Search

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport that is underpinned by the insulin-stimulated delivery of glucose transporter type 4 (SLC2A4, also known and hereafter referred to as GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, and increase cell surface GLUT4 levels. Adipocytokines, such as adiponectin, are secreted via a similar mechanism. We used genome editing to knock out syntaxin-4, a protein reported to mediate fusion between GLUT4-containing vesicles and the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of haemagglutinin (HA)-tagged GLUT4 conjugated to GFP showed that syntaxin-4-knockout cells retain significant GLUT4 translocation capacity, demonstrating that syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells. This article has an associated First Person interview with Hannah L. Black and Rachel Livingstone, joint first authors of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA-GLUT4-GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA-GLUT4-GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells., Competing Interests: Gwyn W. Gould is an Academic Editor for PeerJ., (© 2020 Morris et al.)

Pathways linking activation of the insulin receptor to downstream targets of insulin have traditionally been studied using a candidate gene approach. To elucidate additional pathways regulating insulin activity, we performed a forward chemical-genetics screen based on translocation of a glucose transporter 4 (Glut4) reporter expressed in murine 3T3-L1 adipocytes. To identify compounds with known targets, we screened drug-repurposing and natural product libraries. We identified, confirmed, and validated 64 activators and 65 inhibitors that acutely increase or rapidly decrease cell-surface Glut4 in adipocytes stimulated with submaximal insulin concentrations. These agents were grouped by target, chemical class, and mechanism of action. All groups contained multiple hits from a single drug class, and several comprised multiple structurally unrelated hits for a single target. Targets include the β-adrenergic and adenosine receptors. Agonists of these receptors increased and inverse agonists/antagonists decreased cell-surface Glut4 independently of insulin. Additional activators include insulin sensitizers (thiazolidinediones), insulin mimetics, dis-inhibitors (the mTORC1 inhibitor rapamycin), cardiotonic steroids (the Na + /K + -ATPase inhibitor ouabain), and corticosteroids (dexamethasone). Inhibitors include heterocyclic amines (tricyclic antidepressants) and 21 natural product supplements and herbal extracts. Mechanisms of action include effects on Glut4 trafficking, signal transduction, inhibition of protein synthesis, and dissipation of proton gradients. Two pathways that acutely regulate Glut4 translocation were discovered: de novo protein synthesis and endocytic acidification. The mechanism of action of additional classes of activators (tanshinones, dalbergiones, and coumarins) and inhibitors (flavonoids and resveratrol) remains to be determined. These tools are among the most sensitive, responsive, and reproducible insulin-activity assays described to date., (© 2019 Brewer et al.)

