Metabolic and cardiac adaptation to chronic pharmacologic blockade of facilitative glucose transport in murine dilated cardiomyopathy and myocardial ischemia

Abstract GLUT transgenic and knockout mice have provided valuable insight into the role of facilitative glucose transporters (GLUTs) in cardiovascular and metabolic disease, but compensatory physiological changes can hinder interpretation of these models. To determine whether adaptations occur in response to GLUT inhibition in the failing adult heart, we chronically treated TG9 mice, a transgenic model of dilated cardiomyopathy and heart failure, with the GLUT inhibitor ritonavir. Glucose tolerance was significantly improved with chronic treatment and correlated with decreased adipose tissue retinol binding protein 4 (RBP4) and resistin. A modest improvement in lifespan was associated with decreased cardiomyocyte brain natriuretic peptide (BNP) expression, a marker of heart failure severity. GLUT1 and −12 protein expression was significantly increased in left ventricular (LV) myocardium in ritonavir-treated animals. Supporting a switch from fatty acid to glucose utilization in these tissues, fatty acid transporter CD36 and fatty acid transcriptional regulator peroxisome proliferator-activated receptor α (PPARα) mRNA were also decreased in LV and soleus muscle. Chronic ritonavir also increased cardiac output and dV/dt-d in C57Bl/6 mice following ischemia-reperfusion injury. Taken together, these data demonstrate compensatory metabolic adaptation in response to chronic GLUT blockade as a means to evade deleterious changes in the failing heart

Identification of druggable small molecule antagonists of the Plasmodium falciparum hexose transporter PfHT and assessment of ligand access to the glucose permeation pathway via FLAG-mediated protein engineering

Although the Plasmodium falciparum hexose transporter PfHT has emerged as a promising target for anti-malarial therapy, previously identified small-molecule inhibitors have lacked promising drug-like structural features necessary for development as clinical therapeutics. Taking advantage of emerging insight into structure/function relationships in homologous facilitative hexose transporters and our novel high throughput screening platform, we investigated the ability of compounds satisfying Lipinksi rules for drug likeness to directly interact and inhibit PfHT. The Maybridge HitFinder chemical library was interrogated by searching for compounds that reduce intracellular glucose by >40% at 10 μM. Testing of initial hits via measurement of 2-deoxyglucose (2-DG) uptake in PfHT over-expressing cell lines identified 6 structurally unique glucose transport inhibitors. WU-1 (3-(2,6-dichlorophenyl)-5-methyl-N-[2-(4-methylbenzenesulfonyl)ethyl]-1,2-oxazole-4-carboxamide) blocked 2-DG uptake (IC50 = 5.8 ± 0.6 μM) with minimal effect on the human orthologue class I (GLUTs 1–4), class II (GLUT8) and class III (GLUT5) facilitative glucose transporters. WU-1 showed comparable potency in blocking 2-DG uptake in freed parasites and inhibiting parasite growth, with an IC50 of 6.1 ± 0.8 μM and EC50 of 5.5 ± 0.6 μM, respectively. WU-1 also directly competed for N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) binding and inhibited the transport of D-glucose with an IC50 of 5.9 ± 0.8 μM in liposomes containing purified PfHT. Kinetic analysis revealed that WU-1 acts as a non-competitive inhibitor of zero-trans D-fructose uptake. Decreased potency for WU-1 and the known endofacial ligand cytochalasin B was observed when PfHT was engineered to contain an N-terminal FLAG tag. This modification resulted in a concomitant increase in affinity for 4,6-O-ethylidene-α-D-glucose, an exofacially directed transport antagonist, but did not alter the Km for 2-DG. Taken together, these data are consistent with a model in which WU-1 binds preferentially to the transporter in an inward open conformation and support the feasibility of developing potent and selective PfHT antagonists as a novel class of anti-malarial drugs.</div

Muscle-specific ablation of glucose transporter 1 (GLUT1) does not impair basal or overload-stimulated skeletal muscle glucose uptake

Glucose transporter 1 (GLUT1) is believed to solely mediate basal (insulin-independent) glucose uptake in skeletal muscle; yet recent work has demonstrated that mechanical overload, a model of resistance exercise training, increases muscle GLUT1 levels. The primary objective of this study was to determine if GLUT1 is necessary for basal or overload-stimulated muscle glucose uptake. Muscle-specific GLUT1 knockout (mGLUT1KO) mice were generated and examined for changes in body weight, body composition, metabolism, systemic glucose regulation, muscle glucose transporters, and muscle

