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(Circulation Research. 1997;80:482-489.)
© 1997 American Heart Association, Inc.

Articles

Upregulation of 5'-AMP–Activated Protein Kinase Is Responsible for the Increase in Myocardial Fatty Acid Oxidation Rates Following Birth in the Newborn Rabbit

A-Olufemi Makinde, James Gamble, , Gary D. Lopaschuk

From the Cardiovascular Disease Research Group, the Lipid and Lipoprotein Research Group, and the Department of Pediatrics and Pharmacology, Faculty of Medicine, The University of Alberta, Edmonton, Canada.

Correspondence to Dr Gary D. Lopaschuk, 423 Heritage Medical Research Bldg, The University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail gary.lopaschuk{at}ualberta.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract In newborn rabbits, fatty acid oxidation rates in the heart significantly increase between 1 and 7 days after birth. This is due in part to a decrease in malonyl coenzyme A (CoA) production by acetyl CoA carboxylase (ACC). In other tissues, 5'-AMP–activated protein kinase (AMPK) can phosphorylate and inhibit ACC activity. In this study, we show that 1- and 7-day-old rabbit hearts have a high AMPK activity, with AMPK expression and activity being greatest in 7-day-old hearts. Hearts were also perfused in the Langendorff mode with Krebs-Henseleit buffer containing 0.4 mmol/L [¹⁴C]palmitate and 11 mmol/L glucose±100 µU/mL insulin. In the absence of insulin, fatty acid oxidation rates were significantly higher in 7-day-old hearts compared with 1-day-old hearts. AMPK activity was also greater in 7-day-old hearts compared with 1-day-old hearts (909±60 and 585±75 pmol·min^-1·mg protein^-1, respectively; P<.05). In 1-day-old hearts, the presence of insulin resulted in a significant decrease in AMPK activity, an increase in ACC activity, and a decrease in fatty acid oxidation rates. In 7-day-old hearts, AMPK activity was also decreased by insulin, although ACC activity remained low and fatty acid oxidation rates remained high. Stimulation of AMPK in 7-day-old hearts with 200 µmol/L 5-amino 4-imidazolecarboxamide ribotide resulted in a further decrease in ACC activity and an increase in fatty acid oxidation rates. These data suggest that AMPK, ACC, and fatty acid oxidation are sensitive to insulin in 1-day-old rabbit hearts and that the decrease in circulating insulin levels seen after birth leads to an increased activity of AMPK. This can then lead to a phosphorylation and inhibition of ACC activity, with a resultant increase in fatty acid oxidation rates.

Key Words: 5'-AMP–activated protein kinase • acetyl coenzyme A carboxylase • newborn • fatty acid oxidation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the fetus, the majority of the heart's energy requirements are obtained from lactate oxidation and glycolysis. After birth, myocardial fatty acid oxidation rates rapidly increase and become a major source of energy for the heart.^{1 2} The mechanisms responsible for this increase in fatty acid oxidation are poorly understood. We have recently demonstrated that this is due, in part, to a decrease in malonyl CoA levels,³ which can result in an increase in CPT 1 activity, a key enzyme involved in fatty acid uptake by the mitochondria.⁴ The drop in malonyl CoA levels following birth occurs secondary to a decrease in the activity of ACC,³ the enzyme responsible for cytoplasmic malonyl CoA production.

In 1-day-old rabbit hearts, ACC is markedly stimulated by insulin.³ This has potential significance in the maturation of fatty acid oxidation, since in the immediate newborn period, levels of insulin in the blood decrease dramatically.⁵ As a result, a decrease in insulin has the potential to decrease ACC activity and lower malonyl CoA levels. Experiments in hepatocytes have shown that insulin can stimulate ACC via an inhibition of a novel AMPK.⁶ This kinase phosphorylates and inhibits ACC activity and is important in the phosphorylation control of ACC.^{7 8} Although it has recently been demonstrated that the adult heart has a high AMPK activity^{9 10} and expression,^{11 12 13 14} the involvement of AMPK in regulating cardiac ACC activity is not clearly understood. We have shown that increases in AMPK activity in the adult heart may increase fatty acid oxidation, secondary to a decrease in ACC activity.^{9 10} Since insulin inhibits AMPK in other tissues,⁶ we hypothesized that the drop in insulin levels following birth results in an increase in cardiac AMPK activity. This then results in a decrease in ACC activity and malonyl CoA levels, resulting in an increase in CPT 1 activity and fatty acid oxidation.

