1. The actions of serotonin (5-HT) on pyramidal cells of the CA1 region of the rat hippocampus were characterized using intracellular recording in in vitro brain slices. 2. 5-HT typically evokes a biphasic response consisting of a hyperpolarization which is followed by a longer-lasting depolarization. These effects on membrane potential are accompanied by a decrease in the calcium-activated after-hyperpolarization (a.h.p). 3. Detailed analysis using 5-HT antagonists and agonists indicates that the hyperpolarization is mediated by a 5-HT1A receptor. Spiperone is the most effective antagonist of the response and the selective 5-HT1A agonist, 8-OHDPAT, behaves as a partial agonist at this receptor. In agreement with the distribution of 5-HT1A binding sites, responses to 5-HT were most prominent in the stratum radiatum. 4. The hyperpolarizing response is associated with a decrease in input resistance, is blocked by extracellular barium and intracellular caesium, is unaffected by the chloride gradient, and its reversal potential shifts with the extracellular concentration of potassium as predicted for a response mediated by a selective increase in potassium permeability. 5. The depolarizing response and reduction in the a.h.p. could be studied in isolation by blocking the hyperpolarizing response with either pertussis toxin or spiperone. The pharmacology of these responses did not correspond to that of any of the 5-HT binding sites reported in C.N.S. tissue. Although the depolarization and blockade of the a.h.p. have the same time course it is unclear if they are mediated by the same or different receptors. 6. The depolarization most likely results from a decrease in resting potassium conductance. However, neither a blockade of the M current nor the a.h.p. current can account for the depolarization. 7. Blockade of phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX) did not enhance the depressant action of 5-HT on the a.h.p., making it unlikely that this action is mediated by cyclic AMP. 8. Blockade of the a.h.p. by 5-HT reduces spike frequency adaptation and counteracts the inhibitory action of 5-HT on 5-HT1A receptors. This excitatory action outlasts the hyperpolarizing action. 9. In summary 5-HT acts on at least two distinct receptors on hippocampal pyramidal cells, one coupled to the opening of potassium channels and a second coupled to a decrease in a resting potassium conductance and a decrease in the a.h.p.

AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKalpha2 (AMPKalpha2-CA). Hepatic AMPKalpha2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKalpha2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKalpha2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element-binding protein-1) and ChREBP (carbohydrate response element-binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKalpha2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKalpha2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.

It has been proposed in several eukaryotic systems that the regulation of gene transcription involves phosphorylation of specific transcription factors. We report here that the yeast transcriptional activator ADR1 is phosphorylated in vitro by cyclic AMP-dependent protein kinase and that mutations which enhance the ability of ADR1 to activate ADH2 expression decrease ADR1 phosphorylation. We also show that increased kinase activity in vivo inhibits ADH2 expression in an ADR1 allele-specific manner. Our data suggest that glucose repression of ADH2 is in part mediated through a cAMP-dependent phosphorylation-inactivation of the ADR1 regulatory protein.

Adenosine-3',5'-cyclic phosphate (cyclic AMP) is absolutely required for flagella formation and, hence, motility in cyclic AMP-deficient mutants of Escherichia coli and Salmonella typhimurium.

The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallographic studies of bacterial CBS domains have shown that two CBS domains form an intramolecular dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria, retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can be caused by mutations in CBS domains of, respectively, cystathionine-beta-synthase, inosine 5'-monophosphate dehydrogenase, AMP kinase, and chloride channels. Despite their clinical relevance, it remains to be established what the precise function of CBS domains is and how they affect the structural and/or functional properties of an enzyme, kinase, or channel. Depending on the protein in which they occur, CBS domains have been proposed to affect multimerization and sorting of proteins, channel gating, and ligand binding. However, recent experiments revealing that CBS domains can bind adenosine-containing ligands such ATP, AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains function as sensors of intracellular metabolites.

