Inhibition of cardiac fibroblast proliferation and myofibroblast differentiation by resveratrol

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Prolonged activation of CFs leads to cardiac fibrosis and reduced myocardial contractile function. Resveratrol (RES) exhibits a number of cardioprotective properties; however, the possibility that this compound affects CF function has not been considered. The current study tests whether RES directly influences the growth and proliferation of CFs and differentiation to the hypersecretory myofibroblast phenotype. Pretreatment of CFs with RES (5-25 microM) inhibited basal and ANG II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK kinase activation. This inhibition by RES reduced basal proliferation and blocked ANG II-induced growth and proliferation of CFs in a concentration-dependent manner, as measured by [(3)H]leucine and [(3)H]thymidine incorporation, respectively. RES pretreatment attenuated ERK phosphorylation when CFs were stimulated with 0.2 nM epidermal growth factor (EGF), a concentration at which EGF-induced ERK activation over basal was similar to the phosphorylation induced by 100 nM ANG II. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 microM RES. Pretreatment of CFs with RES also reduced both ANG II- and transforming growth factor-beta-induced CF differentiation to the myofibroblast phenotype, indicated by a reduction in alpha-smooth muscle actin expression and stress fiber organization in CFs. This study identifies RES as an anti-fibrotic agent in the myocardium by limiting CF proliferation and differentiation, two critical steps in the pathogenesis of cardiac fibrosis.     

AICAR stimulates IL-6 production via p38 MAPK in cardiac fibroblasts in adult mice: a possible role for AMPK

Though known as a sensor of energy balance, AMP-activated protein kinase (AMPK) was recently shown to limit damage and apoptotic activity and contribute to the late preconditioning in heart. Interleukin-6 was also reported to involve in anti-apoptosis and cardio-protection in myocardium. Interestingly, both AMPK activity and IL-6 level were increased in response to ischemia, hypertrophy and oxidative stress. To determine whether AMPK activation will promote IL-6 production, cardiac fibroblasts (CFs) from mice were incubated with AMPK activator, 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR). The results demonstrated that AICAR time and dose-dependently stimulated IL-6 production by ELISA and immunofluorescence. Pretreatment with p38 mitogen-activated protein kinase (MAPK) inhibitor blocked AICAR-induced IL-6 production; furthermore, AICAR-activated p38 MAPK phosphorylation by Western blot. To confirm that the increase in IL-6 production is ascribed to AMPK activation, we used another known AMPK activator, metformin. It also dose-dependently potentiated IL-6 production in CFs, and this potentiation could be reversed by p38 MAPK inhibitor. In conclusion, AMPK activation promoted IL-6 production in CFs via p38 MAPK-dependent pathway.     

Activation of AMP-activated Protein Kinase Suppresses Oxidized Low-density Lipoprotein-induced Macrophage Proliferation

Macrophage-derived foam cells play important roles in the progression of atherosclerosis. We reported previously that ERK1/2-dependent granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, leading to p38 MAPK/ Akt signaling, is important for oxidized low density lipoprotein (Ox-LDL)-induced macrophage proliferation. Here, we investigated whether activation of AMP-activated protein kinase (AMPK) could suppress macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages was assessed by [³H]thymidine incorporation and cell counting assays. The proliferation was significantly inhibited by the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and restored by dominant-negative AMPKα1, suggesting that AMPK activation suppressed macrophage proliferation. AICAR partially suppressed Ox-LDL-induced ERK1/2 phosphorylation and GM-CSF expression, suggesting that another mechanism is also involved in the AICAR-mediated suppression of macrophage proliferation. AICAR suppressed GM-CSF-induced macrophage proliferation without suppressing p38 MAPK/Akt signaling. GM-CSF suppressed p53 phosphorylation and expression and induced Rb phosphorylation. Overexpression of p53 or p27^kip suppressed GM-CSF-induced macrophage proliferation. AICAR induced cell cycle arrest, increased p53 phosphorylation and expression, and suppressed GM-CSF-induced Rb phosphorylation via AMPK activation. Moreover, AICAR induced p21^cip and p27^kip expression via AMPK activation, and small interfering RNA (siRNA) of p21^cip and p27^kip restored AICAR-mediated suppression of macrophage proliferation. In conclusion, AMPK activation suppressed Ox-LDL-induced macrophage proliferation by suppressing GM-CSF expression and inducing cell cycle arrest. These effects of AMPK activation may represent therapeutic targets for atherosclerosis.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy homeostasis and response to metabolic stress. Here, we investigated the effect of AICAR, an AMPK activator, on proliferation of various cancer cells and observed that proliferation of all the examined cell lines was significantly inhibited by AICAR treatment due to arrest in S-phase accompanied with increased expression of p21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in vitro growth of cancer cells was mirrored in vivo with increased expression of p21, p27, and p53 and attenuation of Akt phosphorylation. Anti-proliferative effect of AICAR is mediated through activated AMP-activated protein kinase (AMPK) as iodotubericidin and dominant-negative AMPK expression vector reversed the AICAR-mediated growth arrest. Moreover, constitutive active AMPK arrested the cells in S-phase by inducing the expression of p21, p27, and p53 proteins and inhibiting Akt phosphorylation, suggesting the involvement of AMPK. AICAR inhibited proliferation in both LKB and LKB knock-out mouse embryo fibroblasts to similar extent and arrested cells at S-phase when transfected with dominant negative expression vector of LKB. Altogether, these results indicate that AICAR can be utilized as a therapeutic drug to inhibit cancer, and AMPK can be a potential target for treatment of various cancers independent of the functional tumor suppressor gene, LKB.     

