Glucose-6-Phosphate Acts as an Extracellular Signal of SagS To Modulate Pseudomonas aeruginosa c-di-GMP Levels, Attachment, and Biofilm Formation.

In Pseudomonas aeruginosa, the orphan two-component sensor SagS contributes both to transition to biofilm formation and to biofilm cells gaining their heightened tolerance to antimicrobials. However, little is known about the identity of the signals or conditions sensed by SagS to induce the switch to the sessile, drug-tolerant mode of growth. Using a modified Biolog phenotype assay to screen for compounds that modulate attachment in a SagS-dependent manner, we identified glucose-6-phosphate to enhance attachment in a manner dependent on the glucose-6-phosphate concentration and SagS. The stimulatory effect was not limited to the attachment since glucose-6-phosphate likewise enhanced biofilm formation and also enhanced the expression of select biofilm marker genes. Moreover, exposure to glucose-6-phosphate coincided with decreased swarming motility but increased cellular cyclic-di-GMP (c-di-GMP) levels in biofilms. No such response was noted for compounds modulating attachment and biofilm formation in a manner independent of SagS. Modulation of c-di-GMP in response to glucose-6-phosphate was due to the diguanylate cyclase NicD, with NicD also being required for enhanced biofilm formation. The latter was independent of the sensory domain of NicD but dependent on NicD activity, SagS, and the interaction between NicD and SagS. Our findings indicate that glucose-6-phosphate likely mimics a signal or conditions sensed by SagS to activate its motile-sessile switch function. In addition, our findings provide new insight into the interfaces between the ligand-mediated two-component system signaling pathway and c-di-GMP levels.IMPORTANCE Pathogens sense and respond to signals and cues present in their environment, including host-derived small molecules to modulate the expression of their virulence repertoire. Here, we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa responds to glucose-6-phosphate. Since glucose-6-phosphate is primarily made available due to cell lysis, it is likely that glucose-6-phosphate represents a cross-kingdom cell-to-cell signal that enables P. aeruginosa to adapt to the (nutrient-poor) host environment by enhancing biofilm formation, cyclic-di-GMP, and the expression of genes linked to biofilm formation in a concentration- and SagS-dependent manner.

Hexokinase II dissociation alone cannot account for changes in heart mitochondrial function, morphology and sensitivity to permeability transition pore opening following ischemia.

We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6-phosphate at pH 6.3. Prior HK2 dissociation from pre- or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre- and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca2+].

ß-Glucose-1,6-Bisphosphate Stabilizes Pathological Phophomannomutase2 Mutants In Vitro and Represents a Lead Compound to Develop Pharmacological Chaperones for the Most Common Disorder of Glycosylation, PMM2-CDG.

A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, ß-glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. ß-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. ß-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.

An in-vivo pilot study into the effects of FDG-mNP in cancer in mice.

PURPOSE: Previously, fluorodeoxy glucose conjugated magnetite nanoparticles (FDG-mNPs) injected into cancer cells in conjunction with the application of magnetic hyperthermia have shown promise in new FDG-mNPs applications. The aim of this study was to determine potential toxic or unwanted effects involving both tumour cells and normal tissue in other organs when FDG-mNPs are administered intravenously or intratumourally in mice. MATERIALS AND METHODS: FDG-mNPs were synthesized. A group of six prostate-tumour bearing mice were injected with 23.42 mg/ml FDG-mNPs (intravenous injection, n = 3; intratumoural injection into the prostate tumour, n = 3). Mice were euthanized and histological sampling of tissue was conducted for the prostate tumour, as well as for lungs, lymph nodes, liver, kidneys, spleen, and brain, at 1 hour (n = 2) and 7 days (n = 4) post-injection. A second group of two normal (non-cancerous) mice received the same injection intravenously into the tail vein and were euthanised at 3 and 6 months post-injection, respectively, to investigate if FDG-mNPs remained in organs at those time points. RESULTS: In prostate-tumour bearing mice, FDG-mNPs concentrated in the prostate tumour, while relatively small amounts were found in the organs of other tissues, particularly the spleen and the liver; FDG-mNP concentrations decreased over time in all tissues. In normal mice, no detrimental effects were found in either mouse at 3 or 6 months. CONCLUSION: Intravenous or intratumoural FDG-mNPs can be safely administered for effective cancer cell destruction. Further research on the clinical utility of FDG-mNPs will be conducted by applying hyperthermia in conjunction with FDG-mNPs in mice.

