Discovery of novel pyridazine derivatives as glucose transporter type 4 (GLUT4) translocation activators

We report herein the synthesis and structure-activity relationships (SAR) of a series of pyridazine derivatives with the activation of glucose transporter type 4 (GLUT4) translocation. Through a cell-based phenotype screening in L6-GLUT4-myc myoblasts and functional glucose uptake assays, lead compound 1a was identified as a functional small molecule. After further derivatization, the thienopyridazine scaffold as the central ring (B-part) was revealed to have potent GLUT4 translocation activities. Consequently, we obtained promising compound 26b, which showed a significant blood glucose lowering effect in the severe diabetic mice model (10-week aged db/db mice) after oral dosing even at 10 mg/kg, implying that our pyridazine derivatives have potential to become novel therapeutic agents for diabetes mellitus.     

Kaempferitrin inhibits GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes

Insulin stimulated GLUT4 (glucose transporter 4) translocation and glucose uptake in muscles and adipocytes is important for the maintenance of blood glucose homeostasis in our body. In this paper, we report the identification of kaempferitrin (kaempferol 3,7-dirhamnoside), a glycosylated flavonoid, as a compound that inhibits insulin stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. In the absence of insulin, we observed that addition of kaempferitrin did not affect GLUT4 translocation or glucose uptake. On the other hand, kaempferitrin acted as an inhibitor of insulin-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes by inhibiting Akt activation. Molecular docking studies using a homology model of GLUT4 showed that kaempferitrin binds directly to GLUT4 at the glucose transportation channel, suggesting the possibility of a competition between kaempferitrin and glucose during the transport. Taken together, our data demonstrates that kaempferitrin inhibits GLUT4 mediated glucose uptake at least by two different mechanisms, one by interfering with the insulin signaling pathway and the other by a possible competition with glucose during the transport.     

The ubiquitin regulatory X (UBX) domain-containing protein TUG regulates the p97 ATPase and resides at the endoplasmic reticulum-golgi intermediate compartment

p97/VCP is a hexameric ATPase that is coupled to diverse cellular processes, such as membrane fusion and proteolysis. How p97 activity is regulated is not fully understood. Here we studied the potential role of TUG, a widely expressed protein containing a UBX domain, to control mammalian p97. In HEK293 cells, the vast majority of TUG was bound to p97. Surprisingly, the TUG UBX domain was neither necessary nor sufficient for this interaction. Rather, an extended sequence, comprising three regions of TUG, bound to the p97 N-terminal domain. The TUG C terminus resembled the Arabidopsis protein PUX1. Similar to the previously described action of PUX1 on AtCDC48, TUG caused the conversion of p97 hexamers into monomers. Hexamer disassembly was stoichiometric rather than catalytic and was not greatly affected by the p97 ATP-binding state or by TUG N-terminal regions in vitro. In HeLa cells, TUG localized to the endoplasmic reticulum-to-Golgi intermediate compartment and endoplasmic reticulum exit sites. Although siRNA-mediated TUG depletion had no marked effect on total ubiquitylated proteins or p97 localization, TUG overexpression caused an accumulation of ubiquitylated substrates and targeted both TUG and p97 to the nucleus. A physiologic role of TUG was revealed by siRNA-mediated depletion, which showed that TUG is required for efficient reassembly of the Golgi complex after brefeldin A removal. Together, these data support a model in which TUG controls p97 oligomeric status at a particular location in the early secretory pathway and in which this process regulates membrane trafficking in various cell types.     

Isoproterenol inhibits cyclic AMP-mediated but not insulin-mediated translocation of the GLUT4 glucose transporter isoform