Activation of adenosine monophosphate activated protein kinase inhibits growth of multiple myeloma cells

The role of adenosine monophosphate activated protein kinase (AMPK) in regulating multiple myeloma (MM) cell growth is not yet clear. In this study, we show that the AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAr) and D942 inhibit cell growth in MM cell lines. AICAr also induced an S-phase cell cycle arrest in all four tested cell lines and led to phosphorylation and thus activation of AMPK. Furthermore, the inhibition of a nucleoside transporter by nitrobenzyl-thio-9-beta-d-ribofuranosylpurine (NBTI), inhibition of the adenosine kinase by iodotubericidine and inhibition of AMPK by AMPKI Compound C reversed AICAr effects, indicating that the cellular effects of AICAr were mediated by AMPK. Activation of AMPK inhibited basal extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR) and P70S6 kinase (P70S6K) as well as AKT phosphorylation, and blocked IL-6, IGF-1, and HS-5 stromal cell conditioned medium-induced increase of cell growth. Troglitazone, which has previously been shown to activate AMPK, similarly inhibited MM cell growth, activated AMPK, and decreased ERK and P70S6K phosphorylation. Our results suggest that activation of AMPK inhibits MM cell growth despite stimulation with IL-6, IGF-1, or HS-5 stromal cell conditioned medium and represents a potential new target in the therapy of MM.     

Involvement of Akt2/protein kinase B β (PKBβ) in the 8‐Cl‐cAMP‐induced cancer cell growth inhibition

8‐chloro‐cyclic AMP (8‐Cl‐cAMP), which induces differentiation, growth inhibition, and apoptosis in various cancer cells, has been investigated as a putative anti‐cancer drug. However, the exact mechanism of 8‐Cl‐cAMP functioning in cancer cells is not fully understood. Akt/protein kinase B (PKB) genes (Akt1, Akt2, and Akt3) encode enzymes belonging to the serine/threonine‐specific protein kinase family. It has been suggested that Akt/PKB enhances cell survival by inhibiting apoptosis. Recently, we showed that 8‐Cl‐cAMP and 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) inhibited cancer cell growth through the activation of AMPK and p38 MAPK. Therefore, we anticipated that the phosphorylation of Akt/PKB would be decreased upon treatment with 8‐Cl‐cAMP. However, treatment with 8‐Cl‐cAMP and AICAR induced the phosphorylation of Akt/PKB, which was inhibited by ABT702 (an adenosine kinase inhibitor) and NBTI (an adenosine transporter inhibitor). Furthermore, whereas Compound C (an AMPK inhibitor), AMPK‐DN (AMPK‐dominant negative) mutant, and SB203580 (a p38 MAPK inhibitor) did not block the 8‐Cl‐cAMP‐induced phosphorylation of Akt/PKB, TCN (an Akt1/2/3 specific inhibitor) and an Akt2/PKBβ‐targeted siRNA inhibited the 8‐Cl‐cAMP‐ and AICAR‐mediated phosphorylation of AMPK and p38 MAPK. TCN also reversed the growth inhibition mediated by 8‐Cl‐cAMP and AICAR. Moreover, an Akt1/PKBα‐targeted siRNA did not reduce the phosphorylation of AMPK and p38 MAPK after treatment with 8‐Cl‐cAMP. These results suggest that Akt2/PKBβ activation promotes the phosphorylation of AMPK and p38 MAPK during the 8‐Cl‐cAMP‐ and AICAR‐induced growth inhibition. J. Cell. Physiol. 228: 890–902, 2013. © 2012 Wiley Periodicals, Inc.     

Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways

AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by approximately 80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway.     

