Conditioned medium from hypoxia-treated adipocytes renders muscle cells insulin resistant

Adipose tissue hypoxia is an early phenotype in obesity, associated with macrophage infiltration and local inflammation. Here we test the hypothesis that adipocytes in culture respond to a hypoxic environment with the release of pro-inflammatory factors that stimulate macrophage migration and cause muscle insulin resistance. 3T3-L1 adipocytes cultured in a 1% O2 atmosphere responded with a classic hypoxia response by elevating protein expression of HIF-1α. This was associated with elevated mRNA expression and peptide release of cytokines TNFα, IL-6 and the chemokine monocyte chemoattractant protein-1 (MCP-1). The mRNA and protein expression of the anti-inflammatory adipokine adiponectin was reduced. Conditioned medium from hypoxia-treated adipocytes (CM-H), inhibited insulin-stimulated and raised basal cell surface levels of GLUT4myc stably expressed in C2C12 myotubes. Insulin stimulation of Akt and AS160 phosphorylation, key regulators of GLUT4myc exocytosis, was markedly impaired. CM-H also caused activation of JNK and S6K, and elevated serine phosphorylation of IRS1 in the C2C12 myotubes. These effects were implicated in reducing propagation of insulin signaling to Akt and AS160. Heat inactivation of CM-H reversed its dual effects on GLUT4myc traffic in muscle cells. Interestingly, antibody-mediated neutralization of IL-6 in CM-H lowered its effect on both the basal and insulin-stimulated cell surface GLUT4myc compared to unmodified CM-H. IL-6 may have regulated GLUT4myc traffic through its action on AMPK. Additionally, antibody-mediated neutralization of MCP-1 partly reversed the inhibition of insulin-stimulated GLUT4myc exocytosis caused by unmodified CM-H. In Transwell co-culture, hypoxia-challenged adipocytes attracted RAW 264.7 macrophages, consistent with elevated release of MCP-1 from adipocytes during hypoxia. Neutralization of MCP-1 in adipocyte CM-H prevented macrophage migration towards it and partly reversed the effect of CM-H on insulin response in muscle cells. We conclude that adipose tissue hypoxia may be an important trigger of its inflammatory response observed in obesity, and the elevated chemokine MCP-1 may contribute to increased macrophage migration towards adipose tissue and subsequent decreased insulin responsiveness of glucose uptake in muscle.     

PKCε regulates contraction‐stimulated GLUT4 traffic in skeletal muscle cells

The signaling pathways that stimulate glucose uptake in response to muscle contraction are not well defined. Recently, we showed that carbachol, an acetylcholine analog, stimulates contraction of C2C12 myotube cultures and the rapid arrival of myc‐epitope tagged GLUT4 glucose transporters at the cell surface. Here, we explore a role for protein kinase C (PKC) in regulating GLUT4 traffic. Cell surface carbachol‐induced GLUT4myc levels were partly inhibited by the conventional/novel PKC inhibitors GF‐109203X, Gö6983, and Ro‐31‐8425 but not by the conventional PKC inhibitor Gö6976. C2C12 myotubes expressed several novel isoforms of PKC mRNA with PKCδ and PKCε in greater abundance. Carbachol stimulated phosphorylation of PKC isoforms and translocation of PKCδ and PKCε to membranes within 5 min. However, only a peptidic inhibitor of PKCε translocation (myristoylated‐EAVSLKPT), but not one of PKCδ (myristoylated‐SFNSYELGSL), prevented the GLUT4myc response to carbachol. Significant participation of PKCε in the carbachol‐induced gain of GLUT4myc at the surface of C2C12 myotubes was further supported through siRNA‐mediated PKCε protein knockdown. These findings support a role for novel PKC isoforms, especially PKCε, in contraction‐stimulated GLUT4 traffic in muscle cells. J. Cell. Physiol. 226: 173–180, 2010. © 2010 Wiley‐Liss, Inc.     

Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells

Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase–dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)–MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP–MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP–MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA–mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane.     

Synthetic cannabinoids: Analysis and metabolites

Cannabimimetics (commonly referred to as synthetic cannabinoids), a group of compounds encompassing a wide range of chemical structures, have been developed by scientists with the hope of achieving selectivity toward one or the other of the cannabinoid receptors CB1 and CB2. The goal was to have compounds that could possess high therapeutic activity without many side effects. However, underground laboratories have used the information generated by the scientific community to develop these compounds for illicit use as marijuana substitutes. This chapter reviews the different classes of these “synthetic cannabinoids” with particular emphasis on the methods used for their identification in the herbal products with which they are mixed and identification of their metabolites in biological specimens.     

