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Am J Physiol Endocrinol Metab 288: E246-E253, 2005. First published August 24, 2004; doi:10.1152/ajpendo.00211.2004
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The metabolic "switch" AMPK regulates cardiac heparin-releasable lipoprotein lipase

Ding An, Thomas Pulinilkunnil, Dake Qi, Sanjoy Ghosh, Ashraf Abrahani, and Brian Rodrigues

Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada

Submitted 20 May 2004 ; accepted in final form 18 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The "fuel gauge" AMP-activated protein kinase (AMPK) facilitates ATP production to meet energy demands during metabolic stress. Given the importance of lipoprotein lipase (LPL) in providing hearts with fatty acids (FA), the preferred substrate consumed by the heart, the objective of the present study was to investigate whether activation of AMPK influences LPL at its functionally relevant location, the coronary lumen. Hearts from overnight-fasted rats were first perfused with heparin to release LPL, and homogenates from these hearts were then used to measure total and phospho-AMPK-{alpha} by Western blotting. Manipulation of AMPK activity [with drugs like adenine 9-{beta}-D-arabinofuranoside (Ara-A) and insulin (that inhibit) or perhexiline and oligomycin (that stimulate)] and its influence on LPL was also determined. Fasting augmented the activity of both AMPK and luminal LPL on immediate removal of hearts, effects that still remained even after in vitro perfusion of hearts for 1 h. Inhibition of AMPK in fasted hearts using an inhibitor like Ara-A or through provision of insulin markedly lowered the enhanced luminal LPL activity. In contrast, AMPK activators, like perhexiline and oligomycin, produced a significant elevation in heparin-releasable LPL activity. Thus, with fasting or drugs that influence AMPK, a strong correlation between this metabolic switch and cardiac LPL activity was established. Our data suggest that, in addition to its direct role in promoting FA oxidation, AMPK-mediated recruitment of LPL to the coronary lumen could represent an immediate compensatory response by the heart to guarantee FA supply.

isolated hearts; fasting; cardiac metabolism; palmitoyltransferase-1; adenosine 5'-triphosphate; adenosine 5'-triphosphate-activated protein kinase

CARDIAC TISSUE ACQUIRES MOST of its energy from metabolism of two major substrates, glucose and fatty acids (FA) (33). Entry of glucose in the heart is largely insulin dependent and, once inside, it is converted into pyruvate, acetyl-CoA is produced, and ATP is ultimately generated (31). Compared with glucose, FA is the preferred substrate consumed by the heart, contributing ~70% ATP when supplied at physiological levels. FA is transported in mitochondria as acyl-CoA, which undergoes {beta}-oxidation to release acetyl-CoA. In addition to entering the TCA cycle to produce ATP, acetyl-CoA can also be converted to malonyl-CoA under the influence of acetyl-CoA carboxylase (ACC) (11). Malonyl-CoA is an allosteric inhibitor of carnitine palmitoyltransferase-1 (CPT-1), the enzyme that manages the transport of long-chain fatty acyl-CoA from the cytosol to the mitochondria, where they are oxidized.

The heart has no measurable potential to synthesize FA, and thus is dependent upon exogenous supply, a process that is accelerated by transporters like FA transport protein, CD36, and FA-binding protein plasma membrane (24). Exogenous sources of FA include the plasma free FA fraction or FA released during hydrolysis of triglyceride (TG)-rich lipoproteins; the latter is considered to be the principal source of FA for cardiac utilization (2, 41). Lipoprotein lipase (LPL) is the rate-limiting enzyme for lipoprotein TG breakdown. LPL synthesized in cardiomyocytes is secreted as an active enzyme and binds to myocyte cell surface heparan sulfate proteoglycans (HSPG). Subsequently, the enzyme is translocated to comparable HSPG binding sites on the luminal side of the vessel wall where TG lipolysis occurs (6, 7, 38, 39).

The "fuel gauge" AMP-activated protein kinase (AMPK) regulates cellular metabolism (15). During metabolic stresses associated with energy depletion, like ischemia (when manufacture of ATP is hindered) or exercise (when ATP expenditure is augmented), changes in intracellular AMP/ATP levels promote threonine (Thr172) phosphorylation and activation of AMPK, an important regulator of both lipid and carbohydrate metabolism (14, 17). Once stimulated, AMPK switches off energy-consuming processes like TG and protein synthesis, whereas ATP-generating mechanisms are turned on. Thus, in heart and skeletal muscle, phosphorylated AMPK stimulates glucose uptake by inducing GLUT4 recruitment to the plasma membrane (16, 21, 35) and subsequent glycolysis through activation of 6-phosphofructo-2-kinase (25). More importantly, through its control of ACC, AMPK facilitates FA utilization. As ACC catalyzes the conversion of acetyl-CoA to malonyl-CoA, AMPK, by inhibiting ACC, is able to decrease malonyl-CoA and minimize its inhibition of FA oxidation(19, 20).