Isoproterenol is a beta adrenergic agonist whose effects have been attributed to the generation of cAMP. Previous studies have shown that it inhibits glucose transport in adipocytes without changing the number of insulin-responsive glucose transporters (GLUT4) on the cell surface. However, we have shown previously that cAMP stimulates translocation of GLUT4 to the cell surface in adipocytes (Keladaet al. J Biol Chem 267, 7021–7025, 1992). We therefore further investigated the mechanisms involved in isoproterenol regulation of glucose transport. Consistent with the effects of dibutyryl cAMP, we found that a low concentration of isoproterenol (10 nM) stimulated glucose transport and the translocation of GLUT4 from the low density microsomal fraction to the plasma membrane. By contrast, a higher concentration of isoproterenol (1 M) did not stimulate transport or GLUT4 translocation and furthermore inhibited dibutyryl cAMP-stimulated GLUT4 translocation. This inhibitory effect was specific for cAMP since isoproterenol had no effect on insulin-stimulated GLUT4 translocation. We conclude that isoproterenol has a biphasic effect on glucose transport, mediated by acute translocation of GLUT4 at low concentrations and by inhibition of intrinsic activity at high concentration, both of which may be explained by effects of cAMP. It has a further cAMP-independent effect at high concentration to inhibit cAMP-mediated translocation of GLUT4.This work forms portions of the PhD thesis requirements.     

Selective regulation of the perinuclear distribution of glucose transporter 4 (GLUT4) by insulin signals in muscle cells

Insulin regulates glucose transporter 4 (GLUT4) availability at the surface of muscle and adipose cells. In L6 myoblasts, stably expressed GLUT4myc is detected mostly in a perinuclear region. In unstimulated cells, about half of perinuclear GLUT4myc colocalizes with the transferrin receptor (TfR). Insulin stimulation selectively decreased the perinuclear colocalization of GLUT4myc with TfR determined by 3D-reconstruction of fluorescence images. Perinuclear GLUT4myc adopted two main distributions defined morphometrically as 'conical' and 'concentric'. Insulin rapidly reduced the proportion of cells with conical in favor of concentric perinuclear GLUT4myc distributions in association with the gain in surface GLUT4myc. Upon removal of insulin, the GLUT4myc perinuclear distribution and surface levels reversed in parallel. In contrast, hypertonicity (which like insulin elevates surface GLUT4myc) did not elicit perinuclear GLUT4myc redistribution. Insulin also caused redistribution of perinuclear vesicle-associated membrane protein-2 (VAMP2), without alteration of perinuclear TfR and VAMP3. Inhibitory mutants of phosphatidylinositol-3 kinase (Deltap85) or Akt substrate AS160 (AS160-4P) prevented insulin-mediated perinuclear GLUT4myc redistribution. Tetanus toxin expression did not prevent the perinuclear GLUT4myc redistribution, suggesting that redistribution is independent of GLUT4myc fusion with the plasma membrane. We propose that insulin causes selective, dynamic relocalization of perinuclear GLUT4myc and VAMP2 and perinuclear GLUT4myc redistribution is a direct target of insulin-derived signals.     

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD⁺-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.     

Functional roles of BCAR3 in the signaling pathways of insulin leading to DNA synthesis,membrane ruffling and GLUT4 translocation

Breast cancer anti-estrogen resistance 3 (BCAR3) is an SH2-containing signal transducer and is implicated in tumorigenesis of breast cancer cells. In this study, we found that BCAR3 mediates the induction of ERK activation and DNA synthesis by insulin, but not by IGF-1. Specifically, the SH2 domain of BCAR3 is involved in insulin-stimulated DNA synthesis. Differential tyrosine-phosphorylated patterns of the BCAR3 immune complex were detected in insulin and IGF-1 signaling, suggesting that BCAR3 is a distinct target molecule of insulin and IGF-1 signaling. Moreover, microinjection of BCAR3 inhibitory materials inhibited membrane ruffling induced by insulin, while this did not affect insulin-mediated GLUT4 translocation. Taken together, these results demonstrated that BCAR3 plays an important role in the signaling pathways of insulin leading to cell cycle progression and cytoskeleton reorganization, but not GLUT4 translocation.     

