Heteroacene-Based Polymer with Fast-Switching Visible-Near Infrared Electrochromic Behavior.

The electrochromic properties and application of electronically conducting polymers (ECPs) (PTRPZ-EDOT) consisting of a 3,4-ethylenedioxythiophene (EDOT) and the heteroacene-based molecular scaffold, 6H-pyrrolo[3,2-b:4,5-b'] bis [1,4] benzothiazine (TRPZ), are reported. Known for its high electron mobility and conducting properties, the novel TRPZ scaffold was synthesized to possess two EDOT molecules termini affording TRPZ-EDOT. Electropolymerization of TRPZ-EDOT resulted in remarkable spectroscopic and conductive properties suitable for electrochromic device fabrication. Using atomic force microscopy (AFM), the average surface roughness and surface topography of PTRPZ-EDOT polymer thin films were determined. Spectroelectrochemical data showed that the polymer achieved switching times of 4.07 (coloration) and 0.47 s (bleaching) at 539 nm. The PTRPZ-EDOT film exhibits an optical contrast of 36-44% at 539 nm between its neutral and colored states, respectively. The NIR region from 1000 to 1700 nm shows the appearance of charge carrier bands with a 0-1 V potential range. An electrochromic device was successfully fabricated from PTRPZ-EDOT, showcasing the potential and applicability of the polymer material for advanced technologies such as smart windows, flexible electrochromic screens, and energy storage devices.

The ancient drug salicylate directly activates AMP-activated protein kinase.

Salicylate, a plant product, has been in medicinal use since ancient times. More recently, it has been replaced by synthetic derivatives such as aspirin and salsalate, both of which are rapidly broken down to salicylate in vivo. At concentrations reached in plasma after administration of salsalate or of aspirin at high doses, salicylate activates adenosine monophosphate-activated protein kinase (AMPK), a central regulator of cell growth and metabolism. Salicylate binds at the same site as the synthetic activator A-769662 to cause allosteric activation and inhibition of dephosphorylation of the activating phosphorylation site, threonine-172. In AMPK knockout mice, effects of salicylate to increase fat utilization and to lower plasma fatty acids in vivo were lost. Our results suggest that AMPK activation could explain some beneficial effects of salsalate and aspirin in humans.

Aspirin inhibits mTOR signaling, activates AMP-activated protein kinase, and induces autophagy in colorectal cancer cells.

BACKGROUND & AIMS: Aspirin reduces the incidence of and mortality from colorectal cancer (CRC) by unknown mechanisms. Cancer cells have defects in signaling via the mechanistic target of rapamycin (mTOR), which regulates proliferation. We investigated whether aspirin affects adenosine monophosphate-activated protein kinase (AMPK) and mTOR signaling in CRC cells. METHODS: The effects of aspirin on mTOR signaling, the ribosomal protein S6, S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) were examined in CRC cells by immunoblotting. Phosphorylation of AMPK was measured; the effects of loss of AMPKα on the aspirin-induced effects of mTOR were determined using small interfering RNA (siRNA) in CRC cells and in AMPK(α1/α2-/-) mouse embryonic fibroblasts. LC3 and ULK1 were used as markers of autophagy. We analyzed rectal mucosa samples from patients given 600 mg aspirin, once daily for 1 week. RESULTS: Aspirin reduced mTOR signaling in CRC cells by inhibiting the mTOR effectors S6K1 and 4E-BP1. Aspirin changed nucleotide ratios and activated AMPK in CRC cells. mTOR was still inhibited by aspirin in CRC cells after siRNA knockdown of AMPKα, indicating AMPK-dependent and AMPK-independent mechanisms of aspirin-induced inhibition of mTOR. Aspirin induced autophagy, a feature of mTOR inhibition. Aspirin and metformin (an activator of AMPK) increased inhibition of mTOR and Akt, as well as autophagy in CRC cells. Rectal mucosal samples from patients given aspirin had reduced phosphorylation of S6K1 and S6. CONCLUSIONS: Aspirin is an inhibitor of mTOR and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism. These could contribute to its protective effects against development of CRC.

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase.

Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation.

A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) gamma2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca(2+) ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca(2+), and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.

Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP.

