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It is well known that insulin receptor substrates (IRS) act as a mediator for signal transduction of insulin, insulin-like growth factors, and several cytokines. To identify proteins that interact with IRS and modulate IRS-mediated signals, we performed yeast two-hybrid screening with IRS-1 as bait. Out of 109 cDNA-positive clones identified from a human placental cDNA library, two clones encoded 53BP2, p53-binding protein 2 (53BP2S), a short form splicing variant of the apoptosis-stimulating protein of p53 that possesses Src homology region 3 domain, and ankyrin repeats domain, and had been reported to interact with p53, Bcl-2, and NF-kappaB. Interaction of 53BP2S with IRS-1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays in COS-7 cells and 3T3-L1 adipocytes. The Src homology region 3 domain and ankyrin repeats domain of 53BP2S were responsible for its interaction with IRS-1, whereas the phosphotyrosine binding domain and a central domain (amino acid residues 750-861) of IRS-1 were required for its interaction with 53BP2S. In CHO-C400 cells, expression of 53BP2S reduced insulin-stimulated IRS-1 tyrosine phosphorylation with a concomitant enhancement of IRS-2 tyrosine phosphorylation. In addition, the amount of the phosphatidylinositol 3-kinase regulatory p85 subunit associated with tyrosine-phosphorylated proteins, and activation of Akt was enhanced by 53BP2S expression. Although 53BP2S also enhanced Akt activation in 3T3-L1 adipocytes, insulin-induced glucose transporter 4 translocation was markedly inhibited in accordance with reduction of insulin-induced AS160 phosphorylation. Together these data demonstrate that 53BP2S interacts and modulates the insulin signals mediated by IRSs.

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion.

Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly, although the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6. The VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi Network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, whereas knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.

In muscle and adipose cells, the insulin-responsive aminopeptidase (IRAP) is localized to intracellular storage sites and undergoes insulin-dependent redistribution to the cell surface. Following expression, the newly synthesized IRAP protein traffics to the perinuclear insulin-sensitive compartment and acquires insulin sensitivity 6-9 h following biosynthesis. Knockdown of GGA1 by RNA interference prevented IRAP from entering, but not exiting, the insulin-responsive compartment. Mutation of the dileucine motif at positions 76 and 77 (EGFP-IRAP/AA(76,77)), but not the dileucine motif at positions 53 and 54, resulted in the rapid default of the reporter to the cell surface beginning at 3 h following biosynthesis. Alanine substitution of 9 residues amino- or carboxyl-terminal to LL(76,77) did not perturb basal intracellular sequestration or abrogate insulin-stimulated IRAP translocation. Moreover, a dominant interfering GGA mutant (VHS-GAT) potently inhibited insulin-stimulated translocation of EGFP-IRAP/WT but did not block the constitutive exocytotic trafficking of EGFP-IRAP/AA(76,77). In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA(76,77) constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL(76,77) functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.

Upon binding and activating its cell-surface receptor, insulin triggers signaling cascades that regulate many cellular processes. Regarding glucose homeostasis, insulin suppresses hepatic glucose production and increases glucose transport into muscle and adipose tissues. At the cellular level, glucose uptake results from the insulin-stimulated translocation of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Although the signaling molecules that function proximal to the activated insulin receptor have been well characterized, it is not known how the distal insulin-signaling cascade interfaces with and mobilizes GLUT4-containing compartments. Recently, several candidate signaling molecules, including AS160, PIKfyve and synip, have been identified that might provide functional links between the insulin signaling cascade and GLUT4 compartments. Future work will focus on delineating the precise GLUT4 trafficking steps regulated by these molecules.

To examine the acquisition of insulin sensitivity after the initial biosynthesis of the insulin-responsive aminopeptidase (IRAP), 3T3-L1 adipocytes were transfected with an enhanced green fluorescent protein-IRAP (EGFP-IRAP) fusion protein. In the absence of insulin, IRAP was rapidly localized (1–3 h) to secretory membranes and retained in these intracellular membrane compartments with little accumulation at the plasma membrane. However, insulin was unable to induce translocation to the plasma membrane until 6–9 h after biosynthesis. This was in marked contrast to another type II membrane protein (syntaxin 3) that rapidly defaulted to the plasma membrane 3 h after expression. In parallel with the time-dependent acquisition of insulin responsiveness, the newly synthesized IRAP protein converted from a brefeldin A-sensitive to a brefeldin A-insensitive state. The initial trafficking of IRAP to the insulin-responsive compartment was independent of plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis but had no effect on the insulin-stimulated translocation of the newly synthesized IRAP protein.

