Long-chain acyl-CoA esters inhibit phosphorylation of AMP-activated protein kinase at threonine-172 by LKB1/STRAD/MO25
E. B. Taylor,
W. J. Ellingson,
J. D. Lamb,
D. G. Chesser, and
W. W. Winder
Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah
Submitted 28 October 2004
; accepted in final form 4 January 2005
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ABSTRACT
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Activation of the AMP-activated protein kinase (AMPK) results in acute changes in cellular metabolism and transcriptional events that make the cell more robust when encountering an energy challenge. AMPK is thought to be inhibited by glycogen, the major storage form of intracellular carbohydrate. We hypothesized that long-chain acyl-CoA esters (LCACEs) might also inhibit AMPK signaling. Cytosolic LCACEs are available for immediate transport and oxidation within the mitochondria and accordingly may be representative of the lipid energy charge of the cell. We found that LCACEs inhibited phosphorylation of AMPK by the recombinant AMPK kinase (AMPKK) LKB1/STRAD/MO25 in a concentration-dependent manner. Palmitoyl-CoA (PCoA) did not affect the activity of phosphothreonine-172 AMPK. PCoA potently inhibited AMPKK purified from liver. Conversely, PCoA stimulated the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. Octanoyl-CoA, palmitate, and palmitoylcarnitine did not inhibit AMPKK activity. Removal of AMP from the reaction mixture resulted in reduced AMPKK activity in the presence of PCoA. In conclusion, these results demonstrate that the AMPKK activity of LKB1/STRAD/MO25 is substrate specific and distinct from the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. They also demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25 in a specific manner with a dependence on both a long fatty chain and a CoA moiety. These results suggest that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell.
adenosine 5'-monophosphate-activated protein kinase; adenosine 5'-monophosphate-activated protein kinase kinase; diabetes; fatty acid oxidation; STK 11
AMP-ACTIVATED PROTEIN KINASE (AMPK) is a master metabolic regulator of the eukaryotic cell (1416, 56). AMPK is phosphorylated and activated by an AMPK kinase (AMPKK; see Refs. 18 and 49). LKB1 (STK 11) complexed with the scaffold protein mouse protein 25 (MO25), and the pseudokinase STE-related adaptor protein (STRAD) has recently been identified to be a major AMPKK (17, 48, 59). AMPK must be phosphorylated by AMPKK on its activation loop at threonine-172 for full activation. AMPK is also regulated by allosteric modulators that are thought to both increase AMPK activity directly and make AMPK a better substrate for AMPKK and a poorer substrate for phosphatases (6, 18, 49).
Activation of AMPK results in acute cellular responses that allow it to better meet immediate energy demands and transcriptional responses that make it more robust when faced with future energy challenges (13, 35, 44, 53, 55). Two major acute consequences of AMPK activation are 1) an induction of glucose 4 transporter microvesicle cytoplasm to membrane translocation and fusion with a subsequent increase in glucose uptake and 2) an increase in fatty acid oxidation by phosphorylation and inactivation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis (19, 29, 33). The activity of AMPK is classically regulated by the cellular AMP-to-ATP ratio and may also be regulated by the creatine-to-creatine phosphate ratio (40). AMPK is also thought to be inhibited by glycogen, possibly by interacting with a putative glycogen-binding domain on the AMPK
-subunit (26, 39, 57). Glycogen is the major cellular storage form of carbohydrate and thus an additional indicator of cellular energy status. Lipids are the other major energy source for cellular metabolism. Accordingly, it is likely that activity of the AMPK signaling cascade may be modulated by cellular lipid content.
Recently, it was demonstrated that perfusion of heart muscle with physiological levels of palmitate resulted in phosphorylation and activation of AMPK independent of measurable changes in the AMP-to-ATP and creatine-to-creatine phosphate ratios (4). An earlier investigation found that cultured hepatocytes incubated with 27.5 mM glucose, 100 nM dexamethasone, 10 nM insulin, and with 150 µM acetate, octanoate, or palmitate resulted in a 30-fold increase in cytosolic AMP concentration and subsequent AMPK activation (25). The mechanism driving the phosphorylation of AMPK coincident with palmitate infusion of heart muscle under physiological conditions is currently unknown but provides strong evidence that, in addition to being regulated by carbohydrate, AMPK activity may also be regulated by lipids. Additional evidence that AMPK activity is regulated by lipids is provided by the finding that disruption of stearoyl-CoA desaturase 1 in mice increases AMPK activity (7). We hypothesized that activation of AMPK could be inhibited by a high lipid content. Triglyceride is a relatively inert form of lipid and is therefore unlikely to be a regulator of metabolic signal transduction (5). Better candidates would be active lipids like diacylglycerol (DAG), phospholipids, ceramide, and long-chain acyl-CoA esters (LCACEs; see Ref. 20).
