AMP-Activated Protein Kinase Activates p38 Mitogen-Activated Protein Kinase by Increasing Recruitment of p38 MAPK to TAB1 in the Ischemic Heart

Animals

Animals were housed in accordance with guidelines from the American Association for Laboratory Animal Care. All procedures were approved by the Yale University Animal Care and Use Committee.

Heart Muscle Glucose Uptake

Male Sprague-Dawley rats weighing 250 to 300 g were allowed access to standard chow and water ad libitum; 2-deoxy-d-[1-³H]glucose accumulation in left ventricular papillary muscles was performed as previously described.¹⁵ Muscles were incubated with AICAR (1 mmol/L) or the p38 MAPK activator anisomycin (20 to 60 μg/mL) for variable times in oxygenated buffer or under hypoxic conditions in buffer equilibrated with 100% N₂. The p38 MAPK inhibitor SB203580 (10 μmol/L), extracellular signal-regulated kinase (ERK) inhibitor U0126 (10 μmol/L) or their vehicles were added 30 minutes before activators or hypoxia.

Mouse Heart Perfusions

Transgenic male mice (C57BL/6), expressing a kinase-dead (KD) rat α₂ isoform (K45R mutation) in heart and skeletal muscle,¹² were studied at 4 to 6 months of age. Wild-type (WT) littermates were used as controls. Mouse hearts were retrogradely perfused with Krebs-Henseleit buffer (KHB) containing 7 mmol/L glucose, 0.4 mmol/L oleate, 1% BSA, and a low fasting concentration of insulin (10 μU/mL).⁸ Hearts were perfused for 30 minutes at a flow of 4 mL/min, followed by (1) moderate ischemia (0.75 mL/min for 30 minutes), (2) severe ischemia (no-flow for 15 minutes), or (3) continued baseline flow.

In Vivo Regional Ischemia

The mkk3^−/− (MKK3 KO) mice²⁵ and their mkk3^+/+ WT littermates (kindly provided by Drs R. Flavell and P. Lee, Yale University, New Haven, Conn; Dr R. Davis, University of Massachusetts, Worcester, Mass) were anesthetized with IP ketamine (95 mg/kg) and xylazine (40 mg/kg), intubated, and ventilated with a respirator. After thoracotomy, a suture was placed to ligate the proximal left anterior descending coronary artery for 10 minutes.³ Control mice underwent sham thoracotomy. Hearts were then rapidly excised, and the ischemic region of the left ventricle was freeze clamped in liquid nitrogen.

AMPK Activity and Phosphorylation

AMPK activity was measured with the synthetic SAMS peptide,^7,8 after immuno-isolation with AMPK α subunit (AMPKα) antibody¹² (kind gift of Dr M. Birnbaum, University of Pennsylvania, Philadelphia, Pa) or TAB1 polyclonal antibody (kind gift from Drs J. Ninomiya-Tsuji, North Carolina State Univesity, Raleigh, NC and J. Han, The Scripps Research Institute, La Jolla, Calif)^16,26 coupled to protein G/A Sepharose. AMPK phosphorylation was assessed by immunoblotting with an antibody to α subunits containing phosphorylated Thr172 (Cell Signaling, Beverly, Mass).^7,8

Immunoblotting

Immunoblots were performed as previously described.¹⁵ Heart homogenate proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. For reprobing, membranes were stripped with 50 mmol/L Tris-HCl, 2% SDS, and 0.1 mol/L β-mercaptoethanol (pH 6.8). Rabbit polyclonal antibodies against phospho-p38 MAPK, total p38 MAPK, phospho-Akt, total Akt, phospho-p44/p42 ERK and total p44/p42 ERK were purchased from Cell Signaling. Rabbit polyclonal antibody against TAB1 was used as previously described.^16,26

