Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes.
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5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKalpha2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKalpha2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKalpha2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle. (+info)
Effect of fiber type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat muscle.
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AMP-activated protein kinase (AMPK) may mediate the stimulatory effect of contraction and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on glucose transport in skeletal muscle. In muscles with different fiber type composition from fasted rats, AICAR increased 2-deoxyglucose transport and total AMPK activity approximately twofold in epitrochlearis (EPI), less in flexor digitorum brevis, and not at all in soleus muscles. Contraction increased both transport and AMPK activity more than AICAR did. In EPI muscles, the effects of AICAR and contractions on glucose transport were partially additive despite a lower AMPK activity with AICAR compared with contraction alone. In EPI from fed rats, glucose transport responses were smaller than what was seen in fasted rats, and AICAR did not increase transport despite an increase in AMPK activity. AICAR and contraction activated both alpha(1)- and alpha(2)-isoforms of AMPK. Expression of both isoforms varied with fiber types, and alpha(2) was highly expressed in nuclei. In conclusion, AICAR-stimulated glucose transport varies with muscle fiber type and nutritional state. AMPK is unlikely to be the sole mediator of contraction-stimulated glucose transport. (+info)
Activation of GLUT1 by metabolic and osmotic stress: potential involvement of AMP-activated protein kinase (AMPK).
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In the rat liver epithelial cell line Clone 9, the V(max) for glucose uptake is acutely increased by inhibition of oxidative phosphorylation and by osmotic stress. By using a membrane-impermeant photoaffinity labelling reagent together with an isoform-specific antibody, we have, for the first time, provided direct evidence for the involvement of the GLUT1 glucose transporter isoform in this response. Transport stimulation was found to be associated with enhanced accessibility of GLUT1 to its substrate and with photolabelling of formerly 'cryptic' exofacial substrate binding sites in GLUT1 molecules. The total amount of cell surface GLUT1 remained constant. The precise mechanism for this binding site 'unmasking' is unclear but appears to involve AMP-activated protein kinase: in the current study, osmotic and metabolic stresses were found to result in activation of the alpha 1 isoform of AMP-activated protein kinase, and transport stimulation could be mimicked both by 5-aminoimidazole-4-carboxamide ribonucleoside and by infection of cells with a recombinant adenovirus encoding constitutively active AMP-activated protein kinase. The effect of 5-aminoimidazole-4-carboxamide ribonucleoside, as for metabolic stress, was on the V(max) rather than on the K(m) for transport and did not affect the cell-surface concentration of GLUT1. The relevant downstream target(s) of AMP-activated protein kinase have not yet been identified, but stimulation of transport by inhibition of oxidative phosphorylation or by 5-aminoimidazole-4-carboxamide ribonucleoside was not prevented by either inhibitors of conventional and novel protein kinase C isoforms or inhibitors of nitric oxide synthase. These enzymes, which have been implicated in stress-regulated pathways in other cell types, are therefore unlikely to play a role in transport regulation by stress in Clone 9 cells. (+info)
The fixM flavoprotein modulates inhibition by AICAR or 5'AMP of respiratory and nitrogen fixation gene expression in Sinorhizobium meliloti.
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AICAR, a purine-related metabolite, was recently shown to inhibit respiratory and nifA gene expression in Sino-rhizobium meliloti. Here, we demonstrate that AICAR has essentially no or little effect in a wild-type S. meliloti strain and inhibits respiratory and nitrogen fixation gene expression only in specific mutant backgrounds. We have analyzed in detail a mutant in which addition of AICAR inhibited fixK,fixN,fixT and nifA expression. The corresponding gene,fixM, is located just downstream of fixK1 on pSymA megaplasmid and encodes a flavoprotein oxidoreductase. 5'AMP, a structural analogue of AICAR, mimicked AICAR effect as well as the nucleoside precursors AICAriboside and adenosine. The mode of action of AICAR and 5'AMP in vivo was investigated. We demonstrate that AICAR does not affect FixK transcriptional activity and instead regulates fixK and nifA gene expression. We hypothesize that AICAR and 5'AMP may modulate, possibly indirectly, the activity of the FixLJ two-component regulatory system. The possible physiological roles of AICAR, 5'AMP, and fixM in the context of symbiosis are discussed. (+info)
Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome.
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The insulin resistance syndrome is characterized by several risk factors for cardiovascular disease. Chronic chemical activation of AMP-activated protein kinase by the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta -D-ribofuranoside (AICAR) has been shown to augment insulin action, upregulate mitochondrial enzymes in skeletal muscles, and decrease the content of intra-abdominal fat. Furthermore, acute AICAR exposure has been found to reduce sterol and fatty acid synthesis in rat hepatocytes incubated in vitro as well as suppress endogenous glucose production in rats under euglycemic clamp conditions. To investigate whether chronic AICAR administration, in addition to the beneficial effects on insulin sensitivity, is capable of improving other phenotypes associated with the insulin resistance syndrome, obese Zucker (fa/fa) rats (n = 6) exhibiting insulin resistance, hyperlipidemia, and hypertension were subcutaneously injected with AICAR (0.5 mg/g body wt) daily for 7 weeks. Obese control rats were either pair-fed (PF) (n = 6) or ad libitum-fed (AL) (n = 6). Lean Zucker rats (fa/-) (n = 8) served as a reference group. AICAR administration significantly reduced plasma triglyceride levels (P < 0.01 for AICAR vs. AL, and P = 0.05 for AICAR vs. PF) and free fatty acids (P < 0.01 for AICAR vs. AL, and P < 0.05 for AICAR vs. PF) and increased HDL cholesterol levels (P < 0.01 for AICAR vs. AL and PF). AICAR treatment also lowered systolic blood pressure by 14.6 +/- 4.3 mmHg (P < 0.05), and AICAR-treated animals exhibited a tendency toward decreased intra-abdominal fat content. Furthermore, AICAR administration normalized the oral glucose tolerance test and decreased fasting concentrations of glucose and insulin close to the level of the lean animals. Finally, in line with previous findings, AICAR treatment was also found to enhance GLUT4 protein expression and to increase maximally insulin-stimulated glucose transport in primarily white fast-twitch muscles. Our data provide strong evidence that long-term administration of AICAR improves glucose tolerance, improves the lipid profile, and reduces systolic blood pressure in an insulin-resistant animal model. The present study gives additional support to the hypothesis that AMPK activation might be a potential future pharmacological strategy for treating the insulin resistance syndrome. (+info)
Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes.
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Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl-CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAr) or oligomycin, an inhibitor of mitochondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase-dependent mechanism but did not modify AMPK activation. Conversely, the amino-acid-induced activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK activation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino-acid-induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b) both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy-consuming biosynthetic pathways. (+info)
Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis.
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Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis. (+info)
Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells.
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The aim of this work was to study the effect of a sustained activation of AMP-activated protein kinase (AMPK) on liver cell survival. AMPK activation was achieved by incubating FTO2B cells with AICA-riboside, which is transformed into ZMP, an AMP analogue, or by adenoviral transfection of hepatocytes with a constitutively active form of AMPK. Prolonged AMPK activation triggered apoptosis and activated c-Jun N-terminal kinase (JNK) and caspase-3. Experiments with iodotubercidin, dicoumarol and z-VAD-fmk, which inhibited AMPK, JNK and caspase activation, respectively, supported the notion that prolonged AMPK activation in liver cells induces apoptosis through an activation pathway that involves JNK and caspase-3. (+info)