AMP-activated Protein Kinase Catalytic Subunit Research Articles

The active form of the metabolic sensor AMP-activated protein kinase α (AMPKα) directly binds the mitotic apparatus and travels from centrosomes to the spindle midzone during mitosis and cytokinesis

The metabolic rheostat AMP-activated protein kinase (AMPK) is unexpectedly required for proper cell division and faithful chromosomal segregation during mitosis. Although it is conceptually attractive to assume that AMPK-interpreted microenvironmental bioenergetics may strictly engage cell’s energy status, cell grow, and cell division to avoid that energy stresses trigger cell death, the ultimate framework of AMPK activity towards chromosomal and cytoskeletal mitotic regulation is a question that remains unanswered. We herein reveal that the active form of the α-catalytic AMPK subunit (P-AMPKαThr172) -but not its total form (AMPKα)- transiently associates with several mitotic structures including centrosomes, spindle poles, the central spindle midzone and the midbody throughout all of the mitotic stages and cytokinesis in human cancer-derived epithelial cells. At prophase, P-AMPKαThr172 associates with the two asters of microtubules that begin to nucleate from mature centrosomes. The overlapping localization of P-AMPKαThr172 with the mitotic centrosomal Aurora-A kinase is also apparent on the microtubules near the spindle poles in metaphase and in early anaphase. This Aurora A-like centrosomal localization of P-AMPKαThr172 cannot be detected following chromatid separation following anaphase-telophase transition. Rather, toward the end of anaphase and in telophase P-AMPKαThr172 reactivity exhibited a similar but not identical localization to that occupied by the bona fide chromosomal passenger proteins INCENCP and Aurora-B. This localization of P-AMPKαThr172 at the central spindle and midbody persisted during the furrowing process and, at the completion of telophase, a prominent staining of P-AMPKαThr172 as doublet was apparent on either side of the midbody within the intercellular cytokinetic bridge. An identical mitotic geography of P-AMPKαThr172 was observed in cancer cells lacking the AMPK kinase LKB1, in non-cancerous human epithelial cells, and in mouse fibroblasts. The active form of AMPKα bound to the mitotic apparatus may physically tether the bioenergetic state of a cell to the four-dimensional regulation of the chromosomal and cytoskeletal mitotic events, thus suggesting a putative cytokinetic suppressor function. In this newly discovered scenario, we suggest a primordial mitotic role for the α catalytic AMPK subunit in the eukaryotic evolutionary process as it may ensure, at the cell level, an exquisite coordination between sensing of energy resources and the fundamental biological process of genome division.

A Crystallized View of AMPK Activation

AMP-activated protein kinase (AMPK) is a key metabolic regulator. Recent work (Chen et al., 2009) elucidates the structural interaction between the autoinhibitory sequence and the kinase domain of the AMPK catalytic subunit. Enhanced understanding of the molecular mechanics of AMPK activation might lead to novel therapeutic approaches.

Schizosaccharomyces pombe cell division cycle under limited glucose requires Ssp1 kinase, the putative CaMKK, and Sds23, a PP2A‐related phosphatase inhibitor

Calcium/calmodulin-dependent protein kinase (CaMK) is required for diverse cellular functions, and similar kinases exist in fungi. Although mammalian CaMK kinase (CaMKK) activates CaMK and also evolutionarily-conserved AMP-activated protein kinase (AMPK), CaMKK is yet to be established in yeast. We here report that the fission yeast Schizosaccharomyces pombe Ssp1 kinase, which controls G2/M transition and response to stress, is the putative CaMKK. Ssp1 has a CaM binding domain (CBD) and associates with 14-3-3 proteins as mammalian CaMKK does. Temperature-sensitive ssp1 mutants isolated are defective in the tolerance to limited glucose, and this tolerance requires the conserved stretch present between the kinase domain and CBD. Sds23, multi-copy suppressor for mutants defective in type 1 phosphatase and APC/cyclosome, also suppresses the ssp1 phenotype, and is required for the tolerance to limited glucose. We demonstrate that Sds23 binds to type 2A protein phosphatases (PP2A) and PP2A-related phosphatase Ppe1, and that Sds23 inhibits Ppe1 phosphatase activity. Ssp1 and Ppe1 thus seem to antagonize in utilizing limited glucose. We also show that Ppk9 and Ssp2 are the catalytic subunits of AMPK and AMPK-related kinases, respectively, which bind to common beta-(Amk2) and gamma-(Cbs2) subunits.