Cell Surface GLUT4 Labeling

After incubations, heart muscles were rinsed in ice-cold glucose-free KHB and incubated at 4°C in KHB containing 200 μmol/L Bio-LC-ATB-BGPA (4,4-O-[2-[2-[2-[2-[2-[6-(biotinylamino)hexanolyl]-amino]ethoxy]exoxy]-ethoxy]-4–1-azi-2,2,2-trifluoroethyl)benzoyl]amino-1,3-propanediyl]bis-d-glucose),¹⁵ a kind gift from Dr G. Holman (University of Bath, Bath, UK). BGPA was cross-linked to cell surface glucose transporters by UV irradiation.¹⁵ Cell surface GLUT4 was determined by GLUT4 immunoblotting after streptavidin-agarose isolation.¹⁵

Statistical Analysis

Data were expressed as means±SEM. Significance was tested by Student 2-tail t tests with Bonferroni correction or 2-way repeated measures ANOVA with post hoc analysis. A value of P<0.05 was considered significant.

AMPK and p38 MAPK Activation by Hypoxia and AICAR

We first examined the kinetics of AMPK activation in rat heart papillary muscles that were exposed to hypoxia or AICAR. Both treatments stimulated phosphorylation of AMPK and p38 MAPK in an initial parallel and time-dependent manner (Figure 1). The p38 MAPK activation induced by both hypoxia and AICAR decreased after 30 minutes, despite persistent AMPK activation (Figure 1). Neither hypoxia nor AICAR treatment activated ERK or c-Jun N-terminal kinase (data not shown).

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Figure 1. Effect of AICAR and hypoxia on AMPK and p38 MAPK activation. Time course of AMPK and p38 MAPK phosphorylation induced by AICAR (1 mmol/L) or hypoxia (100% N₂) in isolated rat heart papillary muscles. Immunoblots were performed using antibodies for phospho-AMPK (Thr172) (p-AMPK), AMPK α subunits (AMPKα), phospho-p38 (p-p38) MAPK, or total p38 (p38) MAPK. The graphs show phosphorylated AMPK or p38 MAPK relative to the total amount of kinase. Values are means±SE for 4 experiments. *P<0.01 vs control, †P<0.01 vs 30 minutes.

Attenuation of p38 MAPK Activation by Ischemia in AMPK-Deficient Hearts

To define the role of AMPK in the activation of p38 MAPK during ischemia, we examined the extent of p38 MAPK activation in isolated perfused AMPK-deficient transgenic mouse hearts during ischemia. As previously reported,⁸ the KD α₂ AMPK isoform effectively replaced native heart AMPK α isoforms (Figure 2A). AMPK activity increased 3-fold (P<0.01) during low-flow ischemia (Figure 2B) and 5-fold (P<0.01) during no-flow ischemia (Figure 2C) in the WT hearts but failed to increase in the KD hearts (Figure 2B and 2C). Interestingly, p38 MAPK phosphorylation increased 7-fold (P<0.01) during low-flow ischemia in WT hearts but only 2.3-fold in KD hearts (P<0.01 versus ischemic WT) (Figure 2D). Similar results were seen during more severe no-flow ischemia, which activated p38 MAPK 8-fold (P<0.01) in the WT hearts but only 2-fold in the KD hearts (P<0.01 versus ischemic WT) (Figure 2E).

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Figure 2. Effect of AMPK deficiency on ischemic heart p38 MAPK activation. AMPKα catalytic subunit immunoblots (A) of WT and AMPK-deficient (KD) mouse heart homogenates. The KD α₂ isoform migrates more slowly than WT because of its c-myc tag, and WT isoforms that are not incorporated into the heterotrimeric AMPK complex are degraded in the KD hearts.^8,12 AMPK activity in WT and KD hearts after control perfusions and after 30 minutes of moderate low-flow ischemia (B) or 15 minutes of severe no-flow ischemia (C). Stimulation of p38 MAPK phosphorylation in WT and KD hearts during moderate (D) or severe (E) ischemia. Representative immunoblots using antibodies to phospho-p38 (p-p38) MAPK or total p38 (p38) MAPK, with quantification of the content of phosphorylated p38 MAPK relative to the total amount of p38 MAPK. All values are means±SE for 5 experiments. *P<0.01 vs control, †P<0.01 vs WT ischemia.