The majority of studies examining AMPK regulation of FA utilization have focused on FA oxidation. More recently, AMPK has also been implicated in FA delivery to cardiomyocytes through its regulation of CD36 (23). Given the importance of LPL in providing hearts with FA (2, 41), the objective of the present study was to investigate whether activation of AMPK influences LPL at its functionally relevant location, the coronary lumen. We demonstrate that, after AMPK phosphorylation, heparin-releasable LPL activity is amplified, providing an additional mechanism whereby this metabolic "switch" could regulate cellular energy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and the University of British Columbia (UBC). Adult male Wistar rats (220–240 g) were obtained from the UBC Animal Care Unit and were supplied with a standard laboratory diet (PMI Feeds, Richmond, VA) and water ad libitum. Where indicated, some rats were fasted for 16 h (6:00 PM to 10:00 AM). During fasting, food was withdrawn from the animals, but they had free access to water.

Isolated heart perfusion. Hearts were isolated and perfused as described previously (32). Briefly, rats were anesthetized with 65 mg/kg pentobarbital sodium, and the hearts were carefully excised. Rats were not injected with heparin before killing because it displaces LPL bound to HSPG on the capillary endothelium. Consequently, it was necessary to cannulate the heart quickly to avoid clotting of blood in the coronary arteries. After cannulation of the aorta, hearts were secured by tying below the innominate artery and perfused retrogradely with Krebs-Henseleit HEPES buffer containing 10 mM glucose (pH 7.4). Perfusion fluid was continuously gassed with 95% O2-5% CO2. The rate of coronary flow (7–8 ml/min) was controlled by a peristaltic pump.

Coronary lumen LPL activity. To measure coronary endothelium-bound LPL, the perfusion solution was changed to buffer containing 1% FA-free BSA and heparin (5 U/ml). This concentration of heparin can maximally release cardiac LPL from its HSPG binding sites. The coronary effluent (perfusate that drips down to the apex of the heart) was collected in timed fractions (10 s) over 5 or 10 min where indicated and assayed for LPL activity by measuring the hydrolysis of a sonicated [3H]triolein substrate emulsion (34). Retrograde perfusion of whole hearts with heparin results in a discharge of LPL that is rapid (within 0.5–1 min; suggested to represent LPL located at or near the endothelial cell surface) followed by a prolonged slow release (that is considered to originate from the myocyte cell surface; see Ref. 32). Because we were primarily concerned with examining AMPK regulation of LPL at the coronary lumen, only peak LPL activities are shown. LPL activity is expressed as nanomoles oleate released per hour per milliliter. Subsequent to LPL displacement with heparin, hearts were removed rapidly, washed with Krebs buffer, frozen in liquid nitrogen, and stored at –80°C for Western blot assay of AMPK.

Western blotting for AMPK. AMPK phosphorylation increases its activity ~50- to 100-fold. To determine total and phosphorylated AMPK-{alpha}, whole cell homogenates were isolated as described previously (1). Briefly, hearts were ground under liquid nitrogen and 50 mg were homogenized. After centrifugation at 5,000 g for 20 min, the protein content of the supernatant was quantified using a Bradford protein assay. Samples were diluted and boiled with sample loading dye, and 50 µg were used in SDS-PAGE. After transfer, membranes were blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20. Membranes were incubated either with rabbit AMPK-{alpha} or phosopho-AMPK (Thr172) antibody, and subsequently with secondary goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibody, and visualized using an ECL detection kit.

Measurement of cardiac LPL expression. LPL gene expression was measured in the indicated groups using RT-PCR. Briefly, total RNA from hearts (100 mg) was extracted using Tri-Zol (Invitrogen). After spectrophotometric quantification and resolving of RNA integrity using a formaldehyde agarose gel, reverse transcription was carried out using an oligo(dT) primer and superscript II RT (Invitrogen). cDNA was amplified using LPL-specific primers (8) [5'-ATCCAGCTGGGCCTAACTTT-3' (left) and 5'-AATGGCTTCTCCAATGTTGC-3' (right)]. The {beta}-actin gene was amplified as an internal control using 5'-TGGTGGGTATGGGTCAGAAGG-3' (left) and 5'-ATCCTGTCAGCGATGCCTGGG-3' (right). The linear range was found to be between 15 and 30 cycles. The amplification parameters were set at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min for a total of 30 cycles. The PCR products were electrophoresed on a 1.7% agarose gel containing ethidium bromide. Expression levels were represented as the ratio of signal intensity for LPL mRNA relative to {beta}-actin mRNA.

Measurement of LPL protein and activity in cardiomyocytes. Because endothelial cells cannot synthesize LPL, it is manufactured and processed in myocytes and then translocated across the interstitial space on HSPG-binding sites on the luminal surface of endothelial cells (38, 39). LPL protein and activity measurement was done using isolated cardiomyocytes. Ventricular calcium-tolerant myocytes were prepared by a previously described procedure (29, 30). Cardiac myocytes were suspended at a final cell density of 0.4 x 106 cells/ml, medium was separated by centrifugation, and LPL protein and activity were assayed in cell pellets.