Insulin increases glucose uptake by increasing the rate of exocytosis of the facilitative glucose transporter isoform 4 (Glut4) relative to its endocytosis. Insulin also releases Glut4 from highly insulin-regulated secretory compartments (GSVs or Glut4 storage vesicles) into constitutively cycling endosomes. Previously it was shown that both overexpression and knockdown of the small GTP-binding protein Rab14 decreased Glut4 translocation to the plasma membrane (PM). To determine the mechanism of this perturbation, we measured the effects of Rab14 knockdown on the trafficking kinetics of Glut4 relative to two proteins that partially co-localize with Glut4, the transferrin (Tf) receptor and low-density-lipoprotein-receptor-related protein 1 (LRP1). Our data support the hypothesis that Rab14 limits sorting of proteins from sorting (or 'early') endosomes into the specialized GSV pathway, possibly through regulation of endosomal maturation. This hypothesis is consistent with known Rab14 effectors. Interestingly, the insulin-sensitive Rab GTPase-activating protein Akt substrate of 160 kDa (AS160) affects both sorting into and exocytosis from GSVs. It has previously been shown that exocytosis of GSVs is rate-limited by Rab10, and both Rab10 and Rab14 are in vitro substrates of AS160. Regulation of both entry into and exit from GSVs by AS160 through sequential Rab substrates would provide a mechanism for the finely tuned 'quantal' increases in cycling Glut4 observed in response to increasing concentrations of insulin., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (ken = 0.2 min(-1) versus 0.6 min(-1)). Differentiation decreases Glut4 ken 40% (ken = 0.12 min(-1)). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (kex = 0.025-0.038 min(-1)), not through the fast Tf receptor pathway (kex = 0.2 min(-1)). The kex measured in adipocytes after insulin stimulation is similar (kex = 0.027 min(-1)). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (ksort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (kseq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (kfuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Insulin enhances the uptake of glucose into adipocytes and muscle cells by promoting the redistribution of the glucose transporter isoform 4 (GLUT4) from intracellular compartments to the cell surface. Rab GTPases regulate the trafficking itinerary of GLUT4 and several have been found on immunopurified GLUT4 vesicles. Specifically, Rab14 has previously been implicated in GLUT4 trafficking in muscle although its role, if any, in adipocytes is poorly understood. Analysis of 3T3-L1 adipocytes using confocal microscopy demonstrated that endogenous GLUT4 and endogenous Rab14 exhibited a partial colocalisation. However, when wild-type Rab14 or a constitutively-active Rab14Q70L mutant were overexpressed in these cells, the colocalisation with both GLUT4 and IRAP became extensive. Interestingly, this colocalisation was restricted to enlarged 'ring-like' vesicular structures (mean diameter 1.3 µm), which were observed in the presence of overexpressed wild-type Rab14 and Rab14Q70L, but not an inactive Rab14S25N mutant. These enlarged vesicles contained markers of early endosomes and were rapidly filled by GLUT4 and transferrin undergoing endocytosis from the plasma membrane. The Rab14Q70L mutant reduced basal and insulin-stimulated cell surface GLUT4 levels, probably by retaining GLUT4 in an insulin-insensitive early endosomal compartment. Furthermore, shRNA-mediated depletion of Rab14 inhibited the transit of GLUT4 through early endosomal compartments towards vesicles and tubules in the perinuclear region. Given the previously reported role of Rab14 in trafficking between endosomes and the Golgi complex, we propose that the primary role of Rab14 in GLUT4 trafficking is to control the transit of internalised GLUT4 from early endosomes into the Golgi complex, rather than direct GLUT4 translocation to the plasma membrane.

The Akt substrate AS160 (TCB1D4) regulates Glut4 exocytosis; shRNA knockdown of AS160 increases surface Glut4 in basal adipocytes. AS160 knockdown is only partially insulin-mimetic; insulin further stimulates Glut4 translocation in these cells. Insulin regulates translocation as follows: 1) by releasing Glut4 from retention in a slowly cycling/noncycling storage pool, increasing the actively cycling Glut4 pool, and 2) by increasing the intrinsic rate constant for exocytosis of the actively cycling pool (k(ex)). Kinetic studies were performed in 3T3-L1 adipocytes to measure the effects of AS160 knockdown on the rate constants of exocytosis (k(ex)), endocytosis (k(en)), and release from retention into the cycling pool. AS160 knockdown released Glut4 into the actively cycling pool without affecting k(ex) or k(en). Insulin increased k(ex) in the knockdown cells, further increasing cell surface Glut4. Inhibition of phosphatidylinositol 3-kinase or Akt affected both k(ex) and release from retention in control cells but only k(ex) in AS160 knockdown cells. Glut4 vesicles accumulate in a primed pre-fusion pool in basal AS160 knockdown cells. Akt regulates the rate of exocytosis of the primed vesicles through an AS160-independent mechanism. Therefore, there is an additional Akt substrate that regulates the fusion of Glut4 vesicles that remain to be identified. Mathematical modeling was used to test the hypothesis that this substrate regulates vesicle priming (release from retention), whereas AS160 regulates the reverse step by stimulating GTP turnover of a Rab protein required for vesicle tethering/docking/fusion. Our analysis indicates that fusion of the primed vesicles with the plasma membrane is an additional non-Akt-dependent insulin-regulated step.