Association of AMPK With TAB1

To determine the potential mechanisms responsible for AMPK activation of p38 MAPK in the ischemic heart, we first assessed the extent of p38 MAPK activation in ischemic hearts from transgenic mkk3^−/− (MKK3 KO) versus mkk3^+/+ WT controls. MKK3 is a specific upstream kinase of p38 MAPK.²⁵ Both AMPK (Figure 3A) and p38 MAPK (Figure 3B) were normally activated by in vivo regional ischemia in MKK3 KO hearts, confirming that ischemia does not stimulate p38 MAPK through the MKK3 pathway.²⁴

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Figure 3. Mechanisms of p38 MAPK activation in the ischemic heart: interaction of AMPK with TAB1. A, AMPK activation in hearts from WT and MKK3 KO mice following 10 minutes of regional ischemia or control sham operation. Top, Representative phospho-AMPK (Thr172) (p-AMPK) or pan-α AMPK (AMPKα) immunoblots. Bottom, AMPK activity measured after immunoprecipitation with AMPKα antibody. Values are means±SE for 5 experiments. *P<0.01 vs control. B, Representative MKK3, phospho-p38 (p-p38) MAPK, or total p38 (p38) MAPK immunoblots of homogenates from control or ischemic WT or MKK3 KO hearts. C, Representative TAB1 immunoblots of homogenates (first panel) or p38 MAPK immunoblots of TAB1 or nonimmune rabbit IgG immunoprecipitates (second and third panels, respectively). The graph quantifies the effect of ischemia on the relative amount of p38 associated with TAB1 in WT and MKK3 KO hearts. Values are means±SE for 3 experiments. *P<0.01 vs control. D, Representative AMPKα₂ immunoblots of TAB1 or nonimmune rabbit IgG immunoprecipitates (first and second panels, respectively). The graph quantifies the relative amount of AMPKα₂ associated with TAB1 in WT and MKK3 KO hearts. Values are means±SE for 3 experiments. E, AMPK activity contained in TAB1/AMPK complexes after immunoprecipitation with TAB1 antibody. Values are means±SE for 3 experiments. *P<0.01 vs control.

We then examined whether AMPK might interact with TAB1 to increase p38 MAPK activation during ischemia. TAB1 is a scaffold protein that binds components of the transforming growth factor-β–initiated signal transduction cascade and promotes p38 MAPK autophosphorylation.¹⁶ Immunoblots of TAB1 immunoprecipitates demonstrated that TAB1 was associated not only with p38 MAPK (Figure 3C, second panel) but also with AMPK (Figure 3D, first panel). The TAB1 immunoprecipitates contained the α₂ isoform of the catalytic subunit of AMPK (Figure 3D, first panel) but not the lower abundance α₁ isoform (data not shown). Control immunoprecipitates performed with nonimmune rabbit IgG did not reveal any p38 MAPK or AMPK, supporting the specificity of the TAB1 immunoprecipitates (Figure 3C and 3D).

Interestingly, ischemia increased the amount of p38 MAPK associated with TAB1/AMPK complexes (Figure 3C, second panel and bar graph; P<0.01 versus control). Ischemia-stimulated recruitment of p38 MAPK to TAB1/AMPK complexes was not altered in MKK3 KO hearts compared with ischemic WT hearts (Figure 3C, second panel and bar graph). However, the amount of p38 MAPK detected in TAB1 immunoprecipitates from ischemic AMPK-deficient KD hearts was markedly reduced compared with ischemic WT hearts (Figure 4A, second panel and bar graph; P<0.01 versus WT ischemia).