Inter-subject FDG PET Brain Networks Exhibit Multi-scale Community Structure with Different Normalization Techniques.

Inter-subject networks are used to model correlations between brain regions and are particularly useful for metabolic imaging techniques, like 18F-2-deoxy-2-(18F)fluoro-D-glucose (FDG) positron emission tomography (PET). Since FDG PET typically produces a single image, correlations cannot be calculated over time. Little focus has been placed on the basic properties of inter-subject networks and if they are affected by group size and image normalization. FDG PET images were acquired from rats (n = 18), normalized by whole brain, visual cortex, or cerebellar FDG uptake, and used to construct correlation matrices. Group size effects on network stability were investigated by systematically adding rats and evaluating local network connectivity (node strength and clustering coefficient). Modularity and community structure were also evaluated in the differently normalized networks to assess meso-scale network relationships. Local network properties are stable regardless of normalization region for groups of at least 10. Whole brain-normalized networks are more modular than visual cortex- or cerebellum-normalized network (p < 0.00001); however, community structure is similar at network resolutions where modularity differs most between brain and randomized networks. Hierarchical analysis reveals consistent modules at different scales and clustering of spatially-proximate brain regions. Findings suggest inter-subject FDG PET networks are stable for reasonable group sizes and exhibit multi-scale modularity.

Mitochondria-Bound Hexokinase (mt-HK) Activity Differ in Cortical and Hypothalamic Synaptosomes: Differential Role of mt-HK in H2O2 Depuration.

Glucose and oxygen are vital for the brain, as these molecules provide energy and metabolic intermediates that are necessary for cell function. The glycolysis pathway and mitochondria play a pivotal role in cell energy metabolism, which is closely related to reactive oxygen species (ROS) production. Hexokinase (HK) is a key enzyme involved in glucose metabolism that modulates the level of brain mitochondrial ROS by recycling ADP for oxidative phosphorylation (OxPhos). Here, we hypothesize that the control of mitochondrial metabolism by hexokinase differs in distinct areas of the brain, such as the cortex and hypothalamus, in which ROS might function as signaling molecules. Thus, we investigated mitochondrial metabolism of synaptosomes derived from both brain regions. Cortical synaptosomes (CSy) show a predominance of glutamatergic synapses, while in the hypothalamic synaptosomes (HSy), the GABAergic synapses predominate. Significant differences of oxygen consumption and ROS production were related to higher mitochondrial complex II activity (succinate dehydrogenase-SDH) in CSy rather than to mitochondrial number. Mitochondrial HK (mt-HK) activity was higher in CSy than in HSy regardless the substrate added. Mitochondrial O2 consumption related to mt-HK activation by 2-deoxyglucose was also higher in CSy. In the presence of substrate for complex II, the activation of synaptosomal mt-HK promoted depuration of ROS in both HSy and CSy, while ROS depuration did not occur in HSy when substrate for complex I was used. The impact of the mt-HK inhibition by glucose-6-phosphate (G6P) was the same in synaptosomes from both areas. Together, the differences found between CSy and HSy indicate specific roles of mt-HK and SDH on the metabolism of each brain region, what probably depends on the main metabolic route that is used by the neurons.

Frequency and Mechanisms of Spontaneous Fosfomycin Nonsusceptibility Observed upon Disk Diffusion Testing of Escherichia coli.