Regulation of ERK1/2 by ouabain and Na-K-ATPase-dependent energy utilization and AMPK activation in parotid acinar cells

We previously found that the phosphorylation of ERK1/2 by submaximal concentrations of the muscarinic receptor ligand carbachol was potentiated in rat parotid acinar cells exposed to ouabain, a cardiac glycoside that inhibits the Na-K-ATPase. We now report that this signaling phenomenon involves the prevention of negative regulation of extracellular signal-regulated kinase-1/2 (ERK1/2) that is normally mediated by AMP-activated protein kinase (AMPK). Carbachol increases the turnover of the ATP-consuming Na-K-ATPase, reducing intracellular ATP and promoting the phosphorylation/activation of the energy sensor AMPK. Ouabain blocks the reduction in ATP and subsequent AMPK phosphorylation, which is regulated by the AMP-to-ATP ratio. The ouabain-promoted enhancement of ERK1/2 phosphorylation was not reproduced in Par-C10 cells, an immortalized rat parotid cell line that did not respond to carbachol with an ATP reduction and that employs an upstream AMPK kinase (Ca(2+)/calmodulin-dependent protein kinase kinase, CaMKK) different from that (LKB1) in native cells. In native parotid cells, inhibitory effects of AMPK on ERK1/2 signaling were examined by activating AMPK with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), which is converted to an AMP mimetic but does not alter parotid ATP levels. AICAR-treated cells display increases in AMPK phosphorylation and a reduced phosphorylation of ERK1/2 subsequent to activation of muscarinic and P2X(7) receptors, which promote increases in Na-K-ATPase turnover, but not upon epidermal growth factor receptor activation. These results suggest that carbachol-initiated AMPK activation can produce a negative feedback on ERK1/2 signaling in response to submaximal muscarinic receptor activation and that increases in fluid secretion can modulate receptor-initiated signaling events indirectly by producing ion transport-dependent decreases in ATP.     

Involvement of ERK and AKT signaling in the growth effect of arginine vasopressin on adult rat cardiac fibroblast and the modulation by simvastatin

Arginine vasopressin (AVP) has been shown to directly induce neonatal rat cardiac fibroblasts (CFs) proliferation, a major component involved in cardiac hypertrophy. Herein, we explored whether AVP is also a growth factor for adult rat CFs and, if so, whether the growth effect could be inhibited by simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. AVP significantly increased DNA synthesis in adult rat CFs by 73.5 +/- 5.1% (P < or = 0.05), an effect inhibited by V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin. AVP also activated extracellular signal-regulated kinase 1/2 (ERK1/2) as assessed by MBP phosphotransferase activity (5.1 +/- 0.6 fold over basal level, P < or = 0.05) and Western blot analysis, and effects were mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), but abolished by inhibiting cellular PKC through chronic PMA incubation. In addition, AVP induced PKC activation (27.2 +/- 3.8% from a basal value of 9.3 +/- 0.7%, P < or = 0.05). AVP-induced increase in DNA synthesis could be attenuated by the specific inhibitors of ERK1/2 (PD98059), PI3K (LY294002), and AKT (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, HIMO). Simvastatin inhibited the effects of AVP on DNA synthesis, ERK1/2, and PKC activation in a dose-dependent manner. Phosphatidylinositol-3-kinase (PI3K)-dependent AKT activation induced by AVP was also inhibited by simvastatin. The effects of simvastatin on ERK1/2, PKC, and AKT activation and DNA synthesis could be reversed by mevalonate. These results support a growth-inducing effect of AVP on adult rat CFs through ERK and AKT signalings and the growth effect could be attenuated by simvastatin via inhibiting these two pathways.     

AMPK regulation of the growth of cultured human keratinocytes

AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). At concentrations of 10(-4) and 10(-3) M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10(-6) M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D3 (10(-7) and 10(-6) M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D3 is AMPK-independent.     

Role of protein kinase C in arginine vasopressin-stimulated ERK and p70S6 kinase phosphorylation

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)-stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF-R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972-F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP-stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0-60 min stimulated the serine/threonine phosphorylation of PKC isoforms alpha, delta, epsilon, and zeta. The activation of PKC was dependent on EGF-R and phosphatidylinositol 3-kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12-myristate 13-acetate (PMA) inhibited AVP-induced activation of ERK and p70S6 kinase as well as EGF-R phosphorylation. Rottlerin, a specific inhibitor of PKCdelta, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCepsilon translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCzeta (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, G?6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCdelta plays an important role in AVP-stimulated ERK and p70S6 kinase activation and cell proliferation.     