Insulin regulates glucose uptake through effects on the trafficking of the glucose transporter Glut4. To investigate the degree of overlap between Glut4 and the general endocytic pathways, the kinetics of trafficking of Glut4 and the receptors for transferrin (Tf) and α(2)-macroglobulin (α-2-M; LRP-1) were compared using quantitative flow cytometric assays. Insulin increased the exocytic rate constant (k(ex)) for both Glut4 and Tf. However, the k(ex) of Glut4 was 5-15 times slower than Tf in both basal and insulin-stimulated cells. The endocytic rate constant (k(en)) of Glut4 was also five times slower than Tf. Insulin did not affect the k(en) of either protein. In basal cells, the k(en) for α-2-M/LRP-1 was similar to Glut4 but 5-fold slower than Tf. Insulin increased k(en) for α-2-M/LRP-1 by 30%. In contrast, the k(ex) for LRP-1 was five times faster than Glut4 in basal cells, and insulin did not increase this rate constant. Thus, although there is overlap in the protein machineries/compartments utilized, the differences in trafficking kinetics indicate that Glut4, the Tf receptor, and LRP-1 are differentially processed both within the cell and at the plasma membrane. It has been reported that insulin decreases the k(en) of Glut4 in adipocytes. However, the effect of exocytosis on the "internalization" assays was not considered. Because it is counterintuitive, the effect of exocytosis on these assays is often overlooked in endocytosis studies. Using mathematical modeling and simulation, we show that the reported decrease in Glut4 k(en) can be entirely accounted for by the well established increase in Glut4 k(ex).

Insulin stimulates glucose storage and metabolism by the tissues of the body, predominantly liver, muscle and fat. Storage in muscle and fat is controlled to a large extent by the rate of facilitative glucose transport across the plasma membrane of the muscle and fat cells. Insulin controls this transport. Exactly how remains debated. Work presented in this review focuses on the pathways responsible for the regulation of glucose transport by insulin. We present some historical work to show how the prevailing model for regulation of glucose transport by insulin was originally developed, then some more recent data challenging this model. We finish describing a unifying model for the control of glucose transport, and some very recent data illustrating potential molecular machinery underlying this regulation. This review is meant to give an overview of our current understanding of the regulation of glucose transport through the regulation of the trafficking of Glut4, highlighting important questions that remain to be answered. A more detailed treatment of specific aspects of this pathway can be found in several excellent recent reviews (Brozinick et al., 2007 Hou and Pessin, 2007; Huang and Czech, 2007;Larance et al., 2008 Sakamoto and Holman, 2008; Watson and Pessin, 2007; Zaid et al., 2008)One of the main objectives of this review is to discuss the results of the experiments measuring the kinetics of Glut4 movement between subcellular compartments in the context of our emerging model of the Glut4 trafficking pathway.

The relationship between the plasminogen activator system and integrin function is well documented but incompletely understood. The mechanism of uPAR-mediated signaling across the membrane and the molecular basis of uPAR-dependent activation of integrins remain important issues. The present study was undertaken to identify the molecular intermediates involved in the uPAR signaling pathway controlling alpha5beta1-integrin activation and fibronectin polymerization. Disruption of lipid rafts with MbetaCD or depletion of caveolin-1 by siRNA led to the inhibition of uPAR-dependent integrin activation and stimulation of fibronectin polymerization in human dermal fibroblasts. The data indicate a dual role for caveolin-1 in the uPAR signaling pathway, leading to integrin activation. Caveolin-1 functions initially as a membrane adaptor or scaffold to mediate uPAR-dependent activation of Src and EGFR. Subsequently, in its phosphorylated form, caveolin-1 acts as an accessory molecule to direct trafficking of activated EGFR to focal adhesions. These studies provide a novel paradigm for the regulation of crosstalk among integrins, growth-factor receptors and uPAR.

In adipocytes, insulin triggers the redistribution of Glut4 from intracellular compartments to the plasma membrane. Two models have been proposed to explain the effect of insulin on Glut4 localization. In the first, termed dynamic exchange, Glut4 continually cycles between the plasma membrane and intracellular compartments in basal cells, and the major effect of insulin is through changes in the exocytic and endocytic rate constants, k(ex) and k(en). In the second model, termed static retention, Glut4 is packaged in specialized storage vesicles (GSVs) in basal cells and does not traffic through the plasma membrane or endosomes. Insulin triggers GSV exocytosis, increasing the amount of Glut4 in the actively cycling pool. Using a flow cytometry-based assay, we found that Glut4 is regulated by both static and dynamic retention mechanisms. In basal cells, 75-80% of the Glut4 is packaged in noncycling GSVs. Insulin increased the amount of Glut4 in the actively cycling pool 4-5-fold. Insulin also increased k(ex) in the cycling pool 3-fold. After insulin withdrawal, Glut4 is rapidly cleared from the plasma membrane (t((1/2)) of 20 min) by rapid adjustments in k(ex) and k(en) and recycled into static compartments. Complete recovery of the static pool required more than 3 h, however. We conclude that in fully differentiated confluent adipocytes, both the dynamic and static retention mechanisms are important for the regulation of plasma membrane Glut4 content. However, cell culture conditions affect Glut4 trafficking. For example, replating after differentiation inhibited the static retention of Glut4, which may explain differences in previous reports.