For Western blot analysis, 25 µg of total protein were size fractionated in a SDS-polyacrylamide gel and blotted on a nitrocellulose membrane. After being blocked overnight at 4°C, the membrane was transferred to a solution of 1:1,000 diluted primary antibody (5D2, a monoclonal mouse anti-bovine LPL generously provided by Dr. J. Brunzell, University of Washington, Seattle, WA) and kept for 2 h at room temperature with gentle shaking. After being washed with TBS-Tween 20, the membrane was treated with 1:3,000 diluted secondary antibody (sheep anti-mouse IgG HRP conjugated) for 1 h at room temperature and visualized using an ECL detection kit. Measurement of LPL activity in the cell pellet was carried out by a previously described method (32).

Immunolocalization of LPL. Hearts were removed and placed in 10% formalin for 24 h. After formalin fixation and paraffin embedding, 3-µm sections were cut on silane-coated glass slides. Immunostaining was carried out as described before (30). Briefly, after deparaffinization and rehydration, slides were treated with 5% (vol/vol) heat-inactivated rabbit plasma to block nonspecific binding. Slides were then incubated with chicken anti-bovine LPL antibody (1:400 dilution) overnight. After being washed with TBS, slides were incubated with biotinylated rabbit anti-chicken IgG (1:150 dilution; Chemicon) and streptavidin-conjugated Cy3 fluorescent probe (1:1,000 dilution) for 1 h. Slides were visualized using a Bio-Rad Confocal Microscope.

Treatments. Adenine 9-{beta}-D-arabinofuranoside (Ara-A), a precursor of Ara-ATP, is a competitive inhibitor of AMPK. Insulin is also known to inhibit AMPK phosphorylation. Thus fasted hearts were perfused for 1 h with Ara-A or insulin, and AMPK phosphorylation and heparin-releasable LPL activity were measured. To determine whether promotion of AMPK phosphorylation can influence cardiac LPL, isolated control hearts were perfused with perhexiline (1–10 µM), both in the presence or absence of glucose for 45 min, and cardiac LPL activity was subsequently measured. Perhexiline, an anti-anginal agent, has been described to inhibit CPT-1 and myocardial consumption of FA, and in preliminary experiments, in the absence of glucose, has been shown to activate the phosphorylation of AMPK. To deplete intracellular ATP and induce metabolic stress and activation of AMPK, oligomycin (1–5 µM), an inhibitor of the mitochondrial electron transport chain, was perfused through control hearts for 8–30 min, and heparin-releasable cardiac LPL activity was subsequently measured. Oligomycin up to 300 µM has been used previously in cardiomyocytes to activate AMPK (23).

Serum measurements. Blood samples were removed from animals and centrifuged immediately to collect serum that was stored at –20°C until assayed. Diagnostic kits were used to measure glucose, TG (Sigma), and nonesterified fatty acid (Wako).

Statistical analysis. Wherever appropriate, one-way ANOVA followed by the Tukey or Bonferroni tests or the unpaired and paired Student's t-test was used to determine differences between group mean values (as indicated in the legends for Figs. 1–7). The level of statistical significance was set at P < 0.05.



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  		Fig. 1. Effect of fasting on cardiac AMP-activated protein kinase (AMPK) phosphorylation. After fasting for 16 h, total or phosphorylated AMPK was determined, either immediately upon removal of the heart or after a 1-h perfusion with Krebs buffer. Total and phospho-AMPK-{alpha} were measured using Western blotting and either rabbit AMPK-{alpha} or phospho-AMPK (Thr172) antibodies. Data are means ± SE of 4 different hearts in each group. P < 0.05, significantly different from control (*) and significantly different from all other groups (#).

 


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  		Fig. 7. Regulation of cardiac LPL by AMPK. After synthesis and processing of LPL in myocytes, the enzyme is either transported to lysosomes for degradation or delivered to cell-surface heparan sulfate proteoglycan (HSPG) binding sites. From here, LPL is transferred to similar binding sites on the luminal surface of endothelial cells. At this location, the enzyme hydrolyzes the triglyceride core of circulating lipoproteins to free fatty acids, which are then transported to the heart for ATP generation. During metabolic stress, AMPK-mediated recruitment of LPL to the coronary lumen could represent an immediate compensatory response by the heart to guarantee FA supply. One such mechanism by which AMPK does this is through increased secretion/transfer of LPL from the myocyte to the coronary lumen. Additionally, AMPK may potentially reduce intracellular lysosomal degradation.

 
Materials. Oligomycin, Ara-A, and perhexiline were purchased from Sigma. Total AMPK-{alpha} and phospho-AMPK-{alpha} antibodies were obtained from Cell Signaling (Beverly, MA). ECL detection kit was obtained from Amersham.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Fasting influences cardiac AMPK phosphorylation. Previous studies have reported AMPK phosphorylation in the liver from fasted animals (43). In the present study, overnight (16 h) fasting reduced serum glucose and TG levels, with no affect on free fatty acid (Table 1). After fasting, an approximately threefold increase of AMPK phosphorylation was observed on immediate removal of the heart (Fig. 1). Interestingly, perfusion with Krebs buffer for 1 h further augmented AMPK phosphorylation, but only in hearts from fasted animals (Fig. 1).


