The GLUT5 hexose transporter is also localized to the basolateral membrane of the human jejunum

The intestine is a major site of expression of the human GLUT5 hexose transporter, which is thought to be localized exclusively to the brush border membrane (BBM) where its major role is likely to be in the absorption of fructose. In this study we present novel biochemical and morphological evidence showing that the GLUT5 transporter is also expressed in the basolateral membrane (BLM) of the human intestine. BBM and BLM were isolated by fractionation of human jejunum. BBM were enriched with alkaline phosphatase activity by over 9-fold relative to a crude jejunal homogenate and contained immunoreactive sucrase-isomaltase and GLUT5 proteins. By contrast the BBM fraction was substantially depleted of immunoreactive a1 subunits of the Na,K-ATPase and GLUT2 glucose transporters which were abundantly present in the BLM fraction. This BLM fraction was enriched by over 11-fold in potassium-stimulated phosphatase activity relative to the crude homogenate; BLM also reacted to immunological probes for GLUT5 but showed no observable reactivity with antibodies directed against sucrase-isomaltase. Quantitative immunoblotting revealed that the BBM and BLM contained near equal amounts of GLUT5 per mg of membrane protein. Immunogold localization of GLUT5 on ultrathin sections of human jejunum showed that GLUT5 was present in both apical BBM and BLM. This gold labelling was absent when antiserum was pre-incubated with the antigenic peptide corresponding to a specific C-terminal sequence of human GLUT5. Quantitative analyses of the number of gold particles per unit length of BBM and BLM indicated that the mean density of gold labelling was marginally greater in the BBM (0.399 gold particles/micrometer) than in the BLM (0.293 gold particle/micrometer). The localization of GLUT5 in the BLM of the human jejunum may suggest that it specifically participates in the transfer of fructose across the basal membrane of the enterocyte

Metformin reduces airway glucose permeability and hyperglycaemia-induced Staphylococcus aureus load independently of effects on blood glucose

Background Diabetes is a risk factor for respiratory infection, and hyperglycaemia is associated with increased glucose in airway surface liquid and risk of Staphylococcus aureus infection. Objectives To investigate whether elevation of basolateral/blood glucose concentration promotes airway Staphylococcus aureus growth and whether pretreatment with the antidiabetic drug metformin affects this relationship. Methods Human airway epithelial cells grown at air–liquid interface (±18 h pre-treatment, 30 μM–1 mM metformin) were inoculated with 5×105 colony-forming units (CFU)/cm2 S aureus 8325-4 or JE2 or Pseudomonas aeruginosa PA01 on the apical surface and incubated for 7 h. Wild-type C57BL/6 or db/db (leptin receptor-deficient) mice, 6–10 weeks old, were treated with intraperitoneal phosphate-buffered saline or 40 mg/kg metformin for 2 days before intranasal inoculation with 1×107 CFU S aureus. Mice were culled 24 h after infection and bronchoalveolar lavage fluid collected. Results Apical S aureus growth increased with basolateral glucose concentration in an in vitro airway epithelia–bacteria co-culture model. S aureus reduced transepithelial electrical resistance (RT) and increased paracellular glucose flux. Metformin inhibited the glucose-induced growth of S aureus, increased RT and decreased glucose flux. Diabetic (db/db) mice infected with S aureus exhibited a higher bacterial load in their airways than control mice after 2 days and metformin treatment reversed this effect. Metformin did not decrease blood glucose but reduced paracellular flux across ex vivo murine tracheas. Conclusions Hyperglycaemia promotes respiratory S aureus infection, and metformin modifies glucose flux across the airway epithelium to limit hyperglycaemia-induced bacterial growth. Metformin might, therefore, be of additional benefit in the prevention and treatment of respiratory infection

Evaluation of the groundnut model PNUTGRO for crop response to water availability, sowing dates, and seasons

Field experiments were conducted during the 1987, 1991 and 1992 rainy seasons at Patancheru (latitude 17°32′N; longitude 78°16′E; elevation 545 m), Andhra Pradesh, India, to collect data to test and validate the hedgerow version of the groundnut model PNUTGRO for predicting phenological development, light interception, canopy growth, dry matter production, pod and seed yields of groundnut (Arachis hypogaea L.) as influenced by row spacing and plant population. The model was calibrated using the crop growth and phenology data of groundnut (cv. Robut 33-1) obtained from the 1987 and 1991 rainy season experiments. In these experiments groundnut was grown at plant populations ranging from 5 to 45 plants/m2 with and without irrigation. Changes were made in the cultivar-specific coefficients related to the light penetration into the crop canopy and dry matter production. The model was validated against independent data obtained from a 1992 rainy season experiment. In 1992, groundnut was grown at plant populations ranging from 10 to 40 plants/m2 and at row spacings of 20, 30 and 60 cm. The model predicted the occurrence of vegetative and reproductive stages, canopy development, total dry matter production and its partitioning to pods and seed accurately. Maximum leaf area index observed during the season was significantly correlated with simulated values (r2 = 0.95). In spite of some incidence of diseases and pests, the correlation between simulated and observed pod yield was significant (r2 = 0.61). It is concluded from this study that the hedgerow version of the groundnut model PNUTGRO can be used to quantify groundnut growth and yields as influenced by plant population and row spacing

