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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Very-low-density lipoprotein (VLDL) and chylomicrons (CMs) transport triacylglycerol (TAG) to peripheral tissues. Lipoprotein-TAG may gain access to target cells by lipoprotein lipase (LPL) hydrolysis or via receptor-mediated uptake; the principal routes of entry of VLDL and CM into heart are unknown, and different routes of entry may result in different metabolic fates. To examine this, isolated working rat hearts were perfused with rat VLDL and CMs, dual-labelled with [3H]TAG and [14C]cholesterol. Uptake and utilization of CM-TAG were significantly greater than VLDL-TAG, but both were decreased significantly (more than halved) by tetrahydrolipstatin (THL, an inhibitor of lipoprotein lipase). By contrast, uptake of VLDL-cholesterol was much higher than CM-cholesterol (P < 0.01), and suramin (a lipoprotein receptor antagonist) decreased cholesterol uptake of both forms. CM-TAG oxidation rate was more than 4-fold higher than VLDL-TAG oxidation. However, suramin decreased TAG oxidation from both VLDL and CM without affecting TAG uptake or total utilization, suggesting that the TAG gaining access through receptor-mediated pathways is preferentially ‘channelled’ towards oxidation. Most (79%) CM-TAG was oxidized whilst the proportion of VLDL-TAG oxidized was only about half (49%). In the presence of suramin, there was a significant increase in esterification (incorporation of assimilated [3H]TAG into myocardial tissue [3H]lipids, mainly TAG) of assimilated TAG from both VLDL and CMs, again suggesting that receptor-mediated TAG uptake is directed towards oxidation rather than esterification. The importance of this relatively small pool of TAG is indicated by the fact that cardiac mechanical function declined markedly when lipoprotein receptors were inhibited. These results suggest that CMs, most fatty acids of which gain access into cardiomyocytes through LPL-mediated hydrolysis, are the major supplier of TAG for hearts to oxidize; however, the metabolic fate of VLDL was split evenly between oxidation and deposition as myocardial tissue lipid. Most importantly, VLDL may play a regulatory role in heart lipid metabolism through a lipoprotein receptor-mediated mechanism.

Most (∼70%) of the cardiac energy requirement is supplied by fatty acid (FA) oxidation under normal physiological workload conditions (Lopaschuk et al. 1994; Calvani et al. 2000; van der Vusse et al. 2000). FAs are derived from two sources: (1) circulating non-esterified FAs (NEFAs) bound to plasma albumin, derived from adipose tissue lipolysis, which gain access to the cardiomyocytes both passively (Zakim, 1996) and via carrier-mediated pathways (Abumrad et al. 1998; Van Der Vusse et al. 2000) involving at least three proteins, namely fatty acid transport protein (FATP), fatty acid translocase (FAT) and fatty acid binding protein (FABPpm) (Schaap et al. 1998; Luiken et al. 1999); and (2) circulating esterified FA in the form of triacylglycerols (TAGs), which are transported in plasma within lipoproteins: very-low-density lipoprotein (VLDL) and chylomicrons (CMs). VLDLs are synthesized by the liver from endogenous lipids whereas CMs are synthesized by the intestine from exogenous dietary lipid. However, until recently the relative contributions of NEFAs and TAGs to the heart energy requirements were unknown. Although the metabolism of albumin-bound NEFAs by the heart has been studied extensively (Lopaschuk et al. 1994; Lopaschuk, 1997; Wang et al. 1998; Belke et al. 1999), and suggests that albumin–FAs are the primary energy source for the heart, the role of TAGs within VLDLs and CMs (VLDL-TAGs and CM-TAGs) in heart energy supply has been uncertain. Following development of a technique to prepare species-specific radiolabelled VLDL by liver perfusion (Bennett et al. 2000a), myocardial preference for NEFAs, VLDL-TAGs and CM-TAGs has been investigated in rat (Hauton et al. 2001). CMs (prepared by thoracic duct cannulation) are efficient substrates for heart, being utilized to a similar extent to NEFAs, but VLDLs were less so (Hauton et al. 2001). These studies indicated that whilst NEFAs suppress cardiac CM-TAG utilization, VLDL-TAG utilization was not altered by the presence of NEFAs. Furthermore, marked differences in the metabolic fates (oxidation versus tissue lipid deposition) of FA and TAG substrates were noted (Hauton et al. 2001; Mardy et al. 2001). Thus these studies provided preliminary evidence that intracellular channelling of FAs occurs, leading to differing metabolic fates (i.e. distribution between oxidation and incorporation into cellular lipid) according to the FA sources, and this may be due to different routes of FA and TAG uptake.

Hydrolysis of TAG by endothelium-bound lipoprotein lipase (EC 3.1.1.34; LPL) is widely regarded as the initial step for the ‘bulk’ uptake of lipoprotein-TAG by the heart (Braun & Severson, 1992; Goldberg, 1996; Merkel et al. 2002; Augustus et al. 2003). Following LPL-mediated hydrolysis of plasma TAG, the liberated FA product is assimilated by the cardiomyocyte as for NEFA (above). LPL is synthesized in the cardiomyocyte but translocated to its active site on the luminal surface of the capillary endothelium where it is bound to heparan sulphate proteoglycan. Endothelial LPL hydrolyses the triacylglycerol-rich lipoprotein (TGRLP) TAG core, releasing fatty acids which are then taken up into the cardiomyocyte via the endothelial cell by route(s) unknown.