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  		Table 1. General characteristics of the animals

 
Augmentation of heparin-releasable LPL persists in vitro in fasted hearts. AMPK is known to promote cardiac FA oxidation (37). We considered whether the increase in AMPK phosphorylation is also related to increased FA delivery to the heart via LPL action at its functionally relevant location, the coronary lumen. Results from this and a previous study (36) showed that overnight fasting for 16 h caused a 2.5-fold increase in LPL activity at the coronary lumen (Fig. 2A, left). Immunohistochemical studies of myocardial sections were performed to complement our observation that the augmented LPL in fasted hearts was mainly localized at the endothelial cells. Whereas LPL immunofluorescence was found throughout the control and fasted myocardium, capillary blood vessels in the fasted heart demonstrated a more intense LPL immunoreactivity compared with control (Fig. 2B). Overall, our data suggest a correlation between AMPK phosphorylation and amplified endothelial LPL in fasted hearts.



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  		Fig. 2. Alterations in lipoprotein lipase (LPL) activity and immunofluorescence in hearts isolated from fasted animals. On immediate removal of hearts from control and overnight-fasted rats, LPL activity was determined after perfusion with heparin. In a separate experiment, hearts from the two groups were first perfused for 1 h with Krebs buffer in the recirculating mode (and in the absence of heparin). During the 60-min perfusion, basal LPL activity was determined in the buffer reservoir over time (inset). Subsequently, LPL was displaced by heparin, and activity was determined. Results are means ± SE of 4 rats in each group. B: representative photograph showing the effect of fasting on LPL immunofluorescence as visualized by fluorescent microscopy. Heart sections were fixed and incubated with the polyclonal chicken antibody against bovine LPL followed by incubations with biotinylated rabbit anti-chicken IgG and streptavidin-conjugated Cy3 fluorescent probe. *P < 0.05, significantly different from control.

 
To determine whether this increase in LPL is a consequence of changes in gene expression, LPL mRNA was measured. Interestingly, changes in luminal LPL activity were independent of shifts in mRNA levels (Fig. 3A). Additionally, given that after fasting LPL protein (Fig. 3B) and activity (Fig. 3C) remain unchanged in cardiomyocytes, the predominant cell type in the heart responsible for LPL synthesis and processing, our data suggest that LPL increase at the coronary lumen is likely via posttranslational mechanisms.



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  		Fig. 3. LPL gene expression, protein mass, and activity in hearts isolated from fasted animals. Rats were fasted for 16 h; during fasting, food was withdrawn from the animals, but they had free access to water. LPL gene expression in the whole heart was measured using RT-PCR (A). LPL protein (B) and activity (C) measurements were done using isolated cardiomyocytes. Data are means ± SE of 3 different hearts in each group.

 
Given that AMPK phosphorylation increased further in fasted hearts perfused for 1 h, we evaluated whether there would be an additional increase or maintenance of LPL over this period in vitro. Thus hearts from control and fasted animals were perfused with normal Krebs buffer (minus heparin) for 1 h followed by heparin perfusion for 10 min. Interestingly, after the 1-h perfusion, fasted hearts demonstrated no further increase in LPL activity (Fig. 2A, right). The ability of these hearts to maintain their high heparin-releasable LPL in vitro suggested either an increased recruitment from myocytes or decreased displacement from the coronary lumen. To evaluate this further, during the 60-min perfusion with heparin-free buffer in the recirculating mode, LPL activity in the buffer reservoir (total volume 30 ml) was determined at various intervals. Interestingly, fasted hearts showed greater basal release of LPL throughout the perfusion (Fig. 2, inset).

Inhibition of AMPK phosphorylation lowers cardiac LPL. Because in vitro hearts from fasted animals maintain an augmented AMPK phosphorylation and LPL activity, we hypothesized that inhibition of AMPK phosphorylation should decrease LPL activity. Ara-A, a precursor of Ara-ATP, is a competitive inhibitor of AMPK (27). Perfusion of fasted hearts for 1 h with Ara-A decreased AMPK phosphorylation (Fig. 4A), had no effect on the augmented basal enzyme release (1.6-fold higher than control at 60 min), but decreased heparin-releasable LPL activity (Fig. 4B). Because insulin is also known to inhibit AMPK phosphorylation (18), fasted hearts were perfused for 1 h with insulin. Similar to Ara-A, insulin reduced both AMPK phosphorylation (Fig. 4A) and luminal LPL activity (Fig. 4B).