We have recently reported that following initial biosynthesis, the GLUT4 protein exits the Golgi apparatus and directly enters the insulin-responsive compartment(s) without transiting the plasma membrane (Watson, R. T., Khan, A. H., Furukawa, M., Hou, J. C., Li, L., Kanzaki, M., Okada, S., Kandror, K. V., and Pessin, J. E. (2004) EMBO J. 23, 2059–2070). To investigate the structural motifs involved in these initial sorting events, we have generated a variety of loss-of-function and gain-of-function GLUT4/GLUT1 chimera proteins. Substitution of the GLUT4 carboxyl-terminal domain with GLUT1 had no significant effect on the acquisition of insulin responsiveness. In contrast, substitution of either the GLUT4 amino-terminal domain or the large cytoplasmic loop between transmembrane domains 6 and 7 resulted in the rapid default of GLUT4 to the plasma membrane with blunted insulin response. Consistent with these findings, substitution of the amino-terminal, cytoplasmic loop, or carboxyl-terminal domains individually into GLUT1 backbone did not recapitulate normal GLUT4 trafficking. Similarly, dual substitutions of the GLUT1 amino and carboxyl termini with GLUT4 domains or the combination of the cytoplasmic loop plus the carboxyl terminus failed to display normal GLUT4 trafficking. However, the dual replacement of the amino terminus plus the cytoplasmic loop of GLUT4 in the GLUT1 backbone resulted in a complete restoration of normal GLUT4 trafficking. Alanine-scanning mutagenesis of the GLUT4 amino terminus demonstrated that Phe5 and Ile8 within the FQQI motif and, to a lesser extent, Asp12/Gly13 were necessary for the appropriate initial trafficking following biosynthesis. In addition, amino acids 229–271 in the large intracellular loop between transmembrane domains 6 and 7 functionally cooperated with the amino-terminal domain. These data demonstrate that initial trafficking of GLUT4 from the Golgi to the insulin-responsive GLUT4 compartment requires the functional interaction of two distinct domains.

Following biosynthesis, both GLUT1 and VSV-G proteins appear rapidly (2-3 h) at the plasma membrane, whereas GLUT4 is retained in intracellular membrane compartments and does not display any significant insulin responsiveness until 6-9 h. Surprisingly, the acquisition of insulin responsiveness did not require plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) had no effect on the insulin-stimulated GLUT4 translocation. Furthermore, expression of endocytosis-defective GLUT4 mutants or continuous surface labeling with an exofacial specific antibody demonstrated that GLUT4 did not transit the cell surface prior to the acquisition of insulin responsiveness. The expression of a dominant-interfering GGA mutant (VHS-GAT) had no effect on the trafficking of newly synthesized GLUT1 or VSV-G protein to the plasma membrane, but completely blocked the insulin-stimulated translocation of newly synthesized GLUT4. Furthermore, in vitro budding of GLUT4 vesicles but not GLUT1 or the transferrin receptor was inhibited by VHS-GAT. Together, these data demonstrate that following biosynthesis, GLUT4 directly sorts and traffics to the insulin-responsive storage compartment through a specific GGA-sensitive process.

Since the discovery of insulin roughly 80 yr ago, much has been learned about how target cells receive, interpret, and respond to this peptide hormone. For example, we now know that insulin activates the tyrosine kinase activity of its cell surface receptor, thereby triggering intracellular signaling cascades that regulate many cellular processes. With respect to glucose homeostasis, these include the function of insulin to suppress hepatic glucose production and to increase glucose uptake in muscle and adipose tissues, the latter resulting from the translocation of the glucose transporter 4 (GLUT4) to the cell surface membrane. Although simple in broad outline, elucidating the molecular intricacies of these receptor-signaling pathways and membrane-trafficking processes continues to challenge the creative ingenuity of scientists, and many questions remain unresolved, or even perhaps unasked. The identification and functional characterization of specific molecules required for both insulin signaling and GLUT4 vesicle trafficking remain key issues in our pursuit of developing specific therapeutic agents to treat and/or prevent this debilitating disease process. To this end, the combined efforts of numerous research groups employing a range of experimental approaches has led to a clearer molecular picture of how insulin regulates the membrane trafficking of GLUT4.

To examine the structural determinants necessary for TC10 trafficking, localization, and function in adipocytes, we generated a series of point mutations in the carboxyl-terminal targeting domain of TC10. Wild-type TC10 (TC10/WT) localized to secretory membrane compartments and caveolin-positive lipid raft microdomains at the plasma membrane. Expression of a TC10/C206S point mutant resulted in a trafficking and localization pattern that was indistinguishable from that of TC10/WT. In contrast, although TC10/C209S or the double TC10/C206,209S mutant was plasma membrane localized, it was excluded from both the secretory membrane system and the lipid raft compartments. Surprisingly, inhibition of Golgi membrane transport with brefeldin A did not prevent plasma membrane localization of TC10 or H-Ras. Moreover, inhibition of trans-Golgi network exit with a 19 degrees C temperature block did not prevent the trafficking of TC10 or H-Ras to the plasma membrane. These data demonstrate that TC10 and H-Ras can both traffic to the plasma membrane by at least two distinct transport mechanisms in adipocytes, one dependent upon intracellular membrane transport and another independent of the classical secretory membrane system. Moreover, the transport through the secretory pathway is necessary for the localization of TC10 to lipid raft microdomains at the plasma membrane.