LCACEs are available for immediate transport into the mitochondria for oxidation. Conversely, although other molecules like DAG, phospholipids, and ceramide are important second messengers, they are not immediately available for use as fuel. Thus cytosolic LCACEs may be a metabolically active marker of the lipid energy charge of the cell. We hypothesized that AMPK might be inhibited by LCACEs. Additionally, the accumulation of cytosolic LCACEs is associated with obesity, insulin resistance, type 2 diabetes, and the metabolic syndrome (8, 20, 22, 28). AMPK is a potential target for the treatment of type 2 diabetes (36, 43). LCACEs are postulated to inhibit insulin signaling in part by activation of protein kinase C (PKC) and inhibition of hexokinase (31, 52). Because activation of AMPK chemically or by exercise results in enhanced insulin sensitivity (2, 10, 24), inhibition of AMPK activity by LCACEs may be an additional mechanism by which LCACEs contribute to insulin resistance.
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MATERIALS AND METHODS
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Animal care.
All procedures were approved by the Institutional Animal Care and Use Committee of Brigham Young University. Male Sprague-Dawley (SAS:VAF) rats (Sasco, Wilmington, MA) were housed in a temperature controlled (2122°C) room with a 12:12-h light-dark cycle. Rats were fed standard rat chow (Harlan Teklad rodent diet) and water ad libitum. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (48 mg/kg body wt).
Buffers.
The following buffers were used: buffer A: 50 mM Tris·HCl, 250 mM mannitol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, pH 8.4 at 4°C with 1 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and 1 mM dithiothreitol (DTT) added just before use; buffer B: 50 mM Tris·HCl, 250 mM mannitol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 0.02% (wt/vol) Brij-35, 10% (vol/vol) glycerol, pH 7.4, at 4°C, 1 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, 0.2 mM AEBSF, and 1 mM DTT added just before use; buffer C: like buffer B but without NaF, sodium pyrophosphate, benzamidine, soybean trypsin inhibitor, and AEBSF; AMPK phosphorylation buffer: 100 mM HEPES, 200 mM NaCl, 20% glycerol (vol/vol), 2 mM EDTA, 12.5 mM MgCl2, 0.5 mM ATP, 0.5 mM AMP, and 2.0 mM DTT, pH 7.0; AMARA phosphorylation buffer: 40 mM HEPES, 80 mM NaCl, 8% glycerol, 0.8 mM EDTA, 0.8 mM DTT, 5 mM MgCl2, 0.2 mM ATP, 0.2 mM AMP, 0.33 mM AMARA peptide, and 0.05 µCi/µl [
-32P]ATP, pH 7.0; and Laemmli's buffer (30).
Materials.
General reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. LCACEs and other CoA derivatives were obtained from Sigma-Aldrich. Recombinant LKB1/STRAD/MO25 (AMPKK) and LKB1tide (32) were obtained from Upstate (Charlottesville, VA). Phosphothreonine-172 AMPK (P-AMPK) antibody was obtained from Cell Signaling. His-bind nickel-binding resin was obtained from Novagen (Madison, WI). Polyethylene glycol 6000 (PEG) was obtained from Calbiochem (La Jolla, CA). Chromatographic purification was performed using an AKTA FPLC system from Amersham Biosciences (Piscataway, NJ). Columns were obtained from Amersham.
Purification and concentration of AMPKK activity.
Liver AMPKK activity was purified and concentrated as reported previously (18) through the Q-Sepharose step followed by a modified Mono-Q step. Briefly, livers were minced and then homogenized with a glass homogenizer in buffer A in two parts buffer A to one part liver (wt/vol). The pellet from the 610% PEG cut was resuspended in buffer B. Buffer B was used for all column chromatography, including the modified Mono-Q step. For the modified Mono-Q step, active fractions from the Q-Sepharose step were diluted in an equal volume of buffer B with 0 mM NaCl, loaded on the mono-Q column, and eluted over a 20-ml gradient from 100 to 300 mM NaCl. Active fractions were combined, concentrated, and exchanged into buffer C before assays.