AMP-activated protein kinase promotes human prostate cancer cell growth and survival

The molecular mechanisms underlying the development and progression of prostate cancer are poorly understood. AMP-activated protein kinase (AMPK) is a serine-threonine kinase that is activated in response to the hypoxic conditions found in human prostate cancers. In response to energy depletion, AMPK activation promotes metabolic changes to maintain cell proliferation and survival. Here, we report prevalent activation of AMPK in human prostate cancers and provide evidence that inhibition or depletion of AMPK leads to decreased cell proliferation and increased cell death. AMPK was highly activated in 40% of human prostate cancer specimens examined. Endogenous AMPK was active in both the androgen-sensitive LNCaP cells and the androgen-independent CWR22Rv1 human prostate cancer cells. Depletion of AMPK catalytic subunits by small interfering RNA or inhibition of AMPK activity with a small-molecule AMPK inhibitor (compound C) suppresses human prostate cancer cell proliferation. Apoptotic cell death was induced in LNCaP and CWR22Rv1 cells at compound C concentrations that inhibited AMPK activity. The evidence provided here is the first report that the activated AMPK pathway is involved in the growth and survival of human prostate cancer and offers novel potential targets for chemoprevention of human prostate cancer.

Brought in by force: AMPK, TBC1D1, and contraction-stimulated glucose transport in skeletal muscle

transient intracellular calcium oscillations were the first signaling mediator described to regulate contraction-stimulated glucose transport in skeletal muscle ([6][1]). Thirty years later, the discoveries, that 5′-AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-β-

Optimizing Dietary Restriction for Genetic Epistasis Analysis and Gene Discovery in C. elegans

Dietary restriction (DR) increases mammalian lifespan and decreases susceptibility to many age-related diseases. Lifespan extension due to DR is conserved across a wide range of species. Recent research has focused upon genetically tractable model organisms such as C. elegans to uncover the genetic mechanisms that regulate the response to DR, in the hope that this information will provide insight into the mammalian response and yield potential therapeutic targets. However, no consensus exists as to the best protocol to apply DR to C. elegans and potential key regulators of DR are protocol-specific. Here we define a DR method that better fulfills criteria required for an invertebrate DR protocol to mirror mammalian studies. The food intake that maximizes longevity varies for different genotypes and informative epistasis analysis with another intervention is only achievable at this ‘optimal DR’ level. Importantly therefore, the degree of restriction imposed using our method can easily be adjusted to determine the genotype-specific optimum DR level. We used this protocol to test two previously identified master regulators of DR in the worm. In contrast to previous reports, we find that DR can robustly extend the lifespan of worms lacking the AMP-activated protein kinase catalytic subunit AAK2 or the histone deacetylase SIR-2.1, highlighting the importance of first optimizing DR to identify universal regulators of DR mediated longevity.

A newly synthetic chromium complex—Chromium ( d-phenylalanine) 3 activates AMP-activated protein kinase and stimulates glucose transport