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  		Fig. 4. Consequence of AMPK inhibition on heparin-releasable LPL activity in fasted hearts. AMPK phosphorylation in fasted hearts was inhibited using adenine 9-{beta}-D-arabinofuranoside (Ara-A, 500 µM) or insulin (100 µU/ml) perfusion for 1 h. Subsequently, LPL was displaced by heparin, and activity was determined (B), or total and phospho-AMPK-{alpha} were measured using Western blotting (A). Data are means ± SE of 4 different hearts in each group. *P < 0.05, significantly different from control.

 
Promotion of AMPK phosphorylation in isolated control hearts recruits LPL to the luminal surface. In the presence of glucose, perfusion of isolated control hearts with perhexiline did not change either AMPK phosphorylation (Fig. 5A) or LPL activity (Fig. 5B). However, removal of glucose from the perfusion buffer significantly augmented both AMPK phosphorylation and luminal LPL activity (Fig. 5, A and B). With oligomycin, there was a rapid and concentration-dependent increase in both AMPK phosphorylation (Fig. 6A) and coronary luminal LPL (Fig. 6B). Both perhexiline and oligomycin were without effect on LPL mRNA, protein, and activity in cardiomyocytes (data not shown).



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  		Fig. 5. Consequence of carnitine palmitoyltransferase-1 (CPT-1) inhibition on AMPK phosphorylation and heparin-releasable LPL activity in control hearts. Hearts from control animals were perfused either in the presence or absence of perhexiline (5 mM), with or without glucose, for 45 min. Subsequently, LPL was displaced by heparin, and activity was determined (B) or AMPK was measured using Western blotting (A). Data are means ± SE of 4 different hearts in each group. *P < 0.05, significantly different from control.

 


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  		Fig. 6. Effect of inhibiting ATP synthesis on AMPK phosphorylation and heparin-releasable LPL activity. Hearts from control animals were perfused either in the presence or absence of oligomycin (1–5 µM) for 8 min. Subsequently, LPL was displaced by heparin, and activity was determined (B) or AMPK was measured using Western blotting (A). Data are means ± SE of 4 different hearts in each group. *P < 0.05, significantly different from control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
AMPK is the switch that activates pathways that produce ATP while turning off ATP-consuming processes (14, 26). Thus, during fasting, when glucose supply is inadequate and its oxidation is compromised because of PDK4 overexpression (28, 40), AMPK activity is augmented in the liver (37). The present study, for the first time, demonstrates that fasting also increases cardiac AMPK phosphorylation. Because activation of AMPK is known to exaggerate FA utilization in the ischemic reperfused heart (19), our results suggest that a similar relationship between AMPK activity and FA oxidation must also exist after fasting. Indeed, rates of cardiac oleate oxidation do increase after fasting (45).

Given the role of AMPK in regulating FA oxidation, it is conceivable that it could also influence FA delivery. Provision of FA to the heart involves 1) release from adipose tissue and transport to the heart (22), 2) breakdown of endogenous cardiac TG stores, 3) internalization of whole lipoproteins, and 4) hydrolysis of circulating TG-rich lipoproteins to FA by LPL positioned at the endothelial surface of the coronary lumen (7), and AMPK has been implicated in some of these processes. Thus activation of AMPK is known to augment FA uptake in the contracting isolated cardiomyocyte through the recruitment of FA transporters to the plasma membrane (23). Moreover, although its role in cardiac TG lipolysis is unknown, at least in adipocytes, AMPK is suggested to mediate the lipolytic effect of adrenergic stimulation (44). Given that LPL-mediated hydrolysis of lipoproteins was recently suggested to be the principal source of FA for cardiac utilization (2, 41), we examined its relationship to AMPK activity. In agreement with previous studies, we observed an increase of heparin-releasable LPL activity after overnight fasting, and immunostaining revealed that most of the LPL protein was located at the coronary lumen. Our data suggest that AMPK phosphorylation may play a role in increasing cardiac functional LPL. Because changes in luminal LPL activity were independent of shifts in LPL mRNA or alterations in protein and activity in cardiomyocytes, our data imply that this LPL increase at the coronary lumen is likely via posttranslational mechanisms. Other studies have also established that the fasting-induced changes in cardiac LPL activity are likely through posttranslational mechanisms that do not involve alterations in mRNA levels, protein synthesis, or specific activity of the protein (10). Moreover, at least in adipose tissue, downregulation of LPL during fasting is also posttranslational and involves a shift from active to inactive forms of the lipase (5).

To further examine this relationship, hearts from fasted animals were perfused with Krebs buffer for 1 h (in the absence of heparin), and heparin-releasable LPL activity was subsequently determined. After the 1-h perfusion, only fasted hearts showed further increase in AMPK phosphorylation. Given that the fasted heart prefers FA as an energy substrate for ATP generation, it is likely that this added effect on AMPK phosphorylation is the result of lack of albumin-bound FA and circulating lipoproteins in the perfusate. It should be noted that, even though AMPK phosphorylation increased with time in vitro, heparin-releasable LPL activity at the coronary lumen did not expand further. Because previous studies have demonstrated that the coronary lumen of the rat heart has a finite number of LPL-binding sites and that, under normal conditions, only a fraction of these binding sites are occupied by LPL (29), our present data are indicative of a model that suggests that, once AMPK activation fills these sites (after overnight fasting) with the enzyme, no further increase of LPL is possible. Interestingly, throughout the 1-h perfusion, fasted hearts showed greater basal release of LPL. Given that this increased basal LPL release was not followed by a decline in heparin-releasable LPL activity, our data suggest that, in vitro, it is likely that the fasted heart has higher enzyme transfer from the myocyte to the coronary lumen.