Generation and purification of recombinant AMPK.
Bacteria expressing recombinant
2
2
2 AMPK were prepared as previously described (37, 38). Recombinant
2
2
2-AMPK was extracted and purified by nickel affinity chromatography (51). After buffer exchange into buffer C,
2
2
2-AMPK was concentrated to 1 µg/µl before use for AMPKK activity assays.
AMPKK activity assay.
AMPKK activity was measured in a two-step assay (11, 51). During the first step, AMPK is phosphorylated and activated by AMPKK. During the second step, P-AMPK phosphorylates AMARA (18) peptide. Recombinant
2
2
2-AMPK was diluted 1:19 in water. Recombinant LKB1/STRAD/MO25 (0.86 µg/µl) was diluted 1:100 in buffer C. LCACEs and other CoA derivatives were gently dissolved in water at a concentration five times the desired final concentration during the first step of the assay. For the assay, 500 µl polypropylene microcentrifuge tubes were loaded with 4 µl of AMPK phosphorylation buffer, 2 µl of diluted recombinant
2
2
2-AMPK, and 2 µl of 5x CoA derivative, palmitoylcarnitine, or palmitate-BSA solution. Diluted AMPKK was added to start the first step of the reaction, which was incubated for 20 min at 30°C. After the first incubation, 15 µl AMARA phosphorylation buffer were added to start the second step of the assay. After a 10-min incubation, the reaction was stopped by spotting a 1-cm2 piece of Whatman P81 filter paper with 15 µl of the final reaction mix, waiting 20 s for complete absorption, and then placing the filter paper in a beaker with 100 ml of 1% phosphoric acid.
At the conclusion of the assay, five washes with 100 ml of 1% phosphoric acid were followed by a brief rinse with acetone. After the filter papers dried, they were placed in 3 ml of ecolite scintillation fluid and counted for 1 min. Activity assays with liver AMPKK were performed as above except that 2 µl of liver AMPKK were added instead of diluted recombinant LKB1/STRAD/MO25. Assays to measure the inhibitory effect of palmitoyl-CoA (PCoA) on P-AMPK were performed as above but with the following changes: LKB1/STRAD/MO25 was diluted 1:19 rather than 1:100 to completely phosphorylate all AMPK, 2 µl of water rather than 5x PCoA were added before the start of the first step, and the PCoA was added at the start of the second step where P-AMPK phosphorylates AMARA peptide. For assays of LKB1/STRAD/MO25 kinase activity against LKB1tide, 450 µl AMPK phosphorylation buffer, 225 µl of 0.775 mM LKB1tide in water, 135 µl water, and 4 µl of [
-32P]ATP were combined, and 18 µl of this mixture were loaded in 500 µl of polypropylene microcentrifuge tubes. Tubes were loaded with 5x PCoA solution as above, and the reaction was started with the addition of 2 µl recombinant LKB1/STRAD/MO25 diluted 1:25 and stopped by spotting filter papers and continuing as above.
Western blot validation of AMPKK activity assay.
To validate the AMPKK activity assay, the reaction was stopped after the first step, and the reaction mixture was measured for relative quantity of P-AMPK by Western blot. Volumes in the first step were increased to 12 µl of AMPK phosphorylation buffer, 6 µl of diluted (1:19) recombinant
2
2
2-AMPK, 6 µl 5x CoA derivative solution, and 6 µl of diluted (1:100) recombinant AMPKK. After a 20-min incubation, the reaction was stopped by adding 12 µl of 4x Laemmli's buffer to give a total volume of 42 µl. Bio-Rad 7.5% polyacrylamide Tris·HCl gels were loaded with 40 µl of the final reaction mixture. Western blots were performed as previously described, with the first antibody diluted 1:2,000 and the second antibody diluted 1:1,500 (51).
Statistics.
The inhibitory effect of CoA derivatives at different concentrations on the phosphorylation of AMPK by LKB1/STRAD/MO25 and the phosphorylation of AMARA peptide by AMPK were compared by one-way ANOVA. The AMP dependence of phosphorylation of AMPK by AMPKK in the presence of different concentrations of PCoA was compared using two-way ANOVA. When main effects or interactions reached significance, the Newman-Keuls multiple comparison test was used to determine the location. For all statistical tests, significance was set at P < 0.05. All statistical procedures were performed with the NCSS statistical program (Kaysville, UT). All data are reported as means ± SE.