However, besides LPL, plasma TAG may be taken up by the cardiomyocyte through lipoprotein receptor-mediated pathways. The recently described VLDL/apo-E receptor (VLDL-R) (Sakai et al. 1994; Tiebel et al. 1999; Kamataki et al. 2002) and the TGRLP/apo-B48 receptor (Gianturco et al. 1998; Brown et al. 2000) are highly expressed in the heart, skeletal muscle and adipose tissue (Tiebel et al. 1999). The tissue distribution of the VLDL receptor suggests a possible role in the delivery of lipoproteins, especially remnant lipoprotein particles, which are the products of LPL hydrolysis, to peripheral tissues, including heart for lipid metabolism. Furthermore, considering that the tissue distributions of VLDL-R and LPL are similar (Braun & Severson, 1992; Goldberg, 1996; Tiebel et al. 1999), an interaction between them could exist which can regulate TGRLP metabolism.

In the present study, we hypothesized that the pattern of intracellular metabolic fates (oxidation versus tissue esterification; pattern of incorporated tissue lipid distribution) differs between VLDLs and CMs in the heart and that this difference is due to the different routes of uptake of VLDLs and CMs by the heart. To test this hypothesis, we used dual-labelled ([3H]TAG; [14C]cholesterol) VLDLs and CMs to perfuse working rat hearts. Since cholesterol can only gain access to the cardiomyocyte through a receptor-mediated mechanism, this method allowed assessment of possible routes of uptake of TAG by hearts simultaneously. Further, we used tetrahydrolipstatin (THL), an active site inhibitor of LPL, and suramin, which inhibits the binding between lipoproteins and lipoprotein receptors, to assess the roles of LPL and lipoprotein receptors in TAG delivery to the heart.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

The investigation was performed in accordance with the Animals (Scientific Procedures) Act 1986, and approved by the Medical Division Local Ethical Review Committee of Oxford University.

Animals

Male Wistar rats were fed ad libitum on a chow diet (comprising by weight approximately 52% carbohydrate, 21% protein and 4% fat; the residue was non-digestible material (Special Diet Services, Witham, Essex, UK)) with free access to drinking water and were maintained at an ambient temperature of 20 ± 2°C with a 12 h light−12 h dark cycle (light from 07.30 h).

Chemicals

[9,10<n>-3H]Oleic acid, [4-14C]cholesterol and glycerol tri[9,10<n>-3H]oleate were obtained from Amersham Biosciences, Little Chalfont, UK; Waymouth's medium was purchased from Gibco BRL, Life Technologies, Paisley, UK; tetrahydrolipstatin (THL) was obtained from Roche Porducts Ltd, Welwyn Garden City, UK. Suramin hexasodium salt (8,8′-[carbonylbis[imino-3,1-phenylene carbonyl-imino(4-methyl-3,1-phenylene)-carbonylimino]]bis1,3,5-naphthalene trisulphonic acid hexasodium salt) and other biochemicals were obtained from Sigma Chemical Co., Poole, UK.

Preparation of lipid substrates

3H-Labelled sodium oleate (specific activity 485 mCi mmol−1) was pre-bound to fatty acid- and endotoxin-free bovine serum albumin (BSA) (5% w/v) and added to liver perfusate for production of [3H]triolein-VLDL as described (Bennett et al. 2000a). 14C-Labelled cholesterol (specific activity 50 mCi mmol−1) was freshly prepared as follows: [14C]cholesterol was added to cholesterol, lecithin and triolein (65%) dissolved in toluene. The solvent was then evaporated under N2. Glycerol and Krebs-Henseleit bicarbonate solution were then added, and the mixture was sonicated at 4°C in order to incorporate triolein and cholesterol into an emulsion. The [14C]cholesterol-containing emulsion was added to the liver perfusate or to the gastrostomy for production of doubly labelled VLDLs or CMs, respectively (see below).

Preparation of VLDLs.  VLDLs containing 3H-labelled triolein and 14C-labelled cholesterol were prepared by an extended rat liver perfusion technique under aseptic conditions as described by Bennett et al. (2000a). Briefly, rats (400–500 g; fasted for 24 h prior to experimentation) were anaesthetized with intraperitoneal sodium pentobarbitone (60 mg (kg body weight)−1), abolition of reflexes established, and the portal vein and thoracic inferior vena cava were rapidly cannulated; the abdominal inferior vena cava was ligated. Heparin was not used. The liver was perfused in situ for 8 h with a recirculating solution comprising Waymouth's synthetic tissue culture medium supplemented with amino acids (glutamine, serine, alanine) and glucose. Washed red cells were added to give a final haematocrit of 10% (v:v) and the perfusate was gassed with O2: CO2 95: 5% (v:v) at 37°C; [3H]oleate (1.0 mm final concentration) pre-bound to fatty acid-free albumin was added to the perfusate prior to liver perfusion and subsequently also infused into the perfusate for the first 4 h of the perfusion to maintain the circulating NEFA concentration at about 0.4 mm for the first half of the procedure. [14C]Cholesterol was added to the perfusate simultaneously. After the perfusion, the perfusate was filtered through an ultrafilter with molecular weight cut-off at 30 000 Da (Amicon, Stonehouse, Gloucestershire, UK) then ultracentrifuged at 144 500 g to separate the d < 1.006 g ml−1 layer. Thin-layer chromatography of dual-labelled VLDL showed that > 95% of the 3H label was in the triacylglycerol band and > 98% of 14C label was in the cholesterol and cholesterol ester bands. VLDLs were suspended in fatty acid-free bovine serum albumin (5% w/v); TAG and cholesterol contents were assayed with enzymatic colourimetric test kits (see below). The dual-labelled VLDLs were added to the heart perfusate reservoir to give a final concentration of 0.4 mm TAG.