After ischemia and reperfusion, cardiac AMPK activity is activated, an effect that is prevented by insulin (3). Additionally, in skeletal muscle, stimulation of AMPK by 5-aminoimidazole-4-carboxamide-1-{beta}-D-riboturanoside has been shown to be normalized by Ara-A (27). In our study, these agents were effective in lowering AMPK phosphorylation in the fasted heart. More importantly, in these hearts, insulin and Ara-A also reduced the endothelial-bound LPL pool. Our data suggest that, in vitro, the fasted heart is able to maintain its high LPL activity through AMPK, and inhibition of AMPK phosphorylation can lower this high enzyme activity.

A more direct examination of the relationship between AMPK and LPL was realized using compounds that activate AMPK. One mechanism by which AMPK is stimulated includes modulation of intracellular AMP concentrations. It should be noted that, since the effect of AMP can be antagonized by high concentrations of ATP, a higher AMP-to-ATP ratio is more efficient in activating AMPK than a rise in AMP alone (13). Another method that can induce AMPK activation is through inhibition of ATP production that in turn increases intracellular generation of AMP (12). Perhexiline, a CPT-1 blocker that is widely used in the treatment of ischemia, inhibits FA oxidation and switches the heart to utilize glucose. Given that FA is a major substrate contributing ~70% ATP in heart, we predicted that perhexiline would activate AMPK through impaired ATP generation. However, perfusion of control hearts with perhexiline was without effect on cardiac AMPK or LPL activity, suggesting that metabolic switching to utilize glucose may be sufficient to prevent ATP depletion. To test this idea, glucose was removed from our perfusion media when using perhexiline. Interestingly, this adjustment increased AMPK phosphorylation and was able to recruit LPL to the endothelial cell. Oligomycin, an ATP synthase inhibitor, has been shown to markedly change the AMP-to-ATP ratio and AMPK activity in isolated perfused hearts (25) and cardiomyocytes (23). In our study, oligomycin increased AMPK phosphorylation and heparin-releasable LPL activity in a dose-dependent manner. Given the rapid (8 min) augmentation of LPL after oligomycin, and in the absence of any change in LPL mRNA or protein and activity in cardiomyocytes, our data suggest that the regulation of LPL by AMPK is likely through posttranslational mechanisms. One such mechanism could include an increase in secretion of LPL from the myocyte to the coronary lumen. In this regard, despite the high basal release of LPL to the medium from isolated fasted hearts, heparin-releasable LPL activity remained high, suggesting augmented LPL transfer from myocyte to coronary lumen. However, even though enzyme transfer may have increased, LPL protein and activity remain unchanged in myocytes from fasted animals, suggesting that reduced intracellular lysosomal degradation may also be occurring concomitantly. In adipocytes, stimulation of LPL secretion is known to reduce enzyme degradation (9, 42). Additionally, ATP depletion in Chinese hamster ovary cells after incubation with 2-deoxy-D-glucose drastically reduces LPL degradation rate (4). At present, the target for AMPK phosphorylation that controls LPL secretion and degradation is not known.

In summary, using fasting and modulators of AMPK, a strong correlation between this metabolic switch and cardiac LPL activity was established. Given the rapidity by which oligomycin duplicates the above relationship, our data suggest that, in addition to its direct role in promoting FA oxidation, increased AMPK recruitment of LPL from its major storage site, the cardiomyocyte, to the coronary lumen and decreased intracellular degradation could represent an immediate compensatory response by the heart to guarantee FA supply (Fig. 7).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The studies described in this paper were supported by an operating grant from the Canadian Diabetes Association (in honor of the late Lillian I. Dale) and Heart and Stroke Foundation of BC and Yukon program grant. We gratefully acknowledge financial support from the Health Research Foundation/Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada for Graduate Research Scholarships to T. Pulinilkunnil, S. Ghosh, and D. Qi.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Rodrigues, Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The Univ. of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail: rodrigue{at}interchange.ubc.ca)

