Kinetic evidence for unique regulation of GLUT4 trafficking by insulin and AMP-activated protein kinase activators in L6 myotubes.

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of approximately 20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min(-1)) and was decreased during the steady-state responses to insulin (0.18 min(-1)), AICAR (0.16 min(-1)), and A-769662 (0.24 min(-1)). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels

Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice. To dissociate oxidative stress from impaired oxidative phosphorylation and study whether mitochondrial oxidative stress per se can cause insulin resistance, we used mitochondria-targeted paraquat (MitoPQ) to generate superoxide within mitochondria without directly disrupting the respiratory chain. At ≤10 μm, MitoPQ specifically increased mitochondrial superoxide and hydrogen peroxide without altering mitochondrial respiration in intact cells. Under these conditions, MitoPQ impaired insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation to the plasma membrane in both adipocytes and myotubes. MitoPQ recapitulated many features of insulin resistance found in other experimental models, including increased oxidants in mitochondria but not cytosol; a more profound effect on glucose transport than on other insulin-regulated processes, such as protein synthesis and lipolysis; an absence of overt defects in insulin signaling; and defective insulin- but not AMP-activated protein kinase (AMPK)-regulated GLUT4 translocation. We conclude that elevated mitochondrial oxidants rapidly impair insulin-regulated GLUT4 translocation and significantly contribute to insulin resistance and that MitoPQ is an ideal tool for studying the link between mitochondrial oxidative stress and regulated GLUT4 trafficking

Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes.

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes

Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes.

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease

CD-MPR: Quo Vadis?

Lysosomes are membrane-bound organelles that serve in the degradation of many extracellular and intracellular macromolecules. Lysosomal biogenesis depends on the delivery of newly synthesized lysosomal hydrolases. This process requires the acquisition of the lysosomal targeting signal, the mannose 6-phosphate tag that is specifically recognized by mannose 6-phosphate receptors (MPRs) in the TGN. The receptor-ligand complex is subsequently packaged into clathrin-coated vesicles and transported to early endosomes. The lower pH in the endosomal compartment causes the dissociation of the MPR and the ligand. The lysosomal enzymes are transferred to the lysosome, where they are activated, whereas the MPRs are transported from endosomes back to the TGN where they mediate another round of transport. Two distinct MPRs were identified and characterized - the 46 kDa cation-dependent (CD) MPR and the ~300 kDa cation-independent (CI) MPR. This study concentrates on the CD-MPR. The intracellular trafficking of the CD-MPR is mediated by sorting signals located in its cytoplasmic tail of 67 amino acids. The sorting motifs are recognized by specific adaptor proteins that mediate the vesicular transport of the receptor. Although several motifs and their interacting partners were identified in the CD-MPR, the various trafficking steps are not yet fully understood. In this study we focused on the characterization of two motifs of the receptor - the cysteine C30 and C34 which undergo reversible palmitoylation and the acidic cluster of the casein kinase 2 (CK2) phosphorylation site (E55-E56-S57-E58-E59). The CD-MPR is transported efficiently from late endosomes back to the TGN since only a very small percent of receptors are missorted to lysosomes where they are rapidly degraded. This transport step depends on the palmitoylation of C34, and additionally on the diaromatic motif F18W19. The membrane anchoring mediated by the palmitate, 34 amino acids away from the trans-membrane domain, implies a drastic conformational change on the cytoplasmic tail of the CD-MPR. The diaromatic motif is likely to be better exposed to the interacting protein in the palmitoylated than in the non-palmitoylated CD-MPR. Our hypothesis suggests that the reversible palmitoylation regulates the sorting signals in the cytoplasmic tail of the receptor. This would require that the palmitoylation occur enzymatically. In Part I, we show that indeed the palmitoylation depends on a membrane-bound enzyme. This palmitoyltransferase cycles between the plasma membrane and endosomes. Close proximity of the palmitoyltransferase to the site where the palmitoylation of the CD-MPR is required is optimal to ensure the presence of the palmitoylated C34 in late endosomes. Thus, the localization of the palmitoyltransferase supports our hypothesis of palmitoylation as a regulatory mechanism for the sorting signals in the cytoplasmic tail of the receptor. Correct sorting of the CD-MPR from the TGN to endosomes depends on the D61-X-X-L64-L65 sequence, which interacts with GGA (Golgi-localizing, γ-earcontaining, ARF-binding protein), a monomeric adaptor protein that mediates the formation of clathrin-coated vesicles at the TGN. Several substrates of GGA have a CK2 site upstream of the DXXLL motif and in two cases, phosphorylation by CK2 was shown to increase the affinity of GGA1 to cargo. The CD-MPR also contains a CK2 site upstream of the DXXLL motif, but its involvement in GGA1 binding has not been investigated so far. The CK2 site of the CD-MPR was shown to interact with the adaptor protein 1 (AP-1), another protein involved in the sorting of cargo in the TGN, possibly in cooperation with GGA. Previous reports on the requirement of phosphorylation of the CD-MPR for binding to AP-1 were controversial. In Part II, we analyzed the influence of the CK2 phosphorylation site of the CD-MPR in binding to GGA1 and AP-1 and thus, in sorting in the TGN. A mutational analysis revealed that high affinity binding between CD-MPR and GGA1 was dependent on the acidic amino acid E59 and to a lesser extent on E58, while the phosphorylation of the S57 had no influence, indicating that the GGA1 binding site in the CD-MPR extends to E58-E59-X-D61-X-X-L64- L65. In contrast, AP-1 depended on all glutamates surrounding the serine E55, E56, E58, E59 in the CD-MPR for binding, but was also independent of the phosphorylation of S57. Therefore, we revealed that the phosphorylation of S57 is not required for sorting in the TGN. Interestingly, the binding affinity of GGA1 to the CD-MPR was 2.4-fold higher than that of AP-1 to the partially overlapping binding site in the CD-MPR. Thus, we present a modified model for the sorting process in the TGN, involving both GGA1 and AP-1, where the different binding affinities, determine the order of binding to the partially overlapping binding sites in the CD-MPR. First, GGA1 binds to the CD-MPR due to its higher affinity and is subsequently released from the CD-MPR as a result of its autoinhibition caused by phosphorylation. This allows the AP-1 to bind and recruit the remaining components for correct sorting of the CD-MPR in the TGN. With our work we contributed to the understanding of specific transports steps of the CD-MPR and thereby we are advancing towards the goal of fully elucidating the trafficking of the receptor