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RESULTS
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To test the hypothesis that LCACEs inhibit the phosphorylation of AMPK by the AMPKK LKB1/STRAD/MO25, we assayed AMPKK activity in the presence of 50 µM PCoA, CoA, and the CoA derivatives acetyl-CoA, malonyl-CoA, and acetoacetyl-CoA all at a concentration of 100 µM (Fig. 1, n = 56). Addition of PCoA to the reaction mixture resulted in a marked inhibition of the AMPKK activity of LKB1/STRAD/MO25. Addition of the short-chain CoA derivatives had no effect on AMPKK activity.

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Fig. 1. The effect of coenzyme A (CoA) derivatives on the AMP-activated protein kinase kinase (AMPKK) activity of LKB1/STRAD/MO25 was determined by a two-step activity assay. AMPKK activity without the addition of a CoA derivative served as the control and was normalized to 1. Values are expressed as means ± SE (n = 56 experiments). *Palmitoyl-CoA (PCoA) was significantly different from the control. CoA, acetyl-CoA (Acet), malonyl-CoA (Mal), and acetoacetyl-CoA (AcAcet) were not significantly different from the control (P < 0.05).
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Because the AMPKK assay is a two-step assay relying on phosphorylation of AMARA peptide by P-AMPK, it is possible that PCoA could be inhibiting P-AMPK rather than LKB1. Accordingly, we first completely phosphorylated an AMPK preparation and then added PCoA at concentrations of 0, 5, 7.5, 10, 20, and 50 µM at the start of the second reaction beginning with the addition of AMARA peptide. PCoA had no inhibitory effect on the activity of P-AMPK (Fig. 2, n = 56). To validate the AMPKK activity assay by directly measuring the AMPKK activity of LKB1/STRAD/MO25, we incubated LKB1/STRAD/MO25 with AMPK at PCoA concentrations of 0 or 50 µM, stopped the reaction with the addition of Laemmli's buffer before the addition of AMARA peptide, and Western blotted for P-AMPK (Fig. 3, n = 4). Addition of 50 µM PCoA dramatically reduced the quantity of P-AMPK.

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Fig. 2. The effect of PCoA on the activity of phosphothreonine-172 AMPK (P-AMPK) was measured by completely phosphorylating AMPK during the first step of the reaction and then adding PCoA during the second step of the reaction to final concentrations of 5, 7.5, 10, 20, and 50 µM. P-AMPK activity in the absence of PCoA served as the control and was normalized to 1. Values are expressed as means ± SE (n = 56). PCoA did not significantly affect the activity of P-AMPK (P < 0.05).
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Fig. 3. The AMPKK assay was validated by running the first step of the reaction with or without 50 µM PCoA and then stopping the reaction and Western blotting for P-AMPK. The relative quantify of P-AMPK in the absence of PCoA was normalized to 1. Values are expressed as means ± SE (n = 4). *Phosphorylation of AMPK at threonine-172 was greatly and significantly reduced in the presence of PCoA (P < 0.05).
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To investigate whether PCoA inhibited a non-AMPK-specific kinase activity of LKB1/STRAD/MO25, or whether PCoA had a substrate-specific inhibitory effect, we tested LKB1/STRAD/MO25 kinase activity against the peptide substrate LKB1tide at PCoA concentrations of 0, 5, 7.5, 10, 20, and 50 µM. In contrast to the inhibitory effect of PCoA against the AMPKK activity of LKB1, we found that PCoA stimulated the kinase activity of LKB1/STRAD/MO25 against LKB1tide (Fig. 4, n = 6).

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Fig. 4. The kinase activity of LKB1/STRAD/MO25 against the peptide substrate LKB1tide was measured with 0, 5, 7.5, 10, 20, and 50 µM PCoA. LKB1 activity in the absence of PCoA served as the control value and was normalized to 1. Values are expressed as means ± SE (n = 6). *LKB1 activity was significantly greater at the indicated PCoA concentration than LKB1 activity in the absence of PCoA (P < 0.05).
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We tested the effect of PCoA on AMPKK from rat liver to ensure that the inhibitory effect of PCoA was not limited to recombinant LKB1/STRAD/MO25. Rat liver AMPKK was chromatographically purified and assayed for activity in the presence of PCoA at concentrations of 0, 5, 7.5, 10, 20, and 50 µM. As with recombinant LKB1, increasing concentrations of PCoA resulted in increasing inhibition of liver AMPKK activity (Fig. 5, n = 56).

