FFA, and fate of core lipids as analyzed by compartmental modelling.
J Lipid Res
37
:
1022
–1036,
1996
14.
Riemens SC, Sluiter WJ, Dullaart RP: Enhanced escape of non-esterified fatty acids from tissue uptake: its role in impaired insulin-induced lowering of total rate of appearance in obesity and type II diabetes mellitus.
Diabetologia
43
:
416
–426,
2000
15.
Evans K, Burdge GC, Wootton SA, Clark ML, Frayn KN: Regulation of dietary fatty acid entrapment in subcutaneous adipose tissue and skeletal muscle.
Diabetes
51
:
2684
–2690,
2002
16.
Rensen PC, Herijgers N, Netscher MH, Meskers SC, Van Eck M, van Berkel TJ: Particle size determines the specificity of apolipoprotein E-containing triglyceride-rich emulsions for the LDL receptor versus hepatic remnant receptor in vivo.
J Lipid Res
38
:
1070
–1084,
1997
17.
Vogel T, Weisgraber KH, Zeevi MI, Ben-Artzi H, Levanon AZ, Rall SCJ, Innerarity TL, Hui DY, Taylor JM, Kanner D: Human apolipoprotein E expression in Escherichia coli: structural and functional identity of the bacterially produced protein with plasma apolipoprotein E.
Proc Natl Acad Sci U S A
82
:
8696
–8700,
1985
18.
Jong MC, van Ree JH, Dahlmans VE, Frants RR, Hofker MH, Havekes LM: Reduced very-low-density lipoprotein fractional catabolic rate in apolipoprotein C1-deficient mice.
Biochem J
321
:
445
–450,
1997
19.
Zambon A, Hashimoto SI, Brunzell JD: Analysis of techniques to obtain plasma for measurement of levels of free fatty acids.
J Lipid Res
34
:
1021
–1028,
1993
20.
Jong MC, Voshol PJ, Muurling M, Dahlmans VE, Romijn JA, Pijl H, Havekes LM: Protection from obesity and insulin resistance in mice overexpressing human apolipoprotein C1.
Diabetes
50
:
2779
–2785,
2001
21.
Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification.
Can J Biochem Biophys
37
:
911
–917,
1959
22.
Wolfe RR:
Radioactive and Stable Isotope Tracers in Biomedicine.
New York, Wiley-Liss,
1992
23.
Mendes P: Biochemistry by numbers: simulation of biochemical pathways with Gepasi 3.
Trends Biochem Sci
22
:
361
–363,
1997
24.
Mendes P, Kell D: Non-linear optimization of biochemical pathways: applications to metabolic engineering and parameter estimation.
Bioinformatics
14
:
869
–883,
1998
25.
Greif P, Wastney M, Linares O, Boston R: Balancing needs, efficiency, and functionality in the provision of modeling software: a perspective of the NIH WinSAAM Project.
445
:
3
–20,
1998
26.
Tacken PJ, Hofker MH, Havekes LM, van Dijk KW: Living up to a name: the role of the VLDL receptor in lipid metabolism.
Curr Opin Lipidol
12
:
275
–279,
2001
27.
Baker N, Gan-Elepano M, Guthrie BA, Mead JF: Delayed recycling of plasma FFA in mice: revised model of turnover and oxidation.
Am J Physiol
253
:
R746
–R755,
1987
28.
Olivecrona T, Bergo M, Hultin M, Olivecrona G: Nutritional regulation of lipoprotein lipase.
Can J Cardiol
11 (Suppl. G)
:
73G
–78G,
1995
29.
Evans RD, Bennett MJ, Hauton D: Perfused heart studies to investigate lipid metabolism.
Biochem Soc Trans
28
:
113
–120,
2000
30.
Coburn CT, Knapp FFJ, Febbraio M, Beets AL, Silverstein RL, Abumrad NA: Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice.
J Biol Chem
275
:
32523
–32529,
2000
31.
Oakes ND, Kjellstedt A, Forsberg GB, Clementz T, Camejo G, Furler SM, Kraegen EW, Olwegard-Halvarsson M, Jenkins AB, Ljung B: Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer.
J Lipid Res
40
:
1155
–1169,
1999
32.
Sniderman AD, Maslowska M, Cianflone K: Of mice and men (and women) and the acylation-stimulating protein pathway.
Curr Opin Lipidol
11
:
291
–296,
2000

Address correspondence and reprint requests to Peter J. Voshol, TNO Prevention and Health, Gaubius Laboratory, PO Box 2215, NL-2301 CE Leiden, The Netherlands. E-mail: pj.voshol@pg.tno.nl.

Received for publication 16 May 2002 and accepted in revised form 3 December 2002.

B.T. and P.J.V. contributed equally to this work.

B.T. is currently located at NIZO Food Research, PO Box 20, NL-6710 BA Ede, The Netherlands.

CE, cholesteryl ether; FA, fatty acid; FCR, fractional catabolic rate; FFA, free FA; LPL, lipoprotein lipase; Rd, rate of disappearance; TG, triglyceride; TGFA, FA derived from plasma TG; WAT, white adipose tissue.