The purpose of the present study was to characterize AMPK in the newborn heart and determine if changes in AMPK activity are important in the regulation of ACC and fatty acid oxidation. We demonstrate that heart AMPK expression and activity increases after birth. In 1-day-old hearts, insulin inhibits AMPK activity, which is accompanied by an increase in ACC activity and a decrease in fatty acid oxidation. Our results suggest that a decrease in insulin control of AMPK may be important in the increase in myocardial fatty acid oxidation after birth.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Acetyl CoA was obtained from Boehringer-Mannheim. The SAMS peptide used in the AMPK assay was synthesized by the Alberta Peptide Institute. Anti-AMPK catalytic subunit antibody was a gift from Dr L.A. Witters, Hanover, NH. [³²P]ATP, 1-[¹⁴C]palmitate, and [¹⁴C]sodium bicarbonate were obtained from ICN Radiopharmaceuticals. Peroxidase-labeled streptavidin was purchased from Mandel Scientific. Trans-Blot nitrocellulose membrane was obtained from Bio-Rad. ECL Western blotting detection reagents were purchased from Amersham International. X-ray films were purchased from Kodak. All other chemicals were purchased from Sigma Chemical Co.

Heart Perfusions
One- and 7-day-old New Zealand White rabbit hearts were used in the present study. Newborn rabbits (either sex) were injected with sodium pentobarbital: seven-day-old rabbits were injected with 60 mg/kg IV; 1-day-old rabbits were injected with 60 mg/kg IP. Hearts from anesthetized rabbits were quickly removed and placed in ice-cold Krebs-Henseleit buffer. Hearts were then either frozen immediately with tongs cooled to the temperature of liquid N₂, or the aorta was cannulated, and the hearts were perfused in the Langendorff mode as previously described.³ Hearts were perfused with Krebs-Henseleit buffer containing 11 mmol/L glucose and 0.4 mmol/L [1-¹⁴C]palmitate bound to 3% albumin. Hearts were perfused for a 40-minute period in the presence or absence of 100 µU/mL insulin or in the presence or absence of 200 µmol/L AICAR. Perfusate was delivered to the heart at a 60 mm Hg pressure. Steady state palmitate oxidation rates were measured over the 40-minute period by quantitative collection of ¹⁴CO₂ produced by the heart, using techniques described previously.³

Tissue Workup
In both unperfused and perfused hearts, ventricles were quickly frozen with Wollenberger clamps cooled to the temperature of liquid N₂. The frozen ventricular tissue was then weighed and powdered in a mortar and pestle cooled to the temperature of liquid N₂. The remainder of the ventricular tissue was then stored in liquid N₂ for subsequent biochemical analysis. A portion of the powdered tissue was used to determine the dry-to-wet ratio of the ventricles.

Extraction of AMPK and ACC
Approximately 200 mg of frozen tissue was homogenized with a buffer containing Tris-HCl (0.05 mol/L, pH 7.5 at 4°C), mannitol (0.25 mol/L), NaF (50 mmol/L), sodium pyrophosphate (5 mmol/L), EDTA (1 mmol/L), EGTA (1 mmol/L), dithiothreitol (1 mmol/L), and the following protease inhibitors: phenylmethylsulfonyl fluoride (1 mmol/L), soybean trypsin inhibitor (4 µg/mL), and benzamidine (1 mmol/L). Samples were then centrifuged at 14 000g for 20 minutes at 4°C. The supernatant was then brought to 2.5% PEG with 25% (wt/vol) PEG 8000 and agitated for 10 minutes at 4°C. Samples were then spun at 10 000g for 10 minutes at 4°C. The supernatant was then made up to 6% PEG 8000 using the PEG 8000 stock described above and stirred once again for 10 minutes at 4°C. This fraction was then spun at 10 000g for 10 minutes, and the precipitate was washed with homogenization buffer containing 6% PEG 8000. This was followed by a final centrifugation at 10 000g, after which the protein concentration in the supernatant was measured using a Sigma BCA protein kit.