Fosfomycin maintains activity against most Escherichia coli clinical isolates, but the growth of E. coli colonies within the zone of inhibition around the fosfomycin disk is occasionally observed upon susceptibility testing. We aimed to estimate the frequency of such nonsusceptible inner colony mutants and identify the underlying resistance mechanisms. Disk diffusion testing of fosfomycin was performed on 649 multidrug-resistant E. coli clinical isolates collected between 2011 and 2015. For those producing inner colonies inside the susceptible range, the parental strains and their representative inner colony mutants were subjected to MIC testing, whole-genome sequencing, reverse transcription-quantitative PCR (qRT-PCR), and carbohydrate utilization studies. Of the 649 E. coli clinical isolates, 5 (0.8%) consistently produced nonsusceptible inner colonies. Whole-genome sequencing revealed the deletion of uhpT encoding hexose-6-phosphate antiporter in 4 of the E. coli inner colony mutants, while the remaining mutant contained a nonsense mutation in uhpA The expression of uhpT was absent in the mutant strains with uhpT deletion and was not inducible in the strain with the uhpA mutation, unlike in its parental strain. All 5 inner colony mutants had reduced growth on minimal medium supplemented with glucose-6-phosphate. In conclusion, fosfomycin-nonsusceptible inner colony mutants can occur due to the loss of function or induction of UhpT but are rare among multidrug-resistant E. coli clinical strains. Considering that these mutants carry high biological costs, we suggest that fosfomycin susceptibility of strains that generate inner colony mutants can be interpreted on the basis of the zone of inhibition without accounting for the inner colonies.

Kinetic Modelling of Infection Tracers [18F]FDG, [68Ga]Ga-Citrate, [11C]Methionine, and [11C]Donepezil in a Porcine Osteomyelitis Model.

Introduction: Positron emission tomography (PET) is increasingly applied for infection imaging using [18F]FDG as tracer, but uptake is unspecific. The present study compares the kinetics of [18F]FDG and three other PET tracers with relevance for infection imaging. Methods: A juvenile porcine osteomyelitis model was used. Eleven pigs underwent PET/CT with 60-minute dynamic PET imaging of [18F]FDG, [68Ga]Ga-citrate, [11C]methionine, and/or [11C]donepezil, along with blood sampling. For infectious lesions, kinetic modelling with one- and two-tissue-compartment models was conducted for each tracer. Results: Irreversible uptake was found for [18F]FDG and [68Ga]Ga-citrate; reversible uptake was found for [11C]methionine (two-tissue model) and [11C]donepezil (one-tissue model). The uptake rate for [68Ga]Ga-citrate was slow and diffusion-limited. For the other tracers, the uptake rate was primarily determined by perfusion (flow-limited uptake). Net uptake rate for [18F]FDG and distribution volume for [11C]methionine were significantly higher for infectious lesions than for correspondingly noninfected tissue. For [11C]donepezil in pigs, labelled metabolite products appeared to be important for the analysis. Conclusions: The kinetics of the four studied tracers in infection was characterized. For clinical applications, [18F]FDG remains the first-choice PET tracer. [11C]methionine may have a potential for detecting soft tissue infections. [68Ga]Ga-citrate and [11C]donepezil were not found useful for imaging of osteomyelitis.

Roles of electro-acupuncture in glucose metabolism as assessed by 18F-FDG/PET imaging and AMPKα phosphorylation in rats with ischemic stroke.

