AMP-activated protein kinase in metabolic control and insulin signaling.
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The AMP-activated protein kinase (AMPK) system acts as a sensor of cellular energy status that is conserved in all eukaryotic cells. It is activated by increases in the cellular AMP:ATP ratio caused by metabolic stresses that either interfere with ATP production (eg, deprivation for glucose or oxygen) or that accelerate ATP consumption (eg, muscle contraction). Activation in response to increases in AMP involves phosphorylation by an upstream kinase, the tumor suppressor LKB1. In certain cells (eg, neurones, endothelial cells, and lymphocytes), AMPK can also be activated by a Ca(2+)-dependent and AMP-independent process involving phosphorylation by an alternate upstream kinase, CaMKKbeta. Once activated, AMPK switches on catabolic pathways that generate ATP, while switching off ATP-consuming processes such as biosynthesis and cell growth and proliferation. The AMPK complex contains 3 subunits, with the alpha subunit being catalytic, the beta subunit containing a glycogen-sensing domain, and the gamma subunits containing 2 regulatory sites that bind the activating and inhibitory nucleotides AMP and ATP. Although it may have evolved to respond to metabolic stress at the cellular level, hormones and cytokines such as insulin, leptin, and adiponectin can interact with the system, and it now appears to play a key role in maintaining energy balance at the whole body level. The AMPK system may be partly responsible for the health benefits of exercise and is the target for the antidiabetic drug metformin. It is a key player in the development of new treatments for obesity, type 2 diabetes, and the metabolic syndrome. (+info)
Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because alpha-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-beta (ACCbeta) Ser221 and immunoprecipitated alpha(1)-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated alpha(2)-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCbeta and alpha(1)-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 microM), the CaM-competitive inhibitor KN-93 (10 microM), or the SR Ca(2+) release blocking agent dantrolene (10 microM) all inhibited ACCbeta phosphorylation and alpha(1)-AMPK phosphorylation, suggesting that SR Ca(2+) release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca(2+)-activated CaMKK may control alpha(1)-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle. (+info)
AMP-activated protein kinase: a universal regulator of autophagy?
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Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests hat AMPK inhibits mTORC1 and autophagy also in nonstarved cells. Various Ca(2+) mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate MPK via activation of Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta), and his pathway is required for Ca(2+)-induced autophagy. Thus, we propose that an increase in free cytosolic Ca(2+) ([Ca(2+)](c)) induces autophagy via the CaMKK/beta-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected. (+info)
Upregulation of IL-6 mRNA by IL-6 in skeletal muscle cells: role of IL-6 mRNA stabilization and Ca2+-dependent mechanisms.
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Skeletal muscle cells have been established as significant producers of IL-6 during exercise. This IL-6 production is discussed as one possible mediator of the beneficial effects of physical activity on glucose and fatty acid metabolism. IL-6 itself could be the exercise-related factor that upregulates and maintains its own production. We investigated this hypothesis and the underlying molecular mechanism in cultured C(2)C(12) cells. IL-6 led to a rapid and prolonged increase in IL-6 mRNA, which was also found in human myotubes. Because IL-6 has been shown to activate AMP-activated kinase (AMPK), we studied whether, in turn, activated AMPK induces IL-6 expression. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside upregulated IL-6 mRNA expression, which was blocked by knockdown of AMPK alpha(1) and alpha(2) using small, interfering RNA (siRNA) oligonucleotides. However, the effect of IL-6 was shown to be independent of AMPK, since the siRNA approach silencing the AMPK alpha-subunits did not reduce the upregulation of IL-6 induced by IL-6 stimulation. The self-stimulatory effect of IL-6 partly involves a Ca(2+)-dependent pathway: IL-6 increased intracellular Ca(2+), and intracellular blockade of Ca(2+) with a Ca(2+) chelator reduced the IL-6-mediated increase in IL-6 mRNA levels. Moreover, inhibition of Ca(2+)/calmodulin-dependent kinase kinase with STO-609 or the siRNA approach decreased IL-6 mRNA levels of control and IL-6-stimulated cells. A major, STO-609-independent mechanism is the IL-6-mediated stabilization of its mRNA. The data suggest that IL-6 could act as autocrine factor upregulating its mRNA levels, thereby supporting its function as an exercise-activated factor in skeletal muscle cells. (+info)
Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by alpha-lipoic acid in C2C12 myotubes.
