Urocortin 3 transgenic mice exhibit a metabolically favourable phenotype resisting obesity and hyperglycaemia on a high-fat diet.

AIMS/HYPOTHESIS: Urocortins are the endogenous ligands for the corticotropin-releasing factor receptor type 2 (CRFR2), which is implicated in regulating energy balance and/or glucose metabolism. We determined the effects of chronic CRFR2 activation on metabolism in vivo, by generating and phenotyping transgenic mice overproducing the specific CRFR2 ligand urocortin 3. METHODS: Body composition, glucose metabolism, insulin sensitivity, energy efficiency and expression of key metabolic genes were assessed in adult male urocortin 3 transgenic mice (Ucn3(+)) under control conditions and following an obesogenic high-fat diet (HFD) challenge. RESULTS: Ucn3(+) mice had increased skeletal muscle mass with myocyte hypertrophy. Accelerated peripheral glucose disposal, increased respiratory exchange ratio and hypoglycaemia on fasting demonstrated increased carbohydrate metabolism. Insulin tolerance and indices of insulin-stimulated signalling were unchanged, indicating these effects were not mediated by increased insulin sensitivity. Expression of the transgene in Crfr2 (also known as Crhr2)-null mice negated key aspects of the Ucn3(+) phenotype. Ucn3(+) mice were protected from the HFD-induced hyperglycaemia and increased adiposity seen in control mice despite consuming more energy. Expression of uncoupling proteins 2 and 3 was higher in Ucn3(+) muscle, suggesting increased catabolic processes. IGF-1 abundance was upregulated in Ucn3(+) muscle, providing a potential paracrine mechanism in which urocortin 3 acts upon CRFR2 to link the altered metabolism and muscular hypertrophy observed. CONCLUSIONS/INTERPRETATION: Urocortin 3 acting on CRFR2 in skeletal muscle of Ucn3(+) mice results in a novel metabolically favourable phenotype, with lean body composition and protection against diet-induced obesity and hyperglycaemia. Urocortins and CRFR2 may be of interest as potential therapeutic targets for obesity.

Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer.

AMP-activated protein kinase (AMPK) is a cellular energy sensor activated by metabolic stresses that either inhibit ATP synthesis or accelerate ATP consumption. Activation of AMPK in response to an increase in the cellular AMP:ATP ratio results in inhibition of ATP-consuming processes such as gluconeogenesis and fatty acid synthesis, while stimulating ATP-generating processes, including fatty acid oxidation. These alterations in lipid and glucose metabolism would be expected to ameliorate the pathogenesis of obesity, type 2 diabetes and other metabolic disorders. Recently, AMPK has also been identified as a potential target for cancer prevention and/or treatment. Cell growth and proliferation are energetically demanding, and AMPK may act as an "energy checkpoint" that permits growth and proliferation only when energy reserves are sufficient. Thus, activators of AMPK could have potential as novel therapeutics both for metabolic disorders and for cancer, which together constitute two of the most prevalent groups of diseases worldwide.

Potential role of TBC1D4 in enhanced post-exercise insulin action in human skeletal muscle.

AIMS/HYPOTHESIS: TBC1 domain family, member 4 (TBC1D4; also known as AS160) is a cellular signalling intermediate to glucose transport regulated by insulin-dependent and -independent mechanisms. Skeletal muscle insulin sensitivity is increased after acute exercise by an unknown mechanism that does not involve modulation at proximal insulin signalling intermediates. We hypothesised that signalling through TBC1D4 is involved in this effect of exercise as it is a common signalling element for insulin and exercise. METHODS: Insulin-regulated glucose metabolism was evaluated in 12 healthy moderately trained young men 4 h after one-legged exercise at basal and during a euglycaemic-hyperinsulinaemic clamp. Vastus lateralis biopsies were taken before and immediately after the clamp. RESULTS: Insulin stimulation increased glucose uptake in both legs, with greater effects (approximately 80%, p < 0.01) in the previously exercised leg. TBC1D4 phosphorylation, assessed using the phospho-AKT (protein kinase B)substrate antibody and phospho- and site-specific antibodies targeting six phosphorylation sites on TBC1D4, increased at similar degrees to insulin stimulation in the previously exercised and rested legs (p < 0.01). However, TBC1D4 phosphorylation on Ser-318, Ser-341, Ser-588 and Ser-751 was higher in the previously exercised leg, both in the absence and in the presence of insulin (p < 0.01; Ser-588, p = 0.09; observed power = 0.39). 14-3-3 binding capacity for TBC1D4 increased equally (p < 0.01) in both legs during insulin stimulation. CONCLUSION/INTERPRETATION: We provide evidence for site-specific phosphorylation of TBC1D4 in human skeletal muscle in response to physiological hyperinsulinaemia. The data support the idea that TBC1D4 is a nexus for insulin- and exercise-responsive signals that may mediate increased insulin action after exercise.