We synthesized the chromium (phenylalanine) 3 [Cr( d-phe) 3] by chelating chromium(III) with d-phenylalanine ligand in aqueous solution to improve the bioavailability of chromium, and reported that Cr( d-phe) 3 improved insulin sensitivity. AMP-activated protein kinase (AMPK) is a key mediator for glucose uptake and insulin sensitivity. To address the molecular mechanisms by which Cr( d-phe) 3 increases insulin sensitivity, we investigated whether Cr( d-phe) 3 stimulates glucose uptake via activation of AMPK signaling pathway. H9c2 myoblasts and isolated cardiomyocytes were treated with Cr( d-phe) 3 (25 μM). Western blotting was used for signaling determination. The glucose uptake was determined by 2-deoxy- d-glucose- 3H accumulation. HPLC measured concentrations of AMP. The mitochondrial membrane potential (Δ ψ) was detected by JC-1 fluorescence assay. Cr( d-phe) 3 stimulated the phosphorylation of α catalytic subunit of AMPK at Thr 172, as well the downstream targets of AMPK, acetyl-CoA carboxylase (ACC, Ser 212) and eNOS (Ser 1177). Moreover, Cr( d-phe) 3 significantly stimulated glucose uptake in both H9c2 cells and cardiomyocytes. AMPK inhibitor compound C (10 μM) dramatically inhibited the glucose uptake stimulated by Cr( d-phe) 3, while it did not affect insulin stimulation of glucose uptake. Furthermore, in vivo studies showed that Cr( d-phe) 3 also activated cardiac AMPK signaling pathway. The increase of cardiac AMP concentration and the decrease of mitochondrial membrane potential (Δ ψ) may contribute to the activation of AMPK induced by Cr( d-phe) 3. Cr( d-phe) 3 is a novel compound that activates AMPK signaling pathway, which contributes to the regulation of glucose transport during stress conditions that may be associated the role of AMPK in increasing insulin sensitivity.

The Caenorhabditis elegans AMP-activated Protein Kinase AAK-2 Is Phosphorylated by LKB1 and Is Required for Resistance to Oxidative Stress and for Normal Motility and Foraging Behavior

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.

Downregulation of Cellular Energy Sensor during Disease condition results in Alteration of Lipid Homeostasis in Immune Cells

AMP‐activated protein kinase (AMPK), an energy sensing metabolic switch of mammalian cells, was observed to be down regulated during T cells mediated autoimmune disease. A catalytic subunit of AMPK, alpha1, was predominantly expressed in T‐ and antigen presenting cells. During EAE disease, activity of AMPK was significantly downregulated in thymocytes, spleen cells and brain. Decreased AMPK activity results increased lipid biosynthesis and their content in total T cells, adherent and non‐adherent spleen cells at disease state. Increased lipids biosynthesis leads to the higher proliferative state of these cells was evident from active form of Akt, p42/44 and reduced levels of p21 and p27. AMPK activators restored lipid alteration in total spleen cells by reversing the AMPK activity and inhibited production of pro‐inflammatory cytokines under in vitro stimulation. All together, our results demonstrated that downregulation of AMPK‐mediated altered lipid metabolism in immune cells, resulted an inflammatory environment may be one of the critical aspects of pathogenesis during EAE or Multiple Sclerosis and identified AMPK as a novel therapeutic target for autoimmune diseases.Grants support: Mainly by RG 3810‐A‐1 and PP1283 from the MS Society (SG) and in part by (NS‐22576, NS‐34741, NS‐37766 and NS‐40810) from NIH (IS).

Activation of AMP-activated Protein Kinase Induces p53-dependent Apoptotic Cell Death in Response to Energetic Stress