Incubation of isolated GLUT4-containing vesicles with Xenopus oocyte extracts resulted in a guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and sodium orthovanadate stimulation of actin comet tails. The in vitro actin-based GLUT4 vesicle motility was inhibited by both latrunculin B and a dominant-interfering N-WASP mutant, N-WASP/Delta VCA. Preparations of gently sheared (broken) 3T3L1 adipocytes also displayed GTP gamma S and sodium orthovanadate stimulation of actin comet tails on GLUT4 intracellular compartments. Furthermore, insulin pretreatment of intact adipocytes prior to gently shearing also resulted in a marked increase in actin polymerization and actin comet tailing on GLUT4 vesicles. In addition, the insulin stimulation of actin comet tails was completely inhibited by Clostridum difficile toxin B, demonstrating a specific role for a Rho family member small GTP-binding protein. Expression of N-WASP/Delta VCA in intact cells had little effect on adipocyte cortical actin but partially inhibited insulin-stimulated GLUT4 translocation. Taken together, these data demonstrate that insulin can induce GLUT4 vesicle actin comet tails that are necessary for the efficient translocation of GLUT4 from intracellular storage sites to the plasma membrane.

The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface. Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation. We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP. On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin. Here we show that phosphorylated Cbl recruits the CrkII-C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor–mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft–localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP(3). Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).

It has been previously reported that calmodulin plays a regulatory role in the insulin stimulation of glucose transport. To examine the basis for this observation, we examined the effect of a panel of calmodulin antagonists that demonstrated a specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4) but not insulin- or platelet-derived growth factor (PDGF)-stimulated GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or phosphotyrosine antibody immunoprecipitation of phosphatidylinositol (PI) 3-kinase activity was not affected. Despite the marked insulin and PDGF stimulation of PI 3-kinase activity, there was a near complete inhibition of protein kinase B activation. Using a fusion protein of the Grp1 pleckstrin homology (PH) domain with the enhanced green fluorescent protein, we found that the calmodulin antagonists prevented the insulin stimulation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo. Similarly, although PDGF stimulation increased PI 3-kinase activity in in vitro immunoprecipitation assays, there was also no significant formation of PI(3,4,5)P3 in vivo. These data demonstrate that calmodulin antagonists prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo production of PI(3,4,5)P3 without directly affecting IRS1- or phosphotyrosine-associated PI 3-kinase activity. This phenomenon is similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.

To identify the targeting domains of syntaxin 6 responsible for its localization to the trans-Golgi network (TGN), we examined the subcellular distribution of enhanced green fluorescent protein (EGFP) epitope-tagged syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncation/deletion mutants in 3T3L1 adipocytes. Expression of EGFP-syntaxin 6 resulted in a perinuclear distribution identical to endogenous syntaxin 6 as determined both by confocal fluorescence microscopy and subcellular fractionation. Furthermore, both the endogenous and the expressed EGFP-syntaxin 6 fusion protein were localized to a brefeldin A-insensitive but okadaic acid-sensitive compartment characteristic of the TGN. In contrast, EGFP-syntaxin 6 constructs lacking the H2 domain were excluded from the TGN and were instead primarily localized to the plasma membrane. Although syntaxin 4 was localized to the plasma membrane, syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncations containing the H2 domain of syntaxin 6 were predominantly directed to the TGN. Importantly, the syntaxin 6 H2 domain fused to the transmembrane domain of syntaxin 4 was also localized to the TGN, demonstrating that the H2 domain was sufficient to confer TGN localization. In addition to the H2 domain, a tyrosine-based plasma membrane internalization signal (YGRL) was identified between the H1 and H2 domains of syntaxin 6. Deletion of this sequence resulted in the accumulation of the EGFP-syntaxin 6 reporter construct at the plasma membrane. Together, these data demonstrate that syntaxin 6 utilizes two distinct domains to drive its specific subcellular localization to the TGN.