Glut4 exocytosis in adipocytes uses protein machinery that is shared with other regulated secretory processes. Synapsins are phosphoproteins that regulate a ;reserve pool' of vesicles clustered behind the active zone in neurons. We found that adipocytes (primary cells and the 3T3-L1 cell line) express synapsin IIb mRNA and protein. Synapsin IIb co-localizes with Glut4 in perinuclear vesicle clusters. To test whether synapsin plays a role in Glut4 traffic, a site 1 phosphorylation mutant (S10A synapsin) was expressed in 3T3-L1 adipocytes. Interestingly, expression of S10A synapsin increased basal cell surface Glut4 almost fourfold (50% maximal insulin effect). Insulin caused a further twofold translocation of Glut4 in these cells. Expression of the N-terminus of S10A synapsin (amino acids 1-118) was sufficient to inhibit basal Glut4 retention. Neither wild-type nor S10D synapsin redistributed Glut4. S10A synapsin did not elevate surface levels of the transferrin receptor in adipocytes or Glut4 in fibroblasts. Therefore, S10A synapsin is inhibiting the specialized process of basal intracellular retention of Glut4 in adipocytes, without affecting general endocytic cycling. While mutant forms of many proteins inhibit Glut4 exocytosis in response to insulin, S10A synapsin is one of only a few that specifically inhibits Glut4 retention in basal adipocytes. These data indicate that the synapsins are important regulators of membrane traffic in many cell types.

Caspases are the intracellular molecular machinery responsible for apoptotic cell death. The regulation of these critical proteolytic enzymes is known to occur on multiple levels. While their expression as inactive precursors exhibits a primary level of control, other types of regulation such as post-translational modifications also play a role. Nuclear c-Abl, a nonreceptor tyrosine kinase, plays a role in the regulation of apoptosis in response to DNA damage. The function of cytoplasmic c-Abl in cell death is not fully understood. Here, we report c-Abl dependent caspase-3 and caspase-8 activity in response to staurosporine. Despite the presence and apparent activation of the mitochondrial-dependent apoptotic pathway and cellular demise, we find no caspase-3 activity in cells lacking the Abl gene (Abl(-/-)). These findings demonstrate a novel tyrosine kinase dependent regulation of caspase-mediated cell death.

Csk negatively regulates Src family kinases (SFKs). In lymphocytes, Csk is constitutively active, and is transiently inactivated in response to extracellular stimuli, allowing activation of SFKs. In contrast, both SFKs and Csk were inactive in unstimulated mouse embryonic fibroblasts, and both were activated in response to oxidative stress. Csk modulated the oxidative stress-induced, but not the basal SFK activity in these cells. These data indicate that Csk may be more important for the return of Src-kinases to the basal state than for the maintenance of basal activity in some cell types. Csk must be targeted to its SFK substrates through an SH2-domain-mediated interaction with a phosphoprotein. Our data indicate that caveolin-1 is one of these targeting proteins. SFKs bind to caveolin-1 and phosphorylate it in response to oxidative stress and insulin. Csk binds specifically to the phosphorylated caveolin-1 and attenuates its stress-induced phosphorylation. Importantly, phosphocaveolin was one of two major phosphoproteins associated with Csk after incubation with peroxide or insulin. Paxillin was the other. Activation/rapid attenuation of SFKs by Csk is required for actin remodeling. Caveolin-1 is phosphorylated at the ends of actin fibers at points of contact between the actin cytoskeleton and the plasma membrane, where it could in part mediate this attenuation.