Preparation of chylomicrons.  Chylomicrons containing 3H-labelled triolein and 14C-labelled cholesterol were prepared using a rat thoracic duct cannulation technique (Bezman-Tarcher et al. 1965). Rats (350–400 g) were anaesthetized with intraperitoneal Hypnorm (fentanyl and fluanisone; 1 ml (kg body weight)−1) supplemented with halothane (1–2% v/v) in oxygen until reflexes were abolished. A polyethylene catheter was inserted into the lower thoracic duct via an extra-peritoneal loin incision and externalized to continuously collect chyle; a gastrostomy was also performed. Heparin was not used. The animals were maintained in a restraining cage for 12 h with free access to food and water, but were given additional intragastric fluid replacement; postoperative analgesia was provided with intramuscular carprofen (1 ml (kg body weight)−1). After this initial recovery period triolein/[3H]triolein (1.0 g; 22 mCi) and [14C]cholesterol emulsion (see above) were administered into the stomach, and chyle collected for the subsequent 12 h, following which the animal was humanely killed. Dual-labelled chylomicrons were isolated by washing with bovine serum albumin solution and centrifugation. Thin-layer chromatography of the dual-labelled chylomicrons showed that > 95% of the label was in the triacylglycerol band and > 99% of 14C label was in the cholesterol and cholesterol ester bands. Chylomicrons were suspended in fatty acid-free bovine serum albumin (5% w/v) and TAG and cholesterol contents were assayed with their respective enzymatic colourimetric test kits (Boehringer Mannheim GmbH, Lewes, Sussex, UK; Sigma Chemical Co). Dual-labelled chylomicrons were added to the heart perfusate reservoir to give a final concentration of 0.4 mm TAG.

Isolated perfused working heart preparation

All experiments were commenced between 11.00 h and 12.00 h. Hearts were perfused through the left atrium (anterograde) in ‘working’ mode by the method of Taegtmeyer et al. (1980). Fed rats (250–350 g) were anaesthetized with intraperitoneal sodium pentobarbitone (60 mg (kg body weight)−1) until reflexes were abolished. The heart was rapidly excised and briefly placed in ice-cold Krebs-Henseleit bicarbonate saline; it was then cannulated via the aorta (< 2 min from excision) and perfused retrogradely through the coronary arteries in ‘Langendorff’ mode whilst lung, mediastinal, and peri-cardiac brown adipose tissue were excised, right pulmonary arteriotomy performed, and the left atrium separately cannulated after which the apparatus was switched to ‘working’ mode and cardiac perfusion maintained through the left atrium. A recirculating Krebs-Henseleit bicarbonate buffer solution containing CaCl2 (1.3 mm), glucose (11 mm) and endotoxin- and fatty acid-free bovine serum albumin (1% w/v) was filtered through a 5 μm cellulose nitrate filter (Millipore, Bedford, MA, USA) and gassed with O2: CO2 (95: 5) at 37°C. The first 50 ml of coronary effluent were discarded to free the circuit of blood cells; final perfusate volume was 100 ml. Afterload was maintained at 100 cmH2O and preload (atrial filling pressure) at 15 cmH2O. After an initial 15 min stabilization period, lipid was added slowly (2 min) to the reservoir (Time ‘0’). In some experiments, to examine the role of LPL and lipoprotein receptors on the utilization of VLDL and CM by heart, THL (final concentration 10 μm) or suramin (1 mg ml−1) was added to the perfusate separately 5 min before the administration of lipid. Peak systolic pressure (PSP) and heart rate (HR) were measured by a calibrated pressure transducer (Druck Ltd, Groby, Leicestershire, UK) connected to a side arm of the aortic cannula. Aortic flow rate (AFR) was measured by a timed collection of perfusate ejected through the aortic line, and coronary flow rate (CFR) was measured by a timed collection of perfusate effluent dripping from the heart. Measurements were made at time 0 and at 10 min intervals for 60 min. Cardiac output (CO) was calculated as CFR + AFR. Rate–pressure product (RPP) was calculated as HR × PSP. Hydraulic work (HW) was calculated as CO × mean aortic pressure/heart wet wt. After the final measurements at 60 min, heparin (Leo Laboratories Ltd, Princes Risborough, UK) 5 IU ml−1 was added to the perfusate, and after a further 2 min the heart was rapidly excised, freeze-clamped in light alloy tongs cooled in liquid nitrogen, and weighed. A duplicate sample of the post-heparin perfusate was also frozen in liquid nitrogen.

Measurement of lipid oxidation rate

TAG oxidation rate was estimated by measuring 3H2O production from [3H]triolein in the perfusate as described by Evans & Wang (1997); at 10 min intervals, aliquots of perfusate (1.0 ml) were removed and subjected to Folch lipid extraction with chloroform: methanol (2: 1, v/v), and water. An aliquot of the water phase was removed and counted for radioactivity. TAG and cholesterol uptake (disappearance from the perfusate) were measured by assay of TAG and cholesterol in the organic infranatant phase of the Folch extracts of the timed perfusate aliquots following evaporation of the chloroform and resolubilization with ethanol, using respective enzymatic colourimetric assay test kits (see above).