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Figure 4. Effect of AMPK deficiency on the recruitment of p38 MAPK to TAB1 in ischemic hearts. The association among TAB1, p38 MAPK, and AMPK in homogenates from isolated WT or KD mouse hearts after 15 minutes of global no-flow ischemia or control perfusions. A, Representative TAB1 immunoblots of homogenates (first panel) and p38 MAPK immunoblots of TAB1 or nonimmune IgG immunoprecipates (second and third panels, respectively). The graph quantifies the effect of ischemia on the relative amount of p38 MAPK associated with TAB1 in WT and KD hearts. Values are means±SE for 3 experiments. *P<0.01 vs control, †P<0.01 vs WT ischemia. B, Representative AMPKα₂ immunoblots of heart homogenates after immunoprecipitation with TAB1 antibody (first panel) or nonimmune rabbit IgG (second panel). The graph quantifies the relative amount of AMPKα₂ associated with TAB1 in WT and KD hearts. Values are means±SE for 3 experiments. C, AMPK activity associated with TAB1 immunoprecipitate complexes. Values are means±SE for 3 experiments. *P<0.01 vs control, †P<0.01 vs WT ischemia.

The association between AMPK and TAB1 did not appear to depend on the activation of AMPK. There were similar amounts of AMPK in TAB1 immunoprecipitates in control and ischemic hearts (Figures 3D and 4⇑B), as well as in KD and WT hearts (Figure 4B). However, the AMPK activity associated with TAB1/AMPK complexes was greater in ischemic hearts (Figure 3E; P<0.01 versus control). In contrast, there was no AMPK activation in TAB1/AMPK complexes in ischemic KD hearts (Figure 4C; P<0.01 versus WT ischemia).

Taken together, these findings indicate that ischemic activation of AMPK within TAB1/AMPKα₂ complexes may play a critical role in enhancing the recruitment of p38 MAPK to the scaffold protein TAB1, which then promotes p38 MAPK autophosphorylation.

Inhibition of AMPK-stimulated Glucose Transport by p38 MAPK Inhibitor

The AMPK activator AICAR stimulates glucose transport in heart and skeletal muscle,^10,13 and AMPK has an important role in promoting glucose transport during ischemia and hypoxia.^8,12,14 However, the downstream mechanism responsible for increased glucose transport remains uncertain, leading us to examine whether the AMPK-stimulated glucose uptake might be mediated by downstream p38 MAPK activation. AICAR and hypoxia both increased deoxyglucose uptake in isolated rat heart muscles (P<0.01 for each) (Figure 5A). Preincubation with the p38 MAPK inhibitor SB203580 reduced AICAR- and hypoxia-stimulated glucose uptake by 47±5% (P<0.05) and 56±9% (P<0.05), respectively (Figure 5A).

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Figure 5. Inhibition of AICAR- and hypoxia-stimulated glucose uptake and GLUT4 translocation by SB203580. Heart muscles were preincubated for 30 minutes with p38 MAPK inhibitor SB203580 (10 μmol/L) before incubation with or without AICAR (1 mmol/L) for 60 minutes or hypoxia (100% N₂) for 60 minutes. A, 2-Deoxy-[1-³H]glucose was added during the last 30 minutes to measure glucose uptake. Values are means±SE of 4 experiments. *P<0.01 vs control, §P<0.05 vs control, †P<0.05 vs AICAR or hypoxia alone. B, Cell surface GLUT4 was labeled with Bio-LC-ATB-BGPA, isolated on streptavidin-agarose, and immunoblotted with specific GLUT4 antibodies. Representative immunoblots showing cell surface (streptavidin isolates) and total membrane GLUT4 (first and third panels) or as a negative control, α₁-Na/K-ATPase (second and fourth panels). The ratios of surface to total GLUT4 are expressed as means±SE for 3 experiments, each including 2 to 3 pooled muscles. *P<0.05 vs control, †P<0.05 vs AICAR or hypoxia alone.