Reciprocal Regulation of Endocytosis and Metabolism

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe³⁺, K⁺) or efflux (e.g., Na⁺) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis.Interactions between the cell and its environment obligatorily involve events at the plasma membrane. Cell-surface proteins mediate nutrient uptake, product release, and the sensing of environmental changes, including signals from other cells. Appropriate sensing and response to extracellular cues is essential for the individual cell’s survival and for the coordinated cellular behavior in multicellular organisms. Accordingly, maintenance and dynamics of membrane proteins are fundamental mechanisms of cellular homeostasis and survival.Most plasma membrane proteins are in defined equilibria with intracellular endosomal compartments, such that the amount of a given protein at the plasma membrane is determined by the balance of its endocytosis and its recycling back to the cell surface from endosomes and other intracellular compartments (Fig. 1). Changes in the kinetics of membrane protein traffic acutely affect the levels of individual proteins at the cell surface and thereby impact how cells intake nutrients, sense the environment, and respond to external cues.Open in a separate windowFigure 1.Dynamic regulation of the cell-surface content of membrane proteins. Integral membrane proteins found at the cell surface are dynamically localized to the plasma membrane. The amount of any of these proteins at the cell surface is the result of the balance of exocytosis or recycling of vesicles containing that protein from intracellular membrane compartments and the endocytosis of the protein from the cell surface. Regulation of either the rate of exocytosis or endocytosis results in alteration of the cell-surface content of a given protein.Selective molecular mechanisms trigger traffic of plasma membrane proteins through endomembranes. Among them, ubiquitination and phosphorylation stand out as they can directly target the cargo proteins. Ubiquitination is the covalent attachment of the 76-amino acid polypeptide ubiquitin to the ε-amino group of specific lysine residues (reviewed by Miranda and Sorkin 2007; and see also Piper et al. 2014). Ubiquitination of cell-surface proteins is the principal mechanism of control of endocytosis in yeast (MacGurn et al. 2012), whereas in mammals, additional molecular mechanisms regulate the endocytosis of cell-surface proteins, including alterations in conformation that impact interaction with other proteins, and as mentioned, phosphorylation. Each of these modifications can either enhance or reduce the rates internalization, recycling, or degradation of specific proteins, highlighting the complexity of the regulation of endomembrane traffic. The intricate mechanisms that underlie the reciprocal regulation of endocytosis and metabolism are beginning to be understood. Here we discuss the endocytosis mechanisms in the regulation of cellular intake or efflux of iron, cholesterol, Na⁺, and glucose, and in the regulation of receptor signaling relevant to metabolism.     

Biogas manufacture from co-digestion of untreated primary sludge with raw chicken manure under anaerobic mesophilic environmental conditions

This work aimed to co-digest various wastes to assess the best combination of all mixing ratio, also at choosing the best ratio between untreated primary sludge (UPS) singly from two sources, (South valley University (SUPS) and Abu tesht wastewater station (AUPS) and raw chicken manure (RCM) and comparing the results in either case. The co-digestions of untreated primary sludge from Abu tesht wastewater treatment stations with different levels of raw chicken manure (0:100, 10:90, 30:70, 50:50, 90:10, and 100:0) to obtain the best mixtures. Also, co-digestion of untreated primary sludge from south valley university with different levels of raw chicken manure at the same ratios, to obtain the best mixtures. Batch digestion tests were applied in 2.5 L digester with a working volume of 2.0 L. The samples in triplicates were separately loaded into the digesters locally fabricated and kept for 20 days as a retention period and diluted with the same amount of water. Mesophilic under 35 °C was adopted for untreated primary sludge as well as mixtures with raw chicken manure based on total solids (TS) and volatile solid (VS) proportions. The average biogas yields from AUPS/RCM mixture obtained ranged from 8570 to 5600 ml, by the following descending order, 10: 90 > 90:10 and so on >100:0, and the average biogas yields from SUPS/RCM obtained ranged from 6330 to 5635 ml, in the order of 90: 10 > 10:90 and so on >100:0. The results showed highest biogas yield from AUPS/RCM and SUPS/RCM mixtures with mixing ratio of 10:90 and 90:10, respectively, however, the lowest biogas production detected in separate digestion of AUPS and SUPS. The results indicated that co-digestion between the sludge and raw chicken manure could increase total biogas production volume, enhance sludge treatment process, and produce eco-friendly sludge because of co-digestion process than separate processing of each feedstock.     