N-glycosylation is critical for the stability and intracellular trafficking of glucose transporter GLUT4

The facilitative glucose transporter GLUT4 plays a key role in regulating whole body glucose homeostasis. GLUT4 dramatically changes its distribution upon insulin stimulation, and insulin-resistant diabetes is often linked with compromised translocation of GLUT4 under insulin stimulation. To elucidate the functional significance of the sole N-glycan chain on GLUT4, wild-type GLUT4 and a GLUT4 glycosylation mutant conjugated with enhanced GFP were stably expressed in HeLa cells. The N-glycan contributed to the overall stability of newly synthesized GLUT4. Moreover, cell surface expression of wild-type GLUT4 in HeLa cells was elevated upon insulin treatment, whereas the glycosylation mutant lost the ability to respond to insulin. Subcellular distribution of the mutant was distinct from that of wild-type GLUT4, implying that the subcellular localization required for insulin-mediated translocation was impaired in the mutant protein. Interestingly, kifunensine-treated cells also lost sensitivity to insulin, suggesting the functional importance of the N-glycan structure for GLUT4 trafficking. The K(m) or turnover rates of wild-type and mutant GLUT4, however, were similar, suggesting that the N-glycan had little effect on transporter activity. These findings underscore the critical roles of the N-glycan chain in quality control as well as intracellular trafficking of GLUT4.     

ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin activates glucose transport by promoting translocation of the insulin-sensitive fat/muscle-specific glucose transporter GLUT4 from an intracellular storage compartment to the cell surface. Here we report that an optimal insulin effect on glucose uptake in 3T3-L1 adipocytes is dependent upon expression of both PIKfyve, the sole enzyme for PtdIns 3,5-P(2) biosynthesis, and the PIKfyve activator, ArPIKfyve. Small-interfering RNAs that selectively ablated PIKfyve or ArPIKfyve in this cell type depleted the PtdIns 3,5-P(2) pool and reduced insulin-activated glucose uptake to a comparable degree. Combined loss of PIKfyve and ArPIKfyve caused further PtdIns 3,5-P(2) ablation that correlated with greater attenuation in insulin responsiveness. Loss of PIKfyve-ArPIKfyve reduced insulin-stimulated Akt phosphorylation and the cell surface accumulation of GLUT4 or IRAP, but not GLUT1-containing vesicles without affecting overall expression of these proteins. ArPIKfyve and PIKfyve were found to physically associate in 3T3-L1 adipocytes and this was insulin independent. In vitro labeling of membranes isolated from basal or insulin-stimulated 3T3-L1 adipocytes documented substantial insulin-dependent increases of PtdIns 3,5-P(2) production on intracellular membranes. Together, the data demonstrate for the first time a physical association between functionally related PIKfyve and ArPIKfyve in 3T3-L1 adipocytes and indicate that the novel ArPIKfyve-PIKfyve-PtdIns 3,5-P(2) pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport.     

葡萄糖转运子4 转位信号转导通路的研究进展

葡萄糖是大部分细胞主要能量来源,它进入细胞的过程在生命的维持中无疑成为一个重要的步骤。而葡萄糖进入细胞是依赖于这些细胞上的葡萄糖转运子和相应的对其进行调节的因子。葡萄糖转运子4（GLUT4）在糖进入细胞维持血糖平衡中起了重要的作用。近年有关GLUT4的研究文献很多,但却总给人不系统的感觉。本文对GLUT4转位的胰岛素依赖和非胰岛素依赖的信号途径以及其远端过程及机制作一综述,同时分析了GLUT4转位的信号途径的研究中存在的问题和将来研究的方向。     

GLUT4 molecules are recruited at random for insertion within the plasma membrane upon insulin stimulation

Glucose transporter 4 (GLUT4) is efficiently retained intracellularly. Here, we investigated the insulin-induced reduction of retention. While increasing insulin concentrations led to gradual increases in both the amount of recycling GLUT4 molecules and cell surface GLUT4 levels, the kinetics of the increase in time was independent of insulin concentration. To determine whether there are GLUT4 subpools that have a distinct insulin sensitivity, adipocytes were consecutively stimulated twice with a low concentration of insulin while recycling GLUT4 molecules were continuously labeled. This revealed that not the same pool of GLUT4 molecules was mobilized twice and thus that upon insulin stimulation, GLUT4 is likely to be recruited at random for insertion within the plasma membrane.     

Adipsin and the glucose transporter GLUT4 traffic to the cell surface via independent pathways in adipocytes

Insulin increases the exocytosis of many soluble and membrane proteins in adipocytes. This may reflect a general effect of insulin on protein export from the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membrane proteins GLUT4 and transferrin receptors in 3T3-L1 adipocytes. We show that adipsin is secreted from the trans Golgi network to the endosomal system, as ablation of endosomes using transferrin-HRP conjugates strongly inhibited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan-1-ol blocked adipsin secretion and resulted in accumulation of adipsin in trans Golgi network-derived vesicles. In contrast, butan-1-ol did not affect the insulin-stimulated movement of transferrin receptors to the plasma membrane, whereas this was abrogated following endosome ablation. GLUT4 trafficking to the cell surface does not utilise this pathway, as insulin-stimulated GLUT4 translocation is still observed after endosome ablation or inhibition of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D-dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.     