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Fig. 5. The inhibitory effect of PCoA against rat liver AMPKK (LKK) was assayed at PCoA concentrations of 0, 5, 7.5, 10, 20, and 50 µM. LKK activity in the absence of PCoA served as the control value and was normalized to 1. Values are expressed as means ± SE (n = 56). *LKK activity was significantly less at the indicated PCoA concentration than at the control without PCoA (P < 0.05).
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We tested several other LCACEs and one medium-chain acyl-CoA ester at concentrations of 10 (Fig. 6A, n = 56), and 50 (Fig. 6B, n = 56) µM for an inhibitory effect against the AMPKK activity of LKB1/STRAD/MO25. We tested stearoyl-CoA (18:0), oleoyl-CoA (18:1), linoleoyl-CoA (18:2), myristoyl-CoA (14:0), and octanoyl-CoA (8:0). We found significant inhibition at 10 and 50 µM with each LCACE (P < 0.05). The medium-chain acyl-CoA ester octanoyl-CoA did not inhibit AMPKK activity at 10 µM and showed only a trend for inhibition at 50 µM (P > 0.05, P = .074). Because inhibition was dependent on fatty acid chain length and not the presence of CoA, we assayed AMPKK activity in the presence of palmitoylcarnitine (n = 6) and palmitate (n = 3) compared with PCoA (Fig. 7). Palmitoylcarnitine significantly activated AMPKK, whereas palmitate had no effect.

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Fig. 6. The inhibitory effect of the long-chain acyl-CoA esters (LCACEs) stearoyl-CoA (SCoA), oleoyl-CoA (OleCoA), linoleoyl-CoA (LCoA), myristoyl-CoA (MCoA), and the medium-chain acyl-CoA ester octanoyl-CoA (OctCoA) on the AMPKK activity of LKB1/STRAD/MO25 was measured at concentrations of 10 µM (A), and 50 µM (B) and normalized to AMPKK activity in the absence of acyl-CoAs. Values are expressed as means ± SE (n = 56). *AMPKK activity in the presence of the indicated acyl-CoA ester was significantly less than at the control of 0 µM (P < 0.05).
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Fig. 7. The effects of 50 µM palmitoylcarnitine (PCarn), 400 µM palmitate in 2% BSA (PAcid), and 50 µM PCoA normalized to control AMPKK activity in the absence of a palmitate derivative. The control for PAcid was run in 2% BSA. Values are expressed as means ± SE (n = 6 for PCarn and PCoA, n = 3 for palmitate). *PCoA inhibited AMPKK activity compared with control (P < 0.05). PCarn stimulated AMPKK activity compared with control (P < 0.05).
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We also tested the interaction between AMP and PCoA at concentrations of 0, 5, 7.5, 10, 20, and 50 µM PCoA (Fig. 8, n = 56). The absence of AMP resulted in a significantly lower AMPKK activity at all concentrations of PCoA (P < 0.05). With the exception of the 20 and 50 µM points on the curve without AMP, each increase in PCoA concentration resulted in a decrease in AMPKK activity (P < 0.05). Without AMP, maximal inhibition of AMPKK activity occurred at 20 µM, whereas maximal inhibition occurred at 50 µM with AMP (P < 0.05).

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Fig. 8. The inhibitory effect of PCoA at concentrations of 0, 5, 7.5, 10, 20, and 50 µM against the AMPKK activity of LKB1/STRAD/MO25 was measured with and without 200 µM AMP. AMPKK activity with 200 µM AMP in the absence of PCoA served as the control value and was normalized to 1. Values are expressed as means ± SE (n = 56). Removal of 200 µM AMP decreased AMPKK activity at all concentrations of PCoA (P < 0.05). *PCoA inhibited AMPKK activity at the indicated concentration in both the presence and absence of AMP (P < 0.05).
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DISCUSSION
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These results demonstrate that LCACEs inhibit phosphorylation of AMPK by recombinant LKB1/STRAD/MO25 and AMPKK concentrated and purified from rat liver. To our knowledge, this is the first report of a direct lipid-mediated mechanism for inhibition of AMPK signaling. A previous report found that PCoA at a concentration of 200 nM enhanced phosphorylation of AMPK by AMPKK in a semipurified system (3). We tested the effect of PCoA at 200 nM on the AMPKK activity of LKB1/STRAD/MO25 and found no such effect (data not shown).
