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Insulin controls the spatial distribution of GLUT4 on the cell surface through regulation of its postfusion dispersal.

ABSTRACT: While the glucose transporter-4 (GLUT4) is fundamental to insulin-regulated glucose metabolism, its dynamic spatial organization in the plasma membrane (PM) is unclear. Here, using multicolor TIRF microscopy in transfected adipose cells, we demonstrate that insulin regulates not only the exocytosis of GLUT4 storage vesicles but also PM distribution of GLUT4 itself. In the basal state, domains (clusters) of GLUT4 molecules in PM are created by an exocytosis that retains GLUT4 at the fusion site. Surprisingly, when insulin induces a burst of GLUT4 exocytosis, it does not merely accelerate this basal exocytosis but rather stimulates approximately 60-fold another mode of exocytosis that disperses GLUT4 into PM. In contradistinction, internalization of most GLUT4, regardless of insulin, occurs from pre-existing clusters via the subsequent recruitment of clathrin. The data fit a new kinetic model that features multifunctional clusters as intermediates of exocytosis and endocytosis.

SUBMITTER: Stenkula KG

PROVIDER: S-EPMC3427691 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Insulin controls the spatial distribution of GLUT4 on the cell surface through regulation of its postfusion dispersal.

Stenkula Karin G KG Lizunov Vladimir A VA Cushman Samuel W SW Zimmerberg Joshua J

Cell metabolism 20100901 3

While the glucose transporter-4 (GLUT4) is fundamental to insulin-regulated glucose metabolism, its dynamic spatial organization in the plasma membrane (PM) is unclear. Here, using multicolor TIRF microscopy in transfected adipose cells, we demonstrate that insulin regulates not only the exocytosis of GLUT4 storage vesicles but also PM distribution of GLUT4 itself. In the basal state, domains (clusters) of GLUT4 molecules in PM are created by an exocytosis that retains GLUT4 at the fusion site. ...[more]

PMID: 20816091

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Project description:AMP-activated protein kinase (AMPK) enhances glucose transporter GLUT4 regulation. AMPK also suppresses energy-consuming pathways such as cholesterol synthesis. Interestingly, recent in vitro and in vivo data suggest that excess membrane cholesterol impairs GLUT4 regulation. Therefore, this study tested whether a beneficial, GLUT4-regulatory aspect of AMPK stimulation involved cholesterol lowering. Using L6 myotubes stably expressing an exofacial myc-epitope-tagged-GLUT4, AMPK stimulation by 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR; 45 min, 1 mm) or 2,4-dinitrophenol (DNP; 30 min, 200 μm) increased cell surface GLUT4myc labeling by approximately ≈ 25% (P < 0.05). Insulin (20 min, 100 nm) also increased GLUT4myc labeling by about 50% (P < 0.05), which was further enhanced (≈ 25%, P < 0.05) by AICAR or DNP. Consistent with AMPK-mediated suppression of cholesterol synthesis, AICAR and DNP decreased membrane cholesterol by 20-25% (P < 0.05). Whereas AMPK knockdown prevented the enhanced basal and insulin-stimulated GLUT4myc labeling by AICAR and DNP, cholesterol replenishment only blocked the AMPK-associated enhancement in insulin action. Cells cultured in a hyperinsulinemic milieu, resembling conditions in vivo that promote the progression/worsening of insulin resistance, displayed an increase in membrane cholesterol. This occurred concomitantly with a loss of cortical filamentous actin (F-actin) and defects in GLUT4 regulation by insulin. These derangements were prevented by AMPK stimulation. Examination of skeletal muscle from insulin-resistant Zucker rats revealed a similar elevation in membrane cholesterol and loss of F-actin. Lowering cholesterol to control levels restored F-actin structure and insulin sensitivity. In conclusion, these data suggest a novel aspect of GLUT4 regulation by AMPK involves membrane cholesterol lowering. Moreover, this AMPK-mediated process protected against hyperinsulinemia-induced insulin resistance.