Insulin-stimulated GLUT4 protein translocation in adipocytes requires the Rab10 guanine nucleotide exchange factor Dennd4C

Insulin-stimulated translocation of the glucose transporter GLUT4 to the cell surface in fat and muscle cells is the basis for insulin-stimulated glucose transport. Studies in adipocytes strongly support the following molecular mechanism for this process. Insulin-elicited phosphorylation of the GTPase-activating protein TBC1D4 (AS160) suppresses its activity toward Rab10 and thereby leads to an increase in the GTP-bound form of Rab10, which in turn triggers movement of vesicles containing GLUT4 to the plasma membrane and their fusion with the membrane. This process is expected to require the participation of a guanine nucleotide exchange factor (GEF) to generate the GTP-bound form of Rab10, but this GEF has not hitherto been identified. The present study identifies Dennd4C, a recently described GEF for Rab10, as the primary GEF required for GLUT4 translocation. Knockdown of Dennd4C markedly inhibited GLUT4 translocation, and ectopic expression of Dennd4C slightly stimulated it. Dennd4C was found in isolated GLUT4 vesicles. This study thus identifies another key component in the machinery of GLUT4 translocation. Moreover, it provides a potential explanation for the moderate association of a variant in the Dennd4C gene with type 2 diabetes.     

N-Acetylated alpha-linked acidic dipeptidase expressed in rat adipocytes is localized in the insulin-responsive glucose transporter (GLUT4) intracellular compartments and involved in the insulin-stimulated GLUT4 recruitment

The GLUT4-containing vesicles purified from rat adipocyte contain many protein species of unknown identity, some of which are likely to play a critical role in the trafficking of GLUT4. Presently, we describe an 85-kDa protein in GLUT4-vesicles of rat adipocytes as a potential GLUT4 traffic regulatory protein. MALDI-TOF MS, RT-PCR, gene cloning, protein sequence analysis, and immunoreactivity assay have identified this protein as N-acetylated alpha-linked acidic dipeptidase (NAALADase) expressed in rat adipocytes. NAALADase in rat adipocytes was mostly membrane-associated and colocalized in discrete GLUT4-compartments with enrichment in putative GLUT4-sorting endosomes (G4G(L)). Total cell lysates of adipocytes exhibited NAALADase activity. Next, we treated rat adipocytes with 2-[phosphonomethy]pentanedionic acid (2-PMPA), a potent NAALADase inhibitor, and studied its effect on the distribution of GLUT4 and 3-O-methyl glucose (3OMG) flux. In 2-PMPA-treated adipocytes, there was a significant reduction (by 40%) in the insulin-stimulated GLUT4 translocation to the plasma membrane. The 3OMG flux in insulin-stimulated adipocytes was also delayed (51% of control) by 2-PMPA treatment, indicating that 2-PMPA impairs insulin-stimulated GLUT4 recruitment and the uptake of glucose. It is suggested that NAALADase may function as a regulator required for the insulin-stimulated GLUT4 vesicle movement and/or its exocytosis, thus may regulate insulin-induced GLUT4 recruitment in rat adipocytes.     

Gallic acid induces GLUT4 translocation and glucose uptake activity in 3T3-L1 cells

GLUT4, a 12 transmembrane protein, plays a major role in insulin mediated glucose transport in muscle and adipocytes. For glucose transport, the GLUT4 protein needs to be translocated to the plasma membrane from the intracellular pool and it is possible that certain compounds may be able to enhance this process. In the present work, we have shown that gallic acid can increase GLUT4 translocation and glucose uptake activity in an Akt-independent but wortmannin-sensitive manner. Further analysis suggested the role of atypical protein kinase Cζ/λ in gallic acid mediated GLUT4 translocation and glucose uptake.     