AMPK activation of glucose uptake enhances GLUT4 translocation to the plasma membrane,¹³ but there is also evidence that p38 MAPK may activate GLUT4 present on the cell surface, rather than stimulate translocation.^23,27 To distinguish between these possibilities, cell surface GLUT4 content was determined using the exofacial bis-glucose photolabeling reagent Bio-LC-ATB-BGPA.¹⁵ AICAR and hypoxia treatment increased cell surface GLUT4 content 2.4-fold (P<0.05) and 2.5-fold (P<0.05), respectively (Figure 5B). SB203580 reduced AICAR- and hypoxia-stimulated cell surface GLUT4 by 46±5% (P<0.05) and 41±6% (P<0.05), respectively (Figure 5B). Total membrane GLUT4 content was not different between treated muscles and controls (Figure 5B), indicating that GLUT4 translocation was responsible for increased glucose uptake. The plasma membrane marker α₁-Na/K-ATPase was not detectable in the Bio-LC-ATB-BGPA-binding fractions, demonstrating the specificity of proteins cross-linked by this reagent (Figure 5B).

Stimulation of Glucose Transport by the p38 MAPK Activator Anisomycin

To examine whether activation of p38 MAPK stimulated glucose uptake and GLUT4 translocation, heart muscles were incubated with anisomycin, a pharmacological activator of p38 MAPK.²⁸ Anisomycin activated p38 MAPK in a time- and dose-dependent manner (Figure 6A), without stimulating phosphorylation of AMPK or Akt (Figure 6B), known mediators of GLUT4 translocation.¹ Anisomycin significantly increased deoxyglucose uptake (Figure 6C; P<0.05 versus control). SB203580 blocked anisomycin-stimulated p38 MAPK activation (Figure 6D, first panel) and deoxyglucose uptake (Figure 6C). Anisomycin-treatment also increased cell surface GLUT4 content (Figure 6D, second panel and bar graph; P<0.05 versus control), and this action was also blocked by SB203580 (Figure 6D, second panel and bar graph; P<0.05 versus anisomycin alone). The total membrane GLUT4 content did not differ between treated and control muscles (Figure 6D, third panel), indicating that anisomycin-stimulated GLUT4 translocation.

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Figure 6. Anisomycin stimulation of p38 MAPK and glucose transport. A, Effects of incubation time and anisomycin dose on p38 MAPK phosphorylation in rat heart muscles. Representative phospho-p38 (p-p38) MAPK and total p38 (p38) MAPK immunoblots. B, Effects of anisomycin (40 μg/mL) on AMPK and Akt phosphorylation in heart muscles. Representative phospho-AMPK (Thr172) (p-AMPK), total AMPK (AMPKα), phospho-Akt (Ser⁴⁷³) (p-Akt), and total Akt (Akt) immunoblots. As positive controls, heart muscles were incubated for 30 minutes either under hypoxic conditions for p-AMPK or with insulin (10 mU/mL) for p-Akt. C, Heart muscles were incubated with or without anisomycin (40 μg/mL) for 30 minutes with or without preincubation with the p38 MAPK inhibitor SB203580 (10 μmol/L) before measuring 2-deoxy-[1-³H]glucose uptake. Values are means±SE for 5 experiments. *P<0.05 vs control, †P<0.05 vs anisomycin alone. D, Anisomycin-stimulated GLUT4 translocation to cell surface membrane. Heart muscles were incubated with anisomycin (40 μg/mL) for 30 minutes with or without preincubation with SB203580 (10 μmol/L). Representative immunoblot of heart homogenates using phospho-p38 antibody (p-p38,first panel). Cell surface GLUT4 was labeled with Bio-LC-ATB-BGPA, isolated on streptavidin-agarose, and immunoblotted with GLUT4. Representative immunoblots showing cell surface GLUT4 (s-GLUT4, second panel) and total membrane GLUT4 (t-GLUT4, third panel). The graph quantifies the relative amount of surface to total GLUT4. Values are means±SE for 3 experiments, each including 2 to 3 pooled muscles. *P<0.05 vs control, †P<0.05 vs anisomycin alone.