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Am J Physiol Endocrinol Metab 288: E1229-E1235, 2005. First published February 8, 2005; doi:10.1152/ajpendo.00273.2004
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Perfusion of hearts with triglyceride-rich particles reproduces the metabolic abnormalities in lipotoxic cardiomyopathy

Priya Pillutla,1 Yuying C. Hwang,2 Ayanna Augustus,1 Masayoshi Yokoyama,1 Hiroaki Yagyu,1 Thomas P. Johnston,3 Michiyo Kaneko,2 Ravichandran Ramasamy,2 and Ira J. Goldberg1

Departments of 1Medicine and 2Surgery, Columbia University, New York, New York; and 3Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri, Kansas City, Missouri

Submitted 24 June 2004 ; accepted in final form 4 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hearts with overexpression of anchored lipoprotein lipase (LpL) by cardiomyocytes (hLpLGPI mice) develop a lipotoxic cardiomyopathy. To characterize cardiac fatty acid (FA) and triglyceride (TG) metabolism in these mice and to determine whether changes in lipid metabolism precede cardiac dysfunction, hearts from young mice were perfused in Langendorff mode with [14C]palmitate. In hLpLGPI hearts, FA uptake and oxidation were decreased by 59 and 82%, respectively. This suggests reliance on an alternative energy source, such as TG. Indeed, these hearts oxidized 88% more TG. Hearts from young hLpLGPI mice also had greater uptake of intravenously injected cholesteryl ester-labeled Intralipid and VLDL. To determine whether perfusion of normal hearts would mimic the metabolic alterations found in hLpLGPI mouse hearts, wild-type hearts were perfused with [14C]palmitate and either human VLDL or Intralipid (0.4 mM TG). Both sources of TG reduced [14C]palmitate uptake (48% with VLDL and 45% with Intralipid) and FA oxidation (71% with VLDL and 65% with Intralipid). Addition of either heparin or LpL inhibitor P407 to Intralipid-containing perfusate restored [14C]palmitate uptake and confirmed that Intralipid inhibition requires local LpL. Our data demonstrate that reduced FA uptake and oxidation occur before mechanical dysfunction in hLpLGPI lipotoxicity. This physiology is reproduced with perfusion of hearts with TG-containing particles. Together, the results demonstrate that cardiac uptake of TG-derived FA reduces utilization of albumin-FA.

lipotoxicity; triglyceride; fatty acid metabolism; lipoprotein lipase

LONG-CHAIN FATTY ACIDS (FA) comprise the main fuel source for the heart and meet up to 70–80% of cardiac energy needs (1, 18). FA may be supplied to the heart in three ways (2). First, triglycerides (TG) in adipose tissue may be hydrolyzed by hormone-sensitive lipase into FA. These FA circulate as a complex with albumin. Second, myocytes can take up whole lipoprotein particles containing core TG, which can be hydrolyzed intracellularly to yield FA. Finally, local lipolysis of circulating lipoproteins [chylomicrons and very-low-density lipoproteins (VLDL)] by lipoprotein lipase (LpL) within surrounding capillary beds generates free FA (FFA) (9).

The heart normally metabolizes FA immediately; it has little capacity for storage (18). However, excess cardiac lipid is thought to cause cardiomyopathy in human conditions such as inborn errors of metabolism, diabetes, and obesity (4, 8, 13). Animal models of lipotoxic cardiomyopathy have been created that reproduce the abnormalities seen when the heart's ability to oxidize FA is exceeded (5).

Our laboratory has created a novel model of lipotoxic cardiomyopathy, [the hLpLGPI mouse, a mouse that is express-anchored to cardiomyocytes via a glycosylphosphatidylinositol anchor (19).] We have previously reported that these mice have normal plasma lipids but increased FA and cholesterol within the myocardium (19). Although young mice (2–3 mo) do not have cardiac dysfunction, older animals (>4 mo) develop a dilated cardiomyopathy and die prematurely. This model allowed us to determine if cardiac lipid metabolic defects precede cardiomyopathy.