C. Obst et al. provide a welcome opportunity to clarify the difference between environmental-economic cost-benefit analyses (such as ours) and environmental accounting exercises [such as the UN-SEEA ([ 1 ][1], [ 2 ][2]) initiative]. Accounting studies attempt to assess the total value of goods related to ecosystem services in a manner comparable to that used for market-priced goods in national accounts. A decline in the ecosystem services account over time signals a potential need to invest in underlying natural capital. However, such accounts do not indicate the most cost-effective form of that investment. Environmental economic analyses such as ours typically consider changes in value from the status quo that alternative investments provide, and identify those that yield higher value for money. The two approaches are complements rather than substitutes and serve differing but highly compatible elements of the decision-making process. A. Graham et al. criticize our use of “the value of nonmarket goods such as biodiversity.” Although we valued nonmarket greenhouse gas emissions and recreation, we explicitly did not attempt to define biodiversity in terms of economic values; instead, we applied a quantitative constraint prohibiting the degradation of biodiversity within our scenario analyses and examined the costs this would entail, finding them to be minor relative to other values. Graham et al. 's critique that we should not compare nonmarket values for ecosystem services with the market price of agricultural output ignores the fact that, as stated, we are conducting an economic analysis of marginal changes from the status quo and not attempting to assess the total value of food. In such assessments of changes, the use of market prices is standard ([ 3 ][3]). Indeed, there is an argument ([ 3 ][3]) that such analyses should subtract subsidies (including income support), which would reduce agricultural values ([ 4 ][4]). Graham et al. also question the possible increase in yields attributable to climate change. We do believe that UK farming will generally benefit from warmer temperatures but caution that [as detailed in ([ 5 ][5])] within areas of lower rainfall, increased drought could potentially reduce or even reverse these gains. We agree with R. Aspinall and P. Gregory that it would be better to consider net profits rather than farm gate prices, although again this would have reduced estimates of agricultural values. The need to link land use to its ecosystem service impacts favored our use of Agricultural Census ([ 6 ][6]) data, which omits profits. We are currently addressing this through a link to the Farm Business Survey ([ 7 ][7]) database. However, we disagree with the authors' contention that we should have included the added-value of post-farm food processing. Aside from the fact that the UK food processing industry is a major importer of non-UK produce, such an approach would be analogous to valuing timber at the price of fine furniture. It is the raw material value that is relevant here. Similarly, our analysis explicitly links agricultural land use to its impacts on the ecosystem service considered. 1. [↵][8] United Nations, European Commission, Food and Agriculture Organization of the United Nations, International Monetary Fund, Organisation for Economic Co-operation and Development, World Bank, “System of Environmental-Economic Accounting 2012: Central Framework, prepublication (white cover)” (2012); . 2. [↵][9] United Nations, European Commission, Organisation for Economic Co-operation and Development, World Bank, “System of Environmental-Economic Accounting 2012: Experimental Ecosystem Accounting, pre-publication (white cover)” (2013); [http://unstats.un.org/unsd/envaccounting/eea\_white\_cover.pdf][10]. 3. [↵][11] 1. H. M. Treasury , The Green Book: Appraisal and Evaluation in Central Government (The Stationery Office, London, 2003). 4. [↵][12] 1. I. J. Bateman, 2. A. A. Lovett, 3. J. S. Brainard , Applied Environmental Economics: A GIS Approach to Cost-Benefit Analysis (Cambridge Univ. Press, Cambridge, 2003). 5. [↵][13] 1. C. Fezzi 2. et al ., Env. Res. Econ., 10.1007/s10640-013-9663-x (2013). doi:10.1007/s10640-013-9663-x [OpenUrl][14][CrossRef][15] 6. [↵][16] Department for Environment, Food and Rural Affairs, June Agricultural Census (Edina, Manchester, UK, 2010). 7. [↵][17] Rural Business Research, “Farm Business Survey” (2013); [www.fbspartnership.co.uk][18]. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6 [7]: #ref-7 [8]: #xref-ref-1-1 "View reference 1 in text" [9]: #xref-ref-2-1 "View reference 2 in text" [10]: http://unstats.un.org/unsd/envaccounting/eea_white_cover.pdf [11]: #xref-ref-3-1 "View reference 3 in text" [12]: #xref-ref-4-1 "View reference 4 in text" [13]: #xref-ref-5-1 "View reference 5 in text" [14]: {openurl}?query=rft.jtitle%253DEnv.%2BRes.%2BEcon.%26rft_id%253Dinfo%253Adoi%252F10.1007%252Fs10640-013-9663-x%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [15]: /lookup/external-ref?access_num=10.1007/s10640-013-9663-x&link_type=DOI [16]: #xref-ref-6-1 "View reference 6 in text" [17]: #xref-ref-7-1 "View reference 7 in text" [18]: http://www.fbspartnership.co.uk

