Hexose transporter mRNAs for GLUT4, GLUT5, and GLUT12 predominate in human muscle

In the past few years, 8 additional members of the facilitative hexose transporter family have been identified, giving a total of 14 members of the SLC2A family of membrane-bound hexose transporters. To determine which of the new hexose transporters were expressed in muscle, mRNA concentrations of 11 glucose transporters (GLUTs) were quantified and compared. RNA from muscle from 10 normal volunteers was subjected to RT-PCR. Primers were designed that amplified 78- to 241-base fragments, and cDNA standards were cloned for GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, and GAPDH. Seven of these eleven hexose transporters were detectable in normal human muscle. The rank order was GLUT4, GLUT5, GLUT12, GLUT8, GLUT11, GLUT3, and GLUT1, with corresponding concentrations of 404 +/- 49, 131 +/- 14, 33 +/- 4, 5.5 +/- 0.5, 4.1 +/- 0.4, 1.2 +/- .0.1, and 0.9 +/- 0.2 copies/ng RNA (means +/- SE), respectively, for the 10 subjects. Concentrations of mRNA for GLUT4, GLUT5, and GLUT12 were much higher than those for the remainder of the GLUTs and together accounted for 98% of the total GLUT isoform mRNA. Immunoblots of muscle homogenates verified that the respective proteins for GLUT4, GLUT5, and GLUT12 were present in normal human muscle. Immunofluorescent studies demonstrated that GLUT4 and GLUT12 were predominantly expressed in type I oxidative fibers; however, GLUT5 was expressed predominantly in type II (white) fibers.     

Differential localization of glucose transporter isoforms in non-polarized mammalian cells: distribution of GLUT1 but not GLUT3 to detergent-resistant membrane domains

The hexose transporter family, which mediates a facilitated uptake in mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site. However, it remains unknown how these isoforms are functionally organized in the membrane domains. In this report, we describe a differential distribution of the glucose transporter isoforms GLUT1 and GLUT3 to detergent-resistant membrane domains (DRMs) in non-polarized mammalian cells. Whereas more than 80% of cellular proteins containing GLUT3 in HeLa cell lines was solubilized by a non-ionic detergent (either Triton X-100 or Lubrol WX) at 4 degrees C, GLUT1 remained insoluble together with the DRM-associated proteins, such as caveolin-1 and intestinal alkaline phosphatase (IAP). These DRM-associated proteins and the ganglioside GM1 were shown to float to the upper fractions when Triton X-100-solubilized cell extracts were centrifuged on a density gradient. In contrast, GLUT3 as well as most soluble proteins remained in the lower layers. Furthermore, perturbations of DRMs due to depletion of cholesterol by methyl-beta-cyclodextrin (m beta CD) rendered GLUT1 soluble in Triton X-100. Immunostaining patterns for these isoforms detected by confocal laser scanning microscopy in a living cell were also distinctive. These results suggest that in non-polarized mammalian cells, GLUT1 can be organized into a raft-like DRM domain but GLUT3 may distribute to fluid membrane domains. This differential distribution may occur irrespective of the N-glycosylation state or cell type.     

Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues

It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2-6 and 9. The classical expression of GLUT1-5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers.     

Human articular chondrocytes express three facilitative glucose transporter isoforms: GLUT1, GLUT3 and GLUT9

Glucose is an important metabolite and a structural precursor for articular cartilage and its transport has significant consequences for cartilage development and functional integrity. In this study the expression of facilitative glucose transporters (GLUTs) in human chondrocytes was investigated. Results showed that at least three GLUT isoforms (GLUT1, GLUT3 and GLUT9) are expressed by normal chondrocytes. Given the central role of glucose in chondrocyte physiology and metabolism, its regular provision via GLUTs will influence the metabolic activity and survival of chondrocytes in cartilage matrices.     

Caveolins and intracellular calcium regulation in human airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is a key factor in airway smooth muscle (ASM) tone. In vascular smooth muscle, specialized membrane microdomains (caveolae) expressing the scaffolding protein caveolin-1 are thought to facilitate cellular signal transduction. In human ASM cells, we tested the hypothesis that caveolae mediate Ca(2+) responses to agonist stimulation. Fluorescence immunocytochemistry with confocal microscopy, as well as Western blot analysis, was used to determine that agonist receptors (M(3) muscarinic, bradykinin, and histamine) and store-operated Ca(2+) entry (SOCE)-regulatory mechanisms colocalize with caveolin-1. Although caveolin-2 coexpressed with caveolin-1, caveolin-3 was absent. In fura 2-loaded ASM cells, [Ca(2+)](i) responses to 1 microM ACh, 10 microM histamine, and 10 nM bradykinin, as well as SOCE, were attenuated (each to a different extent) after disruption of caveolae by the cholesterol-chelating drug methyl-beta-cyclodextrin. Transfection of ASM cells with 50 nM caveolin-1 small interfering RNA significantly weakened caveolin-1 expression and blunted [Ca(2+)](i) responses to bradykinin and histamine, as well as SOCE, but the response to ACh was less intense. These results indicate that caveolae are present in ASM and that caveolin-1 contributes to regulation of [Ca(2+)](i) responses to agonist.     