Modulation of O(2) sensitive K (+) channels by AMP-activated protein kinase.

Hypoxic inhibition of K(+) channels in type I cells is believed to be of central importance in carotid body chemotransduction. We have recently suggested that hypoxic channel inhibition is mediated by AMP-activated protein kinase (AMPK). Here, we have further explored the modulation by AMPK of recombinant K(+) channels (expressed in HEK293 cells) whose native counterparts are considered O(2)-sensitive in the rat carotid body. Inhibition of maxiK channels by AMPK activation with AICAR was found to be independent of [Ca(2+)](i) and occurred regardless of whether the alpha subunit was co-expressed with an auxiliary beta subunit. All effects of AICAR were fully reversed by the AMPK inhibitor compound C. MaxiK channels were also inhibited by the novel AMPK activator A-769662 and by intracellular dialysis with the constitutively active, truncated AMPK mutant, T172D. The molecular identity of the O(2)-sensitive leak K(+) conductance in rat type I cells remains unclear, but shares similarities with TASK-1 and TASK-3. Recombinant TASK-1 was insensitive to AICAR. However, TASK-3 was inhibited by either AICAR or A-769662 in a manner which was reversed by compound C. These data highlight a role for AMPK in the modulation of two proposed O(2) sensitive K(+) channels found in the carotid body.

AMPK: a key regulator of energy balance in the single cell and the whole organism.

The AMP-activated protein kinase (AMPK) system is a key player in regulating energy balance at both the cellular and whole-body levels, placing it at centre stage in studies of obesity, diabetes and the metabolic syndrome. It is switched on in response to metabolic stresses such as muscle contraction or hypoxia, and modulated by hormones and cytokines affecting whole-body energy balance such as leptin, adiponectin, resistin, ghrelin and cannabinoids. Once activated, it switches on catabolic pathways that generate adenosine triphosphate (ATP), while switching off ATP-consuming anabolic processes. AMPK exists as heterotrimeric complexes comprising a catalytic alpha-subunit and regulatory beta- and gamma-subunits. Binding of AMP to the gamma-subunit, which is antagonized by high ATP, causes activation of the kinase by promoting phosphorylation at threonine (Thr-172) on the alpha-subunit by the upstream kinase LKB1, allowing the system to act as a sensor of cellular energy status. In certain cells, AMPK is activated in response to elevation of cytosolic Ca2+ via phosphorylation of Thr-172 by calmodulin-dependent kinase kinase-beta (CaMKKbeta). Activation of AMPK, either in response to exercise or to pharmacological agents, has considerable potential to reverse the metabolic abnormalities associated with type 2 diabetes and the metabolic syndrome. Two existing classes of antidiabetic drugs, that is, biguanides (for example, metformin) and the thiazolidinediones (for example, rosiglitazone), both act (at least in part) by activation of AMPK. Novel drugs activating AMPK may also have potential for the treatment of obesity.

Mechanisms underlying the metabolic actions of galegine that contribute to weight loss in mice.

BACKGROUND AND PURPOSE: Galegine and guanidine, originally isolated from Galega officinalis, led to the development of the biguanides. The weight-reducing effects of galegine have not previously been studied and the present investigation was undertaken to determine its mechanism(s) of action. EXPERIMENTAL APPROACH: Body weight and food intake were examined in mice. Glucose uptake and acetyl-CoA carboxylase activity were studied in 3T3-L1 adipocytes and L6 myotubes and AMP activated protein kinase (AMPK) activity was examined in cell lines. The gene expression of some enzymes involved in fat metabolism was examined in 3T3-L1 adipocytes. KEY RESULTS: Galegine administered in the diet reduced body weight in mice. Pair-feeding indicated that at least part of this effect was independent of reduced food intake. In 3T3-L1 adipocytes and L6 myotubes, galegine (50 microM-3 mM) stimulated glucose uptake. Galegine (1-300 microM) also reduced isoprenaline-mediated lipolysis in 3T3-L1 adipocytes and inhibited acetyl-CoA carboxylase activity in 3T3-L1 adipocytes and L6 myotubes. Galegine (500 microM) down-regulated genes concerned with fatty acid synthesis, including fatty acid synthase and its upstream regulator SREBP. Galegine (10 microM and above) produced a concentration-dependent activation of AMP activated protein kinase (AMPK) in H4IIE rat hepatoma, HEK293 human kidney cells, 3T3-L1 adipocytes and L6 myotubes. CONCLUSIONS AND IMPLICATIONS: Activation of AMPK can explain many of the effects of galegine, including enhanced glucose uptake and inhibition of acetyl-CoA carboxylase. Inhibition of acetyl-CoA carboxylase both inhibits fatty acid synthesis and stimulates fatty acid oxidation, and this may to contribute to the in vivo effect of galegine on body weight.