Project description:BACKGROUND:The wild radishes, Raphanus raphanistrum and R. pugioniformis (Brassicaceae) are native to the East Mediterranean region. However, whereas R. raphanistrum is widely distributed worldwide, the endemic R. pugioniformis is limited to specific habitats. In R. raphanistrum the diaspores of the indehiscent fruits comprise glabrous, light, single-seeded segments, whereas the intact fruits of R. pugioniformis are heavy and covered with spiny backward-pointing trichomes. We aimed to investigate whether the structure of the diaspores was directly associated with long- and short-range dispersal in R. raphanistrum and R. pugioniformis, respectively. We further surveyed within-population spatial distributions, to test the hypothesis that short- and long-range dispersal contribute to a patchy vs. uniform distribution patterns of R. pugioniformis and R. raphanistrum, respectively. RESULTS:The results indicated that dispersal by wind and run-off water was substantially lower for diaspores of R. pugioniformis than for those of R. raphanistrum diaspores. Supporting the hypothesis that backward-pointing trichomes promote adherence to soil particles, the displacement on soil surface of R. pugioniformis fruits depended on their orientation relative to wind direction. Furthermore, trichome removal from fruits of R. pugioniformis significantly reduced wind velocity needed to remove fruits that were placed on soils typical of the species' natural habitats. The spatial-distribution survey results indicated a patchy distribution of R. pugioniformis populations as compared with the more uniform arrangement in the studied populations of R. raphanistrum; consistent with the unidirectional vs. homogeneous wind dispersal of the respective diaspores, with respect to wind direction. In addition, R. pugioniformis population sizes changed less between years than those of R. raphanistrum. CONCLUSIONS:Overall, our results indicate that fruit structure is strongly linked to dispersal ability and spatial distribution of the two closely related wild radish species. Whereas R. raphanistrum inhabits homogenous sandy soil habitats, the distribution range of R. pugioniformis includes heterogeneous environments in which growth niches are scarcer. We suggest that the different modes of dispersal have evolved as adaptive traits appropriate to the species' specific habitats.

Project description:Ergosterol, the major sterol in fungal membranes, is critical for defining membrane fluidity and regulating cellular processes. Although ergosterol synthesis has been well defined in model yeast, little is known about sterol organization in the context of fungal pathogenesis. We identified a retrograde sterol transporter, Ysp2, in the opportunistic fungal pathogen Cryptococcus neoformans . We found that the lack of Ysp2 under host-mimicking conditions leads to abnormal accumulation of ergosterol at the plasma membrane, invagination of the plasma membrane, and malformation of the cell wall, which can be functionally rescued by inhibiting ergosterol synthesis with the antifungal drug fluconazole. We also observed that cells lacking Ysp2 mislocalize the cell surface protein Pma1 and have thinner and more permeable capsules. As a result of perturbed ergosterol distribution and its consequences, ysp2 Î" cells cannot survive in physiologically-rele-vant environments such as host phagocytes and are dramatically attenuated in virulence. These findings expand our knowledge of cryptococcal biology and underscore the importance of sterol homeostasis in fungal pathogenesis.ImportanceCryptococcus neoformans is an opportunistic fungal pathogen that kills over 100,000 people worldwide each year. Only three drugs are available to treat cryptococcosis, and these are variously limited by toxicity, availability, cost, and resistance. Ergosterol is the most abundant sterol in fungi and a key component in modulating membrane behavior. Two of the drugs used for cryptococcal infection, amphotericin B and fluconazole, target this lipid and its synthesis, highlighting its importance as a therapeutic target. We discovered a cryptococcal ergosterol transporter, Ysp2, and demonstrated its key roles in multiple aspects of cryptococcal biology and pathogenesis. These studies demonstrate the role of ergosterol homeostasis in C. neoformans virulence, deepen our understanding of a pathway with proven therapeutic importance, and open a new area of study.

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Insulin controls the spatial distribution of GLUT4 on the cell surface through regulation of its postfusion dispersal.

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Insulin controls the spatial distribution of GLUT4 on the cell surface through regulation of its postfusion dispersal.

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