We investigated cardiac FA oxidation in LpLGPI transgenic mice. First, we compared uptake and oxidation of [14C]palmitate and [14C]triolein in wild-type and hLpLGPI mouse hearts. Next, we determined FA metabolism in wild-type hearts perfused with [14C]palmitate with or without VLDL or Intralipid; Intralipid is a surrogate for TG-rich lipoproteins. Finally, we assessed the role of LpL in the metabolism of Intralipid using two compounds, heparin and poloxamer 407 (P407; a novel lipase inhibitor). Our data show that hearts from both hLpLGPI and wild-type mice perfused with TG-rich particles have reduced FFA uptake and oxidation. Therefore, hearts use TG-derived FA as an alternative fuel and, in some situations, as the primary source of FA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All experiments were conducted using male C57BL/6 mice that were 2–3 mo old and weighed 20–30 g. Animals were housed in a room undergoing a 12:12-h light-dark cycle and were provided access to standard chow and water ad libitum.

A number of experiments were performed in transgenic mice overexpressing a cardiomyocyte-anchored form of LpL. These mice, termed hLpLGPI, are described in a previous publication (19). hLpLGPI mice develop a lipotoxic cardiomyopathy with age. They have normal circulating levels of lipoproteins and FFA.

Isolated heart preparations. For metabolic studies, an isovolumic isolated Langendorff heart preparation was used, as reported previously (11). Mice were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection. Anticoagulation was not performed, because heparin displaces LpL from its binding site. After deep anesthesia was achieved, a thoracotomy was performed and the heart rapidly excised. Hearts were perfused with modified Krebs-Henseleit buffer containing (in mM) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 5 glucose, 0.4 palmitate, and 0.4 BSA and 70 mU/l insulin. The perfusate was equilibrated with a mixture of 95% O2-5% CO2, which maintained perfusate PO2 at >600 mmHg. Left ventricular developed pressure, heart rate, and coronary perfusion pressure were continuously monitored using an eight-channel AD Instruments physiological recorder, as described previously (11), to ensure the viability of the preparation. There were no differences in these physiological measurements between control and LpLGPI hearts. Additional labeled substrates were then added as indicated, and the heart was perfused in recirculating mode for 60 min, after which perfusion was continued for 5 min with unlabeled buffer in nonrecirculating mode to remove extracellular substrate. The ventricles were frozen, and 4-ml aliquots of perfusate were saved for analysis by combining with 800 µl of 3 N NaOH.

Lipid tracers. Hearts from hLpLGPI and wild-type mice were perfused with [1-14C]palmitate to measure palmitate uptake and oxidation. The recycling heart perfusions contained modified Krebs buffer with BSA, 0.4 mM unlabeled palmitate, and [1-14C]palmitate (80,000 dpm/ml). Steady-state oxidative rates of palmitate were determined by measurement of 14CO2, as described previously (11). Palmitate oxidation rates were expressed as nanomoles of palmitate oxidized per minute per gram of dry weight. In other experiments, oxidation of TG-derived FA was determined by including 0.4 mM [14C]triolein in the same buffer.

In some experiments, an equal molar concentration of 0.4 mM VLDL or Intralipid (Kabi Pharmacia, Clayton, NC), an emulsion containing particles of similar size and shape to chylomicrons (10), was added to the perfusions. VLDL was isolated from fasting blood taken from normolipidemic male subjects (plasma TG levels <150 mg/dl). Plasma was mixed with density 1.006 g/ml buffer and subjected to ultracentrifugation for 24 h at 40,000 rpm in an SW40 rotor. VLDL was aspirated as the floating lipid layer.