Adipocyte insulin resistance can be caused by proximal insulin signaling defects but also from postreceptor mechanisms, which in large are poorly characterized. Adipocytes exposed for 18 h to the HIV protease inhibitor nelfinavir manifest insulin resistance characterized by normal insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate proteins, preserved in vitro phosphatidylinositol 3-kinase (PI 3-kinase) assay activity but impaired activation of PKB/Akt and stimulation of glucose uptake. Here we aimed to assess whether impaired PKB/Akt activation is indeed rate limiting for insulin signaling propagation in response to nelfinavir and the mechanism for defective PKB/Akt activation. Nelfinavir treatment of 3T3-L1 adipocytes impaired the insulin-stimulated translocation and membrane fusion of myc-glucose transporter (GLUT)-4-green fluorescent protein (GFP) reporter. Phosphorylation of PKB/Akt substrates including glycogen synthase kinase-3 and AS160 decreased in response to nelfinavir, and this remained true, even in cells with forced generation of phosphatidylinositol-3,4,5-trisphohphate (PIP3) by a membrane-targeted active PI 3-kinase, confirming that impaired PKB/Akt activation was rate limiting for insulin signal propagation. Cells expressing a GFP-tagged pleckstrin homology domain of general receptors for phosphoinositides 1, which binds PIP3, revealed intact PIP3-mediated plasma membrane translocation of this reporter in nelfinavir-treated cells. However, expression of a membrane-targeted catalytic subunit of PI 3-kinase failed to induce myc-GLUT4-GFP translocation in the absence of insulin, as it did in control cells. Conversely, a membrane-targeted and constitutively active PKB/Akt mutant was normally phosphorylated on S473 and T308, confirming intact PKB/Akt kinases activity, and induced myc-GLUT4-GFP translocation. Collectively, nelfinavir uncovers a postreceptor mechanism for insulin resistance, caused by interference with the sensing of PIP3 by PKB/Akt, leading to impaired GLUT4 translocation and membrane fusion.

Postprandial blood glucose clearance is mediated by GLUT4 (glucose transporter 4) which is translocated from an intracellular storage pool to the plasma membrane in response to insulin. The nature of the intracellular storage pool of GLUT4 is not well understood. Immunofluorescence staining shows that, under basal conditions, the major population of GLUT4 resides in the perinuclear compartment. At the same time, biochemical fractionation reveals that GLUT4 is localized in IRVs (insulin-responsive vesicles). The relationship between the perinuclear GLUT4 compartment and the IRVs is not known. In the present study, we have exchanged the C-termini of GLUT4 and cellugyrin, another vesicular protein that is not localized in the IRVs and has no insulin response. We have found that GLUT4 with the cellugyrin C-terminus loses its specific perinuclear localization, whereas cellugyrin with the GLUT4 C-terminus acquires perinuclear localization and becomes co-localized with GLUT4. This, however, is not sufficient for the effective entry of the latter chimaera into the IRVs as only a small fraction of cellugyrin with the GLUT4 C-terminus is targeted to the IRVs and is translocated to the plasma membrane in response to insulin stimulation. We suggest that the perinuclear GLUT4 storage compartment comprises the IRVs and the donor membranes from which the IRVs originate. The C-terminus of GLUT4 is required for protein targeting to the perinuclear donor membranes, but not to the IRVs.

It is well established that insulin stimulation of glucose uptake requires the translocation of intracellular localized GLUT4 protein to the cell surface membrane. This plasma membrane-redistributed GLUT4 protein was partially co-localized with caveolin as determined by confocal fluorescent microscopy but was fully excluded from lipid rafts based upon Triton X-100 extractability. Cholesterol depletion with methyl-beta-cyclodextrin, filipin, or cholesterol oxidase resulted in an insulin-independent increase in the amount of plasma membrane-localized GLUT4 that was fully reversible by cholesterol replenishment. This basal accumulation of cell surface GLUT4 occurred due to an inhibition of GLUT4 endocytosis. However, this effect was not specific since cholesterol extraction also resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor internalization. To functionally distinguish between caveolin- and clathrin-dependent endocytic processes, we took advantage of a dominant-interfering caveolin 1 mutant (Cav1/S80E) that specifically disrupts caveolae organization. Expression of Cav1/S80E, but not the wild type (Cav1/WT) or Cav1/S80A mutant, inhibited cholera toxin B internalization without any significant effect on transferrin receptor endocytosis. In parallel, Cav1/S80E expression increased the amount of plasma membrane-localized GLUT4 protein in an insulin-independent manner. Although Cav1/S80E also decreased GLUT4 endocytosis, the extent of GLUT4 internalization was only partially reduced ( approximately 40%). In addition, expression of Cav1/WT and Cav1/S80A enhanced GLUT4 endocytosis by approximately 20%. Together, these data indicate that the endocytosis of GLUT4 requires clathrin-mediated endocytosis but that the higher order structural organization of plasma membrane caveolin has a significant influence on this process.