Caveolin-1 is phosphorylated at tyrosine 14 in response to cellular stress. Tyrosine 14 is a consensus Abl phosphorylation site suggesting that caveolin-1 may be an Abl substrate. We report here that expression of c-Abl is required for oxidative stress-induced caveolin-1 phosphorylation. In contrast, c-Src expression is not required. Phosphocaveolin is one of only two phosphotyrosine signals missing in lysates from the Abl(-/-) cells, indicating that these cells still respond to oxidative stress. Oxidative stress-induced tyrosine phosphorylation of caveolin-1 occurs only at the Abl site, tyrosine 14. Caveolin-1 is also a major phosphotyrosine signal detected in cells over-expressing c-Abl. Our results show that Abl activation leads to phosphorylation of caveolin-1 on tyrosine 14. Both Abl and caveolin have been linked to the actin cytoskeleton, and oxidative stress-induced phosphocaveolin is enriched at focal contacts. This suggests that phosphocaveolin regulates these structures, perhaps through recruiting and activating SH2-domain proteins such as Csk., (Copyright 2002 Elsevier Science Inc.)

Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl. To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor-alpha receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation. In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells. Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae. This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae.

PDGF receptors and Src family kinases are concentrated in caveolae, where signal transduction cascades involving these molecules are thought to be organized. The Src family tyrosine kinases are cotransducers of signals emanating from the activated PDGF receptor. However, the Src family kinase substrates that are involved in PDGF-induced signaling remain to be fully elucidated. We have identified a 29-kDa protein in caveolae that was phosphorylated in response to PDGF stimulation. This protein, pp29, was tightly bound to the caveolar coat protein caveolin-1. pp29 was among the most prominent phosphoproteins observed in cells overexpressing Fyn, suggesting that it may be a Fyn substrate. Consistent with this, pp29 was among a specific subset of proteins whose PDGF-stimulated phosphorylation was blocked by expression of kinase inactive Fyn. These data indicate that pp29 lies downstream of Fyn activation in a PDGF-stimulated signaling pathway, and that pp29 is an abundant site for nucleation of signal transduction cascades., (©2002 Elsevier Science (USA).)

Insulin is a potent stimulator of intermediary metabolism, however the basis for the remarkable specificity of insulin's stimulation of these pathways remains largely unknown. This review focuses on the role compartmentalization plays in insulin action, both in signal initiation and in signal reception. Two examples are discussed: (1) a novel signalling pathway leading to the phosphorylation of the caveolar coat protein caveolin, and (2) a recently identified scaffolding protein, PTG, involved directly in the regulation of enzymes controlling glycogen metabolism.

Previous work from this laboratory has shown that insulin stimulates the tyrosine phosphorylation of caveolin in 3T3-L1 adipocytes (Mastick, C. C., Brady, M. J., and Saltiel, A. R. (1995) J. Cell Biol. 129, 1523-1531). This phosphorylation is specific for insulin and involves the activation of a tyrosine kinase downstream of the insulin receptor. We report here that tyrosine phosphorylation of caveolin is detected only in fully differentiated adipocytes, not in fibroblasts (preadipocytes), despite the fact that both cell types express caveolin-1 and active insulin receptor. Caveolin copurifies with caveolin tyrosine kinase activity in both preadipocytes and adipocytes. Accumulating evidence indicates that this kinase is the Src family kinase Fyn. Fyn is expressed in the preadipocytes and the adipocytes and is colocalized with caveolin in low density Triton-insoluble complexes in both cell types. In adipocytes, overexpression of wild type Fyn leads to increased basal phosphorylation of caveolin and hyperphosphorylation of caveolin in response to insulin. In vitro kinase assays suggest that Fyn may be activated in response to insulin through the binding of a tyrosine-phosphorylated insulin receptor substrate protein. Previous work suggested that this protein may be c-Cbl (Ribon, V., and Saltiel, A. R. (1997) Biochem. J. 324, 839-846). In 3T3-L1 adipocytes, Cbl binds to Fyn in an insulin-dependent manner, and Cbl phosphorylation is adipocyte-specific. Here we show that phosphorylated Cbl is translocated into caveolin-enriched Triton-insoluble complexes after insulin stimulation. This may lead to the cell type-specific, compartmentalized activation of Fyn and the specific phosphorylation of proteins in the caveolae in response to insulin in adipocytes.