Tumor suppressor p53-dependent stress response pathways play an important role in cell fate determination. In this study, we have found that glucose depletion promotes the phosphorylation of AMP-activated protein kinase catalytic subunit alpha (AMPKalpha) in association with a significant up-regulation of p53, thereby inducing p53-dependent apoptosis in vivo and in vitro. Thymocytes prepared from glucose-depleted wild-type mice but not from p53-deficient mice underwent apoptosis, which was accompanied by a remarkable phosphorylation of AMPKalpha and a significant induction of p53 as well as pro-apoptotic Bax. Similar results were also obtained in human osteosarcoma-derived U2OS cells bearing wild-type p53 following glucose starvation. Of note, glucose deprivation led to a significant accumulation of p53 phosphorylated at Ser-46, but not at Ser-15 and Ser-20, and a transcriptional induction of p53 as well as proapoptotic p53 AIP1. Small interference RNA-mediated knockdown of p53 caused an inhibition of apoptosis following glucose depletion. Additionally, apoptosis triggered by glucose deprivation was markedly impaired by small interference RNA-mediated depletion of AMPKalpha. Under our experimental conditions, down-regulation of AMPKalpha caused an attenuation of p53 accumulation and its phosphorylation at Ser-46. In support of these observations, enforced expression of AMPKalpha led to apoptosis and resulted in an induction of p53 at protein and mRNA levels. Furthermore, p53 promoter region responded to AMPKalpha and glucose deprivation as judged by luciferase reporter assay. Taken together, our present findings suggest that AMPK-dependent transcriptional induction and phosphorylation of p53 at Ser-46 play a crucial role in the induction of apoptosis under carbon source depletion.

95 MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1) IS REQUIRED FOR THE DEVELOPMENT OF INFLAMMATION, OXIDATIVE DAMAGE AND FIBROSIS IN MURINE STEATOHEPATITIS

as treatments for steatohepatitis. The mechanisms implicated remain poorly understood. Activation of PPARg induces expression of adiponectin, an anti-inflammatory and insulin-sensitising adipocytokine. Upon binding to specific receptors on liver cells, adiponectin activates the AMP-activated protein kinase (AMPK), stimulates b-oxidation and represses hepatic de novo lipogenesis. Also, adiponectin, through PPARa, activates fat oxidation and inhibits inflammatory reaction. Here we tested the hypothesis that the beneficial effect of PGZ on steatohepatitis results from reduced hepatic fat accumulation due to adiponectin-dependent stimulation of AMPK and PPARa. Methods: Mice lacking adiponectin (adipo−/−), the AMPK catalytic subunit alpha1 (AMPKa1−/−) and their respective littermates were fed a methionine and choline (MCD) deficient diet, supplemented or not with pioglitazone 0.01%. Histopathology, hepatic lipid content, serum adiponectin levels were assessed. In liver tissues, we evaluated AMPK (total, a1, a2) activities, proteins and mRNA levels, PPARa mRNA and the expression of downstream targets of AMPK or PPARa. Results: In wt mice, PGZ reduces the severity of MCD diet-induced steatohepatitis, while it increased circulating levels of adiponectin. By contrast, in adiponectin−/− mice, PGZ did not alter the severity of steatohepatitis. AMPKa1−/− mice and their littermates were genetically less sensitive to dietary steatohepatitis than C57 mice. In AMPKa1−/− mice fed the MCD diet, PGZ did not reduce the severity of steatohepatitis, although it significantly increased serum adiponectin levels. In wt mice, PGZ almost completely repressed the expression and activation of SREBP1c, a key transcription factor for de novo lipogenesis and downstream target of AMPK. This effect of PGZ was lacking in adiponectin−/− and in AMPKa1−/− mice. In the strains analysed, PGZ did not alter the expression of PPARa or of its downstream targets implicated in the regulation of lipid oxidation. In conclusion, we showed that the preventive effect of PGZ on MCD-induced steatohepatitis is dependent on the up-regulation of adiponectin. While the adiponectin-PPARa-boxidation axis does not appear to be implicated, our data strongly suggest that adiponectin produces its effect by controlling the expression and activity of SREBP-1c via the activation of the AMPK complex.

Lack of starvation-induced activation of AMP-activated protein kinase in the hypothalamus of the Lou/C rats resistant to obesity