Metabolic control: a new solution to an old problem.

The phenomenon whereby the presence of oxygen regulates the rate of glucose metabolism was first described by Louis Pasteur. A novel mechanism has now been discovered, involving the AMP-activated protein kinase cascade, that can account for the Pasteur effect in ischaemic heart muscle.

Glucose repression/derepression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP:ATP ratio.

BACKGROUND: Genetic studies of Saccharomyces cerevisiae have shown that Snf1p and Snf4p, which together form the SNF1 complex, are essential for gene derepression on removal of glucose from the medium. However the metabolic signal(s) involved, and the exact role of SNF1, have remained enigmatic. Recently, the AMP-activated protein kinase (AMPK) was shown to be the mammalian homologue of SNF1. AMPK is activated by the elevation of the cellular AMP:ATP ratio, which occurs during cellular stress in mammalian cells. The mechanism of activation involves phosphorylation of AMPK by an upstream protein kinase (AMPKK). We have investigated whether a similar mechanism might explain the role of SNF1 in yeast in the response to the stress of glucose starvation. RESULTS: The protein kinase activity of SNF1 was dramatically and rapidly activated by phosphorylation on removal of glucose from the medium. SNF1 was not activated directly by AMP, but could be inactivated by protein phosphatases and reactivated by mammalian AMPKK. We also demonstrated that an endogenous SNF1-reactivating factor, most likely an upstream protein kinase, is present in yeast extracts. Under a variety of different growth conditions, there was a correlation between cellular adenine nucleotide levels and the activation state of SNF1. CONCLUSIONS: Apart from the lack of direct allosteric activation of SNF1 by AMP, the regulation of the mammalian AMPK and yeast SNF1 protein kinase cascades is highly conserved. Adenine nucleotides are now good candidates for metabolic signals which indicate the lack of glucose in the medium, triggering activation of SNF1 and derepression of glucose-repressed genes.

Role of the AMP-activated protein kinase in the cellular stress response.

BACKGROUND: AMP-activated protein kinase is the central component of a protein kinase cascade that phosphorylates and inactivates key regulatory enzymes of several biosynthetic pathways. Elevation of cellular AMP levels activates this kinase, both by allosteric activation, which causes more than 5-fold activation, and by phosphorylation by an upstream kinase kinase, leading to more than 20-fold activation; the result is a greater than 100-fold activation overall. As AMP is usually elevated when cellular ATP is depleted, we have assessed the possibility that the AMP-activated kinase is involved in the cellular response to stress, which is known to lead to ATP depletion. RESULTS: We report that AMP is elevated, and ATP depleted, when isolated rat hepatocytes are subjected to treatments that activate the cellular stress response, namely heat shock or treatment with arsenite. Several events are correlated with these changes in nucleotide levels: first, a large activation of the AMP-activated protein kinase, which can be reversed by treatment with a protein phosphatase; second, phosphorylation and inactivation of one of the known substrates of the AMP-activated kinase, HMG-CoA reductase; and third, inhibition of two of the biosynthetic pathways known to be affected by the AMP-activated kinase, namely sterol and fatty-acid synthesis. CONCLUSIONS: Our results suggest that a major function of the AMP-activated protein kinase is to act protectively, switching off biosynthetic pathways when the cell is subjected to stress that causes ATP depletion, the key signal being a rise in AMP level. By this mechanism, ATP is preserved for processes that may be more essential in the short term, such as the maintenance of ion gradients. This function of the kinase represents a novel role for protein phosphorylation.

Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells.