Similar [DE]XXXL[LI] Motifs Differentially Target GLUT8 and GLUT12 in Chinese Hamster Ovary Cells

The transport of glucose across cell membranes is mediated by facilitative glucose transporters (GLUTs). The recently identified class III GLUT12 is predominantly expressed in insulin-sensitive tissues such as heart, fat and skeletal muscle. We examined the subcellular localization of GLUT12 in Chinese hamster ovary and human embryonic kidney 293 cells stably expressing murine GLUT12. We have previously shown that another class III GLUT8 contains a [DE]XXXL[LI] motif that directs it to late endosomal/lysosomal compartments. Despite also having this highly conserved motif in its amino terminus, GLUT12 does not colocalize with GLUT8. Rather, GLUT12 resides in the Golgi network and at the plasma membrane (PM). Furthermore, GLUT8 and GLUT12 exhibit dramatic differences in trafficking from the PM. Whereas GLUT8 is internalized following its expression at the cell surface, GLUT12 remains largely associated with the PM. To further explore the trafficking mechanisms, we created mutant constructs to explore the potential role of GLUT12's NH₂-terminal dileucine motif in regulating its intracellular sorting. We show that both the GPN and the LL residues within the [DE]XXXL[LI] motif influence the cell surface expression of GLUT12 and conclude that the mechanisms governing the intracellular sorting of GLUT12 are distinct from those regulating the sorting of GLUT8.     

PC12 cells have caveolae that contain TrkA. Caveolae-disrupting drugs inhibit nerve growth factor-induced, but not epidermal growth factor-induced, MAPK phosphorylation

Nerve growth factor (NGF) induces survival and differentiation of the neural crest-derived PC12 cell line. Caveolae are cholesterol-enriched, caveolin-containing plasma membrane microdomains involved in vesicular transport and signal transduction. Here we demonstrate the presence of caveolae in PC12 cells and their involvement in NGF signaling. Our results showed the expression of caveolin-1 by Western blot and confocal immuno-microscopy. The presence of plasma membrane caveolae was directly shown by rapid-freeze deep-etching electron microscopy. Moreover, combined deep-etching and immunogold techniques revealed the presence of the NGF receptor TrkA in the caveolae of PC12 cells. These data together with the cofractionation of Shc, Ras, caveolin, and TrkA in the caveolae fraction supported a role for these plasma membrane microdomains in NGF signaling. To approach this hypothesis, caveolae were disrupted by treatment of PC12 cells with cholesterol binding drugs. Either filipin or cyclodextrin treatment increased basal levels of MAPK phosphorylation. In contrast, pretreatment of PC12 cells with these drugs inhibited the NGF- but not the epidermal growth factor-induced MAPK phosphorylation without affecting the TrkA autophosphorylation. Taken together, our results demonstrate the presence of caveolae in PC12 cells, which contain the high affinity NGF receptor TrkA, and the specific involvement of these cholesterol-enriched plasma membrane microdomains in the propagation of the NGF-induced signal.     

Comparative expression of hexose transporters (SGLT1, GLUT1, GLUT2 and GLUT5) throughout the mouse gastrointestinal tract

Hexose transporters play a pivotal role in the absorption of food-derived monosaccharides in the gastrointestinal tract. Although a basic knowledge of the hexose transporters has already been gained, their detailed distribution and comparative intensities of expression throughout the gastrointestinal tract have not been fully elucidated. In this study, we quantitatively evaluated the expression of SGLT1, GLUT1, GLUT2, and GLUT5 by in situ hybridization and real-time PCR techniques using a total of 28 segments from the gastrointestinal tract of 9-week-old mice. GLUT2 and GLUT5 mRNA expressions were detected predominantly from the proximal to middle parts of the small intestine, showing identical expression profiles, while SGLT1 mRNA was expressed not only in the small intestine but also in the large intestine. Notably, GLUT1 mRNA was expressed at a considerable level in both the stomach and large intestine but was negligible in the small intestine. Immunohistochemistry demonstrated the polarized localization of hexose transporters in the large intestine: SGLT1 on the luminal surface and GLUT1 on the basal side of epithelial cells. The present study provided more elaborate information concerning the localization of hexose transporters in the small intestine. Furthermore, this study revealed the significant expression of glucose transporters in the large intestine, suggesting the existence of the physiological uptake of glucose in that location in mice.     