Targeted energy metabolism balance contributes to neural survival during ischemic stroke. Herein, we tested the hypothesis that electro­acupuncture (EA) can enhance cerebral glucose metabolism assessed by 18F­fluorodeoxyglucose/positron emission tomography (18F­FDG/PET) imaging to prevent propagation of tissue damage and improve neurological outcome in rats subjected to ischemia and reperfusion injury. Rats underwent middle cerebral artery occlusion (MCAO) and received EA treatment at the LI11 and ST36 acupoints or non­acupoint treatment once a day for 7 days. After EA treatment, a significant reduction in the infarct volume was determined by T2­weighted imaging, accompanied by the functional recovery in CatWalk and Rota-rod performance. Moreover, EA promoted higher glucose metabolism in the caudate putamen (CPu), motor cortex (MCTX), somatosensory cortex (SCTX) regions as assessed by animal 18F­FDG/PET imaging, suggesting that three­brain regional neural activity was enhanced by EA. In addition, the AMP­activated protein kinase α (AMPKα) in the CPu, MCTX and SCTX regions was phosphorylated at threonine 172 (Thr172) after ischemic injury; however, phosphorylation of AMPK was further increased by EA. These results indicate that EA could promote AMPKα phosphorylation of the CPu, MCTX and SCTX regions to enhance neural activity and motor functional recovery after ischemic stroke.

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation.

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.

Disrupted brain metabolic connectivity in a 6-OHDA-induced mouse model of Parkinson's disease examined using persistent homology-based analysis.

Movement impairments in Parkinson's disease (PD) are caused by the degeneration of dopaminergic neurons and the consequent disruption of connectivity in the cortico-striatal-thalamic loop. This study evaluated brain metabolic connectivity in a 6-Hydroxydopamine (6-OHDA)-induced mouse model of PD using (18)F-fluorodeoxy glucose positron emission tomography (FDG PET). Fourteen PD-model mice and ten control mice were used for the analysis. Voxel-wise t-tests on FDG PET results yielded no significant regional metabolic differences between the PD and control groups. However, the PD group showed lower correlations between the right caudoputamen and the left caudoputamen and right visual cortex. Further network analyses based on the threshold-free persistent homology framework revealed that brain networks were globally disrupted in the PD group, especially between the right auditory cortex and bilateral cortical structures and the left caudoputamen. In conclusion, regional glucose metabolism of PD was preserved, but the metabolic connectivity of the cortico-striatal-thalamic loop was globally impaired in PD.

Stereotactic Comparison Study of (18)F-Alfatide and (18)F-FDG PET Imaging in an LLC Tumor-Bearing C57BL/6 Mouse Model.

This study aimed to stereotactically compare the PET imaging performance of (18)F-Alfatide ((18)F-ALF-NOTA-PRGD2, denoted as (18)F-Alfatide) and (18)F-fluorodeoxyglucose (FDG) and immunohistochemistry (IHC) staining in Lewis lung carcinoma (LLC) tumor-bearing C57BL/6 mouse model. (18)F-FDG standard uptake values (SUVs) were higher than (18)F-Alfatide SUVs in tumors, most of the normal tissues and organs except for the bladder. Tumor-to-brain, tumor-to-lung, and tumor-to-heart ratios of (18)F-Alfatide PET were significantly higher than those of (18)F-FDG PET (P < 0.001). The spatial heterogeneity of the tumors was detected, and the tracer accumulation enhanced from the outer layer to the inner layer consistently using the two tracers. The parameters of the tumors were significantly correlated with each other between (18)F-FDG SUV and GLUT-1 (R = 0.895, P < 0.001), (18)F-Alfatide SUV and αvß3 (R = 0.595, P = 0.019), (18)F-FDG SUV and (18)F-Alfatide SUV (R = 0.917, P < 0.001), and GLUT-1 and αvß3 (R = 0.637, P = 0.011). Therefore, (18)F-Alfatide PET may be an effective tracer for tumor detection, spatial heterogeneity imaging and an alternative supplement to (18)F-FDG PET, particularly for patients with enhanced characteristics in the brain, chest tumors or diabetes, meriting further study.

2-Deoxy-d-glucose is a potent inhibitor of biofilm growth in Escherichia coli.