Lipids as fuel for energy provision originate from different sources: albumin-bound long-chain fatty acids (LCFA) in the blood plasma, circulating very-low-density lipoproteins-triacylglycerols (VLDL-TG), fatty acids from triacylglycerol located in the muscle cell (IMTG), and possibly fatty acids liberated from adipose tissue adhering to the muscle cells. The regulation of utilization of the different lipid sources in skeletal muscle during exercise is reviewed, and the influence of diet, training, and gender is discussed. Major points deliberated are the methods utilized to measure uptake and oxidation of LCFA during exercise in humans. The role of the various lipid-binding proteins in transmembrane and cytosolic transport of lipids is considered as well as regulation of lipid entry into the mitochondria, focusing on the putative role of AMP-activated protein kinase (AMPK), acetyl CoA carboxylase (ACC), and carnitine during exercise. The possible contribution to fuel provision during exercise of circulating VLDL-TG as well as the role of IMTG is discussed from a methodological point of view. The contribution of IMTG for energy provision may not be large, covering approximately 10% of total energy provision during fasting exercise in male subjects, whereas in females, IMTG may cover a larger proportion of energy delivery. Molecular mechanisms involved in breakdown of IMTG during exercise are also considered focusing on hormone-sensitive lipase (HSL). Finally, the role of lipids in development of insulin resistance in skeletal muscle, including possible molecular mechanisms involved, is discussed.

Exercise training is commonly prescribed for treatment of nonalcoholic fatty liver disease (NAFLD). We sought to determine whether exercise training prevents the development of NAFLD in Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to elucidate the molecular mechanisms underlying the effects of exercise on hepatic steatosis. Four-week-old OLETF rats were randomly assigned to either a sedentary control group (Sed) or a group given access to voluntary running wheels for 16 wk (Ex). Wheels were locked 2 days before euthanasia in the Ex animals, and both groups were euthanized at 20 wk old. Voluntary wheel running attenuated weight gain and reduced serum glucose, insulin, free fatty acids, and triglycerides in Ex animals compared with Sed (P < 0.001). Ex animals exhibited significantly reduced hepatic triglyceride levels and displayed fewer lipid droplets (Oil Red O staining) and reduced lipid droplet size compared with Sed. Wheel running increased by threefold the percent of palmitate oxidized completely to CO(2) in the Ex animals but did not alter AMP-activated protein kinase-alpha (AMPKalpha) or AMPK phosphorylation status. However, fatty acid synthase and acetyl-coenzyme A carboxylase (ACC) content were significantly reduced (approximately 70 and approximately 35%, respectively), and ACC phosphorylation and cytochrome c content were significantly elevated (approximately 35 and approximately 30%, respectively) in the Ex animals. These results unequivocally demonstrate that daily physical activity attenuates hepatic steatosis and NAFLD in an obese rodent model and suggest that this effect is likely mediated, in part, through enhancement of hepatic fatty acid oxidation and reductions in key protein intermediates of fatty acid synthesis.

The innate immune system represents an ancient host defence mechanism that protects against invading microorganisms. An important class of immune effector molecules to fight pathogen infections are antimicrobial peptides (AMPs) that are produced in plants and animals. In Drosophila, the induction of AMPs in response to infection is regulated through the activation of the evolutionarily conserved Toll and immune deficiency (IMD) pathways. Here we show that AMP activation can be achieved independently of these immunoregulatory pathways by the transcription factor FOXO, a key regulator of stress resistance, metabolism and ageing. In non-infected animals, AMP genes are activated in response to nuclear FOXO activity when induced by starvation, using insulin signalling mutants, or by applying small molecule inhibitors. AMP induction is lost in foxo null mutants but enhanced when FOXO is overexpressed. Expression of AMP genes in response to FOXO activity can also be triggered in animals unable to respond to immune challenges due to defects in both the Toll and IMD pathways. Molecular experiments at the Drosomycin promoter indicate that FOXO directly binds to its regulatory region, thereby inducing its transcription. In vivo studies in Drosophila, but also studies in human lung, gut, kidney and skin cells indicate that a FOXO-dependent regulation of AMPs is evolutionarily conserved. Our results indicate a new mechanism of cross-regulation of metabolism and innate immunity by which AMP genes can be activated under normal physiological conditions in response to the oscillating energy status of cells and tissues. This regulation seems to be independent of the pathogen-responsive innate immunity pathways whose activation is often associated with tissue damage and repair. The sparse production of AMPs in epithelial tissues in response to FOXO may help modulating the defence reaction without harming the host tissues, in particular when animals are suffering from energy shortage or stress.