Role of SGK1 kinase in regulating glucose transport via glucose transporter GLUT4

Insulin stimulates glucose transport into muscle and fat cells by enhancing GLUT4 abundance in the plasma membrane through activation of phosphatidylinositol 3-kinase (PI3K). Protein kinase B (PKB) and PKCzeta are known PI3K downstream targets in the regulation of GLUT4. The serum- and glucocorticoid-inducible kinase SGK1 is similarly activated by insulin and capable to regulate cell surface expression of several metabolite transporters. In this study, we evaluated the putative role of SGK1 in the modulation of GLUT4. Coexpression of the kinase along with GLUT4 in Xenopus oocytes stimulated glucose transport. The enhanced GLUT4 activity was paralleled by increased transporter abundance in the plasma membrane. Disruption of the SGK1 phosphorylation site on GLUT4 ((S274A)GLUT4) abrogated the stimulating effect of SGK1. In summary, SGK1 promotes glucose transporter membrane abundance via GLUT4 phosphorylation at Ser274. Thus, SGK1 may contribute to the insulin and GLUT4-dependent regulation of cellular glucose uptake.     

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.     

GLUT4 protein is differently modulated during development of obesity in monosodium glutamate-treated mice

The aim of the present study was to investigate the GLUT4 protein expression during the development of obesity in monosodium glutamate- (MSG) treated mice. Control (C) and neonatally MSG-treated 2-month-old (2-mo), 4-month-old (4-mo) and 7-month-old (7-mo) mice were analyzed. Anthropometric data, basal glycemia and insulinemia were measured; and the GLUT4 protein was assessed by Western blotting in white adipose tissue (WAT), skeletal muscle gastrocnemius (SM) and heart (H). Compared to age-matched C mice, the 2-mo and 4-mo MSG mice were already obese, but metabolically they showed increased or preserved whole-body insulin sensitivity, respectively. At these ages they showed unchanged total GLUT4 content in SM and H. However, in plasma membrane fraction from WAT, the MSG showed increased GLUT4 content at both 2- (by 60%) and 4-month (by 45%) of age. When the GLUT4 protein was expressed by unit of adipocyte surface area the protein amount was increased by 36 and 220% in 2-mo and 4-mo MSG mice, respectively. At 7 months of age, obesity was fully established in MSG mice, showing a strongly insulin resistant condition. Additionally, in the 7-mo MSG-mice the GLUT4 protein was reduced in SM (by 40%), H (by 28%), PM and M fractions of WAT (by approximately 70%), and PM expressed by unit of adipocyte surface area (by 92%). The data demonstrate that early, during the accelerated development of obesity in MSG-treated mice, the GLUT4 content was increased in WAT, and that may play a key role in the development of obesity. Later on, when obesity is fully established, the GLUT4 protein was reduced in SM, heart and WAT, and that may be involved in the insulin resistance present in this condition.     

The fatty acid translocase (FAT)/CD36 and the glucose transporter GLUT4 are localized in different cellular compartments in rat cardiac muscle

The fatty acid translocase (FAT)/CD36 plays an important role in the acute regulation of fatty acid uptake in muscle tissue. We studied the subcellular distribution of FAT/CD36 in rat cardiac muscle after in vivo insulin stimulation by membrane fractionation and immunoisolation of GLUT4- and FAT/CD36-vesicles. FAT/CD36 was equally present in both plasma and microsomal membranes with no effect of insulin on the cellular distribution, whereas GLUT4 increased 2- to 3-fold in the plasma membrane. FAT/CD36 resides in one intracellular pool, whereas GLUT4 is present in two distinct pools. Immunoadsorption of GLUT4-vesicles indicated that FAT/CD36 is undetectable in these vesicles. Likewise, no GLUT4 could be detected in FAT/CD36-vesicles. These vesicles contain a high amount of Rab11 that remained unaffected after insulin stimulation, whereas Rab11 increased about 3-fold in the GLUT4-vesicles in response to insulin. These data show that GLUT4 and FAT/CD36 do not co-localize in cardiac muscle and that FAT/CD36 is not redistributed in response to insulin in the heart. Rab11 may be involved in endosomal recycling of FAT/CD36, however, insulin-associated Rab11 functions appear to be limited to GLUT4-vesicles.