ACC Assay
ACC activity in the PEG 8000 fractions was measured using the CO₂ fixation method.⁶ Briefly, 5 µL of the PEG fraction, containing 20 µg of total protein, was added to a reaction mixture (final volume, 165 µL) containing Tris acetate (60.6 mmol/L), BSA (1 mg/mL), ß-mercaptoethanol (1.32 µmol/L), ATP (2.12 mmol/L), acetyl CoA (1.06 mmol/L), magnesium acetate (5.0 mmol/L), and NaHCO₃ (18.08 mmol/L). Samples were incubated at 37°C for 2 minutes, and the reaction was stopped by adding 25 µL of 10% perchloric acid. Samples were then spun for 20 minutes at 3500 rpm, and 160 µL of supernatant was placed in minivials and dried in a fume hood overnight. H₂O (160 µL), followed by scintillant, was then added to the vials, and the vials were counted. ACC activity was expressed as the amount of malonyl CoA produced per minute per milligram protein.

AMPK Assay
AMPK activity was measured according to the method described by Hardie's group,^{15 16 17} with slight modifications.^{9 10 18} Briefly, 2 µL of the PEG fraction was added to a reaction mixture (final volume, 25 µL) composed of HEPES-NaOH (40 mmol/L), NaCl (80 mmol/L), glycerol (8% [wt/vol]), EDTA (0.8 mmol/L), SAMS peptide (200 µmol/L), dithiothreitol (0.8 mmol/L), [-³²P]ATP (200 µmol/L), MgCl₂ (5 mmol/L), and 0.18% Triton X-100. Samples were also incubated in the presence or absence of 200 µmol/L AMP. This mixture was then incubated for 3 minutes at 30°C. From this incubation mixture, 15 µL was spotted on 1-cm² phosphocellulose paper. The paper was then washed four times for 30 minutes each with 150 mmol/L phosphoric acid, followed by a 20-minute acetone wash. Papers were then dried and counted for radioactivity. AMPK activity was expressed as picomoles ³²P incorporated into the SAMS peptide per minute per milligram protein.

Western Blot Analysis of AMPK
Samples obtained from the 6% PEG extract were subjected to SDS-PAGE, as described previously.³ After the gel electrophoresis using 25 µg protein, the fractionated protein was transferred to nitrocellulose membrane. AMPK protein content was determined using an anti-AMPK catalytic subunit polyclonal antibody kindly provided by Dr L.A. Witters. A chemiluminescence detection method was performed on the membranes using an ECL Western blotting kit, followed by autoradiography.

Statistical Analysis
Data are expressed as the mean±SEM. Comparisons were performed using Student's t test. Where appropriate, ANOVA followed by the Neuman-Keuls test was used to determine statistical significance when more than two group means were involved. Significance was set at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
AMPK Activity and Expression in Newborn Rabbit Hearts
Since AMPK activity or expression had not been previously determined in newborn rabbit hearts, we first extracted AMPK from unperfused 1- and 7-day-old rabbit hearts using a PEG 8000 procedure developed by Grahame Hardie's group.^{15 16 17} Using a two-stage PEG precipitation procedure, the majority of AMPK activity was precipitated in the 6% PEG fraction. AMPK activity in the 6% PEG 8000 precipitate was 1025±95 pmol·min^-1·mg protein^-1 (n=6) compared with 46±6 pmol·min^-1·mg protein^-1 in the 6% PEG 8000 supernatant and 110±1 pmol·min^-1·mg protein^-1 in the 2.5% PEG 8000 supernatant. Using this 6% PEG 8000 precipitate fraction, the time course of AMPK activity was measured in the presence of 200 µmol/L. Rates of incorporation of ³²P into the SAMS peptide were linear up to and beyond 4 minutes (data not shown). As a result, a 3-minute time period was used in all subsequent assays. Fig 1 shows the AMPK activity measured in 1- and 7-day-old unperfused rabbit hearts. Basal AMPK activity was highest in 7-day-old hearts compared with 1-day-old hearts (754±14 versus 490±52 pmol·min^-1·mg protein^-1, respectively; P<.05). Addition of 200 µmol/L AMP to the incubation medium increased AMPK activity in both the 1- and 7-day-old hearts. Under these conditions, AMPK activity remained the highest in PEG 8000 extracts obtained from 7-day-old rabbit hearts.

View larger version (19K): [in this window] [in a new window]   Figure 1. Graph showing AMPK activity measured in 6% polyethylene fractions isolated from unperfused 1- and 7-day-old rabbit hearts. Hearts from 1- and 7-day-old rabbits were used to extract AMPK using PEG 8000 as described in "Materials and Methods," and the activity of AMPK was determined by measuring the incorporation of 32P into the SAMS peptide in the presence or absence of 200 µmol/L AMP. Values shown are the mean±SEM of results from four different experiments. *P<.05 vs the 0 AMP group; P<.05 vs 1-day-old hearts.