The kinase activity of LKB1/STRAD/MO25 may be regulated at the substrate level. LKB1/STRAD/MO25 is a master kinase that phosphorylates and activates 11 of 12 other AMPK-related kinases (32). The kinase activity of LKB1/STRAD/MO25 is not activated by contraction nor are the AMPK-related kinases QSK, QIK, MARK2/3, and MARK4 (46). Because LKB1/STRAD/MO25 is not activated by contraction, but contraction results in substantial phosphorylation of AMPK, it might be expected that the AMPKK activity of LKB1/STRAD/MO25 is regulated at the substrate level and is distinct from other substrate-specific LKB1/STRAD/MO25 kinase activities.
Both our AMPKK assays and Western blots show definitively that the AMPKK activity of LKB1/STRAD/MO25 is inhibited by PCoA and other LCACEs. However, PCoA had an opposite activating effect on the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide (Fig. 4). Thus the LKB1tide-specific kinase activity of LKB1/STRAD/MO25 is unique from the AMPKK activity of LKB1/STRAD/MO25.
The mechanisms regulating the phosphorylation of the other AMPK-related kinases are not well understood. LKB1 appears to be a constitutively active kinase (12). Therefore, it is likely that the kinase activity of LKB1/STRAD/MO25 toward these other kinases is also regulated in a substrate-specific manner. This type of regulation would allow target-specific control over a constitutively active master kinase that regulates cell polarity and proliferation (1, 12).
These results also indicate that LKB1tide is not a reliable substrate with which to measure the AMPKK activity of LKB1/STRAD/MO25. The AMPKK activity of LKB1/STRAD/MO25 at this time may be most reliably measured by using AMPK as the substrate (11, 51). We and others have used a recombinant
1-AMPK subunit truncated at the 312th amino acid (
-1312) as a substrate intermediate to assay AMPKK activity (11, 50, 51). However, caution should be taken with this approach. Because the AMPKK activity of LKB1/STRAD/MO25 appears to be regulated at the substrate level by allosteric modulators, under specific conditions,
-1312 may not interact with LKB1/STRAD/MO25 similarly to heterotrimeric AMPK. We have observed that AMP concentration does not affect the phosphorylation and activity of
-1312 (data not shown), whereas the phosphorylation and activity of
2
2
2 displays AMP dependence (Fig. 8).
Evidence for a specific interaction between LKB1/STRAD/MO25-AMPK and LCACEs is provided by 1) the observation that inhibition of AMPKK by CoA derivatives and fatty acid derivatives is limited to LCACEs and 2) the trend for increasing inhibition at 10 µM observed with increasing fatty chain length. Because LCACEs stimulate phosphorylation of LKB1tide by LKB1/STRAD/MO25, they may be blocking the phosphorylation site on AMPK rather than directly interacting with LKB1/STRAD/MO25. Nonetheless, the direct mechanism for inhibition of AMPKK by LCACEs is unknown. Summarily, these data indicate that the inhibitory effect of LCACEs is dependent on the length of the fatty chain of an acyl-CoA rather than on the CoA or fatty chain moieties themselves.
Reduced activation of AMPK by accumulation of LCACEs in liver may have physiological consequences. Insulin resistance is associated with obesity and can be induced by a high-fat diet in animal models. Feeding rats a high-fat diet for 3 wk increased LCACE concentrations from 7.7 to 26.7 µM in liver and from 4.4 to 8.3 µM in red gastrocnemius muscle, demonstrating that high-fat diets can markedly raise tissue levels of LCACEs (8). Reducing liver LCACE levels by treatment with rosiglitazone resulted in increased AMPK activity (61). Treatment with another thiazolidinedione, pioglitazone, was shown to activate AMPK and reduce ACC activity in rat liver (45).