We have recently cloned from 3T3-L1 adipocytes a novel glycogen-targeting subunit of protein phosphatase-1, termed PTG (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes resulted in a marked increase in PTG expression. Immobilized glutathione S-transferase (GST)-PTG fusion protein specifically bound either PP1 or phosphorylase a. Addition of soluble GST-PTG to 3T3-L1 lysates increased PP1 activity against 32P-labeled phosphorylase a by decreasing the Km of PP1 for phosphorylase 5-fold, while having no effect on the Vmax of the dephosphorylation reaction. Alternatively, PTG did not affect PP1 activity against hormone-sensitive lipase. PTG was not a direct target of intracellular signaling, as insulin or forskolin treatment of cells did not activate a kinase capable of phosphorylating PTG in vivo or in vitro. Finally, PTG decreased the ability of DARPP-32 to inhibit PP1 activity from 3T3-L1 adipocyte lysates. These data cumulatively suggest that PTG increases PP1 activity against specific proteins by several distinct mechanisms.

To investigate the role of N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP)-containing fusion complexes in glucose transporter-4 (GLUT4) membrane trafficking, the subcellular distributions of NSF, alpha-SNAP, and gamma-SNAP in primary rat adipocytes were determined. A large fraction of the NSF and SNAPs were associated with intracellular membranes, distributed between the low-density microsomes (LDM) and high-density microsomes. Very little of the NSF and SNAPs were associated with the plasma membrane fraction. This distribution did not change after insulin stimulation. Approximately 75% of the NSF and SNAPs in the LDM fraction were coimmunoprecipitated with 85% of the GLUT4 and 60% of the vesicle associated membrane proteins (VAMPs; synaptobrevins) VAMP-2 and cellubrevin in anti-GLUT4 immunoadsorptions. In contrast to NSF and the SNAPs, the beta-coatomer protein (beta-COP) found in the LDM fraction was excluded from GLUT4 vesicles. When LDM fractions were solubilized with Thesit (octaethylene glycol dodecyl ether) or Triton X-100, approximately 40% of the alpha-SNAP was colocalized with NSF on glycerol gradients in large (approximately 20S), ATP-sensitive complexes. VAMP-2 and cellubrevin are concentrated in the LDM fractions and in GLUT4 vesicles; both were excluded from these complexes. These data suggest that the steady state association of NSF and the SNAPs with GLUT4 vesicles and cell membranes is independent of the formation of fusion complexes.

The insulin-regulated glucose transporter isotype GlutT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4. However, the tissue distribution of vp165 is much wider than that of GluT4; consequently, vp165 may also function in processes unrelated to insulin action and may serve as a ubiquitous marker for a specialized regulated secretory vesicle.

Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90Rsk, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3'-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 microM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.

The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin-sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin-associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton-insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin-dependent tyrosine phosphorylation of caveolin represents a novel, insulin-specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor.

The precise mechanism by which insulin regulates glucose metabolism is not fully understood. However, it is known that insulin activates two enzymes, phosphatidylinositol 3'-kinase (PI 3'-K) and mitogen-activated protein kinase (MAPK), which may be involved in stimulating the metabolic effects of insulin. The role of these enzymes in glucose metabolism was examined by comparing the effects of insulin, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in 3T3-L1 adipocytes. Treatment of the cells with PDGF or EGF for 5 min increased the MAPK activity 3-5-fold, while insulin treatment produced a 2.5-fold increase. The MAPK activity remained elevated for 1 h after either PDGF or insulin treatment. PDGF and insulin, but not EGF, caused a transient increase in the amount PI 3'-K activity coprecipitated with tyrosine phosphorylated proteins. Although PDGF and insulin caused a similar increase in the activities of these two enzymes, only insulin caused substantial increases in glucose utilization. Insulin increased the transport of glucose and the synthesis of lipid 4- and 17-fold, respectively, while PDGF did not affect these processes significantly. Glycogen synthesis was increased 15-fold in response to insulin and only 3-fold in response to PDGF. Thus, the activation of MAPK and PI 3'-K are not sufficient for the complete stimulation of glucose transport, lipid synthesis, or glycogen synthesis by hormones in 3T3-L1 adipocytes, suggesting a requirement for other signaling mechanisms that may be uniquely responsive to insulin.