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
 

  1. Atkinson LL, Fischer MA, and Lopaschuk GD. Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis. J Biol Chem 277: 29424–29430, 2002.[Abstract/Free Full Text]
  2. Augustus AS, Kako Y, Yagyu H, and Goldberg IJ. Routes of FA delivery to cardiac muscle: modulation of lipoprotein lipolysis alters uptake of TG-derived FA. Am J Physiol Endocrinol Metab 284: E331–E339, 2003.[Abstract/Free Full Text]
  3. Beauloye C, Marsin AS, Bertrand L, Krause U, Hardie DG, Vanoverschelde JL, and Hue L. Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett 505: 348–352, 2001.[CrossRef][ISI][Medline]
  4. Ben-Zeev O, Mao HZ, and Doolittle MH. Maturation of lipoprotein lipase in the endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates. J Biol Chem 277: 10727–10738, 2002.[Abstract/Free Full Text]
  5. Bergo M, Wu G, Ruge T, and Olivecrona T. Down-regulation of adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on. J Biol Chem 277: 11927–11932, 2002.[Abstract/Free Full Text]
  6. Blanchette-Mackie EJ, Dwyer NK, and Amende LA. Cytochemical studies of lipid metabolism: immunogold probes for lipoprotein lipase, and cholesterol. Am J Anat 185: 255–263, 1989.[CrossRef][ISI][Medline]
  7. Blanchette-Mackie EJ, Masuno H, Dwyer NK, Olivecrona T, and Scow RO. Lipoprotein lipase in myocytes, and capillary endothelium of heart: immunocytochemical study. Am J Physiol Endocrinol Metab 256: E818–E828, 1989.[Abstract/Free Full Text]
  8. Brault D, Noe L, Etienne J, Hamelin J, Raisonnier A, Souli A, Chuat JC, Dugail I, Quignard-Boulange A, Lavau M, and Galibert F. Sequence of rat lipoprotein lipase-encoding cDNA. Gene 121: 237–246, 1992.[CrossRef][ISI][Medline]
  9. Cupp M, Bensadoun A, and Melford K. Heparin decreases the degradation rate of lipoprotein lipase in adipocytes. J Biol Chem 262: 6383–6388, 1987.[Abstract/Free Full Text]
  10. Doolittle MH, Ben-Zeev O, Elovson J, Martin D, and Kirchgessner TG. The response of lipoprotein lipase to feeding and fasting. Evidence for posttranslational regulation. J Biol Chem 265: 4570–4577, 1990.[Abstract/Free Full Text]
  11. Dyck JR, Barr AJ, Barr RL, Kolattukudy PE, and Lopaschuk GD. Characterization of cardiac malonyl-CoA decarboxylase and its putative role in regulating fatty acid oxidation. Am J Physiol Endocrinol Metab 275: E2122–E2129, 1998.
  12. Frederich M and Balschi JA. The relationship between AMP-activated protein kinase activity and AMP concentration in the isolated perfused rat heart. J Biol Chem 277: 1928–1932, 2002.[Abstract/Free Full Text]
  13. Hardie DG. Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144: 5179–5183, 2003.[Abstract/Free Full Text]
  14. Hardie DG and Carling D. The AMP-activated protein kinase-fuel gauge of the mammalian cell? Eur J Biochem 246: 259–273, 1997.[ISI][Medline]
  15. Hardie DG and Hawley SA. AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 23: 1112–1119, 2001.[CrossRef][ISI][Medline]
  16. Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters LA, and Goodyear LJ. Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes 49: 527–531, 2000.[Abstract]
  17. Hong SP, Leiper FC, Woods A, Carling D, and Carlson M. Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc Natl Acad Sci USA 100: 8839–8843, 2003.[Abstract/Free Full Text]
  18. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, and Dyck JR. Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278: 39422–39427, 2003.[Abstract/Free Full Text]
  19. Kudo N, Barr AJ, Barr RL, Desai S, and Lopaschuk GD. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5'-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem 270: 17513–17520, 1995.[Abstract/Free Full Text]
  20. Kudo N, Gillespie JG, Kung L, Witters LA, Schulz R, Clanachan AS, and Lopaschuk GD. Characterization of 5'AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion following ischemia. Biochim Biophys Acta 1301: 67–75, 1996.[Medline]
  21. Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ, and Winder WW. 5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 48: 1667–1671, 1999.[Abstract]
  22. Lopaschuk GD, Belke DD. Gamble J, Itoi T, and Schonekess BO. Regulation of fatty acid oxidation in the mammalian heart in health, and disease. Biochim Biophys Acta 1213: 263–276, 1994.[Medline]
  23. Luiken JJ, Coort SL, Willems J, Coumans WA, Bonen A, van der Vusse GJ, and Glatz JF. Contraction-induced fatty acid translocase/CD36 translocation in rat cardiac myocytes is mediated through AMP-activated protein kinase signaling. Diabetes 52: 1627–1634, 2003.[Abstract/Free Full Text]