Inhibition and dissociation of LpL. To assess the role of LpL in the metabolism of Intralipid, we altered LpL actions by use of two different methods. In the first, we added heparin (10 U/ml) to the perfusion buffer (along with [14C]palmitate and 0.4 mM Intralipid) and allowed it to remain in the system throughout recirculation. Heparin releases LpL from its binding sites on the endothelium. In other experiments, we added the lipase inhibitor P407 (12) to the perfusion buffer together with [14C]palmitate and Intralipid. Like Triton, this compound is thought to coat the lipid particle and prevent it from interacting with LpL.

Metabolic studies. Uptake of radiolabeled FA in Intralipid was determined by calculating the amount of FA removed from the perfusion buffer during the experiment. FA oxidation was determined by measuring the formation of [14C]CO2 (15). Aliquots (500 µl) of perfusate were treated with acid or base (500 µl each), and excess CO2 was driven out by treating with nitrogen gas for 5 min. Scintillation fluid (Ecoscint, National Diagnostics) was added, and samples were counted for 14C (Beckman LS500TD). One group of hearts was dried after perfusion to calculate the wet- to dry-weight ratio, which was used to calculate the dry weights of other hearts. This allowed us to normalize the metabolic rates by heart weight. In a separate experiment, ATP was measured in five control and five LpLGPI heart extracts by HPLC methods as described (15).

Tissue lipid analysis. Ventricular tissue was homogenized with a polytron TH-115 (Omni International). Lipids were isolated by extraction into chloroform-methanol (2:1). After centrifugation, scintillation fluid was added to the chloroform extracts, and samples were counted for 14C (Wallac model 1400). The lower chloroform layer was dried down under nitrogen gas, resuspended in 50 µl of chloroform-methanol (2:1), and applied twice onto the thin-layer chromatography (TLC) plate (VWR 5748-7). A neutral solvent system [hexane-ether-acetic acid (70:30:1)] was used to separate extracts into cholesterol esters (CE), triacylglycerols (TG), phospholipids (PL), and FFA. Individual lipid classes were then visualized by I2 and scraped from the plates into scintillation vials.

All values for lipid analysis are presented as means ± SE. Statistical evaluation between two groups was by Student's t-test.

In vivo studies. FA turnover utilized [1-14C]palmitate in ethanol (specific activity 56 mCi/mmol; NEN Life Sciences, Boston, MA) complexed to 6% FA-free BSA (Sigma Aldrich) as described (6). Heart uptake of Intralipid was studied using a nonhydrolyzable core lipid. To prepare this, 20% Intralipid was diluted in sterile PBS to a final 5% concentration and labeled with 40 µCi of [3H]cholesteryl oleoyl ether (Amersham Pharmacia Biotech), as described by van Bennekum et al. (17). Label was added to a small glass vial and slowly evaporated to dryness under N2. Five hundred microliters of a 5% solution of Intralipid were added to the small glass vial and sonicated three times for 20 s at a power level of 40 W to incorporate the labeled CE into the emulsion. The resulting emulsion was stored at 4°C before use in experiments. We injected 300,000 cpm of [14C]palmitate and 1 x 106 cpm of [3H]cholesteryl oleoyl ether simultaneously into each mouse. In addition, human VLDL was double labeled with [3H]cholesteryl ether and [14C]TG by use of cholesteryl ester transfer protein (17) and used as a tracer to compare uptake of a hydrolyzable and nonmetabolized lipid.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Palmitate metabolism in lipotoxic hearts. hLpLGPI mice have a 3.8-fold increase in LpL activity in homogenized heart muscle compared with wild-type hearts and develop cardiomyopathy associated with increased lipid at 4 mo of age but have upregulation of FA oxidation genes (such as carnitine palmitoyltransferase I and acyl-CoA oxidase) and a decrease in glucose transporters at 2 mo (19). To determine whether abnormal FA metabolism tracks with gene expression changes and not with cardiomyopathy, we compared palmitate uptake and oxidation in wild-type and hLpLGPI mouse hearts. Surprisingly, there was a 55% decrease in FA uptake (132 ± 25 vs. 59 ± 9 nmol·g dry wt–1·min–1, P = 0.006) in lipotoxic hearts (Fig. 1A). Correcting for the size of these hearts (hLpLGPI hearts are larger) still resulted in a 46% decrease in [14C]palmitate uptake per heart, significant at P = 0.03. Oxidation was decreased by 76% in the hLpLGPI mouse (68 ± 11 vs. 16 ± 6 nmol·g dry wt–1·min–1, P = 0.01; Fig. 1B). Because the hLpLGPI mouse took up and oxidized less FFA, this suggested that an alternate source of energy was being utilized.