The three-dimensional structure of spinach ferredoxin-NADP+ reductase (NADP+, nicotinamide adenine dinucleotide phosphate) has been determined by x-ray diffraction at 2.6 angstroms (A) resolution and initially refined to an R factor of 0.226 at 2.2 A resolution. The model includes the flavin-adenine dinucleotide (FAD) prosthetic group and the protein chain from residue 19 through the carboxyl terminus at residue 314 and is composed of two domains. The FAD binding domain (residues 19 to 161) has an antiparallel beta barrel core and a single alpha helix for binding the pyrophosphate of FAD. The NADP binding domain (residues 162 to 314) has a central five-strand parallel beta sheet and six surrounding helices. Binding of the competitive inhibitor 2'-phospho-AMP (AMP, adenosine monophosphate) places the NADP binding site at the carboxyl-terminal edge of the sheet in a manner similar to the nucleotide binding of the dehydrogenase family. The structures reveal the key residues that function in cofactor binding and the catalytic center. With these key residues as a guide, conclusive evidence is presented that the ferredoxin reductase structure is a prototype for the nicotinamide dinucleotide and FAD binding domains of the enzymes NADPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADH-cytochrome b5 reductase, and NADH-nitrate reductase. Thus this structure provides a structural framework for the NADH- or NADPH-dependent flavoenzyme parts of five distinct enzymes involved in photosynthesis, in the assimilation of inorganic nitrogen and sulfur, in fatty-acid oxidation, in the reduction of methemoglobin, and in the metabolism of many pesticides, drugs, and carcinogens.

The v-maf oncogene, identified from AS42 avian retrovirus, encodes a nuclear bZip protein. To elucidate the molecular mechanism of cell transformation induced by this oncogene, we determined the specific binding sequences of its product. Maf protein recognized two types of relatively long palindromic consensus sequences, TGCTGACTCAGCA and TGCTGACGTCAGCA, at roughly equal efficiency. The middle parts of these Maf-binding sequences completely match with two binding sequences for AP-1 transcription factor, i.e., phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE) and cyclic AMP responsive element, suggesting partial overlapping of the target genes for Maf and AP-1. Furthermore, Maf efficiently formed heterodimers with the components of AP-1, Fos and Jun, through their leucine zipper structures, and these heterodimers show binding specificities distinct from those for Maf-Maf and Jun-Jun homodimers. Thus, a multiple combination of the dimers should generate a greatly expanded repertoire of transcriptional regulatory potential. DNA data base search for the Maf-binding consensus sequences suggested that some of the TRE-like cis elements reported previously may actually be the targets for Maf family proteins or their heterodimers with other bZip proteins.

p60src, the transforming protein of Rous sarcoma virus (RSV), is a protein kinase that has a strict specificity for tyrosine. The phosphorylation of cellular proteins by p60src (ref. 4) results in transformation. Recently, Barker and Dayhoff discovered that residues 259-485 of p60src have 22% sequence identity with residues 33-258 of the catalytic subunit of cyclic AMP-dependent protein kinase, an enzyme that has a specificity for serine. Because it was necessary to introduce eight gaps to align the two proteins, the question remained as to whether this apparent homology reflected a common evolutionary origin. We demonstrate here that the ATP analogue p-fluorosulphonylbenzoyl 5'-adenosine (FSBA) inactivates the tyrosine protein kinase activity of p60src by reacting with lysine 295. When aligned for maximum sequence identity, lysine 295 of p60src and the lysine in the catalytic subunit which also reacts specifically with FSBA are superimposed precisely. This functional homology is strong evidence that the protein kinases, irrespective of amino acid substrate specificity, comprise a single divergent gene family.

Prostacyclin (PGX) strikingly increases cyclic AMP concentrations in human platelets. Prostacyclin is approximately 10 times more active than PGD2, 30 times more active than PGE1 and more than 1000 times more active than its stable end product, 6-oxo-PGF1alpha. These results correlate well with the anti-aggregating activity of prostacyclin, compared with PGE1 and PGD2.