ICESTARS : integrated circuit/EM simulation and design technologies for advanced radio systems-on-chip

ICESTARS solved a series of critical issues in the currently available infrastructure for the design and simulation of new and highly-complex Radio Frequency (RF) front ends operating beyond 10 and up to 100 GHz. Future RF designs demand an increasing blend of analog and digital functionalities. The super and extremely high frequency (SHF, 3-30GHz, and EHF, 30-300GHz) ranges will be used to accomplish future demands for higher capacity channels. With todays frequency bands of approximately 1 to 3 GHz it is impossible to realize extremely high data transfer rates. Only a new generation of CAD and EDA tools will ensure the realization of complex nanoscale designs. It necessitates both new modeling approaches and new mathematical solution procedures for differential equations with largely differing time scales, analysis of coupled systems of DAEs (circuit equations) and PDEs (Maxwell equations for electromagnetic couplings) plus numerical simulations with mixed analog and digital signals. In ICESTARS new techniques and mathematical models working in highly integrated environments were developed to resolve this dilemma. The ICESTARS research area covered the three domains of RF design: (1) time-domain techniques, (2) frequency-domain techniques, and (3) EM analysis and coupled EM circuit analysis. The ICESTARS consortium comprised two industrial partners (NXP Semiconductors, Infineon Technologies AG), two SMEs (Magwel, AWR-APLAC) and five universities (Upper Austria, Cologne, Oulu, Wuppertal, Aalto), involving mathematicians, electronic engineers, and software engineers

HIV Protease Inhibitors Act as Competitive Inhibitors of the Cytoplasmic Glucose Binding Site of GLUTs with Differing Affinities for GLUT1 and GLUT4

The clinical use of several first generation HIV protease inhibitors (PIs) is associated with the development of insulin resistance. Indinavir has been shown to act as a potent reversible noncompetitive inhibitor of zero-trans glucose influx via direct interaction with the insulin responsive facilitative glucose transporter GLUT4. Newer drugs within this class have differing effects on insulin sensitivity in treated patients. GLUTs are known to contain two distinct glucose-binding sites that are located on opposite sides of the lipid bilayer. To determine whether interference with the cytoplasmic glucose binding site is responsible for differential effects of PIs on glucose transport, intact intracellular membrane vesicles containing GLUT1 and GLUT4, which have an inverted transporter orientation relative to the plasma membrane, were isolated from 3T3-L1 adipocytes. The binding of biotinylated ATB-BMPA, a membrane impermeable bis-mannose containing photolabel, was determined in the presence of indinavir, ritonavir, atazanavir, tipranavir, and cytochalasin b. Zero-trans 2-deoxyglucose transport was measured in both 3T3-L1 fibroblasts and primary rat adipocytes acutely exposed to these compounds. PI inhibition of glucose transport correlated strongly with the PI inhibition of ATB-BMPA/transporter binding. At therapeutically relevant concentrations, ritonavir was not selective for GLUT4 over GLUT1. Indinavir was found to act as a competitive inhibitor of the cytoplasmic glucose binding site of GLUT4 with a KI of 8.2 µM. These data establish biotinylated ATB-BMPA as an effective probe to quantify accessibility of the endofacial glucose-binding site in GLUTs and reveal that the ability of PIs to block this site differs among drugs within this class. This provides mechanistic insight into the basis for the clinical variation in drug-related metabolic toxicity

An Integrated Strategy to Study Muscle Development and Myofilament Structure in Caenorhabditis elegans

A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known

Ligand-Induced Movements of Inner Transmembrane Helices of Glut1 Revealed by Chemical Cross-Linking of Di-Cysteine Mutants