Escherichia coli strain 15 (ATCC 9723), which forms robust biofilms, was grown under optimal biofilm conditions in NaCl-free Luria-Bertani broth (LB*) or in LB* supplemented with one of the non-metabolizable analogues 2-deoxy-d-glucose (2DG), methyl α-d-mannopyranoside (αMM), or methyl α-d-glucopyranoside (αMG). Biofilm growth was inhibited by mannose analogue 2DG even at very low concentration in unbuffered medium, and the maximal inhibition was enhanced in the presence of either 100 mM KPO4 or 100 mM MOPS, pH 7.5; in buffered medium, concentrations of 0.02 % (1.2 mM) or more inhibited growth nearly completely. In contrast, mannose analogue αMM, which should not be able to enter the cells but has been reported to inhibit biofilm growth by binding to FimH, did not exhibit strong inhibition even at concentrations up to 1.8 % (108 mM). The glucose analogue αMG inhibited biofilm growth, but much less strongly than did 2DG. None of the analogues inhibited planktonic growth or caused a change in pH of the unbuffered medium. Similar inhibitory effects of the analogues were observed in minimal medium. The effects were not strain-specific, as 2DG and αMG also inhibited the weak biofilm growth of E. coli K12.

Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb.

[18F]FDG Uptake in the Aortic Wall Smooth Muscle of Atherosclerotic Plaques in the Simian Atherosclerosis Model.

Atherosclerosis is a self-sustaining inflammatory fibroproliferative disease that progresses in discrete stages and involves a number of cell types and effector molecules. Recently, [18F]fluoro-2-deoxy-D-glucose- ([18F]FDG-) positron emission tomography (PET) has been suggested as a tool to evaluate atherosclerotic plaques by detecting accumulated macrophages associated with inflammation progress. However, at the cellular level, it remains unknown whether only macrophages exhibit high uptake of [18F]FDG. To identify the cellular origin of [18F]FDG uptake in atherosclerotic plaques, we developed a simian atherosclerosis model and performed PET and ex vivo macro- and micro-autoradiography (ARG). Increased [18F]FDG uptake in the aortic wall was observed in high-cholesterol diet-treated monkeys and WHHL rabbits. Macro-ARG of [18F]FDG in aortic sections showed that [18F]FDG was accumulated in the media and intima in the simian model as similar to that in WHHL rabbits. Combined analysis of micro-ARG with immunohistochemistry in the simian atherosclerosis model revealed that most cellular [18F]FDG uptake observed in the media was derived not only from the infiltrated macrophages in atherosclerotic plaques but also from the smooth muscle cells (SMCs) of the aortic wall in atherosclerotic lesions.

Evaluation of Avulsion-Induced Neuropathology in Rat Spinal Cords with 18F-FDG Micro-PET/CT.

Brachial plexus root avulsion (BPRA) leads to dramatic motoneuron death and glial reactions in the corresponding spinal segments at the late stage of injury. To protect spinal motoneurons, assessment of the affected spinal segments should be done at an earlier stage of the injury. In this study, we employed 18F-FDG small-animal PET/CT to assess the severity of BPRA-induced cervical spinal cord injuries. Adult Sprague-Dawley rats were randomly treated and divided into three groups: Av+NS (brachial plexus root avulsion (Av) treated with normal saline), Av+GM1 (treated with monosialoganglioside), and control. At time points of 3 day (d), 1 week (w), 2 w, 4 w and 8 w post-injury, 18F-FDG micro-PET/CT scans and neuropathology assessments of the injured spinal roots, as well as the spinal cord, were performed. The outcomes of the different treatments were compared. The results showed that BPRA induced local bleeding and typical Wallerian degeneration of the avulsed roots accompanied by 18F-FDG accumulations at the ipsilateral cervical intervertebral foramen. BPRA-induced astrocyte reactions and overexpression of neuronal nitric oxide synthase in the motoneurons correlated with higher 18F-FDG uptake in the ipsilateral cervical spinal cord during the first 2 w post-injury. The GM1 treatment reduced BPRA-induced astrocyte reactions and inhibited the de novo nNOS expressions in spinal motoneurons. The GM1 treatment also protected spinal motoneurons from avulsion within the first 4 w post-injury. The data from this study suggest that 18F-FDG PET/CT could be used to assess the severity of BPRA-induced primary and secondary injuries in the spinal cord. Furthermore, GM1 is an effective drug for reducing primary and secondary spinal cord injuries following BPRA.