Reduced hepatic AMPK activity resulting from LCACE accumulation could partially account for the absence of ketoacidosis and reduced hepatic fatty acid oxidation in type 2 diabetic subjects. Type 1 diabetes is characterized by high plasma glucagon, high rates of fatty acid oxidation in the liver, and ketoacidosis. Type 2 diabetes is characterized by obesity and reduced rates of whole body fatty acid oxidation. Very recently, perfusion of rat liver with glucagon was shown to result in phosphorylation of LKB1 by protein kinase A (PKA) and a subsequent activation of AMPK (27). Accumulation of LCACEs in the liver of type 2 diabetics may be impairing fatty acid oxidation in the liver by the following two mechanisms: 1) accumulation of LCACEs may directly inhibit basal phosphorylation of AMPK by LKB1/STRAD/MO25 and 2) accumulation of LCACEs may blunt glucagon signaling by disrupting the glucagon-PKA-LKB1-AMPK-ACC axis.
Inhibition of AMPKK activity in the liver may be desirable under certain conditions. Liver LCACEs are intermediates and products in the fatty acid synthesis pathway and therefore may be elevated during periods of increased fatty acid synthesis. Teleologically, it would be energetically inefficient for the cell to be engaged in elevated fatty acid synthesis and fatty acid oxidation simultaneously.
LCACEs in dilute tissue homogenates inhibit enzymes of fatty acid and carbohydrate oxidation. An earlier study demonstrated that the LCACE-mediated inhibitory effect decreased with increasing homogenate concentration, suggesting that unphysiologically high LCACE concentrations would be required to exert a physiologically relevant inhibition (47). Although total LCACE concentrations in tissues may range from 5 to 160 µM, free LCACE concentration has been estimated to be <200 nM, although the actual free LCACE concentration for any tissue is not known (9). Some evidence suggests that bound LCACEs may be active in regulating specific targets (42). The roles of acyl-CoA binding protein and other proteins that may regulate the activity and location of LCACEs, LKB1/STRAD/MO25, and AMPK have not been completely delineated. Additionally, it is unlikely that LCACEs are uniformly distributed throughout the cell. Higher concentrations of LCACEs localized to a particular cellular compartment or protein complex may regulate enzymes within that same compartment or complex.
LCACEs have been postulated to contribute to insulin resistance in skeletal muscle by inhibiting hexokinase and activating particular PKC isozymes directly and indirectly through DAG (31, 52). LCACEs may also contribute to insulin resistance in skeletal muscle by inhibiting phosphorylation of AMPK by LKB1/STRAD/MO25. Both leptin and adiponectin stimulate fatty acid oxidation in skeletal muscle and activate AMPK by an unknown mechanism (34, 60). A reduction in cytoslic LCACEs by transport into the mitochondria may be one mechanism by which leptin and adiponectin activate AMPK in response to an increase in fatty acid oxidation. The previously cited study demonstrating that heart muscle perfusion with palmitate increased phosphorylation of AMPK found that LCACE levels did not change (4). A possible redistribution of LCACEs from the cytosol into the mitochondria was not accounted for, leaving open the possibility that cytosolic LCACE decreased while total LCACE did not change. The LCACE concentration in mitochondria can reach as high as 1 mM, indicating that mitochondria can serve as a sink for LCACEs (23).
A recent study found that human skeletal muscle AMPK signaling is not impaired with obesity although P-AMPK was not measured directly (50). AMPK expression and signaling were found to be no different between obese type 2 diabetics and nondiabetic obese subjects (21). Additionally, both LCACEs and P-AMPK increase in skeletal muscle during a single bout of endurance exercise, but no distinction was made between cytosolic and mitochondrial LCACE pools (54, 58).
The AMPKK activity of LKB1/STRAD/MO25 is likely regulated at the AMPK level by a milieu of allosteric modulators. PCoA and AMP exerted opposing effects on AMPKK activity (Fig. 8). Elimination of AMP from the reaction mixture resulted in a marked reduction in AMPKK activity at all PCoA concentrations. Cellular AMP, ATP, LCACEs, glycogen (39), creatine, creatine phosphate (40), NAD, NADH (41), and other unidentified regulators may coordinately modulate activity of the AMPK signaling cascade.
In conclusion, the results of this study demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25. This result is specific to LCACEs and was not found with shorter-chain CoA derivatives, palmitate, or palmitoyl carnitine. P-AMPK is not inhibited by LCACEs. Thus LCACEs regulate the activity of AMPK by preventing its phosphorylation. These findings suggest that the AMPK signaling cascade is directly regulated by LCACEs and that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell.
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FOOTNOTES
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Address for reprint requests and other correspondence: W. W. Winder, 545 WIDB, Brigham Young Univ., Provo, Utah 84602, (E-mail: william_winder{at}byu.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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