Caveolae, also termed plasmalemmal vesicles, are small, flask-shaped, non-clathrin-coated invaginations of the plasma membrane. Caveolin is a principal component of the filaments that make up the striated coat of caveolae. Using caveolin as a marker protein for the organelle, we found that adipose tissue is the single most abundant source of caveolae identified thus far. Caveolin mRNA and protein are strongly induced during differentiation of 3T3-L1 fibroblasts to adipocytes; during adipogenesis there is also a dramatic increase in the complexity of the protein composition of caveolin-rich membrane domains. About 10-15% of the insulin-responsive glucose transporter GLUT4 is found in this caveolin-rich fraction, and immuno-isolated vesicles containing GLUT4 also contain caveolin. However, in non-stimulated adipocytes the majority of caveolin fractionates with the plasma membrane, while most GLUT4 associates with low-density microsomes. Upon addition of insulin to 3T3-L1 adipocytes, there is a significant increase in the amount of GLUT4 associated with caveolin-rich membrane domains, an increase in the amount of caveolin associated with the plasma membrane, and a decrease in the amount of caveolin associated with low-density microsomes. Caveolin does not undergo a change in phosphorylation upon stimulation of 3T3-L1 adipocytes with insulin. However, after treatment with insulin it is associated with a 32-kD phosphorylated protein. Caveolae thus may play an important role in the vesicular transport of GLUT4 to or from the plasma membrane. 3T3-L1 adipocytes offer an attractive system to study the function of caveolae in several cellular trafficking and signaling events.

Signal transduction pathways stimulated by insulin or insulin-like growth factor-I (IGF-I) were compared in transfected NIH3T3 fibroblast cell lines expressing the human insulin receptor, IGF-I receptor, or a chimeric IGF-I receptor with its carboxy-terminal tail replaced with that of the insulin receptor (approximately 1 x 10(6) receptors/cell). Although receptor autophosphorylation was very similar in the three cell lines overexpressing receptors (EC50 = 1-3 nM), there were differences detected in the protein tyrosine phosphorylation stimulated by insulin and IGF-I in these cells. Although no substrates specific for the insulin receptor were detected, phosphorylation of a 170-kilodalton (kDa; IRS-1) and a 70-kDa protein was 10 times more sensitive to insulin than to IGF-I (EC50 = 1.5-2.5 vs. 14-23 nM). The chimeric receptor stimulated significantly lower levels of phosphorylation of several proteins relative to the wild-type IGF-I receptor. Activation of phosphatidylinositol 3'-kinase paralleled phosphorylation of the 170- and 70-kDa proteins. Despite these differences in protein tyrosine phosphorylation, stimulation of mitogen-activated protein (MAP) kinase and DNA synthesis were very similar in the three cell lines overexpressing receptors. Little difference was detected in Shc phosphorylation or MAP kinase activation through the three receptors, although activation of MAP kinase was more efficiently coupled to the platelet-derived growth factor receptor than to any of the overexpressed receptors. All three receptors stimulated DNA synthesis to levels comparable to 10% serum, with similar sensitivities (EC50 = 1.5-3.5 nM).

GLUT4-enriched vesicles from fat and muscle cells contain a major protein of 165 kDa. In the present study we have prepared GLUT4 vesicles from a large number of rat adipocytes, isolated the 165-kDa protein by SDS-gel electrophoresis, and obtained the sequences of four tryptic peptides. Comparison of the sequences with those in protein data banks showed that the 165-kDa protein had not previously been sequenced. An antibody preparation against one of the peptides immunoblotted the 165-kDa protein. By this means it was found that the 165-kDa protein was concentrated in GLUT4 vesicles, and that insulin treatment of rat adipocytes caused a translocation of the protein to the plasma membrane that paralleled the translocation of GLUT4.

We have previously described the isolation of a Chinese hamster ovary cell line, TfT1.11, that has a pleiotropic, temperature-conditional defect in receptor recycling (Cain, C. C., Wilson, R. B., and Murphy, R. F. (1991) J. Biol. Chem. 266, 11746-11752). These cells show a rapid loss of cell surface receptors upon temperature shift due to a reduction in the rate of receptor recycling. We show here that, in addition to altered receptor recycling, TfT1.11 cells show three defects in lysosome biogenesis. At the nonpermissive temperature, they 1) redistribute at least one lysosomal enzyme from lysosomes to endosomes, 2) fail to transfer fluid-phase material from early endosomes to later compartments, and 3) fail to accumulate fluid-phase markers due to increased efflux of internalized material. The results suggest that the processes of recycling from the endosome and movement of material from endosomes to lysosomes are tightly linked.