The AMP-activated protein kinase (AMPK) is involved in the control of food intake by the hypothalamus. The aim of this work was to investigate if modification of hypothalamic AMPK regulation could be related to the spontaneous food restriction of Lou/C rats, a strain resistant to obesity exhibiting a 40% reduction in caloric intake compared with their lean Wistar counterparts. Three-month-old male Lou/C rats were compared with age-matched male Wistar rats in both fed ad libitum and 24-h food deprivation state. We first confirmed that starvation activated both isoforms of AMPK catalytic alpha subunits and enhanced the phosphorylation state of its downstream targets acetyl-CoA carboxylase and elongation factor 2 in the hypothalamus of Wistar rats. These changes were not observed in the hypothalamus of Lou/C rats. Interestingly, the starvation-induced changes in hypothalamic mRNA levels of the main orexigenic and anorexigenic neuropeptides were also blunted in the Lou/C rats. Analysis of the concentrations of circulating substrates and hormones known to regulate hypothalamic AMPK indicated that the starvation-induced changes in ghrelin, adiponectin and leptin were not observed in Lou/C rats. Furthermore, an increased phosphorylation state of signal transducer and activator of transcription 3 (STAT3), which admittedly mediates leptin signaling, was evidenced in the hypothalamus of the starved Lou/C rats, as well as modifications of expression of the leptin-sensitive genes suppressor of cytokine signaling-3 and stearoyl-coenzyme A desaturase 1. In addition, despite reduced leptin level in fed Lou/C rats, the phosphorylation state of hypothalamic STAT3 remained similar to that found in fed Wistar rats, an adaptation that could be explained by the concomitant increase in ObRb leptin receptor mRNA expression. Activation of hypothalamic AMPK by starvation, which stimulates food intake through changes in (an)orexigenic neuropeptides in the normal rats, was not observed in the spontaneously hypophagic Lou/C rats.

AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside

AICA riboside (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in P(i) (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both alpha1 and alpha2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and P(i). ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and P(i). We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular P(i) depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution.

Stimulation of AMP-Activated Protein Kinase Is Essential for the Induction of Drug Metabolizing Enzymes by Phenobarbital in Human and Mouse Liver

Our previous studies have suggested a role for AMP-activated protein kinase (AMPK) in the induction of CYP2B6 by phenobarbital (PB) in hepatoma-derived cells (Rencurel et al., 2005). In this study, we showed in primary human hepatocytes that: 1) 5'-phosphoribosyl-5-aminoimidazol-4-carboxamide 1-beta-d-ribofuranoside and the biguanide metformin, known activators of AMPK, dose-dependently increase the expression of CYP2B6 and CYP3A4 to an extent similar to that of PB. 2) PB, but not the human nuclear receptor constitutive active/androstane receptor (CAR) ligand 6-(4-chlorophenyl)imidazol[2,1-6][1,3]thiazole-5-carbaldehyde, dose-dependently increase AMPK activity. 3) Pharmacological inhibition of AMPK activity with compound C or dominant-negative forms of AMPK blunt the inductive response to phenobarbital. Furthermore, in transgenic mice with a liver-specific deletion of both the alpha1 and alpha2 AMPK catalytic subunits, basal levels of Cyp2b10 and Cyp3a11 mRNA were increased but not in primary culture of mouse hepatocytes. However, phenobarbital or 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene, a mouse CAR ligand, failed to induce the expression of these genes in the liver or cultured hepatocytes from mice lacking hepatic expression of the alpha1 and alpha2 subunits of AMPK. The distribution of CAR between the nucleus and cytosol was not altered in hepatocytes from mice lacking both AMPK catalytic subunits. These data highlight the essential role of AMPK in the CAR-mediated signal transduction pathway.

AMPK-Mediated AS160 Phosphorylation in Skeletal Muscle Is Dependent on AMPK Catalytic and Regulatory Subunits

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 beta-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (alpha1beta1gamma1 and alpha2beta2gamma1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (alpha2 AMPK knockout [KO], alpha2 AMPK kinase dead [KD], and gamma3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing alpha2 and gamma3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in alpha2 AMPK KO and KD but not gamma3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.