BACKGROUND AND PURPOSE: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (I(amiloride)) and ouabain-sensitive basolateral (I(ouabain)) short circuit current in H441 lung epithelial cells. EXPERIMENTAL APPROACH: H441 cells were grown on permeable filters at air interface. I(amiloride), I(ouabain) and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. KEY RESULTS: Phenformin, AICAR and metformin increased AMPK (alpha1) activity and decreased I(amiloride). The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I(ouabain) across H441 monolayers and decreased monolayer resistance. The decrease in I(amiloride) was closely related to I(ouabain) with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. CONCLUSIONS AND IMPLICATIONS: Activation of alpha1-AMPK is associated with inhibition of apical amiloride-sensitive Na(+) channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I(ouabain), with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component.

The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase.

In addition to acetyl-CoA carboxylase and HMG-CoA reductase, the AMP-activated protein kinase phosphorylates glycogen synthase, phosphorylase kinase, hormone-sensitive lipase and casein. A number of other substrates for the cyclic AMP-dependent protein kinase, e.g., L-pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, are not phosphorylated at significant rates. Examination of the sites phosphorylated on acetyl-CoA carboxylase, hormone-sensitive lipase, glycogen synthase and phosphorylase kinase suggests a consensus recognition sequence in which the serine residue phosphorylated by the AMP-activated protein kinase has a hydrophobic residue on the N-terminal side (i.e., at -1) and at least one arginine residue at -2, -3 or -4. Substrates for cyclic AMP-dependent protein kinase which lack the hydrophobic residue at -1 are not substrates for the AMP-activated protein kinase.

The actions of cyclic AMP on biosynthetic processes are mediated indirectly by cyclic AMP-dependent protein kinase.

Adrenalin and glucagon inhibit glycogen, fatty acid and cholesterol synthesis by elevation of cyclic AMP, activation of cyclic AMP-dependent protein kinase and increased phosphorylation of the rate-limiting enzymes of these pathways. Here, we review recent evidence which indicates that inhibition of these biosynthetic pathways in muscle, adipose tissue and liver is much more indirect than has previously been supposed. In particular, cyclic AMP-dependent protein kinase does not appear to inhibit glycogen synthase, acetyl-CoA carboxylase and HMG-CoA reductase by phosphorylating them directly. It appears to achieve the same end result by inactivation of the protein phosphatases which dephosphorylate these regulatory enzymes in vivo, although this has only been established definitively in the case of glycogen synthesis.

Amino acid sequence around the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase.

Two thermolytic peptides containing the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase have been isolated and sequenched by Edman degradation and fast atom bombardment mass spectrometry. The sequence (V-A-G-Y-S-Y-G) contains the motif G-X-S-X-G found around the reactive serine residue of all known serine proteinases and esterases.

5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase.

Insulin regulates the rate of expression of many hepatic genes, including PEPCK, glucose-6-phosphatase (G6Pase), and glucose-6-phosphate dehydrogenase (G6PDHase). The expression of these genes is also abnormally regulated in type 2 diabetes. We demonstrate here that treatment of hepatoma cells with 5-aminoimidazole-4-carboxamide riboside (AICAR), an agent that activates AMP-activated protein kinase (AMPK), mimics the ability of insulin to repress PEPCK gene transcription. It also partially represses G6Pase gene transcription and yet has no effect on the expression of G6PDHase or the constitutively expressed genes cyclophilin or beta-actin. Several lines of evidence suggest that the insulin-mimetic effects of AICAR are mediated by activation of AMPK. Also, insulin does not activate AMPK in H4IIE cells, suggesting that this protein kinase does not link the insulin receptor to the PEPCK and G6Pase gene promoters. Instead, AMPK and insulin may lie on distinct pathways that converge at a point upstream of these 2 gene promoters. Investigation of the pathway by which AMPK acts may therefore give insight into the mechanism of action of insulin. Our results also suggest that activation of AMPK would inhibit hepatic gluconeogenesis in an insulin-independent manner and thus help to reverse the hyperglycemia associated with type 2 diabetes.

Activation of glucose transport by AMP-activated protein kinase via stimulation of nitric oxide synthase.