Activation of Raf-1 by Ras requires recruitment to the membrane as well as additional phosphorylations, including phosphorylation at serine 338 (Ser-338) and tyrosine 341 (Tyr-341). In this study we show that Tyr-341 participates in the recruitment of Raf-1 to specialized membrane domains called "rafts," which are required for Raf-1 to be phosphorylated on Ser-338. Raf-1 is also thought to be recruited to the small G protein Rap1 upon GTP loading of Rap1. However, this does not result in Raf-1 activation. We propose that this is because Raf-1 is not phosphorylated on Tyr-341 upon recruitment to Rap1. Redirecting Rap1 to Ras-containing membranes or mimicking Tyr-341 phosphorylation of Raf-1 by mutation converts Rap1 into an activator of Raf-1. In contrast to Raf-1, B-Raf is activated by Rap1. We suggest that this is because B-Raf activation is independent of tyrosine phosphorylation. Moreover, mutants that render B-Raf dependent on tyrosine phosphorylation are no longer activated by Rap1.

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (ΔN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/ΔVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.

Glucose is cleared from the bloodstream by a family of facilitative transporters (GLUTs), which catalyze the transport of glucose down its concentration gradient and into cells of target tissues, primarily striated muscle and adipose. Currently, there are five established functional facilitative glucose transporter isoforms (GLUT1-4 and GLUTX1), with GLUT5 being a fructose transporter. GLUT1 is ubiquitously expressed with particularly high levels in human erythrocytes and in the endothelial cells lining the blood vessels of the brain. GLUT3 is expressed primarily in neurons and, together, GLUT1 and GLUT3 allow glucose to cross the blood-brain barrier and enter neurons. GLUT2 is a low-affinity (high Km) glucose transporter present in liver, intestine, kidney, and pancreatic beta cells. This transporter functions as part of the glucose sensor system in beta cells and in the basolateral transport of intestinal epithelial cells that absorb glucose from the diet. A new facilitative glucose transporter protein, GLUTX1, has been identified and appears to be important in early blastocyst development. The GLUT4 isoform is the major insulin-responsive transporter that is predominantly restricted to striated muscle and adipose tissue. In contrast to the other GLUT isoforms, which are primarily localized to the cell surface membrane, GLUT4 transporter proteins are sequestered into specialized storage vesicles that remain within the cell's interior under basal conditions. As postprandial glucose levels rise, the subsequent increase in circulating insulin activates intracellular signaling cascades that ultimately result in the translocation of the GLUT4 storage compartments to the plasma membrane. Importantly, this process is readily reversible such that when circulating insulin levels decline, GLUT4 transporters are removed from the plasma membrane by endocytosis and are recycled back to their intracellular storage compartments. Therefore, by establishing an internal membrane compartment as the default localization for the GLUT4 transporters, insulin-responsive tissues are poised to respond rapidly and efficiently to fluctuations in circulating insulin levels. Unfortunately, the complexity of these regulatory processes provides numerous potential targets that may be defective and eventually result in peripheral tissue insulin resistance and possibly diabetes. As such, understanding the molecular details of GLUT4 expression, GLUT4 vesicle compartment biogenesis, GLUT4 sequestration, vesicle trafficking, and fusion with the plasma membrane has become a major focus for many laboratories. This chapter will focus on recently elucidated insulin signal transduction pathways and GLUT4 vesicle trafficking components that are necessary for insulin-stimulated glucose uptake and GLUT4 translocation in adipocytes.

For policymakers, biodiversity can present more complex challenges than climate change, argue Michel Loreau, Alfred Oteng-Yeboah and their co-authors. So why isn't there an international panel of experts for biodiversity?

Using a crossed-response task, monkeys with neglect induced by frontal lesions appear to have motor rather than sensory neglect. However, the crossed-response task may not reveal sensory neglect (inattention) if no perceptual discrimination is required. We therefore trained two monkeys in a perceptually complex crossed-response task. Following unilateral frontal (arcuate gyrus) ablation, we found no contralateral perceptual disorders or sensory neglect in either monkey but did find a failure to respond with the contralateral arm (motor neglect) in both monkeys. We also found that the monkeys made more incorrect responses with the arm ipsilateral to the lesion than they did with the contralateral arm. The errors made by the ipsilateral arm could be a compensatory strategy or a disinhibition phenomenon. Because these incorrect responses were not rewarded and became more frequent as motor neglect improved, the incorrect responses of the ipsilateral arm are not a compensatory strategy, but rather a defect we term "disinhibition hyperkinesia" or "allokinesia."