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  		Fig. 1. Palmitate uptake and oxidation in wild-type and hLpLGPI mouse hearts. Hearts from wild-type mice and mice overexpressing anchored lipoprotein lipase (LpL) by cardiomyocytes (hLpLGPI, denoted GPI) were perfused in recirculating mode for 60 min, and uptake and oxidation of [14C]palmitate were determined as described in MATERIALS AND METHODS. A: palmitate uptake in nmol·min–1·g dry wt–1 (*P = 0.006). B: palmitate oxidation in nmol·min–1·g dry wt–1 (*P = 0.03).

 
Lipid analysis of hLpLGPI hearts. We next assessed whether palmitate was handled differently by hLpLGPI mouse hearts. The percentage of label recovered as FFA (25 ± 4 vs. 40 ± 11%) tended to increase and PL stores (37 ± 4 vs. 21 ± 9%) to decrease in hearts from hLpLGPI mice. Label incorporation into TG and cholesterol was similar in wild-type and hLpLGPI hearts (Fig. 2).



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  		Fig. 2. Thin-layer chromatography (TLC) analysis of labeled lipids in wild-type and hLpLGPI hearts. After perfusion with radiolabeled palmitate, hearts were analyzed by thin-layer chromatography. Each lipid class was identified using controls. The corresponding spot was scraped from the plates and assessed for radioactivity. CE, cholesterol ester; TG, triglyceride; FFA, free fatty acid; CH, cholesterol; MG, monoglyceride; PL, phospholipid. Data are expressed as %total recovered radioactivity.

 
To determine whether lipotoxic hearts metabolized less FFA for energy production, ATP levels were measured in these hearts. Control hearts contained 18.2 ± 3.7 µmol/g dry wt ATP compared with 10.1 ± 3.4 µmol/g dry wt in hearts from LpLGPI mice (n = 5 in each group, P < 0.04). Therefore, although control and LpLGPI hearts tolerated the perfusion without difficulty, the LpLGPI hearts had reduced energy stores.

TG metabolism. To determine whether the additional energy was supplied by TG, hearts were perfused with [14C]triolein-labeled Intralipid, and the amount of Intralipid-derived FA oxidation was determined (Fig. 3). On average, hLpLGPI hearts oxidized 88% more TG than did the wild-type hearts. Thus the lipotoxicity was associated with more TG and less FFA oxidation.



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  		Fig. 3. Intralipid-TG oxidation by wild-type and hLpLGPI hearts. Intralipid containing radiolabeled TG was perfused through wild-type and hLpLGPI hearts. At the end of the perfusion, oxidation of label was determined as described in MATERIALS AND METHODS.

 
In vivo studies. A possible reason that hLpLGPI hearts had increased TG oxidation despite diminished FFA oxidation was that they incorporated more lipid in vivo before the hearts had been harvested. To test this hypothesis, we assessed particle uptake using cholesteryl ether-labeled Intralipid (Fig. 4). Although plasma decay of the tracer was identical in wild-type and hLpLGPI mice (Fig. 4A), cardiac uptake of particles was greater in the transgenic mice (P = 0.03) (Fig. 4B). Thus, despite decreased FFA oxidation, hLpLGPI hearts appeared to utilize more lipoprotein TG.