Overexpression of CYP2E1 induces HepG2 cells death by the AMP kinase activator 5′-aminoimidazole-4-carboxamide-1-β-<Emphasis Type="SmallCaps">d</Emphasis>-ribofuranoside (AICAR)

5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a phylogenetically conserved serine/threonine protein kinase. AMPK may inhibit cell growth and proliferation and also regulates apoptosis. 5′-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) is a cell-permeable AMPK activator. Activation of AMPK with AICAR has been shown to induce apoptosis of the rat hepatoma cell line FTO2B cells and almost completely inhibited HepG2 cells growth. In this study, a HepG2 cell line, which was transfected with a vector containing human CYP2E1 cDNA (E47 cells), was treated with AICAR. Cell proliferation was blocked, and apoptosis and necrosis were elevated as assessed by cellular morphology, DNA content assay, and lactate dehydrogenase leakage. AICAR treatment significantly increases CYP2E1 activity (20-fold) and expression (5.5-fold) in E47 cells. Iodotubericidin, which inhibits the conversion of AICAR to its activated form AICAR monophosphate, the antioxidants trolox and MnTMPyP, and 4-methylpyrazole, an inhibitor of CYP2E1, all can protect the E47 cells from AICAR-induced necrosis. Production of intracellular reactive oxygen species was increased by AICAR treatment in E47 cells. The cytotoxicity mechanism of AICAR in E47 cells is suggested to include AMPK activation, p53 phosphorylation, p21 expression, overexpression of CYP2E1, and intracellular ROS accumulation.     

AMPK-mediated inhibition of mTOR kinase is circumvented during immediate-early times of human cytomegalovirus infection

Human cytomegalovirus (HCMV) infection increases synthetic rates in infected cells. The resulting increase in energy utilization could potentially increase the AMP:ATP ratio, causing activation of 5'-AMP-activated protein kinase (AMPK). Activated AMPK promotes inhibition of mammalian target of rapamycin (mTOR) kinase, which could be deleterious to the viral infection. Using the AMPK-activating drug 5-amino-4-imidazolecarboxamide ribose (AICAR), we showed that, by 12 h post-HCMV infection, inhibition of mTOR by AMPK is circumvented. However, growth curves showed that progeny virion production is inhibited when AICAR is added, suggesting other inhibitory effects of AICAR or activated AMPK.     

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from AMPK alpha2 knockout (KO), AMPK gamma3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser473 phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr172 (P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46) (P < 0.01) in WT, AMPK alpha2 KO, and AMPK gamma3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46). In conclusion, functional alpha2 and gamma3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.     

Abnormal metabolism flexibility in response to high palmitate concentrations in myotubes derived from obese type 2 diabetic patients

Insulin resistance in type 2 diabetes (T2D) is associated with intramuscular lipid (IMCL) accumulation. To determine whether impaired lipid oxidation is involved in IMCL accumulation, we measured expression of genes involved in mitochondrial oxidative metabolism or biogenesis, mitochondrial content and palmitate beta-oxidation before and after palmitate overload (600 μM for 16 h), in myotubes derived from healthy subjects and obese T2D patients. Mitochondrial gene expression, content and network were not different between groups. Basal palmitate beta-oxidation was not affected in T2D myotubes, whereas after 16 h of palmitate pre-treatment, T2D myotubes in contrast to control myotubes, showed an inability to increase palmitate beta-oxidation (p < 0.05). Interestingly, acetyl-CoA carboxylase (ACC) phosphorylation was increased with a tendency for statistical significance after palmitate pre-treatment in control myotubes (p = 0.06) but not in T2D myotubes which can explain their inability to increase palmitate beta-oxidation after palmitate overload. To determine whether the activation of the AMP activated protein kinase (AMPK)-ACC pathway was able to decrease lipid content in T2D myotubes, cells were treated with AICAR and metformin. These AMPK activators had no effect on ACC and AMPK phosphorylation in T2D myotubes as well as on lipid content, whereas AICAR, but not metformin, increased AMPK phosphorylation in control myotubes. Interestingly, metformin treatment and mitochondrial inhibition by antimycin induced increased lipid content in control myotubes. We conclude that T2D myotubes display an impaired capacity to respond to metabolic stimuli.     

5-Aminoimidazole-4-carboxamide riboside suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of phosphatidylinositol 3-kinase/Akt activation in RAW 264.7 murine macrophages

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.     