Mutation in the γ2-subunit of AMP-activated protein kinase stimulates cardiomyocyte proliferation and hypertrophy independent of glycogen storage.

RATIONALE: AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory γ2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target. OBJECTIVE: We sought to identify the pro-growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage. METHODS AND RESULTS: In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate-stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor-signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth. CONCLUSIONS: Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of γ2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.

An insect gut environment reveals the induction of a new sugar-phosphate sensor system in Bacillus cereus.

Bacteria survive under various conditions by sensing stimuli triggering specific adaptive physiological responses, which are often based on membrane-integrated sensors connected to a cytoplasmic regulator. Recent studies reveal that mucus glycans may act as signal molecules for two-component systems involved in intestinal colonization. Bacillus cereus, a human and insect opportunistic pathogen was used to identify bacterial factors expressed in an insect gut infection model. The screen revealed a promoter involved in the expression of a gene with so far unknown functions. A search for gut-related compounds, inducing its transcription, identified glucose-6-phosphate as an activation signal. The gene is part of a five-gene cluster, including a two-component system. Interestingly such five gene loci are conserved in the pathogenic Bacillus group as well as in various Clostridia bacteria and are with analogy to other multi-component sensor systems in enteropathogenic bacteria, such as E. coli. Thus our results provide insights into the function of two-component and auxiliary sensor systems in host-microbe interactions and opens up possible investigations of such systems in other gut associated bacteria.

α-Bromophosphonate analogs of glucose-6-phosphate are inhibitors of glucose-6-phosphatase.

Glucose-6-phosphatase (G6Pase) is an essential metabolic enzyme that has upregulated activity in Type II diabetes. Synthetic analogs of the G6Pase substrate, glucose-6-phosphate (G6P), may provide new tools to probe enzyme activity, or lead to specific inhibitors of glycosylphosphatase enzymes. Here we have developed synthetic routes to a panel of non-hydrolyzable G6P analogs containing α-bromo, α,α-dibromo, and α-bromo-α,ß-unsaturated phosphonates compatible with a carbohydrate nucleus. We confirm that these functionalities have potency as inhibitors of G6Pase in vitro, providing a series of new phosphate isosteres that can be exploited for inhibitor design.

Depletion of glycolytic intermediates plays a key role in glucose-phosphate stress in Escherichia coli.

In bacteria like Escherichia coli, the accumulation of glucose-6-phosphate (G6P) or its analogs such as α-methyl glucoside-6-phosphate (αMG6P) results in stress that appears in the form of growth inhibition. The small RNA SgrS is an essential part of the response that helps E. coli combat glucose-phosphate stress; the growth of sgrS mutants during stress caused by αMG is significantly impaired. The cause of this stress is not currently known but may be due to either toxicity of accumulated sugar-phosphates or to depletion of metabolic intermediates. Here, we present evidence that glucose-phosphate stress results from depletion of glycolytic intermediates. Addition of glycolytic compounds like G6P and fructose-6-phosphate rescues the αMG growth defect of an sgrS mutant. These intermediates also markedly decrease induction of the stress response in both wild-type and sgrS strains grown with αMG, implying that cells grown with these intermediates experience less stress. Moreover, αMG transport assays confirm that G6P relieves stress even when αMG is taken up by the cell, strongly suggesting that accumulated αMG6P per se does not cause stress. We also report that addition of pyruvate during stress has a novel lethal effect on the sgrS mutant, resulting in cell lysis. The phosphoenolpyruvate (PEP) synthetase PpsA, which converts pyruvate to PEP, can confer resistance to pyruvate-induced lysis when ppsA is ectopically expressed in the sgrS mutant. Taken as a whole, these results provide the strongest evidence thus far that depletion of glycolytic intermediates is at the metabolic root of glucose-phosphate stress.