Determination of specific GTPase (EC 3.6.1.--) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 muM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5'(beta,gamma-imino-triphosphate (App(NH)p) and suppression of the transfer of gamma-32P from GTP to ADP with an ATP regeneration system. Under these conditions catacholamines caused a 30--70% increase in GTP hydrolysis. The stimulation of GTPase activity by catecholamines required the presence of Mg2+ or Mn2+. DIfferent batches of membranes revealed the following specific activities (pmol 32Pi/mg protein min): basal GTPase (determined in the absence of catecholamine), 6-- 11; catecholamine-stimulated TTPase, 3--7; and residual non-specific NTPase 3--5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the beta-adrenergic receptor, and was inhibited by propranolol. The concentrations of DL-isoproterenol which half-maximally activated the GTPase and adenylate cyclase were 1 and 1.2 muM, respectively. The following findings indicate that the catecholamine-stimulated GTPase is independent of the catalytic production of cyclic AMP by the adenylate cyclase. Addition of cyclic AMP to the GTPase assay did not change the rate of GTP hydrolysis. Furthermore, treatment of the membrane with N-ethylmaleimide (MalNEt) at 0 degrees C which caused 98% inhibition of the adenylate cyclase, had no effect on the catecholamine-stimulated GTPase. The affinity and specificity for GTP in the GTPase reactions are similar to those previously reported for the stimulation of the adenylate cyclase. The apparent Km for GTP in the basal and the catecholamine-stimulated GTPase reaction was 0.1 muM. These GTPase activities were inhibited by ITP but not by CTP and UTP. It is proposed that a catecholamine-stimulated GTPase is a component of the turkey erythrocyte adenylate cyclase system.

Protein kinases critically regulate synaptic plasticity in the mammalian hippocampus. Cyclic-AMP dependent protein kinase (PKA) is a serine-threonine kinase that has been strongly implicated in the expression of specific forms of long-term potentiation (LTP), long-term depression (LTD), and hippocampal long-term memory. We review the roles of PKA in activity-dependent forms of hippocampal synaptic plasticity by highlighting particular themes that have emerged in ongoing research. These include the participation of distinct isoforms of PKA in specific types of synaptic plasticity, modification of the PKA-dependence of LTP by multiple factors such as distinct patterns of imposed activity, environmental enrichment, and genetic manipulation of signalling molecules, and presynaptic versus postsynaptic mechanisms for PKA-dependent LTP. We also discuss many of the substrates that have been implicated as targets for PKA's actions in hippocampal synaptic plasticity, including CREB, protein phosphatases, and glutamatergic receptors. Future prospects for shedding light on the roles of PKA are also described from the perspective of specific aspects of synaptic physiology and brain function that are ripe for investigation using incisive genetic, cell biological, and electrophysiological approaches.

Recently, a new method has been described, in which membrane filtration is used to allow the levels of adenine nucleotides in the chloroplast stroma, the cytosol, and the mitochondrial matrix to be measured. This method is now used to investigate the effect of illumination, of respiratory inhibitors, and of uncouplers on the distribution of ATP, ADP, and AMP in wheat (Triticum aestivum var. ;Timmo') leaf protoplasts. (a) The adenine nucleotides are apparently equilibrated by adenylate kinase in the stroma and the cytosol, but not in the mitochondrial matrix. (b) The ATP/ADP quotient in the cytosol is considerably higher than that in the mitochondrial matrix or the chloroplast stroma. (c) A large gradient exists between the ATP/ADP quotients in the cytosol and the mitochondrial matrix in the dark, with a very low ATP/ADP quotient in the mitochondria. This gradient is lowered by uncouplers or respiratory inhibitors showing that, as in animal tissues, it reflects the energization of the mitochondria. (d) In the dark, the stromal ATP/ADP is lower than in the light, and appears to be maintained, at least in part, by import from the cytosol. (e) The cytosolic ATP/ADP, however, actually decreases in the light. This contradicts the widespread assumption, that export of photosynthetically produced ATP from the chloroplast leads to an increase in the cytosolic ATP/ADP, which then inhibits oxidative phosphorylation in the mitochondria. (f) The mitochondrial ATP/ADP increases in the light, and the gradient between the cytosol and mitochondrial matrix falls. This is also difficult to understand in terms of an inhibition of oxidative phosphorylation in the light due to a lack of ADP in the cytosol. (g) The significance of the measured variations in the adenine nucleotide pools are discussed with respect to the diurnal carbohydrate metabolism in a leaf, and to the metabolic function of the chloroplast, the cytosol and the mitochondria.