A Polymorphism in the AMPKα2 Subunit Gene Is Associated With Insulin Resistance and Type 2 Diabetes in the Japanese Population

AMP-activated protein kinase (AMPK) acts as a fuel gauge for glucose and lipid metabolism. The gene encoding the α2 isoform of the catalytic subunit of AMPK (PRKAA2) is located at one of the Japanese type 2 diabetes loci mapped by our previous genome scan (1p36-32). PRKAA2 is, therefore, a good candidate gene for insulin resistance and type 2 diabetes. We screened all nine exons, their exon-intron boundaries, and the 5′ and 3′ flanking regions of PRKAA2 to identify single nucleotide polymorphisms (SNPs), and we genotyped 192 type 2 diabetic patients and 272 nondiabetic subjects to assess possible associations between genotypes or haplotypes and type 2 diabetes. None of the 10 SNPs genotyped was associated with type 2 diabetes, but the haplotype analysis, consisting of six representative SNPs, revealed one haplotype, with the A (minor) allele for rs2051040 and a major allele for the other five SNPs, to be associated with type 2 diabetes (P = 0.009). This finding was confirmed in two larger replication samples (657 case and 360 control subjects, P = 0.021; and 356 case and 192 control subjects from the same area in Japan, P = 0.007) and a significant P value was obtained in the joint haplotype analysis of all samples (1,205 case and 824 control subjects, P = 0.0001). Furthermore, insulin resistance was associated with rs2051040 in nondiabetic subjects, and those with the A (minor) allele had a higher homeostasis model assessment of insulin resistance index than those who did not (initial control subjects [n = 272], P = 0.002; and joint replication control subjects [n = 552], P = 0.037). We speculate that the PRKAA2 gene influences insulin resistance and susceptibility to type 2 diabetes in the Japanese population.

5-Aminoimidazole-4-Carboxamide-1-β-d-Ribofuranoside and Metformin Inhibit Hepatic Glucose Phosphorylation by an AMP-Activated Protein Kinase–Independent Effect on Glucokinase Translocation

AMP-activated protein kinase (AMPK) controls glucose uptake and glycolysis in muscle. Little is known about its role in liver glucose uptake, which is controlled by glucokinase. We report here that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), metformin, and oligomycin activated AMPK and inhibited glucose phosphorylation and glycolysis in rat hepatocytes. In vitro experiments demonstrated that this inhibition was not due to direct phosphorylation of glucokinase or its regulatory protein by AMPK. By contrast, AMPK phosphorylated liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase without affecting activity. Inhibitors of the endothelial nitric oxide synthase, stress kinases, and phosphatidylinositol 3-kinase pathways did not counteract the effects of AICAR, metformin, or oligomycin, suggesting that these signaling pathways were not involved. Interestingly, the inhibitory effect on glucose phosphorylation of these well-known AMPK activators persisted in primary cultured hepatocytes from newly engineered mice lacking both liver alpha1 and alpha2 AMPK catalytic subunits, demonstrating that this effect was clearly not mediated by AMPK. Finally, AICAR, metformin, and oligomycin were found to inhibit the glucose-induced translocation of glucokinase from the nucleus to the cytosol by a mechanism that could be related to the decrease in intracellular ATP concentrations observed in these conditions.

AMP-activated Protein Kinase α2 Activity Is Not Essential for Contraction- and Hyperosmolarity-induced Glucose Transport in Skeletal Muscle

To examine the role of AMP-activated protein kinase (AMPK) in muscle glucose transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive alpha2 (alpha2i TG) and alpha1 (alpha1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In alpha2i TG mice basal alpha2 activity was barely detectable, whereas basal alpha1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK alpha2 activity in alpha2i TG mice, whereas alpha1 activation was attenuated by only 30-50%. Glucose transport was measured in vitro using isolated EDL muscles from alpha2i TG mice. AICAR- and rotenone-stimulated glucose transport was fully inhibited in alpha2i TG mice; however, the lack of AMPK alpha2 activity had no effect on contraction- or sorbitol-induced glucose transport. Similar to these observations in vitro, contraction-stimulated glucose transport, assessed in vivo by 2-deoxy-d-[(3)H]glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in alpha2i TG mice. Thus, AMPK alpha2 activation is essential for some, but not all, insulin-independent glucose transport. Muscle contraction- and hyperosmolarity-induced glucose transport may be regulated by a redundant mechanism in which AMPK alpha2 is one of multiple signaling pathways.