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  		Fig. 4. In vivo uptake of Intralipid core lipid by wild-type and hLpLGPI hearts. Mice were injected with [3H]cholesteryl ether-labeled Intralipid, and plasma decay of the tracer was monitored. Mice were then perfused with saline, and the tracer present in the heart was determined. A: plasma decay of the tracer in the circulation of each mouse was determined by blood taken by retroorbital bleeds. Counts at the 1st time point, 30 s, are shown as 100%. Data are graphed on a semilog scale. B: uptake of tracer into hearts of wild-type and hLpLGPI mice. Data are expressed as %injected dose (ID)/g of heart. *P = 0.03.

 
To test whether there was a difference in uptake of hydrolyzable and other core lipids, double-labeled human VLDL was injected into five control and five LpLGPI mice. The plasma decay of these labels was determined by measuring radioactivity in blood obtained at 30 s and 2, 5, and 15 min. The short interval was utilized because a longer kinetic study would allow time for intracellular metabolism of the acquired label. The plasma clearances of the labels were identical in the control and LpLGPI mice. At the end of the study, 47 ± 12% of the TG and 71 ± 11% of the cholesteryl ether label remained in the plasma of control mice, whereas 54 ± 10% of the TG and 79 ± 7% of the cholesteryl ether remained in the plasma of the LpLGPI transgenic mice. Heart uptake of cholesteryl ether into LpLGPI hearts was 127 ± 20% of control, whereas uptake of labeled TG into the LpLGPI hearts was 131 ± 47% of control. When we normalized the cardiac label to that of the injection, during the 15-min study the hearts obtained 5.3 times as much TG as cholesteryl ether. Thus TG and nonhydrolyzable core lipid were similarly acquired at a greater rate by the lipotoxic hearts. However, cardiac uptake of TG was much greater than that of core lipid.

VLDL competition with fatty acid metabolism. In vivo, hearts are always exposed to both FFA and lipoprotein TG. In an attempt to study this and determine whether the metabolic profile found in hLpLGPI hearts could be due to enhanced TG uptake, we perfused wild-type hearts with isolated human VLDL (0.4 mM TG) added to the perfusate containing 0.4 mM palmitate and [14C]palmitate. VLDL addition led to a dramatic decrease in palmitate uptake (48%) and oxidation (71%) by the heart (Fig. 5). Thus a TG-derived source of FA effectively competed with labeled FFA.



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  		Fig. 5. Very-low-density lipoprotein (VLDL) competition for uptake of palmitate. Recycling perfused hearts from wild-type mice received [14C]palmitate in buffer containing 3% BSA ± 0.4 mM VLDL-TG obtained from normal human subjects. Fatty acid (FA) uptake and oxidation are shown. *Significant differences.

 
[14C]palmitate metabolism with Intralipid. We next perfused wild-type hearts with [14C]palmitate in the presence of Intralipid to determine whether this source of TG would reproduce our finding with VLDL. Intralipid is also a substrate for LpL, although its hydrolysis in this situation might have been reduced due to the lack of a source of apolipoprotein CII, the LpL activator. The addition of 0.4 mM Intralipid to the perfusion buffer reduced [14C]palmitate uptake by 45% (132 ± 25 in control vs. 72 ± 14 nmol·g dry wt–1·min–1 in the presence of Intralipid, P = 0.02; Fig. 6A). FA oxidation decreased 66% (68 ± 6 vs. 23 ± 6 nmol·g dry wt–1·min–1, P = 0.04). Therefore, either the uptake or lipolysis of TG within Intralipid inhibited palmitate metabolism.



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  		Fig. 6. Effects of Intralipid and lipoprotein lipase (LpL) in uptake and oxidation of FFA. Wild-type mice were perfused with radiolabeled palmitate; in some studies, 0.4mM Intralipid was added to replicate the high-TG state found in lipotoxic mice. In other studies, heparin or P407 (an LpL inhibitor) was added to alter the actions of LpL. Uptake and oxidation were measured. A: empty bar, denoting palmitate, shows uptake under control conditions. Palmitate uptake with addition of Intralipid (Intralipid, filled bar), Intralipid and heparin (Heparin, striped bar), and Intralipid + P407 (P407, cross-hatched bar) are shown. *Differences from control P < 0.02. B: [14C]palmitate oxidation under the same conditions as in A.