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Galpha subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Galpha protein Gpal, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpal mutant strains. Physiological studies revealed that the Galpha protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpal and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

The human T-cell leukemia virus type 1 (HTLV-1) transcriptional trans-activator Tax has been demonstrated to have transforming activity in multiple cell culture and transgenic-mouse models. In addition to activating transcription from the viral long terminal repeat (LTR) through the cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors, Tax activates the expression of multiple cellular promoters through the NF-kappaB pathway of transcriptional activation. The Tax mutants M22 and M47 have previously been demonstrated to selectively abrogate the ability of Tax to activate transcription through the NF-kappaB or CREB/ATF pathway, respectively. These mutations were introduced in the tax gene of the ACH functional molecular clone of HTLV-1, and virus produced from the mutant ACH clones was examined for the ability to replicate and immortalize primary human lymphocytes. While virus derived from the clone containing the M47 mutation retained the ability to immortalize T lymphocytes, the M22 mutant lost the ability to immortalize infected cells. These results indicate that activation of the CREB/ATF pathway by Tax is dispensable for the immortalization of T cells by HTLV-1, whereas activation of the NF-kappaB pathway may be critical.

AMP-activated protein kinase (AMPK) has emerged as a key regulator of energy metabolism in the heart. The high energy demands of the heart are primarily met by the metabolism of both fatty acids and glucose, both processes being regulated by AMPK. During myocardial ischaemia a rapid activation of AMPK occurs, resulting in an activation of both glucose uptake and glycolysis, as well as an increase in fatty acid oxidation. This activation of AMPK has the potential to increase energy production and to inhibit apoptosis, thereby protecting the heart during the ischaemic stress. However, at clinically relevant high levels of fatty acids, ischaemic-induced activation of AMPK also stimulates fatty acid oxidation during and following ischaemia. This can contribute to ischaemic injury secondary to an inhibition of glucose oxidation, which results in a decrease in cardiac efficiency. In a number of other non-cardiac tissues, AMPK has been shown to have pro-apoptotic effects. As a result, the question of whether AMPK activation benefits or harms the ischaemic heart remains controversial. The role of AMPK in cardiac hypertrophy is also controversial. Activation of AMPK inhibits protein synthesis, and may be an adaptive response to pathological cardiac hypertrophy. However, none of mouse models of AMPK deficiency (excluding those that may involve the gamma2 subunit mutations) demonstrate increased cardiac mass, suggesting that AMPK is not essential for restriction of cardiac growth. In addition to the potential effects of AMPK on myofibrillar hypertrophy associated with pressure overload, there is also controversy with respect to the cardiac hypertrophy associated with the gamma2 subunit mutations. In the cardiac hypertrophy associated with glycogen overload, both activating and inactivating mutations of AMPK in mice are associated with a marked cardiac hypertrophy. This review will address the issue of whether AMPK activation acts as an enemy or ally to the ischaemic and hypertrophied heart. Resolving this issue has important implications as to whether therapeutic approaches to protect the ischaemic heart should be developed which either activate or inhibit AMPK.

Induction of cyclin D1 gene transcription by estrogen receptor alpha (ERalpha) plays an important role in estrogen-mediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ERalpha has been mapped to an alternative response element, a cyclic AMP-response element at -57, with possible participation of an activating protein-1 site at -954. The action of ERbeta at the cyclin D1 promoter is unknown, although evidence suggests that ERbeta may inhibit the proliferative action of ERalpha. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ERalpha, ERalphaK206A (a derivative that is superactive at alternative response elements), or ERbeta. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ERbeta occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ERalpha, inhibit expression with ERbeta. Strikingly, the presence of ERbeta completely inhibits cyclin D1 gene activation by estrogen and ERalpha or even by estrogen and the superactive ERalphaK206A. The observation of the opposing action and dominance of ERbeta over ERalpha in activation of cyclin D1 gene expression has implications for the postulated role of ERbeta as a modulator of the proliferative effects of estrogen.