Fig 2 shows the levels of the AMPK catalytic subunit expression in hearts from 1- and 7-day-old rabbits. AMPK was expressed in both age groups, with the highest expression observed in 7-day-old rabbit hearts. The increased expression of AMPK paralleled the increase in AMPK activity seen in rabbit hearts obtained 7 days after birth.

View larger version (35K): [in this window] [in a new window]   Figure 2. AMPK catalytic subunit expression in 6% PEG extracts isolated from 1- and 7-day-old rabbit hearts. Western blot analysis was performed as described in "Materials and Methods." SDS-PAGE (9%) was carried out by loading a 25 µg protein sample into each lane. After transfer to nitrocellulose membranes, the catalytic subunit of AMPK was detected using an antibody directed against the catalytic subunit of AMPK.

Phosphorylation Control of AMPK in the Newborn Heart
In a recent study,¹⁰ we showed that incubation of a 6% PEG 8000 extract from adult rat hearts in the presence of protein phosphatase 2A and in the absence of protein phosphatase inhibitors results in a marked decrease in AMPK activity. Table 1 shows the activity of AMPK (measured in the presence of 200 µmol/L AMP) in 6% PEG 8000 extracts from 1-day-old hearts, prepared without sodium fluoride and sodium pyrophosphate in the homogenizing buffer. The absence of sodium fluoride and sodium pyrophosphate during isolation resulted in a decrease in AMPK activity (542±40 pmol·min^-1·mg protein^-1) compared with 6% PEG extracts, in which sodium fluoride and sodium pyrophosphate were present (1025±95 pmol·min^-1·mg protein^-1). A further incubation for 30 or 60 minutes in the absence of sodium fluoride/sodium pyrophosphate resulted in a further decrease in AMPK activity (Table 1). If 200 µmol/L ATP and 5 mmol/L MgCl₂ were present during this incubation, the drop in AMPK activity was not as dramatic. Furthermore, if a phosphatase inhibitor (okadaic acid, 100 nmol/L) was present in the incubation medium, no loss of activity was observed (data not shown). Combined, these data provide indirect evidence that AMPK is under phosphorylation control in the newborn heart.

View this table: [in this window] [in a new window]   Table 1. Effects of Isolation and In Vitro Incubation of 6% PEG 8000 Fractions in the Absence of Phosphatase Inhibitors on AMPK Activity in Unperfused 1-Day-Old Rabbit Hearts

Fatty Acid Oxidation Rates and ACC Activity in 1- and 7-Day-Old Hearts
Fig 3A shows the effects of insulin on fatty acid oxidation rates in isolated perfused hearts from 1- and 7-day-old rabbit hearts. Insulin levels in the immediate newborn period are in the range of 100 µU/mL.⁵ This level of insulin decreases dramatically in the days following birth to negligible levels. Therefore, we perfused 1- and 7-day-old hearts in the present study with or without 100 µU/mL insulin. In the absence of insulin, palmitate oxidation rates were lower in 1-day-old hearts than rates seen in 7-day-old hearts. Addition of insulin (100 µU/mL) resulted in a significant decrease in palmitate oxidation rates in 1-day-old hearts but was without effect on fatty acid oxidation rates in 7-day-old hearts. Under these conditions, palmitate oxidation rates were almost 4-fold higher in 7-day-old hearts compared with 1-day-old hearts. The higher rates of fatty acid oxidation in the 7-day-old hearts were accompanied by lower rates of ACC activity (Fig 3B).

View larger version (15K): [in this window] [in a new window]   Figure 3. Graph showing fatty acid oxidation rates (A) and ACC activity (B) in 1- and 7-day-old isolated rabbit hearts perfused in the presence or absence of insulin. Isolated hearts were perfused with Krebs-Henseleit buffer containing 11 mmol/L glucose and 0.4 mmol/L [14C]palmitate bound to 3% albumin. Fatty acid oxidation rates were measured as described in "Materials and Methods." Insulin, when present, was added to the perfusate at a concentration of 100 µU/mL. Values are the mean±SEM of eight hearts in the control group and six hearts in the insulin group. *P<.05 vs control hearts; P<.05 vs 1-day-old hearts perfused under the same conditions.