Aspergillus nidulans hypB encodes a Sec7-domain protein important for hyphal morphogenesis.

Aspergillus nidulans strains containing the hypB5 temperature sensitive allele have a restrictive phenotype of wide, highly-branched hyphae. The hypB locus was cloned by phenotype complementation using a genomic plasmid library. hypB5 is predicted gene AN6709 in the A. nidulans genome database, which encodes a putative Sec7 domain protein, likely to act early in COPI-mediated vesicle formation for retrograde Golgi to ER transport. The A. nidulans hypB5 allele has a single mutation, cytosine to guanine predicted to cause a nonconservative amino acid change, alanine to proline, in a conserved helix adjacent to the Sec7p nucleotide binding site. This would likely reduce the stability of a highly conserved loop important for nucleotide binding, and is consistent with temperature sensitivity of hypB5 strains. Deletion of AN6709 showed that hypB(Sec7) was not essential. AN6709Delta hyphae resembled the hypB5 restrictive phenotype. As has been shown previously for hypA1 mutants, shifting established hypB5 mutant hyphae from a growth temperature of 28-42 degrees C caused septation in and death of tip cells and growth activation of basal cells.     

Binding of [3H]phencyclidine to membranes from housefly thoracic muscles

The binding of [³H]phencyclidine ([³H]PCP) to a preparation of housefly thoracic muscle membranes was studied. Specific [³H]PCP binding saturated with both time and at concentrations of the radiolabeled ligand greater than 20 nM. One binding site with a K_D of 10.7 nM and a B_max of 3.9 pmol/mg protein was observed. Specific [³H]PCP binding was also readily reversible with a half-time of dissociation (t_1/2) of 13.8 min and varied proportionately with tissue concentration. Of the drugs tested, specific [³H]PCP binding was inhibited by PCP analogs, antipsychotics, antidepressants, Ca²⁺ channel antagonists, and K⁺ channel blockers. [³H]PCP binding was unaffected by addition of carbamylcholine or L-glutamate in absence or presence of ATP, GTP, cAMP, or cGMP. Though the identity of the [³H]PCP binding protein in housefly muscle membranes is still unclear, it is more likely to be an ionic channel such as K⁺ or Ca²⁺ channels than a neurotransmitter receptor.     

Antiprotozoal activity of magnesium oxide (MgO) nanoparticles against Cyclospora cayetanensis oocysts

Outbreaks of Cyclospora cayetanensis infection have been linked to consumption of food and water contaminated by oocysts that can survive both physical and chemical disinfectants. Magnesium oxide (MgO) nanoparticles (NPs) can be potentially used in food as bactericides. In this study, C. cayetanensis pre- and post-sporulated oocysts were exposed to MgO NPs with different doses ranging from 1.25–25?mg/ml. With comparison to control, the antiprotozoal activity of MgO NPs was evaluated by identifying the median effective concentration dose (EC₅₀), lethal concentration dose (LC₉₀), microscopically changes on treated oocysts and rates of sporulation. Among pre- and post-sporulated oocysts, MgO NPs?≥?EC₅₀ was observed after 24?h at concentrations 10 and 12.5?mg/ml, respectively, while?≥?LC₉₀ was observed after 24?h, 48?h and 72?h at concentrations 15, 12.5 and 10?mg/ml, respectively. MgO NPs treated oocysts showed abnormal morphological changes such as an increase in size, wall injury, deposition of vacuolated homogenous particles in the cytoplasm, evacuation of oocyst's contents, and collapse. Sporocysts of treated oocysts were noticed to be peripherally shifted. Sporulation failure of treated oocysts achieved ≥90% after 24?h and 72?h of incubation with 15 and 12.5?mg/ml, respectively, while it was 10.1% among non-treated. All the differences were statistically significant. Our results demonstrated that MgO NPs has a significant anti-Cyclospora effect on both unsporulated and sporulated oocysts, especially considering that it could be biologically synthesized, that way it can be used safely as a preventive agent in food and water disinfectant treatment.