Fibroblast growth factor-21 (FGF21) is a hormone secreted by the liver during fasting that elicits diverse aspects of the adaptive starvation response. Among its effects, FGF21 induces hepatic fatty acid oxidation and ketogenesis, increases insulin sensitivity, blocks somatic growth and causes bone loss. Here we show that transgenic overexpression of FGF21 markedly extends lifespan in mice without reducing food intake or affecting markers of NAD+ metabolism or AMP kinase and mTOR signaling. Transcriptomic analysis suggests that FGF21 acts primarily by blunting the growth hormone/insulin-like growth factor-1 signaling pathway in liver. These findings raise the possibility that FGF21 can be used to extend lifespan in other species.DOI:http://dx.doi.org/10.7554/eLife.00065.001.

Receptors for corticotropin-releasing factor (CRF) are members of a family of G protein-coupled receptors ("Family B") that respond to a variety of structurally dissimilar releasing factors, neuropeptides, and hormones (including secretin, growth hormone-releasing factor, calcitonin, parathyroid hormone, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal polypeptide) and signal through the cyclic AMP and/or calcium pathways. To date, three genes encoding additional CRF-like peptides (urocortins) have been identified in mammals. The urocortins and CRF bind with differential ligand selectivity at the two mammalian CRF receptors. This report was prepared by the International Union of Pharmacology Subcommittee on CRF Receptors, to summarize the current state of CRF receptor biology and to propose changes in the classification and nomenclature of CRF ligands and receptors.

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.

Millions of people regularly obtain insufficient sleep. Given the effect of sleep deprivation on our lives, understanding the cellular and molecular pathways affected by sleep deprivation is clearly of social and clinical importance. One of the major effects of sleep deprivation on the brain is to produce memory deficits in learning models that are dependent on the hippocampus. Here we have identified a molecular mechanism by which brief sleep deprivation alters hippocampal function. Sleep deprivation selectively impaired 3', 5'-cyclic AMP (cAMP)- and protein kinase A (PKA)-dependent forms of synaptic plasticity in the mouse hippocampus, reduced cAMP signalling, and increased activity and protein levels of phosphodiesterase 4 (PDE4), an enzyme that degrades cAMP. Treatment of mice with phosphodiesterase inhibitors rescued the sleep-deprivation-induced deficits in cAMP signalling, synaptic plasticity and hippocampus-dependent memory. These findings demonstrate that brief sleep deprivation disrupts hippocampal function by interfering with cAMP signalling through increased PDE4 activity. Thus, drugs that enhance cAMP signalling may provide a new therapeutic approach to counteract the cognitive effects of sleep deprivation.

BACKGROUND

Low plasma high-density lipoprotein (HDL) is associated with elevated cardiovascular risk and aspects of the metabolic syndrome. We hypothesized that HDL modulates glucose metabolism via elevation of plasma insulin and through activation of the key metabolic regulatory enzyme, AMP-activated protein kinase, in skeletal muscle.

RESULTS

Thirteen patients with type 2 diabetes mellitus received both intravenous reconstituted HDL (rHDL: 80 mg/kg over 4 hours) and placebo on separate days in a double-blind, placebo-controlled crossover study. A greater fall in plasma glucose from baseline occurred during rHDL than during placebo (at 4 hours rHDL=-2.6+/-0.4; placebo=-2.1+/-0.3 mmol/L; P=0.018). rHDL increased plasma insulin (at 4 hours rHDL=3.4+/-10.0; placebo= -19.2+/-7.4 pmol/L; P=0.034) and also the homeostasis model assessment beta-cell function index (at 4 hours rHDL=18.9+/-5.9; placebo=8.6+/-4.4%; P=0.025). Acetyl-CoA carboxylase beta phosphorylation in skeletal muscle biopsies was increased by 1.7+/-0.3-fold after rHDL, indicating activation of the AMP-activated protein kinase pathway. Both HDL and apolipoprotein AI increased glucose uptake (by 177+/-12% and 144+/-18%, respectively; P<0.05 for both) in primary human skeletal muscle cell cultures established from patients with type 2 diabetes mellitus (n=5). The mechanism is demonstrated to include stimulation of the ATP-binding cassette transporter A1 with subsequent activation of the calcium/calmodulin-dependent protein kinase kinase and the AMP-activated protein kinase pathway.

CONCLUSIONS

rHDL reduced plasma glucose in patients with type 2 diabetes mellitus by increasing plasma insulin and activating AMP-activated protein kinase in skeletal muscle. These findings suggest a role for HDL-raising therapies beyond atherosclerosis to address type 2 diabetes mellitus.