Effect of Insulin on AMPK Activity in Newborn Rabbit Hearts
AMPK activity in 1- and 7-day-old hearts perfused in the presence and absence of 100 µU/mL insulin is shown in Fig 4. In the absence of insulin, AMPK activity in the perfused hearts was significantly higher in 7-day-old rabbit hearts compared with 1-day-old hearts. Addition of insulin to the perfusate decreased AMPK activity in both 1- and 7-day-old hearts. If the 6% PEG extracts from the perfused hearts were incubated with protein phosphatase 2A (in the absence of sodium fluoride/sodium pyrophosphate), a complete loss of AMPK activity was seen in all hearts (data not shown). Furthermore, isolation of the 6% PEG fractions in the absence of sodium fluoride/sodium pyrophosphate resulted in a decrease in AMPK activity in all groups to approximately similar levels (Table 2). If AMPK was preincubated with ATP/MgCl₂, an increase in AMPK could be seen in all groups compared with the no-ATP/MgCl₂ groups. This suggests that the changes in AMPK activity observed in the 1- and 7-day-old rabbit hearts in the presence or absence of insulin could be explained by differences in the phosphorylated status of AMPK.

View larger version (21K): [in this window] [in a new window]   Figure 4. Graph showing AMPK activity in hearts from 1- and 7-day-old rabbits perfused in the presence or absence of insulin. AMPK activity was measured in the presence or absence of 200 µmol/L AMP as described in "Materials and Methods." Values shown are the mean±SEM of results from eight control hearts and six hearts perfused with insulin. *P<.05 vs 0 AMP hearts. P<.05 vs 1-day-old hearts perfused under the same conditions.

View this table: [in this window] [in a new window]   Table 2. Effects of Isolation and In Vitro Incubation of 6% PEG 8000 Fractions in the Presence or Absence of ATP/Mg2+ in 1- and 7-Day-Old Rabbit Hearts Perfused in the Presence or Absence of Insulin

Effect of Direct Activation of AMPK on ACC Activity and Fatty Acid Oxidation
Recent studies in hepatocytes and adipocytes have shown that AICAR can stimulate AMPK activity.^{19 20 21 22} To further establish the relationship between AMPK, ACC, and fatty acid oxidation, we perfused a series of 7-day-old rabbit hearts with 200 µmol/L AICAR. As shown in Table 3, perfusion of hearts with AICAR resulted in a significant increase in AMPK activity. Accompanying this increase in AMPK was a significant decrease in ACC activity and a significant increase in palmitate oxidation rates.

View this table: [in this window] [in a new window]   Table 3. Effects of AICAR on AMPK Activity, ACC Activity, and Palmitate Oxidation Rates in 7-Day-Old Rabbit Hearts

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The importance of ACC in the regulation of fatty acid metabolism in the adult and newborn heart has recently been established.^{3 9 10 18} The regulation of ACC in the heart, however, remains poorly understood. Previous studies have shown that liver and adipose isoforms of ACC are under the control of another cytoplasmic enzyme, AMPK (see Reference 7⁷ for review). In the present study, we demonstrate that in the immediate newborn period, rabbit heart ACC activity also appears to be under the control of AMPK. In the presence of insulin, AMPK activity, which is accompanied by a high ACC activity, is low in 1-day-old hearts. We have also previously shown that this high ACC activity is accompanied by a dramatic increase in malonyl CoA levels in the heart.³ In the present study, we demonstrate that lowering insulin levels results in a significant increase in AMPK activity, a lowering of ACC activity, and an increase in fatty acid oxidation rates. This suggests that hormonal control of AMPK by insulin may be an important regulator of fatty acid oxidation rates in the immediate newborn period. We suggest that the drop in circulating insulin levels seen after birth⁵ results in an activation of AMPK and a decrease in ACC activity. The resulting drop in malonyl CoA levels³ will then result in an immediate increase in fatty acid oxidation rates. A proposed scheme describing this is shown in Fig 5.