High glucose-induced RhoA activation requires caveolae and PKCβ1-mediated ROS generation

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We previously showed that RhoA activation by high glucose in mesangial cells (MC) leads to matrix upregulation (Peng F, Wu D, Gao B, Ingram AJ, Zhang B, Chorneyko K, McKenzie R, Krepinsky JC. Diabetes 57: 1683-1692, 2008). Here, we study the mechanism whereby RhoA is activated. In primary rat MC, RhoA activation required glucose entry and metabolism. Broad PKC inhibitors (PMA, bisindolylmaleimide, G?6976), as well as specific PKCβ blockade with an inhibitor and small interfering RNA (siRNA), prevented RhoA activation by glucose. PKCβ inhibition also abrogated reactive oxygen species (ROS) generation by glucose. The ROS scavenger N-acetylcysteine (NAC) or NADPH oxidase inhibitors apocynin and DPI prevented glucose-induced RhoA activation. RhoA and some PKC isoforms localize to caveolae. Chemical disruption of these microdomains prevented RhoA and PKCβ1 activation by glucose. In caveolin-1 knockout cells, glucose did not induce RhoA and PKCβ1 activation; these responses were rescued by caveolin-1 reexpression. Furthermore, glucose-induced ROS generation was significantly attenuated by chemical disruption of caveolae and in knockout cells. Downstream of RhoA signaling, activator protein-1 (AP-1) activation was also inhibited by disrupting caveolae, was absent in caveolin-1 knockout MC and rescued by caveolin-1 reexpression. Finally, transforming growth factor (TGF)-β1 upregulation, mediated by AP-1, was prevented by RhoA signaling inhibition and by disruption or absence of caveolae. In conclusion, RhoA activation by glucose is dependent on PKCβ1-induced ROS generation, most likely through NADPH oxidase. The activation of PKCβ1 and its downstream effects, including upregulation of TGF-β1, requires caveolae. These microdomains are thus important mediators of the profibrogenic process associated with diabetic nephropathy.     

Caveolae Internalization Regulates Integrin-Dependent Signaling Pathways

Integrin-mediated adhesion regulates trafficking of cholesterol-enriched membrane microdomains (CEMM). Upon cell detachment from the extracellular matrix (ECM), CEMMs undergo rapid internalization and are cleared from the plasma membrane. This pathway regulates integrin-mediated Rac membrane targeting, allowing coupling of Rac to downstream effectors. Internalization of CEMMs is mediated by Dynamin-2, a regulator of caveolae dynamics, and caveolin-1, an essential caveolae coat protein. Translocation of tyrosine phosphorylated caveolin-1 from focal adhesions to caveolae upon cell detachment induces CEMM internalization. Notably, integrin-mediated regulation of Erk, phosphatidylinositol-3-OH kinase (PI3K) and Rac pathways is dependent on caveolin-1. These results describe a novel pathway in which integrins prevent downregulation of Erk, PI3K and Rac-dependent pathways by inhibiting caveolin-1-dependent endocytosis. This pathway define a novel molecular mechanism for regulated cell growth and tumor suppression by caveolin-1.     

Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by a facilitative glucose carrier

In most animal cells, transport of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUT). Mammals express at least five distinct transporters (GLUTs 1--5), which are well characterised both functionally and genetically. In contrast, the glucose transport system of fish remains poorly studied. Here we report studies of hexose uptake in carp EPC cells and cloning of a glucose transporter cDNA from these cells. Transport of radio-labelled methylglucose (3-OMG) followed Michaelis--Menten kinetics with a K(m) value (8.5 mM) similar to that of mammalian cells. The inhibition of transport by cytochalasin B and phloretin, but not by phloridzin or cyanide, strongly suggested the existence of a facilitative carrier. D-Glucose, 2-deoxyglucose, 3-OMG, D-mannose and D-xylose were competitive inhibitors of 3-OMG uptake, while L-glucose, mannitol, D-fructose, D-ribose and sucrose did not compete with 3-OMG. We cloned a carp glucose transporter (CyiGLUT1), using RT-PCR and RACE strategies. CyiGLUT1 was different from known carp and zebrafish EST sequences. The complete cDNA (3060 bp) contained one open reading frame encoding a predicted protein of 478 amino acids. The deduced amino acid sequence shared 78% identity with mammalian and avian GLUT1 proteins. Key amino acids involved in substrate selection and catalysis of mammalian GLUTs were conserved in the carp transporter.     

Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms

We describe the functional expression of three members of the family of human facilitative glucose transporters, the erythrocyte-type transporter (GLUT 1), the liver-type transporter (GLUT 2), and the brain-type transporter (GLUT 3), by microinjection of their corresponding mRNAs into Xenopus oocytes. Expression was determined by the appearance of transport activity, as measured by the transport of 3-O-methyl-D-glucose or 2-deoxy-D-glucose. We have measured the Km for 3-O-methyl-D-glucose of GLUTs 1, 2, and 3, and the results are discussed in light of the possible roles for these different transporters in the regulation of blood glucose. The substrate specificity of these transporter isoforms has also been examined. We show that, for all transporters, the transport of 2-deoxy-D-glucose is inhibited by D-but not by L-glucose. In addition, both D-galactose and D-mannose are transported by GLUTs 1-3 at significant rates; furthermore, GLUT 2 is capable of transporting D-fructose. The nature of the glucose binding sites of GLUTs 1-3 was investigated by using hexose inhibition of 2-deoxy-D-glucose uptake. We show that the characteristics of this inhibition are different for each transporter isoform.     

心肌肌浆网膜含有膜质微区结构

细胞膜质微区(microdomain)是细胞膜上特殊的结构域,在细胞信号转导和物质运输过程中起着非常重要的作用.绝大多数膜质微区来源于全细胞膜,即包括质膜和细胞器膜.最新研究表明细胞器膜如高尔基体膜也有膜质微区,因此分离了猪心肌浆网膜的膜质微区.首先获得了没有质膜污染的猪心肌浆网,用去污剂TritonX-100处理该肌浆网,获得了去污剂不溶的质膜微区(SR-DRM),该微区富集胆固醇和鞘磷脂.质膜微区的标记脂和蛋白质:神经节苷脂GM1和Caveolin-3也在该区富集.同时还研究了心肌浆网Ca2 -ATPase(SERCA2a)的分布,结果表明,相当数量的SERCA2a分布在膜质微区,并且有正常的生理功能.上述研究结果表明,在心肌浆网膜上有膜质微区的存在,进一步证明膜质微区不仅存在于细胞质膜,也普遍存在于细胞器膜.     

Energy metabolism and its linkage to intracellular Ca2+ and pH regulation in rat spermatogenic cells

Energy metabolism and intracellular adenine nucleotides of meiotic and postmeiotic spermatogenic cells are highly dependent on external substrates for oxidative phosphorylation and glycolysis. Using fluorescent probes to measure the changes in cytosolic [Ca2+] ([Ca2+]i) and pH (pHi), we were able to demonstrate that changes in energy metabolism of meiotic and postmeiotic spermatogenic cells were rapidly translated into changes of pHi and [Ca2+]i in the absence or presence of external Ca2+. Under these conditions, mitochondria were gaining cytosolic calcium in these cells. Our results indicate that Ca2+ mobilised by changes in metabolic energy pathways originated in thapsigargin-sensitive intracellular Ca2+ stores. Changes in intracellular adenine nucleotides, measured by HPLC, and a likely colocalization of ATP-producing and ATP-consuming processes in the cells seemed to provide the linkage between metabolic fluxes and the changes in pHi and [Ca2+]i in pachytene spermatocytes and round spermatids. Glucose metabolism produced an increase of [Ca2+]i in round spermatids but not in pachytene spermatocytes, and a decrease in pHi in both cell types. Hence, glucose emerges as a molecule that can differentially modulate [Ca2+]i and pHi in pachytene spermatocytes and round spermatids in rats.     

Involvement of caveolin-2 in caveolar biogenesis in MDCK cells

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis.     

GLUT7: a new intestinal facilitated hexose transporter

The very last member of the SLC2A gene family of facilitated hexose transporters to be cloned was SLC2A7 (hGLUT7). It has been assigned to the class II of the GLUT family on the basis of sequence similarity, and its closest family member is GLUT5, an intestinal fructose transporter. GLUT7 is primarily expressed in the small intestine and colon, although mRNA has been detected in the testis and prostate as well. The protein is expressed in the apical membrane of the small intestine and colon, and it has a high affinity (<0.5 mM) for glucose and fructose. The abundance of the protein in the small intestine does change in parallel with the dietary carbohydrate. However, the distribution of GLUT7 along the small intestine does not entirely match with the availability of glucose and fructose, suggesting that the physiological substrate for this transporter has yet to be identified. Unlike GLUT13, the proton-coupled myoinositol transporter (HMIT), there is no evidence for the coupling of protons to the hexose movement via GLUT7. One area of study in which GLUT7 has provided a useful comparison with GLUT1 has been in the development of the hypothesis that the facilitated hexose transporters may have a selectivity filter at the exofacial opening of the translocation pore, which helps to determine which hexoses can be transported. If substantiated, the elucidation of this mechanism may prove useful in the design of hexose analogs for use in cancer imaging and therapeutics.     