Activation of AMPK (AMP-activated protein kinase) by phosphorylation at Thr172 is catalysed by at least two distinct upstream kinases, i.e. the tumour suppressor LKB1, and CaMKKbeta (Ca2+/calmodulin-dependent protein kinase kinase-beta). The sequence around Thr172 is highly conserved between the two catalytic subunit isoforms of AMPK and the 12 AMPK-related kinases, and LKB1 has been shown to act upstream of all of them. In the present paper we report that none of the AMPK-related kinases tested could be phosphorylated or activated in intact cells or cell-free assays by CaMKKbeta, although we did observe a slow phosphorylation and activation of BRSK1 (brain-specific kinase 1) by CaMKKalpha. Despite recent reports, we could not find any evidence that the alpha and/or beta subunits of AMPK formed a stable complex with CaMKKbeta. We also showed that increasing AMP concentrations in HeLa cells (which lack LKB1) had no effect on basal AMPK phosphorylation, but enhanced the ability of agents that increase intracellular Ca2+ to activate AMPK. This is consistent with the effect of AMP on phosphorylation of Thr172 being due to inhibition of dephosphorylation, and confirms that the effect of AMP is independent of the upstream kinase utilized.

5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside acutely stimulates skeletal muscle 2-deoxyglucose uptake in healthy men.

Activation of AMP-activated protein kinase (AMPK) in rodent muscle by exercise, metformin, 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR), and adiponectin increases glucose uptake. The aim of this study was to determine whether AICAR stimulates muscle glucose uptake in humans. We studied 29 healthy men (aged 26 +/- 8 years, BMI 25 +/- 4 kg/m(2) [mean +/- SD]). Rates of muscle 2-deoxyglucose (2DG) uptake were determined by measuring accumulation of total muscle 2DG (2DG and 2DG-6-phosphate) during a primed, continuous 2DG infusion. The effects of AICAR and exercise on muscle AMPK activity/phosphorylation and 2DG uptake were determined. Whole-body glucose disposal was compared before and during AICAR with the euglycemic-hyperinsulinemic clamp. Muscle 2DG uptake was linear over 9 h (R(2) = 0.88 +/- 0.09). After 3 h, 2DG uptake increased 2.1 +/- 0.8- and 4.7 +/- 1.7-fold in response to AICAR or bicycle exercise, respectively. AMPK alpha(1) and alpha(2) activity or AMPK phosphorylation was unchanged after 20 min or 3 h of AICAR, but AMPK phosphorylation significantly increased immediately and 3 h after bicycle exercise. AICAR significantly increased phosphorylation of extracellular signal-regulated kinase 1/2, but phosphorylation of beta-acetyl-CoA carboxylase, glycogen synthase, and protein kinase B or insulin receptor substrate-1 level was unchanged. Mean whole-body glucose disposal increased by 7% with AICAR from 9.3 +/- 0.6 to 10 +/- 0.6 mg x kg(-1) x min(-1) (P < 0.05). In healthy people, AICAR acutely stimulates muscle 2DG uptake with a minor effect on whole-body glucose disposal.

Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction.

Recent studies indicate that the LKB1 tumour suppressor protein kinase is the major "upstream" activator of the energy sensor AMP-activated protein kinase (AMPK). We have used mice in which LKB1 is expressed at only approximately 10% of the normal levels in muscle and most other tissues, or that lack LKB1 entirely in skeletal muscle. Muscle expressing only 10% of the normal level of LKB1 had significantly reduced phosphorylation and activation of AMPKalpha2. In LKB1-lacking muscle, the basal activity of the AMPKalpha2 isoform was greatly reduced and was not increased by the AMP-mimetic agent, 5-aminoimidazole-4-carboxamide riboside (AICAR), by the antidiabetic drug phenformin, or by muscle contraction. Moreover, phosphorylation of acetyl CoA carboxylase-2, a downstream target of AMPK, was profoundly reduced. Glucose uptake stimulated by AICAR or muscle contraction, but not by insulin, was inhibited in the absence of LKB1. Contraction increased the AMP:ATP ratio to a greater extent in LKB1-deficient muscles than in LKB1-expressing muscles. These studies establish the importance of LKB1 in regulating AMPK activity and cellular energy levels in response to contraction and phenformin.

PD98059 and U0126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway.

The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.

Regulation of hormone-sensitive lipase activity and Ser563 and Ser565 phosphorylation in human skeletal muscle during exercise.