 
Role of LpL in Intralipid metabolism. Next, we examined whether local LpL actions were responsible for competition between [14C]palmitate and Intralipid. Heparin releases LpL from the capillary lumen (9) but allows the enzyme to retain activity while circulating in the perfusate. This permits TG lipolysis to continue, but at a location distant from the endothelium, and abolishes LpL-bridging actions. Addition of heparin to the perfusion buffer increased [14C]palmitate uptake by 320%, more than restoring FA uptake to baseline perfusion with palmitate only (Intralipid 72 ± 14 vs. heparin 323 ± 22 nmol·g dry wt–1·min–1, P < 0.001; Fig. 6A). This suggests that localization of LpL on the capillary endothelium is necessary for Intralipid-mediated inhibition of palmitate uptake.

Next, we added the LpL inhibitor P407 to the perfusion medium. Uptake of [14C]palmitate was increased by 360%, again restoring FA uptake (Intralipid 72 ± 14 vs. heparin 341 ± 38 nmol·g dry wt–1·min–1, P < 0.001; Fig. 6A). Therefore, heparin and P407 led to identical effects. The reasons that both treatments led to uptakes even greater than those found under control conditions might reflect the loss of competition due to plasma lipoproteins in the hearts before their removal from the animals.

Although both heparin and P407 restored palmitate uptake by the heart, there was still a marked inhibition of FA oxidation; palmitate oxidation remained ~26% (with heparin) or 57% (with P407) of that found in the absence of Intralipid (Fig. 6B). Presumably, in the presence of heparin and P407, uptake of some intact Intralipid continued, and some of this intracellular lipid was also oxidized. If this is true, then the acquired labeled palmitate might be diverted to a different intracellular pool.

TLC analysis of cardiac lipids. Three major classes of lipids extracted from the heart are shown in Fig. 7. Compared with hearts perfused with only palmitate, Intralipid-treated hearts had more radioactivity in FFA (25 ± 4.2 vs. FFA: 45 ± 4.7% CPMTotal, P = 0.03) and less in PL (37 ± 3.6 vs. 12 ± 0.07% CPM, P = 0.02). TG content did not vary. These differences in radioactivity recovered from the perfused hearts were similar to those found when hLpLGPI and wild-type hearts were compared. There were no statistical differences in cardiac labeled FFA content between Intralipid-perfused hearts and hLpLGPI hearts perfused with palmitate only (P = 0.72 for FFA, P = 0.48 for PL). Thus perfusion of wild-type hearts with Intralipid appeared to have replicated the metabolic phenotype found in the hLpLGPI lipotoxic heart.



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  		Fig. 7. TLC of heart lipids from Intralipid and LpL-inhibited hearts: Hearts that had been perfused with radiolabeled palmitate ± Intralipid or with P407 were analyzed by TLC. Compared with hearts perfused with only palmitate, Intralipid-treated hearts had more radioactivity in FFA (P = 0.03) and less in PL (P = 0.02), replicating the metabolic profile found in hLpLGPI hearts perfused with palmitate. Inhibition of LpL with P407 decreased intracellular FFA (P = 0.003) and increased PL. *Significant differences, P = 0.0008.