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose (K(m) = 0.3 mM) and fructose (IC(50) = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 microM d-glucose was not inhibited by 200 microM phloretin or 100 microM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.     

Reggie-1 and reggie-2 localize in non-caveolar rafts in epithelial cells: cellular localization is not dependent on the expression of caveolin proteins

Reggie-1 and reggie-2 are highly conserved and widely expressed proteins associated with membrane rafts. The molecular function of reggies remains to be clarified, but recent data indicate that they are involved in various cellular processes such as insulin signaling, phagocytosis and actin remodeling. However, there is discrepancy in the literature if reggies are associated with caveolae or non-caveolar rafts. Reggies are expressed and raft associated also in many cells which do not contain caveolae, such as neurons and lymphocytes. However, it is not clear if the function or localization of reggies are dependent on the presence of caveolae and expression of caveolin-1 protein. In this study, we directly addressed this question in epithelial cells. We could show that ectopic expression of caveolin-1 does not result in any change in the cellular localization of reggie-1, which is present at the plasma membrane also in the absence of caveolin-1. On the other hand, caveolin-2, which localizes in caveolae, is dependent on caveolin-1 expression in order to be localized at the plasma membrane. Although reggie-1 and reggie-2 strongly interact with each other, we did not detect a direct interaction between caveolin-1 and reggies by means of a yeast two-hybrid assay, nor could reggies be co-immunoprecipitated with caveolin-1. Furthermore, endogenous reggie-1 and -2 were found not to colocalize with caveolin-1 in epithelial cells. Thus, our data indicate that reggies are localized in microdomains different from caveolae, and the function of reggies is different from and independent of caveolin-1.     

Insulin-like growth factor-I activation of Akt survival cascade in neuronal cells requires the presence of its cognate receptor in caveolae

Insulin-like growth factor-I (IGF-I) plays important roles in survival of neurons. Caveolae, cholesterol-rich microdomains of plasma membrane, act as platforms for some neurotrophic factors. In this study, we examined a possible role of caveolae in IGF-I signal transduction in pheochromocytoma PC12 cells. IGF-I treatment attenuated serum withdrawal-induced apoptosis, which was reversed by treatment with methyl-beta-cyclodextrin (CD) that removes cholesterol from plasma membrane. Immunocytochemical and subcellular fractionation analyses revealed that IGF-I receptor (IGF-IR) was colocalized with caveolin-1, a major protein component in caveolae, and that CD treatment reduced IGF-IR contents in caveolae. Consistent with these findings, IGF-I phosphorylation of insulin receptor substrate-1 and Akt was impaired, and cholesterol supply restored the IGF-I action. Furthermore, experiments using small interfering RNA revealed that the reduction of caveolin-1 expression impaired the IGF-I action. In addition, the colocalization of IGF-IR with caveolin-1, and the caveolae-dependent IGF-I action were duplicated in primary culture of rat cerebellar granule neurons. These results demonstrate that the presence of IGF-IR in caveolae is required for the neuroprotective action of IGF-I.     

Activation of the glucose transporter GLUT4 by insulin.

The transport of glucose into cells and tissues is a highly regulated process, mediated by a family of facilitative glucose transporters (GLUTs). Insulin-stimulated glucose uptake is primarily mediated by the transporter isoform GLUT4, which is predominantly expressed in mature skeletal muscle and fat tissues. Our recent work suggests that two separate pathways are initiated in response to insulin: (i) to recruit transporters to the cell surface from intracellular pools and (ii) to increase the intrinsic activity of the transporters. These pathways are differentially inhibited by wortmannin, demonstrating that the two pathways do not operate in series. Conversely, inhibitors of p38 mitogen-activated protein kinase (MAPK) imply that p38 MAPK is involved only in the regulation of the pathway leading to the insulin-stimulated activation of GLUT4. This review discusses the evidence for the divergence of GLUT4 translocation and activity and proposed mechanisms for the regulation of GLUT4.