Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type

Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.     

Mobility of the spin-labeled side chains of some novel antifolate inhibitors in their complexes with dihydrofolate reductase

Four spin-labeled inhibitors of dihydrofolate reductase (DHFR) have been synthesized, each of which has the 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) reporting group at a different distance from the 2,4-diaminopyrimidine moiety by which the inhibitors are anchored and oriented in the active site. Inhibitors in which the TEMPO group is attached by a short side chain are weakly bound to DHFR from bacteria (Streptococcus faecium and Lactobacillus casei), to the bovine enzyme and to recombinant human DHFR. However, binding is sufficiently tight, especially in the ternary complexes with NADPH, for recording of the EPR spectra of the bound ligands. The spectra indicate that when these inhibitors are bound to the enzyme the TEMPO group is highly immobilized with correlation time, tau c, 4-20ns. Inhibitors that have the reporter group attached to the glutamate moiety of methotrexate bind to all four DHFRs more tightly than the inhibitors with shorter side chains by factors of up to 10(6). However, in most complexes formed by the inhibitors with longer side chains immobilization of the TEMPO group is slight (tau c 0.2-4 ns). These results are in general agreement with predictions from X-ray crystallographic results including thermal factors but there are some unanticipated differences between some results for bacterial and eukaryotic enzymes. Three of the splin-labeled inhibitors would provide good probes for distance measurements in and around the active site of mammalian DHFR.     

Angiotensin II potentiates responses of the rabbit basilar artery to adrenergic nerve stimulation

Angiotensin II has little contractile effect on the isolated rabbit basilar artery; however, it markedly potentiates contractile responses to adrenergic nerve stimulation. This is not a post-synaptic effect of angiotensin, as responses to exogenous norepinephrine are not altered. Angiotensin increases stimulation-evoked release of norepinephrine, and this effect probably accounts for the increased response to adrenergic nerve stimulation. Since sympathetic stimulation may protect the cerebral circulation from hypertensive damage, increased responsiveness to adrenergic nerve activity produced by angiotensin may have a beneficial effect.     

The role of arachidonate lipoxygenase in the release of SRS-A from guinea-pig chopped lung

The immunological release of SRS-A was investigated in guinea-pig chopped lung. A number of unsaturated fatty acids, all of which are substrates for arachidonate lipoxygenase were found to potentiate the release of SRS-A. This potentiation was enhanced by indomethacin, a cyclo-oxygenase inhibitor, and completely reversed by nordihydroguaiaretic acid (NDGA) and eicosatetraynoic acid (ETA) which inhibit lipoxygenase. This suggests that some aspect of arachidonate lipoxygenase action stimulates release of SRS-A and that release of SRS-A is increased by redirection of arachidonic acid (AA) metabolism via the lipoxygenase pathway (Hamberg, 1976). However, although exogenous ¹⁴C-AA increased SRS-A output it was not incorporated into SRS-A.     

Analysis of larger than tetrameric poly(adenosine diphosphoribose) by a radioimmunoassay in nuclei separated in organic solvents.

Anitibodies were prepared against poly(adenosine diphosphoribose) of an average chain length of 40 adenosine diphosphoribose units by repeated injection of the polymer mixed with methylated albumin and adjuvants into rabbits. The antibody was present mainly in the 7 S fraction of the immunoglobulins. A membrane binding assay was developed, and its specificity determined for the detection of (adenosine diphosphoribose)ngreater than4 in organs. The method is suitable for the study of the variation of the polymer content of nuclei. The size recognition of the anti-poly(adenosine diphosphoribose) globulin fraction was the same for polymers composed of 4--40 adenosine diphosphoribose units, but smaller oligomers were not detectible. A quantitative extraction technique was developed and applied for radioimmunoassay of nuclear (adenosine diphosphoribose)n greater than 4. Organs were freeze-clamped, freeze dried, broken into subcellular fragments in a colloid mill, and the nuclear fraction was subsequently separated in organic solvents in order to preserve the polymer. Nicotinamide and nicotinic acid, when administered in vivo, augmented the (adenosine diphosphoribose)n greater than 4 content of rat liver and heart. Tissues of infant pigeons contained larger quantites of (adenosine diphosphoribose)ngreater than4 than tissues of adult rats.     

Simulated warming does not impair seedling survival and growth of Nothofagus pumilio in the southern Andes

It has been predicted that subalpine forests will be negatively affected by global warming; however, direct responses to experimental warming have been scarcely examined in these systems. In this study we evaluated the effects of higher temperatures with and without water addition on the survival and growth of recently emerged (small) and large seedlings of the widely distributed species Nothofagus pumilio in subalpine forests of the southern Chilean Andes. We also examined the variations in seedling traits related to carbon balance in order to infer the causal mechanisms of survival and growth responses. Treatments of open top chambers (OTCs) were combined with watering in two locations with differing climates: Antillanca (40°S, humid) and Cerro Castillo (46°S, drier). OTCs increased mean and maximum air temperatures by 0.6 °C and 2–3 °C, respectively, and decreased soil humidity by 56% in Antillanca and 30% in Cerro Castillo, fulfilling methodological expectations and climate model predictions. After two complete growing seasons, the survival, relative growth rate (RGR), biomass, and a suite of seedling traits were measured and analyzed using mixed-effects models. Warming and warming in combination with watering significantly increased large seedling survival in Cerro Castillo. In Antillanca, warmer conditions increased the height, biomass, and leaf area of small seedlings, and the RGR of large seedlings. In this location, warming also caused lower leaf carbon isotopic composition in both age classes and higher specific leaf area in small seedlings, suggesting whole-plant carbon gain improvements; warming did not produce any drought effects. Our results indicate that warming produces positive effects on the seedling establishment of N. pumilio in the southern Andes, highlighting the importance of site-specific effects in response to climate change in widespread species. Site-specific effects can most likely explain the discrepancies between the results of this study and the predictions outlined by previous studies for these forests.     

Monoclonal antibody disulfide reduction during manufacturing

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.