 
Inhibition of LpL with P407 decreased intracellular FFA (45 ± 4.7 vs. 15 ± 3% CPM, P < 0.01) and increased PL (12 ± 0.07 vs. 22 ± 0.9% CPM, P < 0.01). In addition, there was a trend toward an increase in radioactivity found in TG. Thus LpL activity modulated intracellular FA accumulation, presumably by providing competing substrates for oxidation. When local LpL activity was blocked by P407, a greater percentage of FA may have been diverted to TG synthesis.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Lipotoxic hLpLGPI mice develop dilated cardiomyopathy after 4 mo of age; young mice (used in this study) have normal cardiac function as assessed by echocardiography. By comparing these young mice with wild-type mice, we have determined the following. 1) hLpLGPI hearts have a defect in FA uptake and oxidation; this precedes the development of heart dysfunction. 2) Intralipid-associated core lipid uptake, traced with cholesteryl ether, was greater in hLpLGPI mice. This suggests increased uptake of whole lipoprotein particles. By use of labeled VLDL, TG uptake was similarly increased; however, more than fivefold more TG than cholesteryl ether was found in the heart. 3) VLDL and Intralipid addition inhibited uptake and oxidation of perfusate palmitate in wild-type hearts. This proves that TG-derived FA compete with FFA for heart uptake and oxidation. Moreover, addition of TG-containing particles to the perfusion recapitulated the defects found in the hLpLGPI mice. 4) This process required LpL activity and its association with endothelial cells.

We first compared FA oxidation in wild-type and hLpLGPI hearts by using a Langendorff perfusion model. The hLpLGPI hearts had a striking reduction in FA uptake and oxidation. Although a similar finding might be expected in failing hearts that tend to shift from FA to glucose oxidation (3), the genetic profile of these hearts had suggested the opposite; genes involved in FA oxidation were increased and glucose transporters decreased (19). Thus the data suggested that hLpLGPI hearts used an alternative FA source.

In the perfused heart, FA uptake represents FA transferred into the cardiomyocyte, esterified with acyl-CoA, and then either stored as cellular lipid or oxidized. A reduction in any of these steps could have resulted in the observed reduction in uptake. Because the percentage of palmitate that was oxidized after it was taken up by hLpLGPI hearts was reduced, it suggested that more was converted to cellular lipids. Such a hypothesis would be compatible with the reduced ATP levels observed in hLpLGPI hearts. Using TLC analysis of heart tissue, we attempted to define this pathway. Although there was significant variation between hearts, hLpLGPI hearts tended to retain more of the label as unesterified FA. This would occur if there were a defect in long-chain acyl-CoA synthetase (ACS) or competition for the actions of this enzyme. Gene expression of ACS was unchanged in the hLpLGPI hearts (20). Another option is that palmitate oxidation was reduced because these hearts had greater stores of intracellular FFA and TG. We showed that hLpLGPI hearts internalized greater amounts of Intralipid core lipids both in vivo and during in vitro perfusions. We hypothesize that these metabolic alterations, which precede the development of cardiomyopathy, reduce the dependence of the heart on FFA. Thus, despite reductions in FFA oxidation and decreased glucose transporter expression, hLpLGPI mouse hearts from young mice did not have significant alterations in cardiac function.

If uptake or competition by lipoprotein lipid was the reason for reduced palmitate uptake and oxidation in hLpLGPI hearts, we hypothesized that a similar situation would be reproduced in vitro if VLDL were added to the palmitate-containing perfusate. Indeed, in wild-type hearts, FA uptake and oxidation were reduced by VLDL addition. It should be noted that the concentrations of VLDL used (0.4 mM, ~32 mg/dl) are significantly less than those found in normal human plasma. Thus it is likely that in vivo lipoprotein-derived TG compete with FFA for uptake by the heart.

We then studied the effects of a second source of TG, Intralipid, on heart uptake and metabolism of palmitate. Intralipid is metabolized by the heart in vivo (2), although much of its uptake is via whole particle uptake (10). Intralipid, like VLDL, reduced palmitate uptake and oxidation. Moreover, the intracellular content of radiolabeled FA was increased. These data explain the metabolic derangement in the hLpLGPI hearts and clearly show that lipoprotein lipid delivered as VLDL or Intralipid effectively competes with FA-associated with albumin. Thus these data further establish an important role for lipoproteins, and not just FFA, as a cardiac energy source.