Incorporation of exogenous lipid into myocardial lipid

Myocardial [3H]- and [14C]lipid contents were estimated by grinding frozen myocardium to powder under liquid N2 and extracting the lipids from an aliquot with chloroform: methanol (Folch). After repeated washing, the lipids were resolubilized in chloroform and separated by thin-layer chromatography using a hexane–diethylether–acetic acid system with standards co-run. 3H and 14C radioactivity were measured in the various lipid bands after visualization with rhodamine 6G under ultraviolet light. Lipid utilization was the sum of lipid oxidation and tissue lipid incorporation.

LPL activity

LPL activity was estimated in duplicate samples by using a 3H-labelled triolein substrate emulsion containing starved rat serum as a source of apolipoprotein-CII to maximize LPL detection (Nilsson Ehle & Schotz, 1976); the serum was pre-treated by heating to 56°C to inactivate non-specific plasma lipases. Radioactivity in evolved fatty acids was counted following extraction in methanol–chloroform–heptane. Heparin-releasable LPL activity was measured by adding postheparin perfusate taken at 62 min directly in the above assay system without modification (expressed as nmol fatty acid released per min per g wet wt of heart). Tissue residual LPL activity was measured in acetone–diethyl ether-dried tissue powders ground from the working hearts frozen in liquid nitrogen; a duplicate sample of frozen heart tissue was weighed, dried down with acetone–ether in parallel with the samples and re-weighed to correct expression of activity (from nmol fatty acid released per min per mg of acetone dried powder to nmol fatty acid released per min per g wet wt of heart). ‘Total’ LPL activity in these experiments was ‘heparin-releasable’+‘residual’ LPL activities (nmol fatty acid released per min per g wet wt of heart).

Statistics

Results are expressed as means ±s.e.m. Statistical analysis was performed by ANOVA for repeated measurements, with post hoc correction for multiple comparisons, where appropriate. Statistical significance was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Uptake of lipoprotein TAG and cholesterol by working rat heart

Uptake of chylomicron-TAG by perfused working rat hearts was significantly greater (almost double) than that of VLDL-TAG (both present at 0.4 mm), although both were decreased significantly (more than halved) by the administration of the LPL inhibitor THL. Suramin had no significant effect on the uptake of TAG in the form of either VLDL or CMs (Fig. 1). The pattern of 14C-labelled cholesterol and cholesterol ester incorporation into the cardiac tissue (Fig. 2A) closely resembled cholesterol uptake (data not shown), indicating bulk transport into the cell of lipoprotein particles, and suggesting that export and/or recycling of cholesterol/cholesterol ester was not occurring to a significant extent in the time course of the current experiments. By contrast to TAG uptake, uptake of cholesterol from the medium and incorporation into tissue [14C]cholesterol/cholesterol ester was significantly greater (double) when presented in the form of VLDL compared to CMs (Fig. 2A). THL did not alter cholesterol uptake from either VLDL or CMs but suramin significantly decreased cholesterol uptake from both VLDL and CMs (Fig. 2A). Since the cholesterol content of different lipoproteins varies, we calculated the equivalent apoprotein-B (apo-B) uptake that this cholesterol uptake represented. Taking VLDL (cholesterol + cholesterol ester) content as 10% of total VLDL mass (Tam & Breckenridge, 1983; Y.-G. Niu & R. D. Evans, results not shown) and VLDL apo-B content as 0.5% of total VLDL mass (Tam & Breckenridge, 1983), and taking CM (cholesterol + cholesterol ester) content as 3.5% of total CM mass (Bhattacharya & Redgrave, 1981; Y.-G. Niu & R. D. Evans, results not shown), and CM apo-B content as 0.1% of total CM mass (Imaizumi et al. 1978; Bhattacharya & Redgrave, 1981), the difference between VLDL and CM uptake was even greater – about 4-fold (Fig. 2B).

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Figure 1. Triacylglycerol uptake in the form of very-low-density lipoprotein or chylomicrons by isolated working rat heart Isolated rat hearts were perfused with rat very-low-density lipoprotein (VLDL) or rat chylomicrons (CM) (0.4 mm TAG) with tetrahydrolipstatin (THL; 10 μm), suramin (1 mg ml−1) or vehicle (control). TAG uptake was measured as disappearance of TAG from the perfusate; for further details see text. Results are expressed as means ±s.e.m.(n= 8 per group). Statistically significant differences between VLDL and CMs are indicated: **P < 0.01; significant differences between THL-treated and control hearts are indicated: ##P < 0.01.

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image

Figure 2. Assimilation of lipoprotein cholesterol and apo-B by isolated working rat heart A, incorporation of rat very-low-density lipoprotein (VLDL) and rat chylomicron (CM) [14C]cholesterol into cardiac tissue [14C]cholesterol and [14C]cholesterol ester. Isolated rat hearts were perfused with VLDL or CMs (0.4 mm) with or without THL (10 μm) or suramin (1 mg ml−1) for 1 h and tissue lipids extracted; cholesterol assimilation was estimated as recovery of myocardial 14C-labelled cholesterol/ester. B, calculation of apo-B mass assimilated by hearts. See text for further details. Results are expressed as means ±s.e.m.(n= 8 per group). Statistically significant differences between VLDL and CM are indicated: *P < 0.05, **P < 0.01; significant differences between suramin-treated and control hearts are indicated: +P < 0.05, ++P < 0.01.