View larger version (8K): [in this window] [in a new window]   Figure 5. Scheme showing proposed pathway by which a decrease in circulating insulin levels may increase fatty acid oxidation rates in the newborn heart. The drop in circulating insulin levels that occurs after birth (Reference 5) results in an activation of AMPK (possibly due to phosphorylation of AMPK by an AMPK kinase). This results in a phosphorylation and inhibition of ACC. This then results in a decrease in myocardial malonyl CoA levels (Reference 3), which relieves the inhibition of CPT 1, the rate-limiting enzyme involved in fatty acid transport into the mitochondria (Reference 4). The result is an increase in fatty acid oxidation, which now provides a greater proportion of the heart's ATP demands (References 1 through 3).

Further evidence supporting a role for AMPK in regulating fatty acid oxidation is provided by the AICAR results. AICAR is a cell-permeable activator of AMPK, which is taken up by the cell and converted to 5-amino-4-imidazolecarboximide ribotide.²⁰ Recent studies in liver and adipose tissue have shown that this results in an activation of AMPK and an inhibition of ACC.^{19 20 21 22} In 7-day-old rabbit hearts, we also observed an increase in AMPK and a decrease in ACC activity following AICAR administration. This was also accompanied by a significant increase in fatty acid oxidation rates, providing further evidence for a role of AMPK in regulating fatty acid oxidation in the newborn heart.

To our knowledge, this is the first study to demonstrate not only that AMPK is expressed in the newborn heart but that the content and activity of AMPK increase after birth. Immunoblot analysis revealed that AMPK protein content was higher in 7-day-old hearts compared with 1-day-old hearts. This was accompanied by a greater activity of AMPK in 7-day-old hearts under basal conditions (ie, no insulin in the perfusate). Since AMPK from both 1- and 7-day-old rabbits could be inhibited by insulin, it appears that insulin control of the enzyme persists after birth. Therefore, it is conceivable that the dramatic drop in insulin levels following birth may result in the removal of a physiological inhibitor of the AMPK enzymatic activity (ie, insulin). This would be expected to increase or enhance AMPK activity in vivo. Other studies have also suggested that removal of an inhibition may result in a significant activation. For instance, Massillon et al²³ reported that activation of glycogen synthase results from inhibition of glycogen synthase kinase (phosphorylation of glycogen synthase by this kinase results in the inhibition of glycogen synthase). As a result, we suggest that changes in insulin levels, as opposed to insulin control of AMPK, are responsible for the increase in AMPK activity and the decrease in ACC activity following birth.

At birth, insulin levels are in the range of 100 µU/mL and then rapidly drop in the days after birth.⁵ Therefore, we used 100 µU/mL insulin in our heart perfusion studies. Whereas this concentration is physiological for a 1-day-old rabbit heart, it could be considered as a suprapharmacological dose in a 7-day-old rabbit heart. However, we felt that it was important to use similar insulin concentrations in the performance of these studies. The 7-day-old hearts perfused in the absence of insulin are closest to the physiological level of insulin seen in vivo, whereas the 1-day-old hearts perfused with insulin are closest to what would be seen physiologically in 1-day-old rabbits. Of interest is that these two perfusion conditions resulted in the greatest differences in AMPK, ACC, and fatty acid oxidation rates between the two age groups, supporting the concept that changes in circulating insulin levels, and therefore AMPK and ACC activity, are important in the developmental changes in fatty acid oxidation in the newborn period.

Our data also show that AMPK in the newborn heart can be rapidly activated or inhibited, which probably involves a change in the phosphorylated status of AMPK. AMPK is a heterotrimeric protein consisting of 63-kD catalytic subunit and two other regulatory subunits (40 and 38 kD). Phosphorylation of AMPK by an upstream AMPKK will activate AMPK, whereas dephosphorylation of AMPK by phosphatases (particularly protein phosphatases 2A and 2C) will decrease AMPK activity.⁷ AMP is able to increase AMPK activity by direct allosteric activation of AMPK, by facilitating AMPKK phosphorylation of AMPK, by directly activating AMPKK, or by inhibiting dephosphorylation of AMPK.^{24 25} Our data provide indirect evidence that cardiac AMPK is also under phosphorylation control and that insulin decreases the phosphorylated state of AMPK. Although inhibition of AMPK by insulin has been demonstrated in liver,⁷ the mechanism by which insulin inhibits AMPK has not been established. Whether AMP plays a role in insulin effects on AMPK is also not clear. Insulin would be expected to have the opposite effect of stress on cellular AMP levels, although in the aerobically perfused heart insulin does not have discernible effects on AMP levels, which are already very low.