The relative orientation and proximity of the pseudo-symmetrical inner transmembrane helical pairs 5/8 and 2/11 of Glut1 were analyzed by chemical cross-linking of di-cysteine mutants. Thirteen functional di-cysteine mutants were created from a C-less Glut1 reporter construct containing cysteine substitutions in helices 5 and 8 or helices 2 and 11. The mutants were expressed in Xenopus oocytes and the sensitivity of each mutant to intramolecular cross-linking by two homobifunctional thiol-specific reagents was ascertained by protease cleavage followed by immunoblot analysis. Five of 9 mutants with cysteine residues predicted to lie in close proximity to each other were susceptible to cross-linking by one or both reagents. None of 4 mutants with cysteine substitutions predicted to lie on opposite faces of their respective helices was susceptible to cross-linking. Additionally, the cross-linking of a di-cysteine pair (A70C/M420C, helices 2/11) predicted to lie near the exoplasmic face of the membrane was stimulated by ethylidene glucose, a non-transported glucose analog that preferentially binds to the exofacial substrate-binding site, suggesting that the binding of this ligand stimulates the closure of helices at the exoplasmic face of the membrane. In contrast, the cross-linking of a second di-cysteine pair (T158C/L325, helices 5/8), predicted to lie near the cytoplasmic face of the membrane, was stimulated by cytochalasin B, a glucose transport inhibitor that competitively inhibits substrate efflux, suggesting that this compound recruits the transporter to a conformational state in which closure of inner helices occurs at the cytoplasmic face of the membrane. This observation provides a structural explanation for the competitive inhibition of substrate efflux by cytochalasin B. These data indicate that the binding of competitive inhibitors of glucose efflux or influx induce occluded states in the transporter in which substrate is excluded from the exofacial or endofacial binding site

A Phosphatidylinositol 3-Kinase/Protein Kinase B-independent Activation of Mammalian Target of Rapamycin Signaling Is Sufficient to Induce Skeletal Muscle Hypertrophy

Overexpression of Rheb activates mTOR signaling via a PI3K/PKB-independent mechanism and is sufficient to induce skeletal muscle hypertrophy. The hypertrophic effects of Rheb are driven through a rapamycin-sensitive (RS) mechanism, mTOR is the RS element that confers the hypertrophy and the kinase activity of mTOR is necessary for this event

Integrin α PAT-2/CDC-42 Signaling Is Required for Muscle-Mediated Clearance of Apoptotic Cells in Caenorhabditis elegans

Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin α subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2–mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin α subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180)/CED-12 (ELMO) or CED-6 (GULP) respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level

Elevated phosphorylation and activation of PDK-1/AKT pathway in human breast cancer

Activation of kinases signalling pathways contributes to various malignant phenotypes in human cancers, including breast tumour. To examine the possible activation of these signalling molecules, we examined the phosphorylation status in 12 protein kinases and transcription factors in normal primary human mammary epithelial cells, telomerase-immortalised human breast epithelial cell line, and two breast cancer lines, MDA-MB-468 and MCF-7, using Kinexus phosphorylated protein screening assays. The phosphorylation of FAK, mTOR, p70S6K, and PDK-1 were elevated in both breast cancer cell lines, whereas the phosphorylation of AKT, EGFR, ErbB2/Her2, PDGFR, Shc, and Stat3 were elevated in only one breast cancer line compared to normal primary mammary epithelial cells and telomerase-immortalised breast epithelial cells. The same findings were confirmed by Western blotting and by kinase assays. We further substantiated the phosphorylation status of these molecules in tissue microarray slides containing 89 invasive breast cancer tissues as well as six normal mammary tissues with immunohistochemistry staining using phospho-specific antibodies. Consistent findings were obtained as greater than 70% of invasive breast carcinomas expressed moderate to high levels of phosphorylated PDK-1, AKT, p70S6K, and EGFR. In sharp contrast, phosphorylation of the same proteins was nearly undetectable or was at low levels in normal mammary tissues under the same assay. Elevated phosphorylation of PDK-1, AKT, mTOR, p70S6K, S6, EGFR, and Stat3 were highly associated with invasive breast tumours (P<0.05). Taken together, our results suggest that activation of these kinase pathways by phosphorylation may in part account for molecular pathogenesis of human breast carcinoma. Particularly, moderate to high level of PDK-1 phosphorylation was found in 86% of high-grade metastasised breast tumours. This is the first report demonstrating phosphorylation of PDK-1 is frequently elevated in breast cancer with concomitantly increased phosphorylation of downstream kinases, including AKT, mTOR, p70S6K, S6, and Stat3. This finding thus suggested PDK-1 may promote oncogenesis in part through the activation of AKT and p70S6K and rationalised that PDK-1 as well as downstream components of PDK-1 signalling pathway may be promising therapeutic targets to treat breast cancer