Objective: In humans and monkeys, the intraparietal sulcus separates the superior parietal lobule from the inferior parietal lobule (IPL). Whereas in humans Brodmann's area 7 is above this sulcus, in monkeys it is below and therefore part of the IPL. In humans, the IPL consists of Brodmann's areas 39 and 40. Some investigators contend that the monkey homologue of the human IPL (areas 39 and 40) is the monkey's IPL (area 7). Others contend that it is, at least in part, in the monkey's superior temporal sulcus (STS). In humans, IPL lesions induce neglect. Although IPL lesions in monkeys also have been reported to induce neglect, the STS was involved in these lesions. We sought to learn which of these two areas, when ablated, produces neglect. Design: Study of five adult stump-tailed macaque monkeys by making five isolated STS and six IPL lesions. Results: Inferior parietal lobule lesions were associated with misreaching but not with unilateral neglect. Neglect was observed in association with five of the six STS lesions. Conclusions: With regard to neglect, STS may be the monkey homologue of the human IPL. Animals with STS lesions and humans with IPL lesions may manifest unilateral neglect because these areas are necessary for normal awareness of external stimuli. This awareness may result from the integration of the areas important in stimulus localization (the "where is it?" system) and stimulus identification (the "what is it?" system), as well as the areas important in defining the biologic importance of stimuli, such as the frontal lobes and limbic areas.

Insulin recruits two transmembrane proteins, GLUT4 and IRAP, to the plasma membrane of muscle cells and adipocytes. The subcellular trafficking and localization of GLUT4, and to a lesser extent IRAP, have been intensely studied, yet the molecular mechanisms responsible for their insulin-responsive compartmentalization remain unknown. Herein we have investigated the endocytosis and recycling of IRAP from the cell surface back to the insulin-responsive compartment (IRC). Our results show that a key dileucine motif at position 76,77 (LL76,77), although required for the initial biosynthetic entry of IRAP into the IRC, is dispensable for entry into the IRC via the endosomal system. Indeed, we found that an AA76,77 mutant of IRAP is fully capable of undergoing endocytosis and is correctly routed back to the IRC. To verify that the AA76,77 mutant enters the bona fide IRC, we show that the internalized IRAP-AA76,77 construct is sequestered in an IRC that is insensitive to brefeldin A yet sensitive to a dominant-interfering mutant of AS160 (AS160-4P). In addition, we show that the GGA clathrin adaptors are not required for the re-entry of IRAP from the cell surface back into the IRC, whereas the Q-SNARE syntaxin 6 is required for this process.

The principal sources of input to rat medial precentral cortex (PCm), as revealed by iontophoretically applied horseradish peroxidase, are: the ventral lateral, mediodorsal, central lateral and ventromedial thalamic nuclei; contralateral PCm; ipsilateral cortical areas related to vision, somatic sensation and audition; retrosplenial and ventrolateral orbital cortex. These results are compared to findings in monkeys and discussed in relation to the phenomenon of polymodal neglect of hemispace.

This study of the rostral part of medial agranular cortex (AGm) was undertaken with two principal aims in mind. First, to delineate the efferent connections of AGm and compare these with the pattern of afferents defined by us in a previous study. Second, to provide a firmer basis for anatomical and functional comparisons with cortical regions in monkeys. Autoradiographic, horseradish peroxidase, and fiber degeneration techniques were used. Rostral AGm has a variety of corticocortical connections--with lateral agranular motor cortex (AGl); visual, auditory, and somatic sensory regions; and limbic/paralimbic areas including orbital, insular, perirhinal, entorhinal, retrosplenial and presubicular fields. The projections to orbital, perirhinal and entorhinal cortices are bilateral. Thalamic projections of rostral AGm are concentrated in the ventral lateral, central lateral, paracentral, mediodorsal and ventromedial nuclei. Moderate terminal fields are consistently seen in the reticular, anteromedial, central medial, gelatinosus, parafascicular, and posterior nuclei. More caudal projections reach the central gray, superior colliculus and pontine gray. The efferents of the adjacent AGl were also examined. Although many of these overlapped those of rostral AGm, there were no efferents to visual or auditory cortex and limbic/paralimbic projections were reduced. Thalamic projections were more focused in the ventral lateral and posterior nuclei and there were no terminal fields in the central gray or superior colliculus. Based on its afferent and efferent connections, role in contralateral neglect, and the results of microstimulation studies, rostral AGm can be viewed as a multimodal association area with strong ties to the motor system. On these structural and functional grounds, rostral AGm bears certain striking resemblances to the frontal eye field, supplementary motor, and arcuate premotor areas of monkey cortex.