It should be noted that AMPK activity measured in freshly excised hearts had higher enzymatic activity than in tissue extract from hearts that had undergone 30 or 40 minutes of aerobic perfusion. It is unlikely that this was due to an unstable isolated heart preparation, since heart function was stable throughout the perfusion period, and if hearts were stressed, an increase in AMPK would be expected compared with unperfused hearts. One reason for these differences may relate to differences in the hormonal milieu in isolated hearts compared with that seen in vivo. It is also possible that during extraction of the unperfused heart from the rabbit, some nonspecific phosphorylation occurs, resulting in an activation of AMPK.

A number of studies^{7 15 16 17 20 21 26 27} have provided convincing evidence that AMPK acts as a "fuel gauge" in anabolic tissues, such as liver and adipose tissue. Activation of AMPK in times of metabolic stress results in a phosphorylation of biosynthetic enzymes such as 3-hydroxy-3-methylglutaryl CoA reductase and ACC. Although ACC in liver, adipose tissue, and mammary glands is primarily involved in the synthesis of fatty acids, in heart ACC functions primarily to regulate fatty acid oxidation. We propose that in cardiac muscle, AMPK also acts as a fuel gauge, although in heart AMPK increases the production of energy from fatty acid oxidation in times of metabolic stress. Decreases in insulin levels will result in a decrease in the contribution of glucose metabolism as a source of energy in the heart. As a result, the demand for fatty acid oxidation increases. Our data suggest that AMPK increases under conditions of low insulin, resulting in an inhibition of ACC and an acceleration of fatty acid oxidation.

The mammalian ACC enzyme is a large, complex, multifunctional enzyme that catalyzes the carboxylation of acetyl CoA to form malonyl CoA.²⁸ Extensive characterization has been carried out on the 265-kD isoenzyme at the level of both the protein and the gene. In tissues such as the liver, ACC can undergo rapid reversible phosphorylation, resulting in an inhibition of enzyme activity.^{29 30} The 265-kD ACC protein contains a number of sites that can be phosphorylated, with AMPK phosphorylating ACC on the Ser⁷⁹ site.³⁰ Phosphorylation of hepatic ACC by AMPK is accompanied by marked decreases in the ACC activity. Until recently, it was not known whether this same phenomenon occurred in the heart. The adult heart does express a significant amount of AMPK mRNA and protein,^{11 12 13 14} , which is primarily the ₂ catalytic subunit of AMPK.³¹ As shown in Fig 2, the newborn rabbit heart also expresses AMPK, and this expression, which is accompanied by an increase in AMPK activity, increases after birth (Fig 1). Recently, two isoforms of the AMPK catalytic subunit have been identified: an ₁-isoform, which appears to be predominantly expressed in the liver, and an ₂-isoform, which is highly expressed in the heart and skeletal muscle. Whether the increase in expression of AMPK was due to the ₁- or ₂-isoform of the AMPK was not determined in the present study. It is also not clear whether the role of these two isoforms differs in the heart. Experiments are now being performed to determine whether the increase in AMPK gene expression seen in 7-day-old hearts represents an increase in the ₁- or ₂-isoform of AMPK.

We have recently shown that myocardial ACC activity is inversely correlated with both AMPK activity and levels of fatty acid oxidation in hearts subjected to ischemia followed by reperfusion,^{9 10} providing strong evidence for a role of AMPK in regulating myocardial fatty acid oxidation via phosphorylation and thus inhibition of ACC. Data from this study suggest that AMPK phosphorylation of ACC in the newborn heart also has an important role in regulating fatty acid oxidation and that an increase in AMPK phosphorylation of ACC may be largely responsible for the rapid increase in the fatty acid oxidation rates following birth.

It should be noted that insulin did not increase ACC activity in 7-day-old rabbit heart to the same extent as in the 1-day-old hearts. One possible explanation for this may be that the higher expression of AMPK in 7-day-old hearts may result in an AMPK activity that is high enough to phosphorylate and inhibit ACC even in the presence of insulin. A second possibility is that a change in the control of ACC dephosphorylation in 7-day-old hearts occurs (ie, a decreased activity of the phosphatases involved in ACC dephosphorylation). However, to date, no information exists as to which phosphatases are important in the control of ACC in the heart. Finally, it is possible that the ACC expressed in 7-day-old hearts may be under a phosphorylation control different from the ACC expressed in 1-day-old hearts. Ki-Han Kim's group^{8 28 29} has reported the existence of an mRNA for the 265-kD isoform of ACC that codes for a protein that would not be as readily phosphorylated at the Ser¹²⁰⁰ site. However, to date, complete sequence information on the 280-kD isoform of ACC (which predominates in the heart) has not been obtained.