Hormone-sensitive lipase (HSL) catalyses the hydrolysis of myocellular triacylglycerol (MCTG), which is a potential energy source during exercise. Therefore, it is important to elucidate the regulation of HSL activity in human skeletal muscle during exercise. The main purpose of the present study was to investigate the role of 5'AMP-activated protein kinase (AMPK) in the regulation of muscle HSL activity and Ser565 phosphorylation (the presumed AMPK target site) in healthy, moderately trained men during 60 min bicycling (65%). Alpha2AMPK activity during exercise was manipulated by studying subjects with either low (LG) or high (HG) muscle glycogen content. HSL activity was distinguished from the activity of other neutral lipases by immunoinhibition of HSL using an anti-HSL antibody. During exercise a 62% higher (P < 0.01) alpha2AMPK activity in LG than in HG was paralleled by a similar difference (61%, P < 0.01) in HSL Ser565 phosphorylation but without any difference between trials in HSL activity or MCTG hydrolysis. HSL activity was increased (117%, P < 0.05) at 30 min of exercise but not at 60 min of exercise. In both trials, HSL phosphorylation on Ser563 (a presumed PKA target site) was not increased by exercise despite a fourfold increase (P < 0.001) in plasma adrenaline. ERK1/2 phosphorylation was increased by exercise in both trials (P < 0.001) and was higher in LG than in HG both at rest and during exercise (P = 0.06). In conclusion, the present study suggests that AMPK phosphorylates HSL on Ser565 in human skeletal muscle during exercise with reduced muscle glycogen. Apparently, HSL Ser565 phosphorylation by AMPK during exercise had no effect on HSL activity. Alternatively, other factors including ERK may have counterbalanced any effect of AMPK on HSL activity.

CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations.

CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (gamma 2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine beta-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine beta-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives.

A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that is activated by low cellular energy status and affects a switch away from energy-requiring processes and toward catabolism. While it is primarily regulated by AMP and ATP, high muscle glycogen has also been shown to repress its activation. Mutations in the gamma2 and gamma3 subunit isoforms lead to arrhythmias associated with abnormal glycogen storage in human heart and elevated glycogen in pig muscle, respectively. A putative glycogen binding domain (GBD) has now been identified in the beta subunits. Coexpression of truncated beta subunits lacking the GBD with alpha and gamma subunits yielded complexes that were active and normally regulated. However, coexpression of alpha and gamma with full-length beta caused accumulation of AMPK in large cytoplasmic inclusions that could be counterstained with anti-glycogen or anti-glycogen synthase antibodies. These inclusions were not affected by mutations that increased or abolished the kinase activity and were not observed by using truncated beta subunits lacking the GBD. Our results suggest that the GBD binds glycogen and can lead to abnormal glycogen-containing inclusions when the kinase is overexpressed. These may be related to the abnormal glycogen storage bodies seen in heart disease patients with gamma2 mutations.

Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes.

In type 2 diabetes, insulin activation of muscle glycogen synthase (GS) is impaired. This defect plays a major role for the development of insulin resistance and hyperglycemia. In animal muscle, insulin activates GS by reducing phosphorylation at both NH(2)- and COOH-terminal sites, but the mechanism involved in human muscle and the defect in type 2 diabetes remain unclear. We studied the effect of insulin at physiological concentrations on glucose metabolism, insulin signaling and phosphorylation of GS in skeletal muscle from type 2 diabetic and well-matched control subjects during euglycemic-hyperinsulinemic clamps. Analysis using phospho-specific antibodies revealed that insulin decreases phosphorylation of sites 3a + 3b in human muscle, and this was accompanied by activation of Akt and inhibition of glycogen synthase kinase-3alpha. In type 2 diabetic subjects these effects of insulin were fully intact. Despite that, insulin-mediated glucose disposal and storage were reduced and activation of GS was virtually absent in type 2 diabetic subjects. Insulin did not decrease phosphorylation of sites 2 + 2a in healthy human muscle, whereas in diabetic muscle insulin infusion in fact caused a marked increase in the phosphorylation of sites 2 + 2a. This phosphorylation abnormality likely caused the impaired GS activation and glucose storage, thereby contributing to skeletal muscle insulin resistance, and may therefore play a pathophysiological role in type 2 diabetes.

Activity modes and modulation of the peptide-gated Na(+) channel of Helix neurones.

Unitary currents activated by steady-state applications of FMRFamide analogues have been recorded in outside-out patches from the C2 and F2 neurones of Helix aspersa. The bimodal conductance distribution ranged from 4 pS to 6 pS in patches in each neurone. Application of up to 10 microM cytochalasin B or D to 12 of the 41 inside-out patches tested stimulated activity of the larger conductance events and/or increased the amplitude of smaller conductance events, indicating a cytoskeletal influence on the channels. FMRFamide-gated currents were blocked by external Ca(2+) and Mg(2+), both with an IC(50) of about 1 mM. During continuous application of FMRFamide, clustering of events and/or the appearance of a mode of activity with a lower probability of being open (P(open)) suggested two different types of desensitization. More rarely, exceptionally high P(open) activity with low FMRFamide concentrations was seen. In some cases, there were long periods of high activity during which the channels gated normally with regular short sojourns in the fully closed state. In others, isolated long openings were seen which interrupted the normal gating pattern of the channels. Amiloride and its analogue 5-(N-ethyl- N-isopropyl) amiloride (EIPA) consistently caused channel block, but in some patches with a low background activity an increased P(open) was also observed in the presence of each drug.