Neglect is a disorder in which the response to stimulation is diminished or absent on the side of the body contralateral to the lesion in the absence of an elemental sensory or motor defect. Most cases of neglect in humans are induced by cortical damage, but there have been no investigations of the pharmacologic basis of neglect induced by cortical damage. We examined the role of the dopamine system in polymodal neglect caused by a unilateral lesion of the medial precentral prefrontal cortex of the rat. A dose-response examination of the effect of apomorphine on neglect revealed that apomorphine, at 0.5 mg/kg, the highest dose examined, significantly improved the orientation scores of subjects in all modalities tested and significantly decreased the total number of allesthetic responses. The therapeutic effect of apomorphine was mediated by dopamine receptors as the therapeutic effect of apomorphine was blocked by prior administration of spiroperidol. These results demonstrate the important role of disruption of dopamine mechanisms in neglect induced by a lesion of medial precentral cortex.

The extent of electroencephalographic (EEG) abnormalities in 23 consecutive patients with unilateral neglect was compared with the extent of EEG abnormalities in 20 matched aphasic controls. In the neglect group, 22 patients showed EEG slowing that extended over the entire hemisphere. In the control group only 8 patients showed similar hemispheric slowing. This study demonstrates that patients with unilateral neglect from discrete lesions may have EEG slowing in regions of the hemisphere that are remote from the lesion. This study also provides support for the hypothesis that neglect is a unilateral arousal defect.

• The evoked potential was studied in Macaca speciosa before and after they received a lesion inducing neglect. Significant evoked potential changes associated with neglect were seen only in late components (N 2 , P 3 ). These observations support the hypothesis of the arousalattention mechanism of neglect.

Discrete unilateral mesencephalic reticular formation lesions were made in four female stump-tailed macacques (Macaca speciosa ). Control lesions of identical size were placed in the left medial dorsal thalamic nucleus, left ventral posterolateral thalamic nucleus, and left pontine nucleus. Experimental subjects showed profound tactile, visual and auditory neglect, in addition to ipsilateral hemispheric slowing on EEG. Controls did not demonstrate either phenomenon. This study suggests that neglect results from any lesion along the corticolimbic-reticular activating loop, and that the basis of neglect is a unilateral defect in the alerting response to sensory stimuli because of a disruption of this loop.

• Four monkeys were trained to open a door with either the right or left hand in response to a tactile stimulus to either leg. After unilateral frontal arcuate ablation inducing unilateral neglect, the response time on this task increased most when the monkey responded with the hand contralateral to the lesion, but also increased when the monkey used the hand ipsilateral to the lesion. The side of stimulation had no effect on response time. Control (anterior superior temporal) lesions did not cause neglect and only affected response time slightly in one monkey (using the limb contralateral to the lesion). We conclude that response time is increased in animals with unilateral neglect and that the increase results from a defect in intention to act (motor neglect) rather than from sensory neglect.

Monkeys trained to perform with the extremity contralateral to a stimulus had unilateral neglect induced by a parietotemporal lesion. Their performance was normal postoperatively when stimulated on the side contralateral to the lesion, but they made errors when stimulated on the normal side (the side ipsilateral to the lesion), often failing to use the extremity on the neglected side (the side contralateral to the lesion). Although we expected parietotemporal lesions to induce sensory neglect (failure to respond to contralateral stimuli), we could show only that these animals had a defect of intention (contralateral hypokinesia). In this respect, neglect induced by parietotemporal lesions is similar to that induced by frontal or medial thalamic lesions.

Five monkeys trained to perform with the extremity contralateral to a stimulus had unilateral neglect induced by frontal and reticular formation lesions. Postoperatively the performance of the animals was abnormal only on ipsilateral stimulation, which suggests that the mechanism underlying neglect in these subjects is not deafferentation of sensory inattention but a defect of intention.

The cerebral hemispheres may be mutually inhibitory. An injured hemisphere may continue to be inhibited while the uninjured hemisphere is released from inhibition, inducing attentional bias. Patients with neglect might therefore benefit from transection of the corpus callosum. To test this hypothesis, we ablated the frontal arcuate gyrus alone or several months after corpus callosum transection in monkeys. Neglect was significantly worse in animals with a corpus callosum transection. Therefore, the hemispheres may be mutually excitatory or compensatory, and a callosal transection would not be therapeutic. Because the rate of recovery in the two groups was not different, recovery seemed to be an intrahemispheric phenomenon.

To learn whether animals with parietotemporal lesions have sensory inattention or defective intention, we trained monkeys to respond with the contralateral limb to a threshold tactile stimulus. After parietotemporal lesions that induced neglect, the monkeys continued to respond normally to threshold stimuli on the side opposite the lesion (the neglected side), but made errors when stimulated on the normal side (ipsilateral to the lesion), often failing to use the contralateral extremity. On this learned task, there was no abnormality of sensory input or sensory attention. The problem was attributed to an impaired preparation to respond (intention).