The newborn rabbit heart expresses both a 265-kD isoenzyme and a 280-kD isoenzyme of ACC.³ Unfortunately, unlike the 265-kD isoenzyme of ACC, the 280-kD ACC has not been extensively characterized at either the protein or gene level. Trumble et al³² have recently performed a kinetic characterization of affinity-purified ACC from rat skeletal muscle that is expressed exclusively as a 272-kD protein. Kinetic constants were found to differ from the kinetic constant of adipose tissue ACC, which expresses the 265-kD isoenzyme of ACC. In addition, on the basis of recent structural evidence of both the 280-kD ACC protein³³ and potential molecular cloning,³⁴ it appears that the 280-kD isoenzyme of ACC is most likely the product of a distinct gene. Although it is not known whether the phosphorylation sites on 265-kD ACC are also present in 280-kD ACC, it has been suggested that the 280-kD ACC in liver is more readily phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.³⁴ We have also shown indirectly^{9 10} and directly³⁵ that phosphorylation of ACC inhibits enzyme activity.

In the absence of insulin, AMPK activity was significantly higher in 7-day-old rabbit hearts compared with 1-day-old hearts. Paralleling these changes, it was also evident that ACC activity was lower and that fatty acid oxidation rates were higher compared with the levels in 1-day-old hearts. Insulin decreased AMPK activity by a similar proportion in both the 1- and 7-day-old rabbit hearts (ie, 60%). Low insulin levels in 7-day-old rabbits may therefore result in high AMPK activity, low ACC activity levels, low levels of malonyl CoA, and a resultant acceleration of fatty acid oxidation rates. Of interest, although the addition of insulin to the perfusate inhibited AMPK activity in 7-day-old hearts, no change in ACC activity was observed. This may be explained by the fact that ACC activity was already very low in these hearts and that by 7 days circulating insulin levels were not as high; in fact, insulin levels had dropped to a very low level. We have recently demonstrated that AMPK will directly phosphorylate and inhibit purified 265- and 280-kD ACC from adult rat heart.³⁵ However, to date, it has not been determined directly whether AMPK has differing effects on phosphorylation and inhibition of ACC from 1- and 7-day-old rabbit hearts.

Our data also suggest that perfusion of hearts with insulin is capable of decreasing AMPK activity, possibly because of a decrease in the phosphorylation of AMPK. The mechanisms responsible for this are not clear. Insulin could either be stimulating a phosphatase that dephosphorylates and inactivates AMPK, or it could be inhibiting the phosphorylation and activation of AMPK. Weekes et al³⁶ have recently characterized an AMPKK that phosphorylates and activates AMPK. Whether the activity of this AMPKK is inhibited by insulin remains to be determined.

Summary
The results from the present study demonstrate that AMPK activity and gene expression increased in the immediate newborn period. Increased AMPK activity may be involved in the regulation of ACC in the newborn rabbit heart. It would appear that the rapid decline in insulin levels after birth results in an increased AMPK activity, with resultant increased phosphorylation and inhibition of ACC activity. This could lead to low levels of malonyl CoA and facilitate translocation of activated fatty acyl CoA into the mitochondria for increased ß-oxidation of fatty acids.

	Selected Abbreviations and Acronyms

 

ACC = acetyl CoA carboxylase AICAR = 5-amino 4-imidazolecarboxamide ribotide AMPK = 5'-AMP–activated protein kinase AMPKK = AMPK kinase CoA = coenzyme A CPT 1 = carnitine palmitoyltransferase 1 ECL = enhanced chemiluminescence PEG = polyethylene glycol SAMS = synthetic peptide substrate for 5'-AMPK with the amino acid sequence HMRSAMSGLHLVKRR

	Acknowledgments

 
This study was supported by a grant from the Heart and Stroke Foundation of Alberta. Dr Lopaschuk is a Medical Research Council Scientist and an Alberta Heritage Foundation for Medical Research Senior Scholar. A.M. Makinde and J.G. Gamble are graduate trainees of the Alberta Heritage Foundation for Medical Research and the Heart and Stroke Foundation of Canada.

Received August 26, 1996; accepted December 18, 1996.

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