GSK3-mediated raptor phosphorylation supports amino acid-dependent Q2 mTORC1-directed signalling

The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser(859). We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation

Enhanced insulin sensitivity associated with provision of mono and polyunsaturated fatty acids in skeletal muscle cells involves counter modulation of PP2A

International audienceAims/Hypothesis: Reduced skeletal muscle insulin sensitivity is a feature associated with sustained exposure to excess saturated fatty acids (SFA), whereas mono and polyunsaturated fatty acids (MUFA and PUFA) not only improve insulin sensitivity but blunt SFA-induced insulin resistance. The mechanisms by which MUFAs and PUFAs institute these favourable changes remain unclear, but may involve stimulating insulin signalling by counter-modulation/repression of protein phosphatase 2A (PP2A). This study investigated the effects of oleic acid (OA; a MUFA), linoleic acid (LOA; a PUFA) and palmitate (PA; a SFA) in cultured myotubes and determined whether changes in insulin signalling can be attributed to PP2A regulation. Principal Findings: We treated cultured skeletal myotubes with unsaturated and saturated fatty acids and evaluated insulin signalling, phosphorylation and methylation status of the catalytic subunit of PP2A. Unlike PA, sustained incubation of rat or human myotubes with OA or LOA significantly enhanced Akt-and ERK1/2-directed insulin signalling. This was not due to heightened upstream IRS1 or PI3K signalling nor to changes in expression of proteins involved in proximal insulin signalling, but was associated with reduced dephosphorylation/inactivation of Akt and ERK1/2. Consistent with this, PA reduced PP2Ac demethylation and tyrosine 307 phosphorylation-events associated with PP2A activation. In contrast, OA and LOA strongly opposed these PA-induced changes in PP2Ac thus exerting a repressive effect on PP2A.Conclusions/Interpretation: Beneficial gains in insulin sensitivity and the ability of unsaturated fatty acids to oppose palmitate-induced insulin resistance in muscle cells may partly be accounted for by counter-modulation of PP2A

The catalytic subunit of the system L1 amino acid transporter (S<i>lc7a5</i>) facilitates nutrient signalling in mouse skeletal muscle

The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/-) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass

Luminous WISE-selected Obscured, Unobscured, and Red Quasars in Stripe 82

We present a spectroscopically complete sample of 147 infrared-color-selected active galactic nuclei (AGNs) down to a 22 μm flux limit of 20 mJy over the ~270 deg^2 of the Sloan Digital Sky Survey Stripe 82 region. Most of these sources are in the QSO luminosity regime (L_(bol) ≳ 10^(12) L⊙) and are found out to z ≃ 3. We classify the AGNs into three types, finding 57 blue, unobscured Type-1 (broad-lined) sources; 69 obscured, Type-2 (narrow-lined) sources; and 21 moderately reddened Type-1 sources (broad-lined and E(B − V) > 0.25). We study a subset of this sample in X-rays and analyze their obscuration to find that our spectroscopic classifications are in broad agreement with low, moderate, and large amounts of absorption for Type-1, red Type-1, and Type-2 AGNs, respectively. We also investigate how their X-ray luminosities correlate with other known bolometric luminosity indicators such as [O III] line luminosity (L_([O III])) and infrared luminosity (L_(6μm)). While the X-ray correlation with L_([O III]) is consistent with previous findings, the most infrared-luminous sources appear to deviate from established relations such that they are either underluminous in X-rays or overluminous in the infrared. Finally, we examine the luminosity function evolution of our sample, and by AGN type, in combination with the complementary, infrared-selected, AGN sample of Lacy et al. (2013), spanning over two orders of magnitude in luminosity. We find that the two obscured populations evolve differently, with reddened Type-1 AGNs dominating the obscured AGN fraction (~30%) for L_(5μm) > 10^(45) erg s^(−1), while the fraction of Type-2 AGNs with L_(5μm) < 10^(45) erg s^(−1) rises sharply from 40% to 80% of the overall AGN population