Adiponectin inhibits neutrophil apoptosis via activation of AMP kinase, PKB and ERK 1/2 MAP kinase

Neutrophils are abundant, short-lived leukocytes that play a key role in the immune defense against microbial infections. These cells die by apoptosis following activation and uptake of microbes and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Adiponectin exerts anti-inflammatory effects on neutrophil antimicrobial functions, but whether this abundant adipokine influences neutrophil apoptosis is unknown. Here we report that adiponectin in the physiological range (1–10 μg/ml) reduced apoptosis in resting neutrophils, decreasing caspase-3 cleavage and maintaining Mcl-1 expression by stabilizing this anti-apoptotic protein. We show that adiponectin induced phosphorylation of AMP-activated kinase (AMPK), protein kinase B (PKB), extracellular signal-regulated kinase (ERK 1/2) and p38 mitogen activated protein kinase (MAPK). Pharmacological inhibition of AMPK, PKB and ERK 1/2 ablated the pro-survival effects of adiponectin and treatment of neutrophils with an AMPK specific activator (AICAR) and AMPK inhibitor (compound C) respectively decreased and increased apoptosis. Finally, activation of AMPK by AICAR or adiponectin also decreased ceramide accumulation in the neutrophil cell membrane, a process involved in the early stages of spontaneous apoptosis, giving another possible mechanism downstream of AMPK activation for the inhibition of neutrophil apoptosis.     

alpha2 But not alpha1 AMP-activated protein kinase mediates oxidative stress-induced inhibition of retinal pigment epithelium cell phagocytosis of photoreceptor outer segments

Oxidative stress causes retinal pigment epithelium (RPE) cell dysfunction and is a major risk factor leading to the development of dry-type age-related macular degeneration. Taking pharmacological and genetic approaches, we address the mechanisms by which sublethal oxidative stress inhibits RPE cell phagocytosis. Sublethal oxidative stress dose-dependently inhibited RPE cell phagocytosis of photoreceptor outer segments (POS) and activated AMP-activated protein kinase (AMPK) as determined by increased Thr172 and Ser79 phosphorylation of AMPKalpha and its substrate acetyl-CoA carboxylase, respectively. Similar to oxidative stress, 5-aminoimidazole-4-carboxamide riboside (AICAR), a pharmacological activator of AMPK, inhibited RPE cell phagocytosis of POS in a dose-dependent manner. Inhibition of RPE cell phagocytosis by AICAR was fully reversed by blockade of AICAR translocation into cells by dipyridamole or inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor 5-iodotubercidin. In agreement, AICAR-induced activation of AMPK was abolished by preincubation with dipyridamole or 5-iodotubercidin. Knock-out experiments further revealed that alpha2 but not alpha1 AMPK was involved in RPE cell phagocytosis and that activation of alpha2 AMPK contributed to the inhibition of RPE cell phagocytosis by oxidative stress. Inhibition of RPE cell phagocytosis by activation of alpha2 AMPK was associated with a dramatic increase in acetyl-CoA carboxylase phosphorylation. In comparison, AMPK had no role in oxidative stress-induced breakdown of RPE barrier function. Taken together, reduction in POS load under oxidative stress might direct RPE cells to a self-protected status. Thus, activating AMPK could have therapeutic potential in treating dry macular degeneration.     

Activation of AMP-activated protein kinase inhibits protein synthesis associated with hypertrophy in the cardiac myocyte

A necessary mediator of cardiac myocyte enlargement is protein synthesis, which is controlled at the levels of both translation initiation and elongation. Eukaryotic elongation factor-2 (eEF2) mediates the translocation step of peptide-chain elongation and is inhibited through phosphorylation by eEF2 kinase. In addition, p70S6 kinase can regulate protein synthesis by phosphorylating eEF2 kinase or via phosphorylation of ribosomal protein S6. We have recently shown that eEF2 kinase is also controlled by phosphorylation by AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. Moreover, the mammalian target of rapamycin has also been shown to be inhibited, indirectly, by AMPK, thus leading to the inhibition of p70S6 kinase. Although AMPK activation has been shown to modulate protein synthesis, it is unknown whether AMPK could also be a regulator of cardiac hypertrophic growth. Therefore, we investigated the role of AMPK activation in regulating protein synthesis during both phenylephrine- and Akt-induced cardiac hypertrophy. Metformin and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside were used to activate AMPK in neonatal rat cardiac myocytes. Activation of AMPK significantly decreased protein synthesis induced by phenylephrine treatment or by expression of constitutively active Akt. Activation of AMPK also resulted in decreased p70S6 kinase phosphorylation and increased phosphorylation of eEF2, suggesting that inhibition of protein synthesis involves the eEF2 kinase/eEF2 axis and/or the p70S6 kinase pathway. Together, our data suggest that the inhibition of protein synthesis by pharmacological activation of AMPK may be a key regulatory mechanism by which hypertrophic growth can be controlled.