  24. Luiken JJ, Turcotte LP, and Bonen A. Protein-mediated palmitate uptake, and expression of fatty acid transport proteins in heart giant vesicles. J Lipid Res 40: 1007–1016, 1999.[Abstract/Free Full Text]
  25. Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den Berghe G, Carling D, and Hue L. Phosphorylation, and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischemia. Curr Biol 10: 1247–1255, 2000.[CrossRef][ISI][Medline]
  26. Muoio DM, Seefeld K, Witters LA, and Coleman RA. AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target. Biochem J 338: 783–791, 1999.
  27. Musi N, Hayashi T, Fujii N, Hirshman MF, Witters LA, and Goodyear LJ. AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle. Am J Physiol Endocrinol Metab 280: E677–E684, 2001.[Abstract/Free Full Text]
  28. Pilegaard H, Saltin B, and Neufer PD. Effect of short-term fasting and refeeding on transcriptional regulation of metabolic genes in human skeletal muscle. Diabetes 52: 657–662, 2003.[Abstract/Free Full Text]
  29. Pulinilkunnil T, Abrahani A, Varghese J, Chan N, Tang I, Ghosh S, Kulpa J, Allard M, Brownsey R, and Rodrigues B. Evidence for rapid "metabolic switching" through lipoprotein lipase occupation of endothelial binding sites. J Mol Cell Cardiol 35: 1093–1103, 2003.[CrossRef][ISI][Medline]
  30. Pulinilkunnil T, Qi D, Ghosh S, Cheung C, Yip P, Varghese J, Abrahani A, Brownsey R, and Rodrigues B. Circulating triglyceride lipolysis facilitates lipoprotein lipase translocation from cardiomyocyte to myocardial endothelial lining. Cardiovasc Res 59: 7887–7897, 2003.
  31. Randle PJ, Hales CN, Garland PB, and Newsholme EA. The glucose fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1: 785–789, 1963.[ISI][Medline]
  32. Rodrigues B, Cam MC, Jian K, Lim F, Sambandam N, and Shepherd G. Differential effects of streptozotocin-induced diabetes on cardiac lipoprotein lipase activity. Diabetes 46: 1346–1353, 1997.[Abstract]
  33. Rodrigues B and McNeill JH. The diabetic heart: metabolic causes for the development of a cardiomyopathy. Cardiovasc Res 26: 913–922, 1992.[ISI][Medline]
  34. Rodrigues B, Spooner M, and Severson DL. Free fatty acids do not release lipoprotein lipase from isolated cardiac myocytes or perfused hearts. Am J Physiol Endocrinol Metab 262: E216–E223, 1992.[Abstract/Free Full Text]
  35. Russell RR 3rd, Bergeron R, Shulman GI, and Young LH. Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol Endocrinol Metab 277: E643–E649, 1999.
  36. Sambandam N, Abrahani MA, St Pierre E, Al-Atar O, Cam MC, and Rodrigues B. Localization of lipoprotein lipase in the diabetic heart: regulation by acute changes in insulin. Arterioscler Thromb Vasc Biol 19: 1526–1534, 1999.[Abstract/Free Full Text]
  37. Sambandam N and Lopaschuk GD. AMP-activated protein kinase (AMPK) control of fatty acid and glucose metabolism in the ischemic heart. Prog Lipid Res 42: 238–256, 2003.[CrossRef][ISI][Medline]
  38. Saxena U, Klein MG, and Goldberg IJ. Transport of lipoprotein lipase across endothelial cells. Proc Natl Acad Sci USA 88: 2254–2258, 1991.[Abstract/Free Full Text]
  39. Stins MF, Maxfield FR, and Goldberg IJ. Polarized binding of lipoprotein lipase to endothelial cells. Implication for its physiological actions. Arterioscler Thromb 12: 1437–1446, 1992.[Abstract/Free Full Text]
  40. Sugden MC, Greenwood GK, Smith ND, and Holness MJ. Peroxisome-proliferator-activated receptor-alpha (PPARalpha) deficiency leads to dysregulation of hepatic lipid and carbohydrate metabolism by fatty acids and insulin. Biochem J 364: 361–368, 2002.[CrossRef][ISI][Medline]
  41. Teusink B, Voshol PJ, Dahlmans VE, Rensen PC, Pijl H, Romijn JA, and Havekes LM. Contribution of fatty acids released from lipolysis of plasma triglycerides to total plasma fatty acid flux and tissue-specific fatty acid uptake. Diabetes 52: 614–620, 2003.[Abstract/Free Full Text]
  42. Vannier C and Ailhaud G. Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport. J Biol Chem 264: 13206–13216, 1989.[Abstract/Free Full Text]
  43. Witters LA, Gao G, Kemp BE, and Quistorff B. Hepatic 5'-AMP-activated protein kinase: zonal distribution and relationship to acetyl-CoA carboxylase activity in varying nutritional states. Arch Biochem Biophys 308: 413–419, 1994.[CrossRef][ISI][Medline]
  44. Yin W, Mu J, and Birnbaum MJ. Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis in 3T3–L1 adipocytes. J Biol Chem 278: 43074–43080, 2003.[Abstract/Free Full Text]
  45. Young ME, Guthrie PH, Razeghi P, Leighton B, Abbasi S, Patil S, Youker KA, and Taegtmeyer H. Impaired long-chain fatty acid oxidation and contractile dysfunction in the obese Zucker rat heart. Diabetes 51: 2587–2595, 2002.[Abstract/Free Full Text]



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