Several investigations have previously shown that TG-rich lipoproteins can provide lipid for the heart. More than three decades ago, Fielding (7) demonstrated that perfused rat hearts accumulated chylomicron lipids. The tracer used in those experiments was cholesteryl ester, a marker for core lipids. Because TG is the other major lipoprotein core lipid, Fielding’s experiments, like ours, suggest that, in addition to FFA, hearts acquire lipid contained in the core of lipoproteins. The relative amounts of FFA obtained by the heart from whole particle uptake vs. TG lipolysis probably vary as a function of the composition of the circulating lipoproteins.

How important is lipoprotein-TG compared with albumin-associated FFA? In one study in which chylomicrons and palmitate were added to heart perfusions, Mardy et al. (14) found that unlabeled palmitate did not significantly alter oxidation of chylomicron-derived FA. Why did FFA not alter chylomicron TG uptake in these studies? When TG-rich lipoproteins are hydrolyzed along the capillary wall (step 1 in Fig. 8), it is likely that the local FA concentration becomes very high, much greater than the 0.4 mM that was added to the perfusate as a competitor.



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  		Fig. 8. Competing sources of cardiac FA. Although FA associated with albumin can provide lipid to the heart, their oxidation is challenged at 2 steps. LpL located on the capillary lumen produces high local concentrations of FA that compete for uptake by the cell (step 1). Lipoprotein-derived TG may also be converted into FA within the cell (step 2). The source of this lipid might be partially metabolized lipoproteins, denoted remnant, produced from chylomicrons (CM) and VLDL.

 
A second pathway for lipid uptake is via internalization of whole lipoprotein particles and generation of FA intracellularly (step 2 in Fig. 8); this is the process that might have been mimicked with Intralipid. Our experiments using both VLDL and Intralipid suggest that heart acquisition of lipid as either locally liberated FA or TG competes with the utilization of FA. These data are in agreement with a recent analysis of in vivo kinetic and tissue uptake data that also concluded that lipoprotein-TG, and not FFA, is the primary lipid metabolic fuel of the heart (16).

Our final set of experiments tested the roles of local LpL actions in the Intralipid effects. Heparin treatment reduced the Intralipid-mediated inhibition of palmitate uptake; presumably local LpL is required to effectively compete for cardiac uptake of albumin-FA. Inhibition of LpL actions by P407 had the same effect on palmitate uptake in the presence of Intralipid. Thus LpL actions provide a lipoprotein source of FA that competes with FFA.

In addition to oxidation, FA are stored and used for cellular components such as phospholipids. Our TLC data suggest that, when provided with excess lipoprotein lipid, hearts responded by utilizing less FFA and diverting more of this lipid to other pathways.

In summary, we have provided new data to understand the role of lipoprotein-derived FA in cardiac metabolism and how this may be altered in lipotoxic hearts overexpressing LpL. Failing hearts normally switch from FA to glucose utilization and reduce expression of genes required for FA metabolism. In our lipotoxic model, hearts do not appear to switch to greater glucose oxidation; rather, these hearts compensate by reducing oxidation of FFA. This is due, at least in part, to greater dependence on lipoprotein-TG as a source of FA. The hLpLGPI hearts accumulate more intracellular FA, in part because lipoprotein-derived FA may have saturated the esterification and oxidation pathways. A similar metabolic alteration was reproduced by adding VLDL and Intralipid to perfused wild-type hearts. Most importantly, this experiment illustrated the normal physiology of hearts in vivo; albumin-FA are constantly competing with FA produced by LpL actions on circulating lipoproteins.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
P. Pillutla was supported by a fellowship from the Stanley J. Sarnoff Endowment. I. J. Goldberg's laboratory is supported by National Heart, Lung, and Blood Institute Grants HL-45095 and HL-73029, and R. Ramasamy is supported by Grants HL-61783, and HL-68954.


   ACKNOWLEDGMENTS
 
This work was performed during P. Pillutla's matriculation at New York University School of Medicine.

P. Pillutla's current address is Department of Medicine, University of California, San Francisco, CA 94143.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. J. Goldberg, Dept. of Medicine, Columbia Univ., 630 West 168th St., New York, NY 10032 (E-mail: ijg3{at}columbia.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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