Insulin-stimulated glucose uptake involves the recruitment of the glucose transporter 4 isoform (GLUT4) from an intracellular location to the plasma membrane of fat and muscle cells. Although the activation of the PI3-kinase/protein kinase B (PKB) pathway is central to this effect of insulin, the key substrates for PKB that are involved require identification. Here we report that serine318 on the FYVE domain-containing PtdIns(3)P 5-kinase (PIKfyve) is a novel substrate for PKB, and show that phosphorylation stimulates the PtdIns(3)P 5-kinase activity of the enzyme. We also demonstrate that PIKfyve is phosphorylated on serine318 in intact cells in response to insulin, in a PI3-kinase-dependent manner, and that PIKfyve colocalises with a highly motile subpopulation of insulin-regulated aminopeptidase (IRAP)/GLUT4 vesicles. Finally, we demonstrate that overexpression of a PIKfyve[S318A] mutant in 3T3-L1 adipocytes enhances insulin-stimulated IRAP/GLUT4 vesicle translocation to the plasma membrane suggesting a role for PKB-dependent phosphorylation of PIKfyve in insulin-regulated IRAP/GLUT4 trafficking. The phosphorylation and activation of PIKfyve by PKB provides a novel signalling paradigm that may link plasma membrane-localised PtdIns(3,4,5)P3 signals via a protein kinase cascade to regulated PtdIns(3,5)P2 production, and thereby to the control of trafficking of other membrane cargos.

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble Nethyl-maleimide-sensitive factor attachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulinstimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.In muscle, adipose tissue, and 3T3-L1 adipocytes, insulin primarily increases glucose uptake by promoting the trafficking of vesicles containing GLUT4 (glucose transporter isoform 4) to the plasma membrane (reviewed in Refs. 1 and 2). In the resting state, the majority of GLUT4 resides in vesicles distributed throughout the cell interior, with a fraction of GLUT4 undergoing cycling between the plasma membrane and several intracellular sorting compartments. Insulin triggers a substantial translocation of GLUT4-containing vesicles to the plasma membrane, a phenomenon that can largely explain the increase in V max of the glucose uptake observed. A detailed molecular description of how insulin promotes this translocation is presently lacking, although some of the components involved in the signaling process and the fusion events have been identified.Binding of insulin to its receptor activates the integral tyrosine kinase, which then elicits a cascade of cellular signaling responses, including phosphorylation of the cytosolic proteins of the insulin-receptor-substrate family (reviewed in Ref.3). As a consequence of tyrosine phosphorylation, insulin-receptor substrates-1 and -2 bind several effectors; the resultant activation of phosphatidylinositide (4,5)-bisphosphate kinase (PI3-kinase) 1 is of particular importance because it is known to play a key role in transducing the insulin signal leading to GLUT4 vesicle translocation (4 -6). The lipid product of this enzyme, namely phosphatidylinositide 3,4,5-trisphosph...

PURPOSE.A large body of research has linked macular lutein and zeaxanthin to reduced risk of degenerative eye disease. The earliest published hypothesis for the role of the pigments was not based on chronic protection but immediate function. Recent data on macular pigment (MP) have shown that screening the foveal cones from short-wave light does, in fact, result in improvements in photostress recovery (PR), glare disability (GD), and chromatic contrast (CC). This study examined those relations on a larger sample. METHODS.A total of 150 young healthy subjects were assessed. Plasma samples were obtained from 100 subjects for HPLC quantification of serum xanthophylls. MP density was measured using customized heterochromatic flicker photometery. GD, PR, and CC were measured in Maxwellian view using a broadband xenon light source. GD was measured by increasing the intensity of an annulus until it veiled a central target. PR was measured as the time necessary to regain sight of a central target after a 5-second exposure to an intense bleaching light. CC was measured as the amount of light necessary in a 460-nm background to lose sight of a central target.RESULTS. MP density was significantly related to serum lutein and zeaxanthin combined (r ¼ 0.31, P ¼ 0.002), GD (r ¼ 0.24, P ¼ 0.0015), PR (r ¼ À0.18, P ¼ 0.01), and CC (r ¼ 0.46, P ¼ 0.00005).CONCLUSIONS. These results confirm earlier reports of a significant relation between variation in macular pigment optical density and immediate effects on visual function. As with many species, intraocular yellow filters in humans appear to improve many aspects of the visual stimulus. (ClinicalTrials. gov number, NCT00909090.) (Invest Ophthalmol Vis Sci.