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Metabolic fate of TAG in the form of VLDL and CMs

Metabolic fate of assimilated [3H]TAG was examined by measuring oxidation rate and deposition of tissue 3H-labelled lipids. Chylomicron-TAG oxidation rate was more than 4-fold higher than VLDL-TAG oxidation (70 ± 12 versus 15 ± 2 nmol fatty acid min−1 (g wet wt)−1, respectively, P < 0.01) (Fig. 3), reflecting its greater TAG uptake (Fig. 1). THL and suramin both decreased TAG oxidation markedly from both VLDL and CMs (Fig. 3). Despite their differences in uptake (Fig. 1) and oxidation (Fig. 3), total deposition of exogenous [3H]TAG label into endogenous myocardial tissue [3H]lipids was strikingly similar to uptake for VLDL (Fig. 4) and CMs (Fig. 5); the pattern of incorporation of assimilated VLDL-TAG and CM-TAG into various tissue lipid classes was also similar. Most of the [3H]TAG incorporated into tissue lipids was recovered as TAG; however, the percentage of TAG in the total tissue [3H]lipids was significantly different between VLDL (Fig. 4) and CMs (Fig. 5) (65 ± 2%versus 46 ± 1%, P < 0.01). Consistent with the decreased lipoprotein-TAG uptake and oxidation, THL decreased total myocardial tissue [3H]lipid incorporation whereas, surprisingly, suramin increased it significantly for both VLDL (Fig. 4) and CMs (Fig. 5). Interestingly, however, both THL and suramin increased the percentage of TAG recovered among the total tissue [3H]lipid spectrum significantly in both lipoproteins at the expense of the other tissue lipid classes (Figs 4 and 5).

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Figure 3. Oxidation rate of lipoprotein triacylglycerol by the isolated working rat heart Isolated rat hearts were perfused with [3H]triolein-labelled rat very-low-density lipoprotein (VLDL) or rat chylomicrons (CM) (0.4 mm) with tetrahydrolipstatin (THL; 10 μm), suramin (1 mg ml−1) or vehicle (control). TAG oxidation was measured as 3H2O production. See text for further details. Results are expressed as means ±s.e.m.(n= 8 per group). Statistically significant differences between VLDL and CM are indicated: ***P < 0.001; significant differences between THL-treated and control hearts are indicated: ##P < 0.01; significant differences between suramin-treated and control hearts are indicated: +P < 0.05, ++P < 0.01.

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image

Figure 4. Incorporation of VLDL-[3H]triolein into myocardial tissue lipids Isolated rat hearts were perfused with [3H]triolein in the form of rat very-low-density lipoprotein (VLDL; 0.4 mm) with tetrahydrolipstatin (THL; 10 μm), suramin (1 mg ml−1) or vehicle (control). Lipids were extracted from myocardial tissue and separated by thin-layer chromatography according to lipid class to estimate incorporation of VLDL-[3H]triolein into tissue [3H]lipids. For further details see text. Results are expressed as total mass of [3H]lipid incorporated, and proportion incorporated into each lipid class. PL, phospholipid; DAG, diacylglycerol; FA, fatty acid; TAG, triacylglycerol; CE, cholesterol ester. Data are presented as means ±s.e.m.(n= 8 per group). Statistically significant differences between THL-treated and control hearts are indicated: ##P < 0.01; significant differences between suramin-treated and control hearts are indicated: +P < 0.05, ++P < 0.01.

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Figure 5. Incorporation of chylomicron-[3H]triolein into myocardial tissue lipids Isolated rat hearts were perfused with [3H]triolein in the form of rat chylomicrons (CM; 0.4 mm) with tetrahydrolipstatin (THL; 10 μm), suramin (1 mg ml−1) or vehicle (control). Lipids were extracted from myocardial tissue and separated by thin-layer chromatography according to lipid class to estimate incorporation of CM-[3H]triolein into tissue [3H]lipids. For further details see text. Results are expressed as total mass of [3H]lipid incorporated, and proportion incorporated into each lipid class. PL, phospholipid; DAG, diacylglycerol; FA, fatty acid; TAG, triacylglycerol; CE, cholesterol ester. Data are presented as means ±s.e.m. (n= 8 per group). Statistically significant differences between THL-treated and control hearts are indicated: ##P < 0.01; significant differences between suramin-treated and control hearts are indicated: ++P < 0.01.

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Most (79%) of the TAG derived from chylomicrons was oxidized whilst the proportion of VLDL-TAG oxidized was only about half (Fig. 6; P < 0.01). THL and suramin both altered the metabolic fate of TAG from VLDL and CMs (i.e. changed the distribution profile between oxidation and deposition as tissue lipid): both decreased the proportion oxidized (Fig. 6).

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Figure 6. Metabolic fate of TAG from VLDL and chylomicrons in working rat heart Rat hearts were perfused with [3H]triolein (0.4 mm) in the form of very-low-density lipoprotein (VLDL) or chylomicrons (CM), together with tetrahydrolipstatin (THL; 10 μm), suramin (1 mg ml−1) or vehicle (control). Metabolic fate was calculated as the percentage of both [3H]TAG oxidation (3H2O production) and tissue [3H]lipid incorporation relative to the total utilization of [3H]TAG. For further details see text. Results are presented as means ±s.e.m.(n= 8 per group). Statistically significant differences between VLDL and CM are indicated: *P < 0.05; significant differences between THL-treated and control hearts are indicated: ##P < 0.01; significant differences between suramin-treated and control hearts are indicated: ++P < 0.01.