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alpha-Lipoic acid (ALA) widely exists in foods and is an antidiabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by the activation of AMP-activated protein kinase (AMPK) in skeletal muscle, but the underlying mechanism for AMPK activation is unknown. Here, we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. Incubation of C2C12 myotubes with 200 and 500 microM ALA increased the activity and phosphorylation of the AMPK alpha-subunit at Thr(172). Phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC), at Ser(79) was also increased. No difference in ATP, AMP, and the calculated AMP-to-ATP ratio was observed among the different treatment groups. Since the upstream AMPK kinase, LKB1, requires an alteration of the AMP-to-ATP ratio to activate AMPK, this data showed that LKB1 might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased the intracellular Ca(2+) concentration measured by fura-2 fluorescent microscopy (P < 0.05), showing that ALA may activate AMPK through enhancing Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) signaling. Indeed, chelation of intracellular free Ca(2+) by loading cells with 25 microM BAPTA-AM for 30 min abolished the ALA-induced activation of AMPK and, in turn, phosphorylation of ACC at Ser(79). Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA-stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser(79). In addition, ALA treatment increased the association of AMPK with CaMKK. To further show the role of CaMKK in AMPK activation, short interfering RNA was used to silence CaMKK, which abolished the ALA-induced AMPK activation. These data show that CaMKK is the kinase responsible for ALA-induced AMPK activation in C2C12 myotubes. (+info)
Transgenic mice expressing an inhibitory truncated form of p300 exhibit long-term memory deficits.
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The formation of many forms of long-term memory requires several molecular mechanisms including regulation of gene expression. The mechanisms directing transcription require not only activation of individual transcription factors but also recruitment of transcriptional coactivators. CBP and p300 are transcriptional coactivators that interact with a large number of transcription factors and regulate transcription through multiple mechanisms, including an intrinsic histone acetyltransferase (HAT) activity. HAT activity mediates acetylation of lysine residues on the amino-terminal tails of histone proteins, thereby increasing DNA accessibility for transcription factors to activate gene expression. CBP has been shown to play an important role in long-term memory formation. We have investigated whether p300 is also required for certain forms of memory. p300 shares a high degree of homology with CBP and has been shown to interact with transcription factors known to be critical for long-term memory formation. Here we demonstrate that conditional transgenic mice expressing an inhibitory truncated form of p300 (p300Delta1), which lacks the carboxy-terminal HAT and activation domains, have impaired long-term recognition memory and contextual fear memory. Thus, our study demonstrates that p300 is required for certain forms of memory and that the HAT and carboxy-terminal domains play a critical role. (+info)
Sex-dependent up-regulation of two splicing factors, Psf and Srp20, during hippocampal memory formation.
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Gene transcription is required for long-term memory (LTM) formation. LTM formation is impaired in a male-specific manner in mice lacking either of the two Ca(2+)/calmodulin-dependent kinase kinase (Camkk) genes. Since altered transcription was suggested to cause these impairments in LTM formation, we used microarrays to screen for CaMKKbeta-dependent gene expression changes. Here we show that the hippocampal mRNA expression of two splicing factors, splicing factor arginine/serine-rich 3 (Sfrs3/Srp20) and polypyrimidine tract-binding protein-associated splicing factor (Psf), is altered in CaMKKbeta-deficient males. In wild-type (WT) mice, the basal expression level in the hippocampus is higher in males than in females, and the sex difference in Srp20 expression is detectable before puberty. Training in two hippocampus-dependent learning tasks, the spatial version of the Morris water maze (MWM) and background contextual fear conditioning, increases the hippocampal mRNA expression of both splicing factors in WT males. However, the increase in Srp20 mRNA expression occurs only in males and not in females, whereas the up-regulation of Psf expression occurs in both sexes. Importantly, control experiments demonstrate that the up-regulation of both splicing factors is specific for the learned associations after contextual fear conditioning. In summary, we provide the first evidence for a regulation of splicing factors during LTM formation and we suggest that alternative splicing contributes to sex differences in LTM formation. (+info)
Activity-dependent synaptogenesis: regulation by a CaM-kinase kinase/CaM-kinase I/betaPIX signaling complex.
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