Citation: Hammond BR, Fletcher LM, Roos F, Wittwer J, Schalch W. A double-blind, placebo-controlled study on the effects of lutein and zeaxanthin on photostress recovery, glare disability, and chromatic contrast. Invest Ophthalmol Vis Sci. PURPOSE.Past studies have shown that higher macular pigment optical density (MPOD) and lutein (L) and zeaxanthin (Z) supplementation are related to improvements in glare disability, photostress recovery, and chromatic contrast. This study assessed those links using a randomized, double-blind, placebo-controlled design.METHODS. The visual effects of 1 year of supplementing L (10 mg/d) and Z (2 mg/d) were investigated. One hundred fifteen young, healthy subjects were recruited and randomized into the study (58 received placebo, 57 LþZ). Several dependent measures were collected at baseline and then once every 3 months: serum L and Z measured by HPLC chromatography; MPOD measured using customized heterochromatic flicker photometry; photostress recovery assessed by measuring the time needed to recover visual acquisition of a grating target after 30 seconds of an intense xenon white flash exposure; glare disability evaluated as the energy in a surrounding annulus necessary to veil a central grating target; and chromatic contrast assessed by measuring thresholds for a yellow grating target superposed on a 460-nm background.RESULTS. Macular pigment optical density increased significantly versus placebo at all eccentricities (10, 30, 60, and 105 minutes from the center of the macula). Serum L and Z also increased significantly by the first follow-up visit (at 3 months), and remained elevated throughout the intervention period of 1 year. Chromatic contrast and photostress recovery time improved significantly versus placebo. Glare disability was correlated with macular pigment density throughout the study period but did not increase significantly in the treated group.CONCLUSIONS. Daily supplementation with LþZ resulted in significant increase in serum levels and MPOD and improvements in chromatic contrast and recovery from photostress. These results are consistent with past studies showing that increasing MPOD leads to improved visual performance. (ClinicalTrials.gov number, NCT00909090.)

Background: Past studies have suggested that higher lutein (L) and zeaxanthin (Z) levels in serum and in the central nervous system (as quantified by measuring macular pigment optical density, MPOD) are related to improved cognitive function in older adults. Very few studies have addressed the issue of xanthophylls and cognitive function in younger adults, and no controlled trials have been conducted to date to determine whether or not supplementation with L + Z can change cognitive function in this population. Objective: The purpose of this study was to determine whether or not supplementation with L + Z could improve cognitive function in young (age 18–30), healthy adults. Design: A randomized, double-masked, placebo-controlled trial design was used. Fifty-one young, healthy subjects were recruited as part of a larger study on xanthophylls and cognitive function. Subjects were randomized into active supplement (n = 37) and placebo groups (n = 14). MPOD was measured psychophysically using customized heterochromatic flicker photometry. Cognitive function was measured using the CNS Vital Signs testing platform. MPOD and cognitive function were measured every four months for a full year of supplementation. Results: Supplementation increased MPOD significantly over the course of the year, vs. placebo (p < 0.001). Daily supplementation with L + Z and increases in MPOD resulted in significant improvements in spatial memory (p < 0.04), reasoning ability (p < 0.05) and complex attention (p < 0.04), above and beyond improvements due to practice effects. Conclusions: Supplementation with L + Z improves CNS xanthophyll levels and cognitive function in young, healthy adults. Magnitudes of effects are similar to previous work reporting correlations between MPOD and cognition in other populations.