The palmitoyltransferase of the cation-dependent mannose 6-phosphate receptor cycles between the plasma membrane and endosomes

The cation-dependent mannose 6-phosphate receptor (CD-MPR) mediates the transport of lysosomal enzymes from the trans-Golgi network to endosomes. Evasion of lysosomal degradation of the CD-MPR requires reversible palmitoylation of a cysteine residue in its cytoplasmic tail. Because palmitoylation is reversible and essential for correct trafficking, it presents a potential regulatory mechanism for the sorting signals within the cytoplasmic domain of the CD-MPR. Characterization of the palmitoylation performing an in vitro palmitoylation assay by using purified full-length CD-MPR revealed that palmitoylation of the CD-MPR occurs enzymatically by a membrane-bound palmitoyltransferase. In addition, analysis of the localization revealed that the palmitoyltransferase cycles between endosomes and the plasma membrane. This was identified by testing fractions from HeLa cell homogenate separated on a density gradient in the in vitro palmitoylation assay and further confirmed by in vivo labeling experiments by using different treatments to block specific protein trafficking steps within the cell. We identified a novel palmitoyltransferase activity in the endocytic pathway responsible for palmitoylation of the CD-MPR. The localization of the palmitoyltransferase not only fulfills the requirement of our hypothesis to be a regulator of the intracellular trafficking of the CD-MPR but also may affect the sorting/activity of other receptors cycling through endosomes

Protein Kinase Cε Modulates Insulin Receptor Localization and Trafficking in Mouse Embryonic Fibroblasts

<div><p>We have previously shown that deletion of protein kinase C epsilon (PKCε) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCε-deficient (PKCε^−/−) mice to examine this mechanistically. PKCε^−/− MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCε deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCε^−/− cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.</p> </div

Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes.

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease

Proximal insulin signalling in WT and PKCε^−/− MEFs.

<p>A. Insulin receptor expression was assessed by immunoblotting, after cells under basal conditions were extracted and equal amounts of protein were subjected to SDS-PAGE. Data are means from 3 independent experiments. B,C. Insulin receptor and IRS-1 protein stability in the presence of chronic insulin were determined in WT (closed circles) and PKCε^−/− (open circles) MEFs treated with 1 µM insulin in the presence of 50 µg/mL cycloheximide for the indicated times. Data are means from 5 independent experiments carried out in duplicate. For the investigation of acute signalling, WT (closed circles) and PKCε^−/− (open squares) cells were serum-starved for 2 h and stimulated with 100 nM insulin for the indicated times, prior to cell lysis and immunoblotting. D. Phospho-Y1162/1163 and total insulin receptor; t-test: area under the curve WT vs PKCε^−/− MEFs P<0.001). E. Phospho-Y612 and total IRS-1. F. Phospho-S473 and total Akt. G. Phospho-S636/639 IRS-1. Data are means ± SEM from 3 or more independent experiments in 3 sets of MEF cell lines. H. MEFs were preincubated without (open symbols) or with (closed symbols) phosphatase inhibitors for 20 min prior to insulin stimulation, and insulin receptor Y1162/1163 phosphorylation determined by immunoblotting. Results shown are means from 2 independent experiments.</p