Glucose transport in skeletal muscle is stimulated by two distinct stimuli, insulin and exercise. The mechanism by which exercise stimulates glucose transport is not known, although it is distinct from the insulin-mediated pathway. Recently, it has been shown that AMP-activated protein kinase (AMPK) is activated by exercise in skeletal muscle, whereas pharmacological activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAR) leads to increased glucose transport. It has been postulated, therefore, that AMPK may be the link between exercise and glucose transport. To address this, we have examined the signaling pathway involved in the stimulation of glucose uptake after activation of AMPK. Here we show that activation of AMPK by AICAR in rat muscle and mouse H-2Kb muscle cells activates glucose transport approximately twofold. AMPK in H-2Kb cells is also activated by hyperosmotic stress and the mitochondrial uncoupling agent, dinitrophenol, both of which lead to increased glucose transport. In contrast, insulin, which activates glucose transport two- to-threefold in both rat muscle and H-2Kb cells, has no effect on AMPK activity. A previous study has shown that AMPK phosphorylates and activates endothelial nitric oxide synthase (NOS). We show here that NOS activity in H-2Kb cells is activated after stimulation of AMPK by AICAR. Treatment of H-2Kb cells or rat muscle with NOS inhibitors completely blocks the increase in glucose transport after activation of AMPK. In addition, an inhibitor of guanylate cyclase also blocks activation of glucose transport by AICAR in H-2Kb cells. These results indicate that activation of AMPK in muscle cells stimulates glucose transport by activation of NOS coupled to downstream signaling components, including cyclic GMP.

Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1α.

Glucocorticoid levels rise dramatically in late gestation to mature foetal organs in readiness for postnatal life. Immature heart function may compromise survival. Cardiomyocyte glucocorticoid receptor (GR) is required for the structural and functional maturation of the foetal heart in vivo, yet the molecular mechanisms are largely unknown. Here we asked if GR activation in foetal cardiomyocytes in vitro elicits similar maturational changes. We show that physiologically relevant glucocorticoid levels improve contractility of primary-mouse-foetal cardiomyocytes, promote Z-disc assembly and the appearance of mature myofibrils, and increase mitochondrial activity. Genes induced in vitro mimic those induced in vivo and include PGC-1α, a critical regulator of cardiac mitochondrial capacity. SiRNA-mediated abrogation of the glucocorticoid induction of PGC-1α in vitro abolished the effect of glucocorticoid on myofibril structure and mitochondrial oxygen consumption. Using RNA sequencing we identified a number of transcriptional regulators, including PGC-1α, induced as primary targets of GR in foetal cardiomyocytes. These data demonstrate that PGC-1α is a key mediator of glucocorticoid-induced maturation of foetal cardiomyocyte structure and identify other candidate transcriptional regulators that may play critical roles in the transition of the foetal to neonatal heart.

Phosphorylation and inactivation of HMG-CoA reductase at the AMP-activated protein kinase site in response to fructose treatment of isolated rat hepatocytes.

We have previously shown that incubation of isolated hepatocytes with fructose leads to elevation of AMP and activation of the AMP-activated protein kinase. We now show that this treatment causes marked inactivation of HMG-CoA reductase. Using immunoprecipitation from the microsomal fraction of 32P-labelled cells, we also show that this treatment leads to a 2.6-fold increase in the phosphorylation of the 100 kDa subunit of HMG-CoA reductase. Successive digestion of this 32P-labelled subunit with cyanogen bromide and endoproteinase Lys-C confirmed that Ser-871, the site phosphorylated in cell-free assays by the AMP-activated protein kinase, was the only site phosphorylated under these conditions.

The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase.

Acetyl-CoA carboxylase purified from isolated hepatocytes is activated dramatically by protein phosphatase treatment, concomitant with a reduction of the phosphate content from 3.7 to 1.1 mol/subunit. Glucagon treatment of the cells produces a further inactivation of the enzyme that is totally reversed by phosphatase treatment, and is associated with an increase in phosphate content of 0.8 mol/subunit, distributed in two peptides which contain the sites phosphorylated in vitro by the cyclic AMP-dependent and AMP-activated protein kinases. Sequencing of these peptides shows that the low activity of acetyl-CoA carboxylase is due to phosphorylation by the AMP-activated protein kinase, and not cyclic AMP-dependent protein kinase, even after glucagon treatment.

Evidence that the acyl-O-esters are intermediates in the catalysis. The mechanism of rabbit mammary fatty acid synthase.

The sequence acetyl-CoA leads to acetyl-O-enzyme leads to acetyl-S-acyl carrier protein has for the first time been demonstrated directly with a multifunctional (mammalian) fatty acid synthase. This was achieved by blocking of the active-site thiols of rabbit mammary fatty acid synthase with iodoacetamide. The modified enzyme was incubated with [14C]acetyl-CoA to form acetyl-O-enzyme, and acetyl-CoA was removed rapidly by centrifuge desalting. We were then able to demonstrate transfer of the acetyl group from [14C]acetyl-O-enzyme to the pantetheine thiol in a fragment of rabbit mammary fatty acid synthase containing the phosphopantetheine group, and to E. coli acyl carrier protein.