Phenformin and 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) activation of AMP‐activated protein kinase inhibits transepithelial Na+ transport across H441 lung cells

Active re-absorption of Na+ across the alveolar epithelium is essential to maintain lung fluid balance. Na+ entry at the luminal membrane is predominantly via the amiloride-sensitive Na+ channel (ENaC) down its electrochemical gradient. This gradient is generated and maintained by basolateral Na+ extrusion via Na+,K+-ATPase an energy-dependent process. Several kinases and factors that activate them are known to regulate these processes; however, the role of AMP-activated protein kinase (AMPK) in the lung is unknown. AMPK is an ultra-sensitive cellular energy sensor that monitors energy consumption and down-regulates ATP-consuming processes when activated. The biguanide phenformin has been shown to independently decrease ion transport processes, influence cellular metabolism and activate AMPK. The AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) also activates AMPK in intact cells. Western blotting revealed that both the alpha1 and alpha2 catalytic subunits of AMPK are present in Na+ transporting H441 human lung epithelial cells. Phenformin and AICAR increased AMPK activity in H441 cells in a dose-dependent fashion, stimulating the kinase maximally at 5-10 mm (P = 0.001, n = 3) and 2 mm (P < 0.005, n = 3), respectively. Both agents significantly decreased basal ion transport (measured as short circuit current) across H441 monolayers by approximately 50% compared with that of controls (P < 0.05, n = 4). Neither treatment altered the resistance of the monolayers. Phenformin and AICAR significantly reduced amiloride-sensitive transepithelial Na+ transport compared with controls (P < 0.05, n = 4). This was a result of both decreased Na+,K+-ATPase activity and amiloride-sensitive apical Na+ conductance. Transepithelial Na+ transport decreased with increasing concentrations of phenformin (0.1-10 mm) and showed a significant correlation with AMPK activity. Taken together, these results show that phenformin and AICAR suppress amiloride-sensitive Na+ transport across H441 cells via a pathway that includes activation of AMPK and inhibition of both apical Na+ entry through ENaC and basolateral Na+ extrusion via the Na+,K+-ATPase. These are the first studies to provide a cellular signalling mechanism for the action of phenformin on ion transport processes, and also the first studies showing AMPK as a regulator of Na+ absorption in the lung.

AMP-activated protein kinase: balancing the scales

AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that plays a key role in the regulation of energy control. AMPK is activated in response to an increase in the ratio of AMP:ATP within the cell. Activation requires phosphorylation of threonine 172 within the catalytic subunit of AMPK by an upstream kinase. The identity of the upstream kinase in the cascade remained frustratingly elusive for many years, but was recently identified as LKB1, a kinase that is inactivated in a rare hereditary form of cancer called Peutz–Jeghers syndrome. Once activated, AMPK initiates a series of responses that are aimed at restoring the energy balance within the cell. ATP-consuming, anabolic pathways, such as fatty acid synthesis and protein synthesis are switched-off, whereas ATP-generating, catabolic pathways, such as fatty acid oxidation and glycolysis, are switched-on. More recent studies have indicated, that AMPK plays an important role in the regulation of whole body energy metabolism. The adipocyte-derived hormones, leptin and adiponectin, activate AMPK in peripheral tissues, including skeletal muscle and liver, increasing energy expenditure. In the hypothalamus, AMPK is inhibited by leptin and insulin, hormones which suppress feeding, whilst ghrelin, a hormone that increases food intake, activates AMPK. Furthermore, direct pharmacological activation of AMPK in the hypothalamus by 5-aminoimidazole-4-carboxamide ribose increases food intake in rats, demonstrating that AMPK plays a direct role in the regulation of feeding. Taken together these findings indicate that AMPK has a pivotal role in regulating pathways that control both energy expenditure and energy intake.