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TAG utilization in the working rat heart

Assuming no other metabolic fate of TAG taken up from the perfusate by the working heart, utilization of TAG was expressed as TAG oxidation plus total tissue [3H]lipid accumulation (Fig. 7). A similar pattern to TAG uptake (disappearance of TAG from the medium) was noted (Fig. 1): CM-TAG utilization was greater than VLDL-TAG utilization and THL, but not suramin, decreased VLDL-TAG and CM-TAG utilization significantly (Fig. 7).

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Figure 7. Utilization of triacylglycerol in the form of very-low-density lipoprotein or chylomicrons by isolated working rat heart Isolated rat hearts were perfused with rat very-low-density lipoprotein (VLDL) or rat chylomicrons (CM) (0.4 mm TAG) with tetrahydrolipstatin (THL; 10 μm), suramin (1 mg ml−1) or vehicle (control). Utilization was calculated as the sum of oxidation and total tissue deposition; for further details see text. Results are expressed as means ±s.e.m.(n= 8 per group). Statistically significant differences between VLDL and CM are indicated: *P < 0.05; significant differences between THL-treated and control hearts are indicated: #P < 0.05; ##P < 0.01.

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Lipoprotein lipase activity

LPL activity was measured in the perfusate at the end of the 60-min perfusion period following heparin administration (heparin-releasable portion, corresponding to the physiologically active endothelial enzyme), and in the heart tissue (tissue residual portion, nascent). There was no significant difference in LPL activity between hearts perfused with VLDL and chylomicrons (Fig. 8). As expected, 10 μm THL decreased the activity of heparin-releasable LPL significantly; interestingly, tissue residual (recruitable) LPL activity increased simultaneously with the inhibited endothelial activity (Fig. 8), confirming that THL does not have significant intracellular access. Suramin had no effect on LPL activity (Fig. 8).

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Figure 8. Lipoprotein lipase activity in the isolated working rat heart Lipoprotein lipase (LPL) activity was measured in post-heparin perfusate (heparin-releasable) and in myocardial tissue (tissue-residual) samples following 60 min perfusion with rat very-low-density lipoprotein (VLDL) or rat chylomicrons (CM) (0.4 mm TAG) together with tetrahydrolipstatin (THL; 10 μm), suramin (1 mg ml−1) or vehicle (control). Total LPL represents the sum of both heparin-releasable and tissue-residual LPL activities. For further details see text. Results are expressed as means ±s.e.m.(n= 8 per group). Statistically significant differences between THL-treated and control hearts are indicated: #P < 0.05; ##P < 0.01.

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Cardiac mechanical performance

Under conditions of moderate preload and afterload, hearts perfused with chylomicrons tended to slightly increased hydraulic work compared to those perfused with VLDL (P= 0.058). Suramin at 1 mg ml−1 significantly decreased the hydraulic work of all hearts whilst THL significantly decreased cardiac mechanical function in hearts perfused with CM to levels comparable to hearts perfused with VLDL (Fig. 9).

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Figure 9. Cardiac mechanical performance in isolated working rat hearts Isolated rat hearts were perfused with [3H]triolein-labelled rat very-low-density lipoprotein (VLDL) or rat chylomicrons (CM) (0.4 mm) with tetrahydrolipstatin (THL; 10 μm), suramin (1 mg ml−1) or vehicle (control) at 15 cmH2O pre-load and 100 cmH2O afterload. For further details see text. Results are expressed as means ±s.e.m.(n= 8 per group). Statistically significant differences between THL-treated and control hearts are indicated: #P < 0.05; significant differences between suramin-treated and control hearts are indicated: +P < 0.05, ++P < 0.01.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Very-low-density lipoprotein and chylomicrons are the two major lipoprotein carriers of triacylglycerol in plasma. TAG is probably an important cardiac substrate (Wang et al. 1998; Bennett et al. 2000b; Sambandam et al. 2000; Hauton et al. 2001; Mardy et al. 2001; Augustus et al. 2003): LPL knockout mice expressing the enzyme only in heart are rescued and have normal plasma triacylglycerols (Levak Frank et al. 1999). However, compared with extensive studies on cardiac metabolism of non-esterified FAs little is known about cardiac assimilation, utilization and metabolism of TAG-rich lipoproteins.

Lipoprotein uptake

At least two pathways are involved in TGRLP uptake by heart (Hauton et al. 2001; Kamataki et al. 2002; Augustus et al. 2003): LPL-catalysed hydrolysis of the TAG core (Eckel, 1989; Braun & Severson, 1992; Merkel et al. 2002), and lipoprotein receptor-mediated endocytosis (Takahashi et al. 1995; Masuzaki et al. 1996; Kamataki et al. 2002). The relative importance of the two mechanisms in cardiac TAG utilization is unknown. LPL is considered the rate-limiting enzyme for TAG hydrolysis, and is present in high activity in the heart (Enerback & Gimble, 1993) producing FAs and remnant lipoprotein particles. FAs are the primary oxidative fuel for heart metabolism (Calvani et al. 2000; Van Der Vusse et al. 2000; Hauton et al. 2001). The function of the VLDL-R remains unknown, as does the contribution of lipoprotein receptors to TAG uptake by the heart. There is some correlation between LPL-mediated and lipoprotein receptor-mediated mechanisms (VLDL-R deficient mice have decreased LPL activity (Yagyu et al. 2002), suggesting a regulatory function for the VLDL-R pathway), and this may enable LPL and lipoprotein receptors to regulate lipoprotein metabolism more efficiently (Chawla et al. 2001). This is supported by the similar tissue-specific expression and distribution of the VLDL-R and LPL (Auwerx et al. 1992; Kamataki et al. 2002). Furthermore, LPL bound to the surface of the cardiomyocyte plays an important role in the uptake of lipoprotein-TAG by heart (Yagyu et al. 2003), probably due to its effect of ‘bridging’ remnant lipoprotein particles to the receptor (Beisiegel et al. 1991; Hultin et al. 1995; Goldberg, 1996).

We used two experimental strategies to examine the routes of uptake, and subsequent metabolic fates of the two TGRLPs: (1) doubly radio-labelled ([3H]TAG; [14C]cholesterol) lipoprotein particles, permitting each to be independently tracked (cholesterol is assimilated solely by receptor-mediated uptake; TAG is assimilated by receptor- and LPL-mediated mechanisms) and (2) selective LPL and lipoprotein receptor inhibition by THL (Boren et al. 1998; Krebs et al. 2000; Strauss et al. 2002; Milosavljevic et al. 2003) and suramin (Brown et al. 1986; Huettinger et al. 1992; Martins et al. 2000), respectively. Thus, [3H]TAG and [14C]cholesterol dual-labelled VLDL and CMs were used to perfuse the working rat heart with or without antagonists. Suramin non-specifically inhibits all receptors of the LDL-receptor superfamily, preventing intracellular uptake of lipoproteins. The perfusion protocol was limited to 60 min to avoid the formation of myocardial oedema: metabolic integrity of the isolated heart is maintained for about 90 min but declines rapidly thereafter (Taegtmeyer et al. 1980). Furthermore, maintenance of hydraulic work is an extremely sensitive index of cardiac function, and decreases rapidly with oedema; cardiac mechanical function was well-preserved in these experiments (Fig. 9) and there was no difference in dry: wet wt ratios (data not shown).

CM-TAG uptake (Fig. 1) and oxidation (Fig. 3) were significantly greater than VLDL-TAG uptake, and CMs supported improved cardiac work (Fig. 9). This confirms our previous work (Hauton et al. 2001), which suggests that CMs are optimal substrates for LPL-mediated TAG lipolysis and that CM-TAG is an important cardiac fuel. Intralipid (similar in size to CM) is readily assimilated by mouse heart in vivo (more than mouse VLDL) and uptake of Intralipid-TAG by heart is greatly decreased by THL or in LPL knockout mice rescued by liver LPL expression (Augustus et al. 2003). Chylomicrons have a greater affinity for LPL than has VLDL because they are larger (Karpe & Hultin, 1995; Potts et al. 1995; Goldberg, 1996; Xiang et al. 1999). However, significant amounts of VLDL-TAG still enter the cardiomyocyte through LPL hydrolysis, as indicated by its inhibition by THL (Fig. 1). When THL was added to the perfusate to inhibit LPL activity, the TAG oxidation rate and tissue [3H]lipids, from both VLDL and CMs, decreased significantly (Figs 3, 4 and 5). This is unsurprising if LPL is the major pathway for TAG uptake by heart. Furthermore, because TAG taken up by LPL-mediated hydrolysis is mostly oxidized, THL can decrease the percentage of oxidized TAG (especially in the form of CMs). Thus LPL is a quantitatively more important route of ‘bulk’ TAG uptake than lipoprotein receptor-mediated pathways: uptake and utilization of TAG, either in the form of VLDL or CMs, was significantly inhibited by THL. However, although suramin tended to decrease TAG uptake, this was not significant (Fig. 1). It further suggests that FAs derived from LPL hydrolysis of lipoprotein-TAG are an important cardiac energy source. Since some TAG uptake persisted in the presence of THL, and despite low LPL activity (Fig. 8), this confirmed that THL does not interfere with LPL-heparin binding or bridging ability (Lookene et al. 1994). Since cholesterol can only be assimilated by the cardiomyocyte through receptor-mediated lipoprotein particle uptake, uptake of both lipoprotein particles by lipoprotein receptors was estimated by examining the uptake and metabolic fate of [14C]cholesterol.

In contrast to TAG uptake, VLDL-cholesterol uptake was greater than CM-cholesterol uptake; suramin significantly decreased cholesterol uptake, indicating that suramin was able to block lipoprotein receptors (Fig. 2A), whereas THL had no effect. Hence cholesterol is indeed taken up by heart through a suramin-sensitive lipoprotein receptor, and the VLDL particle or its remnant is an efficient substrate for receptor-mediated endocytosis. This effect was confirmed by measurement of [14C]cholesterol deposition into myocardial tissue [14C]cholesterol and [14C]cholesterol ester, which closely agreed with uptake data and indicated good recovery of label (Fig. 2A) (assuming no export or recycling of the [14C]-cholesterol/cholesterol ester). Since the cholesterol content varies between lipoprotein classes, the equivalent content and hence uptake of apo-B (apo-B48; apo-B100) was calculated using our own and other published data on lipid: apoprotein ratios in rat VLDL and CMs derived from liver perfusion and thoracic duct cannulation techniques; apo-B content can be calculated from the measured cholesterol content, and since VLDL and CMs both contain one copy of apo-B (Elovson et al. 1988), the apo-B content corresponds to lipoprotein particle number. Calculated apo-B uptake was even greater for VLDL than CMs, confirming the greater affinity and uptake of VLDL by lipoprotein receptors than CMs. CM particles are larger than VLDL (VLDL TAG: apo-B = 170: 1; CM TAG: apo-B = 950: 1; Y.-G. Niu & R. D. Evans, unpublished data); since the same TAG concentration (0.4 mm) of VLDL and CMs was added to the perfusate, there were greater numbers of VLDL particles in the perfusate. This may partly account for the greater uptake of VLDL by lipoprotein receptors. VLDL is an important transport form for cholesterol. However, VLDL-R expression is increased with a switch from glucose to fatty acids as energy substrates (such as in the fasting state; Masuzaki et al. 1996; Kamataki et al. 2002), suggesting a role for VLDL-R in myocardial energy metabolism or its regulation.

Metabolic fate of lipoproteins

TAG-FAs assimilated into cardiomyocytes have two principal metabolic fates: (1) oxidation, providing ATP for the heart's energetic needs; (2) esterification, forming the structural lipids (such as phospholipids) of heart and providing a limited energy storage resource (e.g. cellular TAG) (Saddik & Lopaschuk, 1991; Hauton et al. 2001; Mardy et al. 2001). TAG oxidation (Fig. 3) broadly reflected its uptake (Fig. 1). However, suramin inhibited TAG oxidation, but not uptake or overall utilization (Fig. 7), suggesting that the TAG that does enter the cardiomyocyte through receptor (suramin-inhibitable) pathways is preferentially channelled to oxidation. This was supported by examination of tissue [3H]lipids, which again suggests that receptor-mediated TAG uptake is directed towards oxidation, since suramin treatment increased the proportion of exogenous fatty acid incorporated into tissue lipids (Figs 4 and 5) at the expense of oxidation. Suramin decreased cholesterol uptake, but FA uptake was unaffected, suggesting a compensatory increase in functional LPL activity (although this was not found by direct LPL measurement); such a mechanism could explain the observed shift from oxidation to tissue lipid incorporation. This effect was associated with impaired cardiac function (Fig. 9), suggesting that the provision of TAG-FA for oxidation via receptors is vital. Attempts to increase the concentration of suramin were unsuccessful due to severe inhibition of cardiac function (data not shown). This may indicate that lipoprotein receptor function or targeted lipid uptake, though quantitatively less than LPL-mediated lipid uptake, is essential for myocardial contraction (or may indicate a direct cardiotoxic effect of the inhibitor). Conversely the data further suggest that whilst some LPL-mediated (THL-inhibitable) TAG uptake is directed towards oxidation, LPL-mediated TAG uptake is also an important contributor to the tissue lipid pool(s). Nearly 80% of CM-TAG was oxidized whilst only about 50% of VLDL-TAG was oxidized, the remainder being recovered in tissue lipids (Fig. 6). This demonstrates that the metabolic fates of VLDL-TAG and CM-TAG are different and suggests differential intracellular trafficking of FAs derived from CMs or VLDL. A putative mechanism for this effect would be the differing route of TGRLP-TAG uptake, with specific uptake pathways ‘channelling’ TRGLP-TAG-derived FAs to particular metabolic fates. Support for this proposal exists: Mardy et al. (2001) found different metabolic fates of NEFAs and CMs; the authors attributed this to the requirement of LPL for CM uptake. Our own work also demonstrated a disparity between TAG and NEFA metabolic fate, putatively attributable to differing uptake mechanisms (Hauton et al. 2001).

In conclusion, FAs derived from TAG-rich lipoproteins are an important source of substrate for the heart. CM-TAGs are mainly assimilated into the cardiomyocyte through a LPL-mediated mechanism. Most of the CM-TAG-derived FAs are oxidized, and contribute to maintenance of efficient cardiac mechanical function. By contrast, VLDLs are taken up into cardiac tissue to a greater extent by a lipoprotein receptor-mediated mechanism; most of the VLDL-TAG-derived FAs are incorporated into tissue lipids, in contrast to the greater oxidation of CM-TAGs. Differential inhibition studies indicate that whilst the LPL route of uptake supplies FAs for both oxidation and esterification (tissue lipid deposition), lipoprotein receptors tend to direct assimilated lipid towards oxidation; although quantitatively minor, this latter pathway may be important since its inhibition causes a marked decline in cardiac mechanical function. Despite this, in the face of limited TAG-FA supply, tissue incorporation of exogenous lipid is preferentially preserved at the expense of oxidation, suggesting a vital role for these newly formed tissue lipid pools. Further studies are necessary to investigate the details of channelling mechanisms between the two routes of uptake for TAG in order to elucidate their significance.

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  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix
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Appendix

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Acknowledgements

The authors are grateful to Professor David Severson, University of Calgary, Alberta, Canada, for advice, and to Oxford Surgical Sciences Trust Fund and the Intavent Foundation for financial support. Y.-G.N. was in receipt of a Sino-British Trust Fellowship.